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PARATHYROID GLAND FUNCTION AND CALCIUM REGULATION IN HEALTHY AND SEPTIC HORSES
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Ramiro E. Toribio, D.V.M., M.S.
*****
The Ohio State University 2001
Dissertation Committee:
Dr. Catherine W. Kohn, Co-Adviser Approved by
Dr. Thomas J. Rosol, Co-Adviser Co-Adviser
Dr. Charles C. Capen
^(^Adviser Dr. Gustavo W. Leone Department of Veterinary Clinical Sciences UMI Number 3031276
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ABSTRACT
Extracellular calcium (Ca^^ concentrations are tightly regulated by complex
homeostatic mechanisms, including parathyroid hormone (PTH), calcitonin, and 1,25-
dihydroxyvitamin D (calcitriol). PTH is secreted by chief cells of the parathyroid gland,
and is considered the most important calcium-regulating hormone. In vitro studies in
parathyroid chief cells from different species have improved our understanding on the
physiology and pathophysiology of the parathyroid gland. However, there is limited
information on parathyroid gland function in horses. Horses have unique features with
regard to calcium metabolism, including high serum Ca^^ concentrations, high urinary
fractional clearance of calcium, low serum concentrations of vitamin D (1,25-
dihydroxyvitamin D, 25-hydroxyvitamin D), and an increased Ca^^ set-point (Ca^^
concentration at which PTH secretion is 50% maximal). Several pathological conditions
in horses are associated with abnormal calcium homeostasis. We have determined that in
the horse (as oca|B jn # $ ^ p ^ e s l # Ca^^^ sigmoidal, andjhe Ca^^
set-point is higher than in other domestic animals and man, which may, in part, explain
the high serum calcium concentrations of the horse. Hypocalcemia occurs frequently in
humans with sepsis. We found Aat sepsis (enterocolitis) in horses was associated with
hypocalcemia, and that some horses did not have the expected increase in serum PTH
concentrations for the degree of hypocalcemia, suggesting impaired parathyroid gland
u fimction. When equine parathyroid cells were studied in vitro for 30 days, we determined that, over time in culture there was a decrease in PTH secretion, and in PTH and calcium- sensing receptor (CaR) mRNA expression. Furthermore, equine parathyroid cells exposed to an inflammatory mediator, interleukin-1 P (IL-IP), had decreased PTH secretion and PTH mRNA expression, and increased CaR mRNA expression compared to controls, indicating that IL-ip may play a role in regulating chief cell function in the horse. We cloned, sequenced, and determined the tissue expression of calcitonin, calcitonin gene-related peptide (CGRP)-I, and CGRP-I in the horse. Information from these genes will be valuable to further study of the role of the calcitonin gene family in calcium regulation in healthy.and sick horses. Calcium homeostasis is a complex process that involves many regulatory factors. Disturbances of calcium regulation are frequent in human and veterinairy medicine. With our studies we have provided additional information to understand calcium regulation in horses.
Ill Dedicated to my wife and children
iv f- ' ■' -.■- :' " ' : [ : .:’, AW
ACKNOWLEDGMENTS
I want to especially thank my co-adviser. Dr. Catherine W. Kohn, for her trust and support during my residency program in Equine Internal Medicine and my doctoral studies at The Ohio State University. I am very thankful to my co-adviser. Dr. Thomas J. Rosol, for his friendship, great ideas, and suggestions, and for changing my perspective of molecular biology and clinical research. 1 am also grateful to the Distinguished University Professor, Dr. Charles C. Capen, for sharing his knowledge on calcium metabolism in animals. I want to acknowledge Dr. Gustavo Leone for his expertise and technical advice in molecular genetics. I want to express my gratitude to Drs. Deborah Groppe, Bruce LeRoy, Virgile Richard, Rani Sellers, and Sarah Tarmehill-Gregg for their technical assistance and friendship. I am grateful to Drs. Kenneth Hinchcliff, Stephen Reed, Dennis Chew, and Richard Sams for their assistance during my clinical studies on calcium regulation in horses. I am indebted to Drs. James D. Smith, Fairfield T. Bain, and T. Douglas Byars at Hagyaid-Davidson-McGee Associates, Lexington, Kentucky, for recommending me to the College of Veterinary Medicine of The Ohio State University. Their trust in me has _madea.betteLfiituieJbLipeLand my family. ______I want to express my appreciation to Lori Kipp, Marge Hauer, Kris Bums, and Robin King for locating horses to be used in our studies. I thank the C. Gletm Barber fund of the College of Veterinary Medicine, The Ohio State University, for providing me with financial support. Without their assistance my woric would not have been possible. I also thank the Equine Research Funds of the College of Veterinary Medicine, The Ohio State University, for providing resources to carry out these studies.
1
:::: VITA
November 16,1963. ...Bom - Colon, Panama
1982. ...B.S., Agricultural Sciences, Mexico-Panama Technological Institute, Panama
1988. ..D.V.M., National University of Rio Cuarto, Argentina
1988-1990 ..Large Animal Veterinarian, Panama
1991-1993 ..Assistant Professor, Large Animal Anatomy, Physiology and Reproduction, Technological Institute of Costa Ûca
1993 ..M.S., Animal Endocrinology, National University of Costa Rica
1993-1996 ..Equine Veterinarian, Panama
1996-present Large Animal Internal Medicine Resident/Graduate Research Associate, College of Veterinary Medicine, The Ohio State University
PUBLICATIONS
Research Publications
1. Toribio RE, Kohn CW, Chew DJ, Capen CC, Rosol TJ. Cloning and sequence analysis of the complementary DNA for feline preproparathyroid hormone. Am J Vet Res 2001 ; 62:xxx-xxx. In press. f.- vi
A Î *: f'-j }vA # a K'»:? 2. Sellers RS, Toribio RE, Blomme EA. Idiopathic systemic granulomatous disease and macrophage expression of PTHrP in a miniature pony. J Comp Pathol 2001; 125:214-218.
3. Toribio RE, Kohn CW, Chew DJ, Sams RA, Rosol TJ. Comparison of serum parathyroid hormone and ionized calcium and magnesium concentrations and fractional urinary clearance of calcium and phosphorus in healthy horses and horses with enterocolitis. Am J Vet Res 2001 ; 62:938-947.
4. Toribio RE, Kohn CW, Lawrence AE, Hardy J, Hutt JA. Thoracic and abdominal blastomycosis in ahorse. Vet Med Assoc 1999; 214:1357-1360.
5. Toribio RE, Bain FT, Mrad DR, Messer NT 4th, Sellers RS, Hinchcliff KW. Congenital defects in newborn foals of mares treated for equine protozoal myeloencephalitis during pregnancy, Vet Med Assoc 1998; 212:697-701.
6. Toribio RE, Molina JR, Forsberg M, Kindahl H, Edqvist LE. Effects of calf removal at parturition on postpartum ovarian activity in Zebu {Bos indicus) cows in the humid tropics. Acta Vet Scand 1995; 36:343-352.
7. Toribio RE, Molina JR, Bolafros JM, Kindahl H. Blood levels of the prostaglandin F2 alpha metabolite during the postpartum period in Bos indicus cows in the humid tropics.ZentralbiattEurVeterinannedizinrReiheAiJ VetMed^A} 1994;41:630-639.
8. Toribio RE, Bolafros JM, Kindahl H. Cambios en los niveles de prostaglandins Fze durante el ciclo e s ^ y la prefrez de vacas cebuinas. Ciencias Veterinarias 1993; 15:27-33.
■ ■ . . ■ " . V 9. Toribio RE. A handbook o f embryology, histology, anatomy, and physiology of farm animals. Costa Rica: Technological Institute of Costa Rica Press, 1992; 294 pp.
vu 10. Toribio RE. Niveles de prostaglandina p 2a medidos a través de su metabolito (PGFM) durante el periodo posparto en vacas cebuinas (Bos indicus) en el trôpico hùmedo de Costa Rica. Notas Veterinarias, 1992; 2:9-11.
FIELDS OF STUDY
Major Field: Veterinary Clinical Sciences Equine Endocrinology Clinical Field: Equine Internal Medicine
Vlll -'..h
TABLE OF CONTENTS
Page Abstract...... ii
Dedication ...... iv
Acknowledgments...... v
Vita...... vi
List of Tables...... xi
List of Figures...... xii
Chapters:
1. Introduction...... 1
2. Comparison of serum parathyroid hormone and ionized calcium and magnesium concentrations and fractional urinary clearance of calcium and phosphorus in healthy horses and horses with enterocolitis ...... 11
Abstract...... 11 Introduction...... 12 Materials and Methods ...... IS Results...... 22
-—— ------— - — — ------1 discussion — References...... 36
3. Hysteresis and calcium set-point during stepwise changes in serum ionized calcium in healthy horses ...... 47
Abstract...... 47 Introduction...... 48 Materials and Methods ...... 51 Results...... 61 Discussion...... 68 References...... 76
.. . • ;n'. " .>■ V : -,J
ft:. 4.^ i P ^A ym id hormone (PJH) sécrétion, PTH mRNA and calcium-sensing receptor # ,ÿ mRNA expression in equine parathyroid cells, and effects of interleukin-1 on K- : equine parathyroid cell hmctibn ...... 89
rv Abstract...... 89 Introduction...... 90 Materials and Methods ...... 93 Results...... 99 Discussion...... 103 References...... 107
S. Molecular cloning and expression of equine calcitonin, calcitonin gene-related peptide-I, and calcitonin gene-related peptide-II ...... 119
Abstract...... 119 Introduction...... 120 Materials and Methods ...... 122 Results...... 126 Discussion...... 129 References...... 133
Bibliography ...... 148
c': 3 .. --I';
LIST OF TABLES
îaW ê £âgs ■'T.-■ 2.1 Results (meaii,± SD) of serum biochemical analyses for healthy horses and horses with eÀeroooÜtis...... 42 AkC : '* 2.2 Mew ± SD fractional urinary clearance of calcium (FCa) and phosphorus (FP) in heaitfiy horses and horses with enterocolitis ...... 43
5.1 Nucleotide and amino acid identity (%) of equine preprocalcitonin (preproCT), procalcitonin (proCT), calcitonin (CT) and katacalcin to other species ...... 136
XI LIST OF FIGURES
Figure Page
2.1 Box plot of fiactional urinary clearance of calcium (FCa; top) and phosphorus (FP; bottom) in healthy horses (n = 20) and horses with enterocolitis (n = 20). Mean FCa was significantly (P<0.05) less and FP significantly ^<0.0S) greater in horses with enterocolitis. • = Sth and 95th percentiles ...... 44
2.2 Bland-Altman bias plots illustrating differences in serum intact parathyroid hormone (PTH) concentrations for 40 (top) and 35 (bottom) horses determined by use of an immimochemiluminometric assay (ICMA) and an immunoradiometric assay versus the mean of these 2 values for each sample. To obtain the plot depicted in the bottom panel, the 5 serum samples that yielded >50 pmol of PTH/L by use of the ICMA were excluded andysis. Notice that bias is reduced in this plot, compared with the plot depicted in the top panel. Solid top and bottom lines represent ± 2 SD..;...... 45
2.3 Scatterplot of serum intact PTH concentrations versus serum ionized c a lc i^ (Ca?*) concentrations in 62 healthy horses (o [open circles]) and ‘ é4 horses with enterocolitis (closed symbols). On die basis of Ca and PTH concentrations, horses with enterocolitis were assigned to normocalcemic (♦[closed diamonds]; n = 13), hypocalcémie and nonresponder (▼ [closed upside-down triangle]; n = 5), hypocalcémie and mid-responder (■ [closed squares]; n = 26), and hyptxialcemic and high responder (A [closed triangles]; n = 10) groups. The vertical dashed line indicates the lower limit of the reference range used to determine normocalcemia...... 46
3.1 Intact PTH and Ca^* concentrations plotted against time in 12 healthy horses in which hypocalcemia was induced prior to hypercalcemia (Experiments 1 and 4). NazEDTA infusion resulted in increased serum PTH concentrations, while calcium gluconate infusion resulted in decreased serum PTH concentrations. Horses o f Experiments 1 and 4 had higher serum PTH and PTHmax concentrations when compared to horses in which hypercalcemia was induced prior to hypocalcemia (Fig. 3.2). The area under the time concentration curve (AUC) for serum PTH was xii significantly greater in horses of Experiments 1 and 4 than in horses o f ___ Experiments 2 and 3 (Fig. 3.2.). There was no statistical difference in the AUC for Ca^^ concentrations in Experiments 1 and 4 as compared to Experiments 2 and 3 (Fig. 3.2) ...... 81
3.2 Intact PTH and Ca^^ concentrations plotted against time in 12 healthy horses in which hypercalcemia was induced prior to hypocalcemia (Experiments 2 and 3). The AUC for serum PTH was significantly less in horses during Experiments 2 and 3 as compared to Experiments 1 and 4. There was no significant difference in the AUC for serum Ca^^ concentration in Experiments 2 and 3 as compared to Experiments 1 and 4...... 82
3.3 Intact PTH and Ca^ concentrations plotted against time in 4 healthy horses (Experiment S, control). The horses were infused with 0.9% NaCl, and had no significant changes in serum PTH and Ca^^ concentrations occurred during the experiment ...... 83
3.4 Intact PTH concentrations (% of PTHmax) plotted against serum Ca^^ concentrations in healthy horses (Experiments 1 and 4). Induction of hypocalcemia (I) resulted in a Ca^/PTH sigmoidal curve (closed squares). During the recovery period (2) PTH concentrations were lower (hysteresis), and the PTH/Ca^^ relationship displayed linearity (open squares). Once serum Ca^^ reached the reference range (1.55-1.67 mmol/L) hypercalcemia was induced (3) (closed circles). During the recovery period from hypercalcemia (4) PTH concentrations were greater (open circles) than during induction of hypercalcemia (hysteresis) ...... 84
3.5 Intact PTH concentrations (as a % of mean PTHmax from Experiments 1 and 4) plotted against serum Ca^* concentrations in healthy horses (Experiments 2 and 3). The induction (1; closed circles) and recovery (2; open circles) from hypercalcemia displayed hysteresis. The induction of hypocalcemia (3; closed squares) was not as sigmoidal and serum PTH — ------— eoncentrations- were nof as-highas-in^Experiments-T and4, but there was hysteresis (4; open squares) ...... 85
3.6 Relationship between serum Ca^^ and PTH concentrations in 12 healthy horses. PTH concentrations were expressed as a percentage of PTHmax for each horse. The sigmoidal relationship between serum Ca^^ and PTH concentrations was calculated using the four-parameter logistic model (dotted line) and the Emax sigmoidal model (Hill’s equation; solid line). The Ca^^ set-point was calculated as 50% of PTlfrnax for each horse. Both regression lines overlapped indicating that alternative mathematical methods to calculate the relationship between serum Ca^^ and PTH concentrations can be applied ...... 86
xiii I ; 3.7 Induction of ÊypocAéemiâ (Experiments 1 and 4) resulted in an increase of FCa firom 2JSFÆ 0X% (time 0) to 32.3±8% at the end of NazEDTA infusion (I20in^^*|«^ p^ m c te a ^ from 0.1±0.1% to 7.5 ± 1.3% at the end of the NàzHyf iiâuslon. At the end o f the recovery period from :hypocâlcWnà F W décrêweiftaSW àlS^, and FP decreased to 1.9±0.2%. Induction of hypercdcemia resulted in an increase of FCa to 59.1 ±7.3%, while FP continued to decline to values similar to baseline (0.17±0.10%)...... 87
3.8 During the induction of hypercalcemia (Experiments 2 and 3) FCa increased from 3.8 ± 0.4% to 63.0 ± 13.2% (P<0.05) at 120 min, and FP increased from 0.03 ± 0.04% to 0.9 ± 0.6% at 120 min. Dining the recovery from hypercalcemia FCa decreased to 11.4 ± 5.5%, which is higher than baseline values (P<0.05), and FP decreased to 0.01±0.01%. During hypocalcemia FCa increased^ to 38.0 ± 5.7% at the end of the NazEDTA infusion, and FP increased to 5.2 ± 0.9% ...... 88
4.1 Phase contrast microscopy of parathyroid chief cells. Cells in suspension varied from single cells to clumps of cells (a, 40X). The cells were polygonal, growing in colonies and spreading out to form a monolayer (days 3-5) (b,c, lOOX). The number of spindle-shaped cells on day 2, presumably fibroblasts (arrow), was minimal (c)...... I ll
4.2 PTH secretion of equine parathyroid chief cells cultured in a low Ca^^ (0.8 voM) medium. PTH secretion decreased over time. On day 5 of culture, PTH secretion was 68%, on day 10 was 33%, and after day 13, PTH secretion was below 18% compaied to day 0. To determine the doubling times, chief cells were harvested every 5 days for 30 days. Doubling time increased from 28 h on day 2 to 68 h on day 30 (insert graph) ...... 112
4.3 Equine parathyroid chief cells on day 0 (collection day) were exposed to different Ca^^ concentrations for 3 h. A sigmoidal relationship between PTH and Ca^^ concentrations was displayed. Calcium concentrations greater than 2.0 m M did not result in an additional decrease in PTH ------secretuNvand-Ci^ c(mcentrations-lower-than-0.8~iiiAf^di(f not resufr m additional secretion of PTH. The Ca^^ set point, calculated as the Ca^^ concentration at which PTH concentration was 50% of PTHmax was calculated to be 1.3 mM ...... 113
4.4 Reduction in responsiveness of equine parathyroid chief cells to changes in Ca^^ concentrations. By day 5 of culture chief cells remained responsive to changes in Ca^^; however, there was a rightward shift in the Ca^^ set- point from 1.38 m M (day 0) to 1.57 mM (dotted line) because higher Ca^^ concentrations were req u ie ^ to inhibit PTH secretion. After day 10 of culture there was no significant difference in PTH secretion between low and high Ca^^ concentrations. Control (100% PTH secretion) for each day was the PTH secreted at a Ca^^ concentration of 0.8 mM...... 114 xiv 4.5 Effect o f low (0.8 mM) and high (2.0 xaM) concentrations on CaR mRNA and! PTH niR^IA expression. A significant effect of low or high Ca^^ concentrations on CaR mRNA expression was not detected on either " day 1 of 2. The decline of CaR mM4A was time dependent and not statistically associated to Ca^^ concentrations. No significant difference between low and high Ca^^ concentrations and PTH mRNA expression was found on day 1. However, on day 2 chief cells exposed to high Ca^^ concentrations had lower PTH mRNA expression than cells exposed to low Ca^^ concentrations *(P<0.05) ...... 115
4.6 PTH and CaR mRNA expression, and PTH secretion in parathyroid chief cells cultured for 30 days in a low Ca^^ medium. PTH secretion, PTH and CaR mRNA expression decreased over time when compared to day 0. By day 2 o f culture, CaR mRNA expression decreased to 41%, by day 5 to 31%, and by day 30 to 16% of the controls (day 0). PTH mRNA expression decreased, but not as rapidly as CaR mRNA expression. By day 2 PTH mRNA expression was 78%, by day 5 it was 43%, by day 10 it was 35%, and by day 30 to 25% of the controls (day 0) ...... 116
4.7 Equine parathyroid chief cells incubated with human recombinant interleukin-ip (IL-IP) (0-2000 pgAnL) and human recombinant IL-1 receptor antagonist (IL-lra) (100 ng/ml). Parathyroid cells incubated with IL-ip had a significant (P<0.05) decrease in both PTH secretion (75%) and PTH mRNA expression (73%) (P<0.05) when IL-ip concentration was 2000 pg/ml. No significant difference in PTH secretion and PTH mRNA expression was detected at lower IL-ip concentrations; however, a trend for low PTH secretion and PTH mRNA expression was present. IL- ip concentrations greater than 200 pg/ml resulted in a significant (P<0.05) overexpresion of CaR mRNA (up to 142%). The effects of IL-ip on PTH secretion, and PTH mRNA and CaR m ^ A expression were blocked when an IL-lra was added to the culture medium prior to IL-ip. No effect of IL-lra on PTH secretion, and PTH and CaR mRNA was detected when compared to controls ...... 117
5.1 cDNA and deduced amino acid sequences of equine preprocalcitonin (clone A4, GenBank No. AF249307). Numbers on the left indicate nucleotides. Numbers on the right in^cate amino acids ftom the start methionine. The ATG and TAA (*) codons specifying the 423 bp open reading frame and the AATAAA polyadenylation signal are indicated by black boxes. The sequence for the mature calcitonin peptide (32 aa) is underlmed. Equine katacalcin amino acid sequence (21aa) is in a shaded box...... 137
XV
... , V-, - 5.2 Alignment of the aa sequences of preprocalcitonin from different species. NCBI accession numbers: equine, AF249307; human, M64486; rat, VO1229; mouse, X97991; ovine, M98053; salmon, Y00765; chicken, X03012; and canine, AJ271090. Amino acids are shown in one letter code. Amino acid numbers from the start methionine are indicated on the right margin. Dots represent amino acids identical to equine preproCT. Mature calcitonin peptide aa sequence (32aa) is in a shaded box. Katacalcin (CCP) sequence is in a black box...... 138
5.3 Alignment of mature calcitonin aa sequences from different species. Amino acids identical to equine CT are presented with dots. Asterisks represent cdnàerved aa (>90%). The overall homology and NCBI accession numbers are shown on the right columns. Amino acids 1,4-7,9, 28, and 32 are identical in all known calcitonin sequences. All species have leucine (L) at position 16, except humans, which have phenylalanine (F)...... :...... 139
5.4 Phylogeny dendrogram for calcitonin. The dendrogram was constructed using multi-way alignment with Align plus. Equine calcitonin was placed in the mouse, rat, and human class, with which it has the highest homology. Also shown in Fig. 5.3 ...... 140
5.5 Alignment of the signal and N-terminal peptides of clones A4 (CT), B1 (CGRP-I), and A1 (CGRP-U). Dots represent identical amino acids. There is 100% homology between clones A4 and Bl, indicating that the gene encoding for these clones is equivalent to the CALC-I gene in humans, which encodes for CT and CGRP-I. Clone A1 sequence is the product of a different gene; the aa homology between clone Al, and clones A4 and Bl is 65%...... 141
5.6 cDNA and deduced amino acid sequence (127aa) of the equine preproCGRP-I cDNA (clone Bl, GenBank No. AF257471). The single letter aa is shown under the nt sequence. Numbers on the left indicate nucleotides.-Numbers on-the-right-indieate amino-acids from the start methionine. The ATG and TGA specifying the 384 bp open reading frame and the AATAAA polyadenylation signal are indicated by black boxes. The sequence for the CGRP Bl mature peptide (37 aa) is underlined 142
5.7 Comparison of the deduced or chemically-determined aa sequences of COM (and human amylin) in different species to equine CGRP-I (clone Bl). Alignment was performed with Align plus software. Nucleotide and amino acid sequences were obtained from the NCBI Oittp://www.ncbi.nlm.nih.gov). Asterisks represent conserved aa (>90%) among species. Dots represent amino acids identical to equine CGRP-I (clone Bl). Amino acids DF (14-15) are only present in chicken, bullfrog, phyllomedusa, and salmon. The degree of homology (%) of clone Bl to xvi other species is low (<59%). Amino acids 2-5, 7,9, 11-13, 16, 27, 30, and 33 are identical between equine CGRP-I (clone Bl) and human amylin (35%)...... 143
5.8 Alignment of the deduced or chemically determined aa sequences of CGRP (and human amylin) in different species compared to equine CGRP-II (clone Al). Unlike equine CGRP-I, the aa sequence of equine CGRP-n is more conserved when compared to other species. Asterisks represent conserved aa (>90%). Identical amino acids to that of equine CGRP-II (Al) are presented in dots. Amino acids 2-7,9,11-13,16,19,30, and 32-34 are identical between equine CGRP-II (clone Al) and human amylin (43%). The homology at the N-terminus may be responsible for some overlap in function between amylin and CGRP ...... 144
5.9 cDNA and deduced amino acid sequence (129aa) of the equine CGRP-II cDNA (clone Al, GenBank No. AF257470). The single letter aa is shown under the nt sequence. Numbers on the left indicate nucleotides. Numbers on the right indicate amino acids ftom the start methionine. The ATG and TGA specifying the 390 bp open reading frame and the polyadenylation signal are indicated by black boxes. The sequence for the CGRP mature peptide (37 aa) is underlined ...... 145
5.10 Phylogeny dendrogram for CGRP. The dendrogram was constructed with the Align plus software. Two major branches representing mammalian and low vertebrate CGRPs are present. One additional branch representing clone Bl (equine CGRP-1) suggest an early divergence of this gene when compared to clone AI (equine CGRP-II) ...... 146
5.11 Tissue-specific expression o f equine calcitonin, CGRP-1, and CGRP-11 mRNA. The blots were probed as described in section 2.4. (A) Ethidium bromide stained agarose gel to demonstrate RNA loading and integrity. The tissues are l=thyroid gland, 2=basal ganglia, 3=hippocampus, 4=rostral brain, 5=caudal cortex, 6=hypothalamus, 7=thalamus, ------8=^Mtuita^— gimid, 9=medulla— obkmgata, 40=cerebellar vermis. ll=cerebellar hemisphere, 12=spinal cord, 13=dorsal root ganglia, I4=lung, 15=small intestine, 16=adrenal gland, 17=parathyroid gland, 18=spleen, 19=liver, 20=kidney. Phosphorimages for equine calcitonin (B), CGRP-1 (C), and CGRP-H (D) ...... 147 CHAPTER I
INTRODUCTION
Calcium is one of the most important minerals in the body, with diverse physiological functions such as muscle contraction, hormone secretion, enzyme activation, cell division, cell membrane stability, neuromuscular excitability, and blood coagulation (Rosol and Capen, 1997). Due to the importance of calcium in normal intracellular and extracellular processes, maintenance of a steady state concentration of calcium is extremely important.
Calcium is found in three main compartments: the skeleton, soft tissues, and the extracellular fluid. The skeleton contains approximately 99% of the total body calcium as hydroxyapatite crystals. The remaining calcium is present in the cell membrane, endoplasmic reticulum (0.9%), and in the extracellular fluid (0.1%) (Rosol and Capen,
1997). In plasma, calcium exists as a free or ionized form (50%), bound to proteins
(40%), and complexed to anions (10%) (Hurwitz, 1996).
Regulation of extracellular ionized calcium (Ca^^ concentrations is controlled by a complex homeostatic system that includes three major hormones: parathyroid hormone
(PTH), calcitonin (CT), and 1,25-dihydroxyvitamin D (calcitriol) (Hurwitz, 1996; Mundy and Guise, 1999). Parathyroid hormone-related protein (PTHrP) is a newly discovered protein (Suva et al., 1987) that shares considerable homology with PTH, binds and
1 activates the PTH-I receptor, and is important in cell growth and, differentiation and in
calcium transport aodsS^inSlBBrraes (Wysoimerski and Stewart, 1998). However, in
normal adults, under '^Sysiological conditions, PTHrP has no effect on calcium
homeostasis (Mundÿ arid Guise, 1999). Excessive secretion of PTHrP by some tumors
results in humoral hypercalcemia of malignancy.
The biological functions of PTH include stimulation of osteoclastic bone
resorption, thereby increasing Ca^^ release into the blood, stimulation of calcium
reabsorption and inhibition of phosphate reabsorption in the renal tubules, and
stimulation o f calcitriol synthesis in the kidney, which then increases intestinal calcium
and phosphate absorption and inhibits PTH secretion from the parathyroid gland (Rosol
and Capen, 1997).
PTH is secreted by the chief cells of the parathryroid gland in response to small
changes in extracellular concentrations. There is an inverse sigmoidal relationship
between serum Ca^^ concentrations and PTH secretion (Brown, 1983; Chapter 3),
represented by a four parameter model (Brown, 83), which enables the parathyroid gland
to rapidly respond to changes in Ca^^ concentrations. Changes in extracellular Ca^^ concentrations are detected by a calcium-sensing G protein-linked cation receptor (CaR) in the parathyroid cells (Brown et al., 1993).
Abnormal calcium homeostasis frequently occurs in horses with various pathological conditions. However, our knowledge of equine calcium regulation and parathyroid gland function is limited. Horses have unique features with regard to calcium metabolism, including high serum total and ionized calcium concentrations (Toribio et al., 2001; Chapter 2), high urinary fractional clearance of calcium (Toribio et al., 2001; p ï ï ' ? ’'' ■ '■
Chapter 2). lowi^MhEhRmcentradons of vitaminLjD..(1^25-dihYÉ-Qxyyi
hydroxyvitamin D) (M&nptââ et al., 1988; Breidenbach et al., 1998), and an increased
Ca^^ set-point (Chaptér 3). Pathological conditions of the horse characterized by
abnormal, parathyroid gland function include idiopathic hypocalcemia of foals (Beyer et
al., 1997), primary hyperparathyroidism (Frank et al., 1998), nutritional secondary
hyperparathyroidism (Ronen et al., 1992), pseudohyperparathyroidism (Marr et al.,
1989), hypoparath^idism (Couëtil et al., 1998), vitamin D toxicity (Harrington and
Page, 1983), renal disease (Elfers et al., 1986), exercise-induced hypocalcemia (Aguilera-
Tejero et al., 2001), and sepsis (Toribio et al., 2001; Chapter 2).
