University of the Pacific Scholarly Commons
University of the Pacific Theses and Dissertations Graduate School
1976
The functional development of the adrenal-sympathetic nervous system in neonatal and adolescent swine
Sidney Koon Hung Woo University of the Pacific
Follow this and additional works at: https://scholarlycommons.pacific.edu/uop_etds
Part of the Animal Sciences Commons
Recommended Citation Woo, Sidney Koon Hung. (1976). The functional development of the adrenal-sympathetic nervous system in neonatal and adolescent swine. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/435
This Thesis is brought to you for free and open access by the Graduate School at Scholarly Commons. It has been accepted for inclusion in University of the Pacific Theses and Dissertations by an authorized administrator of Scholarly Commons. For more information, please contact [email protected]. THE FUNCTIONAL DEVELOPHENT OF THE ADRENAL SYMPATHETIC NERVOUS SYSTm-1 IN NEONATAL AND ADOLESCENT SWINE
A 'l'hesis Presented to the Faculty of the Graduate School University of t.he Pacific
~ --
In Partial Fulfillment of the Requirements for L:he Degree Master of Science
by
Sidney I\. Vloo December 1976 DEDICATION
The author dedicates this ·thesis to his mother, who is an infinite source of inspiration to her children.
ii ACKNOWLEDGMENTS
The author wishes to express his sincere gratitude to Dr. Hubert C. Stanton for his invaluable guidance and support throughout his encouragement in this research; Dr. Donald Y. Shirachi, for his encouragement, guidance, and advice; Dr. Marvin H. Malone for his supervision and advice in the preparation of this thesis; Dr. Charles W. Roscoe and Dr. Horach H. Loh for generously agreeing to act as members of his thesis committee. The author is also grateful to Mr. Raymond L. Mueller and Dr. Harold J. Mersmann of Shell Development Company for their interest a~d ~elE i~ this research. Finally, the author wishes to express his apprecia tion to Mrs. Carol Sarnoff for her technical assistance in the preparation of this thesis.
iii TABLE OF CONTENTS
Page LIST OF TABLES .. vii LIST OF FIGURES ix
Chapter
I INTRODUCTION ...... 1 Statement of the Problem 1 Review of the Literature 5 Catecholamines and the Adrenal Medulla . . . 5 Ontogenesis of the Sympathetic N~rvous System. . . • . . . . 22 Perinatal Survival of the Swine. 25
II !4ATERIALS AND METHODS. 30
------Animals. . . • • . • . . 30 Estimation of Medullary and Cortical Volumes ...... 30 Determination of Catecholamines. 32 Determination of Catecholamine Biosynthetic Enzymes Activities 34
','· Tyrosine Hydroxylase (TH). 36 DOPA Decarboxylase (DDC) : Method 1 •.....•.. 39 DOPA Decarboxylase (DDC): Method 2 • • • • • • • • • • 39 Dopamine-B-hydroxylase (DBH) 42 Phenethanolamine-N-Methyltrans ferase (PNMT) . -: ...... 45 Biuret Method...... • . . 48 Estimation of Maximal Velocity (Vmax) and Michaelis Constant ( Km) • • • • • • • • • • • • • 49
Determination of Tyrosine Concen·tra·tions in Adrenal Gland and Plasma . . . • 49 iv v
Chapter Page
II Tyrosine Extraction from Adrenal Glands .. ' •..••.• 49 Tyrosine Extraction from Plasma. so Fluorometric Determination of Tyrosine Concentration •••.. so In Vitro Biosynthesis of Catecholamines -rrom Tyrosine by Adrenal Slices . 51 Amine Uptake by Adrenal Vesicles • 52 Amine Efflux from Adrenal Vesicles 58 III RESULTS •...... 59 Growth of the Adrenal Gland: General Aspects • • . . . • • . . • • • 59
Relationship Between Adrenal and Body Weight . . • • 59 Growth of the Medulla in Relation- ship to the Cortex. . . • • . . • 59 Relationship Bet1-1een Catecholamine Content and Piglet Age. . . . . 62
Ontogenic Patterns of Catecholamine Biosynthetic Enzymes. . • • • . • . 69
~-- Tyrosine Hydroxylase (TH) Activity ancf PTgleE Age~- . -: :- . ; ;- . -:- . DOPA Decarboxylase (DDC) Activity and Piglet Age ....•..••. 69 Dopamine-S-Hydroxylase (DBH) Activity and Piglet Age . • • • • 73 Phenethanolamine-N-Methyltransferase (PNM1') Activity and Piglet Age •. 73 Apparent Kinetic Patterns of the Catecholamine Biosynthetic Enzymes and Piglet Age...... • • 73
Synthesis of Catecholamines by Adrenal Slices in vitro . • ...... • 78
Effect of a-Methyl-p-Tyrosine (aMpT) on the in vitro Biosynthesis of Catecholamines...... • . 78 Adrenal and Plasma Tyrosine Con- centrations and Piglet Age. . . 80 Ontogenesis of in vitro Catechol- amine Biosynthesis. • . . . • • . 80 vi
Chapter Page
III Ontogenesis of in vitro Amine Uptake and Efflux by Isolated Adrenal Vesicles. . • 83 Relationship of Amine Uptake with Piglet Age...... 83 Relationship of Amine Efflux with Age. 88
IV DISCUSSION. 92 v SUMMARY 106 BIBI,IOGRAPHY. . 108 LIST OF TABLES
Table Page
I Piglet .1-!ortality at Five Locations. 3 II Diet Compositions, Percent...... 31 III Percent of Medullary Volume in Terms of Cortical Volume as Related to Species and Age .•••..•••• . . . 61 IV Mean Body Weights and Adrenal Gland Catecholamine (CAl Content Correlated with Piglet Age. . • • . • • . • . . . 63
v Mean Specific Activity of Tyrosine Hydroxy lase·in Adrenal Homogenates Correlated with Pigl~t Age. . . . . • . . . . • . . 70
VI Mean Specific Activity of DOPA Decarboxylase in Adrenal Homogenates Correlated with __ Pigle_t age. . __ • ~ ·'--··. __. . • ,__ ._. ,___. __. __ 70_
VII Mean Specific Activity of Dopamine-B Hydroxylase in Adrenal Homogenate Cor- related with Piglet Age...... 74
VIII Mean Specific Activity of Phenelthanolamine N-Methyltransferase in Adrenal Homogenate Correlated with Piglet Age . • . . • . . 74
IX Apparent Michaelis-Menten Kinetic Parameters j of Catecholamine Biosynthetic Enzymes in ' Adrenal Homogenate Correlated with Piglet Age . • • . . . . . · · · · · 77
X Mean Tyrosine Concentration in Plasma and Adrenal Glands Correlated 1>/i th Age • 82
XI Mean Catecholamine Synthesis Correla·ted with Tyrosine Concentration and Piglet Age. 84
vii viii
.LIST OF TABLES l (continued) 1 Table Page XII Effect of Piglet Age on Vesicular Up- take of Epinephrine (10 min. incuba tion) in the Presence or Absence of Reserpine...... 85
XIII Effect of Piglet Age on Vesicular Up take of Metaraminol (10 min. incuba tion) in the Presence or Absence of Reserpine...... 86 XIV Slopes of Uptake Curves Correlated with Age ...... 89
XV Efflux (60 minutes) of Epinephrine and Metaraminol Correlated with Piglet Age_ • . . • • • • . • . • . . • • • . 91 LIST OF FIGURES
Figure Page
1 Norepinephrine standard curve. 35
2 Epinephrine standard curve • . 35
3 Determination of optional amount of homo genate, incubation time, and substrate concentration for the assay of tyrosine hydroxylase. . • . . . . • ••• 37
4 Determination of optional amount of homo genate, incubation time and substrate concentration for the assay of DOPA decarboxylase. • • • • . . •• 40
5 Determination of optional amount of homo gena::e, incubation time and substrate concentration for the assay of dopamine- S-hydroxylase...... 43
------6 Determination of optional amount of homo- genate, incubation time and substrate concentration for the assay of phenethanolamine-N-methyltransferase. 46
7 Elution pattern of norepinephrine and normethanephrine...... ••. 53
8 Determination of optimal parameters of the amine uptake by isolated adrenal vesicles • • 55
9 Relationship between body and adrenal weights...... • . • . . • • 60
10 The change of norepinephrine content as a function of age...... •.. 64
11 The change of epinephrine content as a function of age. • . • • . . . • 66
ix X
Figure Page 12 The change of catecholamine content as a function of age. • • . • . . 67
13 The change of epinephrine content as a function' of age...... 6 8
14 The change of specific activity of tyrosine hydroxylase as a function of age • • • 71
15 The change of specific activity of DOPA decarboxylase as a function of age • • 72
16 The change of specific activity of DOPA-S- hydroxylase as a function of age. . • • . 75 17 The change of specific activity of phenethanolamine-N-methyltransferase as a function of age. . . • . . • 76.
18 In vitro inhibition of tyrosine hydroxyl- -ase- by a-methy1-E-tyrosine . • . . • . 79
19 In vitro inhibition of catecholamine bio- synthesis by a-methyl-E···tyrosine. • . . 81
20 Amine uptake by adrenal vesicles of 0-day- _o:J,.g swi_ne_witl:Land_wij:hQut_rE!SE!;:p_;i.ne .__ ._. _ 87 Chapter I
INTRODUCTION
Statement of the Problem
Birth is a severe environmental change. The neonate is abruptly exposed to the harsh extrauterine environment and must seek food and shelter on its own.. Furthermore, it must maintain homeostasis successfully in order to sur vive. The condition of homeostasis has been defined by Cannon (1939) as an internal state of equilibrium estab: lished by automatic adjustmen-ts when the animal experiences
-i- environmental changes.• _These internaL adjustments are_ brought about by a number of coordinated physiological and biochemical mechanisms. The development of the animal after birth is marked by complex changes. Certain periods are particularly im por·tant and have been defined as "critical periods" (Krecek, 1971). Two processes are important to the sur- vival of the animal during these times. The process of "adapta·tion" involves the maturation of specific physiological systems according to the needs of the neonatal animal, and is dic·tated by the external environment. The process of
1 2 "tolerance" re.nders the neonate relatively unresponsive to· a stressor as compared to the adult and protects the animal from over-responding and depleting vital resources until it is physiologically mature. An example of tolerance is the resistance to anoxia of immature neonates. The newborn pig has a high rate of mortality. The number of pigs born 114 days after conception represent · only about 55% of the original ova released. Besides having a large number of embryo losses during the gestation period, 13-25% of the live-born piglets die before weaning as shown
_j in Table I. The low viability of the neonatal piglets is un- doubtedly related to their inability to maintain homeostasis during stress. The' sympathetic division of the autonomic nervous system is a major coordinator of homeostasis. There-
------fore, a study of the development of the sympathetic nervous system could provide some clues to explain the high mortal ity rate of the newborn pigs. Since the coordinating ability of the sympathetic nervous system is not only dependent on the maturity of the system, but also on the development of t.he effector organs and their responsiveness to activa- tion, it is logical to examine their relative development. Three possibilities can exist: (1) The sympathetic nervous system in neo- natal pigs is underdeveloped relative to its effector organs. Homeostasis of the animal would then be poorly T4\-BLE I
Piglet Mortality at Five Locations
Death Prior Farrowings, Litter Size, Stillborn, to weaning, Total Loss, Location N Mean Percent Percent Percenta
Minnesotab 5,263 9.2 5.6 20.5 26.1 c Indiana 1,302 9.7 5.5 l3.0 18.5
United Kingdomd 90 10.8 7.9 24.4 32.3 e Canada 773 8.9 7.2 16.4 23.6 f Illinois 21,536 9.8 7.0 19.7 26.7 a U.S.A. average loss = 20-30% of live-borb pigs farrowed {Stanton and Carroll, 1974). The end result is 7.5 pigs/litter weanedl bKernka111p , 19 6 5 . dEnglish, 1968., fLeman, 1972. cBauman, 1966. eFahmy and Bern~rd, 1971.
w 4
coordinate? when the piglet encounters
environmental stressors. Consequently,
the neonate would have difficulty in
adaptation.
(2) The sympathetic nervous system in neo
natal pigs develops at the same rate
as its effector organs. Homeostasis
would then be maintained in harmony
and all the physiological systems would
be fully utilized.
(3) The effector organs in neonatal pigs
are underdeveloped relative to the
sympathetic nervous system. The co- ' ordinating activi·ty of the sympathetic
nervous system would then be hampered
because of the low responsiveness of
these organs. Furthermore, the neonate
would be unable to tolerate environ-
mental changes because the sympa·thetic
nervous system could exert excessive de-
mands on the effector organs, thereby
depleting the vital resources of the
animal.
It is logical to postulate that the low capability of the neonatal piglets to cope with environmental stressors is due to a "dyssynchrony" of the development of the sym- pathetic nervous system and its effector organs. A number ·-,,-
5 of deveiopmental studies on one type of effector organ-- the metabolic depots--have been reported on the swine. The following studies were undertaken to determine the func- tiona! maturation of the sympathetic nervous system in order to allow this hypothesis to be tested. These studies were conducted using adrenal glands. The justification for this comes from the following observations: (1) When the sympathetic nervous system is activated by the cold, epinephrine has been observed to increase more markedly than norepinephrine during the period of initial challenge, suggesting a marked discharge from the adrenal medulla (Bertin and Chevillard, 1967).
(2), The high percentage of epinephrine found in newborn swine exposed to cold stress implies significant contributions of catechol- amines from the adrenal medulla (Stanton et al., 1972). (3) Severe stress activates the adrenal medulla ~ in most animals (Yuwiler, 1971).
Review of ·the Literature
Catecholamines and the - Adrenal Medulla Structure of the Adrenal Medulla. The adrenal medulla is on·togenically, structurally and functionally similar to 6 the sympathetic ganglia. The nerves supplying the adrenal medulla terminate in deeply invaginated synapses in con- tinguity with the chromaffin cells. ·Host of these fibers are believed to be cholinergic. Horphological evidence for this claim was initially based upon observations upder light microscopy (Coupland and Holmes, 1958) and more re- cently demonstrated by electron histochemistry (Lewis and
Shute, 1969). As these nerves are myelinated preganglionic fibers, the chromaffin cells are believed to be similar in anatomy and function to the postganglionic sympathetic neurops. ·.In addition, it has been demonstrated that the norepinephrine-containing cell populations are innervated by postganglionic adrenergic fibers from an as yet unidenti- fied ganglionic source (Prentice and Wood, 1975).
The adrenal medulla is composed of groups and columns of chromaffin cells interspersed with groups of isolated nerve cells and permeated with venous sinusoids. All the chromaffin cells are of the large columnar type arranged in rows one cell thick along the margins of the sinusoids.
The bases of these cells point away from the sinusoids to the extracellular spaces where they synapse with the nerve terminals (Al-Lami, 1970). The adrenal gland is highly vascular, supplied by arteries derived from the abdominal aorta, the inferior phrenic and renal arteries (Coupland,
1965a). The venous sinusoids drain into the suprarenal vein, which emerges from the hilus of the adrenal gland :, '
7 and joins the inferior vena cava on the right and the left renal vein on the left.
Histology of the Chromaffin Cell. The fine struc tures of adrenal chromaffin cells have been described over the years for various mammalian species (Coupland, 1965a; Yates, 1963). A full spectrum of subcellular organelles is known to occur in these cells. These organelles are well developed, indicating the presence of high metabolic activity (Al-Lami, 1970). They include the mitochondria (slightly elongated), the Golgi complex and lysosomes. The chromaffin cells of young animals have numerous ribo somes arranged in groups which may have a helical or rosette form (Coupland and Weakley, 1968, 1970). Newborn animals have concentric arrays of rough endoplasmic reticulum _:whereas in older _animals_ these ar_e more _thinly_ distributed __ or stacked in small groups (Coupland and Weakley, 1970). There is also evidence for the presence of secretory tubules and an intracellular cytoskeleton separating the catechol amine vesicles (.Rahwan et al., 1973). The chromaffin cells have been classified into two types, namely the epinephrine and the norepinephrine-storing cells (Coupland and Hopwood, 1966). The peripheral sympathetic nerve ending, in con trast, contains exclusively norepinephrine. Adult adrenal chromaffin cells store either epinephrine or norepinephrine granules under normal circums·tances. However, granules from both of these catecholamines may be observed within the same 8
cell during fetal life ~nd shortly after birth (Coupland and Weakley, 1970). The organelles of importance in the chromaffin cells are the membrane-bound, electron-dense vesicles which are the major storage sites for the catechol- amines (Blaschke and Welch, 1953). The composition of these vesicles includes protein, lipids, catecholamines, adenosine phosphates, calcium, magnesium and water (Hillarp, 1959). The soluble components of the vesicles contain adenine nucleotides with ATP being the major one. In addition, two of the soluble proteins have been identified. They are 1 dopamine-S-hydroxylase and chromogranin A, which may be -j I chemically related ·since purified bovine adrenal medullary chromogranin A has been reported to have the identical amino acid sequence' to a subunit of soluble vesicular dopamine-S-hydroxylase (Aunis et al., 1975). The membrane
------of the chromaffin vesicle is made up of proteins which include dopamine-S-hydroxylase, ATPases, cytochrome B 561 and a cytochrome B:NADH reductase. It also contains almost all of the lipids in the vesicle (Kirshner, 1974).