Hypocalcemia is a common finding in humans and horses with sepsis (Carlstedt et
al., 1998; Toribio et al., 2001; Chapter 2). Hypocalcemia may result in neuromuscular,
cardiovascular, and gastrointestinal tract dysfunction. Muscle weakness, tetany,
synchronous diaphragmatic flutter, cardiac arriiythmias, ileus, and convulsions have been
observed in hypocalcémie horses (Grubb et al., 1996). Severe hypocalcemia can also
result in death. Clinical observation has revealed that hypocalcemia is common in horses
with severe gastrointestinal disease (colic, enterocolitis). To determine the prevalence of
hypocalcemia, and to evaluate parathyroid gland function in septic horses, we studied
'homes âdnutfea"fo~Thê Ohio SfafirtM vefsi^Conege o r Veterinary Medicine with a
history of severe enterocolitis (diarriiea). We found that hypocalcemia is frequent in
horses with severe diarrhea (80%). Furthermore, some horses with sepsis and
hypocalcemia have low serum PTH concentrations for the degree of hypocalcemia,
indicating an inappropriate response of the parathyroid gland to low serum Ca^^
concentrations (Chuter 2). We suspect that horses with inappropriate parathyroid gland
response to hypocalcemia have an abnormally low Ca^^ set-point. 3 The role of ionized magnesium (Mg^^ has received less attention than that of
Cs?* in critically ill patients. Serum Mg^^ concentration can influence Ca^^ concentration,
and Mg^^ deficiency can result in hypocalcemia attributable to impaired PTH secretion as
well as altered renal and skeletal responsiveness to PTH (Fatemi et al., 1991). No data
were available regarding serum Mg^^ concentrations in healthy or ill horses. We
determined that hypomagnesemia was frequent in horses with enterocolitis (78%)
(Toribio et al., 2001; Chapter 2). We speculate that low serum Mg^^ concentrations in
some horses witfr h^ocalcemia may have resulted in inappropriately low PTH
concentrations wiA respect to degree of hypocalcemia, due to impairment of PTH
secretion or synthesis. Most humans with Mg^^ depletion and hypocalcemia have
inappropriately low seruih PTH concentrations (Abbott and Rude, 1993).
To better understand equine parathyroid gland frmction in healthy and sick horses,
we evaluated the response of the parathyroid gland to changes in serum Ca^^
concentrations in healthy horses (Chapter 3). The Ca^^ set-point, defined as the Ca^"^
concentration corresponding to 50% of maximal PTH concentrations (Felsenfeld and
Llach, 1993), or the midpoint in the PTH/Ca^^ sigmoidal curve (Brown, 1983), is
considered a measure of parathyroid gland sensitivity to changes in Ca^^ concentrations
(Cunningham, 1996; Felsenfed and Llach, 1993). Abnormalities of the Ca^^ set-point
have been reported in humans with chronic renal frdlure (Slatopolsky and Delmez, 1996)
and primary hyperparathyroidism (Schwarz et al., 1992). We found that the Ca^^ set- point in healthy horses was higher (1.37 mmol/L) than in dogs and humans (l.D-1.2 mmol/L) (Chapter 3). Serum total and ionized calcium concentrations in horses are greater than those in other domestic animals and humans (Kaneko et al., 1997). The high % '5. ... . r V -' ......
Ca^^ set-point in the horse may result in high serum concentrations. We also
speculate that horses are not as sensitive as other species to the suppressive effects of
Ca^^ on PTH secretion, which results in a high Ca?* set-point.
We determined that the phenomenon of hysteresis (differing PTH concentrations
for one Ca^^ concentration value depending on the direction of change in Ca^^
concentration) was present in healthy horses. This finding indicates that the parathyroid
gland of the horse, in addition to detect absolute serum Ca^^ concentrations, can also
detect the direction of change in serum Ca^^ concentrations (Chapter 3).
In vitro studies o f parathyroid cells from different species have improved our
understanding of the physiology and pathophysiology of the parathyroid gland (Brown et
al., 1976; Liu et al., 2001; Sakaguchi et al., 1987; Fasciotto et al., 1989). However, the
number of studies on equine parathyroid gland function is limited (Roussel et al., 1987;
Estepa et al., 1998; Aguilera-Tejero et al., 1998; Toribio et al., 2001; Chapter 2), and no
study on equine parathyroid cell function in vitro has been reported (Chapter 4). To
expand our knowledge o f equine parathyroid gland physiology we evaluated in vitro
equine parathyroid cell morphology and proliferation, PTH secretion, PTH mRNA and
calcium-sensing receptor (CaR) mRNA expression. There was an inverse sigmoidal
relationship between PTH secretion and Ca^^ concentrations as occurs in vivo (Chapter 3;
Chapter 4). Over time in cell culture, parathyroid cell PTH secretion, and PTH and CaR
mRNA expression decreased, and parathyroid cells became less sensitive to changes in
Ca^^ concentrations (Chapter 4).
Inflammatory mediators such as interleukin (IL)-1, lL-6, and tumor necrosis
factor (TNF)-a are increased in horses with sepsis (Seethanathan et al., 1990; Barton et CaRjnRNi
expression and decrease PTH secretion in bovine parathyroid cells (Nielsen et al., 1997;
Carlstedt et al 1999). When the effect of IL-ip on equine parathyroid chief cell was
evaluated in vitro, there was a decrease in PTH secretion and mRNA expression, and an
increase in CaR mRNA expression (Chapter 4). Increased CaR mRNA expression results
in lower PTH secretion, decreasing the Ca^^ set-point, and may be a reason that
parathyroid glands of septic horses secrete inappropriately low PTH concentrations for
the degree of hypocalcemia. We believe that IL-1 plays an important role in regulating
PTH secretion in the horse.
Calcitonin (CT) is a calcium-lowering hormone synthesized by the C-cells
(parafollicular cells) of the thyroid gland (Copp, 1994). Studies on the calcitonin gene
family have revealed alternative splicing of the primary transcripts (Rosenfeld et al.,
1981; Amara et al., 1985) resulting in mRNA products for calcitonin, calcitonin gene-
related peptide (CGRP)-I, and CGRP-II. CGRPs are important in a diverse array of physiological process^ including inhibition of bone resorption, modulation of neurotransmitter actnnfy,. vasodilation, relaxation of smooth muscle, alteration of gastrointestinal motility, neurogenic inflammation, and anti-inflammatory activity (Brain and Cs^bndge, 1996). Senim calcitonin, procaJcitohin, and CGRP concentrations have been reported to be increased in human patients with hypocalcemia and sepsis (Chesney et al., 1983; Sperber et al., 1990; Parida et al., 1998; Lind et al., 2000). The importance of peptides of the calcitonin gene family in the pathogenesis of hypocalcemia in septic human patients and horses is unknown. We have cloned and sequenced the cDNA encoding for equine calcitonin, CGRP-I, and CGRP-II (Chapter 5), in order to study the importance of these peptides in the pathogenesis of hypocalcemia in horses. In our studies we describe calcium homeostasis in healthy and septic hofs^ . _fo
healthy horses, we determined that the relationship between serum Ca^^ and PTH
concentrations was sigm ot^, aqd that the Ca^^ set-point was higher than in humans and
dogs. We also found that the phenomenom of hysteresis in the Ca /PTH relationship was *’ < t present in healthy horses. We cultWed equine parathyroid chief cells, and evaluated their
function in vitro, in order to support future studies on equine parathyroid physiology and pathophysiology. We determined that interleukin-1 P may alter equine parathyroid cell function in vitro. We found that hypocalcemia is frequent in septic horses, and that many horses with hypocalcemia had abnormal parathyroid gland function. We cloned 3 different products of the equine calcitonin gene family to study their role in the pathogenesis of hypocalcemia in the horse. In conclusion, the information generated from these studies will be valuable for future research on calcium metabolism in the horse, and in understanding parathyroid gland function in healthy and sick horses. W ^ ;
, V
#% RefcrCTCcs
Abbott LG, Rude RIC; Clinical manifestations of magnesium deficiency. M iner Electrolyte Metdb ; ||^ r 9 : 3 14-322.
Amara SO, Arnza jL,;Lef^SE, Swanson LW, Evans RM, Rosenfeld MG. Expression in bt^^of^a,messenger RNA encoding a,novel neuropeptide homologous to calcitonin gbA&#^tW#)tide.Science 1985; 22^:loiM-1097.
Barton MH, Bruce EH, Moore JN, Norton N, Anders B, Morris DD. Effect of tumor necrosis factor antibody given to horses during early experimentally induced endotoxemia. Am J Vet Res 1998; 59:792-797.
Brain SD, Cambridge H. Calcitonin gene-related peptide: vasoactive effects and potential therapeutic role. Gen Pharmacol 1996; 27:607-611.
Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC. Cloning and characterization of an extracellular Ca(2+)- sensing receptor from bovine parathyroid. Nature 1993; 366:575-580.
Brown EM. Four-parameter model of the sigmoidal relationship between parathyroid hormone release and extracellular calcium concentration in normal and abnormal parathyroid tissue. J Clin Endocrinol Metab 1983; 56:572-581.
Bueno AC, Seaborn TL, Comick-Seahora J, Horohov DW, Moore RM. Plasma and urine nitric oxide concentrations in horses given below a low dose of endotoxin. Am J Vet Res 1999; 60:969-976.
Carlstedt E, Ridefelt P, Lind L, Rastad J. Interleukin-6 induced suppression of bovine parathyroid hormone secretion. Biosci Rep 1999; 19:35-42.
Chesney RW, McCarron DM, Haddad JG, Hawker CD, DiBella FP, Chesney PJ, Davis JR. Pathogenic mechanisms^ of-the- hypocalcenmr o f the staphylococcaf toxic-shock syndrome. JLab Clin Med 1983; 101:576-585.
Copp DH. Calcitonin: discovery, development, and clinical application. Clin Invest Med 1994; 17:268-277.
Cunningham J. Parathyroid pathophysiology in uraemia. Nephrol Dial Transplant 1996; ll(Suppl 3):106-110.
Estepa JC, Aguilera-Tejero E, Mayer-Valor R, Almaden Y, Felsenfeld AJ, Rodriguez M. Measurement of parathyroid hormone in horses. Equine Per J 1998; 30:476-481. Apiilera-Teieio E. Garfia^B. Estepa JC. Lopez L Maver-Valor R. Rodriguez M. E flfe c J s, of exercise and EDTA administration on blood ionized calcium and parathyroid hormone in horses. Am J Vet Res 1998; 59:1605-1607.
Fatemi S, Ryzen E, Flores J, Endres DB, Rude RK. Effect of experimental human magnesium depletion on parathyroid hormone secretion and 1,25-dihydroxyvitamin D metsboMsm. J Clin Endocrinol Metab 1991; 73:1067-1072.
Felsenfeld AJ, Llach F. Parathyroid gland function in chronic renal failure. Kidney Int 1993; 43:771-789.
Grubb TL, Foreman JH, Benson GJ, Thurmon JC, Tranquilli WJ, Constable PD, Olson WO, Davis LE. Hemodynamic effects of calcium gluconate administered to conscious horses. J Vet Intern Med 1996; 10:401-404.
Hurwitz S. Homeostatic control of plasma calcium concentration. Crit Rev Biochem Mol Biol 1996; 31:41-100;
Kaneko JJ, Harvèÿ*JW; Bhiss ML. Appendixes. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical biochemistry o f domestic animals. 5“* ed. San Diego: Academic Press, 1997; 885-905.
Lind L, Carlstedt F, Rastad J, Stierhstrom H, Stridsberg M, Ljunggren O, Wide L, Larsson A, Heilman P, Ljunghall S. Hypocalcemia and parathyroid hormone secretion in critically ill patients. Crit Care Med 2000; 28:93-99.
Mundy GR, Guise TA. Hormonal control of calcium homeostasis. Clin Chem 1999; 45:1347-1352.
Nielsen PK, Rasmussen AK, Butters R, Feldt-Rasmussen U, Bendtzen K, Diaz R, Brown EM, Olgaard K. Inhibition of PTH secretion by interleukin-1 beta in bovine parathyroid glands in vitro is associated with an up-regulation of the calcium-sensing receptor mRNA. Biochem Biophys Res Commun 1997; 238:880-885.
Parida SK, Schneider DB, Stoss TD, Pauly TH, McGillis JP. Elevated circulating calcitonin gene-related peptide in umbilical cord and infant blood associated with maternal and neonatal sepsis and shock. Pediatr Res 1998; 43:276-282.
Rosenfeld MG, Amara SG, Roos BA, Ong ES, Evans RM. Altered expression of the calcitonin gene associated with RNA polymorphism. Nature 1981; 290:63-65.
Rosol TJ, Capen CC. Calcium-regulating hormones and diseases of abnormal mineral (calcium, phosphorus, magnesium) metabolism. In: Kaneko JJ, Harvey JW, Brass ML, eds. Clinical biochemistry o f domestic animals. 5th ed. San Diego: Academic Press, 1997; 619-702. • ' ■ ' . . : * j T
_RousseLJÜ. ijnJï:C^Strait_JEL_Modransky_PD._R&dioimmunoassay for parathyroid hormone in equids. Am J Vet Res 1987; 48:586-589.
Schwarz P, Sorensen HA, Momsen G, McNair P, Transbol I. Normal pattern of parathyroid response to blood calcium lowering in primary hyperparathyroidism: a citrate clamp study. Clin Endocrinol (Oxf) 1992; 37:344-348.
Seethanathan P, Bottoms GD, Schafer K. Characterization of release of tumor necrosis factor, interleukin-1, and superoxide anion from equine white blood cells in response to endotoxin. Am J Vet Res 19%; 51:1221-1225.
Slatopolsky E, Delmez JA. Pathogenesis of secondary hyperparathyroidism. Nephrol Dial Transplant 1996; ll(Suppl 3):13Q-135.
Sperber SJ, Blevins DD, Francis JB. Hypercalcitoninemia, hypocalcemia, and toxic shock syndrome. Rev/n/êcrD» 1990; 12:736-739.
Wysolmerski JJ, Stewart AF. The physiology of parathyroid hormone-related protein: an emerging role as a developmental factor. Anna Rev Physiol 1998; 60:431-460.
, 10 CHAPTER 2
COMPARISON OF SERUM PARATHYROID HORMONE AND IONIZED
CALCIUM AND MAGNESIUM CONCENTRATIONS AND FRACTIONAL
URINARY CLEARANCE OF CALCIUM AND PHOSPHORUS IN HEALTHY
HORSES AND HORSES WITH ENTEROCOLITIS
Abstract
Objective—To evaluate calcium balance and parathyroid gland function in healthy horses and horses with enterocolitis and compare results of an inununochemiluminometric assay (ICMA) with those of an inununoradiometric assay
(IRMA) for determination of serum intact parathyroid hormone (PTH) concentrations in horses.
Animals—64 horses '^ tlf enterocolitis and 62 healthy horses.
Procedures—BIooiTand urine samples for determination of serum total calcium, ionized •• calcium. (Ca^^ and magnesium (Mg^^, phosphorus, BUN, total protein, creatinine, albumin, and PTH concentrations, venous PCO2 and PO 2 , and fractional urinary clearance of calcium (FCa) and phosphorus (FP) were collected when horses entered the study.
Serum concentrations of PTH were measured in 40 horses by use of both the IRMA and
ICMA.
11 RèswK»—^Most (48/64;75%) horses witfi enterocolitis had decreased serum total calcium,
Ca^\ and M g^ concentrations and increased phosphorus concentrations, compared with
healthy horses. Serum PTH concentration was increased in most (36/51; 70.5%) horses
with hypocalcemia. In adclition, FCa was significantly decreased and FP significantly
increased in horses widfien&r^oiitis, compared with healthy horses. Results of ICMA
were in agreement with results of KMA.
Condnslons and Clinical Rdevance^EnterocoHtis in horses is often associated with hypocalcemia; 79.7% of horses in the present study had ionized hypocalcemia. Because FCa was low, it is unlikely that renal calcium loss was the cause of hypocalcemia. Serum PTH concentrations varied in horses with enterocolitis and hypocalcemia. However, we believe low PTH concentration in some hypocalcémie horses may be the result of impaired parathyroid gland function.
Introduction
The primary function of the parathyroid gland is to prevent hypocalcemia by rapid
secretion of parathyroid hormone (PTH) when serum ionized calcium (Ca^^ concentrations decrease (Rosol and Capen, 1996). In several species, PTH induces an acute increase in blood calcium concentration by enhancing renal reabsoiption and bone release of calcium, and a chronic increase by facilitating intestinal calcium absorption
(Agus et al., 1973; McSheehy and Chambers, 1986; Nemere and Norman, 1986; Hurwitz,
1996). For reasons not yet understood, serum Ca^^ concentrations decrease in humans and other animals with certain pathologic conditions (eg, endotoxemia, sepsis, severe bums) (Zaloga and Chemow, 1987; Zaloga et al., 1988; Nakamura et al., 1998; Murphey
12 .£L^al«Jl(KMÛ_.HMçh__oftenjrsmlt5_ijLja»_iiiçisæsLJ^ concentation. and.
subsequent return of serum concentration to within the physiologic range (Sibbald et
al., 1977; Nakamura et al., 1998). '' Clinical observation has revealed that many horses with severe enterocolitis are
hypocalcémie. Hypocalcenua may result in neuromuscular, cardiovascular, and
gastrointestinal tract dysfunction. Muscle weakness, tetany, synchronous diaphragmatic
flutter, cardiac arrhythmias, ileus, and convulsions have been observed in hypocalcémie
horses (Grubb et al., 1996). Severe hypocalcemia can result in death. Supplementation
with calcium added to replacement fluids appears to benefit horses with enterocolitis and
hypocalcemia. Although the contribution o f calcium to improvement in gastrointestinal
tract and cardiac function in such horses has not been defined, we believe that restoration
of calcium homeostasis is an important aspect of managing horses with enterocolitis.
We hypothesized that horses with severe enterocolitis and hypocalcemia would
also have high serum PTH concentrations. High PTH concentrations would result in
increased renal resorption of calcium, decreased factional urinary clearance of calcium
(FCa), and increased fiactional urinary clearance of phosphorus (FP) (Agus et al., 1973;
Kinoshita et al., 1986; Strewler and Rosenblatt, 1995).
The role of ionized magnesium (Mg^^ has received less attention than that of
Ca^^ in critically ill patients. In humans, serum concentration can influence Ca^^
concentration (Rosol and Capen, 1996; Rosol and Capen, 1997), and Mg^^ deficiency can
result in hypocalcemia attributable to impaired PTH secretion as well as altered renal and
skeletal responsiveness to PTH (Rosol and Capen, 1996). Treatment with magnesium
restores these deficiencies (Fatemi et al., 1991). 1,25-Dihydroxyvitamin D metabolism is
13
. .. more sensitive to depletion than either PTH secretion or PTH-mediated osteoclastic bone resorption (Rude et al., 1985). The effects of on mineral metabolism may be more important in chronically ill patients than has been appreciated (Fatemi et al., 1991).
Few data are available regarding serum Mg^^ concentrations in healthy or ill horses.
There are multiple radioimmunoassays for use in measuring PTH concentrations.
The assays recognize either the amino-, carboxyl-, or midregion Augment of PTH or the
Aill-length (intact) hormone (Felsenfeld and Llach, 1993). Results of radioimmunoassays for the carboxyl- and midregion fragments do not reflect biological activity of the hormone; only assays for the N-terminal region or intact hormone measure biologically active PTH (Rodriguez et al., 1991). The introduction of 2-site immunometric assays to ' A*- measure intact PTH 1 ^ lâulted in an important advance, because these assays provide greater reliability arid precision than earlier assays (Brown et al., 1987; Nussbaum et al., ' ^ i 1987).Two-site immunometric assays,for.PTH use 2 antibodies, one to the N-terminal region and the other to the carboxyl-terminal region. Intact PTH is captured between these 2 antibodies. The inununoradiometric assay (IRMA) for PTH has been shown to be useful in dogs (Torrance and Nachreiner, 1989), cats (Barber et al., 1993), cows
(Yamagishi and Naito, 1997), and horses (Martin et al., 1996; Beyer et al., 1997; Estepa et al., 1998; Aguilera-Tejero et al., 1998; Frank et al., 1998). In horses, measurement of serum PTH concentration has been used to aid in the diagnosis of idiopathic hypocalcemia o f foals (Beyer et al., 1997), primary hyperparathyroidism (Roussel et al.,
1987; Peauroi et al., 1998; Frank et al., 1998), nutritional secondary hyperparathyroidism
(Ronen et al., 1992; Clarke et al., 1996), pseudohyperparathyroidism (Roussel et al.,
1987; Marr et al., 1989), hypoparathyroidism (Couëtil et al., 1998), vitamin D toxicosis
14 ..
I ■ iwL- - . M» T .3 I I -T y .^ - TP* ^ " — - . ' W#WF V .* i" ■ ♦’rt.,
(Harrington and Page EH, 1983)^ and renal disease (Elfers et al., 1986). Evaluation of
serum PTH concentration has also been used to investigate the relationship between
serum Ca^^ and PTH concentrations in healthy horses (Roussel et al., 1987; Martin et al.,
1996; Estepa et al., 1998; Aguilera-Tejero et al., 1998).
Recently, an automated solid-phase 2-site inununochemiluminometric assay
(ICMA) for quantitative determination of intact PTH has become available. Results of this assay and those of the IRMA are in good agreement for human serum (Chen and
Tsai, 1996; Michelangeli et al., 1997). However, the ICMA can detect lower serum PTH concentrations and is more sensitive and more reliable than the IRMA (Michelangeli et al., 1997).
The purposes of the study reported here were to compare the ICMA and IRMA for measurement of intact PTH in horses and to evaluate calcium balance and parathyroid gland function in healthy horses and horses with enterocolitis by measuring serum total calcium and Ca^\ phosphorus, Mg^^, and intact PTH concentrations and FCa and FP.
Materials and Methods
Animals—This study was approved by the Ohio State University Institutional Laboratory
Animal Care and Use Committee. The affected group consisted of 64 (39 sexually intact females, 18 castrated males, 7 sexually intact males) horses with enterocolitis that were selected over a 1-year period from horses admitted to The Ohio State University
Veterinary Teaching Hospital with a history of severe diarrhea of < 48 h duration. Horses in this group were between 1 and 22 years of age (6.3±5.1 years) and comprised several
15 Thoroughbreds, 11 Standardbreds, and 23 other breeds).
Affected horses had received no calcium and no more than 5 L of fluid, administered IV
or PO, prior to admission. Complete physical examinations, including cardiovascular,
respiratory tract, and gastrointestinal tract assessment and assessment of rectal
temperature, hydration status, and mentation, were performed immediately afler
admission. Clinical evidence of acute, profuse, watery diarrhea and dehydration were
required for entry into the study.
The control group consisted o f62 (42 sexually intact females, 14 castrated males,
6 sexually intact males) healthy horses between 2 and 21 years of age (mean ± SD, 6.9 ±
3.6 years). Healthy horses were selected from the Ohio State University College of
Veterinary Medicine teaching herd during a 1-year period and comprised several breeds
(35 Standardbreds, 15 Thoroughbreds, and 12 other breeds). Horses in this group were
fed a diet of grass and grass hay, had no history of illness for the 3 months preceding the
study, and had received no treatments for the month preceding the study. Horses were determined to be healthy on the basis of results of complete physical examinations. In addition, results of CBC and biochemical analyses, including determination of serum total calcium and Ca^^ concentrations and plasma fibrinogen concentration, were within reference ranges.
Venous blood samples from three horses with chronic renal failure and hypercalcemia (one thoroughbred mare of 10 years of age, one Thoroughbred gelding of
7 years of age, and one Quarter horse gelding of 10 years of age) were used to determine serum intact PTH concentrations for PTH assay validation.
16 Sample collection—Venous blood samples for determination of serum total calcium.
Ca^\ total protein, albumin, creatinine, BUN, phosphorus, and Mg^^ concentrations were collected into tubes without additives. Venous blood samples for CBC were collected into tubes with EDTA, and for determination of plasma fibrinogen concentration, into tubes with sodium citrate. Venous blood samples were also collected under anaerobic conditions into hepârinized syringes for determination of Pvcoj and PvOz. Urine was collected by free catch pr catheterization for urinalysis and urine biochemical analyses.
Blood samples for serum biochemical analyses were collected at the same time as urine.
Hematologic and biochemical analyses—Complete blood counts were performed, using an automated system." Serum total calcium and protein, creatinine, BUN, albumin, and phosphorus concentrations were determined by use of colorimetric reactions in an automated analyzer.*’ Plasma fibrinogen concentrations were determined, using a nephelometric analyzer.*’ Partial pressures of oxygen and carbon dioxide in venous blood were measured, using a blood gas analyzer,** and pH was corrected for rectal temperature.
Blood samples for Ca^^ and Mg^^ determinations were processed immediately, using
Ca^ - or Mg^^-selective electrodes.®
Fractional clearance of calcium and phosphorus—Urine was acidified with 6 N HCl to dissolve calcium salts. Urine calcium and creatinine concentrations were measured as described for serum. Fractional clearances of calcium and phosphorus were calculated, using serum and urine calcium, phosphorus, and creatinine concentrations, as described by Roussel et al. (1993).
17 ».
PcterinihatioB of intact PTH concentratioii—Venous blood, collected into tubes
without additives, was centrifuged at 4 **€ immediately after clotting, and serum was
stored at —20 °C for batch analysis. Serum PTH concentration was measured by use of 2-
site IRMA^ for human intact PTH that was validated for use in horses (Estepa et al.,
1998) and a 2-site ICMA^for human intact PTH.
Ten serum samples for which PTH concentrations were measured by the Nichols
Institute^ IRMA and the ICMA _ . .. (ICMA _ PTH range for these samples was 3.5 to 77.0 pmol/L) were selected and sent to a different laboratory** to determine serum PTH
concentrations using the 2-site DPC PTH IRMA.' This laboratory routinely determines
PTH concentration in horses using this assay and we wanted to compare their PTH results
with our results within this PTH range. Intact PTH concentrations are reported in pmol/L
(pmol/L= pg/ml X 0.105).
The ICMA is a solid-phase 2-site assay. The solid phase, a polystyrene bead in the
test unit, is coated with an affinity-purified goat polyclonal antibody against amino acids
44 to 84 o f human PTH. Serum samples and alkaline phosphatase-conjugated affinity-
purified goat polyclonal antibody against amino acids I to 34 of human PTH were
incubated for 60 min at 37 °C in the test unit,^ with intermittent agitation. Intact PTH in
the sample was bound and formed an antibody sandwich complex. The assay system*
automatically handled sample and reagent additions, incubation and separation steps, and
measurement of photon output by use of a temperature-controlled luminometer. Results
were calculated from an observed signal, using a stored master curve (Babson, 1991).
The woridng range was O.I to 263 pmol/L (1 to 2,500 pg/ml), and the sample volume was
50 pi.
18
..:,v :.P'- ______The PTH IRMA (like the ICMA) is an immunometric assay based on two
antibodies, in which a solid phase is coated with a high affinity-purified goat polyclonal
antibody against the mid-region and C-terminal PTH. The other antibody is prepared to
bind N-terminal PTH, and this antibody is labeled for detection with Only intact
PTH forms the sandwich complex necessary for detection. This assay has been
previously validated for intact PTH determination in horses (Estepa et al., 1998).
Sensitivity of the ICMA was defined as the smallest single value that could be
distinguished from 0 at the 95% confidence limit, using equine serum. For the ICMA and
IRMA, the manufacturers report a calculated sensitivity of 0.1 pmol of intact PTH/L for
human serum. We determined a sensitivity of 0.12 pmol of intact PTH/L for equine
serum by use of the ICMA.
The intra-assay coefficient of variation for the ICMA was determined by
measuring 5 aliquots each of equine serum with low (2.5 pmol/L), medium (19.0
pmol/L), and high (180.0 pmol/L) PTH concentrations. The interassay coefficient of
variation for the ICMA was determined by comparison of results of 5 replicated measures
of equine serum with low (8.1 pmol/L), medium (20.5 pmol/L), and high (170.0 pmol/L)
PTH concentrations. Equine serum samples with low (6.2 pmol/L), medium (26.8
pmol/L), and high ( 180.0 pmol/I) PTH concentrations were assayed undiluted and diluted
1:2, 1:4, and 1:8, and results were evaluated for dilutional parallelism by comparing
measured (observed) values with expected (calculated) values. Two additional dilutions
were performed (1:16 and 1:32) for the sample with high PTH concentration. Expected
values were calculated by multiplying measured values by the dilution factor.
Correlations between observed PTH and calculated PTH concentrations were determined.
' 19 Precision of the IRMA was not evaluated, because this assay has been validated for use in Ü ■ ^gliw-m-'H --i- .4.-* tfT*— .4 4- ^ «&-4-.T4;j N& 4_ #..# ~V 44l^^=* horses (Estepa et al., 1998).
Comparison of results between the IRMA and ICMA—Serum PTH concentrations
were determined by use of both the ICMA and IRMA for 32 horses with enterocolitis, 3
horses with chronic renal failure and hypercalcemia, and 5 clinically normal horses.
Results were compared between tests.
Statistical analyses—Initial analysis consisted of descriptive statistics for all variables.
Data were compared by use of an unpaired Mest or the Mann-Whimey rank test between affected and healthy horses (Fisher and Van Belle, 1993). All analyses were performed with the assistance of commercially available software.'^
On the basis of serum Ca^^ concentration, horses with enterocolitis were assigned to a normocalcemic or hypocalcémie (serum Ca^^ concentration < 6.0 mg/dl) group. The serum Ca^^ concentration cutoff used to define hypocalcemia was derived from results of a previous report of Ca^^ concentrations in healthy horses (Kohn and Brooks, 1990), and data from healthy horses in the present study. Calcium concentrations less than the minimum limit defined by the 95% confidence interval were considered below normal.