Catecholamine Biosynthesis. Catecholamines are biogenic amines which possess a 3,4-dihydroxyphenyl (catechol) nucleus. The three most commonly found catecholamines in animals and plants are epinephrine, norepinephrine and dopamine. Recent investigations have indicated that a fourth, namely epinine, is found in bovine andporcine adrenal tis- sues (Laduron et al., 1974). 9 Epinephrine was the first catecholamine to be dis- covered (Abel and Crawford, 1897) and synthesized (Stolz, 1904). Norepinephrine was discovered and simultaneously synthesized by Stolz (1904). Following the discovery of DOPA decarboxylase (Holtz et al., 1938), the formation of norepinephrine was suggested to be part of the synthetic pathway of epinephrine (Blaschke, 1939). The hypothetical pathway of conversion of tyrosine into epinephrine was later confirmed by isotope experiments (Udenfriend and Wyngaarden, 1956). All the enzymes involved in the biosyn- thesis of catecholamines had been discovered and their regulatory mechanisms elucidated by 1964 (Udenfriend, 1966). Recent investigations in catecholamine biosynthesis sug- gest that epinine may' be an important intermediate in the pathway (Laduron et al., 1974). This ca-techolamine was
------first proposed as the immediate precursor of epinephrine by Halle in 1906. Subsequently in vitro studies have shown the formation of epinine from dopamine by adrenal N- methyltransferase (Laduron, 1972) and the conversion of epinine into epinephrine by dopamine-(3-hydroxylase (Bridgers and Kaufman, 1972). The first step in the biosynthesis of epinephrine from tyrosine is the conversion of the amino acid into 3,4-dihydroxyphenylalanine (DOPA). The reaction is catalyzed by the enzyme tyrosine hydroxylase (TH), which was first isolated and characterized by Nagatsu et al. (1964). TH appears ·to be localized in the soluble fraction of bovine :, .
10
adrenal chromaffin cells (Wurzburger and Musacchio, 1971).
The natural cofactor of this enzyme is 6-methyltetra-
hydropterin (Lloyd et al., 1971). Activity of the enzyme
requires molecular oxygen as a substrate (Daly et al.,
1968), whereas the role of ferrous ion as a cofactor has
not yet been established. Nevertheless, there are indica-
tions that ferrous ion not only stabilizes the enzyme but
also stimulates the reaction (Nagatsu, 1973). The hydroxyla-
tion of tyrosine to DOPA in the guinea pig heart has been
shown to be slmver than all the subsequent steps in the
biosynthetic pa·thway of epinephrine; therefore, this has
been established to be the rate-limiting step (Levitt,
1965).
The second step in the biosynthesis of catechol-
amines involves the conversion of DOPA into dopamine and
is catalyzed.by the enzyme DOPA decarboxylase (DDC). This
enzyme was first discovered by Holtz et al. in the mammalian
kidney in 1938. Subsequent characterization of the enzyme
indicates that it is not specific for DOPA, but decar-
boxylates a variety of aromatic ~-amino acids (Levenberg
-~ et al., 1962). DDC is widely distributed in a number of
mammalian organs including adrenal glands, stomach and
liver, and is present almost entirely in the soluble sub-
cellular fraction (Blaschke et al., 1955). A number of
observations suggest that pyridoxal-5-phosphate is the
prosthetic group of this enzyme (Holtz and Bachman, 1952)
and that it needs at least one free sulfhydryl group 11 for activity (Christenson et al., .1970). Dopamine-13-hydroxylase (DBH) is the enzyme respon- sible for the conversion of dopamine into norepinephrine. It was first isolated and characterized from adrenal medul- lary extracts (Levin et al., 1960) and subsequently puri fied to a single homogeneous protein (Friedman and Kaufman, 1965). Approximately one-half of the enzyme is membrane- bound in the adrenal chromaffin cell, while .the other half is localized in ·the soluble fraction (Axelrod, 1973). DBH is released along with catecholamines during vesicular secre- tion (Kirshner, 1974). The enzyme contains copper andre- j quires ascorbic acid as cofactor in this particular step -J (Kaufman, 1966). Enzyme activity is stimulated by the pres- 1 ence of dicarboxylic acids (Goldstein et al., 1965). DBH is also implicated in ·the 13-hydroxylation of epinine, but ·~·· it does not require ascorbic acid as a fac.tor in this re- action (Bridgers and Kaufman, 1962). Recently, this enzyme i was found to have a 24-hour circadian rhythm in rat adrenal 1 ' glands and that this could be abolished by pinealectomy 1 (Banerji and Quay, 1976). The last step in the biosynthesis of epinephrine is catalyzed by the enzyme phenethanolamine-N-methyltrans ferase (PNMT). The activity of this enzyme converting norepinephrine into epinephrine was first described by Bulbring (1949). The enzyme later was partially purified 12
and characterized by Axelrod (1962), who found that s- adenosyl methionine was the required cofactor and that PNMT was dependent on intact sulfhydryl groups. for activity. The enzyme also shows high specificity towards phenethanol amines and is highly localized in the soluble fraction of
the adrenal chromaffin tissues (Axelrod, 1966). A non- specific N-methyl transferase 1vas proposed to be the enzyme responsible for the methylation of dopamine to epinine
(Laduron, 1972). Regulation of catecholamine biosynthesis comprises a number of mechanisms which can be divided into two 1 categories: short- and long-term. Short-term regulation I occurs in the cytosol of the chromaffin cell where feed- back inhibition is exerted by catecholarnines on tyrosine hydroxylase, the proposed rate-limiting enzyme. There is
cytoplasmic pool of catecholamines (Alousi and Weiner,
1966) which may exert their feedback action by competing with the tetrahydrobiopterin cofactor of the enzyme (Nagatsu
~tal., 1972). Other potential sites of regulation include the membrane of the chromaffin vesicle as this structure regulates the access of dopamine into the vesicle to be
hydroxylated (Molinoff et al., 1974). Endogenous inhibi·tion of DBH has been identified in rat adrenal glands and may limit the rate of dopamine hydroxylation in intact tissues
(Orcutt and Molinoff, 1976). Long-term regulation is controlled by affecting the 13
formation of new enzymes. This can be achieved in a number
of ways. It has been observed in recent years that the
catecholamine biosynthetic enzymes can be induced to increase
in activity after catecholamine depletion, which led to the
concept of transsynaptic induction. There is considerable
evidence that tyrosine hydroxylase, dopamine-fl-hydroxylase
and phenethanolamine-~-methyltransferase are partially con-
I trolled by this type of regulation (Mueller et al., 1969a; j Thoenen et al., 1970; Ciaranello and Black, 1971). Acetyl-
I choline is believed to be the immediate mediator of trans- j synaptic induction, based on the fact that ganglionic -j blocking agents abolish tyrosine hydroxylase induction
(Mueller et al., 1970) whereas carbachol can mimic the in j duction .effect (Guidotti and Costa, 1973). The attachment of acetylcholine to a receptor on the postsynaptic nerve 1 ------r cell membrane after its release from a presynaptic nerve ending induces a specific subcellular effect. Cyclic j adenosine 3',5'-monophosphate (cAMP) has been proposed as J I ~ i a mediator between the receptor and the synthetic machinery ' for the catecholamine biosynthetic enzymes (Guidotti et al.,
' 1973). However, the involvemen·t of cyclic nucleotide in _,i i the induction of these enzymes is controversial at present:,
since such induction has been shown to occur without: a pre-
ceding increase in cAl1P (Otten et al., 1973). Protein
synthesis is implicated in transsynapt:ic induction as
the entire process can be abolished if actinomycin is 14 administered immediately after catecholamine depletion by reserpine (Mueller et al., 1969b). Adrenal cortical hormones also play an important role in the long-term regulation. The enzyme most sig nificantly influenced by these hormones is PNMT. Coupland -1 (1953) observed that norepinephrine was the main amine in those species of animals where the chromaffin cells were not in contact with adrenal cortical tissues. He pro- posed that the corticosteroids were important in the methylation of norepinephrine and this proposal has been proven to be correct. PNMT levels are markedly reduced by hypophysectomy but can be restored by the administration of adrenal cortical trophic hormone (ACTH) or glucocorticoids (Wurtman and Axelrod, 1966). Hypophysectomy also causes small but significant changes in tyrosine hydroxylase and
---- dopamine-S-hydroxylase. The administration of ACTH can restore the activity of these enzymes (Wurtman ei_: al., 1972), and recently glucocorticoids have been reported to be effec- tive as well (Axelrod, 1975). Actinomycin D or puromycin can block the increase in PNMT activity induced in vivo by dexamethasone. This suggests that corticosteroids in- crease enzyme activity by inducing their .synthesis (Wurtman et al., 1972).
Catecholamine Secretion. Very little is known at presen·t about the molecular events that couple stimulation to secretion in the adrenal medulla. Both nicotinic and 15 muscarinic receptors were identified in the adrenal medulla and acetylcholine has been shown to be the normal physi ological mediator of excitation of the adrenal medulla (Feldberg and Minz, 1934). The release of neurotrans mitters from the terminal varicosities of a neuron initi ated by the arrival of action potentials is called the stimulus-secretion coupling. The process is calcium de pendent (Douglas, 1968). It is not known how this divalent cation exerts its effect in the chromaffin cell (Stjarne, 1972). There are two schools of thought as to how the vesicles travel from the cytoplasm to .the plasma membrane of the chromaffin cell: (1) Random displacement by Brownian movement carries the vesicles to the membrane, pro vided the viscosity of the cytoplasm is
····not-unusually hTgh -(Mattnews~ r970T. (2) The vesicles are transported by micro tubules to the plasma membrane. Support for this theory includes the observation that colchicine, a drug which destroys the micro·tubules, inhibits the release of norepinephrine and DBH from the adrenal medulla (Douglas and Sorimachi, 1972). Small amounts of lysolecithin can bring about the fusion of cell membranes (Lucy, 1970). Since .this phospho lipid comprises 17% of the adrenal vesicles (Lagercrantz, 1971), it is highly probable that this compound is involved 16 in the attachment of the vesicle to the plasma membrane. Low concentrations of ATP and magnesium may in certain media accelerate the spontaneous release of catecholamines as well as that of ATP and protein (Poisner and Trifaro, 1967). This has led to the proposition that the ATP-Mg may be involved in a step of the stimulated secretion of the vesicles from chromaffin cells (Stjarne, 1972). Lyse- lecithin may also be involved directly in the secretory pro- cess by inflicting local damage to the vesicular membrane (Blaschke et al., 1967; Douglas, 1968). The process of exocytosis has been proposed as a mechanism for the release of catecholamines ·on the basis of electron microscopic studies (DeRobertis and Sabatini, 1960). Later, unequiv- ocal microscopic evidence' of exocytosis in the adrenal medulla of the hamster was reported (Diner, 1967). The phenomenon, with the vesicle releasing its entire soluble content to the exterior of the cell (Slotkin and Kirshner, 1973). It appears that the membrane of the secretory vesicles after exocytosis does not remain fused to the plasma membrane of the chromaffin cell, but is specifically re- trieved (Benedeczky and Smith, 1972). The process of retrieval involves the formation of a bristle-coating on the cytoplasmic face of the vesicular membrane, followed by the fission ·Of the membrane into several smaller vesicles as it separates from the plasma membrane (Smith, 1973). 17
Catecholamine Uptake and Storage. Eeuptake is an
important route of inactivation of catecholamines after
their release. Some investigators claim that.more than 90%
of the released transmitters are recaptured (Haefely et al.,
1964). The neuronal uptake of norepinephrine (Uptake ) is 1 mediated by a high affinity and a stereochemically selective
transport system located in the axonal membrane of the
adrenergic neuron (Iversen, 1971). This system is highly
dependent on the presence of sodium ions and is blocked
by cocaine and tricyclic anti-depressants. In addition,
a second uptake system (Uptake ) is present in various 2 glandular and smooth-muscle tissues (Gillespie, 1973) •
. This system is membrane-bound bu·t differs from Uptake in 1 having a lower affinity for norepinephrine and a higher
affinity for N,-substituted catecholamines such as
epinephrine (Iversen, 1974). The catecholamines are taken
up by storage vesicles after having been transported
through the neuronal membrane.
Early studies of the distribution of tracer amounts
of radioactive-labelled catecholamines have indicated that
the adrenal medulla .is highly efficient in accumulating the
labelled material (Axelrod e~ al., 1959; Whitby et al.,
1961). This led to later studies which demonstrated the
existence of a catecholamine uptake system in isolated
adrenal medullary vesicles (Kirshner, 1962; Carlsson et al.,
1962). Numerous experiments have been done since then to 18 characterize this system. It has been found to be tem perature-sensitive: the incorporation of amines at 37°C . t . .h 0 ~s w~ce t at at 30 C. It is magnesium-ATP dependent as 1 the presence of these substances enhance the uptake several fold. The system is inhibited by reserpine and JI j certain other drugs including sulfhydryl agents such as N-ethylmaleimide and chelating agents such as EDTA 1 (Kirshner, 1974) .. The uptake process is quite specific. Serotonin is preferred over catecholamines which, in turn, are preferred over related phenethylamines (Slotkin, 1973a) • The process can be saturated. Uptake of epinephrine in bovine adrenal vesicles has an estimated K of 4-8 x 10-4M m (Lundborg, 1966). Recent advances in the knowledge of the uptake sys- tern allows one to regard uptake and storage as two distinct processes. Slotkin (1971) observed that serotonin was incorporated to a greater extent than epinephrine despite the fact that it was not stored as stably as epinephrine. It was also observed that although competition occurred among related amines for the same uptake system, their com- petitive potency was unrelated to subsequent storage stability (Slotkin and Kirshner, 1971) .. A model proposed by Slotkin (1974) suggests the attachment of a catecholamine molecule to a carrier a·t the outside surface of the vesicu- lar membrane. The amine-carrier complex subsequently dis- sociates on the inside surface by transphosphoryla·tion wi·th ATP. 19
Some amines su~h as metaraminol may be taken up by a second uptake system (Lundborg, 1966). This system is independent of magnesium and ATP and is relatively insensitive to the inhibition by reserpine in vitro (Lundborg and Stitzel, 1967). Metaraminol appears to be preferred over tyramine which is preferred over epin- ephrine (Slotkin and Kirshner, 1971). In vivo experiments, however, have demonstrated that norepinephrine is concen- trated and stored by this second system after the Mg-ATP system has been blocked by reserpine (Lundborg and Stitzel, 1967). Amine efflux from bovine vesicles occurs at different rates ·with the two different uptake systems. •rypical efflux curves obtained with metaraminol are bi- phasic, with a rapid and a slow component. Seventy five percent of the labeled metaraminol is released with a ~------half-time of about 5 minutes and 25% with a half-time of about 42 minutes. Epinephrine has a different efflux pattern as .22% of the amine is released with a half-time of about 5 minutes and 78%.with a half-time·of about 160 minutes (Slatkin and Kirshner, 1971). Therefore, one can conclude that metaraminol is distributed primarily into the fast pool while epinephrine is distributed primarily into the slow pool. The two pools may reflect the existence of two populations of storage vesicles or two different pools within the same vesicle which do not exchange amines
with each other (Slotkin ~!:. al., 1971). It has been ob served that reserpine does not alter the half-times of 20 either the fast or the .slow pool, indicating that this drug exerts its effect through the blockade of the uptake pro- cess rather than by disrupting the storage complex (Slotkin et al., 1971).
_j Epinephrine is believed to be bound to a matrix I composed of ATP and chromogranins after entry into the vesicle (Kirshner, 1974). Calcium may also be part of the binding complex (Borowitz, 1970). Physical chemical studies indicate that these components interact with each other through weak intermolecular forces which are rapidly made -l and broken •. A complex is thus formed as a result of the j statistical kinetics of these interactions (Kirshner, 1974). I Enzymatic c_a·tabolism. The catabolism of catechol- amines generally involves two different enzymes: a mono-
e.mi_11e o_xig_asg (Ml\.0) _ wbi_gh de Ontogenesis of th~ Sympathetic Nervous System 'l'he primitive cellular precursors of the sympathetic nervous sys·tem are believed to arise from ectodermal ele- ments in the neural crest region of the embryo (Coupland, 1965b). These stem cells subsequently differentiate into neuroblasts and pheochromoblasts. During develop- ment, the stem cells of the neuroblasts migrate to peripheral tissues and form the paravertebral and preaortic sym pathetic ganglia, from which ·the postganglionic sympathetic neurons finally emerge. Those stem cells which are destined to become pheochromocytes either give rise to extra-adrenal chromaffin cells or they invade the adrenal cortex to form the primordial adrenal medulla (Levine and Landsberg, 1974). The sympathetic nervous system matures both mor- phologically and functionally. Fluorescent microscopic studies indicate that the mature neuron contains a larger 23 number of varicosities with higher concentrations of catecholamines than the immature ones (Schiebler and Heene, 1968). The presence of catecholamines has been demonstrated during rat development as early as the fer tilized ovum stage prior to and during cleavage (Burden and Lawrence, 1973). Generally, the content of catechol amines of most adrenergically innervated mammalian tissues is low during fetal and early postnatal life (Iversen, 1967), though there is some species variation. Several phylogenetic studies have demonstrated a correlation be tween. the development of the heart and increments in catecholamine concentration in the cardiac tissue (Mirkin, 1972). The unique exception is the calf heart, >-lhich centains the same concentration of norepinephrine as the adult bovine heart (Vogel et al., 1969). enzymes represent the development of the functional aspect of the sympathetic nervous system. Tyrosine hydroxylase was the first to be documented using in vivo studies in the chick embryo (Ignarro.and Shideman, 1968). The in crease of DBH was correlated to the development of central adrenergic neurons (Coyle and Axelrod, 1972). The sub cellular distribution of these enzymes shifts from the cell bodies of the neurons to the nerve terminals during maturation (Coyle, 1973). The activity of the catabolic enzymes also appears to increase with development. MAO and COMT initially were 24 detected in the embryonic chick on the fourth day of incu bation and reached maximal levels by the nineteenth day (Ignarro and Shideman, 1968). The activities of these two enzymes in s.wine kidney and liver increased with age. The development was more variable in other tissues (Stanton et al., 1975). The development of the uptake capability of the sympathetic neuron generally parallels the appearance of endogenous catecholamines in the innervated tissue. Ex cellent correlation between these two events has been noted in the heart and spleen (Iversen et al., 1967). The stor age of catecholamines follows a similar pattern. Catechol- amine storage is preceded by the storage of ATP during de velopment of the adrenal medulla. Biochemical analysis of the adrenal medulla in rabbits and rats during pre- --arm postnatal storages l:fas indicated- that the -molar ra-t:io of catecholamines to ATP increases many fold from the seven- teenth day after conception to adult age (O'Brien et al., . -- 1972). This suggests that the fetal adrenal medulla con- tains vesicles which mainly store_ATP and which gradually take up catecholamines during the course of maturation. Microscopic findings confirm these biochemical results (Pletscher et al., 1973). The vesicles of the rat are denser at birth than in older animals. It has been sug- gested that the rate of synthesis of the storage vesicles is low at birth but increases rapidly during maturation (Slatkin, 1973b). 25 Epinephrine and norepinephrine have both been observed to be present in the adrenal medulla of the fetal rabbit (Brundin, 1965). Epinephrine- and norepinephrine- containing cells are found in the fetal swine adrenal medulla at about nine weeks of age (Podgornaya, 1968). It is also of interest to note that transsynaptic enzyme induction appears to play an important part in the development of the sympathetic nervous system. Denerva- tion of the adrenal medulla prevents age-dependent increases in enzyme activity and catecholamine content (Patrick and Kirshner, 1972). The appearance of transsynaptic enzyme induction seems to reflect the maturation of neuronal tissues of the rat. Toxic doses of reserpine induced tyrosine hydroxylase in the adult rat but did not do so in neonates during the first three weeks of life (Black and Reis, 1975). Perinatal Survival of the Swine The large number of stressors the perinatal piglet encounters may be partially responsible for the high mor- tali ty rate-. - The swine is a polytocous species; therefore, the fetuses face nutritional competition in utero (Pomeroy, 1960). The survival of the fetus is also dependent on its position in the uterus because of the differential trans- port of nutrients and g~ses to different parts of the uteru~ (Dawes, l968). The maternal environment, often in fluenced by external factors, may play an important part 26 in determining the viability of the fetus (Stanton and Carroll, 1974). Asphyxia due to reduced respiratory-gas exchange between the fetus and the placenta during partura- tion imposes a severe survival problem (James et al., 1958). Prenatal asphyxia is not only associated with pre- natal death, but with postnatal morbidity as well (Curtis, 1974). The pig also faces competition for food with its I litter mates during the neonatal period. In addition, it I is no longer sheltered in the uterus and is exposed to j -l many hazards such as chilling, infection, crushing and i trampling by ·the sow . • Chilling is particularly insidious and is a major cause of neonatal death. It may not only cause death directly, but chilling also predisposes the neonates to crushing, starvation, and disease since the chilled pig- let is lethargic and may not nurse or seek the sow. The newborn piglet is highly susceptible to cold, with a thermo neutral temperature of 32 - 35°C. This is high compared to many other species including the lamb and the calf which have thermoneutral temperatures of about 29°C and 13°C, respectively (Curtis, 1974). The marked intolerance to cold in the neonatal piglet is partially due to the large surface area and the sparse pelage of the animal (Mount, 1968). The absence of brown fat deprives the pig- let of one o:E the important heat generating mechanisms-- nonshivering thermogenesis (Bruck, 1970). The total fa·t 27 content of the pig constitutes only about l% of the body weight at birth (Widdowson, 1950); therefore, the insula- tive capacity is minimal in a pig of this age. The immaturity of the immune system in the neonatal piglet predisposes the animal to infections. Measurable developments in the imn1une system do not occur until the piglet has reached 10 to 14 days of age. The neonate is completely dependent before this time on the colostrum supplied by its mother which contains the necessary im- munoglobulins (Wilson, 1974). -l i Neonatal piglets are deficient in their capability l for the S-oxidation of fatty acids. This may be related to the low number of mitochondria present in the liver for the first 48 hours of life (Mersmann, 1974). Hepatic gluconeogenesis also -occurs- at a-much -lower rcrte than with - older ages. The cardiovascular system of the neonatal piglet is quite immature. The heart rate of these animals is highly susceptible to changes in blood glucose concentra tion. Hypoglycemia is associated with a rapid fall of heart rate, a decrease in body temperature and frequently death. Newborn piglets have a heart rate of 200-300./minute which decreases in rate exponentially with growth (Engelhardt, 1966). Blood hemoglobin levels in pigs under 6 weeks of age is highly sensitive to inadequate dietary iron (Bustad, 1966) and parenteral iron frequently must be provided. 28 What is the role of the sympathetic nervous sys tem of a perinatal piglet exposed to stressors? Prenatal and intrapartum anoxia have been shown to directly acti vate the adrenosympathetic system of the bovine fetus (Comline and Silver, 1966b). However, the catecholamines released may also exert several actions harmful to the neo nate. Epinephrine and norepinephrine infusions given to newborn piglets have been shown to decrease blood volume and increase interstitial volume (Cheek and Rowe, 1966). Mobilization of carbohydrate stores during parturation may reduce the physiological.reserves of the newborn and seriously hamper the animal's ability to withstand further stress (Stanton and Carroll, 1974). The piglet crouches and shivers violently in cold bu·t cannot generate heat by ~onshive_ring metabolic means. Altl1o11gh t:he sy_mpa~!1etic system does not seem to be directly involved in shivering, it plays an important role in reducing heat loss by induc ing peripheral vasoconstriction (Stanton and Mueller, 1973a). Catecholamine-induced mobilization of lipids, though less important for cold adaptation of the newborn pigs, is part of a thermoregulatory response to cold in pigs older than 50 hours (Mersmann, 1974). The neonatal piglet responds to starvation by mobilizing liver glycogen stores (Stanton and Mueller, 1974). It may also be very dependent on glycolysis as in dicatced by increased plasma lactate levels during starva tion (Stanton et al., 1973). 29 'Thus, activation of the sympathetic nervous system constitutes part of the adaptive response of the neonatal piglet to many of the environmental stressors. Any dyssynchrony in the system should result in a lowered capability of the animal to withstand stress. Chapter II MATERIALS AND METHODS Animals l The crossbred swine (Hampshire x Yorkshire x Duroc) j used in these studies were born and reared in temperature- -1 controlled facilities and handled according to standard swine husbandry practices at the Shell Development Com- pany Biological Sciences Research Center in Modesto, California. The animals were transported in temperature- controlled vehicles a short distance from the barns to the laboratory and were kept near thermoneutrality (33 - 35°C for newborn and 27°C for older animals) .. Food and water were given ad libitum. The diets of the pig varied with age and are presented in Table II. The newborn and 14-day-bld pigs were stunned with a blm1 on the head by a hammer while the 70-day-old animals were stunned with a captive-bolt pistol. The animals were exsanguinated and the adrenal glands quickly removed and placed on ice. Estimation of Medullary and Cortical Volumes Adrenal glands were rapidly removed from the animals 30 ------~~~-~--~'----'-~·-'-- . ·-- ~t~---'-~------....__.___._._. __ ,_ _...,_~"~------'-----'---· ~~~--~~- -~"~ TA)3LE II Diet Compositions, Percent Baby Pig, Grower, Finisher, Sow, Sow, 21% Protein 18% Protein 15% Protein Component Gestation Lact?ttion (0-34 days) (35-60 days) (60 days-adult) Milo -- -- 14.75 .1. 66 1. 6.6 Corn grain 77.42 72.50 46.11 71.67 78.86 Soybean oil meal 7.85 9.62 20.00 15.00 9.50 Bran 2.80 3.46 Alfalfa meal 3.90 ~.79 2.03 5.00 4.00 Cottonseed meal -- L_ 2.93 Meat and bone meal 5.60 6.88 5.00 Fish soluble 0.60 0.68 2.67 5.00 4.00 1\l"ney (delactose) -- --i 6.67 Salt (iodized) 0.60 b.68 0.40 0.50 0.50 Vitamin premix 0.60 0.68i Limestone 0.60 e.68 -- 0.70 0.90 Trace mineral supplement 0.03 e.o3 Vitamin and mineral premix -- -- 0.20 I__ Vitamin and mineral premix -- -- 0.17 0.16 w Dicalcium phosphate -- ,--' -- 0.30 0.40 1-' !2_. , :!::_. methionine -- ~-- 0.04 Modified from Siers et al., 1976. 32 and immersed in formaldehyde for three days. The left gland of each pair was divided longitudinally into three or four sections, depending upon the size of the gland. . ' Four centimeter-thick sections were embedded in paraffin blocks and cut into five or six thinner slices. These slices were subsequently treated with hematoxylin-eosin stain and mounted on glass (slides prepared by Miller Clinical Laboratory, Modesto, California). The areas of the medulla and the cortex were es·timated from representa tive slices of each section with a grid. The slices were enlarged twenty times with a Nikon Profile projector (Nikon Inc., Instrument Division, Gardea City, New York), and. the grid was attached to the screen. Volumes of the medulla and the cortex were calculated by integrating the .<'treas estimated from the slices over the whole l<=ngth_ of the adrenal gland. Determination of Catecholamines The method of this determination was modified from the procedure of Anton and Sayer (1962). Adrenal glands _j were.trimmed, weighed and homogenized in ice cold 0.4 N perchloric acid (PCA) in a concentration of 100 mg per 4 ml using a Brinkman Polytron homogenizer. The homogenates were centrifuged at 40,000 g for 20 minutes in a Sorvall RC2-B refrigerated centrifuge. The supernatants were diluted to 2.5 mg/ml 1·lith 0.4 N PCA and transferred to 50- ml beakers. Norepinephrine and epinephrine standards I 33 contained! ml (5 Jlg/mll each of the respective catechol lI amine. The blank contained 1 ml of 0.4 N PCA; 4 ml of water and 15 ml of- 0.1% EDTA were subsequently added to each beaker. The solutions were adjusted to pH 6.5 with 0.5 M K2co 3 and placed on Bio-Rex 70 carboxylic acid resin columns (BioRad Lab., Richmond, California). The columns were washed with 20 ml of water and the catecholamines were eluted with 5 ml of 0.5 N HCl. Two separate aliquots of 0.6 ml from each sample were used for the analysis. One set of the aliquots was used for the measurement of norepinephrine at pH 6.5 and the other set for the measure- ment of epinephrine at pH 2.4. For norepinephrine, 0.1 ml of 1 M phosphate buffer and 0.2 ml of 0.15 N NaOH were added to each sample immediately before the assay. The samples w~re_ then O_?:i buffer and sodium hydroxide before the assay to keep the pH at 2.4_. The excitation and emission wavelengths were 414 and 522 m~, respectively. The samples and norepin- ephrine standard were read against a setting of epinephrine standard at 100 and the blank at 0. Standard curves for the catecholamines are shown in Figures 1 and 2. Determination of Catecholamine Biosynthetic Enzymes Activities The adrenal glands were trimmed and homogenized in ice-cold isotonic 0.15 M KCl using a Brinkman Polytron homogenizer. The homogenates were used for the analysis of the catecholamine biosynthetic enzymes. Before using, the homogenates were dialyzed overnight a·t 4°C against 1 L . -- - --100- volumes-of-.5 mM T-ris=Cl-bu-ffer at--pH- 7-.-0 to remove-- catecholamines and other endogenous inhibitors of small molecular weight. The dialysis did not change the activity of either DOPA decarboxylase or phenethanolamine-~- methyl transferase. Two methods of DOPA decarboxylase assay were used. The first method was not satisfactory because we were unable to saturate the enzyme. The results reported in Table VI were assayed by this method. Data from the two .DOPA decarboxylase methods correlate very well with each other. In the assay for Dopamine-13-hydroxylase, N- ethylmaleimide did not block the activity of endogenous _L__ -~~~- ·-~~~~- ~~~~~"-•- ,,ic~--! ~---•- --•- -~~~~-~~~-~- 100 ~ 100 90 90 :::.!?0 :::.!?0 ~ ~ 0.625 L25 2.5 0.625 1.25 2.5 5 Norepinephrine, 1-19 Epinephrine, 1-19 w U1 Figure l. Norepinephrine standard 'curve Figure 2. Epinephrine Standard curve 36 inhibitors in swine adrenal tissues, but dialysis removed their inhibition. Copper was added in the incubation mixture to replace that which was removed by dialysis. The protein of the adrenal tissues was determined by the Biuret method. Tyrosine Hydroxylase (TH) The assay method was modified from the procedure of Nagatsu et .al. (1964). Figure 3 shows .the determina-. tion of optimal amount of homogenate, time of incubation and substrate concentration for this enzyme. The incuba- tion mixture for the enzyme assay contained: 0.1 ml of 1M Tris-maleate buffer (pH 5.7), 0.1 ml of 5 mM Fe(NH l 4 2 (so4 >2 , 0.1 ml of 10 mM 6,7-dimethyl-5,6,7,8-tetrahydro pterine (DMPH ) dissolved in 1 M mercaptoethanol, 0.4 ml 4 of tissue homogenate, 0.1 ml of 1 roN L-tyrosine con 3 taining 10-20 !lCi/ml of 3,5- H-tyrosine and 0.1 ml of glass-distilled water. The mixture was incubated for ex actly 10 minutes at 37°C with constant shaking. The re action was stopped by adding 0.1 ml of 30% trichloroacetic acid and the precipitated protein was removed by centri- fugation of the sample at 1000 g for 15 minutes. Five- tenths ml of the supernatant followed by 1 ml of water was placed on a 5 x 30 rom Dowex 50 H+ column (100- w-x4 200 mesh). The radioactivity of 0.5 ml of the eluabe was determined in a liquid scintillation spectrometer using 15 ml of Bray's scintillation medium. '~~~~~,-~~~-~,,~,,; L_,, ,,, ,,, ,,,,~~,, , I'',,~~~,,.,,~,,,~,~,_,,,,,,, "~~~, !