The hypocalcémie group was further divided into 3 subgroups on the basis of serum PTH concentration. Affected horses with serum PTH concentrations within reference range were considered nonresponders, whereas horses with midrange PTH concentrations were considered mid-responders and horses with high PTH concentrations, high-responders.
Results were compared among these 3 subgroups by use of a Kruskal-Wallis 1-way
20 ANOVA on ranks, with pair-wise multiple comparisons performed by use of the Dunn
method (Glantz, 1997).
For affected horses, data regarding serum PTH concentrations failed the
Kolmogorov-Smimov test for normality (Fisher and Van Belle, 1993). Therefore,
Spearman rank tests for correlation (Fisher and Van Belle, 1993) were used to calculate
correlation (p) between serum PTH concentration in affected horses and total calcium,
Ca^\ Mg^"^, and phosphorus concentrations and FCa and FP. A Pearson product moment
test (Fisher and Van Belle, 1993) was used to calculate correlation (r) between PTH
concentration and total calcium, Ca^\ Mg^\ and phosphorus concentrations and FCa and
FP in healthy horses.
Because PTH concentrations in horses with enterocolitis were not normally
distributed and the test for equal variance between serum PTH concentration in healthy
horses and horses with enterocolitis failed, a Mann-Whitney rank sum test (Fisher and
Van Belle, 1993) was used to compare PTH concentrations between healthy and affected
horses. Fractional urinary clearance of calcium and FP were compared by use of a r-test
between these 2 groups. For all tests, P values < 0.05 were considered significant.
Bland-Altman bias plots, in which differences between results of ICMA and
IRMA for each sample were plotted against the mean of these 2 values (Bland and
Altman, 1986), were^ used to determine agreement between the 2 PTH assays. Bias was defined as the mean difference between values obtained for each sample by the 2 different methods, and error or variability was defined as the SD of these differences.
21 Results ______
Physical exaniination results—Mean ± SD duration of enterocolitis before admission
was 27.3 ± 15.4 h for the 64 affected horses in our study. Physical examination on
admission revealed that heart rate was significantly higher in horses with enterocolitis (71
±21 beats/min), compared with healthy horses (40 ± 1 beats/min). Respiratory rate, rectal
temperature, and ca^Hl^yLrefill'^time were also significantly higher in horses with
enterocolitis (35 ± 17 bfteths/^min, 38.2 ± 0.9 °C, and 3 ± 1 seconds, respectively),
compared with healthy horses (16 ± 3 breaths/min, 37.6 ± 0.4 °C, and < 2 seconds,
respectively). *
Serum analytes—Serum total calcium and Ca^^ concentrations were significantly less in
horses with enterocolitis than in healthy horses (Table 2.1). Differences in the Ca^^-to-
total calcium concentration ratio (Ca^VtCa) were not detected between groups. Total
calcium concentrations < 10.9 mg/dl were considered less than reference range, and 48 of
64 (75.0%) horses with enterocolitis had total hypocalcemia. In addition, Ca^^ concentrations < 6.0 mg/dl were considered less than reference range, and 51 of 64
(79.7%) horses with enterocolitis had ionized hypocalcemia. Serum phosphorus concentration was significantly higher in horses with enterocolitis than healthy horses, whereas Mg^^ concentration was significantly less. Ionized magnesium concentrations <
0.47 mmol/L were considered less than reference ran^; 50 of 64 (78.0%) horses with enterocolitis had ionized hypomagnesemia.
22 i p i f :
■i.. Fractional urinary clearance of calcium and phosphorus—Urine and blppd samples
were collected simultaneously from 20 healthy horses and 20 horses with enterocolitis
and hypocalcemia. Fractional urinary clearance o f calcium was significantly less and FP
significantly greater in horses with enterocolitis than in healthy horses (Table 2.2).
Fractional urinary clearance of calcium in all horses with enterocolitis was less than the
mean FCa of the control group (Fig 2.1).
Evaluation of the ICMA—The ICMA had intra-assay coefficients of variation of 5.6,
6.3,’ and*7.7^& for serum samples with low (2.5 pmol/L), medium (19.0 pmol/L), and high
(180 pmol/L) concentrations of PTH, respectively. The interassay coefficients of
variation for serum samples with low (8.1 pmol/L), medium (20.5 pmol/L), and high (170
pmol/L) PTH concentrations were 6.4, 6.3, and 5.9%, respectively. Determination of
PTH concentration using serial dilutions of serum samples containing low (6.2 pmol/L),
medium (26.8 pmol/L), and high (180 pmol/L) concentrations revealed parallelism
between measured (observed) and expected (calculated) values (r = 0.99).
Comparison of the IRMA and ICMA—When results were compared by use of the
Mann-Whitney test, significant differences were not detected between assays (P = 0.80).
Results of these 2 tests were correlated (p = 0.98). The 2 assays were also compared by
use of difference or bias plots (ie, Bland-Altman plots). Analysis of results for all serum
samples suggested that differences between methods were proportionally greater as PTH
concentration increased (Fig. 2.2). However, all but 3 data points were within 2 SD of the
mean difference. The bias over the whole range o f values was 1.9 pmol/L, and the error
23 (variability) was 6.9 pmol/L. When the 5 serum samples with PTH cqncen#adpns > 50
pmol/L were excluded from analysis, the bias was 0.18 pmol/L and error (variability) was
2.5 pmol/L, confirming our impression that there was proportional bias in these data.
Results of the ICMA were usually greater than those of the IRMA for serum samples
with high PTH concentrations. In addition, at high PTH concentrations, the difference in
results between the 2 assays was greater than at lower concentrations. Our interpretation
o f these analyses was that, despite some proportional bias, agreement between results of
ICMA and IRMA was good, and differences between values obtained by these 2 methods
are not likely to be clinically relevant.
Serum intact PTH concentrations—In healthy horses (n = 62), serum PTH
concentration as measured by use o f the ICMA was 5.8 ± 5.7 pmol/L (median, 3.7
pmol/L; range 0.12, td.21.9 pmol/L). Horses with enterocolitis had a wide range of serum
PTH concentrations (median, 29.1 pmol/L; range, 0.4 to 290 pmol/L). On the basis of
serum Ca^^ concentration, 13 of these affected horses were further assigned to the
normocalcemic group, and 51 to the hypocalcémie group.
Affected horses with ionized hypocalcemia (n = 51) were then assigned to 3
subgroups on the basis of serum intact PTH concentration (Fig 2.3). Fifteen horses were classified as nonresponders; these horses had hypocalcemia and PTH concentrations within our reference range (0.6 to 21.9 pmol/L). Twenty-six were mid-responders; horses in this group had hypocalcemia and a moderately high serum PTH concentration (range,
23.3 to 121 pmol/L). Finally 10 horses were considered high responders. These horses had high serum PTH concentrations (184 to 290 pmol/L). Significant differences in
24 K' ç-:-' ■ ■
serum total calciüm, Ca^\ phosphorus, creatinine, and BUN concentrations were not
detected among these 3 subgroups (Table 2.1). ; In control horses, serum Ca^^ and intact PTH concentrations were significantly (r
= -0.46) correlated. These values were also significantly (p = -0.54) correlated in horses
with enterocolitis. Serum concentration was significantly less in horses with
enterocolitis than healthy horses.
A positive correlation between serum PTH concentration and FP was detected in
healthy horses (r = 0.52) and in horses with enterocolitis (p = 0.37). However, no
association between PTH concentration and FCa was detected in horses with
enterocolitis.
Discussion
The majority (36/51; 79.7%) of horses with enterocolitis in the present study had
ionized hypocalcemia; however, parathyroid gland responses to hypocalcemia were
variable. Possible causes of hypocalcemia in these horses included renal loss of calcium
(Zaloga et al., 1988), sequestration of calcium in the lumen of the gastrointestinal tract as
a result of loss or poor absorption associated with inflanunation (Nakamura et al., 1998),
impairment in calcium mobilization (Sperber et al., 1990; Assicot et al., 1993; Dandona
et al., 1994), tissue sequestration of calcium (Zaloga et al., 1988), and impairment of
calcium release by the target tissue in response to PTH (Zaloga et al., 1988).
Renal loss as a cause of hypocalcemia in horses in our study was unlikely; mean
FCa was significantly less in affected horses, compared with healthy horses. Reduction in
25 FCa is an appropriate homeostatic response to hypocalcemia. Low FCa has also been
reported in association with hypocalcemia in other species (Zaloga et al., 1988). Low
urinary calcium excretion may be explained by a slow rate of calcium filtration at the
glomerulus or by efficient reabsorption of filtered calcium in the renal tubules. The
mechanism of renal tubular cell reabsorption of calcium in humans and other animals
with hypocalcemia has not been well described but may be PTH-mediated via an increase
in tubule intracellular cAMP concentration (Sibbald et al., 1977). An association between
high PTH and intracellular cAMP concentrations has been described in some humans
with sepsis and hypocalcemia (Sibbald et al., 1977). Alternatively, renal reabsorption of
calcium may be mediated by non-PTH dependent mechanisms. It has been suggested that
the cell membrane Ca^^-sensing receptor plays an important role in the control of renal
calcium reabsorption in the thick ascending limb of the loop of Henle via phospholipase
A:, adenylate cyclase, and the sodium-potassium-chloride cotransporter (Brown et al.,
1999).
Impairment of calcium mobilization may be the result of impaired parathyroid
gland secretion of PTH (Taylor et al., 1978; Sibbald et al., 1978), poor osteoclast response to PTH (Zaloga et al., 1988), or high serum concentrations of calcitonin and procalcitonin (Sperber et al., 1990; Assicot et al., 1993; Dandona et al., 1994; Oberhoffer et al., 1999). Secretion of PTH may have been impaired in the nonresponder group of affected horses in the present study. Serum PTH concentrations are low in some humans with sepsis and hypocalcemia (Amaud et al., 1971) or hypocalcemia and severe bums
(Klein et al., 1977) and in sheep with bums and hypocalcemia (Murphey et al., 2000).
PTH secretion in our nonresponder horses could have resulted fiom
26 \ :■!.
uprejgulation o f the Ca^%sensing receptor on parathyroid gland chief cells (Nielsen et al..
1997). Stimulation of the Ca^^-sensing receptor results in inhibition of cellular cAMP via I inhibitory G-proteins (Brown et al., 1999), and consequently, in decreased PTH secretion. P:: Inflammatory mediators that may increase in concentration in horses with enterocolitis Kr. V >.P* • (eg, interleukin- [IL-] 1, IL-6, and tumor necrosis factor- [TNF-] a) may induce increased
expression of Ca^^-sensing receptors on chief cells (Nielsen et al., 1997). Endotoxin,
another important mediator of enterocolitis in horses, has been shown to increase IL-1,
IL-6, and TNF-a concentrations in horses (Seethanathan et al., 1990; Barton et al., 1998;
Bueno et al., 1999) and;i&âeforë, may indirectly suppress PTH secretion (Nielsen et al.,
1997). A diminished" os^oé(hstip response to PTH could result in inadequate calcium
mobilizâtiôfl^frotii éoiiè. H i^ -re ^ n d é r ho'i^s in our study had high serum PTH
concentrations but remained hypocalcémie.
Tissue sequestration as a cause of hypocalcemia has been studied in humans and
other animals but was not evaluated in the present study. In humans with sepsis,
intracellular calcium concentrations are high in hepatocytes, aortic smooth muscle cells,
and blood cells (Hotchkiss and Karl, 1996), whereas in rats with sepsis, skeletal muscle
calcium uptake is increased (Sayeed, 1996). Calcium accumulates in abdominal fluid and
liver of pigs with endotoxemia (Carlstedt et al., 2000). Carlstedt et al. (2000) suggested
that because interstitial fluid volume is much greater than blood volume, interstitial
accumulation o f calcium could result in hypocalcemia.
Several mechanisms have been suggested for increased calcium entry into cells of
septic patients, including lL-1-mediated increased flux across the cell membrane
(Dinarello, 1984), depletion of intracellular stores of calcium that results in a retrograde
27 signal and activates influx across the membrane (Hotchkiss and Karl, 1996), insulin
resistance (Sayeed, 1996), and impaired Ca^^-dependent ATPase activity (Massry and
Fadda, 1993). It has been shown that during sepsis and endotoxemia in rats, there is an
increase in cytosolic Ca^^ that can cause cell toxicosis and death by activation of
proteases, phospholipases, and other enzymes (Song et al., 1993). We do not believe that
influx of Ca^^ from the extracellular to the intracellular compartment in our horses can
explain the observed ionized hypocalcemia, because intracellular concentrations are low
(100 nM) and ammcrg^e in mtracellular Ca concentrations sufficient to disrupt cellular
functions woul^be^ unlikely to cause a major change in extracellular ionized calcium
cone«t%tioo.^ *
In' most species, the action of vitamin D to increase calcium absorption in the
proximal portion of the small intestine is an important homeostatic response to
hypocalcemia. Zaloga and Chemow (1987) reported that some humans with sepsis
caused by infection with Gram-negative bacteria also have renal la-hydroxylase
deficiency, vitamin D deficiency, and acquired calcitriol resistance. We did not assess
serum concentrations of vitamin D in our horses with enterocolitis and hypocalcemia.
Plasma concentrations of calcidiol and calcitriol are reported to be much lower in horses than most mammals and birds, and la-hydroxylase activity in the renal cortex of horses is not detectable (Breidenbach et al., 1998). The importance of vitamin D and its metabolites in the regulation of calcium metabolism in horses requires further study.
Although hypoproteinemia and hypoalbuminemia were common findings in horses with enterocolitis, we did not detect a significant difference in Ca^VtCa between control and affected horses. This finding indicates that, overall, total calciiun and Ca^^
28 concentrations changed proportionally in the same direction in affected horses. Although a decrease in serum total calcium concentration is expected in the face of hypoalbuminemia, we did not expect that Ca^^ concentration would decrease as a result of low serum albumin concentration. Low Ca^^ concentrations in affected horses with hypoalbuminemia were likely the result of impaired calcium homeostasis.
Serum concentration was significantly decreased in horses with enterocolitis and hypocalcemia, compared with healthy horses. Unlike calcium ion concentration, for which complex regulatory mechanisms safeguard homeostasis, regulation of the magnesium ion concentration is dependent on gastrointestinal tract absorption and renal reabsorption with little endocrine control (Kayne and Lee, 1993). The low serum Mg^^ concentrations that we observed in our affected horses may, therefore, have been the result of poor Mg^^ absorption fiom the lumen of inflamed bowel or diminished renal reabsorption of magnesium resulting in excessive urinary losses. Because we did not determine fractional furinary clearance of magnesium, we could not assess urinary ^0 «1 * * • . I Y .
* i ' *T magnesium losses. ^
The role o f in critically ill patients has received less attention than that of
Cà^i If has been suggested that Mg^^ may serve as a Ca^^ antagonist and prevent Ca^^ entry into cells during sepsis or endotoxemia (Salem et al., 1995). Magnesium may also have a protective effect in endotoxemia, because magnesium deficiency predisposes humans with endotoxemia to a poor outcome (Salem et al., 1995).
A major complication in humans with moderate to severe Mg^^ deficiency is hypocalcemia (Abbott and Rude, 1993). Most humans with Mg^^ depletion and hypocalcemia have inappropriately low serum PTH concentrations with respect to degree
29 of hypocalcemia, suggesting impainnent in PTH secretion or synthesis. Administration of
to these padmitg^hes^s in an increase in serum PTH concentration, whereas in
clinically normal hum ^rM g^^ administration results in a decrease in PTH concentration
(Abbott and Rude, .1993). Although horses with enterocolitis in the present study had
signifrejuSly lower serum Mg^^ concentrations than did healthy horses, serum Mg^^
concentrations also decreased in affected horses as PTH concentrations increased. The
importance of this observation is unknown. However, some humans with Mg^^
deficiency and hypocalcemia have high serum PTH concentrations, suggesting end-organ
resistance (renal or skeletal) to PTH (Abbott and Rude, 1993). Both renal and skeletal
PTH resistance have been detected in humans with hypomagnesemia (Abbott and Rude,
1993). In addition, serum 1,25-dihydroxyvitamin D concentrations were low in humans
with hypomagnesemia, suggesting that formation of vitamin D is sensitive to Mg^~^
depletion (Rude et al., 1985). Experimentally induced Mg^^ depletion in humans resulted
in renal resistance to PTH-induced 1,25-dihydroxyvitamin D synthesis (Fatemi et al.,
1991). The role of magnesium and its interactions with calcium in horses with
enterocolitis deserve further study.
The increase in FP in horses with enterocolitis and hypocalcemia was consistent
with high PTH concentrations in these horses. However, the correlation between PTH
concentration and FP was poor (p = 0.37), indicating that factors other than PTH are
likely important in regulating renal phosphorus clearance in horses with enterocolitis and hypocalcemia.
In 56% (36/64) of our horses with enterocolitis, serum Ca^^ concentrations were low and PTH concentrations high. However, a subgroup of these horses had low serum
30 concentrations of PTH despite concomitant hypocalcemia. We spwulate that this low
PTH response may have been attributable to suppression of PTH secretion or synthesis by inflammatory mediators such as IL-1, IL-6, and TNF-a. The mechanisms by which cytokines might alter PTH release remain to be determined. Inflammatory cytokines in horses with hypocalcemia may facilitate over expression of the Ca^^-sensing receptor on parathyroid cells and permit increased concentrations of intracellular calcium, thus indirectly decreasing PTH release. Additionally, it is known that the effects of Ca^^ and
Mg^'*' on PTH secretion are interdependent. It is possible that hypomagnesemia in some horses may have permitted intracellular accumulation of calcium and reduced cAMP production in the parathyroid gland, resulting in decreased PTH synthesis or release.
Moreover, low serum Mg^^ concentrations may have resulted in renal and skeletal PTH resistance.
A second subgroup of affected horses with hypocalcemia had high serum PTH concentrations (high responders). The high PTH concentrations in these horses could représenta supraphysiologic response of the parathyroid gland to the combined effects of hypocalcemia and increased concentrations of inflammatory mediators. In humans and rats, hyperphosphatemia may directly stimulate PTH release (Almaden et al., 1996; Kates et al., 1997). High PTH concentrations in the high-responder horses were associated with high serum phosphorus concentrations; mean phosphorus concentration in high-responder horses was 7.2 ± 2.5 mg/dl, whereas mean phosphorus concentrations in the mid- and nonresponders were 5.7 ± 2.8 and 5.5 ± 2.8 mg/dl, respectively. However, the difference in phosphorus concentrations among groups was not significant (P = 0.3). Serum concentrations o f PTH in the high-responder group may have been high because target
- ; i.A ^ \ -1^1 ■' Y:
Jbypomagnesemia may induce JPTH.
resistance. Alternatively, high-responder horses may have had low serum concentrations
of 1,25-dihydroxyvitamin D. Low 1,25-dihydroxyvitamin D concentrations appear to be
permissive for enhanced PTH secretion (Kates et al., 1997).
Some (13/64; 20.3%) horses with enterocolitis had serum Ca^^ concentrations
within reference range. We believe that in these horses, calcium homeostatic mechanisms
were efficient enough to maintain extracellular Ca^^ concentration within the physiologic
range. Another explanation might be that these horses were assessed early in the disease
process. In addition, 3 affected horses with normocalcemia had serum PTH
concentrations greaterthah the' iÿper limit of our reference range. It is possible that this
increase in serum PTH concentration was the direct result of inflammatory mediators or
endotoxins acting oh the parathyroid gland. Alternatively, efficient PTH release may
have resulted in correction of hypocalcemia, but serum PTH concentration remained
high. Nakamura et al. (1998) found that endotoxin infusion in rats resulted in an increase
in serum PTH concentration in some animals.
Methods for describing agreement between results of 2 tests, such as the ICMA
and the IRMA, are controversial when neither test is considered the gold standard. We
included assessments of correlation and bias between the 2 tests. The Spearman
correlation coefficient was high (p = 0.98), indicating a good association in results
between tests The Bland-Altman plot for all data revealed that bias over the whole range
o f values was 1.9 pmol/L, indicating that the ICMA yielded PTH concentrations 1.9
pmol/L greater thanthe IRMA. However, inflection of the Bland-Altman plot for all data
that this assessment may have overestimated the bias for low PTH
32 a r r-yr M m m - i - : . ' ■ concentrations and underestimated the bias for high concentrations (Stôckl et al., 1996).
For this reason, we examined a second Bland-Altman plot in which data were restricted
to those obtained by analysis of serum samples that yielded PTH concentrations < 50
pmol/L by use of the ICMA. In the second plot, the bias was reduced to 0.2 pmol/L.
Agreement between results of the 2 tests was thus better at concentrations < 50 pmol/L,
and inspection of the second plot indicates that only 2 values were outside 2 SD of the
mean of the differences. Serum PTH concentrations > 50 pmol/L are 2 times greater than
the upper limit of our reference range for the ICMA. We believe that PTH values higher
s. ■' than 50 pmol/L are unlikely to provide relevant information when making clinical
decisions on case management. Enhanced sensitivity, shorter incubation time,
automation, and lack of radioactivity are important advantages of the ICMA over the
IRMA.
Mean and reference range values for serum PTH concentrations in the healthy
horses of this study, measured by use of the ICMA, (mean ± SD, 5.8 ± 5.7 pmol/L; range,
0.12 to 21.9 pmol/L) were greater than those reported by Estepa et al. (1998), measured
by use of the human intact PTH IRMA (mean ± SD, 3.3 ± 2.4 pmol/L; range, 0.8 to 8.7
pmol/L) and the rat PTH IRMA (mean ± SD, 4.6 ± 3.1 pmol/L; range 1.1 to 10.1
pmol/L). Breidenbach et al. (1998), who used the human intact PTH IRMA to measure
plasma PTH concentrations in horses, reported a higher mean PTH value than ours (mean
± SD, 22.9 ± 19.0 pmol/L). Serum PTH concentrations for clinically normal horses have
been determined by use of C-terminal assays; values were similar to or greater than those
of the present study (Roussel et al., 1987; Van Heerden et al., 1990; Enbergs et al., 1996).
The PTH response of healthy horses to acute hypocalcemia was evaluated by Roussel et
33 /.V-- ■ Ù* * jküK';:Y;v : ’ i al>. (1987). usinga C-terminal immunoassay. The reference range for PTH concentrations in that study was greater than values we determined for healthy horses in the present
study.; This is likely attributable to the prolonged serum half-life of the C-terminal portion
of PTH (Arnaud et al., 1974). We do not recommend the use of the older C-terminal PTH
assays, because they do not measure intact hormone. With the new 2-site immunometric
assays for intact PTH, it is now possible to accurately determine concentrations of
biologically active PTH in horses. We believe that the variation among results of
previous studies is related to assay variation. Differences in the calcium and phosphorus
content of diets among studies may also have contributed to these apparent differences.
Most (51/64; 79.7%) horses with enterocolitis in the present study also developed
ionized hypocalcemia. The expected response of the parathyroid gland to hypocalcemia is
to increase secretion of PTH. Serum PTH concentrations in some (36/51; 70.5%) of the
affected hypocalcémie horses was high. However, in others (15/51; 29.5%), serum PTH
concentrations did not increase in proportion to the degree of hypocalcemia. Reasons for
this variation in response to hypocalcemia among horses with enterocolitis are not clear.
We found that renal'c^lum loss (le, FCa) was significantly lower in horses with
enterocolitis than in healthy horses. We believe that impaired calcium mobilization Aom
jbjone, s^pstration or loss of cdcium ^ m the gastrointestinal tract, or failure of the
parathyroid gW d to secrete adequate PTH are the most likely causes of hypocalcemia in
horses with enterocolitis.
34 - . - .y '■>'
Endnotes
"Coulter Counter Model S-plus, Coulter Electronics, Inc, Hialeah, Fla.
^oehringer Mamilièini/HîÈÿhi 911 system, Boehringer Mannheim Corp, Indianapolis, ■ u . • Ind.
"ACL 200 Automated Coagulation Laboratory, Instrumentation Laboratory, Lexington,
Mass.
‘‘a BL 500, Radiometer Copenhagen, Radiometer Medical A/S, Copenhagen, Denmark.
"Nova 8, Nova Biomedical, Waltham, Mass.
'intact PTH kit, Nichols Institute Diagnostics, San Juan Capistrano, Calif.
‘hnmulite intact PTH assay. Diagnostic Products Corporation, Los Angeles, Calif.
""Michigan State University Animal Health Diagnostic Laboratory, Lansing, Mich.
'Coat-A-Count intact PTH IRMA, Diagnostic Products Corporation, Los Angeles, Calif.
^Immulite System, Diagnostic Products Corporation, Los Angeles, Calif.
'"SigmaStat 2.01 for Windows, SPSS Inc, Chicago, HI.
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Circ Shock 1988; 24:143-148. 41 ------r — Enterocolitis* : I ' - Hypocakemic (n > 51) Healthy : All Nortnocakemk Nonresponder Mid-responder High-responder Varialrle (n>62) (n -6 4 ) (n “ 13| (n«15) (n -2 6 ) (n»IO) Total calcium (mg/dl) ll.9± 0;5t 10.084:1.6 11.34:0.9 9.7±1.16 9.541.5 9.442.3 C af (mg/dl) 6.6±0:3it ^.5±0.0 6.440.3 5.340.4 5.140.5 4.740.9 Ca^VtCa 55.3 ± 1 * . >55.443,9 57.8±6.1 56.24:7.4 54.445.1 53.744.7 MgTfmmol/L) 0.51±0.lt ' 0.4h^.l# ^ 0.540.1 0.4540.08 0.3840.1 0.3540.05 Phosphorus (mg/dl) 3.2±1.0t 5.9t2.8 1 4.042.4 5.5442.8 5.742.8 7.242.5 PTHfpmolÆ.) 5.8*5.-^ 55.64:733 12.84:13.6 12.24:5.6 50.6426.4 230.4439.6 BUN (mg/dl) I4.l± 2.èt ^40.8±29.0 26.74:18.2 36.9433.8 41.4425.4 50.2430.4 Creattnine (mg/dl) 1.3±0.2t 3.64:1.8 26.74:18.2 3.14:1.6 3.341.9 4.641.8 Total protein (g/dl) 6.9±0.6t - 5.94:1.5 6.14:1.3 5.84:1.8 6.141.4 6.041.2 Albumin (g/dl) 3.2±0.2it - 2.740.7 2.940.6 2.540.7 2.740.7 2.940.7 pH 7.4±0.cil % 7.3540.08 7.3640.06 7.3440.11 7.3740.07 7.3440.05 Total C02(mEo/L) 26.4 ±2i4 23.844.9 24.244.6 23.246.2 25.044.5 23.644.0 *Horses with enterocolitis were assipied to nonnocalcemic and hypocalcémie groups on the basis of serum Ca^* concentration (hypocalcémie, (ja^* concentration < 6.0 mg/dl). Horses iij the hypocalcémie group were assigned to 3 groups on the basis of serum PTH concentrations (nonresponder, P|TH concentration range of 0.6 to 21.9 pn^l/L; mid-responder, range of 23.3 to 121 pmol/L; high responder, range of 184 to 290 pmol/L). fSignificantly (P < 0.05) different from value for all horses with enterocolitis. t 1 Ca^* = Ionized calcium. Ca^VtCa = Ratio of ionized calcium to total calcium concentration. Mg^* = Ionized magnesium. PTH = Parathyroid hormone; Table 2.1—Results (mean± SP) of serum biochemical analyses for healthy horses and horses with enterocolitis W :: -■ ' --- - .... - "T ! < ' Enterocolitis* 1 ' Hypocakemic Healthy { All Normocaiceinic Nonresponder Mid-rraponder High responder Variable (n -2 0 ) I (n -2 0 ) (n -4 ) (n -6 ) (n -6 ) (n -4 ) FCa(%) 3.4±2.6t ! 0.8 ±0.8 2.1 ± 1.8 H 0.5 ±0.46 0.8 ±0.7 0.3 ±0.1 FP(%) 0.08±0.2t i 1.9 ±2.6 1.8 ±0.6 ^ 3.4 ±3.6 1.0 ±1.9 0.3 ±0.0 See Table 2.1 for key. i )i Table 2.2—Mean ± SD Aactiopal urinary clearance of calcium (FCa) and phosphorus (FP) in healthy horses and horses witli I enterocolitis ! t 7 6 5 4 3 IL. 2 1 0 Healthy Enterocolitis IL Healthy Enterocolitis Figure 2.1. Box plot of fractional urinary clearance of calcium (FCa; top) and phosphorus (FP; bottom) in healthy horses (n = 20) and horses with enterocolitis (n = 20). Mean FCa was significantly (P < 0.05) less and FP significantly (P < 0.05) greater in horses with enterocolitis. • = 5th and 95th percentiles. 4 4 3 0 25 20 • 15 • •• • Zero bias I -10 ■ -15 0 50 100 I ISO Mean PTH(pmoUL) 10 8 6 4 2 0 Zero bias -2 • # # •4 -6 "1— I I 10 20 30 40 SO Mean PTH (pmol/L) Figure 2.2. Bland-Altman bias plots illustrating differences in serum intact parathyroid hormone (PTH) concentrations for 40 (top) and 35 (bottom) horses determined by use of an immunochemiluminometric assay (ICMA) and an immunoradiometric assay versus the mean o f these 2 values for each sample. To obtain the plot depicted in the bottom panel, the 5 serum samples that yielded > 50 pmol of PTH/L by use of the ICMA were excluded from analysis. Notice that bias is reduced in this plot, compared with the plot depicted in the top panel. Solid top and bottom lines represent ± 2 SD. 45 350-1 300 - 250 - ^I 200 - o . 150 Ë 100 50 4 0 T T- 1------3 4 5 8 Ca^* (mg/dl) Figure 2.3. Scatterplot of serum intact PTH concentrations versus serum ionized calcium (Ca^^ concentrations in 62 healthy horses (o [open circles]) and 64 horses with enterocolitis (closed symbols). On the basis of Ca^^ and PTH concentrations, horses with enterocolitis were assigned to nonnocalcemic (4 [closed diamonds]; n = 13), hypocalcémie and nonresponder (▼ [closed upside-down triangle]; n = 15), hypocalcémie and mid-responder (■ [closed squares]; n = 26), and hypocalcémie and high responder (A [closed triangles]; n = 10) groups. The vertical dashed line indicates the lower limit of the reference range used to determine normocalcemia. 46 CHAPTERS HYSTERESIS AND CALCIUM SET-POINT DURING STEPWISE CHANGES IN SERUM IONIZED CALCIUM IN HEALTHY HORSES Abstract The parathyroid gland is designed to respond to changes in extracellular ionized calcium (Ca^^ concentrations by controlling parathyroid hormone (PTH) secretion. Abnormalities in Ca^^ homeostasis are reported in horses with several pathological conditions; however, there is little information on the regulation of calcium in horses. The objectives of thd^pre&epf^Wy were to determine the Ca^^ set-point (the calcium concentration correspoA&rg' to 50% of maximal PTH secretion) in healthy horses, to determine whether ihe Ca /PTH response curves during hypocalcemia and hypercalcemia were characterized by hysteresis (different PTH concentrations for the same serum Ca^* concentration depending on the direction of changes in Ca^^ concentrations), and to determine if the order o f experimentally-induced hypocalcemia or hypercalcemia had an effect on PTH secretion (area under the time concentration curve, AUC). The Ca^ set-point and hysteresis were determined in 12 healthy horses in a cross over design by infusing NazEDTA (30-90 mg/kg/h) and calcium gluconate (4-16 m ^ ^ ) . Four healthy horses were infused with 0.9% NaCl to serve as controls. 47 The CsL^* set-point was 1.37 ± 0.05 mmol/L. which is higher than values, teported for humans and dogs (1.0-1.2 mmol/L). In horses in which hypercalcemia was induced first the Ca^^ set point was lower (1.30 ± 0.05 mmol/L). We demonstrated that the phenomenon of hysteresis was present during experimentally-induced hypocalcemia and hypercalcemia. At the same serum Ca^^ concentration, the serum PTH concentration was higher when the serum Ca2+ concentration was falling (induction of hypocalcemia and recovery from hypercalcemia) than when the serum Ca^^ was rising (induction of hypercalcemia and recovery from hypocalcemia). Horses in which hypocalcemia was followed by hypercalcemia secreted more (?<0.05) PTH (7440 ± 740 pmol min/L) than horses in which hypercalcemia was followed by hypocalcemia (5990 ± 570 pmol min/L). In conclusion, this study has demonstrated that the Ca^^ set-point in the horse is higher than in other domestic animals and man. We have shown that the Ca^^/PTH relationship in ^ tS e s ÿ s i^ o id a l and displays hysteresis during both hypocalcemia and hypercalcemia, andihat extracellular Ca^^ concentrations may affect the response of the - pyathyroid gland to hypobalcemia. Defining the Ca set-point in horses may help to explain the pathophysiology of hypo and hypercalcemia in this species. Introduction Parathyroid hormone (PTH) is secreted by the chief cells of the parathryroid gland, and plays an important role in calcium homeostasis. Parathyroid hormone secretion is altered by small physiological changes in extracellular ionized calcium (Ca^^ concentrations, and there is an inverse sigmoidal relationship between serum Ca^^ 48 t3 u - A -'b -.t" - ' ■ *. ■ • ~ ^ concentrations and PTH secretion flBrown. 1983; Maver and Hurst. 1978; Brent et al., 1988; Aguilera-Tejero et al., 1996). This relationship is represented by a four-parameter model (Brown, 83), which enables the parathyroid gland to rapidly respond to hypocalcemia. The parathyroid chief cells detect changes in extracellular Ca^^ concentrations by a calcium-sensing G protein-linked cation receptor (CaR) (Brown et al., 1993). The calcium set-point is defined as the serum Ca^^ concentration corresponding to the midpoint PTH value between maximal PTH concentration (PTHmax) and minimal PTH concentration (PTHmm) (Brown, 1983), or the Ca^*^ concentration corresponding to 50% of PTHmax (Felsenfeld and Llach, 1993). The Ca^^ set-point is considered an indicator of the serum Ca^^ concentration at which PTH secretion is stimulated (Felsenfed and Llach, 1993). Determining the Ca^^ set-point in the horse will provide useful information in understanding the response of the parathyroid gland when calcium homeostasis is disturbed. Abnormalities of the Ca^^ set-point have been reported in humans with chronic renal failure (Slatopolsky and Delmez, 1996), hemodialysis (Pahl et al., 1996), and primary hyperparathyroidism (Schwarz et al., 1992). Conditions of horses characterized by abnormal calcium homeostasis include idiopathic hypocalcemia of foals (Beyer et al., 1997), primary hyperparathyroidism (Frank et al., ^199^ nutritional secondary hyperparathyroidism (Ronen et al., 1992), pseudohyperparathyrôîdism (Marr et al., 1989), hypoparathyroidism (Couetil et al., 1998), vitamin D toxîcîty (Hairrington and Page, 1983), renal disease (Elfers et al., 1986), and se p ^ P'oribio et al., 2001). . 49 We have reported that some horses with sepsis and ionized hypocalcemia do not have the expected increase in serum PTH concentrations for the degree of hypocalcemia (Toribio et al., 2001). Such horses may have impaired parathyroid gland function associated with an abnormally low Ca^^ set-point. The Ca^^ set-point has been determined in humans, dogs, and horses using immunoradiometric assays (IRMA) for intact PTH (Felsenfeld and Llach, 1993; Aguilera-Tejero et al.,1996; Estepa et al., 1998). Hysteresis in the Ca^^/PTH relationship refers to the fact that, for the same serum concentration, the PTH concentration is different during the induction of either hypocalcemia or hypercalcemia than during the recovery periods (Conlin et al., 1989). Hysteresis has been demonstrated in humans (Conlin et al., 1989) and dogs (Aguilera- Tejero et al., 1996), and is thought to result from depletion of PTH stores as a consequence o f prolonged or severe hypocalcemia (Schwarz et al., 1992; Kwan et al., 1993; Sanchez et al., 1996), or from the ability of the parathyroid gland to sense changes in the direction of extracellular Ca^^ concentrations (Aguilera-Tejero et al., 1996). Because the parathyroid gland is designed to respond to either hypocalcemia or hypercalcemia, and there is a sigmoidal relationship between serum Ca^^ and PTH concentrations (Mayer and Hurst, 1978; Brown, 1983; Aguilera-Tejero et al., 1996), it is possible that the extracellular Ca^^ concentrations at the time of serum Ca^^ change may affect the response of the parathyroid gland to hypocalcemia or hypercalcemia. We speculate that PTH secretion in horses with experimentally-induced hypocalcemia followed by hypercalcemia will be different compared to PTH secretion from horses with experimentally-induced hypercalcemia followed by hypocalcemia. 50 . f ■ The objectives of this study were 1) to determine the set-point in healthy horses, 2) to determine if induction and recovery from hypocalcemia and hypercalcemia results in hysteresis of serum PTH concentration in healthy horses, and 3) to determine if the order o f induction of abnormal calcium concentration (hypocalcemia followed by hypercalcemia, or hypercalcemia followed by hypocalcemia) affects PTH secretion. Materiab and Methods Horses Sixteen healthy horses (12 females and 4 castrated males), aged 3 to 12 (7.7 ± 2.8) years were selected from The Ohio State University College of Veterinary Medicine teaching herd. All horses were fed a diet of grass and grass hay, had no history of illnesses, and had received no treatments for one month prior to sampling. To assure health, a complete physical examination was performed. Values for a complete blood count (CBC), serum chemistry profile, serum total calcium (tCa), ionized calcium (Ca^^, ionized magnesium (Mg^^, PTH (1-84), phosphorus, and plasma fibrinogen concentrations, blood gases, as well as urinalysis and urine chemistry profile were within the reference ranges for all horses. The horses had experimentally-induced hypocalcemia followed by hypercalcemia, or hypercalcemia followed by hypocalcemia, and were randomly assigned to each experiment. One week later the same horses were submitted to the alternative experiment. The Ohio State University Institutional Laboratory Animal Care and Use Committee approved this study. 51 m m Ê s m m m Induction o ifhypocalcemia and hypercalcemia To induce hypocalcemia, the horses were infused with NazEDTA (5% solution in 5% dextrose), at a rate of 30-90 mg/kg/h. To induce hypercalcemia the horses were infused with elemental calcium (calcium gluconate 23% solution; calcium gluconate hemicalcium salt with 9.3% elemental calcium), at a rate of 4-16 mg/kg/h. The goal of the NazEDTA and calcium gluconate infusions was to change serum Ca^^ concentrations by ± 0.85 mmol/L (± 3.4 mg/dL) from the normal serum Ca^^ concentrations (1.6S±0.1 mmol/L, 6.6 ± 0.3 mg/dL), in order to maximally stimulate or suppress PTH secretion. Na:EDTA was infused intravenously to decrease serum Ca^^ concentrations to 0.8 nunol/L, and calcium gluconate was infused to increase serum Ca^^ concentrations to 2.5 nunol/L in 120 min. To monitor for side-effects of hypocalcemia and hypercalcemia, heart rate, respiratory rate, capillary refill time, and electrocardiogram (lead II) were evaluated every 15-20 min during the experiments. Ejqteriment /. Inductfim^f li^ocalceinia followed by hypercalcemia After baseline sampling, 6 healthy horses (4 females and 2 castrated males) were infiised with NazEDTA at a rate of 30 mg/kg/h (time 0). The rate of infusion was increased by 10 mg/kg/h every 15 min, to a final rate of 90 mg/kg/h (90-120 min). The horses were infused with NazEDTA for 120 min, and then the infusion was discontinued until serum returned to normal values (recovery period). Recovery to normocalcemia was not assisted by infusion o f calcium. During the recovery period. 52 _bl(KxiJ^aîLcj;uiC(ælEatiQiisj^€æ£LjdëL^W^^ cqncenb@dons_ reached the reference range (1.55-1.67 mmol/L). When blood Ca^^ concentration was within the reference range, horses were infused with elemental calcium at a rate of 4 mg/kg/h. The infiision rate was increased by 2 mg/kg/h every 15 min to a final rate of 16 mg/kg/h (90-120 min). Elemental calcium infusion rates of 4-8 mg/kg/h are within the rates that we routinely use in critically ill hypocalcémie horses admitted to the Ohio State University College of Veterinary Medicine Equine Intensive Care Unit. Higher infusion rates of calcium gluconate were used to maximally suppress PTH secretion. After the calcium gluconate infusion was discontinued (recovery period), blood samples were taken every 10 min until blood Ca^^ concentrations reached the reference range, then every 4 h for 16 h to monitor serum Ca^^ and PTH concentrations. Blood samples were collected by venipuncture at 24 and 48 h for a CBC, a serum chemistry profile, and serum Ca^* and PTH concentrations. The time required for serum Ca^^ concentrations to return to the reference range after induction of either hypocalcemia or hypercalcemia was estimated as the time interval from the discontinuation of the infusion of either NaiEDTA or calcium gluconate to the time whe Ae first serum Ca^^ value within the reference range was documented. Serum Ca^^ and PTH values obtained during the induction of hypocalcemia in this experiment were used to calculate the Ca^^ set-point and to model the Ca^^/PTH relationship. Serum Ca^^ and PTH concentrations obtained during induction and recovery 6om hypocalcemia, and during the induction and recovery from hypercalcemia were used to determine hysteresis. Four horses fiom this group were used to calculate the urinary fractional clearance o f calcium (FCa) and phosphorus (FP). 53 ■*< - , Ejoferiment 2. Induction of hypercalcemia followed by hypocalcemia One week following Experiment 1, horses were submitted to experimentally- induced hypercalcemia followed by hypocalcemia. Serum chemistry profile, CBC, serum PTH, tCa, Ca^\ Mg^\ and phosphorus concentrations, as well as urine profiles and urinalyses were within the reference ranges for all horses prior to Experiment 2. Horses were infused with elemental calcium at a rate of 4 mg/kg/h (time 0) with 2 mg/kg/h increases every 15 min, to a final rate of 16 mg/kg/h (90-120 min). The horses were infused with calcium gluconate for a total of 120 min. During the recovery period, blood Ca^^ concentrations were measured every 10 min until Ca^^ concentration reached reference range values. When Ca^^ concentration was within the reference range, the horses were infused with NazEDTA at a rate of 30 mg/kg/h with 10 mg/kg/h increases every IS min, to a final rate of 90 mg/kg/h (90-120 min). After NazEDTA infusion was discontinued, blood samples were taken every 10 min until reached reference range values, then every 4 h for the next 16 h to monitor serum Ca^^ and PTH concentrations. Serum Ca^^ and PTH concentrations during induction and recovery from hypercalcemia and hypocalcemia were used to determine hysteresis, and serum Ca^^ and PTH values finm the induction of hypercalcemia were used to model the Ca^^/PTH sigmoidal relationship. Experimen ts. Induction of hyp«ircafcemi«-followed by hypocalcemia In six additional healthy horses (4 females and 2 castrated males) experimental hypercalcemia followed by hypocalcemia was induced as described in Experiment 2. Serum Ca^* and PTH concentrations from the induction and recovery from hypercalcenua and hypocalcemia were used to calculate hysteresis, and serum Ca^^ and PTH concentrations from the induction of hypercalcemia were used to model the Ca^^/PTH sigmoidal relationship. Four horws fronfthis group were used to calculate FCa and FP. '' : , / / Ejqp^ment ^ Induction of hypnadcemii^ followed by hypercalcemia One week following Experiment 3, horses were submitted to experimentally- induced hypocalcemia followed by hypercalcemia as described in Experiment I. Values for serum chemistry profiles, CBC, serum PTH, tCa, Ca^\ Mg^^, and phosphorus concentrations, as well as urine profiles and urinalyses were within the reference ranges prior to Experiment 4. Serum Ca^^ and PTH values during the induction of hypocalcemia were used to calculate the Ca^^ set-point and to model the Ca^^/PTH relationship. Serum Ca^* and PTH concentrations during the induction and recovery from hypocalcemia and hypercalcemia were used to determine hysteresis. 55 Fout healthy horses infused with 0.9% NaCl at a rate of 1 ml/kg/h for 120 min. The infusion was then discontinued for 120 min, followed by a further 120 min of infusion, and then discontinued. Blood samples were taken every 20 min from time 0 for 8 h, then every 4 h for the next 16 h to measure serum Ca^^ and PTH concentrations. Two horses from this group were used to calculate FCa and FP. Venous blood samples to determine serum phosphorus, total calcium, and creatinine concentrations to calculate the FCa and FP were collected every 60 min for 8 h. For all experimental horses, the catheters were removed after the infusions were discontinued. Venous blood samples for a CBC, a serum chemistry profile, and serum tCa, Ca^^ and PTH concentrations were collected by venipuncture at 24 and 48 h. Urine profiles and analyses were also performed at 24 and 48 h to assess renal function. Sampling Before the experiments, venous blood samples for serum chemistry profile, and tCa, Ca^\ Mg^^, phosphorus, and PTH concentrations were collected in tubes with no additives. Venous blood samples for a CBC and fibrinogen concentration were collected in EDTA or sodium citrate, respectively. For blood gas determinations, venous blood samples were collected anaerobically in heparinized syringes. An intravenous catheter was placed aseptically in each jugular vein. The left catheter was used for the infusion of 56 ■calcimiLglucionate^NaîEDTA^ot Q^% NaCl. and the right catheter was used for blood_ sample collection. Catheter patency was maintained by administration of 0.9% NaCl solution at a slow rate (0.5 ml/kg/h). For serum PTH measurement, the blood samples were allowed to clot at 4 °C for one hour, centrifuged at 4 °C immediately after clotting, and stored at -20 °C until batch analysis. Four female horses from Experiment 1, four female horses from Experiment 3, and two female horses from Experiment 5 were used to calculate FCa and FP every 60 min for 8 h. An indwelling 28 French Foley urinary catheter was left in place for 8 h. The bladder was emptied 10 min before every urine collection. Hematology and blood analytes Complete blood cell counts were performed by an automated system (Coulter Counter Model S-plus, Coulter Electronics, Inc, Hialeah, Florida, USA). Serum profile, tCa, and phosphorus were measured using colorimetric reactions in an automated analyzer (Boehringer MMuiheim/Hitachi 911 system, Boehringer Mannheim Corp, Indianapolis, Indiana, USA). Plasma fibrinogen concentrations were determined using a nephelometric analyzer. (ACL 200 Automated Coagulation Laboratory, Instrumentation Laboratory, Lexington, Massachusetts, USA). Blood gases were measured using a blood gas analysis system (ABL 500, Radiometer Cqsenhagen, Radiometer Medical A/S, Copenhagen, Denmark). The blood samples for Ca^^ and Mg^^ concentrations were collected under anaerobic conditions and measured immediately using Ca^ - or Mg^- selective electrodes (Nova 8, Nova Biomedical, Waltham, Massachusetts, USA). Serum 57 I * :: .. S " : intact PTH concentrations were determined using an immunochemiluminometric assay^ (bnmulite intact PTH assay. Diagnostic Products Corporation, Los Angeles, California, USA) validated for the horse by our laboratory (Toribio et al., 2001). Fractional urinary clearance of calcium and phosphorus Urine was acidified with 6 N HCl to dissolve calcium salts. Urine calcium, phosphorus and creatinine concentrations were measured as described for serum. FCa and FP were calculated using serum and urine total calcium, phosphorus and creatinine concentrations as described by Roussel et al. (1993). The calcium set-poiut The Ca^^ set-point was calculated as the serum Ca^^ concentration at which serum PTH concentrations were 50% of maximal PTH concentration during the induction of hypocalcemia (F$ilsenfeld and Llach, 1993). The Ca^^ set-point was calculated for every animal using Ca^^ and PTH concentrations from the induction of hypocalcemia of Experiments 1 and 4. Values of and PTH from the recovery period after hypocalcemia were not included in the calculation of the Ca^^ set-point because different values of PTH were expected (hysteresis) for the same Ca^* value. Because hypercalcemia may have affected the response of the parathyroid gland to hypocalcemia, we also calculated the Ca^^ set-point in the horses hom Experiments 2 and 3. 58 The Cm'/PTH relatlonshfp To determine the relationship between serum and PTH concentrations, serum Ca^^ and PTH concentration from the induction of hypocalcemia (Experiments 1 and 4) and the induction of hypercalcemia (Experiments 2 and 3) were used. Serum Ca^^ and PTH values from the recovery periods were not included because hysteresis was expected. The Ca^^/PTH relationship was determined by the four-parameter logistic model previously described by Brown (1983), and by the Emax sigmoidal model with a baseline value (or Hill’s equation) (Gabrielsson and Weiner, 1997). In the four-parameter logistic model, y = - ~ ; ~ +d 1 +(fj y=PTH released, o=maximum PTH concentration measured during hypocalcemia (PTHmax). <#=minimumPTH concentration measured during hypercalcemia (PTHmin). x=serum Ca^^ concentration, c=Ca^* set-point, and b=slope. In the Emax sigmoidal model (Hill’s equation). F r ' ” E = E .+ ' E C so + C E=Effect (PTH released), £o=PTHmin. £ ’m E C ‘s(f=Ca. * set-point, n=slope, sigmoidicity factor, or Hill coefficient. 59 The serum PTH area under the time concentration curve (AUC) _ ____ To detennine if the order ofNazEDTA or calcium infusion had an effect on PTH secretion, non-compartmental analysis (linear trapezoidal method) (Gabrielsson and Weiner, 1997) was used to calculate the area under the time concentration curve (AUC) for serum PTH concentrations. AUC (n-n’+l) is the area between two time points. Ci and Ci+7 are the corresponding serum PTH concentrations, and A/ is the time interval (10 min). ti 2 To calculate the AUC per horse over the entire range of PTH secretion, the following formula was used, 0 S 2 Statistical analyses Results were expressed as meapSD. PTHmax was defined as the maximal PTH concentration during NazEDTA-induced hypocalcemia, and PTHmin as the minimal PTH concentration during calcium-induced hypercalcemia. Comparisons between groups were made using the r-test or the Mann-Whitney rank test. PTH values during induction of and recovery fix>m hypocalcemia in Experiments 1-4 were compared using the Kruskal- Wallis one-way ANOVA (Fisher and Van Belle, 1993), and multiple comparisons were 60 ------ made with the Dunn’s test (Fisher and Van Belle, 1993). Similarly, Kruskal-Wallis one way ANOVA was used&tp compare PTH concentrations during induction of and recovery from hypercalcemi^ with mWtiple comparisons made with the Dunn’s test. Spearman rank (p) correlation was used to determine associations between serum Ca^^ and PTH concentrations during the different experiments (Fisher and Van Belle, 1993). Hysteresis was defined as statistically different serum PTH values for the same 2+ 2+ serum Ca value depending on the direction of change of serum Ca concentration. P values less than 0.05 were considered significant. Results There were no significant differences in serum Ca^^ and PTH concentrations between Experiments 1 and 4, nor were there differences in the time required time required for serum Ca^^ concentrations to return to the reference range after induction of either hypocalcemia or hypercalcemia. Therefore, these two experiments were pooled (n=12). There were no significant differences when serum Ca^^ and PTH concentrations from Experiments 2 and 3 were compared, nor were there statistical differences in the time required for serum Ca^^ concentrations to return to the reference range after induction of hypercalcemia or hypocalcemia. Therefore, the experiments were pooled (n=12). For calculation of the Ca^^ set-point and hysteresis serum PTH values were expressed as a percentage of PTHmax because there was heterogeneity in serum PTH concentrations between horses. 61 Baseline values for serum tCa, Ca^\ P, and PTH concentrations were not statistically different among experimental groups. Infusion of NaaEDTA resulted in a decrease in serum Ca^^ concentrations and an increase in serum PTH concentrations, and infusion of calcium gluconate increased serum concentrations and decreased serum PTH concentrations (Figs. 3.1, 3.2). During hypocalcemia and hypercalcemia the correlation (p) between serum Ca^* and PTH concentrations ranged 6om *0.5 to -0.9 (P Ejqferiments 1 and 4 During NazEDTA-induced hypocalcemia, serum Ca^^ concentration decreased to 0.85 ± 0.10 nunol/L (3.5 ± 0.40 mg/dL) (normal^ 1.65 ± 0.10 mmol/L, 6.6 ± 0.3 mg/dL), and serum PTH concentration increased to 53.0 ± 11.0 pmol/L (normal=4.4 ± 2.5 pmol/L) (Fig. 3.1). The mean PTHmax in Experiments 1 and 4 was higher than that in Experiments 2 and 3.^KEHZ(#y^ues in our experiments were greater than those reported in previous studies in horses«where an immunoradiometric assay for PTH was used (Aguilera-Tejero et/dl , 1998;, Estepa et al., 1998). After the NazEDTA infusion was ^ w O ' discontinued, senim Ca retiunedto the reference values in 106 ± 10 min (Fig. 3.1). 62 ' C,. ' ' \".'