~=~- '''--·--'-'--'---""'-""'-~=='-'=--'-"""- I Figure 3. Determination of, optional amount of homogenate, incubation time, and sub strat~ concentration for the assay of tyros1ne hydroxylase. 38 0 C\J ft Q) 0 E 1- c 0 0 tO -c (X) ~ .c ft :::J ~ 0 U) c I 0 tO tO 0 C\i )( C\J ~ a 01 x lA.Id::> 0 0 c 'AW•!P'It GSDiAXOJpiiH SUJSOJ•~J. ' ft ~ ·-E-tO 0 tO I'll ...... d Cl> G01 X Vlld::l E , I( l!fl) P'i1 0 C\J ~ d ft .... tO 0 tO 0 a 01 X lAJd::l 'At)ll)~:>'if GSDJAlWJpAH SUJSOJiiJ. 39 DOPA Decarboxylase {DDC) : Method 1 This method was modified from the procedure of Creveling and Daly {197la). The incubation mixture con- tained: 0.3 ml of 1.08 M NaH Po , 0.23 M Na B o buffer 2 4 2 4 7 {pH 6.9), 0.025 ml of 3.2 mM pyridoxal 5 1 -phosphate, 0.025 ml of 0.03 M tranylcypromine sulfate, 0.15 ml of glass distilled water, 0.2 ml of tissue homogenate, 0.05 ml of 0.4 mM 3,4-dihydroxy ~-phenylalanine {~-DOPA) con taining 10 ~Ci/ml of Q,~- 14 c DOPA. The mixture was in cubated for exactly 20 minutes at 37°C with constant shaking. The reaction was stopped by chilling the sample to 0°c in an ice bath. Ten ml of acid-free butanol was added and the mixture shaken for 5 minutes followed by centrifug- ing at 100 g for 10 minutes. One ml of the upper organic 1_9-yE)r_waQ ac1c'!ed to_lO_ ro:L Qf_B:t:" DOPA Decarboxylase {DDC): Method 2 This assay was modified from the procedure of McCaman et al. {1972). Figure 4 shows the optimal deter- mination of amount of homogenate, time of incubation and substra·te concentration for this enzyme. The incuba- tion mixture contained: 0.05 ml of premixed buffer con- taining 0. 4 M NaH 2P04(pH 7. 0), 4 mM tranylcypromine sul phate and 0.4 mM pyridoxal phosphate; 0.05 ml glass-dis tilled water, 0.5 ml of homogenate and 0.05 ml of 0.4 m!1 3,4-dihydroxy ~-phenylalanine {~-DOPA) containing 10 ~Ci/ml. -"--'--'----~··~--"---'-'--_.__;...'-"-'----'"-="-- '--'-'·--~-~··----~·-"'"""'=--'~--'·-- . ======"-'·-~--~~"-'---'--'-"''-'·'-- =~"--'-"'''-. Figure 4. Determination of optional amount of homogenate, inct'tbation time and sub strate concentration for the assay of DOPA decarboxylase. I 41 vI() 0v I() Ul rt) Q) 0 ::J rt) -c I() :::E (\J ft Q) 0 (\J E 1- !Q :::E c: (\J .2 ft 0 ~ -0 IQ .0 I ;:, I I() (J 0 c )( I ...... I . ft Q) 0 -, -c ! Q) 01 0 E 0 J: L_ _J__ ro •.D v L'\1 0 £01 X li\ld::> ',{l!A!P\1 action, 0. 2 ml of ice cold 0. 05 M NaH 2PO 4(pH 7. 0} fol lowed by 0.2 ml of glass-distilled water was added. Dopamine, the reaction product, was extracted by q.dding 0.2 ml of 0.1 M bisdiethylhexylphosphoric acid (DEHPA) in CHC1 3. The mixture was vortexed for 15 seconds and cen trifuged at 1000 g for 10 minutes. The top aqueous layer was aspirated and discarded. An additional 0.2 ml of 0.05 M NaH 2Po4 was .added and the separation procedure repeated. One tenth ml of the lower organic phase was subsequently dissolved in 1 ml of 0. 3 M hyamine in methanol. The solution was added to 15 ml of Bray's scintillation medium for the determination of radioactivity. This method was modified from the procedure of Creveling and Daly (197lb). Figure 5 shows the optimal determination of amount of homogenate, time of incubation and substrate concentration for this enzyme. The incuba- tion mixture contained: 0.2 ml of 1 M KH 2Po4 buffer (pH 5.5), 0.05 ml of 0.2 M fumarate, 0.05 ml of 0.2 M ascorbate, 0.05 ml of 0.1 M tranylcypromin~ sulfate, 0.05 ml of 0.4 mM cupric sulfate, 0.05 ml of catalase (75 mg/ml) 0.15 ml of glass-distilled water, 0.2 ml of tissue homogenate, 0.2 ml of 1 mM tyramine containing 10 llCi/ml of 3H-tyramine. The mixture was incubated for ~~~~~ '~C~C·"--''-'-'"-'-'"--'--'----'---'-'-'---' -·~----"'------'-'-0,- --""' -"'- Figure 5. Determination ofloptional amount of homogenate, incubation time and sub strate concentration for the assay of :dopamine-13-hydroxylase. '· 44 ,...,0 It) C\1 ...."'Q) ::l 0 c: C\1 ::E ~ It) Q) E i= 0 c: ....0 Cl ..0 It) ::l 0c: 0 C\1 ::E ~ 0 q q ,....,~ r<) C\1 I £01 X Wd:J 0 )( ' A1!1\1 p \1 HSO ...... 0 0 .:: 0 1 u 1 Q) 0 1- (() -,--.:::---- "'"0 10 ..c"' J E ::l ~ C\1 en ~ E ...... q 0\ E 0 £01 )( VIJ d:) 0 C\1 ...... 'At!fi!P'V HBO 0 or 0 ·-c: Q) 0\ 0 0 E :r:0 '<1: 0 1.0 0 q 0 J'() r<"i C\1 C\i £01 )( lfiJd:J 'At !II! P\1 HEJa 45 exactly'l5 minutes at 37°C with constant shaking. There action was stopped with the addition of 1 ml of 7% per- chloric acid. The precipitated protein was removed after the sample was centrifuged at 1000 g for 10 minutes. One ml of the supernatant was mixed with 0.3 ml of 12.45 N ammonium hydroxide and 0.3 ml of 2% sodium periodate. The mixture was incubated for exactly 4 minutes at room tern- perature. Three tenths ml of 10% NaHso and 1 ml of 5 N 3 HCl were added and 1 ml of the mixture was removed and I placed in screw-capped culture tubes with teflon caps -I (16 x 100 mm) along with 5 ml of toluene. The mixture was l centrifuged at 1000 g for 10 minutes. Four ml of the top j organic layer was delivered to the counting vial along with 10 ml of 0.56% omnifluor-toluene solution for the §et_ennina_tion ()_J: rAdiQac_tivj,ty_._ Phenethanolamine-N-Methyltransferase (PNMT) This method was modified from the procedure of Creveling and Daly (1971) •. The dialyzed homogenate was centrifuged at 40,000 g for exactly l'hour in a Sorvall i RC2-B refrigerated centrifuge before incubation. Figure --j 6 shows ·the optimal determination of amount of homogenate, time of incubation and substrate concentration for this enzyme. The incubation mixture was placed in screw-capped culture tubes {16 x 100 mm) with Teflon-lined caps and contained: 0.1 ml of 0.5 M Tris buffer (pH 8.5), 0.1 ml of 0.15 M KCl, 0.2 ml of the supernatant, 0.05 ml of 0.4 mM ' L_ ___ , !, ~~~~~~~~~""""·""~"~ ·· ..~.. ~ ··~ Figure 6. Determination of ,optional amount of homogenate, incubation time and sub strate concentration for the assay of phenethanolamine-N-methyltransferase. !- I ~~--~·~--~~~. --"- ~'-- ""~--"---U.....~-~--.c.___.___.~~-"~-~---""'--'---'---··'-·~~~-"'-'--~-· IS.Or i r<>· 15.0r ~ ;:.. .::: 0 .=:- .., -o -- (.) ::£ X 10.0 - 0.025 0.05 0.1 0.2 0.3 0.4 0.5 0.6 10 20 30 40 50 60 I Homogenate, (100 mg/ml), ml Incubation Time, Minutes 12.5 Phenethanolomine )a ~ 10.0 -c::: ;:.. .:::- r<> -0 (.) - 1 4 ' Substrate Cone. (x 10- ), M I 48 I I S-adenosylmethionine containing 1 ).JCi/ml of 14c s-adenosyl methionine, 0.05 ml of 30 rnl'1 phenethanolamine. The mix ture was incubated for exactly 30 minutes at 37°C with constant shaking. The reaction was stopped by adding 0.5 ml of 0.5 M sodium borate buffer (pH 10) followed by 6 ml of toluene-isoamyl alcohol (30:1). ·The tubes were then capped and shaken vigorously. The organic layers were separated by centrifuging at 1000 g for 10 minutes. Four ml of the top organic layer was added to 10 ml of 0.56% omnifluortoluene solution for the determination of radioactivity. Biuret Method The method was modified from the procedure of Gornall e~ al. (1949). The biuret reagent contained: 1.5 L g -of ·SH 6~-o g of NaK <:: B406-;-4H -csoaiumpotassium cuso4 2o-, 4 2o 1 ' tartrate), 300 ml. of 10% NaOH and glass-distilled water was added to make 1 liter. The incubation mixture for the samples contained: 0.2 ml of homogenate, 1.1 ml of glass-distilled water and 0.2 ml of 10% deoxycholate (DOCA). The standard mixture contained: 1.0 ml of bovine serum albumin (1 mg/ml), 0.3 ml of glass-distilled water and 0.2 ml of 10% DOCA. The blank mixture contained: 1.3 ml of glass-distilled water and 0.2 ml of 10% DOCA. The sample, standard and blank mixtures were incubated for ex actly 15 minutes at 37°C. One and five tenths ml of the biuret reagent was added to each mixture, which was again 49 incubated for 15 minutes at 37°c. Optical density for each mixture was read at 540 m~ with a Gilford 350 N micro-sample spectrophotometer. Estimation of Maximal Ve1oci t (Vmax and MiChaelis Constant (KM) The Vmax and Km for the enzyme kinetic curves were both estimated by the non-linear least-squares method (Cleland, 1967; Wilkinson, 1961). The calculations were performed on.a Hewlett-Packard model 9830A programmable calculator (Hewlett Packard, Palo Alto, California). Determination of Tyrosine Concentrations in Adrenal Gland and Plasma Tyrosine Extraction from Adrenal Glands Adrenal glands were homogenized in 0.4 N perchloric acid solution in a concentration of 100 mg/ml. The homogen- ate was centrifuged at 40,000 g for 20 minutes in a Sorvall RC2-B refrigerated centrifuge. Two ml of the supernatant was combined with 8 ml of 0.1% EDTA and the mixture was titrated to pH 6.5 with 2 N K2co3 . The solution was placed on a Bio-Rex 70 carboxylic acid resin column (BioRad Lab., Richmond, California) followed by 20 ml of glass-distilled water.· The eluates were combined and titrated to pH 2.0 with 6 N HCl. This solution was placed on a BioRad AGSW-XB resin column (100-200 mesh) followed by 20 ml of glass-distilled water and the eluates were discarded. Ten ml of 1 N NH oH. 4 .., ' 50 was added to the column and the eluate collected was dried overnight under a steady stream of air. The residue ob- tained was redissolved in 0.5 ml of 0.01 N HCl solution. Tyrosine Extraction from Plasma The method was modified from the procedure of Wong et al. (1964). The blood sample was centrifuged at 1000 g for 20 minutes. One volume of the plasma was added to an equal volume of 6 N trichloroacetic acid. The mixture was centrifuged at 1000 g for 5 minutes and the super natant was saved for fluorometric determination. Fluorometric Determination of 'l'yrosine Concentration The method was modified from the procedure of Wong - et aL (~964-) ·-- The tyrosine extracts were-assayed in mix=- tures each containing 0.1 ml of 1-nitroso-2-naphthol in 95% ethanol (3 mg/ml), 0.5 ml of an acid nitrite solution containing 3 mg of sodium nitrite per ml of 1.5 N nitric acid, 0.05 ml of the extract. The standard mixture con- tained 0.05 ml of !!-tyrosine solution (0.025 mg/ml) in- -e stead of the extract. The mixture was incubated for ex- actly 30 minutes at 37°c with constant shaking. The reac- tion was stopped by adding 4 ml of glass-distilled water and 5 ml. of ethylene dichloride. The resultant mixture was vigorously shaken for 5 minutes followed by 5 minutes of centrifugation of 1000 g. Four ml of the top aqueous 51 layer were transferred to a cuvette for fluorometric determination. Samples were read against a standard set- ting of 100 and blank setting of 0 in an Aminco-Bowman spectrofluorometer. The excitation and emission wave- lengths were 460 and 570 mJ.!, respectively. In Vitro Biosynthesis of Catecholamines from Tyrosine by Adrenal Slices Adrenal glands were trimmed and sliced with a Mcilwain mechanical tissue chopper (Brinkman Instruments, - I New York) to a thickness of 0.5 mm. Slices from both ends of the .glands were discarded. A combination of slices j from various sections of the gland weighing 200 mg was in- ' cuba ted .in 2 ml of Krebs bicarbonate buffer. 'rhe buffer (pH 7. 4) 1 contained: 0.154 .. N_ac~, _(1.15~ M_ K<:l, ~0~5 _M thereof. The buffer was saturated with 95% o 2 and 5% co2 just prior to the experiment. The tissue slices were in _j cubated for exactly .2 hours at 37°C under a constant pres- sure of 95% o and 5% co . Duplicate controls were kept 2 2 on .ice. Incubation of the slices was stopped by drying them on tissue papers and immersing them in 2 ml of 0.4 N perchloric acid. They were homogenized by a Brinkman Polytron and centrifuged at 40,000 g for 20 minutes. The 52 supernatant was placed ,on aio-Rex 70 carboxylic acid resin soluinn and washed with 20 ml of water. Catechol- amines were separated by the method of Minard and Grant (1972). They were eluted from the column in five continu- ous 5 ml fractions of 2% boric acid followed by five continuous 5 ml fractions of 0.2 N HCl. The elution pattern of norepinephrine and its ~-methylated metabolite normetanephrine is shown in Figure 7. Five tenths ml of elute from each fraction was added to 15 ml of Bray's medium for the determination of radioactivity. -l Amine UJ2take by Adrenal Vesicles The method was modified from the procedure of Slatkin (1973b). Adrenal glands were trirrmed, weighed and placed in ice cold 0. 3 M1)UC£O~-Hepe~. m_c=dim!l (pH_'7. 4_) • The glands were minced with scissors and homogenized in a 1 I Potter Elvehjem homogenizer at a concentration of 1 g/20 ml. Three ml of the crude homogenate were removed for tissue and vesicular catecholamine determination. The remainder of the crude homogenate was centrifuged at 800 g in a Sorvall RC2-B refrigerated centrifuge for 10 minutes. Figure 8 shows the optimal parameters for the experimental procedure. The incubation mixture contained: 0.6 ml of sucrose-Hepes buffer containing 0.3 M sucrose, 0.05 M Hepes, 0.05 mM iproniazid (pH 7.4); 0.1 ml of 0.05 M ATP-Mg, 0.2 ml of supernatant and 8 )lg of epinephrine or metaraminol contaJ.nJ.ng· · 1 )lCJ.' o f 14 C-epJ.nep' h rJ.ne ' or 3H-me t aramJ.no . 1 or _j ~"-·'----'-'=-'~'-'=-=--'-'~"-'-" "~~~~ '-I"'.> '--'·-' -- "'-'--'--"'"0-C~'·'"-'L""'''-"- Figure 7. Elution pattern !of norepinephrine and normethanephrine. I Norepinephrine: :o--o ! Normetanephrine =i /';- -/'; E = sample eluat:e W = 20 ml water !eluate B = 2% boric acid elu.ite in 5 continuous fractions of', 5 ml each subscript de- notes fracti'on number. I H = 0. 2 N HCl eluate in 5 continuous frac- tion of 5 ml1 each. 54 ' 10 £01 x wdo .•O,-=....l'"-'-'-'--'--'------'.'-!-''--~'""------'-' ·-"--'.='C"'----"----'-"'"---0'-'---'C--.O'-'------'-'------'-=--'=-==.....:.:..~'=""'<"' ----·c-' I Figure 8. Determination of optimal parameters of the amine uptake by isolated adrenal vesicles Met= Metaraminoli Epi =Epinephrine, The parameters are obtained by one determination. 1 ,I ~-----.:.~. ... '---~--·-~- ·~~~~-----~-,-~-~~~---~-~-'"-~~~-- 2.5 0 1.5 ~- 0> :::1. 0 37"c 0 0 1.0 ...... 0> 37°C ::1. .!: G.> 0.5 ""'0 0.- -:::> Met Met Epi Epi ·With Without With Without, Minutes of Concentration ATP ATP · ATP ATP ' Incubation of Tissue U1 "' 57 appropriate dilutions thereof. The suspension of vesicles was incubated for exactly 10 minutes at 37°C with constant shaking. Duplicate controls were kept on ice. Incuba- tion was stopped by diluting the suspension with 9 ml of ice cold 0.3 M sucrose. The sample was centrifuged at 26,000 g for 20 minutes in the Sorvall RC2-B. The super- natant was decanted and the pellet resuspended in 3 ml of ice-cold 0.3 M sucrose. The washing procedure was re- peated once. The pellet was resuspended in 2 ml of ice cold glass-distilled water and vortexed after the second wash. The lysed vesicles were centrifuged at 26,000 g for 20 minutes. Five tenths ml of the supernatant was added to 15 ml of Bray's medium for the determination of radio- activity. i 1 j- - .Tissue__ catecholamines _wer_e_extracted by adding -1 I ml of the crude homogenate to 1 ml of 0.8 N perchloric acid and centrifuged at 40,000 g for 20 minutes. The supernatant was used for the fluorometric determination of catecholamine content. Extravesicular catecholamines were extracted by adding the supernatant from the 26,000 g centrifuged crude homogenate to an equal volume of 0.8 N perchloric acid. The precipitated protein was removed by centrifuging at 40,000 g for 20 minutes and the super- natant was used for the fluorometric determination. _The units of uptake were expressed as pg/1000 ~g of total catecholamines. 58 · Amine Efflux from Adrenal Vesicles The method was modified from the procedure of Slatkin et al. (1971). Two steps were involved in this experiment. The first step involved the uptake of radioactively labeled amines by the vesicles. The methods for this has already been described in the previous section. Sixteen ~g of epinephrine and metaraminol containing 1 ~Ci of the radio- active label were used. The experiment on efflux started immediately after the second wash of the vesicles with 0. 3 M sucrose in the uptake procedure. The pellet was resus- pended in 5 ml of buffer containing: 0.3 M sucrose, 0.05 M Hepes, 0. 05 mM iproniazid and 50 ml-1 ATP-Mg (pH 7. 4) • Each suspension was divided into 1 ml aliquots for the epinephrine efflux and 2 ml aliquots for the metaraminol efflux. The samples were incubated at 37°C with constant shaking. One sample of each amine was kept on ice for 60 minutes as controls. At the end of 5, 10, 20, 30, 40 and 60 minutes, duplicate samples were removed from the water bath and efflux was stopped by the addition of 2 I _J volumes of ice cold 0.3 M sucrose. The samples were cen- t:r:ifuged at 26,000 g for 10 minutes and the pellets lysed in 1 ml of glass-distilled water. They were centrifuged at 26,000 g for 20 minutes and 0.5 ml of the supernatant from each sample was added to 15 ml of Bray's medium for the determination of radioactivity. Chapter III RESULTS Growth of the Adrenal Gland: General Aspects Relationship Between Adrenal and Body Weight The relationship between the growth of the adrenal glands and the overall growth of piglets was investigated by plotting the log of adrenal weights as a function of the log of body v1eights as shown in Figure 9. Data from a total of 2 95 piglets were used ranging· from 0. 5 kg to l1- __ 11_._0 kg J.n hody_ weights.- 'I'he -CU-J;"Ve-indica-toes-a lcinear -re~ j lationship between the two parameters with a coefficient 2 of determination (r ) equal to 0.8372. The equation of the line was found to be: log y = 2.30 + 0.628 (log x). 