. ■ ., •■ ■ . . %; ______Üsing the values for 50% of FTH^mr the set-point was calculated to he 1.17 j: 0.05 nunol/L (5.5 ± 0.3 mg/dL) (Figs. 3.4, 3.6), which is greater than the Ca^^ set-point reported for humans and dogs (Felsenfeld and Llach, 1993; Aguilera-Tejero et al., 1996). Induction of hypocalcemia resulted in a sigmoidal relationship between serum Ca^^ and PTH concentrations (Figs. 3.4, 3.6). Maximal PTH concentrations were attained with a mild decrease in serum Ca^^ concentration, and serum Ca^^ concentrations below 1.25 mmol/L did not result in an additional increase in serum PTH concentrations. Furthermore, at serum Ca^^ concentrations below 1.10 nunol/L, serum PTH concentrations decreased in 4 horses to approximately 80% of PTHmax- Hysteresis was present in the hypocalcémie phase of the experiments. For the same serum Ca^^ concentration, PTH concentrations were higher during the induction of hypocalcemia than during recovery (P<0.05), and when NazEDTA infusion was discontinued, serum PTH concentrations decreased sharply, in a linear fashion despite the severe hypocalcemia (Fig. 3.4). During the hypercalcémie phase of Experiments 1 and 4, serum PTH concentrations decreased to very low levels or were below the detection limit of the assay (Figs. 3.1, 3.4), and serum Ca^^ concentration increased to 2.45 ± 0.20 mmol/L (9.8 ± 0.9 mg/dL)- at th& e n d ^ f the calcium gluconate infusion (Fig.^ 3.1). After the calcium gluconate infusion was discontinued, serum Ca^^ returned to baseline values in 86 ± 11 min (Figt 3.1). For a ACa^^ of 0.85 mnwl/L, it took approximately 20 min longer to recover fronf hypocalcemia than hypercalcemia. Immediately after calcium gluconate infusion was discontinued, serum PTH concentrations remamed low, but once serum Ca^^ concentration was below 2.0 mmol/L serum PTH concentrations increased to greater values (P<0.05) than those during the infusion phase (hysteresis; Fig. 3.4). 63 4 /h'f"» - •. >, Æjqstes^ÊOisJLmLS—^ During the induction of hypercalcemia, serum Ca^^ concentrations increased to 2.42 ± 0.20 mmol/L (9.7 ± 0.8 mg/dL), and serum PTH decreased to low concentrations or were below the limit of detection of the PTH assay (Fig. 3.2). When the calcium gluconate infusion was discontinued, serum Ca^^ concentrations returned to normal values in 89 ± 18 min. Serum PTH concentrations remained low when serum Ca^^ concentrations were greater than 2.0 mmol/L, but when serum Ca^^ concentrations decreased below 2.0 mmol/L, serum PTH concentrations increased to values greater (P<0.0S) than those during the induction of hypercalcemia (hysteresis; Fig. 3.5). PTH concentrations of figure 3.5 are expressed as a percentage of the mean PTHmax from Experiments 1 and 4 (Fig. 3.4). When serum Ca^^ concentration was within the reference range, hypocalcemia was induced. Serum Ca^^ concentration decreased to 0.83 ± 0.05 nunol/L (3.5 ± 0.2 mg/dL), and serum PTH concentration increased to 42.2 ± 8.0 pmol/L (Fig. 3.2). In contrast to the induction of hypocalcemia in Experiments 1 and 4, in which the Ca^/PTH relationship was sigmoidal, the induction of hypocalcemia in Experiments 2 and 3 resulted in a more linear Ca^/PTH relationship, and serum PTH did not reach a plateau in 8 of 12 horses (Fig. 3.5). For the same time interval and rate of NazEDTA infusion, induction of hypocalcemia resulted in significantly lower serum PTH concentrations in Experiments 2 and 3 than in Experiments 1 and 4 (P<0.05; Figs. 3.1-3.2, 3.4-3.5). When NazEDTA infusion was discontinued, serum PTH concentrations decreased in a pattern similar to that of Experiments 1 and 4, but it took longer for serum Ca^^ concentrations to 64 return to normal values (116 ± 18min) than in Experiments I tmd 4: however, the ^ e difference was not statistically significant. For the same serum Ca^^ concentration, serum PTH concentrations were significantly higher during the induction of hypocalcemia and during the recovery Aom hypercalcemia than during recovery from hypocalcemia and induction of hypercalcemia (hysteresis; Fig. 3.5). For a of 0.85 nunol/L, it took approximately 25 min longer to recover from hypocalcemia than hypercalcemia. Induction of hypocalcemia and hypercalcemia resulted in mild changes in serum phosphorus concentrations; however, these changes were not significantly different from baseline, nor in Experiments 1 and 4 as compared to Experiments 2 and 3. The Ca^^ set-point for horses of Experiments 2 and 3 was significantly lower (1.3 ± 0.05 mmol/L, 5.2 j=<0.2 mg/dL) than the set-point for horses of Experiments 1 and 4 (P<0.05). " Serum PTH and PTHmax concentrations from Experiments 1 and 4 were significantly greater than serum PTH and PTHmax concentrations from Experiments 2 and 3 (P<0.05) during induction of hypocalcemia. However, serum PTH concentration from the recovery of hypocalcemia from Experiments 1 and 4 were not statistically different than those of Experiments 2 and 3. PTHmax concentrations in horses of Experiments 1 and 4 were 6 to 22-fbld greater than the baseline PTH concentrations (mean was 11.3 ± 5.1- fold increase), while PTHmax concentrations in Experiments 2 and 3 were 5 to 12 greater than the PTH baseline concentrations (7.7 ± 2.0-fold increase). No statistical difference in serum PTH concentrations was found during the recovery from hypercalcemia between experiments. 65 Experiments 5 In the control horses (Experiment 5, Fig. 3.3), there was no significant difference fit)m baseline values iri*SMum Ca^^ and PTH concentrations at any time point during the NaCl infusion. Serupi Ca and PTH concentrations remained within the reference range during the infusion of 0.9% NaCl and during the recovery period. The relationship between serum Ca * and PTH concentrations The serum Ca^^ concentration and PTH concentration (expressed as a percentage of PTHmax (100%) for each horse) during the induction of hypocalcemia (Experiments 1 and 4), and the induction of hypercalcemia (Experiments 2 and 3) were plotted and analyzed by the four-parameter logistic model and the Emax sigmoidal model. The resulting functions were sigmoidal and almost identical (Fig. 3.6). The relationship between serum Ca^* and PTH concentrations in the horse is therefore demonstrated to be inverse and sigmoidal as described for other species (Mayer and Hurst, 1978; Brown, 1983; Aguilera-Tejero et al., 1996). The Ca^^ set-point was 1.37 ± 0.05 mmol/L (Fig. 3.6). The area under the PTH-time concentration curve (AUC) When the AUC for PTH production in Experiments 1 and 4 was compared to that of Experiments 2 and 3, horses in Experiments 1 and 4 secreted more PTH (7440 ± 740 66 % Y . \ r ■ V . ' - ■ I:pmQi roia/L)..thaa-hoi:ses fiom Expcrimeats 2 and L.CSS§a^JJaLpniQLminÆ)^(lJPS)v_ débité receiving the same dose of NaiEDTA and calcium gluconate (Figs. 3.1-3.2). * Fractional urinary clearance of calcium and phosphorus Induction of hypocalcemia in Experiments 1 and 4 resulted in increased FCa and FP. FCa increased &om 2.9 ± 0.8% (time 0) to 32.3 ± 8% at the end of NazEDTA infusion (120 min), and FP increased from 0.1 ± 0.1% to 7.5 ± 1.3% at the end of the NazEDTA infusion (Fig. 3.7). At the end o f the recovery from hypocalcemia, FCa decreased to 8.9 ± 2.9%, which was greater than the baseline value (P<0.05), and FP decreased to 1.9 ± 0.2%, which was greater than the baseline value (?<0.05). Induction of hypercalcemia resulted in an increase of FCa to 59.1 ± 7.3%, while FP continued to decline to values similar to baseline (0.17 ± 0.1%) (Fig. 3.7). Calcium gluconate infusions resulted in higher FCa than NajEDTA infusions, and NazEDTA infusions resulted in higher FP than calcium gluconate infusions. In Experiments 2 and 3, during the induction of hypercalcemia, FCa increased from 3.8 ± 0.4% to 63.0 ± 13.2% (P<0.05) at 120 min, and FP increased from 0.03 ± 0.04% to^ 0.9- ± 0.6% a t -120—min-(P hypercalcemia, FCa decreased to 11.4 t 5.5%, a value statistically higher than the baseline value (P<0.05),JÈ(4^ FP decreased to 0.01 ± 0.01%. Induction of hypocalcemia resulted in an increase p-FCa tq 38.0 ± 5.7% at the end of the NazEDTA infusion, and an increase in FP to 5J2 0.9%. C?aljpium gluconate infusions resulted in higher FCa than NazEDTA infusions, and NazEDTA infusions resulted in higher FP than calcium gluconate infusions. 67 — ■*»-—— -^-™- *r-— —«’w—fc ■■!■■- - ■ -j - ■ I I IIH — M __ _ ^ ^■.M>'—- .,- ----^ - - -»-" - . ».£ ?i^. . ' KU In this study we have detennined that horses have a higher Ca^^ set-point (1.37 ± 0.05 mmol/L) than human beings and dogs (l.O-i.2 mmol/L) (Felsenfeld and Llach, 1993; Aguilera-Tejero et al., 1996). Serum total and ionized calcium concentrations in horses are greater than those in other domestic animals and humans (Kaneko et al., 1997). The high serum Ca^^ concentration in the horse may be the result of a high Ca^^ set-point. We speculate that horses are not as sensitive as other species to the suppressive effects of Ca^^ on PTH secretion. There is also evidence that the Ca^^ set-point is dependent upon basal Ca^^ concentrations, and an increase in basal Ca^^ concentrations was associated with an increase in the Ca^^ set-point, suggesting an adaptation of the set-point to existing serum Ca^^ concentrations (Rodriguez et al., 1997; Caravaca et al., 1995; Pahl et al., 1996). Increased extracellular Csl^* concentrations have been associated with increased intracellular calcium concentrations, and an increase in the Ca^^ set-point (Wallfelt et al., 1988). The Ca^^ set-point is determined in part by factors that regulate PTH gene expression. Calcitriqir(l,25-^hydroxyvitamin D) and extracellular Ca^^ have negative regulatory effects @%tlK PTH gene (Slatopolsky and Delmez, 1996). Calcitriol increases the sensitivity of'the parathÿcbid gland to extracellular Ca by increasing the expression ■ Jt\ ’» ■' ' -T'' . = ■ ■ , of the C ^ a n d megalin/^330/LRP-2 mRNA. This results in low PTH production, and shifts the Ca^^/PTH sigmoidal curve (and the Ca^^ set-point) to the left (down) (Slatopolsky and Delmez, 1996; Brown et al., 1996; Liu et al., 1998). Plasma 68 ♦T., jtanjL^toilon$JpLvÜ9miïl Ilm D) in the horse are considerably lower than those in other domestic animals and humans (Mâenpââ et al., 1988; Breidenbach et al., 1998). Because horses have low serum calcitriol concentrations, and calcitriol inhibits PTH synthesis and secretion in other species (Slatopolsky and Delmez, 1996), it is possible that higher serum Ca^^ concentrations are required to suppress PTH secretion in the horse, resulting in a higher Ca^^ set-point. A low number of vitamin D receptors (VDR) in equine parathyroid chief cells could reduce the regulatory effect of calcitriol on PTH synthesis and secretion. There is a direct relationship between serum calcitriol concentrations and parathyroid gland VDR protein and mRNA expression in other species (Naveh-Many et al., 1990; Beckerman and Silver, 1999). Parathyroid glands from humans and rats with chronic renal failure and uremia contained 30-50% of the number of VDR found in controls (Korkor et al., 1987; Merke et al., 1987). Based on the vitamin D/VDR relationship in the parathyroid glands in other species, we speculate that horses may have a relatively low number of VDR in their parathyroid glands. The physiology of regulation of VDR in the horse parathyroid gland has not been reported. Brown et al. (1993) cloned a calcium-sensing receptor (CaR) in parathyroid chief cells that binds Ca^"^ and other cations and regulates PTH secretion by sensing changes in phosphoinositide metabolism and cytosolic Ca^^ concentration. Activation of the CaR results in decreased PTH secretion, and a lower Ca^^ set-point, while decreased expression or activity of the CaR results in an increased Ca^^ set-point (Kifor et al., 1996). Humans with uremia have a 50% decrease in the number o f CaR in the parathyroid chief cells (Kifor et al 1996), which would be sufficient to account for the abnormally increased Ca^^ set-point in these patients. Other proteins such _a_s_ megalin/a>330/LRP-2, which is an endocytic receptor with calcium-sensing properties in the parathyroid gland and other tissues (Lundgren et al., 1994; Hjâlm et al., 1996), may play an important role in the regulation of PTH synthesis and secretion. Decreased mRNA expression and protein synthesis for megalin/gp330/LRP-2 have been demonstrated in parathyroid adenomas and are associated with an increased in Ca^^ set- point in these patients (Famebo et al., 1998). Whether the high set-point in the horse results from a relatively low expression and/or activity of proteins in calcium-sensing system remains to be determined. An increased Ca^^ set-point has been reported in association chronic renal failure in humans (Brown et al., 1982), and is likely the cause of the hypercalcemia in affected individuals. The Ca^* set-point has not yet been studied in sick horses. In our laboratory, we have studi^sl^tic hofses with low Ca^^ concentrations that do not display an increase in serum PTH concentration of the magnitude expected based on the severity of ; _ y "4 the hypocalcemia (Toribio et al 2001). We hypothesize that a low Ca^^ set-point in these horses could reduce parathyroid gland synthesis/secretion of PTH. Possible mechanisms for reduction in PTH synthesis/secretion in this setting include increased activity of the calcium-sensing system and/or effects of local and systemic factors including inflammatory mediators (Nielsen et al., 1997; Carlstedt et al., 1999). Although hysteresis has been reported in humans (Conlin et al., 1989) and dogs (Aguilera-Tejero et al., 1996), this it the first study to report the presence of hysteresis in the Ca^^/PTH relationship in horses. We found that, at the same serum Ca^^ concentrations, PTH values were higher during induction of than during recovery from 70 JiyiKM:akeiniaw^aiuLjHaæ^lûglu;x-diinfig_j:Qç^ inductLomM hypercalcemia. We believe, based on the results of this study, that equine parathyroid glands are able to detect the direction of change in serum Ca^^ concentration during both hypocalcemia and hypercalcemia. Although it is not clear why the parathyroid gland responds differently to changes in calcium concentrations depending upon the direction of concentration change, previous studies have suggested that PTH release is not determined only by the absolute serum Ca^^ concentration, but also by the rate of serum Cat^^ change (kwan et al., 1993; Brent et al., 1988; Grant et al., 1990; Cunningham et al., 1989). An "anticipatory response” of the parathyroid gland is expected to be advantageous, enabling the parathyroid gland chief cells to respond more rapidly to changes in serum Ca^^ concentrations. A decrease in the rate of PTH secretion during hypocalcemia (hysteresis) may result from extracellular calcium-dependent intracellular PTH degradation (Kwan et al 1993, Habener et al 1984). However, this mechanism has been questioned because alterations in intracellular PTH degradation in response to changes in extracellular calcium concentrations require at least 40 min (Habener et al 1984) and PTH secretion decreased within minutes o f an increase in extracellular Ca^^ concentration in ours and other studies (Conlin et al., 1989; Aguilera-Tejero et al., 1996). Parathyroid fatigue, as a result of rapid depletion of intracellular PTH stores, has been considered a possible cause of hysteresis during hypocalcemia (Kwan et al., 1993). However, in the present study, we used a slow (120 min) induction model of hypocacenua, and most horses were capable of maintaining increased serum PTH concentrations throughout the experiment. A previous study in dogs, using variable rates of induction of hypocalcemia, found no differences in 71 PTH secretion during induction and recovery from hypocalcemia, nor in maximal PTH concentrations (Aguilera-Tejero et al., 1996). Likewise, humans had comparable maximal PTH concentrations whether hypocalcemia was induced in 60 or 120 min (Brent et al 1988). In addition, Aguilera-Tejero et al. (1996) found that hysteresis was a reproducible phenomenon in dogs after two sequential cycles of hypocalcemia indicating that that hysteresis may not be completely explained by PTH depletion. All horses with induced hypocalcemia (Experiments 1 and 4) reached a serum PTH concentration plateau. Four of these horses had a decline in serum PTH concentration to approximately 80% of PTHmax when serum concentrations declined below 1.1 mmol/L, and before the NazEDTA infusion was discontinued. Parathyroid gland exhaustion or fatigue may have been responsible for the decreased PTH secretion in these horses. It is also possible that this decline in serum PTH may have resulted from impaired PTH synthesis and/or secretion due to other regulatory processes such as alterations in the calcium-sensing system. Hysteresis is a phenomenon now reported in 3 species. Hysteresis is the response of the parathyroid gland to reductions in the rate of change of the serum Ca^^ concentration or to a reversal in the direction of change in the serum Ca^^ concentration -(Lewk^et-aL1995; Gtan4et^al.,1990;-Aguilera-tejero-et^aL,-1996; Conlin et al., It may also be a manifestation of parathyroid gland exhaustion during prolonged or severe hypocalcemia (Kwan et al., 1993). Hysteresis may be a mechanism designed to protect against over correction of t|^ serum Ca^^ concentrations. When hypocalcemia is abating, the parathryroid chief ^çel&*may decrease PTH secretion to reduce the compensatory v - V'"* ' calcium-conserving effeçts of PIH on the kidney and its calcium mobilizing effects on bone CAt{uilerarTej!OT et al., 1996). .' "'T : . ' 72 The lowest serum concentration in our studies was attained at 110-120 min of NazEDTA infusion. Based on information from other species, this interval may have been long enough for the parathyroid gland to synthesize new PTH. No information on the time required for equine parathyroid cells to synthesize and secrete PTH is available. Bovine parathyroid cells require approximately 30 min to synthesize and secrete PTH (Habener et al., 1984), and chief cell PTH stores are thought to be sufficient to maintain maximal (hypocalcemia-stimulated) PTH secretion for up to 1.5 h in calves (Mayer and Hurst, 1978). Change in PTH mRNA expression in response to a change in extracellular Ca^^ concentrations requires up to 3 h in rats (Yamamoto et al., 1989) and 12 h in cattle (Russell et al., 1983). This response is too slow to support new synthesis of PTH in the short term. However, it is possible that in our horses, a combination of PTH release from cytoplasmic stores, and new synthesis of PTH was sufficient to sustain increased serum PTH concentrations during experimental hypocalcemia. We, like others (Conlin et al., 1989), found that, during the induction of hypercalcemia, serum PTH concentrations were lower than during the recovery period (Figs. 3.4 and 3.5). It is possible that higher serum PTH concentrations during recovery from hypercalcemia are important to reduce renal loses of calcium. The reason that recovery period serum PTH concentrations remained as low as induction period values when serum Ca^^ concentrations were greater than 2.0 mmol/L is unclear. We speculate that in the horse, sehun Ca^^ concentrations higher than 2.0 nunol/L have no additional stimulatory ef%ct on the calcium-sensing system, and therefore chief cells cannot differehHate directional changes in serum Ca^^ concentrations at these high serum Ca^^ concentrations. We also speculate that it is advantageous for the parathyroid gland to 73 Ü s i ^ ' “ ~ .^ # L ;• sense the absolute sàrdm concentrations, as well as the direction of change in serum t« ^ ‘ concentrations, because thisprovides better calcium homeostatic control. In addition to determining that hysteresis was present during both hypocalcemia and hypercalcemia in horses, we found that the order in which hypocalcemia or hypercalcemia is induced affects PTH production by the parathyroid gland. Horses in which hypocalcemia was followed by hypercalcemia (Experiments 1 and 4) secreted more PTH (P<0.05) while hypocalcémie than horses in which hypercalcemia was followed by hypocalcemia (Experiments 2 and 3). Moreover, the sigmoidal relationship between serum Ca^^ and PTH concentrations in horses in which hypocalcemia was induced first was not present when hypocalcemia was preceded by hypercalcemia. It is unclear why horses in Experiments 2 and 3 produced less PTH (for the same dose of NazEDTA and at the same serum Ca^^ concentrations), than horses in Experiments 1 and 4. It is possible that modulation of PTH secretion by the Ca^^-sensing system may vary according to the extracellular concentrations to which the parathyroid cells are exposed. Although the present study was not designed to sustain (clamp) high serum Ca^* concentrations, our results were similar to those reported by Sanchez et al. (1996) in which hypercalcemia was sustained in dogs for 90 min. In our study, we increased serum Ca^^ concentrations to almost twice baseline values, which may have been similar to clamping serum Ca^^ at high concentrations. Exposure of chief cells to high extracellular Ca^^ concentrations may have modified Ca^^-sensing mechanisms such that PTH secretion in response to subsequent hypocalcemia was reduced in both the canine and the equine models. An increase in serum Ca^^ concentrations resulted in an increase in FCa with minimal changes in FP. We believe that inhibition of serum PTH secretion as a result of 74 hypercalcemia, as well as activation of the CaR in the_dLstal convoluted tobules_ of kidney (Brown, 1999), were the mechanisms responsible for the increased FCa in these horses. Induction of hypocalcemia resulted in an increase in FCa despite high serum concentrations of PTH, and may have reflected excretion of calcium-EDTA chelated complexes into the urine. The increased FP during hypocalcemia probably resulted from PTH-mediated inhibition of phosphate reabsorption in the proximal tubules (Rosol and Capen, 1997). In conclusion, this study has demonstrated that the Ca^^ set-point in the horse is higher than in other domestic animals and man. We have shown that the Ca^^/PTH relationship in horses is sigmoidal and displays hysteresis during both hypocalcemia and hypercalcemia, and that extracellular Ca^^ concentrations may affect the response of the parathyroid gland to hypocalcemia. Defining the Ca^^ set-point in horses may help to explain the pathophysiology of hypo and hypercalcemia in this species. 75 References Aguilera-Tejero E, Sanchez J, Almaden Y, Mayer-Valor R, Rodriguez M, Felsenfeld AJ. Hysteresis of the PTH-calcium curve during hypocalcemia in the dog: effect of the rate and linearity of calcium decrease and sequential episodes of hypocalcemia. J Bone Miner Res 1996; 11:1226-1233. Beckerman P, Silver J. Vitamin D and the parathyroid. Am J Med Sci 1999; 317:363-369. Beyer MJ, Freestone JF, Reimer JM, Bernard WV, Rueve ER. Idiopathic hypocalcemia in foals. J Vet Intern Med 1997; 11:356-360. Breidenbach A, Schlumbohm C, Harmeyer J. 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Four-parameter model of the sigmoidal relationship between parathyroid hormone^ release and extracellular calcium-concentration-m normaf and abnormal parathyroid tissue. 7 C/i/t Endocrinol Metab 1983; 56:572-581. Brown EM. Physiology and pathophysiology of the extracellular calcium-sensing receptor. Am J Med 1999; 106:238-253. Caravaca F, Cubero JJ, Jimenez F, Lopez JM, Aparicio A, Cid MC, Pizarro JL, Liso J, Santos 1. Effect of the mode of calcitriol administration on PTH-ionized calcium relationship in uraemic patients with secondary hyperparathyroidism. Nephrol Dial Transplant 1995; 10:665-670. Carlstedt E, Ridefelt P, Lind L, Rastad J. Interleukin-6 induced suppression of bovine parathyroid hormone secretion. Biosci Rep 1999; 19:35-42. 76 Couëtil LL. Soika JE. Nachreiner RF. Primary hvpoDarathvroidism in a horse. J Vet Intem Med 1998; 12:45-49. Conlin PR, Fajtova VT, Moctensen RM, LeBoff MS, Brown EM. Hysteresis in the relationsh^ between serum ionized calcium and intact parathyroid hormone during recovery fiom induced hyper- and hypocalcemia in normal humans. J Clin Endocrinol Metab 1989; 69:593-599. Cunningham^ Ji Altmann P, Gleed JH, Butter KC, Marsh FP, O'Riordan JL. Effect of direction and. rate of change o f calcium on parathyroid hormone secretion in uraemia. Nephrol Dial Transplant 1989; 4:339-344. HRns RS, Bayly WM, Brobst DF, Reed SM, Liggitt HD, Hawker CD, Baylink DJ. AlWadons in calcium, phosphorus and C-terminal parathyroid hormone levels in equine acute renal disease. Cornell Vet 1986; 76:317-329. Estepa JC, Aguilera-Tejero E, Mayer-Valor R, Almaden Y, Felsenfeld AJ, Rodriguez M. Measurement of parathyroid hormone in horses. Equine VetJ 1998; 30:476-481. Famebo F, Hoog A, Sandelin K, Larsson C, Famebo LO. Decreased expression of calcium-sensing receptor messenger ribonucleic acids in parathyroid adenomas. Surgery 1998; 124:1094-1098. Felsenfeld AJ, Llach F. Parathyroid gland function in chronic renal failure. Kidney Int 1993; 43:771-789. Fisher LD, Van Belle G. Biostatistics: a methodology fo r the health science. New York: John Wiley & Sons Inc, 1993; 304-495. Frank N, Hawkins JF, Couëtil LL, Raymond JT. Primary hyperparathyroidism with osteodystrophia fibrosa of the facial bones in a pony. J Vet Med Assoc 1998; 212:84- 86 . -GekmxAasextiyNf^sitet-Di-Pharmaeoldnetic/PharmacodyTtamic Data AnalysxsT Concepts and applications. 2nd ed. Stockholm, Sweden: Swedish Pharmaceutical Press, 1997:58- 250. Grant FD, Conlin PR, Brown EM. Rate and concentration dependence of parathyroid hormone dynamics during stepwise changes in serum ionized calcium in normal humans. J Clin Endocrinol Metab 1990; 71:370-378. Hjâlm G, Murray E, Cnunley G, Harazim W, Lundgren S, Onyango I, Ek B, Larsson M, Juhlin C, Heilman P^JDq^H, Akerstrom G, Rask L, Morse B. Cloning and sequencing of human s>330, a^ é|^^-b in d in g receptor with potential intracellular signaling properties. Eur JBiochei»-l9§6; 239:132-137. " / ' 77 Habener JF. Rosenblatt M, Potts JT Jr. Parathyroid hormone: biochemical aspects of biosynthesis, secretion, action, and metabolism. Physiol Rev 1984; 64:985-1053. Harrington DD, Page EH. Acute vitamin D3 toxicosis in horses: case reports and experimental studies of the comparative toxicity of vitamins D2 and D3. J Am Vet Med Assoc 1983; 182:1358-1369. Kaneko JJ, Harvey JW, Bruss ML. Appendixes. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Clinical biochemistry o fdomestic animals. 