10 10 Growth of the Medulla in . Relationslup to the Cortex. Table III presents a comparison of medullary volume as a percentage of the cortical volume in relationship to piglet age. The data indicate no marked change in per centage volume of the medulla between piglets of dif ferent ages. The percentage medullary volume of 0-day-old piglets reported in the taple is an average of two observa tions, ranging from 26 to 37 percent. Hence the 10% 59 60 1000 900 800 700 600 500 Ol. -..c::: 400 01 Q) == c c: 300 ..Q) -l '0 ct 1 200 1 I 1 1 I rl j IQOL---·------L------L---~--L--L~~~~ I 2 3 4 5 6 7 8 9 10 Body. Weight, kg _J Figure 9. Relationship between body and adrenal weight. 61 TABLE III Percent of Medullary Volume in. Terms of Cortical Volume as Related to Species and Age Species N Age Percentage Swine 2 0 days 32 2 3 days 33 2 7 days 37 2 14 days 20 1 70 days 24 Sheep 1 Adult 24a Cat Adult 5b Dog Adult 20b Rat Adult 5b . - - b- Rabbit Adult 2.5 Guinea pig Adult 1.5b aThis statistic was obtained in this laboratory; pro cedures were similar to that used for the swine. bModified from West, 1955. 62 difference between the newborn and 14-day-old piglet may not be significant as the percentage volumes are quite variable between piglets of the same age. The same table also shows interspecies comparisons of percent medullary volumes. The data on the dog, cat, rat, rabbit and guinea pig are acquired from West (1955). The swine, sheep and dog have similar size adrenal medullae and have the highest percentages. The cat and rat rank second, with percent medullary volumes about 1/4 of those in swine. The rabbit follows and the guinea pig has the smallest percent medullary volume. Relationship Between Catecholamine Content and Piglet Age The catecholamine content and percentage of epin- ephrine _in_ adrena~ _gl_gnclf3 of neOrl_Cita_1_ and jJlVeni].§ swine_ are presented in Table IV. More than one litter of pigs were used among all the ages studied. This took into account the variability existing between litters. The patterns of ontogenesis of the catecholamines are illus- _trated in the following figures where the ordinate of each is expressed as a percentage of the mean values of the 150- day-old pigs. Figure 10 shows the pattern of norepinephrine changes with age. The norepinephrine content was about 1.7 times higher at birth than at 150 days of age. There was an increase in content from birth to a peak at 14 days of age. A gradual decline followed and the content ~ ~ ·---~-~~~---"-· ··--~-· ····~---· TABLE iV Mean Body Weights and Adrenal Gland Catecholamine (CA) Conten~a Correlated with Piglet Age Piglet Litters, ·Piglets, Bpdy wt., Total CA, Eprinephrine Age N N kg :±_ 1 S.E.M. mg/g :.!: 1 S.E.M. % + 1 S.E.M. 0 day 4 9 1.12 + 0.12 2.29 + 0.14 28 + 1 - 3 days 4 9 1.6'3 + 0.13 2.57 + 0.09 30 + 2 7 days 4 9 2.114 + 0.14 2.73 + 0.15 36 + 2 - 14 days 4 9 3.418 + 0.18 3.69 + 0.17 36 + 1 39 days 6 11 8.419 + 0.82 4.16 + 0.29 48 + 1 70 days 3 5 38.6,0 + 0.50 2.92 + 0.40 43 + 2 ! - - - 150 days 3 5 78.60 + 4.00 2.15 + 0.28 56 + 3 I I aCatecholamine content is expressed as per unit of whole gland (cortex + medulla) ~ "'w I 64 250 -c 230 Q) c 210 -0 u 190 >- 0 Cl I 170 0 I() 150 -0 130 ;,.!! 0 110 Q) ------.------c .... 90 .t:: c. _7Q __ -----j----- w e c. 50 Q)... z0 30 10 0 ~o----~~o----2~o----~3~0----4~0----5~o----s~o----7~o Age, Days Figure 10. The change of norepinephrine content as a function of age. 65 at 70 days of age was approximately equal to that at birth. Figure 11 shows the changes in pattern·of epinephrine content with age. The epinephrine content at birth was I 1 approximately 50% of that at 150 days of age. An increase in content from birth to a peak level at 39 days of age was followed by a gradual decline. The epinephrine content at 70 days of age was two times higher than that at birth. Figure 12 shows the pattern of total catecholamine changes with age. This figure represents the composite changes of norepinephrine and epinephrine. The total catecholamine content at birth was approximately equal to that at 150 days of age. There was a marked increase in content between birth and 14 days of age. This was fol- 39 days of age doubling the content at birth. The content at 70 days of age decreased to a slightly higher level than that at birth. Figure 13 shows the pattern of percentage changes in epinephrine with age. The percentage epinephrine con- tent at 150 days of age was twice that at birth. An in- crease in content from birth to 7 days of age was followed by a more gradual rise to reach a plateau at 39 days of age. The content at 70 days of age was slightly lower than that at 39 days of age. 66 250 230 c: -Q) 210 c: -0 u 190 > 0 170 0 I -1 0 150 I 10 130 -0 0~ 110 I . I Q) 90 c: ] .... ___J __ - -· .c.. ]Q . a. Q) c: 50 a. w 30 10 0 0 10 20 30 40 50. 60 70 Age, Days Figure 11. The change of epinephrine content as a function of age. I 67 l 1 .... 250 c ....Q) c 230 0 u 210 > a 0 190 I 0 l() 170 l ..... -j 0 150 I I 0~ 130 ~ 1 (/) Q) 110 j ·-c.: I E 90 I I a 0 I .,C 70 0 1- Q) a 50 I -u 1 I 30 -] ....a 0 1- 10 0~----~~--L----~----~--~L---~----~ 0 10 20 30 40 50 60 70 Age, Days Figure 12. The change of catecholamine content as l a function of aqe. 68 100 ------.------ 90 c: -Q) c: 80 -0 l u i;' 70 0 I 0 U) 60 50 A Q) -I ...c: 40 .c; 0. Q) c: 30 0. lLJ +- c: 20 -cu ....0 Q) a. 10 0~----~----~--~----~----~--~----~ 0 10 20 30 40 50 60 70 Age, Days _j Figure 13. The change of epinephrine content as a function of age. 69 Ontogenic Patterns of Catecholamine Biosynthetic Enzymes Tyrosine Hydroxylase (TH) Activity and Piglet Age Table V presents the mean specific activity of TH correlated with piglet age. The ontogenic pattern of the activity of this enzyme is illustrated in Figure 14. The activity of TH was lower at birth than that at 150 days of age. There was an increase in activity from birth to reach a peak at 3 days of age. This was followed by a -1 rapid decline between 3 and 14 days of age and a more I gradual decline between 14 and 70 days of age. The activity at 70 days of age was approximately 50% of that at 150 days of age. DOPA Decarboxylase (DDC) Activ1ty and P1glet Age 1 Table VI presents the mean specific activity of DDC j correlated with piglet age. The ontogenic pattern of the j activity of this enzyme is illustrated in Figure 15. The activity of DDC was slightly lower at birth than that at 150 days of age. There was a gradual increase in ac tivity from birth to 70 days of age. The activity at 70 days of age was about 1.5 times higher than that at 150 l days of age. 70 TABLE V Mean Specific Activity of Tyrosine Hydroxylase in Adrenal Homogenates Correlated with Piglet Age. Mean Specific Activity of DOPA Decarboxylase in Adrenal Homogenates Correlated with Piglet Age Piglet Litters, Piglets, Nanomoles product/ Age N N g/hr ::':. 1 S.E.M. 0 day 4 8 6860 -+ 422 3 days 4 8 8358 -+ 624 7 days 4 8 7403 -+ 457 14 days 4 8 8062 -+ 477 39 days 6 6 8983 +- 934 70 days 3 8 11643 +- 393 150 days 3 5 8351 +- 936 71 I 150 I 140 1 ;::.. 1 -> 130 -j -0 120 ~ Q) 70 C/) 0 ;::.. 60 X 0... "'0 50 ;::.. ::c 40 Q) c 30 C/) __ !o.,;;0 ____ ~- -- >- 2U 1- 10 0 0 10 20 30 40 50 60 70 Age, Days i -~ Figure 14. The change of specific activity of tyrosine hydroxylase as a function of age. I 72 I i l 150 >- 140 -> 130 0 - Figure 15. The change of specific activity of DOPA ~decarboxylase as a function of age. 73 Dopamine~S-Hydroxylase (DBH) Activity and Piglet Age Table VII presents the mean specific activity of DBH correlated with piglet age. The ontogenic pattern of the activity of this enzyme is illustrated in Figure 16. The activity of DBH at birth was about 60% of that at 150 days of age. There was a marked increase in activity be tween birth and 3 days of age. The increase reached a plateau at about 14 days of age and remained the same to 150 days of age. Phenethanolamine-N-Methyltransferase (PNMT) Activity and Piglet Age Table VIII presents the mean specific activity of PNMT correlated with piglet age. The ontogenic pattern j +-- of the activity of this enzyme is illustrated in Figure 17. The activity of PNMT at birth was about 60% of that j at 150 days of age. There was a marked increase in activity between birth and 3 days of age. This was followed by a more gradual rise reaching a peak at 39 days of age. The activity of the enzyme had decreased to the 3-day-old level by 70 days of age. ~parent Kinetic Patterns of the Catecholamine Biosynthetic Enzymes and Piglet Age Michaelis-Menten kinetic parameters of the catechol- amines biosynthetic enzymes were measured on adrenal homo- genates in order to further characterize their ontogenic changes in activity. Table IX summarizes the results . TABLE_VIII _ -· -- Mean Specific Activity of Phenelthanolamine-N- Methyltransferase in Adrenal Homogenate - Correlated \vith Piglet Age Piglet Litters, Piglets, Nanomoles product/ Age N N g/hr ±. 1 S.E.M. 0 day 4 8 316 16 -+ 3 days 4 8 461 + 41 7 days 4 9 472 -+ 24 14 days 4 10 496 + 24 -~ - 39 days 6 6 625 -+ 42 70 days 3 5 440 +- 33 150 days 3 4 528 -+ 38 75 150 140 130 120 >- 110 ·- -> 100 ------0 Figure 16. The change of specific activity of DOJ>A-S hydroxylase as a function of age. 76 1 150 140 130 120 110 ....>o ....> 100 0 <( 90 >o 0 80 0 I 0 70 lt'l· 60 0 - 50 0~ ~: 40 ::1! z 30 a. ·- - 20 10 0 L----~----~L-----L-----~-----L----~----~-- 0 10 20 30 40 50 60 70 Age, Days Figure 17. The change of specific activity of phenethanolamine-N-methyltransferase as a function of age. L -~~~~ r-~~---~" '-'--'-- TABLE IX Apparent Michaelis-.l-1enten K,inetic Parameters of Catecholamine Biosynthetic Enzymes in Adrenal Homogenate Correlated with Piglet Agea Age, I b Enzyme vmax ±_1 S.E.M. Km + 1 S.E.M. Days 1 -4 Tyrosine :hydroxylase 0 84.3 + 6.7 2.23 + 0.35 X 10 M - - -4 14 65.2 + 1.4 1.37 + 0.08 X 10 M - -4 70 40.9 + 0.8 1.31 + 0.06 X 10 M -4 DOPA decarboxylase 0 244.0 + 18.8 1.94 + 0.50 X 10 M - -4 14 248.7 + 12.7 3.50 + 0.41 X 10 M 4 70 389.1 + 33.8 5.31 0.94 X 10- M - ~ 3 Dopamine-S-hydroxylase 0 274.4 + 16.2 1.68 + 0.24 X 10- M - -3 14 314.4 + 2.2 1.38 + 0.03 X 10 M 3 70 349.7 + 4.4 1.28 ~ 0.05 X 10- M -4 Phenylethanolamine-~- 0 1.2.6 + 0.5 1.68 + 0.20 X 10 M - - -4 methyl transferase 14 17.2 + 0.8 1.29 + 0.16 x 10 M 4 70 14.0 + 0.7 1.60 + 0.23 X 10- M - " aBoth Km and Vmax are estimated by the method of non-linear least squares. Each mean represents a pooled average of 16 piglet~ from 4 litters for the 0-day-olds, 8 piglets from 4 litters for the 14-day-olds, and ~ piglets from 4 litters for the 70-day-olds. bunits of Vmax are in nanomoles product/mg protein/hr. I 78 obtained from these studies. The Vmax of TH decreased with age; the Vmax of TH in the 70-day-old pig was about 50% that of the 0-day-old piglet. The apparent Km of TH decreased about 35% between birth and 14 days of age but stayed at the same level be- tween 14 and 70 days of age. There was no change in the Vmax of DDC between 0 and 14 days of age, but the Vmax was increased at 70 days of age. The apparent Km of DDC increased with age; the apparent Km of the 70-day-old pigs was about 3 times higher than that of the 0-day-old piglets. The Vmax of DBH increased gradually between birth ! and 70 days of age. The apparent Km of DBH stayed constant throughout the three ages. There was a slight increase in ·the Vmax of PNMT T ! between birth and 14 days of age. The Vmax decreased to approximately the level of that at birth at 70 days of age. The apparent Km of PNMT stayed at approximately the same level among the three ages. Synthesis of Catecholamines by Adrenal Slices in vitro Effect of a-1-!ethyl-p-Tyrosine (O:MpT) on the in vitro Biosynthesis of Catecholamines Figure 18 illustrates the in vitro effect of aMpT on the activity of 'l'H in adrenal homogenate from a 14-day-old 79 100 90 80 ~ 0 70 R c: 0 60 ..c- 50 .c c: 40 30 -2o - -. 10 o(- Methyi-,E-Tyrosine Cone., M Figure 18. In vitro inhibition of 'tyrosine hydroxylase l)y a-methyl-p-tyrosine. (The inhibition is expressed as-a percent of the activity of the enzyme without a-methyl-p-tyrosine.) . it 80 piglet. · The drug inhibited 20% of the enzyme activity at 7 5 x 10- M and 100% at 1 x 10-3M. To determine the effect of aMpT on catecholamine biosynthesis, adrenal slices from a 14-day-old piglet were incubated with the drug. Figure 19 illustrates the results of this study. The catecholamines isolated in the 21 boric acid fraction were expressed as a percentage of the control value. The inhibitor, UMpT, caused an 80% inhibition of the bio 6 synthesis of catecholamines at 1 x 10- M. The inhibition was complete at 1 x 10-3M. Adrenal and Plasma Tyrosine Concentrations and Piglet Age Adrenal and plasma tyrosine concentrations were measured with age to determine the amount of tyrosine to be used in the in vitro catecholamine biosynthesis study. The results are presented in Table X. There were sub- stantial differences of tyrosine concentrations between ages in both the adrenals and the plasma. Adrenal tyrosine concentration decreased with age. The tyrosine level at birth was 3.5 times higher than that at 70 days of age. _j Plasma tyrosine concentration followed a similar pattern, I " but with more marked differences. The tyrosine level of the newborns was 6 times higher than that of 70-day-old pigs. Ontogenesis of in vitro Catechol_amine B1osynthesis Tyrosine concentrations determined from plasma were ';,-. 81 100 90 80 0:.$! 70 ft 1 c:: 60 ....0 .c 50 .&:. I c 40 l 30 20 10 oC.-Methyl-p-Tyrosine Conc.,M Figure 19. In vitro inhibition of catecholamine bio synthesis by a-methyl-p-tyrosine. (The inhibition is expresse~ as a percent of the amount of catecholamine synthesized without a-methyl-p-tyrosine.) ~~~"~ ~--- L__ ~~~~---~-'"'-~~~~ ~~~~~ TABLE X Mean Tyrosine Conbentration in Plasma and Adrenal GlandsiCorrelated with Age Age Litters, Piglets, Adrenal Tyrosine, Piglets, Plasma Tyrosine, Days N N mg/100 mll ±_ 1 S.E.M. N mg/100 ml + 1 S.E.M. - I 0 2 6 6 7.12 + 0.79 5.35 ±.1 0.66 - 14 2 6 3.73 +! 0.23 6 2.89 + 0.15 70 2 4 1.54 + 0.06 3 1.20 + 0.04 Q) 1\) 83 used to incubate with the adrenal slices. The results are summarized in Table XI. There was no significant dif ference between the amount of catecholamines·synthesized by adrenal slices from 0- or 14-day-old piglets. However, the amount of catecholamines synthesized by the slices from these two ages were significantly higher than that synthesized by the slices from 70-day-old pigs. Adrenal slices of the 0- and 14-day-old piglets synthesized 5 times more catecholamines than the 70-day-old pigs at a tyrosine concentration of 7.12 mg/100 ml. Increasing the concentra- tion of tyrosine in the incubation medium tended to in crease the amount of catecholamines synthesized by the adrenal slices of the 14- and 70-day-old pigs. Ontogenesis of in vitro Amine Uptake -1- and Eff-lux by Isolated-Adrenal VesicTes j Relationship of Amine Uptake with Piglet Age j Table XII summarizes the results of the uptake of epinephrine with and without reserpine by adrenal vesicles of 0-, 14- and 70-day-old pigs. The results of the uptake of metaraminol with and without reserpine by adrenal vesicles of similar aged pigs are presented in Table XIII. A composite graph of the uptake of epinephrine and met araminol with and without reserpine by the vesicles of the 0-day-old piglets is shown in Figure 20. The uptake of both amines increased in a linear fashion with increasing L .. ~ "~· ~· "-~·"·-~ ~~~·~~··-- TABLE XI ! Mean Catecholamine synthesis Correlated with Tyrosine Concentration1 and Piiglet Age a Age Litters, Tyrosine Cone., I Piglets, Nanomoles of Catecholamines/ Days N mg/100 ml N g tissue/hr ~ 1 S.E.M. 0 2 7.12 4 1437.25 + 156.38 14 2 2.80 4 1358.00 + 111.69 2 7.12 4 1421.50 + 64.06 70 2 1.20 4 135.20 + 11.67 2 2.89 2 160.00 + 1.00 2 7.12 2 265.00 + 39.00 i aThe concentrations are expressed in mg/1000 ml of adrenal extracts. I """"' ~~~.·~~~~--'-·---·~~~~~-·-· ~-~-~~--~~---~.-~~~-~ ~~·--~~- TABLE XII I Effect of Piglet Age on Vesicular Uptake of Epinephrine (10 min .. incubatibn) in the Presence or Absence. of Reserpinea Reserpine Presence/ Litters, Epinephrine Absence N 0 Day 14 Days 70 Days 1 g 0 2 529j. 3 + 29.8 788.1 + 87.7 872.6 + 65.5 + 2 24'.4 + 4.6 11.9 + 2.6 11.7 + 2.5 2 g 0 2 1014.2 + 18.2 1474.1 + 151.9 1754.4 + 125.6 I - + 2 251.7 + 6.6 32.0 + 6.0 26.1 + 2.4 I 4 g 0 2 23041.3 + 69.0 2469.6 + 242.3 3332.0 + 230.6 + 2 7~.9 + 6.1 47.6 + 15.2 55.1 + 5.6 8 g 0 39o4i.8 + 247.1 3790.3 + 350.1 5841.5 + 1018.4 2 - + 2 157!.6 :!:: 49.3 126.0 + 42.4 111.5+ 24.1 co U1 aData for .o day piglets represent 3 de-qerminations each of which is a pool of tissues from 4 piglets. Units of uptake are expressed in nanograms/1000 ~g of endogenous catecholamines. · ~L __. __ ,, ~~~~~ 1] TA~LE XIII ,.-.,. Effect of Piglet Age on Vesicular Uptake of Metaraminol (10 min. incubation) in the Presence or Absence 1of Reserpinea Reserpine Presence/ Litters, Metaraminol Absence N O!Day 14 Days 70 Days 1 1lg 0 2 161.2 + 19.7 301.5 + 41.5 256.5 +. 4.3 + 2 llll7 + 7.6 102.1 + 24.6 127.8 + 19.5 2 ].lg . 0 2 340.8 + 16.9 660.7 + 73.3 427.2 + 8.7 ' - + 2 254!. 7 + 13.4 205.0 + 43.3 292.4 + 14.9 4 ].lg 0 2 6501.2 + 4.3. 3 1160.2 + 59.1 1033.2 + 83.0 I + 2 503.8 + 12.0 414.5 + 96.9 541.3 + 13.3 8 ].lg 0 2 1331!.6 + 143.5 2264.3 + 202.1 1882.3 + 169.7 + 2 11091.0 + 76.8 877.0 + 225.1 1132.1 + 115.9 aData for. 0 day piglets represent 3 det;erminations each of which is a· pool of co tissues from 4 piglets. Units of uptake are expressed in nanograms/1000 1lg "' of endogenous catecholamines. I 87 4.0 0 3.0 «J Q) ~ c 2.0 -Q, ::l 1.