5"* ed. San Diego: Academic Press, 1997; 885-905. Kifor O, Moore FD Jr, Wang P, Goldstein M, Vassilev P, Kifor I, Hebert SC, Brown EM. Reduced immunostaining for the extracellular Ca2+-sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab 1996; 81:1598-1606. Korkor AB. Reduced binding of [3H] 1,25-dihydroxyvitamin D3 in the parathyroid glands of patients with renal failure. N E nglJM ed 1987; 316:1573-1577. Kwan JT, Beer JC, Noonan K, Cunningham J. Parathyroid sensing of the direction of change of calcium in uremia. Kidney Int 1993; 43:1104-1109. Lewin E, Nielsen PK, Olgaard K. The calcium/parathyroid hormone concept of the parathyroid glands. Curr Opin Nephrol Hypertens 1995; 4:324-333. Liu W, Yu WR, Carling T, Juhlin C, Rastad J, Ridefelt P, Akerstrom G, Heilman P. Regulation of gp330/megalin expression by vitamins A and D. Eur J Clin Invest 1998; 28:100-107. Lundgren S, Hjâlm G, Heilman P, Ek B, Juhlin C, Rastad J, Klareskog L, Akerstrom G, Rask L. A protein involved in calcium sensing of the human parathyroid and placental cytotrophoblast cells belongs to the LDL-receptor protein superfamily. Exp Cell Res 1994; 212:344-350. Mâenpââ PH, Koskinen^T, Koskmen~E; Serum profiles of vitamins A, E and D in mares^ and foals during different seasons. JAnim Sci 1988; 66:1418-1423. Marr CM, Love S, Pirie HM. Clinical, ultrasonographic and pathological findings in a horse with splenic lymphosarcoma and pseudohyperparathyroidism. Equine Vet J 1989; 21:221-226. Mayer GP, Hurst JG: Sigmoidal relationship between parathyroid hormone secretion rate and plasma calciuihi concentration in calves. £>tdocri>to/oigv 1978; 102:1036-1042. M i^e J,,Hugel'U, Zlotkbyirski ^ Szalw A, Bommer J, Mall G, Ritz E. Diminished pÀÀ^yfoîd'l,25(OH)2D3 recqitors in experimental uremia. K id n ^ Int 1987; 32:350- 353. - 78 i ' J # " ' ...... Naveh-Many T, Marx, R, Keshet E, Pike JW, Silver J. Regulation of 1,25- dihydroxyvitamin DS receptor gene expression by 1,25-dihydroxyvitamin D3 in the parathyroid iiTvivb/y Ç/iii 7/^,5/ 1990; 86:1968-1975. Niehen PK,‘Rasmussen AK, Butters K Feldt-Rasmussen U, Bendtzen K, Diaz R, Brown EM, Olgaard K. Inhibition of PTH secretion by interleukin-1 beta in bovine parathyroid glands in vitro is associated with an up-re^ation of the calcium-sensing receptor mRNA. Biochem Biophys Res Commun 1997; 238:880-885. Pahl M, Jara A, Bover J, Rodriguez M, Felsenfeld AJ. The set-point of calcium and the reduction of parathyroid hormone in hemodialysis patients. Kidney Int 1996; 49:226-231. Rodriguez M, Caravaca F, Fernandez E, Borrego MJ, Lorenzo V, Cubero J, Martin-Malo A, Betriu A, Rodriguez AP, Felsenfeld AJ. Evidence for both abnormal set-point of PTH stimulation by calcium and adaptation to serum calcium in hemodialysis patients with hyperparathyroidism, y Rone M/ter Res 1997; 12:347-355. Ronen N, van Heerden J, van Amstel SR. Clinical and biochemistry findings, and parathyroid hormone concentrations in three horses with secondary hyperparathyroidism. JSA fr Vet Assoc 1992; 63:134-136. Rosol TJ, Capen CC. Calcium-regulating hormones and diseases of abnormal mineral (calcium, phosphorus, magnesium) metabolism. In: Kaneko JJ, Harvey JW, Bruss ML, eds. Ciinical biochemistry o f domestic animais. 5"* ed. San Diego: Academic Press, 1997; 61^702. Roussel AJ, Cohen ND, Ruoff WW, Brumbaugh GW, Schmitz DO, Kuesis BS. Urinary indices of horses after intravenous administration of crystalloid solutions. J Vet Intem Med 1993; 7:241-246. Russell J, Lettieri D, Sherwood LM. Direct regulation by calcium of cytoplasmic messenger ribonucleic acid coding for pre-proparathyroid hormone in isolated bovine parathyroid cells, y C/m Invest 1983; 72:1851-1855. Sanchez J, Aguilera-Tejero E, Estepa JC, Almaden Y, Rodriguez M, Felsenfeld AJ. A reduced PTH response to hypocalcemia after a short period of hypercalcemia: a study in dogs Kidney Int Suppl 1996; 57:818-22. Schwarz P, Sorensen HA, Momsen G, McNair P, Transbol 1. Normal pattern of parathyroid response to blood calcium lowering in primary hyperparathyroidism: a citrate clamp study. Clin Endocrinol (Oxf) 1992; 37:344-348. Slatopolsky E, Delmez JA. Pathogenesis of secondary hyperparathyroidism. Nephrol Dial Transplant 1996; ll(Suppl 3):I30-135. 79 Toribio, RE, Kohn, CW, Chew, DJ. Sams, RA, Rosol, TJ. Comparison of serum parathyroid hormone and ionized calcium and magnesium concentrations and fractionai urinary clearance of calcium and phosphorus in healthy horses and horses with enterocolitis. Am J Vet Res 2001; 62:938-947. Wallfelt C, Gylfe E, Larsson R, Ljunghall S, Rastad J, Akerstrom G. Relationship between external and cytoplasmic calcium concentrations, parathyroid hormone release and weight of parathyroid glands in human hyperparathyroidism. J Endocrinol 1988; 116:457-464. Yamamoto M, Igarashi T, Muramatsu M, Fukagawa M, Motokura T, Ogata E. Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat. JClin Invest 1989; 83:1053-1056. 80 70 N ajE D T A Ca^ Gluconate r 3.0 60 - 2.5 SO I 40 - 2.0 & X 30 % - 1.5 I 20 I 10 - 1.0 0 - -10 1------r —— I------1------1------1------1------1------1------1------1 I------0.5 •50 0 50 100 150 200 250 300 350 400 450 500 550 minutes Figure 3.1. Intact PTH and Ca^^ concentrations plotted against time in 12 healthy horses in which hypocalcemia was induced prior to hypercalcemia (Experiments 1 and 4). J4aiEDTA infusion resulted in_ increased serum_PTH concentrations^ while calcium gluconate infusion resulted in decreased serum PTH concentrations. Horses of Experiments 1 and 4 had higher serum PTH and PTHmax concentrations when compared to horses in which hypercalcemia was induced prior to hypocalcemia (Fig. 3.2). The area under the time concentration curve (AUC) for serum PTH was significantly greater in horses of Experiments 1 and 4 than in horses of Experiments 2 and 3 (Fig. 3.2.). There was no statistical difference in the AUC for Ca^^ concentrations in Experiments 1 and 4 as compared to Experiments 2 and 3 (Fig. 3.2). 81 3.0 70 Ca^ Gluconate NajEDTA 60 -2 .5 50 4 o 2 40 4 L-2.0 I I 30 - CL - 1.5 % J 20 I 10 H 1.0 0 -10 I I I I------1 I I------1------1------1------1------r 0.5 •50 0 50 100 150 200 250 300 350 400 450 500 550 minutes Figure 3.2. Intact PTH and Ca^^ concentrations plotted against time in 12 healthy horses in which hypercalcemia was induced prior to hypocalcemia (Experiments 2 and 3). The AUC for serum jPTH was significantly less in horses during Experiments 2 and 3 as compared to Experiments 1 and 4. There was no significant difference in the AUC for serum Ca^^ concentration in Experiments 2 and 3 as compared to Experiments 1 and 4. 82 nmy:i 70 r 3.0 NaCI NaCI 60H - 2.5 50 Ë 2 40 H 2.0 I 30 - % 1.5 I 20 - I 10 ■ - 1.0 0 - -10 I I I------1------1------,------1 I------r.....— 0.5 •50 0 50 100 150 200 250 300 350 400 450 500 550 minutes Figure 3.3. Intact PTH and concentrations plotted against time in 4 healthy horses (Experiment 5, control). The horses were infused with 0.9% NaCI, and had no significant changes in serum PTH and Ca^^ concentrations occurred during the experiment. 83 120 1 Induction of Hypocalcemia 110 Recovery from Hypocalcemia 100 Induction of Hypercalcemia Recovery from Hypercalcemia 80 70 60 - Set-Point 5 0 - 40 • 30 2 0 - 1 0 - 0 - 3 -10 -1------r 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 Ca^*(minol/L) Figure 3.4. Intact PTH concentrations (% of PTHmax) plotted against serum concentrations in healthy horses (Experiments 1 and 4). Induction of hypocalcemia (1) resulted in a Ca^^/PTH sigmoidal curve (closed squares). During the recovery period (2) PTH concentrations were lower (hysteresis), and the PTH/Ca^^ relationship displayed linemrity (cq^iT squares). Oiïcë^lenim reached the reference range (1.55-1.67 mmol/L) hypercalcemia was induced (3) (closed circles). During the recovery period from hypercalcemia (4) PTH concentrations were greater (open circles) than during induction of hypercalcemia (hysteresis). 84 120 -, Induction of Hypocalcemia 110 - Recovery from Hypocalcemia Induction of H ypei^cem ia 100 - Recovery from Hypercalcemia 90 - S 80 ■ 70 - 60 - *s 50 - set-point ^ 40 - t 30 - 20 - 10 - -10 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 Ca^*(mmol/L) Figure 3.5. Intact PTH concentrations (as a % of mean PTHmax from Experiments I and 4) plotted against serum Ca^^ concentrations in healthy horses (Experiments 2 and 3). The induction (1; closed circles) and recovery (2; open circles) from hypercalcemia displayed hysteresis. The induction of hypocalcemia (3; closed squares) was not as sigmoidal and serum PTH concentrations were not as high as in Experiments 1 and 4, but there was hysteresis (4; open squares). 85 y/: 100 1 90 - 80 - ^ 70 - • • E 60 - set-point I so *S 5g 40- X 30 - 20 ■ normal range 10 - 0150 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 Ca^* (mmol/L) Figure 3.6. Relationship between serum and PTH concentrations in 12 healthy horses.. PTH concentrations were^ e^qxressed as & percentage of PTHmw for eack horse. The sigmoidal relationship between serum Ca^^ and PTH concentrations was calculated using the four-parameter logistic model (dotted line) and the Emax sigmoidal model (Hill’s equation; solid line). The Ca^^ set-point was calculated as 50% o f PTHmax for each horse. Both regression lines overlapped indicating that alternative mathematical methods to calculate the relationship between serum Ca^^ and PTH concentrations can be applied. 86 80 -I Ca Gluconate r 10 70- - a 60 - 50- « 40 - - 4 & • 30 - I 2 0 - 10 - - 0 -50 0 50 100 150 200 250 300 350 400 450 500 550 minutes Figure 3.7. Induction of hypocalcemia (Experiments 1 and 4) resulted in an increase of TC*rhôm Z3rt^O:8%^(timê Oriô 3Z 3^8% artB ë éhd^ôfNâzEDTA ihfusfon (120 mih), and FP increased from 0.1 ± 0.1% to 7.5 ± 1.3% at the end of the NazEDTA infusion. At the end of the recovery period from hypocalcemia FCa decreased to 8.9 ± 2.9%, and FP decreased to 1.9 ± 0.2%. Induction of hypercalcemia resulted in an increase of FCa to 59.1 ± 7.3%, while FP continued to decline to values similar to baseline (0.17 ± 0.1%). 87 ’:- -fu 80 1 Ca Gluconate r 10 70 - - a 60 - - 6 i t I - 2 20 - - 0 10 - -50 0 50 100 150 200 250 300 350 400 450 500 550 minutes Figure 3.8. During the induction of hypercalcemia (Experiments 2 and 3) FCa increased from 3.8 ± 0.4% to 63.0 ± 13.2% (P 88 I. , 5. ■ V; WHS . : : •r CHAPTER 4 ^c. ' “=• ’ . EQUINE PÂRATÏTOOID CHIEF CELLS IN VITRO: SECRETION OF ' PAR^TlAROID HORMONE (PTH), PTH AND CALCIUM-SENSING RECEPTOR mRNA EXPRESSION, AND EFFECTS OF INTERLEUKIN-1 Abstract Parathyroid hormone (PTH) is secreted by the chief ceils of the parathyroid gland in response to changes in extracellular ionized calcium (Ca^^ concentrations. In vitro studies on parathyroid cells from different species have improved our understanding of the physiology and pathophysiology of the parathyroid gland. Several conditions in the horses are associated with abnormal parathyroid gland function. In a previous study we found that hypocalcemia and abnormal parathyroid gland function were present in some horses with sepsis. In the present study we evaluated equine parathyroid cell morphology and proliferation, PTH secretion, PTH mRNA and calcium-sensing receptor (CaR) mRNA expression. We also evaluated the effect of interleukin-1 P (IL-IP), a cytokine known to be increased during sepsis in humans and animals, on PTH secretion, PTH and CaR mRNA expression. There was an inverse sigmoidal relationship between PTH secretion and concentrations on day 0. PTH secretion in a low Ca^^ (0.8 vaM) medium decreased from 100% (day 0) to 13% (day 30). The inhibitory effect of high 89 concentrations on PTH secretion also declined. By day S of culture, higher Ca^^ concentrations were required to inhibit PTH secretion, and there was a rightward shift (increase) of the Ca^^ set-point. After day 10 of culture, there was no significant difference in PTH secretion between low (0.8 mhf) and high (2.0 mM) concentrations. Parathyroid cells exposed to high concentrations had lower PTH mRNA expression (P CaR mRNA (up to 142%). The effects of IL-ip were blocked by an lL-1 receptor antagonist (IL-lra). We conclude that the decreased responsiveness of parathyroid cells to Ca^^ from 0 to 30 days could be explained in part by the reduced CaR expression. We also believe that IL-1 plays an important role in regulating PTH secretion in the horse. Introduction rSr V VO- Parathyroid hormone (PTH) is secreted by the chief cells of the parathryroid gland, ^ .j d a y s an important mlp in calcium homeostasis. Parathyroid hormone secretion is altered by small physfological changes in extracellular ionized calcium (Ca^^ concentrations, and there is an inverse sigmoidal relationship between serum Ca^^ concentrations and PTH secretion (Brown, 1983; Mayer and Hurst, 1978; Brent et al., 90 pm? 1988; Aguilera-Tejero et ai., 1996; Chapter 3), represented by a four-parameter model (Brown, 83), which enables the parathyroid chief cells to rapidly respond to hypocalcemia. These changes in concentrations are detected by a calcium-sensing system which includes a G protein-linked calcium receptor (CaR) and gp330/megalin/LRP-2 (a glycoprotein of the LDL-receptor superfamily) (Brown et al., 1993; Lundgren et al., 1994; Hjâlm et al., 1996). In vitro studies on parathyroid cells from different species have improved our understanding of the physiology and pathophysiology of the parathyroid gland (Brown et al., 1976; Liu et al., 2001; Sakaguchi et al., 1987; Fasciotto et al., 1989). However, no in vitro study on equine parathyroid cells has been reported. Horses have unique features with regard to calcium metabolism, including high serum total and ionized calcium concentrations compared to other species (Toribio et al., 2001), high urinary fractional clearance of calcium (Toribio et al., 2001), low serum concentrations of vitamin D (1,25- dihydroxyvitamin D, 25-hydroxyvitamin D) (Mâenpâa et al., 1988; Breidenbach et al., 1998), and an increased Ca^^ set-point (Chapter 3). Conditions of the horse characterized by abnormal parathyroid gland function include idiopathic hypocalcemia of foals (Beyer et al., 1997), primary hyperparathyroidism (Frank et al., 1998), nutritional secondary hyperparathyroidism (Ronen et al., 1992), pseudohyperparathyroidism (Mart et al., 1989), hypoparathyroidism (Couëtil et al., 1998), vitamin D toxicity (Harrington and Page, 1983), renal disease (Elfers et al., 1986), exercise-induced hypocalcemia (Aguilera-Tejero et al., 2001), and sepsis (Toribio et al., 2001). 91 rHÿt>6ciâfcéima iÿa éomWin fin^gm-'Kumans and horses with sepsis (Carlstedt et al., 1998; Toribio et al., 2001). bi a previous study we found that some horses with sepsis and hypocalcemia had low serum PTH concentrations for their degree of hypocalcemia, indicating an inappropriate response of the parathyroid gland to low serum Ca^^ concentrations (Toribio et al., 2001). We believe that this abnormal parathyroid gland function may have resulted in part from increased serum concentrations of inflammatory mediators. Inflammatory mediators known to be increased in horses with sepsis, such as interleukin (IL)-1 and lL-6 (Seethanathan et al., 1990; Barton et al., 1998; Bueno et al., 1999) have been shown in other species to increase the expression of the CaR mRNA in parathyroid cells and decrease PTH secretion (Nielsen et al., 1997; Carlstedt et al 1999). Because sepsis in horses is often associated with increased concentrations of IL-1 and IL- 6, we believe that these inflammatory mediators may directly or indirectly suppress PTH secretion. The objectives of this study were, 1) to evaluate equine parathyroid cell morphology and proliferation in vitro, 2) to determine the functionality (measured as PTH secretion) of chief cells to changes in extracellular Ca^^ concentrations, 3) to measure PTH secretion, PTH mRNA and CaR mRNA expression in equine chief cells for 30 days, and 4) to assess the effect o f interleukin-1 on PTH secretion, PTH mRNA and CaR mRNA expression. 92 >■ /■ Materials and Methods __ _ Preparation of Parathyroid Ceils Equine parathyroid glands were obtained within 15 min of euthanasia from horses donated to the Ohio State University College of Veterinary Medicine for humane destruction. The glands were transported in ice-cold phosphate buffered saline (PBS) containing penicillin (100 lU/ml), streptomycin (lOOpg/ml), and gentamicin (20pg/ml). The time from collection to transport to the laboratory was 15 min. The glands were trimmed of fat and connective tissue, and minced in DMEM/F12 medium, pH 7.4, containing CaClz (1.0 mM), MgCb (0.5mM), and 0.5% ESA. Dissociated cells were obtained by collagenase P (2 mg/ml, Roche Molecular Biochemicals, Indianapolis, IN, USA) and Dnase I (50pg/ml, Sigma, St. Louis, MO, USA) digestion o f the minced tissue in DMEM/F12 medium, pH 7.4, containing penicillin (10 lU/ml), streptomycin (lOpg/ml), gentamicin (4 pg/ml), CaClz (1.0 mAi), MgCb (0.5mM), and 0.5% BSA, in a 5% CO 2 incubator (Forma Scientific Inc, Marietta, OH, USA) for 45-60 min at 37°C, with continuous shaking (100 rpm), and vigorous pipetting every 15 min using a 60 cc syringe. The cell suspension was then filtered through a 100 pm cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ, USA), and sedimented two times by centrifugation at lOOg for 5 min to reduce cellular debris and fibroblast contamination. Cell viability after processing, assessed by trypan blue dye exclusion was estimated around 95%. The cells were subsequently washed in DMEM/F12 medium supplemented with 0.5% BSA. The final parathyroid culture medium was DMEM/F12, pH 7.4, 93 ...... supplemented with penicillin (10 lU/ml), streptomycin (10 ng/ml), gentamicin (4 pg/ml). ITS (insulin, transferrin, selenous acid, BSA, and linoleic acid), and variable concentrations of Ca^^ (O.S-3.0 mM). To maintain the parathyroid cells in suspension we replaced fetal bovine %rum (FES) with ITS, because based on preliminary experiments using 5-10% FBS and from a report (Racke and Nemeth, 1993), the absence of FBS in the medium resulted in decreased attachment of the parathyroid cells to the culture plates, making their collection easier. We used low concentrations of streptomycin and gentamicin (polycations) to reduce intracellular Ca^^ mobilization, which inhibits PTH secretion (Katz et al., 1992). Morphological studies For morphological studies, the parathyroid cells were cultured in parathyroid medium containing a Ca^^ concentration of 0.8 mM and 5% FBS to increase cell attachment. Phase contrast light microscopy (40X, lOOX, and 200X) was used to examine equine parathyroid chief cell morphology and growth. The proliferative life span of parathyroid cells was determined by subculturing the cells every 5 days as previously described by Brandi et al. (1986). The cells were seeded at a density of 1x10^ cells per well in 35mm multiwell plates. Twenty-four, 48, 72, and 96 (day 30) h after seeding, the cells were detached with trypsin/0.5% EDTA and counted in triplicates using a hemocytometer. 94 a-:.: : - . \ The Ca**/PTH relationship in vitro—the Cm ^ set point The effect of varying concentrations on PTH secretion was evaluated on day 0 (collection day) using parathyroid chief cells in suspension. The cells (1x10^ cells) were incubated in triplicate at 37 "C, 5% CO 2, for 3 h in parathyroid culture medium (Ca^^=0.5-3.0 mM) on a shaker at 100 rpm. At the end of the incubation time and after centrifugation at SOOg for S min, the medium was aspirated and frozen at -20 °C until analysis for intact PTH (1-84), and the cells were lysed to determine cell protein concentration. PTH production was expressed as % of maximal PTH concentration (PTHmax) measured, and normalized to cell protein content. The Ca^^ set point was calculated as the Ca^^ concentration at which intact PTH concentration was 50% o f PTHmax- PTH secretion and PTH and CaRmRNA expression The effect of low Ca^^ concentration on PTH secretion was determined in triplicate every 2-3 days for 30 days. Parathyroid chief cells (1x10^) were incubated in parathyroid culture medium with a Ca^^ concentration of 0.8 m M for 3 h. At the end of the incubation time, and after centrifugation at SOOg for 5 min, the medium was aspirated and fix>zen at -20 °C until analysis for intact PTH, and the cells were lysed to determine protein concen^tion. . . -^Thc effect o f varying concentrations (0.8-2.0 mM) on PTH secretion was determined in triplicate wells of parathyroid chief cells (1x10^ cells) incubated for 3 h on 95 days 0. 5. 10. 15. and_30. PTH concentrations were normalized for protein content^ a n d expressed as % of controls (Ca^^=0.8 mM) for each day. The effect of low (0.8 mM) and high (2.0 mM) Ca^* concentrations on CaR and PTH mRNA expression was evaluated in duplicate using 0.5x10^ chief cells on days 1 and 2. The expression of PTH and CaR mRNA were determined in duplicate using 0.5x10^ parathyroid chief cells (Ca^^=0.8 mM) by Northern blot analysis on days 0,1, 2, 5, 10, 15, 20, 25, 30, and expressed as PTH mRNA/GAPDH mRNA or CaR mRNA/GAPDH mRNA ratio. Controls consisted of PTH mRNA or CaR mRNA/GAPDH mRNA expression on day 0 (100% expression). The effect of IL-ip on PTH secretion, and PTH and CaR mRNA expression The effect of human recombinat IL-ip (R&D systems Inc., Minneapolis, MN, USA) (20,200, and 2000 pg/ml) oh PTH secretion, and PTH and CaR mRNA expression was examined in parathyroid chief cells (0.5x10^ in duplicate during incubation for 24 h in low Ca^^ (0.8 mM) medium. The concentrations of IL-1 ^ used in this study were chosen from previous reports based on the effect of IL-ip on bovine parathyroid cell function (Nielsen et al., 1997). To determine if the effect of IL-ip on PTH secretion, and PTH and CaR mRNA expression was mediated through an lL-1-specific receptor, a human recombinant lL-1 receptor antagonist (IL-lra, Amgen Inc., Thousand Oaks, CA, USA) at 100 ng/ml was added to the medium (Ca^^=0.8 mM) 2 h previous to the addition of IL-ip (2000 96 K g ^ :*w ^ py/mlV The cells were incubated for 24 h after adding IL-lB. In addition, parathyroid i ' i - * '' « chief cells were also incubated with IL-lra alone (100 ng/ml). PTH, CaR, and GAFDH mRNA expression To measure PTH mRNA expression, a 399-base pair probe was generated by pol^erase chain amplification o f canine PTH cDNA (Rosol et al., 1995) (primers: 5'- GTGTGTGAAGATGATGTCTGC; 5-TTGTTGCCCTATGCTGTCTA). For CaR mRNA expression, a 1.6 kb CaR cDNA was generated by reverse transcription polymerase chain reaction (primers: 5 -TGGAACTGGGTGGGCACAAT, 5’- CCAGCAGGAACTGCAGGTTGAG) of equine parathyroid gland total RNA, subcloned into pCR4-TOPO (Invitrogen, Carlsbad, CA, USA), sequenced, and used as a probe. For GAPDH mRNA expression, a 780-bp fragment of human GAPDH cDNA was used. The probes were labeled with [^^P]- dATP (NEN life Science Products, Boston, MA, USA) using a random primers DNA labeling system (Life Technologies, Grand Island, New York, USA) to 1 x 10® cpm/pg of DNA. Total RNA was extracted fiom 0.5x10^ parathyroid chief cells using guanidinium thiocyanate and silica-based filters (RNAqueous-4PCR kit, Ambion, Austin, TX, USA). Total RNA (lOpg) was electrophoresed on denaturing formaldehyde (6%) agarose (1%) gels. After electrophoresis for 3 h at 70 volts, the RNA was transferred onto nylon membranes (Stratagene, La Joya, CA, USA). The membranes were UV irradiated with a Stratalinker UV Crosslinker 1800 (Stratagene), prehybridized in Ultrahyb solution (Ambion) for 1 h at 42 °C and hybridized in Ultrahyb solution with denatured herring 97 ...... ' ■"'^ ' ._ ; ^ _ _ '; ; ^ '- _ 'at-y-'-. ^ ’ '- spAm DM&*md cDNA probe (t x"10* cpmAinl of hybridization solution) for 16 h. After hybridization the membranes were washed twice for S min in 2X SCC/0.1% SDS at room temperature and twice with O.IX SCC/0.1% SDS at 42 °C for 15 min. Quantitation of mRNA was performed using a Phosphorhnager 445 SI (Molecular Dynamics, Sunnyvale, CA, USA), and ImageQuant software (Molecular Dynamics). The cDNA probes were stripped from the membranes by boiling twice in O.IX SCC/0.1 SDS for 10 min. The same membranes were sequentially hybridized with the PTH, CaR, and GAPDH probes. Measurement of PTH Intact PTH (1-84) was measured with a two site immmunochemiluminonetric assay (fanmulite intact PTH assay. Diagnostic Products Corporation, Los Angeles, CA, USA), specific for human intact PTH, and previously validated to measure equine serum intact PTH concentrations (Toribio et al., 2001; Chapter 2). PTH concentrations were normalized to cell protein content. Protein determination Ceil protein was extracted from the parathyroid cells using RIPA cell lysis buffer (PBS, 1% NP40, 0.1% SDS, 1% deoxycholate, 2mAf EDTA). Protein concentrations were measured using the Coomassie brilliant blue binding protein assay (Bradford, 1976) using bovine serum albumin as the protein standard. 98 Electrolytes measurements Electrolytes (Ca^\ Mg^\ Na% and pH in the parathyroid culture medium were measured using a NOVA 8 analyzer (Nova Biomedical, Waltham, MA, USA). Statistical analyses r < Results are presented as mean ± SD. Comparisons between groups were made using the /-test or the Mann-Whitney rank test. Comparisons among groups were made using one-way ANOVA with Tukey’s test for multiple comparisons, or the Kruskal- Wallis one-way ANOVA with Dunn’s test for multiple comparisons depending on the data distribution (Fisher and Van Belle, 1993). When analyzing the effect of time and different Ca^^ concentrations on PTH secretion, and PTH and CaR mRNA expression, two-way ANOVA with Tukey’s test for multiple comparisons was used. Statistical analyses were performed with SigmaStat 2.01 for Windows (SPSS Inc, Chicago, IL, USA). P values less than 0.05 were considered statistically significant. Results Morphological studies Parathyroid cells in suspension varied from single cells to clumps of cells (Fig. 4.1a). When 5% of FBS was added to DMEM/F12 medium (Ca^^ = 0.8 mM), attachment 99 _QLthfiLparathyroid_chieflcells,tCLCultiitfiLdishes.»^as_eyidenta^^ 6 h of iacubation^and by „ day 2 of culture approximately 90% of the cells were attached to the plates (Fig. 4.1b,c). The cells were polygonal, grew in colonies and spread out to from a monolayer. The number of spindle-shaped cells on day 2, presumambly fibroblasts, was minimal (<1%) (Fig. 4.1c). Parathyroid cells were harvested every 5 days for 30 days to measure doubling times. On day 5 the doubling time was estimated to be 38 ± 8 h, on day 10 it was 48 ± 10 h, and on day 30 it was 68 ± 15 h (Fig. 4.2). The Ca /PTH relationship in vitro - the Ca ^ set point When equine parathyroid chief cells in suspension were exposed to varying Ca^^ concentrations for 3 h, the relationship between PTH and Ca^^ concentrations was sigmoidal (Fig. 4.3). PTH secretion ranged from 455 ± 102 ng/mg of cell protein (Ca^^ = 0.8 mAf, 100% PTH secretion) to 55 ± 27 ng/mg of cell protein (Ca^^ = 3.0 mM). Calcium concentrations greater than 2.0 m M did not result in an additional decrease in PTH secretion, and Ca^^ concentrations lower than 0.8 mM did not result in additional secretion of PTH. Subsequent experiments were performed with Car* concentrations of 0.8-2.0 mM. The Ca^^ set point in fresh parathyroid cells in suspension, calculated as the Ca^^ concentration at which PTH concentration was 50% of PTHmax was 1.30 ± 0.05 mM (Fig. 4.3), which is similar to the Ca^^ set point that we measured in healthy horses (1.37 i 0.05 roM) (Chapter 3). ^ 1 0 0 PTH secretion, and PTH and CaR mRNA expression PTH secretion in a low (0.8 vaM) medium decreased over time. On day 0 PTH secretion was 385 ± 52 ng/mg of cell protein (100%). By day 5 of culture PTH secretion declined to 68 ± 12%, by day 10 to 33 ± 6%, and by day 30 to 13 ± 3% to that of day 0 (Fig. 4.2). The inhibitoy effect of high Ca^^ concentrations on PTH secretion also declined over time. By day 5 of culture, high Ca^^ concentrations inhibited PTH secretion; however, there was a rightward shift (increase) of the Ca^^ set-point (Fig. 4.4) because higher Ca^^ concentrations were required to inhibit PTH secretion. After day 10 of culture there was no significant difference in PTH secretion between low and high Ca^^concentrations (Fig. 4.4). When the effect of low (0.8 mM) and high (2.0 mM) Ca^^ concentrations on CaR mRNA expression wa^evaluated on days 1 and 2, no effect of low (0.8 mM) or high (2.0 mM) Ca^^ concehtratidns on CaR mRNA expression was detected (Fig. 4.5). There was a decline in CaR mRNA expression that was time-dependent, but was not statistically associated with Ca^^ concentrations. PTH mRNA expressions on day 1 was not significantly different between low and high Ca^^ concentration experiements; however, on day 2, parathyroid chief cells exposed to high Ca^^ concentrations had lower PTH mRNA expression than cells exposed to low Ca^^ concentrations (P<0.05) (Fig. 4.5). CaR and PTH mRNA expression declined over time when compared to day 0. By day 2 of culture, CaR mRNA expression declined to 41 ± 7%, by day 5 to 31 ± 6%, by day 10 to 21 ± 7%, and by day 30 to 16 ± 6% of controls (day OXFig. 4.6). PTH mRNA expression declined, but not as rapidly as CaR mRNA. By day 2, PTH mRNA expression 101 was 79 i: 5%. by day 5 it Mais 43 ± 13%. by day 10 it was 35 ± 11%, and by day 30 Jt was_ 25 ± 8% of controls (da^^ OXFig. 4.6). The elh^t'''n olr PTH secretion, andf FMI and CaR mRNA expression When parathyroid cells were incubated with increasing concentrations of human recombinant IL^l^, only an IL-1 g concentration Of 2000 pg/ml decreased both PTH secretion (75%) and PTH mRNA expression (73%) (P<0.05) (Fig. 4.7). No statistically significant difference in PTH secretion and PTH mRNA expression at lower IL-lp concentrations was detected when compared to controls; however, a trend for reduced PTH secretion and mRNA expression was present at 200 pg/ml of IL-ip (Fig. 4.7). In contrast, IL-ip concentrations of > 200 pg/ml resulted in a significant (P<0.05) overexpression of CaR mRNA (up to 142%). When IL-lra was added to the culture medium 2 h prior to adding IL-ip (2000 pg/ml), there was no significant difference in PTH secretion, and PTH and CaR mRNA expression when compared to controls (no IL- IP), suggesting that the effects of IL-ip on PTH secretion, and PTH and CaR mRNA -expiesskm-were- mediate 102 <«r. W r - ' - r - ^ : 5 *■ «rt- : O ff.'* '". In this study we demonstrated that equine parathyroid chief cells cultured in vitro were responsive to changes in extracellular concentrations for up to 5 days after collection. We also found that PTH secretion in adherent parathyroid chief cells when compared to cells in suspension (day 0) was significantly lower, and the Ca^^ set-point was shifted rightwards on day 5, due to decreased responsiveness to high Ca^^ concentrations. These findings suggest that equine parathyroid chief cells must be used early after collection when evaluating their responsiveness to changes in extracellular Ca^^ concentrations. Similar findings were reported with bovine parathyroid cells, in which there was decreased parathyroid cell responsiveness (PTH secretion) to changes in Ca^^ concentrations over time (Mithal et al., 1995). Equine parathyroid cells had a rapid decrease in CaR mRNA expression in vitro, and by day 5, CaR mRNA expression was reduced to 31%. We speculate that the decreased sensitivity of equine parathyroid cells to high extracellular Ca^^ concentrations resulted, in part, from decreased CaR mRNA expression and protein synthesis, as previously reported in bovine parathyroid cells (Mithal et al., 1995). Mithal et al. (1995) demonstrated that over time, bovine parathyroid chief cells in culture had decreased expression of CaR mRNA, and decreased immunostaining for the extracellular domain of CaR. By day 4 of culture, CaR mRNA expression decreased to 21% and CaR immunostaining decreased to 8-18% in bovine chief cells. It is important to note that, in our study, despite the low expression of CaR mRNA (day 5), the cells were still responsive to changes in extracellular Ca^^ concentrations, although PTH secretion on 103 day 5 was 68% and PTH mRNA expression was 43% of day 0. We speculate that even with a 70% reduction in CaR mRNA expression, there was sufficient CaR to suppress PTH secretion, or possibly the megalin/gp330/LRP-2 calcium-sensing protein (or other calcium-sensing mechanisms) may be regulating PTH secretion in these cells (Lundgren et al., 1994; Hjâlm et al., 1996). Decreased mRNA expression and protein synthesis for megalin/gp330/LRP-2 may contribute to determining the Ca^^ set-point (Faraebo et al., 1998). CaR protetn's^mthësis was not evaluated in this study, although it would have been expected to be low) as previously reported by Mithal et al. (1995). PTH mRKÀ expression d e cree d over time, but not as rapidly as CaR mRNA expression, suggesting that CaR expression may be more critical for PTH secretion in equine parathyroid chief cells in culture compared to PTH mRNA expression. In addition to decreased CaR mRNA expression, there was a rightward shift of the Ca^^ set-point, which we believe resulted from the decreased expression of CaR mRNA, as previously reported (Kifor et al., 1996). It is unclear why there was a decrease in CaR mRNA expression in equine chief cells, but we believe that the lack of growth factors (and calcitriol) may have resulted in parathyroid cell dedifferentiation, and subsequently in decreased calcium-sensing ability (Brown et al., 1996; Nielsen et al., 1997). No effect o f changes in medium Ca^^ concentrations on CaR mRNA expression was found. These results are similar to those of Brown et al. (1996) who found that CaR mRNA was not regulated by Ca^^ concentrations, but rather by calcitriol. The effect of calcitriol on CaR mRNA and PTH mRNA expression was not evaluated in this study. We also determined that PTH mRNA expression was inversely related to Ca^^ concentrations; PTH mRNA expression was significantly lower on day 2 in high Ca^^ 104 concentration medium as compared to low Ca^* concentration medium. In corroborating experiments, Moallem et al. (1998) found that hypocalcemia (and hyperphosphatemia) increased PTH mRNA stability by changing the ability of cytosolic proteins to bind the PTH mRNA 3 -untranslated region in rats. Factors not evaluated in this study, that may affect PTH mRNA expression, synthesis, and secretion, include endothelin 1, PTH fragments, chromogranin A and its bioactive peptides, phosphate, vitamin D, and other calcium-sensing proteins like me^in/gp330/LRP-2 (Fujii et al., 1991; Fujimi et al., 1991; Lundgren et al., 1994; Kilav et al., 1995; Slatopolsky and Delmez, 1996; Hjâlm et al., 1996; Fasciotto et al., 2000). Interleukin-1 is a cytokine with neuroendocrine, endocrine, metabolic, cardiovascular, and proinflammatory functions (Dinaiello, 2000). Our study showed that IL-ip inhibits PTH secretion, decreases PTH mRNA expression, and increases CaR mRNA expression in equine parathyroid cells cultured for 24 h. These effects were blocked when parathyroid cells were incubated with an lL-1 receptor antagonist, indicating that equine parathyroid cells have specific receptors for IL-I. In a previous study with bovine parathyroid cells, Nielsen et al. (1997) found that the effect of IL-ip to inhibit PTH secretion and increase CaR expression, was present in parathyroid tissue slices but not in dispersed parathyroid cells. The effect of lL-13 on equine parathyroid tissue slices was not evaluated in the present study. Although equine parathyroid cells became less responsive to changes in extracellular Ca^^ over time in culture as occurs with bovine parathyroid cells (Mithal et al., 1995), we found that equine parathyroid cell function could still be altered by IL-ip. We speculate that increased serum concentrations of IL-I in septic horses may result in CaR mRNA overexpression, lowering the Ca^^ set- point, and reducing the response of the parathyroid gland to hypocalcemia. 105 Several conditions in the horse are associated with abnormal calcium homeostasis (Harrington and Page, 1983; Elfers et al., 1986; Marr et al., 1989; Ronen et al., 1992; Dart et al., 1992; Beyer et al., 1997; Frank et al., 1998; Couëtil et al., 1998; Toribio et al., 2001; Aguilera-Tejero et al., 2001). However, there is limited information on equine parathyroid gland function. We evaluated equine parathyroid cell proliferation, PTH secretion, PTH and CaR mRNA expression, as well as the effect of interleukin-ip on PTH secretion, PTH and CaR mRNA expression. In conclusion, we have demonstrated that equine parathyroid chief cells cultured in vitro are responsive to changes in extracellular Ca^^ concentrations for up to 5 days in vitro. There was a time-dependent decrease in PTH secretion, and PTH and CaR mRNA expression. Based on the rapid decrease in CaR mRNA expression, we believe that CaR mRNA expression is more critical in determining the in vitro responsiveness of equine parathyroid chief cells to changes in extracellular Ca^^ than PTH mRNA expression. We found that IL-ip, an inflammatory mediator known to be increased in septic horses, inhibited PTH secretiqit^^wd mRNA expression, and increased CaR mRNA expression. We consider that overrapresrion of CaR from the IL-ip treatment could have increased Ca^]^4 sa*itivi^ in the equine parathyroid chief cells, resulting in decreased PTH secretion. These findings indicate that IL-ip may play a role in regulation of equine parathyroid gland function in vivo. The information generated from this study will be valuable for future research on calcium metabolism in the horse, and in understanding parathyroid gland function in healthy and sick horses. 106 JBi&EüiSiS- Éi : Aguilera-Tejero E, EsjefMC^C^l^pez I, Bas S, Garfia B, Rodriguez M. Plasma ionized calcium and parathyfbid hormone concentrations in horses after endurance rides. J Am Vet Med Assoc Aguilera-Tejero E, Sanchez J, Alinaden Y, Mayer-Valor R, Rodriguez M, Felsenfeld AJ. Hysteresis gf’the PTH-ca)cium cuiye during hypocalcemia in the dog: effect of the rate and linearity of calcium decrease and sequential episodes of hypocalcemia. J Bone Miner Res 1996; 11:1226-1233. Barton MH, Bruce EH, Moore JN, Norton N, Anders B, Morris DD. Effect of tumor necrosis factor antibody given to horses during early experimentally induced endotoxemia. Am J Vet Res 1998; 59:792-797. Beyer MJ, Freestone JF, Reimer JM, Bernard WV, Rueve ER. Idiopathic hypocalcemia in foals. J Vet Intern Med 1997; 11:356-360. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing die principle ofprotein-dye binding. Anal Biochem 1976; 72:248-254. Brandi ML, Fitzpatrick LA, Coon HG, Aurbach GD. Bovine parathyroid cells: cultures maintained for more than 140 population doublings. Proc Natl Acad Sci U SA 1986; 83:1709-1713. Breidenbach A, Schlumbohm C, Harmeyer J. Peculiarities of vitamin D and of the calcium and phosphate homeostatic system in horses. Vet Res 1998; 29:173-186. Brent GA, LeBoff MS, Seely EW, Conlin PR, Brown EM. Relationship between the concentration and rate of change of calcium and serum intact parathyroid hormone levels in normal humans. 7 C/m Endocrinol Metab 1988; 67:944-950. Brown-EM; Four-parameter model~of the-sigmoidaFtckrtionship between parathyroid hormone release and extracellular calcium concentration in normal and abnormal parathyroid tissue. JClin Endocrinol Metab 1983; 56:572-581. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC. Cloning and characterization o f an extracellular Ca(2+)- sensing receptor from bovine parathyroid. Nature 1993; 366:575-580. Brown AJ, Zhong M, Finch J, Ritter C, McCracken R, Morrissey J, Slatopolsky E. Rat calcium-sensing receptor is regulated by vitamin D but not by calcium. Am J Physiol 1996; 270:F454-F460. 107 Bueno AC. Seahom TLr.6 Caravaca F, Cubero JJ, Jimenez F, Lopez JM, Aparicio A, Cid MC, Pizarro JL, Liso J, SrmtW L Efifect of the mode of calcitriol administration on PTH-ionized calcium relationslup. in uraemic patients with secondary hyperparathyroidism. Nephrol Dial Transplant 1995; 10:665-670. Carlstedt E, Ridefelt P, Lind L, Rastad J. Interleukin-6 induced suppression of bovine parathyroid hormone secretion. Biosci Rep 1999; 19:35-42. Carlstedt F, Lind L, Rastad J, S^emstrom H, Wide L, Ljunghall S. Parathyroid hormone uad ionized calcium levels are related to the severity of illness and survival in critically ill patients. Eur JClin Invest 1998; 28:898-903. Couëtil LL, Sojka JE, Nachreiner RF. Primary hypoparathyroidism in a horse. J Vet Intern 1998; 12:45-49. Dinarello CC. Proinflammatory cytokines. Chest 2000; 118:503-508. Elfers RS, Bayly WM, Brobst DF, Reed SM, Liggitt HD, Hawker CD, Baylink DJ. Alterations in calcium, phosphorus and C-terminal parathyroid hormone levels in equine acute renal disease. Cornell Vet 1986; 76:317-329. Famebo F, Hoog A, Sandelin K, Larsson C, Famebo LO. Decreased expression of calcium-sensing receptor messenger ribonucleic acids in parathyroid adenomas. Surgery 1998; 124:1094-1098. Fasciotto BH, Gorr SU, DeFranco DJ, Levine MA, Cohn DV. Pancreastatin, a presumed product of chromogranin-A (secretory protein-1) processing, inhibits secretion from porcine parathyroid cells in culture. £n ——Faseiotto-BHrDenity-J€rGreeley-GH-Jfr€ Fisher LD, Van Belle G. Biostatistics: a methodology for the health science. New York: John Wiley & Sons Inc, 1993; 304-495. Frank N, Hawkins JF, Couëtil LL, Raymond JT. Primary hyperparathyroidism with osteodystrophia fibrosa of the tiicial bones in a pony. J A m Vet Med Assoc 1998; 212:84- 86 . Fujii Y, Moreira JE, Orlando C, Maggi M, Aurbach GD, Brandi ML, Sakaguchi K. Endothelin as an autocrine factor in the regulation of parathyroid cells. Proc Natl Acad Sci U S A 1991; 88:4235-4239. 108 —Fujimi T, Baba H. Fukase M. Fuiita T. Direct inhibitory —m "WTTgi effect Tr - _ '■-* of' "' -- — amino-terminal '— —I wTW ■ ~ ■ ~i om— Mil I III parathyroid hormone fragment [PTH(l-34)] on PTH secretion from bovine parathyroid primary cultured cells in vitro. AocAent BiopA!y.s i?er Commun 1991; 178:953-958. Harrington DD, Page EH. Acute vitamin D3 toxicosis in horses: case reports and experimental studies of the comparative toxicity of vitamins D2 and D3. J A m Vet M ed Assoc 1983; 182:1358-1369. Hjalm G, Murray E, Crumley G, Harazim W, Lundgren S, Onyango 1, Ek B, Larsson M, Juhlin C, Heilman P, Davis H, Akerstrom G, Rask L, Morse B. Cloning and sequencing of human gp330, a Ca(2+)-binding receptor with potential intracellular signaling properties. Eur J Biochem 19%; 239:132-137. Katz CL, Butters RR, Chen CJ, Brown EM. Stnicture-fimction relationships for the effects of various tmwoglycoside antibiotics on dispersed bovine parathyroid cells. Em/ocriuo/oigyd9%Kl% 903-910. Kifor O, Moore Fl5 Jf/Wan^ P, Goldstein M, Vassilev P, Kifor I, Hebert SC, Brown EM. Reduced iinmunoslamtp^fbs the extracellular Ca2+-sensing receptor in primary and u tg ^c ^ q iy la iy hÿperpàra^ C/m Memb 1996; 81:1598-1606. Kilav Rj Silver J, Naveh-Many T. Parathyroid hormone gene expression in h)^phosphatemic rats. JC lin Invest 1995; %:327-333. Lundgren S, Hjalm G, Heilman P, Ek B, Juhlin C, Rastad J, Klareskog L, Akerstrom G, Rask L. A protein involved in calcium sensing of the human parathyroid and placental cytotrophoblast cells belongs to the LDL-receptor protein superfamily. Exp Cel! Res 1994; 212:344-350. Mâenpaâ PH, Koskinen T, Koskinen E. Serum profiles of vitamins A, E and D in mares and foals during different seasons. JA nim Sci 1988; 66:1418-1423. Marr CM, Love S, Pirie HM. Clinical, ultrasonographic and pathological findings in a -hofse-with-splenie-lyR^diosaiecNnft-and-pseudohyperparathyroidism; Equine^ FeKA1989; 21:221-226. Mayer GP, Hurst JG. Sigmoidal relationship between parathyroid hormone secretion rate and plasma calcium concentration in calves. £m/ocnno/ogy 1978; 102:1036-1042. Mithal A, Kifor O, Kifor 1, Vassilev P, Butters R, Knqicho K, Simin R, Fuller F, Hebert SC, Brown EM. The reduced responsiveness of cultured bovine parathyroid cells to extracellular Ca2+ is associated with marked reduction in the expression of extracellular Ca(2+)-sensing receptor messenger ribonucleic acid and protein. Endocrinology 1995; 136:3087-3092. 109 ^ "'>i'4' ■’ ^ fc. Moallem E* Kilav R. Silver J. Naveh-Many T. RNA-Protein binding and post- tianscriptional regulation of parathyroid hormone gene expression by calcium and phosphate. y^ib/Orew 1998;273:5253-5259. Nielsen PK, Rasmussen AK, Butters R, Feldt-Rasmussen U, Bendtzen K, Diaz R, Brown EM, Olgaarà K. Inhibition of PTH secretion by interleukin-1 beta in bovine parathyroid glands in vitro is associated with an up-re^ation of the calcium-sensing receptor mRNA. Biochem Biophys Res Commun 1997; 238:880-885. Racke FK, Nemeth EF. Cytosolic calcium homeostasis in bovine parathyroid cells and its modulation by protein kinase C. J Physiol 1993; 468:141-162. Ronen N, van Heerden J, van Amstel SR. Clinical and biochemistry findings, and parathyroid hormone concentrations in three horses with secondary hyperparathyroidism. JSAJr Vet Assoc 1992; 63:134-136. Rosol TJ, Steinmeyer CL, McCauley LK, Grone A, DeWille JW, Capen CC. Sequences of the cDNAs encoding canine parathyroid hormone-related protein and parathyroid hormone. Gene 1995; 160:241-243. Sakaguchi K, Santora A, Zimering M, Curcio F, Aurbach GD, Brandi ML. Functional epithelial cell line cloned from rat parathyroid glands. Proc Natl Acad Sci U SA 1987; 84:3269-3273. Seethanathan P, Bottoms GD, Schafer K. Characterization of release of tumor necrosis factor, interleukin-1, and superoxide anion from equine white blood cells in response to endotoxin. Am J Vet Res 19%; 51:1221-1225. Slatopolsky E, Delmez JA. Pathogenesis of secondary hyperparathyroidism. Nephrol Dial Transplant 1996; Suppl 3:130-135. Toribio, RE, Kohn, CW, Chew, DJ, Sams, RA, Rosol, TJ. Comparison of serum parathyroid hormone and ionized calcium and magnesium concentrations and fractional -urinary clearance o f cakium and-phosfrfiorus in-healthy horses and horses with enterocolitis. Am J Vet Res 2001 ; 62:938-947. _V. • 110 Figure 4.1. Phase contrast microscopy of parathyroid chief cells. Cells in suspension varied from single cells to clumps of cells (a, 40X). The cells were polygonal, growing in colonies and spreading out to form a monolayer (days 3-5) (b,c, lOOX). The number of spindle-shaped cells on day 2, presumably fibroblasts (arrow), was minimal (c). Ill 120 1 90 110 - 100 - 60 90 - 40 80 - 30 70 - 60 - I 50 - Days 40 - 30 - 20 - 10 ■ 10 15 20 25 30 Days Figure 4.2. PTH secretion of equine parathyroid chief cells cultured in a low (0.8 toM) medium. PTH secretion decreased over time. On day 5 of culture, PTH secretion was 68%, on day 10 was 33%, and after day 13, PTH secretion was below 18% compared to day 0. To determine the doubling times, chief cells were harvested every S days for 30 days. Doubling time increased from 28 h on day 2 to 68 h on day 30 (insert graph). 112 130 1 y . 1 2 0 - 110 - 100 - 90 ■ 80 - 70 ■ X 60 ■ k set-point 50 - - 40 - 30 ■ 20 ■ 10 - 0.0 0.5 1.0 1.5 2.0 2.5 3.0 C a " (m *0 Figure 4.3. Equine parathyroid chief cells on day 0 (collection day) were exposed to differenl-€«^^ concentrations for 3^ hr A- sigmoidal- relationship^ between^ PTH and concentrations was displayed. Calcium concentrations greater than 2.0 mM did not result in an additional decrease in PTH secretion, and Ca^^ concentrations lower than 0.8 mM did not result in additional secretion of PTH. The Ca^^ set point, calculated as the Ca^^ concentration at which PTH concentration was 50% of PTHmax was calculated to be 1.3 mM. 113 120 n 110 - 100 - 90 - 80 - 70 - g I % - Day 0 Day 5 Day 10 20 - Day 15 Day 30 10 - 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 C a'* (m/M) Figure 4.4. Reduction in responsiveness of equine parathyroid chief cells to changes in Ca^^ concentrations. By day 5 of culture chief cells remained responsive to changes in Ga^^rhoweveF,-there-was a-rightwttfd-shift-i!Hhe-€^- set-pmnt^Aom l.3&mM(day 0>to- 1.57 mM (dotted line) because higher Ca^^ concentrations were requiered to inhibit PTH secretion. After day 10 of culture there was no significant difference in PTH secretion between low and high Ca^^concentrations. Control (100% PTH secretion) for each day was the PTH secreted at a Ca^^ concentration of 0.8 mM 114 •Ta.. .. 110 - 100 - 90 • 80 • 1 70 ■ PTH i CaR a 60 - 50 ■ QAPDH 1 E 40 ■ CaR mRNA and low Ca^* CaR mRNA and high Ca'* 30 ■ PTH mRNA and low Ca** PTH mRNA and highCa** 20 - -p- -I 0 2 Days Figure 4.5. Effect of low (0.8 niA^ and high (2.0 mM) concentrations on CaR mRNA and PTH mRNA expression. A significant effect of low or high Ca^* -eoneentrations 115 120 -1 10 15 20 25 30 110 ■ 100 90 - GAPDH 80 - 70 - 60 50 - 40 - 30 ■ 20 ■ PTHmRNA CaRmRNA 10 - PTH secretion 0 —T— -r- 0 5 10 15 20 25 30 Days Figure 4.6. PTH and CaR mRNA expression, and PTH secretion in parathyroid chief cells-cultured for 30 days-ina low Ca?^ inediuin. PTR secretion, PTH and CaR mRNA expression decreased over time when compared to day 0. By day 2 of culture, CaR mRNA expression decreased to 41%, by day 5 to 31%, and by day 30 to 16% of the controls (day 0). PTH mRNA expression decreased, but not as rapidly as CaR mRNA expression. By day 2 PTH mRNA expression was 78%, by day 5 it was 43%, by day 10 it was 35%, and by day 30 to 25% of the controls (day 0). 116 T.C:'-'- :-; : - ';t: '^- . ■ ■-■■■/■ ■ ■ PTH ■ PTHnfiNA 200 2 0 0 0 2000+IL-1m IL-Ira pg/ml Figure 4.7. Equine parathyroid chief cells incubated with human recombinant IL-ip (0- 2000 pg/mL) and human recombinant lL-1 receptor antagonist (IL-lra) (100 ng/ml). Parathyroid cells incubated with IL-ip had a significant (P 117 Acknonvledgments Support for R.E. Toribio was provided by the C. Glenn Barber fund of the College of Veterinyy Medipine, The Ohio State University. Funds for this study were providwLby The Ohio State University College of Veterinary Medicine Equine Research Funds. The authors acknowledge Drs. E. F. Nemeth (NFS Phamaceuticals, Inc., Salt Lake City, Utah) and E. M. Brown (Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts) for their suggestions on parathyroid cell culture. We thank Amgen, Inc. for providing the human recombinant interleukin-1 receptor antagonist. We are thankful to Drs. Deborah Groppe, Sunee Kunakomsawat, Brace LeRoy, Virgile Richard, Rani Sellers, and Sarah Tannehill-Gregg for their technical assistance. We are grateful to Ms. Lori Kipp for locating horses to be used in this study. 118 '3: CHAPTER 5 MOLECULAR CLONING AND EXPRESSION OF EQUINE CALCITONIN, CALCITONIN GENE-RELATED PEPTIDE-I, AND CALCITONIN GENE-RELATED PEPTIDE-n Abstract In this study we describe the cloning of equine calcitonin (CT), calcitonin-gene related peptide (CGRPH, and CGRP-II cDNA. We also describe a divergent form of CGRP (CGRP-0. The nucleotide sequence for equine preproCT (835 bp) consisted of a 102 bp 5’ UTR, a 423 bp coding region for a 140-amino acid protein, and a 310 bp 3’ UTR. Equine calcitonin has greatest homology (>85%) to human, rat and mouse subgroups of calcitonins. Equine preproCGRP-I (702 bp), consisted of a 102 bp 5* UTR, a 384 bp coding region for a 127-amino acid protein, and a 216 bp 3’ UTR. Equine CGRP-I (37aa) has low homology (<59%) to CGRPs of other species. The signal and terminal peptides for equine calcitonin and CGRP-I were identical, indicating that these peptides are encoded by a gene equivalent to the human CALC-I gene. Equine preproCGRPH (960 bp), consisted of a 69 bp 5’ UTR, a 390 bp coding region for a 129- amino acid protein, and a 501 bp 3’ UTR. Equine CGRP-II (37 aa) has >80% homology to chicken, human, rat, ovine, swine, and bovine CGRPs. The homology between equine 119 CGRP-I and CGRP-II is low (56%). THe hijÿi homology of equine CGRP-II, and the low . '■.■■■, - I homology o f equine CGRP-I to CGRP in other species were unexpected findings, because the homology of all known CGRP peptides was reported to be high in mammals. Northern blot analysis o f equine tissues revealed that expression of calcitonin mRNA was restricted to the thyroid gland, while expression of CGRP-I and CGRP-II mRNA was present in several regions of the nervous system and other tissues. Introduction Studies on calcitonin gene expression have revealed alternative splicing of the primary transcript (Rosenfeld et al., 1981; Amara et al., 1985; Nelkin et al., 1988; Cumaraswamy et al., 1993). Calcitonin (CT), a calcium-lowering hormone synthesized by the C-cells (parafollicular cells) of the thyroid gland (Copp et al., 1962), is a single chain peptide of 32 aa residues whose amino-terminus contains a cyclic structure involving seven amino acids bridged by a disulfide bond (Becker et al., 1996). The human polypeptide precursor o f calcitonin, preprocalcitonin (preproCT), contains 141 amino acids (Le Moullec et al., 1984). Human procalcitonin (PCT) contains 116 amino acid residues, and consists o f a centrally positioned immature CT that contains 33 amino acids, a 57-amino acid N-terminal peptide, and a 21-amino acid carboxyl terminal peptide (CCP or katacalcin) (Becker et al., 1996). In 1981, Rosenfeld et al. demonstrated that the calcitonin gene was the source of an alternative mRNA, and the corresponding peptide was named calcitonin gene-related peptide (CGRP). 120 >«31; f 2 Human CGRP-I (or CGRPj;ftLis a 3_7_anMno-acid_peptide whiclL differs from. human CGRP-II (or CGRP-P) by three amino acids (Amara et al., 1985; Steenbergh et al.,1985). In humans, both CT and CGRP-I are encoded by the C41C-/ gene (Becker et al., 1996). CGRP-II originates from the CALC-H gene, for which there is no known alternative splicing of the primary transcript (Steenbergh et al.,1986; Becker et al.,1996). Alevizaki et al. (1986), have identified a region within the CALC-H gene which has the potential to encode a novel calcitonin-like peptide. However, no corresponding mRNA has been identified, and it is unclear whether this sequence can be expressed. CGRPs are pleiotropic neuropeptides found in the central and peripheral nervous system (Brain and Cambridge, 1996; Van Rossum et al. 1997), and also are present in C-cells of the thyroid gland (Sabate et al., 1985; Becker et al., 1996; Van Rossum et al. 1997). CGRPs have been implicated in a diverse array of physiological processes including neurotransmitter activity, vasodilation, relaxation of smooth muscle, cardiac acceleration, synthesis and function of nicotinic acetylcholine receptor, nociception, carbohydrate metabolism, gastrointestinal motility, neurogenic inflammation, gastric acid secretion, inhibition of bone resorption, and anti-inflammatory activity by reducing TNF-a concentrations (Goltzman and Mitchell, 1985; Brain and Cambridge, 1996; Valentijn et al 1997; Feng et al., 1997; Monneret et al., 2000; Gangula et al., 2000). Hypocalcemia is a common finding in human patients with sepsis, and serum calcitonin, procalcitonin, and CGRP concentrations have been reported to be increased in many of j^qse cases (Chesney et al., 1983; Sperber et al., 1990; Parida et al., 1998; Lind et al., 2000). Similarly, hypocalcemia is common in horses with sepsis and endotoxemia (Toribio et al., 2001). The importance of peptides of the calcitonin gene family in the 121 pathogenesis o f hypocalcemia in septic human patients and horses is unknown. Because there were no published studies on the equine calcitonin gene family, we cloned and sequenced the cDNA encoding for equine calcitonin, CGRP-I and CGRP-II. bivestigations of CT, PCT, and CGRP in horses with enterocolitis and endotoxemia will provide useful information on the pathogenesis of hypocalcemia associated with sepsis and endotoxemia in animals and man. , ‘^ -4 ' -, Materials and Methods "V* ' A- V t cDNA synthesis and tibrary construction Total RNA was extracted from equine thyroid tissue with Trizol (Gibco ERL, Grand Island, NY, USA). Poly (A)^ mRNA was isolated using oligo(dT)-cellulose columns (Amersham Pharmacia Biotech, Piscataway, NJ, USA). First strand cDNA synthesis was performed using the Great Lengths cDNA synthesis kit (Clontech, Palo Alto, CA, USA). The cDNA was then cloned into XTriplEx (Clontech, Palo Alto, CA, USA) and packaged with the Gigapack 111 Gold Packaging Extract (Stratagene, La Joya, CA, USA). The directionally-cloned equine thyroid cDNA library contained 1.2 x 10^ recombinants, and the cDNA insert size ranged from 0.5-4.0 kb. 122 vÉUM Luria-Bertani/agar/MgS 0 4 top agarose dishes (150mm) were plated with approximately 5 x 10^ pfu per dish, using E. coli XL-1 blue as the phage host. The plates were incubated at42"C for4 h and at room temperature overnight. When plaques were confluent the plateà^were stored at 4°C. Nylon membranes (Osmonics, Minnetonka, MN, USA) were placed omthe tc^ agarose for 2-3 min. Subsequently the cDNA was denatured in 1.5M NaCl/D.SM NaOH, neutralized in 1.5M NaCl/0.5M Tris-HCl pH 8.0, and rinsed in 0.2M Tris-HCl pH 7.5, 2X SSC. The nylon membranes were dried, and the DNA was bound to the membranes by UV crosslinking in a Stratalinker UV Crosslinker 1800 (Stratagene, La Joya, CA, USA). A 301 bp probe was generated by PCR of ovine calcitonin cDNA (Cumaraswamy et al., 1993). Primers (5- CTCGACCTCTCCTGAAACTC, 5-CTCCAGAGCTAAGCGGTGCA) were designed to amplify exons 1-3, and the amplified product hybridized to common regions of CT and CGRP. The probe was radiolabeled with (**'32P)dATP (NEN life Science Products, Boston, MA, USA) using a DNA-labeling kit (Gibco ERL, Grand Island, NY, USA) to 1.5 X 10® cpm/pg of DNA. The nylon membranes were prehybridized in 50% fbrmamide/5X SSPE/5X Denhardt’s solution/0.1% SDS/100 pg/mL denatured herring sperm DNA for 4 h at 42 °C. The prehybridization solution was removed, and new solution and probe (5 x 10^ cpm/nylon membrane) were added. The nylon membranes were incubated at 42"C for 20 h. The membranes were washed once in 2X SSC/0.5% SDS for 20 min at room temperature, and twice in IX SSC/0.1% SDS at 60 °C for 15 min, and then were dried and wrapped in plastic film. Initial screening to detect positive 123 clones was perfotmed using a Phosphorlmager 445 SI (Molecular Dynamics, S unnyy^ CA, USA). When positive clones were detected, the membranes were exposed to autoradiography film (Eastman Kodak Company, Rochester, NY, USA) at -80 °C. Positive clones were screened two additional times to obtain single clones. The phage DNA was isolated with the Wizard Plus Lambda DNA purification system (Promega Corporation, Madison, WI, USA). Plasmids (pTriplEx) were generated by cre- recombinase-mediated site-specific recombination in £ . coli BM2S.8 (Clontech, Palo Alto, CA, USA). The plasmid was purified with Wizard Plus Minipreps DNA purification system (Promega Corporation, Madison, WI, USA). DNA sequence analysis Approxhnately h x 10 clones were screened. Four clones hybridized to the CT/CGRP probe. The phage and plasmids inserts were sequenced with X TriplEx-specific sequencmg primers (Clontech, Palo Alto, CA, USA), the T7 promoter primer, and equine CT and CGRP-specific primers, using dideoxy nucleoside triphosphates and an ABI PRISM 373XL DNA sequencer (PE Biosystems, Foster City, CA, USA). The nucleotide and protein sequences were analyzed and compared to other species using Align Plus software (Sci-ed Software, Durham, NC, USA). 