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Exogenous Epi or Met (!Jg/ml) Figure 20. Amine uptake by adrenal vesicles of 0-day-old swine with and without reserpine. aUnits of uptake are expressed as nanograms/1000\lg of endogenous catecholamines. 88 I concentrations of the amines. All of the curves pass through 0. The uptake of metaraminol at 8 11g/ml was 34.% of that of epinephrine. Reserpine inhibited 95% of the up- take of epinephrine but only 30% of that of metaraminol. Since the uptake of the amines as a function of concen- tration has been shown to be linear, the slopes should reflect the rate of uptake of amines by the vesicles. An ontogenic comparison of the rate of uptake of epineph- rine and metaraminol are presented in Table XIV. There was no significant difference in the uptake of epineph rine between 0- and 14-day-old piglets. However, the up- take of epinephrine by the 70-day-old pigs was significantly higher than the upt,ake by the 0- and 14-day-old piglets. 1'here was no difference between the 14- and 70-day-old pigs in_the uptake of _metar_aminol, _Wh_er~as t_he__ O=-day-_ old pigs exhibited a lower rate of metaraminol uptake. Reserpine exerted a more profound effect on the uptake of metaraminol by 14- and 70-day-old than by the 0-day-old pigs. Relationship of Amine Efflux with Age The storage of amines in. vesicles involves the up- take minus the efflux of the amines. Therefore, the efflux component was studied in order to characterize the storage process. The results were expressed as the log of the percentage of aminesremaining in the vesicles as a function ~~~~~~~~~-L._. 1·'·--~-·--- ~~~-· I TABLE XIV Slopes of Uptake Curves Correlatedi w1th~ Age Slopea + 1 S.E.M. Amine 0 Day I 14 Days 70 Days Epinephrine 0.485 + 0.027 0.417 + 0.043 0.702 + 0.089 - I Epinephrine + Reserpine 0.020 + 0.004 0.016 + 0.004 0.014 + 0.002 - ! Metaraminol 0.166 :J:: O.Ql3 0.276 + 0.019 0.237 + 0.017 Metaraminol + Reserpine 0.142 + 0.007 0.111 + 0.021 0.142 + 0.010 - ! - aThe slopes are calculated by the least squares method; the unit of each slope is expressed as 1,000 )Jg total t:atecholamines-1 co "' 90 I of time. They.were analyzed by linear regression and presented in Table XV. There was no difference in the 1 j efflux of epinephrine by the vesicles between the ages. j The efflux of metaraminol by the vesicles were also similar between the ages. Hence, the uptake of einephrine and metaraminol is a good indication of the storage capacity of the amines in the pig. It J ~ l ~~~"·~·~-·~-'--~t""--"7-'-· TABLE XV I Efflux (60 minutes) of Epinephrine and Metaraminol Correlated withiPiglet Age -,-- Ainine Age, Cone. at 2 Days Amine t;i.me oa Slope:!:. S.E.M.b r 0 Epinephrine 2942.6 -0.0107 + 0.00216 0.86 Metaraminol 948.1 -0.0156 + 0.00390 0.76 14 Epinephrine 5680.6' -0.0125 + 0.00207 0.88 I Metaraminol 1182.0 -0.0161 + 0.00431 0.74 I 70 Epinephrine 68p5.6 -0.0124 + 0.00282 0.80 Metaraminol 22s1.o -0.0128 + 0.00348 0.73 I aEach figure represents a single determination on a pooled sample from 4 piglets (0 days, 14 days) or 2 piglets (70 days). Concentrations are expressed as nanograms I 1000 g ofI endogenous catecholam1nes.• I bThe units of the slope are log % remaining/unit of time. I ID 1-' Chapter IV DISCUSSION I Three ages of pigs were chosen for comparison in this study. They were 0, 14 and 70 days of age, repre- senting respectively the birth, preweaning and postweaning stages of development of the pig. Approximately 25% of the postnatal. deaths occur within one day of birth and over 75% occur within one to two weeks after birth. There is little change in viability between weaning at 21-56 days and market at 154 days of age (Stanton and Carroll, 1974). These three ages, therefore, represent important phases in the survival of the piglet. r Most of the parameters for comparison in the studies were based on content per gram (wet weight) of the whole gland. Perhaps it might have been better to make com- parisons on a total-content-per-gland basis because the water content of the glands may vary with age. However, this could not be done because whole gland weights were not always recorded. Since the percentage medullary volume in terms of the cortex was about the same in all the ages studied and the gland weight increased linearly with body weight, the comparisons based on content per gram (we·t weight) should reflect changes with age, although these 92 93 comparisons may not be as apparent as whole gland levels. The percentage volume of the adrenal medulla in terms of the cortex varies widely from species to species (Table III). Therefore, interspecies comparison of hor mone and enzyme levels per unit of whole glands may be misleading unless corrections for percent medullary volumes are made. For _example, interspecies comparison of catecholamine levels based on content per gram of whole glands has indicated that the swine and the sheep have higher levels than that of the cat and the dog. The latter two species, in turn, have higher levels than that of the rat, rabbit and guinea pig. However, after adjusting 1 the levels of percent medullary volume, catecholamine contents are similar in all the species. -The-patterns-of-ontogenic changes-in-catecholamine t content of swine adrenal glands indicate that they reach a maximum during late neonatal or juvenile periods and gradually decline to lm..rer stable levels in the adult ~I 1 (Figures 10-12). Similar patterns have also been found in the rabbit, guinea pig, calf and fowl (Shepherd and West, 1951; Comline and Silver, 1966b; Comline and Silver, 1971). A different pattern has been reported in cats and dogs where catecholamine levels of these two species continuedto rise from birth to adulthood (Shepherd and West, 1951). The catecholmaine levels in rat adrenal glands increase steadily from 4 to 15 weeks of age (Patrick and Kirshner, 1972). All of the species discussed resemble 94 each other to the extent thattheepinephrine:norepinephrine ratio increases during development. Tyrosine hydroxylase has been established as the rate-limiting step of norepinephrine biosynthesis in the guinea pig heart (Levitt et al., 1965). There is evidence in this thesis that TH may be the rate-limiting enzyme in the swine adrenal gland as well, although the definitive experiments comparing apparent Km and Vmax in purified prep- arations have not yet been done. The apparent Km of the enzymes presented in Table IX shows that TH needs a les- ser amount of substrate than DBH .and DDC to reach half- maximal velocity. In addition, TH has the lowest Vmax compared to DDC and DBH. aMpT, which abolished the activity of Til at 1 x 10-3M concentration (Figure 18), also inhibited 98% of the synthesis of catecholamines (Figure 19). However, it is interesting to note that l aMpT inhibited 82% of the biosynthesis of catecholamines while only blocking 25% of the specific activity of TH 1 at the .same concentration. Therefore, the inhibitory 1 action of aMpT on catecholamine synthesis may not be con- i -~ fined to Til alone. PNMT has approximately the same ap- parent Km as TH but has a markedly lower Vmax. Hence, PNMT appears to be the rate-limiting enzyme of epinephrine biosynthesis in the swine adrenal medulla. This postula tion is also supported by the parallel pattern of ontogenesis of PNMT and epinephrine (Figures 11 and 17). Since aMpT inhibited catecholamine biosynthesisby 82% and only 25% j ! 95 of TH activity as disc~ssed above, it is possible that MpT has an inhibitory effect on PNMT, as epinephrine is I one of the major catecholamines in the swine adrenal gland. The specific activity of TH in the pig declined from birth through 70 days of age and returned at 150 days of age to the level observed at term (Figure 14). The ontogenic changes of the activity of this enzyme in the rat follows a different pattern. TH activity increases steadily with age between 4 and 15 weeks, which parallels the increase of catecholamines (Patrick and Kirshner, 1972). No differences have been observed in TH activity between fetal and adult sheep (Friedman, 1972). However, only two ages of sheep were studied by this investigator. It is difficult, therefore, to establish a developmental pat·tern for this species. The apparent Km of TH in the newborn piglet was found to be higher than that of the 14- and 70-day-old pigs (Table IX). Possible explanations include the presence of higher amounts of inhibitors in the. newborn adrenals, the presence of activators in the adrenals of the older pigs, and the different rates of development of possible isozymes. Since the apparent Km was estimated from crude preparations, these results repre- sent trends only and may not mimic the pattern seen with more purified preparations. The specific activity of DDC in the pig increased gradually with age from birth through 70 days of age and returned to neonatal levels at 150 days of age (Figure 15). 96 The apparent Km of this, enzyme also increased \~ith age (Table IX). DDC has a higher apparent KM and Vmax com- pared to TH and PNMT. Hence this enzyme apparently has no obvious regulatory role on catecholamine biosynthesis in the swine adrenal gland. The specific activity of DBH increased markedly from birth to a maximum during late neonatal age and re- mained stabel through 150 days of age in the pig (Figure 16). The ontogenic pattern of this enzyme in the rat, however, is quite different. There is a gradual increase in activity between 4 and 15 weeks of age which parallels the increase in catecholamines (Patrick and Kirshner, 1972). A similar pattern has been observed between 1- and 50-day- old rats by Slotkin' (1973). Although the data in Table VII represents the total activity of the enzyme, they may also reflect the population of catecholamine storage vesicles per gram of adrenal tissue at each age. This is because a ]_arge proportion of DBH is known to be bound to the vesicular membrane and the enzyme has been used as a ' : ... marker for stroage vesicles in the rat (Viveros et al. , 1969). The specific activity of PNMT in the pig increased markedly from birth in the early neonatal period. This ' j was followed by a gradual increase to reach a peak in the late neonatal period. A decline ensued through 70 days of age but returned to neonatal levels at 150 days of age (Fig-ure 17). Parallel ontogenic patte:t;ns between.PNMT and 97 epinephrine levels are·apparent by comparing Figures 11 and 17. The appearance of PNMT activity in the fetal rat has been correlated with the beginning of .adrenocortical function (Fuller and Hunt, 1967). However, there is ap- parently no relationship between the development of adrenal PNMT activity observed in our piglet studies and the ontogenic changes in plasma 17-hydroxycorticosteroid producing capacity of the swine adrenal cortex by Dvorak (1972). This investigator reported a rapid decrease in adrenocortical function after birth reaching a low level l at 25 days and maintained through 75 days of age. Un- I fortunately, no information is available on the ontogenesis · of the venous output of corticosteroids from the adrenal cortex of the swine to enable a more definititve com- ] __ -pa-rison. -- The -ra-t has- been-o:Sser-ved -to--fol-low- a--cUfferent-- ontogenic pattern of PNMT activity as the enzyme decreases in activity with age (Philpott et al., 1969). The determination of tyrosine levels made possible the use of physiological levels of this amino acid in the j in vitro biosynthesis of.catecholamines. Levels of tyrosine ! i between 0, 14 and 70 days of age show marked differences in both plasma and adrenal glands of the swine (Table X). The content of tyrosine decreased with age in both. All of -' the levels of plasma tyrosine are nevertheless within the same range as that observed in the rat (Dairman, 1971). The difference in diet between the .newborn and the 70-day-old pig undoubtedly accounts for part of the large difference in 98 plasma tyrosine concentration between the two ages. The. baby pig received twice as much tyrosine content in its diet than the 70-day-old pig (approximated from Siers et al., 1976). The difference may also reflect the development of enzyme systems such as .tyrosine transaminase, which me tab- olize tyrosine in the liver. Although there is no significant difference between 0 and 14 days of age, adrenal slices from these ages were five times higher in their ability to synthesize catechol- amines than those from 70 days of age (Table XI). More- over, there appears to be a relationship between an age- dependent decrease in the synthesis of catecholamines and plasma tyrosine concentration. This may reflect a lower transport of tyrosine into the chromaffin cell, or a lower -catecholamine hiosynthe.tic_ability. in the_older pigs_. _The_ amount of catecholamines synthesized in the 0- and 14,-,day- old piglets approach a plateau with higher tyrosine concen trations. This indicates that TH operates near maximal velocity in the neonatal swine adrenal gland. A similar observation has been made on the rat where the tyrosine concentration is saturating (Levenberg and Victor, 1974). The chromaffin storage vesicles play an important role in recapturing the catecholamines intraneuronally in the adrenal gland. Two apparently distinct systems are present within these vesicles. The one v1hich has a high affinity for epinephrine in the adrenal medulla is identified as the slow pool whereas the one which is 99 relatively nonspecific and takes up metaraminol is known as the fast pool (Slatkin and Kirshner, 1971). The swine adrenal vesicles exhibit a higher rate to take up 1 epinephrine at 70 days of age than at preweaning ages I (Table XIV). Increased metaraminol uptake appeared at I 14 days of age, indicating an earlier maturation of the nonspecific uptake system or the fast pool. There was no difference in the rate of efflux of amines between the ages, suggesting that the stability of binding of the amines to the storage complex remained the same (Table XV). Thus, the uptake of the amine also reflects the storage capacities of the amines in the swine. The rat adrenal vesicles exhibit an increase inthe uptake of epinephrine between 20 and 30 days' of age; a decrease between 30 and 1- 40 days of age and an increase to the 30-day level at 50 days of age. The metaraminol uptake remains stable between 1 1 10 and 30 days of age but declines gradually through 50 days of age. (Slatkin, 1973b). The efflux of epinephrine -~ remains stable from 20 to 50 days of age. Therefore, the uptake of epinephrine reflects storage between 20 and 50 days of age in the rat. There are no data available on the efflux of metaraminol from rat adrenal vesicles. No change has been observed in the absolute extent of inhibi- tion by reserpine in both uptake systems in the swine. This indicates the irreversible characteristic of the inhibition since an increased capacity for uptake in the older pigs failed to change the amount of inhibition by reserpine. 100 The ontogenesis· of the catecholamine catabolic enzymes in swine adrenal glands have been studied in our laboratory previously. The activity of COMT increases markedly between birth and 14 days of age in the swine. This is followed by a gradual decline to the level at term and remains relatively stable between 39 and 150 days of age (Stanton et al., 1975). MAO activity in the swine increases gradually from birth to 70 days of age and re- mains stable through 150 days of age (Stanton et al., 1975). Recently, the development of MAO in the rat has been reported to follow a different pattern (Blatchford et al., 1976). The activity of MAO in this species is low and stable between 5 and 10 days of age, but rises markedly ' between 10 and 80 days of age to 5 times the activity at· 1-- the--s~day-oi:d-level.-- ] The high specific activity of TH in the adrenal homogenates and::the large amount of catecholamines syn- thesized by the adrenal slices suggest a high biosynthetic capability of the adrenal medulla in the newborn pig. This high capability is apparently maintained throughout the neonatal period as similar data are obtained from the 14-day-old pig. However, the comparatively small rate of uptake by the adrenal vesicles of the neonate indicates a low catecholamine storage capacity in the piglet. Other supportive evidence includes the low catecholamine content and DBH activity in the adrenal glands of the neonatal piglet. Therefore, the high biosynthetic capability may be viewed 101 as a compensatory mechanism for the relatively low re- serves of catecholamines. The amount of catecholamines synthesized by the pig is apparently also influenced by diet. Insufficient dietary tyrosine could lower the amount produced (Table XI). On the other hand, excess tyrosine in the diet of the neonate would have a less significant effect as the amount of catecholamines synthe- sized by the adrenal slices approaches a plateau with in- creasing tyrosine concentrations. The proportion of epinephrine present in the adrenal gland is comparatively low at birth and in the early neonatal period as it is for most other species. This poses a problem for ·the newborn piglet which will be discussed later. There are few ontogenic data available at present in regard to the catecholamine secretory function of the adrenal medulla in swine. Nevertheless, newborn pigs have been shown to respond to insulin-induced hypoglycemia by releasing catecholamines via reflex neuronal stimulation 1! (Stanton et al., 1974). This action is in contrast to newborn rats which do not respond to insulin (Slatkin, 1973b). The overall function of the adrenal medulla in the neonatal pig is thus relatively mature compared to other species. However, the coordinating ability of the adreno sympathetic nervous system is not only dependent on the 102 maturity of the system, but also on the development of the effector organs and their responsiveness to activation as well. It is logical, therefore, to relate the ontogeny of the adrenosympathetic system to the development of the effector organs. Unfortunately, developmental informa tion is lacking at present for many of these organs in the swine. Nevertheless, adequate knowledge on the ontogeny of one type of effector organ--the metabolic I depots--provides useful information regarding the coordin I ating ability of the adrenosympathetic nervous system I I during stress. Adipose tissue, a major depot for metabolic resources in times of stress, is underdeveloped in the neonatal piglet as it accounts for only 1% of the total lipolytic rate is low at birth and increases after 2 days of age (Mersmann et al., 1975). The adipose tissue in J ·the neonatal piglet is not able to contribute as an energy -1 source during stress; this is supported by the observation that plasma nonesterified fatty acids are not significantly elevated by catecholamines in piglets of this age .(Stanton and Mueller, 1973a). Consequently, the newborn piglet is highly dependent on the glycogen reserves it has in the 1 liver and skeletal muscle. However, the neonate is less . responsive than older pigs to the hyperglycemic effects of catecholamines, especially norepinephrine (Stanton and • Mueller, l973b). It has been observed that norepinephrine \ ,_ '' 103 does not increase the metabolic rate of piglets less than 1 week of age, while epinephrine is active at birth (Stanton and Mueller, 1973a). The low percentage of epinephrine in the adrenal medulla of the neonatal pig- let, therefore, may reduce the efficiency of its response to stress. Defective g.luconeogenesis and poor oxidative metabolic capacity in the liver adds to the survival problem faced by the neonatal piglet. Starvation accounts for 18% of the deaths of neo- natal swine (Kernkamp, 1965). Mild fasting depletes liver glycogen in neonates of all ages, and the blood glucose· level is significantly reduced in piglets less than 1 day old. It has been observed that liver glycogenolysis alone may not be adequate to balance glucose utilization during fasting (Stanton and Mueller, 1974). Another important contribution to neona.tal mortality is chilling, which accounts for 5% of the deaths (Kernkamp, 1965). Cold ex- posure produces mobilization of glucose with a concommitant -1 'I decrease in skeletal muscle glycogen but not in liver glycogen (Curtis and Rogler, 1970). It is doubtful that skeletal muscle glycogen contributes significantly to blood glucose since this tissue has insignificant glucose- 6-phosphatase activity (Scrutton and Utter, 1968). There fore, the piglet is primarily dependent on shivering and peripheral vasoconstriction to maintain body temperature .(Bruck, 197.0; Stanton and Mueller, 1973a). These mechanisms may not be enough to keep the piglet warm since 104 insulation of the animal is minimal due to the lack of adipose tissue and scanty hair. Other principal causes of death include disease (18%) due partly to the lack of antibodies in the neonatal piglet and its dependence on the colostrum of the sow. An additional problem in this respect is the low quality of maternal care the sow gives to her litter. Crushing of piglets by the sow contributes to 31% of the neonatal deaths (Kernkamp, 1965). Thus the survival of the neonatal piglet is threatened by a number of stressors--many of which it finds difficult to withstand partly because of inherent deficien- cies in metabolic reserves. The stressors may also enhance the threat of each other. For example, chilling may weaken the p_igl~t or make it_lethargi~ so a£_ t()_ impair its ~bility to compete for food, thereby causing the animal to starve. Starvation may predispose the animal to infection because of inadequate acquisition of antibodies from the colostrum 1 of the sow during the first two days of life. The weakness created by these stressors.may also predispose the piglet j to trampling and crushing because a chilled, lethargic pig j would be unable to avoid the sow as effectively as an alert ' animal. The adrenosympathetic system in the pig is function- al at birth and would be expected to operate as a major coordina- tor in the maintenance of homeostasis--a process allowing the animal to resist some of the stressors it encounters. Hov;ever, ·:') 105 this coordinating ability may be severely hampered by the general underdevelopment and low responsiveness of the metabolic reserves to adrenergic stimulation. These observations suggest that the "dyssynchrony" in the de velopment between the adrenosympathetic nervous system and certain of its effector organs, namely the metabolic depots, may be responsible for the low capability of neonatal pig lets to withstand stress. There is very little information in regard to the development of other effector organs of the sympathetic nervous system .such as the heart and the lungs. Moreover, the possibility of the sympathetic nervous system exerting excessive demands on the effector organs and depleting their vital reserves has not been examined to any extent. Therefore, investigation into these __twg__areas; rnay yielC!so_rne valuable information on the overall coordinating ability of the adrenosympathetic nervous sys tem in relationship with age. This may eventually lead to the establishment of a tenable theory in explaining the low capability of neonatal swine to withstand.stress. Chapter V SUMMARY An ontogenic characterization of the swine adrenal medulla.has been conducted in this study. The purpose was to test the hypothesis that a "dyssynchrony" in the develop ment of the adrenosympathetic nervous system with respect to its effector organs could be partly responsible for the low capability of neonatal piglets to withstand stress. A number of developmental studies on one type of effector organ--the metabolic depots--have been reported on the swine. This study is, ·therefore, primarily concerned ·wit!Ytlie -functional ontugeny-o"f- the·adrenosympathetic nervous system. The catecholamine content and specific activities of catecholamine biosynthetic enzymes in amine uptake and efflux by the·addrenal medulla, as well as in vitro catecholamine biosynthesis, has been examined in pigs representing different phases of development. A cross species comparison of the functional ontogeny of the adrenal medulla has also been made in the Discussion. Results obtained indicate that the adrenosympathetic nervous system in the pig is well developed at birth. However, 1 106 ,, ' 107 reports in the literature on the sympathetic metabolic effector organs such as the fat and glycogen depots in dicate that they are relatively underdeveloped in the neonatal pig. Therefore, the coordinating action of the adrenosympathetic nervous system is severely hampered by dyssynchrony iri development of this system and its effec tor organs. The failure to maintain homeostasis by the neonatal pig as a result of this dyssynchrony may severely impair its ability to withstand environmental stress. _] __ _ BIBLIOGRAPHY 1. Abel, J. I. and Crawford, A. c. (1897) : On the blood pressure-raising constituent of the suprarenal capsule. Bull. Johns Hopkins Hosp., ~: 151-157. 2. Al-Lami, F. (1970) Follicular arrangements in. hamster adreno-medullary cells: light and electron microscopic studies. Anat. Rec., 168: 161-178. 3. Alousi, A. and Weiner, N. (1966) The regulation of norepinephrine synthesis in sympathetic nerves: effect of nerve stimulation, cocaine, and catecholamine releasing agents. Proc. Nat. Acad. Sci. (Washington), 56: 1491-1496. 4. Anton, A. H. and Sayre, D. F. (1962) A study of the factors affecting the aluminum oxide. A tri hydroxyindole procedure for the analysis of catechol amines. J. Pharmacal. Exp. Ther., 138: 360-375. - 5-. -Aunis ,_D. ,_Allard_, _D._,_ Miras~PortugaL,__ M. __ a:n_d Mande1,__ P. (1975) : Bovine adrenal medullary chromogranin A: studies on the structure and further evidence for identity with dopamine-S-hydroxylase subunit. Biochem. Biophy. Acta, 393: 284-295. 6. Axelrod, J. (1957) 0-methylation of epinephrine and other catechols in-vitro and in vivo. Science, 126: 400-401. 7. Axelrod, J. (1959) The metabolism of catecholamines in vivo and in vi·tro. Pharmacal. Rev., 11: 402-408. 8. Axelrod, J. (1962) Purification and properties of phenylethanolamine-N-methyltransferase. J. Biol. Chern. 237: 1657-166lf. 9. Axelrod, J. (1966) : Methylation reactions in the formation and metabolism of catecholamines and other biogenic amines. Pharmacal. Rev., 18: 95-113. 108 109 10. Axelrod, J. (1973) Dopamine-S-hydroxylase: regula tion of its synthesis and release from nerve terminals. ibid., 24: 233-243. 11. Axelrod, J. (1975) Relationship between catechol amines and other hormones. Recent Prog. Hormone Res., 31: 1-27. 12. Axelrod, J. a:nd Tomchick, R. (1958) : Enzyma-tic a methylation of catecholamines. J. Biol. Chern., 233: 697-701. 13. Axelrod, J., Weil-Malherbe, H. and Tomchick, R. (1959) The physiological disposition of 3H-epinephrine and its metabolite metamephine. J. Pharmacal. Exp. Ther., 127: 251-256. 14. Banerji, T. K. and Quay, w. B. (1976) : Adrenal dopamine-13-hydroxylase activity: 24-hour rhythmicity and evidence for pineal control. Experientia, 32: 253-255. 15. Bauman, R. H., Kadlec, J. E. and Powlen, P. A. (1966) : Some factors affecting loss in baby pigs. Res. Bull. No. 810, Purdue University, Lafayette. 16. Benedeczky, I. and Smith, A. D. (1972) Ultra- ~tructural __ st\}_cHes on __the _ad_ren~l medulla of golden hamster: origin and fate of secretory granule8:' z. Zellforsch Hikrosk. Anat., 124: 367-386. 17. Bertin, R. and Chevillard, L. (1967) Urinary elimina- tion of catecholamines in male Long Evans rats during adaptation to various temperatures. Ccimp. Rend. Soc. Biol., 161: 248-251. 18. Black, I. B. and Reis, D. J. (1975) Ontogeny of the induction of tyrosine hydroxylase by reserpine in the superior cervical ganglion, nucleus locus coeruleus and adrenal gland. Brain Res.,!:!_!: 269-278. 19. Blaschke, H. (1939) The specific action of L-DOPA decarboxylase. g. Physiol., ~: 50p-5lp. 20. Blaschke, H., Firemark, H., Smith, A. D., and Winkler, H. (1967) : Lipids of the adrenal medulla: lyso lecethin, a characteristic constituent of chromaffin granules. Biochem. J., 104: 545-549. 21. Blaschke, H., Hagen, P. and Welch, A. D. (1955) Observations on the intracellular particles of the adrenal medulla. J. Physiol., 1~: 27-49. ·I'' 110 22. Blaschke, H., Richter, D. and Schlossmann, H. (1937) : The oxidation of adrenaline and other amines. Biochem. ~·· 31: 2187-2196. 23. Blaschke, H. and Welch, A. D. (1953) : Localization of adrenaline in cytoplasmic particles of bovine adrenal medulla. Nauyn-Schmiedeberg's Arch. Exp. Path. Pharmak., 219: 17-22. 24. Blatchford, D., Holzhauer, N., Grahame-Smith, D. G. and Youdim, M. B. H. (1976) Ontogenesis of enzyme systems deaminating different monoamines. Brit. J. Pharmacal., 57: 279-293. 25. Borowitz, J. L. (1970) Catecholamine binding com plex in bovine adrenal medulla--possible involvement of calcium. Biochem. Pharmacal., 19: 2475-2481. 26. Bridgers, w. F. and Kaufman, S. (1962) The enzymatic conversion of epinine to epinephrine. J, Biol. Chern., 237: 526-588. 27. Brock, o. J. and Fonnum, F. (1972) : The regional and subcellular distribution of catechol-0-methyltransferase in the rat grain. J. Neurochem., 19:-2049-2055. 28. Bruck, K. (1970) : Nonshivering thermogenesis and brown adipose tissue in relation to age, and their lj integration in the thermoegulatory system. In: t- :B~i'own AdiposeTl-ssue. -- (0~ :Lindoerg, ed:T: Jl.rne-ric-an i Elsev~er, New York. 29. Brundin, T. (1965) Catecholamines in adrenals from fetal rabbits. Acta Physiol. Scand., 63: 509-510. 30. Biilbring, E. · (1949) : Methylation of noradrenaline by minced suprarenal tissue. Brit. J. Pharmacal., 4: 234-244. . 31. Burden, H. w. and. Lawrence, I. E. (1973) : Presence .J of biogenic amines in early rat development. Am. J. Anat., 136: 251-256. 32. Cannon, W. B. (1939) The wisdom of the body. 2nd ed. Norton, New York. 33. Cheek, D. B. and Rowe, R. D. (1966) Aspects of sympathetic activity in the newborn, including the respiratory d i s tr e s s syndrome . ::.P.:::ec:d:.:•:__C:::l=i=nc:•__::N;.;:o:..:r=-t:::h:.o Amer., 13: 863-877. 111 34. Christenson, J. G., Dair~~n, W. and Udenfriend, S. (1970) : Preparation and properties of a homogeneous aromatic 1-amino acid decarboxylase. Arch. Biochem. Biophys., 141: 356-367. 35. Ciaranello, R. D. and Black, I. B. (1971) : Kinetics of glucocorticoid mediated induction of phenethanol amine-N-methyltransferase in the hypophysectomized rat. Biochem. Pharmacal., 20: 3529-3530. · 36. Cleland, W. W. (1968) : Statistical analysis of enzyme kinetic data. Adv. Enzymology, ~: 1-32. 37. Caroline, R. S. and Silver, M. (1966a) : Development of activity in the adrenal medulla of the foetus and newborn animal. Brit. Hed. Bull., 22: 16-20. 38. Caroline, R. s. and Silver, M. (1966b) : The develop ment of the adrenal medulla of the foetal and new~ born calf. J. Physiol., 183: 305-340. 39. Caroline, R. s. and Silver, M. (1971) : Catechol amine secretion by the adrenal medulla of the foetal and newborn foal. ibid., 216: 659-682. 40. Coupland, R. E. (1953) : On the morphology and adrenaline-noradrenaline content of chromaffin tissue. J. Endocrinol., i= 194-203. j-- 41. Coupland, R. E. (1965a) : Electron microscopic obser vation on the structure of the rat adrenal medulla. II. Normal innervation. J. Anat. (Lond.), 99: 255-272. 42. Coupland, R. E. (1965b) : The natural history of the chromaffin cell. Longmans, Green & Co., Ltd., London. ! 43. Coupland, R. E. and Holmes, R. L. (1958) : The dis i tribution of cholinesterase in the adrenal glands of the rat, cat and rabbit. J. Physiol. (Lond.), 141: 97-106. 44. Coupland, R. E. and Hopwood, D. (1966) : The mechanism of the differential staining reaction of adrenaline and noradrenaline-storage granules in tissues fixed in glutaraldehyde. J. Anat. (Lond.), 100: 227-243. 45. Coupland, R. E. and Weakley, B. s. (1968) : Developing chromaffin tissue in the rabbit: an electron micro scopic study. ibid., 102: 425~455. 112 46. Coupland, R. E. and Weakley, B. S. (1970) : Electron microscopic observations on the adrenal medulla and extra-adrenal chromaffin tissue of the post natal rabbit. ibid., 106: 213-231. 47. Coyle, J. T. and Axelrod, J. (1972) : Tyrosine hydroxy lase in rat brain: developmental characteristics. J. Neurochem., 19: 1117-1123. 48. Coyle, J. T. (1973) : The development of cat'echol aminergic neurons of the central nervous system. Neurosci. Res.,~: 35-52. 49. Creveling, C. R. and Daly, J. W. (1971) : Assay of enzymes of catecholamine biosynthesis and metabolism. In:Methods of biochemical analysis. (Glick, ed.) Interscience Publishers, New York, pp. 153-182. 50. Crout, J. R. (1961) : Effect of inhibiting both catechol-0-methyltransferase and monoamine oxidase on cardiovascular responses to norepinephrine. Proc. Soc. Exp. Biol. (New York), 108: 482-484. 51. Curtis, s. E. (1974) : Responses of the piglet to perinatal stressors. J. Animal Sci., ~: 1031-1036. 52. Curtis, S. E. and Rogler, J. c. (1970) : Thermo regulatory ontogeny·in piglets: sympathetic and g,di]2ok~i_net_ic~:rei>pon_se<; _to_cold. _Am. J.. PhysioL, _ 1~ 218: 149-152. 53. Dairman, w. (1972) : Catecholamine concentration and 1 the activity of tyrosine hydroxylase after an increase l in the concentration of tyrosine in rat tissues. Brit. J. Pharmacal., 44: 307-310 •. 54. Daly, J, w., Levitt, M., Guroff, G. and Udenfriend, s. (1968) Isotope studies on the mechanism of action of adrenal tyrosine hydroxylase. Arch. Biochem. Biophys., 126: 593-598. 55. Dawes, G. s. (1968) : Foetal and neonatal physiology. Yearbook Medical Publishers Inc., Chicago. 56. Denenberg, v. H. (1964) : Critical periods, stimulus input and emotional reactivity: a theory of infantile stimulation. Psycho!. Rev., 71: 335-351. 57. De Robertis, E. D. and Sabatini, D. D. (1960) : Sub microscopic analysis of the secretory process in the adrenal medulla. Fed. Proc., 19: 70-78. 113 58. Diner, 0. (1967) :·The expulsion of adrenal medullary granules in the hamster. c. R. Acad. Sci. (Paris), . 265: 616-619. 59. Douglas, W. w. (1968) : Stimulus-secretion coupling: the concept and clues from chromaffin and other cells. Brit. J. Pharmacal., l!= 451-474. 60. Douglas, W. W. and Sorimachi, M. (1972) : Colchicine inhibits adrenal medullary secretion evoked by acetyl choline without affecting that evoked by potassium. ibid., ~: 129-132. . 61. Dvorak, M. · (1972) : Adrenocortical function in foetal, neonatal and young pigs. J. Endocrinol., 54: 4 7 3-481. 62. Engelhardt, w. v. (1966) : Swine cardiovascular physiology--a review. In: Swine in biomedical research. (L. K. Bustad and R. 0. McClellan, ed.) Frayn Printing Co., Seattle. p. 307. 63. English, P. R. (1968) : A study of the apparent rela tive contribution of factors to pig mortality. Pr.ell.m~nary report of PIDA, School of Agriculture, Oniversity of Aberdeen, Aberdeen, Scotland. 