124 Northern btotanaf^b Total RNi^ (20pg) from *20 different equine tissues was electrophoresed on denaturing formaldehyde (6%) agarose (1%) gels. After electrophoresis for 3h at 65 volts, the RNA was transferred onto nylon membranes (Stratagene, La Joya, CA, USA). The membranes were UV irradiated (Stratagene, La Joya, CA, USA), prehybridized in QuikHyb solution (Stratagene, La Joya, CA, USA) for 20 min at 68"C and hybridized in QuikHyb solution with denatured herring sperm DNA and the cDNA probe (1.25 x 10^ cpm/ml of hybridization solution) for 1 h. For equine calcitonin a 298 bp probe was generated by PCR of exon 4 (primers, 5-CTGGGCACATACACGCAGGA; 5'- TGCTCTCTCTCCTCCCATCTG). For equine CGRP-I (clone Bl) a 224 bp probe was generated by PCR of exon 5 (primers, 5-TGCTGGGAGTATGGCGAACA; 5- GAGTGATTTCAATGTCCAGAAGCAT). For equine CGRP-II (clone A1), a 318 bp probe was generated by PCR of exon 6 (primers, 5'- TGACAACAGCTTGGTGAGAAGG; 5 -AGGCATAGCATCACCGTTACCA). There was no homology between the CGRP-II probe and CGRP-I. Initial screening of the membranes was performed using a Phosphorlmager 445 SI (Molecular Dynamics, Sunnyvale, CA, USA). When hybridization bands were detected, the membranes were exposed to autoradiography film (Eastman Kodak Company, Rochester, NY, USA) at -80 °C. 125 Results Cloning o f equine calcitonin and CGRPs Screening of approximately 1 x 10^ clones revealed 4 clones (Al, A4, Bl, and 82) that hybridized with the CT/CGRP-specific probe, and contained complete cDNA sequences for calcitonin and CGRP. Sequence analysis revealed 3 different cDNA products, with a length of 835 bp for calcitonin (A4), 702 bp for CGRP-1 (Bl, B2), and 960 bp for CGRP-II (Al). Clone B2 sequence was 100% identical to clone Bl, therefore only clone Bl was analyzed. Nucleotide and deduced amino acid sequences were compared to sequences available in the NCBI GenBank fhttp://www.ncbi.nlm.nih.eovI Equine calcitonin sequence analysis Clone A4 (835 bp), contained the cDNA sequence for equine preprocalcitonin (Fig. 5.1). The preprocalcitonin cDNA consisted of 102-bp of the 5’ UTR, the 423-bp coding region, and 310-bp of the 3* UTR, including a 19 bp poly (A)^ tail. The 423 bp coding region encoded for a 140 aa peptide (preprocalcitonin, MW=15,357 Da). Preprocalcitonin, procalcitonin, calcitonin and katacalcin nucleotide and amino acid sequences comparisons aré'presented in table 5.1. The deduced aa sequence for equine preproCT was aligned with human, rat, mouse, dog, sheep, salmon, and chicken preproCT sequences, using the BLOSUM 62 scoring matrix in Align plus (Fig. 5.2, table 126 : 5.1). There are conserved amino acids in the signal and N-terminal peptides, and mature CT across species. However, there is lack of homology in the carboxyl terminal peptide (CCP, katacalcin). Equine CT signal peptide has 25 amino acids, the N-terminal peptide has 56 amino acids, and katacalcin has 21 aa (MW=2,310 Da). Between the N-terminal peptide and the mature CT there is a dibasic amino acid cleavage site (KR).The predicted equine procalcitonin aa sequence has 115 amino acids (MW=12,507 Da). Equine CT (32aa, MW=3,385 Da) amino acid sequence was compared to human, rat, mouse, dog, salmon, zebrafish, cattle, pig, chicken, sheep, and bullfiog calcitonin, using the BLOSUM 62 scoring matrix in Align plus (Fig. 5.3, table 5.1). A phylogeny dendrogram for calcitonin was constructed using Align plus (Fig. 5.4). The dendrogram has 3 classes, and equine CT was placed in the human, rat, and mouse class. The signal and N-terminal peptide aa sequences of clone A4 were compared to clones Al and Bl (Fig. 5.5). There is 100% homology between clones A4 and 81; however the homology between clones A4 (and Bl) to clone Al is 65%, indicating that clone Al is encoded by a different gene. Equine CGRP sequence anafysis The cDNA sequences for the signal and N-terminal peptides of clone Bl were identical to clone A4 (Fig. 5.5), which indicated that these clones were derived Aom the same gene. We named this product equine CGRP-I. Clone Bl (702 bp) contained the complete sequence for equine preproCGRP-1 (Fig. 5.6). Equine preproCGRP-1 consisted o f a 102 bp 5* UTR, a 384 bp coding region, and a 216 bp 3’ UTR, including a 25 bp 127 poly (A)^ tail. The 384 bp coding region encoded for a 127 aa peptide (preproCGRP-I, MW=13,863 Da). The deduced aa sequence for mature equine CGRP-I (37aa, MW= 3,770 Da) was aligned to CGRP aa sequences from other species (Figs. 5.7-5 8). The greatest homology was found with swine CGRP (59%) and human CGRP-11 (59%). The homology with equine clone Al CGRP was 56%. The signal peptide fbr equine CGRP-1 has 25 aa residues, the N-terminal peptide has 54 aa residues and the C-terminal peptide has 4 aa residues. Between the N-terminal peptide and the mature CGRP-1 (37aa) there is a dibasic amino acid cleavage site (KR). Ao'The cDNA sequence for the signhl and N-terminal peptides of clone Al has 65% homology with clones A4 and Bl. Clone AI cDNA likely represents a gene equivalent to the CALC-H gene in humans, which encodes for human CGRP-11. We named the peptide encoded by clone Al equine CGRP-11. Clone Al (960 bp) contained the complete sequence for equine preproCGRP-U (Fig. 5.9). Equine preproCGRP-U consisted of a 69 bp 5’ UTR, a 390 bp coding region, and a 501 bp 3’ UTR, including a 25 bp poly (A)^ tail. The 390 bp coding region encoded for a 129 aa peptide (preproCGRP-11, MW=13,924 Da). The mature equine CGRP-11 (37 aa, MW=3,782) aa sequence was aligned to CGRP sequences from other species (Fig. S.7-5.8). Unlike equine CGRP-1, equine CGRP-11 has higher homology to other mammalian and non-mammalian CGRPs (ranging from 81-97% for mammals; Fig. 5.8). The signal peptide for equine CGRP-11 has 25 aa residues, the N-terminal peptide has 56 aa residues and the C-terminal peptide has 4 aa residues. A dibasic amino acid cleavage site (KR) is present between the N- terminal peptide and the mature CGRP (37aa), as seen in clones A4 and Bl. The C- terminal peptides for clones Al and Bl are identical, and both are preceded by a 5 aa 128 cleavage site (GRRRR). A phylogeny dendrogram for CGRPs from different species was constructed using Align plus (Fig. 5.10). Sequence information was submitted to tite NCBI with accession numbers AF249307 (calcitonin), AF257471 (CGRP-1), and AF257470 (CGRP-11). Northern filot anafysis Northern blot analysis of equine tissues revealed that expression of calcitonin mRNA was restricted to the thyroid gland (Fig. 5.11). In contrast, the expression of equine CGRP-1 and CGRP-11 mRNA was present in multiple tissues (Fig. 5.11). Expression of CGRP-I was detected in the thyroid gland, hippocampus, pituitary gland, medulla oblongata, dorsal spinal root ganglia, lung, small intestine, and kidney. Expression of CGRP-II was detected in the thyroid gland, basal ganglia, hypothalamus, thalamus, medulla qblongata, spinal cord, dorsal spinal root ganglia, and kidney. The expression of CGRP in the kidney was an unexpected finding. «X ■ ■ Dlscusslom In this study we found three different transcripts encoding for peptides of the equine calcitonin gene family. In humans, calcitonin is encoded by the CALC-I gene (Becker et al., 1996). The CALC-I gene also encodes for CGRP-1 (or CGRP-a) by alternative splicing of the primary transcript (Rosenfeld et al., 1981). Other genes of the calcitonin family include CALC-H, encoding fbr CGRP-11 (or CGRP-P), CALC-HI (a 129 pseudogene), and CALC-IV, encoding for amylin (Becker et al., 1996). It has been . •# ■■•• f suggested that CT, CGRP and amylin were encoded by a single gene early in evolution, and that these genes are the result of duplication (Becker et al., 1996). Furthermore, gene duplidation, resulting in the CALC-I and CALC-II genes may have resulted in greater biological diversity (Steenbergh et al., 1986). The equine CT signal and N-terminal peptides were identical to that of equine CGRP-I indicating that clones A4 (CT) and Bl (CGRP-I) resulted from alternative splicing of the same primary transcript. Based on the homology of the signal and N- terminal peptides of clone Bl to that of clone A4, we named the deduced aa sequence from clone Bl equine CGRP-I. The degree of homology of mature equine CGRP-1 to CGRP in other species is low. The signal and N-terminal peptides of clone Al have a low homology to that of clones A4 and Bl (65%). We believe that clone Al is encoded by a different gene (equivalent to human CALC-II). We have named this peptide equine CGRP-II. An unexpected finding was that the mature equine CGRP-11 has more homology with CGRPs of other species than equine CGRP-1, and that the homology between equine CGRP-I and CGRP-11 was low. The homology between human CGRP-1 and human CGRP-II is 91%, while the homology between equine CGRP-1 and equine CGRP-U is 56%. We speculate, based on the homology of equine CGRP-11 to CGRPs of other species, that CGRP-II in the horse may have assumed many of the functions attributable to CGRP-I in other species (neutransmitter, pain mediator, smooth muscle relaxation), or that equine CGRP has functions other those of a neutransmitter. An example is the expression of equine CGRP-I and CGRP-11 in the kidney. To our knowledge, this is the first study that reports expression of a product of the calcitonin 130 gene family in renal tissue. Bioassay studies would be appropriate to elucidate the different roles that CGRP may play in the horse. Furthermore, studies on this novel equine CGRP-I would be o f interest from the evolutionary perspective. This is the first publication on the nucleotide and amino acid sequences of calcitonin and CGRP in the horse. We have determined that mature equine CT is similar to CT of other species CT. Equine CGRP-I has low homology to CGRPs of other species, and we believe, based on the identity of the signal and N-terminal peptides, that equine CGRP-I is encoded by a gene equivalent to human CALC-I gene. We are studying the role o f the calcitonin gene family in horses with certain pathological conditions (sepsis, endotoxemia, hypocalcemia). In humans, procalcitonin may be involved in the pathogenesis of sepsis and inflammation (Oberhoffer et al., 1999). Measurement o f human procalcitonin has been possible by the development of two-site immunoassays, which require one antibody to calcitonin or to the N-terminal peptide and a second antibody to the carboxyl terminal peptide (CCP, katacalcin). Unfortunately, katacalcin is not conserved across species, making the use of human procalcitonin assays too unreliable to measure equine procalcitonin. With the cDNA and amino acid sequences for equine preprocalcitonin, preproCGRP-I and preproCGRP-lI available, it is now possible to study the role of the peptides of the calcitonin gene family in healthy and diseased horses. 131 Acknowledgments The authors are grateful to Drs. A. Thiagalingam and B. Nelldn at the John Hopkins University Oncology Center for providing us with the sheep calcitonin cDNA (GenBank Accession No. M980S3). The authors are also grateful to Drs. D. Groppe, B. LeRoy, V. Richard, R. Sellers, and S. Tannehill-Gregg for their technical assistance. Funded by The Ohio State University College of Veterinary Medicine Equine Research Fund No. 520039. Support for Dr. Toribio was provided by the C. Glenn Barber Fund of the Ohio State Universi^X^llege of Veterinary Medicine. 132 References Alevizaki M, Shiraishi A, Rassool FV, Ferrier GJ, MacIntyre I, Legon S. The calcitonin like sequence of the beta CGRP gene. FEBS Lett 1986; 206:47-52. 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' ' 135 ,.r; ' Species preproCT proCT CT Katacalcin nt aa nt aa nt aa nt aa Human 83 73 83 74 91 90 66 38 Rat 75 68 74 68 86 90 56 33 Mouse 75 66 73 66 85 87 54 28 Sheep 75 59 73 57 68 50 66 38 Salmon 58 45 58 48 63 59 40 22 Chicken 56 40 59 44 66 53 45 12 Canine 75 63 73 62 79 65 40 23 Zebrafish NA NA NA NA 60 56 NA NA Bovine NA NA NA NA NA 53* NA NA Swine NA NA NA NA NA 53* NA NA Bullfrog NA NA NA NA NA 50* NA NA NA: Incomplete or no sequence available. *From aa sequencing. Table 5.1. Nucleotide and amino acid identity (%) of equine preprocalcitonin (preproCT), procalcitonin (proCT), calcitonin (CT) and katacalcin to other species. 136 1 CGACGCCAGAGATACTTCGAGAGTGCCTTTCCCTGACTTCGAÇGCTGCTGCTGCCACTGT 61 ctctgttccaggctacctgctagtacttgcccgaaaggcgtc ^ | qgcttctggaagttc M G F W K p 6 121 TCCCCCTTCCTGCCTCTCAGCATCTTGGTCCTGTACCAGGTGGGCATCATCCAGGCAGCA SPFLPLSILVLYQVGIIQAA 26 18 1 CCATTCAGGTCTGCCTTGGAGAGCCTCCCAGATCCTGCTGTACTCCCTGAGGAGGAATCG PFRSALBSIiPDPAVLPBEBS 46 2 4 1 CGACTCCTACTGGCTGCGCTGGTGAAGGACTATGTGCAGATGAAGGTCAGGGCGCTGGAG RLI.LAALVKDYVQMKVRALE 66 3 0 1 CAGGAGCAGGAGACAGGGGGCGCCAGCCTGGACAGCCCCAGAGCTAAGCGGTGCAGTAAT QEQETG GASLDS P R A K R C S N 86 3 6 1 CTGAGTACCTGTGTGCTGGGCACATACACGCAGGACCTCAACAAGTTTCACACGTTCCCT LSTCVL GTYTQDLWKFHTFP 106 4 2 1 CAGACTGCAATTGGGGTCGGAGCACCTGGGAAGAAAAGGGTCATGGCCAGAGGCTTGGAG QTAIGVGAP G K K R ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ I 126 4 8 1 agagaccacggccctcacattggcacgtcccaggatgccta ^ B gcccctccctctcct HHHHHHHBIHÉHHHHHHHHHH * 140 541 TTCTAATTTCTTTTCyriSlg^CTTTCCTATAAGTTAATGCATGTGGTCCTCTCTGCTTGT 6 0 1 TCTTCGGGCTGGTÀTGAG.T6GCTTTTTTTGTGGCAGTCGATGTCTGGAAGCTCAGATGGG 6 6 1 AGGAGAGAGAGCAGGACC^CAGACCAGAAGAGAATCACCCAGGAAGAGATCAGAGAGAG 7 2 1 AGAGGGCAGCCCTGTGAGTdCÇTGAGAATTTCATAGCAGAGCTrCTCACTCCAGCTTCTG 7 8 1 aatgtgcttgtcattgggg B ^ B agtatatttccaaaaaaaaaaaaaaaaaaa Figure 5.1. cDNA and deduced amino acid sequences of equine preprocalcitonin (clone A4, GenBank No. AF249307 ). Numbers on the left indicate nucleotides. Numbers on the right indicate amino acids from the start methionine. The ATG and TAA (*) codons specifying the 423 bp open reading frame and the A AT AAA polyadenylation signal are indicated by black boxes. The sequence for the mature calcitonin peptide (32 aa) is underlined. Equine katacalcin anruno acid sequence (21aa) is in a shaded box. 137 ?TT , i'-' EQUINE MGPWKFSPFLPLSILVLYQVGIIQAAPPRSALESLP-DPAVLPEEESRLLLAALVKDYVQ 59 HUMAN ...... ^V...... L. *SLH...... S. D ...... Q... . 60 RAT .. .L ...... W . . .L .. .ACGL. .V.L..T...S. -GM T.S •. A .. - . .QN.M. 58 MOUSE ...L ...... W ...L...ACSL..V.L..1 ...S.-GM.T.S...V..- . .Q..M. 58 CANINE ..L ..S ....AP. a...C. A. GL ...... G .. — .. TA .S.K.G...... A. . . 59 OVINE . . .G.S....AP.....C.A.SL..T.L.... T.. — ..GA.S.K.G...... A... 59 SALMON .VMM.L.AL.IAYP. .IC.MYSSH. . .A.TG.. .MT-.QVT.TDY.A.R. N I..EP.. 59 CHICKEN ------VYA. .VC.MDSP------V.PG. . .IT- .RVT.SDY.A.R. N . . .EPI. 48 EQUINE MKVRA-LEQB—QE 113 HOMAN ..ASE- 114 RAT ....E —....EE.. 114 MOUSE . A. E— . . . * 114 CANINE R.NELEQ.-.,.^. . .E.S .AE 114 OVINE R.TNBi. . . .V É.( SA.WK.. .NY.RYSGMGP .PE 116 SALMON .TSEE-.t/—-A . . .R.MS. , • • • » • .KLS.E.H.LQ.Y.R.NT .S. 112 CHICKEN .TAEE-...A-tS. ...R.IS.,. .AS. • • • * » .KLS.E.H.LQ.Y.R.DV .A. 100 EQUINE ______140 HOMAN;; c .D.SSD.. . . ------:R. .NHCBEESL 141 RAT .D..KD..TN------.H.YP.N 136 MOUSE .DV.KD..TN------.QS.F.N 136 CANINE T. , .DI.S ... .G*"“•R"~"-“ 130 OVINE T . , .DI.NS..K.------LSS.P.VPT..N 143 SALMON T. , .SLP---.SN------RYASY.D.y.GI 136 CHICKEN T. , .NVLND.DHBRYANY.ETL.NN 127 Figure 5.2. Alignment of the aa sequences o f preprocalcitonin from different species. NCBI accession numbers: equine, AF249307; human, M64486; rat, V01229; mouse, -X9799I;_ canine, -AJ2I1090^ ovine,-M980S2; salmon, Y0Q765; and chicken, X03D12. Amino acids are shown in one letter code. Amino acid numbers from the start methionine are indicated on the right margin. Dots represent amino acids identical to equine preproCT. Mature calcitonin peptide aa sequence (32aa) is in a shaded box. Katacalcin (CCP) sequence is in a black box. 138 m r n m . . . r I Homology NCBI * ***** * * * * % N o. E q u in e CSNLSTCVLGTYTQDLNKFHTFPQTAIGVGAP AF249307 Human .6 . . M...... P ...... 90 M64486 Rat .G.. .M...... S...... 90 V 01229 M ouse .G . . M...... S.. .B. . 87 X 97991 C am ine ...... SK.. N------SGIGF.ABT. 6 5 X 56994 S alm o n ------KLS.B.H.LQ.Y.R.NT.S.T. 59 Y00765 Z e b r a f i s h . .S . ------KLS.B.H.LQ.y.R.NV.A.T. 56 A F 026271 B o v in e .. .SA.WK.. .NY.R.SGMGF.FBT. 53 TCBO S w in e . . .SA.WRB. .N. .R.SatGF.PBT. 53 TCPG C h ic k e n .AS. ----- KLS.B.H.LQ.Y.R.DV.A.T. 53 X 03012 O v in e .. .SA.WK.. .NY.RYSGMGP.PBT. 50 M98053 B u l l f r o g . .G. . . ;A.MKLS. . HR.NSY.R.NV.A.Y. 50 Yoshida et al. 1997 Figure 5.3. Alignment o f mature calcitonin aa sequences from different species. Amino acids identical to equine CT are presented with dots. Asterisks represent conserved aa (>90%). The overall homology and NCBI accession numbers are shown on the right columns. Amino acids 1, 4-7, 9, 28, and 32 are identical in all known calcitonin sequences. All species have leucine (L) at position 16, except humans, which have phenylalanine (F). 139 r— Equine Human ■ C ^Rat Mouse Canine Bovine c Ovine Swine Salmon Zebrafish c — Chicken Bullfrog Figure 5.4. Phytogeny dendrogram for calcitonin. The dendrogram was constructed using multi-way alignment with Align plus. Equine calcitonin was placed in the mouse, rat, and human class, with which it has the highest homology. Also shown in Fig. 5.3. 140 S;;;' V . V . •■: : s..'. 40(i'' A4 MGFWKFSPFLPLSILVLYQVGIIQAAPFRSALBSLPDPAVLPBEESRLLIiAALVKDYVQH (60) B1 ...... (60) AX >..G.P.S. • AP• •. ..C.A.Sli. .Q.L. ,S...... A.S.K.G. A. . .R (60) A4 KVRALEQEQBTGGAS (75) BX ...... (75) AX .T H E QEMBG (75) Figure 5.5. Alignment of the signal and N-terminal peptides of clones A4 (CT), B1 (CGRP-1), and A1 (CGRP-II). Dots represent identical amino acids. There is 100% homology between clones A4 and Bl, indicating that the gene encoding for these clones is equivalent to the CALC-I gene in humans, which encodes for CT and CGRP-I. Clone A1 sequence is the product of a different gene; the aa homology between clone Al, and clones A4 and Bl is 65%. V* > 141 1 AAGTGCCACAQATACTTCGAGAGTGCCTTTCCCTGACTTCGACGCTGCTGCTGCCACTGT 61 CTCTGTTCCAGGCTACCTGCTAGTACTTGCCCGAAAGGCGTcHIjQGCrrCTGGAAGTTC M G F W K F 6 1 2 1 TCCCCCTTCCTGCCTCTCyVGCATCTTGGTCCTGTACCAGGTGGGCATCATCCAGGCAGCA SPFLPLSILVLYQVGIIQAA 26 1 8 1 CCATTCAGGTCTGCCTTGGAGAGCCTCCCAGATCCTGCTGTACTCCCTGAGGAGGAATC6 PFRSALBSLPDPAVLPEEBS 46 2 4 1 CGACTCCTACTGGCTGCGCTGGTGAAGGACTATGTGCAGATGAAGGTCAGGGCGCTGGAG RLLLAALVKDYVQMKVRALE 66 3 0 1 CAGGAGCASGAGACAGGGGGGGCCAGCATCACTGCCGAGAAaAGATCCTGCAACACTGCC Q E QBTGGAS ITAQKR S C N T A 86 3 61 AGCT6CTT6ACCCATCGGCTGGCAGGCTTGCTGAGCAGTGCTGGGAGTATGGCGAACAGC S C LTHRLAGLLSSAGSMAWS 106 4 2 1 AACTTGCT6CCCACTGAGATGGGCTTCAAAGTCTCTGGCCGTCGCCGCAGGGACCTTCAG N L LPTEMGFKVS GRRRRDLQ 1 26 4 8 1 GC^HpCAGTGAAATGACTCCAGGAAGAAGATCACCATGAAGCTGAACTCTACTTCCTT A * 1 27 5 4 1 TACTTCATAGTGAAAGCAACTTATTGGACAGGGAGCATGGAAGATACACCTATATGCATG CTTCTGGACATTGAAATCACTCTTTrCTGTTTAAAATAAACTAAAGCTAAATGCAGlHI 6 6 1 ■ atcattgcaagtaccaaaaaaaaaaaaaaaaaaaaaaaaa (teduc^ am ipQ acid ^pgnce ( 127aa) of the equine preproCGRP-I cDNA (clone B it jOenBank No. AF257471). The single letter aa is shown under the nt sequence. Numbers on the left indicate nucleotides. Numbers on the right indicate amino acids from the start methionine The ATG and TGA specifying the 384 bp open reading frame and the-AATAAA polyadenylation signaL are, indicated by black, boxes. The. sequence for the CGRP Bl mature peptide (37 aa) is underlined. 142 Homology NCBI t No. C lo n e Bl SCNTASCLTHRLAGLLSSAGSMANSNLLPTBMGFKVS - “ AF257471 B ovine ...T.V...... RS.GWK. .FV. .NV.SEAF 56 227702 Swine ...T.V...... RS.G.VK. .FV. .DV.SBAP 59 P30880 O vine ...T.V...... RS.GWK. .FV. .NV.SQAF 56 P30881 R at I ...T.V...... RS.GWKD.FV. .NV.SEAF 54 L29188 R at I I ...T.V... ___ RRS.GWKD.FV. .NV.S.AF 54 A44173 R a b b it G . . T.V...... RS.G.VK. .FV. .NV.SEAF 56 227771 Human-1 A D..T.V...... RS.GWKN.FV. .NV.S.AF 51 X15943 Human. 2 A ...T.V...... RS.G.VK. .FV. .NV.S.AF 59 P10092 C hicken A ...T.V... .DF. .RS.GVGKN.FV. .NV.S.AF 48 P10286 Bullfrog A . . .T.V*.. .DP. .RS.G. -KN.FV. .NV.S.AF 54 P31888 Salm on I A ...T .V ... .DF.NRS.G.G. . .FV. .NV.A.AF 54 S40497 Salm on IV A . . .T.V... .DF..RS.G.G.. .FV..NV.A.AF 56 U71287 Phyllomedusa D.ST.A.Q. .DF..RS.GIGSPDPV..DVSANSF 35 P81564 Human Amylin K . . .T.A.Q. .NF. VHSSNNFGAI.SS.NV.SNTY 35 M27503 C lone A l ...T.V...... RS.GWK. .FV. .DV.SEAP 56 AF257470 < Figuré 5^7. Comparison of the deduced'or chemically-determined aa sequences of CGRP (and human amylin) in different species to equine CGRP-I (clone Bl). Alignment was performed with Align plus software. Nucleotide and amino acid sequences were obtained from the NCBI (httD://www.ncbi.nlm.nih.govt. Asterisks represent conserved aa (>90%) among species. Dots represent amino acids identical to equine CGRP-I (clone Bl). Amino acids DF (14-15) are only present in chicken, bullfrog, phyllomedusa, and salmon. The degree of homology (%) o f clone Bl to other species is low (<59%). Amino acids 2-5, 7, 9 ,11-13, 16,27,30, and 33 are identical between equine CGRP-1 (clone Bl) and human amylin (35%). 143 v / ' . ■ Homology NCBI ************ ****** ***** ** ** No. C lone Al SCNTATCVTHRLAGLLSRSGGWKSNPVPTDVGSEAP AP257470 B ovine 97 227702 Swine 97 P30880 Ovine Q 94 P30881 R at I 94 L29188 R at 11 K 89 A44173 R a b b it G 91 227771 Human-1 A K 86 X15943 Human-2 A M K 89 P10092 C hicken A DP .G.N K 81 P10286 B u llfro g A DP MA.N K 78 P31888 Salmon 1 A DP AK 72 S40497 Salmon IV A DP MGN .AK 75 U71287 Phyllomedusa DP IGSPD SANS 59 P81564 Human A m ylin K VH . .NTY 43 M27503 Clone Bl .SA.SMAN..LL I.PKVS 56 AP257471 Figure 5.8. Alignment of the deduced or chemically determined aa sequences of CGRP (and human amylin) in different species compared to equine CGRP-II (clone Al). Unlike equine CGRP-1, the aa sequence of equine CGRP-II is more conserved when compared to other species. Asterisks represent conserved aa (>90%). Identical amino acids to that of equine CGRP-II (Al) are presented in dots. Amino acids 2-7, 9, 11-13, 16, 19, 30, and 32-34 are identical between equine CGRP-II (clone Al) and human amylin (43%). The homology at the N-terminus may be responsible for some overlap in function between amylin and CGRP. 144 1 CGCGGCCGCGTOGACCGGGTGGACAGGCCGCTCGCGCTCTCCTGCAACTCGGGTCACCAG 61 AGAGGCATCHjGGCTTCGGCAAGCCCTCCTCCTTCCTGGCTTTCAGCATCTTGGTCCTG MGFGKPSSFLAFSILVL 17 121 TGCCAGGCAGGC^GCCTCCAGGCGCAACCACTCAGGTCCTCTTTGGAGAGCCTCCCAGAC C Q AQPLRSSLESLPD 37 181 CCGGCTG(AC%P^TGAGAAGGAAGGGCGCCTCCTGCTGGCTGCACTGGTGAAGGCATAT P A A jB KEGRLLLAALVKAY 57 2 41 GTGCAGAGGAÀGACCAATGAGCTGGAGCAGGAGCAGGAACAGGAGATGGAGGGCTCCAGC V Q R " B L E Q E Q E Q E M E G S S 77 301 CTCACTGCCXZAQAAGAGATCCTGCAACACTGCCACCTGTGTGACCCATCGGCTGGCAGGC ‘ 'il . T» ' A Q K R S 'C W-T; A TCVTHRLAG 97 361 CTGÇTGAGCAGATCTGGAGGGGTGGTGAAGAGCAACTTTGTGCCCACCGATGTGGGCTCT L ' L S R S GG’VVKSNFVPTDVGS 117 42 1 GAAGCCTTCGGCCGTCGCCGCAGGGACCTTCAGGCq^pCAGTGAAATGACTCCAGGAA E A F GRRRRDLQA* 129 48 1 GAAGGTTACCATGAAGCTGAACTCACCACTTCTATTAATTTCTGTTGACAACAGCTTGGT 5 4 1 GAGAAGGCCCTAGGAAGATATATGTGTTTGCATCCTAATAGATATTGAAAAAAGCACCTT 60 1 TGTCCCTTGAAAGGAATGAAACTGAATGCAAAATAAGCTAATTCCATATCTGTTGTGCAT 661 CCTTTTTACATTTGATTCCATGTGCAGTAGATGTGATGGCATCTCTCATCAGCTTATCTG 721 GTAGCAAACTGGTTCTTTGAGAGCCATCCTGTTGATCGCACCAGCTCTGCCAAACCTTAG 781 GGGACAAGAAATCGCTGCCTCTTGTGGCCTCTGGGGACACATGGTAACGGTGATGCTATG 841 CCTTGTTGTCTAAGAACMGATTOTATAATTTGTTTAAGGAAATGTCAATATTGTGCCAT 901 ttgtgaccttcatcaag ^ H H aacatattttggaaaaaaaaaaaaaaaaaaaaaaaaa Figure 5.9. cDNA and deduced amino acid sequence (129aa) of the equine CGRP-II cDNA (clone A l, GenBank No. AF257470). The single letter aa is shown under the nt sequence. Numbers on the left indicate nucleotides. Numbers on the right indicate amino acids from the start methionine. The ATG and TGA sj^cifying the 390 bp open reading frame and the polyadenylation signal are indicated by black boxes. The sequence for the CGRP mature peptide (37 aa) is underlined. 145 m M C h ic k e n B u l l f r o g — S alm o n S alm o n IV Phyllomedusa Human 1 Humeui 2 I— Equine CGRP-II ( c lo n e A l) '— S w in e I— R a t I JT ------R a t I I U 0O v in e T— B o o ' v in e R a b b it Equine CGRP-I (clone Bl) Figure 5.10. Phylogeny dendrogram for CGRP. The dendrogram was constructed with the Align plus software. Two major branches representing mammalian and low vertebrate CGRPs are present. One additional branch representing clone Bl (equine CGRP-I) suggest an early divergence of this gene when compared to clone Al (equine CGRP-11). 146 Figure 5.11. Tissue-specific expression o f equine calcitonin, CGRP-I, and CGRP-II mRNA. The blots were probed as described in section 2.4. (A) Ethidium bromide stained agarose gel to demonstrate RNA loading and integrity. The tissues are l=thyroid gland, 2=basal ganglia, 3=hippocampus, 4=rostral brain, 5=caudal cortex, 6=hypothalamus, 7=thalamus, 8=pituitary gland, 9=medulla oblongata, 10=cerebellar vermis, LWcerebellaiL hemiqiherer l^ q iinal cord, l3F-dorsaLrool ganglia, 14=?lung,^ L5=smalL intestine, 16=adrenal gland, 17=parathyroid gland, 18=spleen, 19=liver, 20=kidney. Phosphorimages for equine calcitonin (B), CGRP-I (C), and CGRP-II (D). 147 BIBLIOGRAPHY Abbott LG, Rude RK. Clinical manifestations of magnesium deficiency. Miner Electrolyte Metab 1993; 19:314-322. Aguilera-Tejero E, Estepa JC, Lopez 1, Bas S, Garfia B, Rodriguez M. Plasma ionized calcium and parathyroid hormone concentrations in horses after endurance rides. J Am Vet Med Assoc 2001; 219:488-490. Aguilera-Tejero E, Garfia B, Estepa JC, Lopez 1, Mayer-Valor R, Rodriguez M. 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