64. Fahmy, M. H. and Bernard, c. (1971) : Causes of --mort-al±ty--in-¥or-kshire-pigs. -from- bi-rth- to- 20~weeks of age. Can. J. Anim. Sci., 51: 351-359. 65. Feldberg, w. and Minz, B. (1934) : The occurrence of an acetylcholine-like substance in adrenal venous blood by stimulation of the splanchnic nerves. ~fliigers Arch. Gesamte Physiol. Henschen, 233: 657-682. 66. Friedman, s. and Kaufman, S. (1965) : 3, 4-Dihydroxy phenylethylarnine /3-hydroxylase. Physical properties, copper content, and role of copper in the catalytic activity. J. Biol. Chern., 240: 4763-4773. 67. Friedman, w. F. (1972) : The intrinsic physiologic properties of the developing heart. Frog. Cardiovasc. Res., 15: 88-111. 68. Fuller, R. w. and Hunt, J. M. (1967) : Ac·tivity of phenethanolamine-N-methyltransferase in the adrenal glands of foetal and neonatal rats. Nature, 214: 190. 69. Gillespie, J. s. (1973) : Uptake of noradrenaline by smoothmuscle. Brit. Med. Bull., 29: 136·-147. 114 70. Goldstein, M., Lauber, E. and McKereghan, M. R. (1965) : Studies on the purification and charac terization of 3,4-dihydroxyphenylethylamine hydroxylase. J. Biol. Chern., 240: 2066-2072. 71. Gornall, A. G., Bardawill, C. J. and David, M. M. (1949) : Determination of serum proteins by means of the Biuret reaction. J. Biol. Chern., 177: 751-766. - 72. Guidotti, A., Zivkovic, B., Pfeiffer, R. and Costa, E. (1973) : Involvement of 3' ,5'-cyclic adenosine menophosphate in the increase of tyrosine hydroxylase ·activity elicited by .cold exposure. Nauyn-Schmiedeberg's Arch. Pharmacal., 278: 195-206. 73. Guidotti, A. and Costa, E. (1973) Involvement of adenosine 3' ,5' monophosphate in the activation of tyrosine hydroxylase elicited by drugs. Science, 179: 902-904. 74. Haefely, W., Hurliman, A. and Thoenen, H. (1964) A quantitative study of the effect of cocaine on the response of the cat nictitating membrane to nerve stimulation and to injected noradrenaline. Brit. J. Pharmacal., 22: 5-21. 75. Halle, w. L. (1906) : About the formation of adrenalin __ in organisms ..Beitr .... Chem .. Ph¥sio .. Path,_,_ ~=- 27_6~280_. __ 76. Hillarp, N. A. (1959) : FUrther observations on the state of the catecholamines stored in adrenal medullary granules. Acta Physiol. Scand., !Z_: 271-279. 77. Holtz, P. and Bachmann, F. (1952) : Activation of DOPA decarboxylase from the adrenal medulla during adrenal cortical extraction. Naturwissenschaften, 39: 116-120. I _J 78. Holtz, P., Heise, R. and Ludtke, K. (1938) : Fermenta tive degradation of L-dioxyphenylalanine (DOPA) in the kidney. Arch. exp. Path. Pharmakol., 191: 87-118. 79. Hare, M. L. (1928) : Tyramine oxidase. I. A new enzyme i system in liver. Biochem. J., ~: 968-979. j 80. Igaue, I., Gomes, B. and Yasunobu, K. (1967) : Beef mitochondrial monoamine oxidase, a flavin dinucleo tide enzyme. Biochem. Biophys. Res ... Comm•, 29: 562-570. 115 81. Ignarro, L, S. and. Shideman, F. E. ( 19 6 8) : Appearance and concentrations of catecholamines and their bio synthesis in embryonic and developing chick. J. Pharmacol. Exp. Therap., 159: 38-58. 82. Inscoe, J. E., Daly, J. and Axelrod, J. (1965) : Factors affecting the enzymatic formation of Q methylated dihydroxy derivatives. Biochem. Pharmacol., 14: 1257-1263. 83. Iversen, L. L. (1971) : Role of transmitter uptake mechanisms in synaptic transmission. Brit. J. Pharmacol., 41: 571-591. 84. Iversen, L. L. (1974) : Uptake mechanisms for neuro transmitter amines. Biochem. Pharmacol., 23: 1927- 1935. 85. Iversen, L. L., De Champlain, J., Glowinski, J. and Axelrod, J, (1967) : Uptake, storage and metabolism of norepinephrine in tissues of the developing rat. J. Pharmacol. Exp. Ther., 157: 509-516. 86. James, L. S., Weisbrot, I. M., Prince, C. E., Holaday, D. A. and Apgar, V. (1958) : The acid base status of'human infants in relation to birth asphyxia and the onset of respiration. J. Pediat., I1 - -- - 52: 379-394. 87. Kaufman, s. (1966) : Coenzymes and hydroxylases: ascorbate and dopamine-S-hydroxylase, tetrahydro pteridines and phenylalanine and tyrosine hydroxy I lases. Pharmacol. Rev., 18: 61-69. 88. Kernkamp, H. C. H. ( 19 6 5) : Birth and death statistics ~ on pigs of preweaning age. J. Amer. Vet. Med. Assoc., i 146: 337-340. 89. Kirshner, N. (1962) Uptake of catecholamines by a particulate fraction of the adrenal medulla. J. Biol. Chern., 237: 2311-2317. 90. Kirshner, N. (1974) : Function and organization of chromaffin vesicles. Life Sci., 14: 1153-1167. 91. Kopin, I. J. (1972) : Metabolic degradation of cate cholamines. The relative importance of different pathways under physiological conditions and after administration of drugs. Handbk. Exp. Pharmacal., I 33: 270-282. 116 93. Krecek, J. (1971) ; The theory of critical develop mental periods and postnatal development of endocrine functions. In: Biopsychology of development. (Toback, ed.) Academic press, New York, pp. 233-248. 94. Laduron, P. (1972) · : N-methylation of dopamine to epinine in adrenal medulla: a new model for the bio synthesis of adrenaline. Arch. Int. Pharmacodyn., 195: 197-208. 95. Laduron, P., Van Gompel, P., Leysen, J. and Claeys, M. (1974) : In vivo formation of epinine in adrenal medulla: a possible step for adrenaline biosynthesis. Nauyn-Schmiedeberg's Arch. Pharmacal., 386: 227-238. 96. Lagercrantz, H. (1971) : Isolation and characteriza tion of sympathetic nerve trunk vesicles. Acta Physiol. Scand., 366 (suppl.): 4-44. 97. LaBlanc, J. and Hount, L. E. (1969) Effects of noradrenaline and adrenaline on oxygen consumption rate and arterial blood pressure in the newborn pig. Nature (Lond.), 217: 77-78. 98. Leman, A. D., Knudson, C., Rodeffer, H. E. and Mueller, A. G.' (1972) : Reproductive performance of swine on 76 Illinois farms. J. Amer. Vet. Med. Assoc., 161: 1248-1250. 1- 99. Levin, E. Y., Levenberg, B. and Kaufamn, S. (1960) : The enzymatic conversion of 3,4-dihydroxyphenylethyl amine to norepinephrine. J. Biol. Chern.; 235: 2080. 100. Levine, R. J. and Landsberg, L. (1974) : Catecholamines and the adrenal medulla. In: Duncan's Diseases of Metabolism: Endocrinology. (P. K. Bondy and L. E. Rosenberg, ed.) w. B. Saunders Co., Philadelphia, pp. 1181-1224. 101. Levitt, M., Spector, S., Sjoerdsma, A. and Udenfriend, s. (1965) : Elucidation of the rate limiting step in nbrepinephrine biosynthesis in the perfused guinea-pig ear. J. Pharmacal. Exp. Ther., 148: 1-8. 102. Lewis, P. R. and Shute, C. c. (1969) : An electron microscopic study of cholinesterase distribution in the rat adrenal medulla. J. Microscopy, ~: 181-194. 103. Lloyd, T. and Weiner, N. (1971) : Isolation and characterization of a. tyrosine hydroxylase cofactor from bovine adrenal medulla. Molec. Pharmacal., 7: 569-580. 117 104. Levenberg, W.and Victor, S. J. (1974) Regulation of tryptophan and tyrosine hydroxylase. Life Sci. , 14: 2337-2353. 105. Levenberg, W., Weissback, H. and Udenfriend, s. (1962) : Aromatic L-amino acid decarboxylases. J. Biol. Chern., 237: 89-93. 106. Lucy, J. A. (1970) : The fusion of biological mem branes. Nature (Lond.), 227: 814-817. 107. Lundborg, P. (1966) : Uptake of metaraminol by the adrenal medullary granules. Acta Phvsiol. Scand., 67: 423-429. 108. Lundborg, P. and Stitzel, R. (1967) : Uptake of bio genic amines by two different mechanism present in adrenergic granules. Brit. J. Pharmacal. Chemother., 29: 342-349. 109. Matthews, E. K. (1970) : Calcium and hormone release. In: A Symposium on Calcium and Cellular Function. (A.· w. Cuthbert, ed.) Macmillan, London, pp. 163- 182. 1- 110. McCaman, M. W., McCaman, R. E. and Lees, G. J. (1972) j Liquid cation exchange--a basis for sensitive ~adio J metric assays for aromatic amino acid decarboxylases. j- -An 112. Mersmann, H. J., Houk, M., Phinney, G., Underwood, M.- c. and Brown, L. J. (1973) : Lipogenesis by in vitro liver and adipose tissue preparation from- neonatal swine. Am. J. Physiol., 224: 1123-1129. 113. Mersmann, H. J. Phinney, G., Brown, L. J. and Arakelian, M. c. (1975) : Factors influencing the lipolytic response in swine (Sus domesticus) adipose tissue. Gen. Pharmacal., ~: 193-199. 114. Minard, F. N. and Grant, D. S. (1972) : A convenient method for the chromatographic analysis of norepine phrine, dopamine and serotonin. Biochem. Med., 6: 46-52. 115. I 116. Molinoff, P. B., Nelson, D. L. and Orcutt, J. c. (1974) : Neuropsychopharmacology of monoamines and their regulatory enzymes. (E. Usdin, ed.) Ravin Press, New Yo~k, pp. 95-104. 117. Mount, L. E. (1968) : The climatic physiology of the pig. Williams and Wilkins Co., Baltimore, p. 78. 118. Mueller, R. A., Thoenen, H •. and Axelrod, J. (1969a) : Adrenal tyrosine. hydroxylase: compensatory increase in activity after chemical sympathectomy. Science, 158: 468-469. 119. Mueller, R. A., Thoenen, H. and Axelrod, J. (1969b) : Inhibition of trans-synaptically increased tyrosine hydroxylase activ~ty by cycloheximide and actinomycin D. Molec. Pharmacal.,~: 463-469. 120. Mueller, R. A., Thoenen, H. and Axelrod, J. (1970) : Inhibition of neuronally induced tyrosine hydroxylase by nicotinic receptor blockade. Eur. J. Pharmacal., 10: 51-56. 121. Nagatsu, T. (1973) : Biosynthesis of catecholamines. In: Biochemistry of catecholamines. University of Park Press, Baltimore, pp. 9-48. J j 122. Nagatsu, T., Levitt, M. and Udenfriend, S. (1964a) -Tyros-ine--hydroxyla-se:-th-e irri t·ial-step-in-norepinephrine - r- synthesis. J. Biol. Chern., 239: 2910-2917. , 123. Nagatsu, T., Levitt, M. and Udenfriend, s. (1964b) A rapid and simple radio assay for tyrosine hydroxylase activity. Anal. Biochem., 2_: 122-126. 124. Nagatsu, T., Mitzutani, K., Nagatsu, I., Matsuura, S. and Sugimoto, T. (1972) : Pteridines as cofactor or inhibitor of tyrosine hydroxylase. Biochem. Pharmacal., 21: 1945. -~ 125. O'Brien, R. A., Da Prada, M. and Pletscher, A. (1972) 1 The ontogenesis of catecholamine and adenosine-5 - triphosphate in the adrenal medulla. Life Sci., 11: 749-759. 126; Orcutt, J. c. and Molinoff, P. B. (1976) : Endogenous Inhibition of dopamine-8-hydroxylase in rat organs. Biochem. Pharmacal., 25: 1167-1174. 119 127. Patrick, R. L. an~ Kirshner, N. (1972) : Develop mental changes in rat adrenal tyrosine hydroxylase, dopamine-S-hydroxylase and catecholamine levels: ef fect of denervation. Dev. Biol., ~: 204. 128. Philpott, J. E., Zarrow, M. X., Denenberg, v. H., Lu, K. H., Fuller, R. w. and Hunt, J. M. (1969) : Phenenthanolamine N-methyltransferase and adrenocort ical activity in the neonatal rat. Life sci., 8: 367-371. 129. Pletscher, A., DaPrada, M., Steffen, H., Lutold, B. and Berneis, K. H. (1973) Mechanisms of catecholamine accumulation in adrenal chromaffin granules. Brain Res.,~: 317-326. 130. Podgornaya, G. A. (1968) Differentiation of cells of the adrenal medullary substance in embryogenesis in the pig. Arkh. Anat. Gistol. Embriol., ~: 50-56. 131. Poisner, A. M. and Trifaro, J. M. (1967) : The role of ATP and ATPase in the release of catecholamines from the adrenal medulla. I. ATP-evoked release of catecholamines, ATP and protein from isolated chromaffin grapules. Molec. Pharmacal., 1_: 561-571. 132. Pomeroy, R. W. (1960) : Infertility and neonatal mortality in the sow: IV. Further observations -artd-conclu.s-icrn:s-;-- -J. -Agr .--sc-i.-, 4-3-: !>6-o6. -- 133. Prentice, F. D. and Wood, J. G. (1975) : Adrenergic innervation of cat adrenal medulla. Anat. Rec., 181: 689-704. 134. Rahwari, R. G., Borowitz, J. L. and Hinsman, E. J. (1973) : Evidence for secretory tubules in the adrenal medulla. Arch. Int. Pharmacodyn., 206: 234-351. 135. Schiebler, T. H. and Heene, R. (1968) : Evidence of catecholamine in the hearts of rats during development. Histochemie, 14: 328-334. 136. Scrutton, M. C. and Utter, M. F. (1968) : The regula tion of glycolysis and gluconeogenesis in animal tissues. Ann. Rev. Biochem., 22= 249-302. 137. Shepherd, D. M. and West, G. B. (1951) : Noradrenaline and the suprarenal medulla. Brit. J.. Pharmacal. , 6: 665-674. 120 138. Si~rs, D. G., Dekay, D. E., Mersmann, H. J., Brown, L. J. and Stanton, H. C. (1976) : Late gestation feeding of Dichlorvos: a psychosocial characterization of the neonate and a growth survival response. J. Animal Sci., 42: 381-392. 139. Slotkin, T. A. (1973a) : Hypothetical model of catecholamine uptake into adrenal medullary storage vesicles. Life Sci., 13: 675-683 •. 140. Slotkin, T. A. (1973b) : Maturation of the adrenal medulla. I. Uptake and storage of amines in isolated storage vesicles of the rat. Biochem. Pharmacol•, 22: 2023-2032. 141. Slotkin, T. A., Ferris, R. M. and Kirshner, N. (1971) : Compartmental analysis of amine storage in bovine adrenal medullary granules. Molec. Pharmacol., 7: 308-316. 142. Slotkin, T. A. and Kirshner, N. (1971) : Uptake, storage and distribution of amines in bovine adrenal medullary vesicles. ibid., 7_: 581-592. 143. Slotkin, T. A. and Kirshner, N. (1973) : All-or-none secretion of adrenal medullary storage vesicle con tent in the rat. Biochem. Pharmacal., 22: 205-219. - -JA4-. Smi-th ,--A-·- c. -(r973-) : -Mechanisms- invo-lved- in the-re- lease of noradrenaline from sympathetic nerves. Brit. Med. Bull., ~: 123-129. 145. Stanton, H. C., Brown, L. J. and Mueller, R. L. (1973) Irrelationships between ma.ternal and neonatal factors and thermoregulation in·fasted neo natal swine. Comp. Biochem. Physiol., 44A: 97-105. 146. Stanton, H. C. and Carroll, J. K. (1974) : Potential mechanisms responsible for prenatal and perinatal mortality or ·low viability of swine. J. Animal Sci., 38: 1037-1044. 147. Stanton, H. c., Cornejo, R. A ..Mersmann, H. J., Brown, L. J. and Mueller, R. L. (1975) Ontogenesis of monoamine oxidase and catechol-0-methyltransferase in various tissues of domestic swine. Arch. Int. ?harmacodyn. Therap., ~: 128-144. · 1 I j 1 I 121 148. Stanton, H. C., Mueller, R. L. and Bailey, C. L; (1972) Adrenal c'atecholamine levels and synthesizing enzyme activities in newborn swine exposed to cold and 6-hydroxydopamine. Proc. Soc. Exp. Biol. Med., 141: 991-995. 149. Stanton, H. C. and Mueller, R. L. (1973a) : Meta bolic responses to cold and catecholamines as a function of age in swine. Comp. Biochem. Physiol., 45A: 215-225. 150. Stanton, H. C. and Mueller, R. L. (1973b) Metabolic responses induced in neonatal swine by norepinephrine, epinephrine, and isoproterenol. Proc. Soc. Exp. Biol. Med., 143: 492-494. 151. Stanton, H. C. and Mueller, R. L. (1974) Epinephrine induced hyperglycemia and glycogenolysis in fed and fasted neonatal swine. Biol. Neonate, ~: 307-319. 152. Stanton·, H. C., Woo, S. K. , Cornejo, R. A. and Mueller, R. L. (1974) Catecholamine release in newborn pigs. ·Pharmacologist, 16: 191. 153. Stjarne, L. (1972) : The synthesis, uptake and star age of catecho~amines in the adrenal medulla. The effect of drugs. Handbk. Exp. Pharmacal., 33: 231-269. - ------154. Stolz, F. (1904) : About adrenalin and alkylaminoace tobenzcatechin. Ber. d. deutsch .. Chern. Gesellsch., 37: 4149-4154. 155. Tipton, K. F. (1973) : Biochemical aspects of mono amine oxidase. Brit. Med. Bull.,~: 116-119. 156. Thoenen, H., Mueller, R. A. and Axelrod, J. (1970) Neuronally dependent induction of adrenal phene thanolamine'-N-methyltransferase by 6-hydroxydopamine. Biochem. Pharmacal., 19: 66.9-673. 157. Udenfriend, S. (1966) : Biosynthesis of the sympathetic neurotransmitter, norepinephrine. In: The Harvey Lectures, Series 60. Academic Press, New York, p. 57. 158. Udenfriend, S. and Wyngaarden, J. B. (1956) : Pre cursors of adrenal epinephrine and norepinephrine in vivo. Biochim. Biophys. Acta, 20: 48-52. 122 159. Verity, H. A., su,. c. and Bevan, J. A. (1972) : Transmural and subcellular localization of mono amine oxidase and catechol-0-methyl transferase in rabbit aorta. Biochem. Pharmacal., 21: 193-201. 160. Viveros, o. H., Arqueros, L., Connett, R. J., and Kirshner, N. (1969) : Hechanism of secretion from the adrenal medulla. IV. The fate of the storage vesicles following insulin and reserpine administra tion. Malec. Pharmacal.,~: 69-82. 161. Vogel, J. H. K., Jacobowitz, D; and Chidsey, c. A. (1969) : Distribution of norepinephrine in the fail ing bovine heart. Circ. Res.,~: 71-84. 162. Whitby, L. G., Axelrod, J., Weil-Melherbe, H. (1961): The fate of 3H-norepinephrine in animals. J. Pharmacal. Exp. Ther., 132: 193-201. 163. Widdowson, E. M. (1950) Chemical composition of newly born animals. Nature (Land.), 166: 626-628. 164. Wilkinson, G. N. (1961) : Statistical estimation in enzyme kinetics. Biochem. J., 80: 324-332. 165. Wong, P. w. K.', O'Flynn, M. E. and Inouve, T. (1964) Micromethods for measuring phenylalanine ar,d tyro sine in serum. Clin. Chern., 10: 1098-1104. 166. Wurtman, R. J. and Axelrod, J. (1966) : control of enzymatic synthesis of adrenaline in the adrenal medulla by adrenal cortical steroids. J. Biol. Chern., 241: 2301-2305. 167. Wurtman, R. J. Pohorecky, L. A. and Baliga, B. S. (1972) : Adrenocortical control biosynthesis of epinephrine and proteins in the adrenal medulla. Pharmacal. Rev.,~: 411-426. 168. Wurzburger, R. J. and Musacchio, J. M. (1971) : Subcellular distribution and aggregation of bovine adrenal tyrosine hydroxylase. J. Pharmacal. Exp. Ther., 177: 155-168. 169. Yates, R. D. (1963) : An electron microscopic study of the effects of reserpine on adreno-medullary cells of the Syrian hamster. Anat. Rec., 146: 29-45. 170. Youdim, M. B. H., Collins, G. G. S. and Sanaller, M. (1969) : Multiple forms of rat brain monoamine oxidase. Nature (Land.), 223: 626-628.