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Modulation of brain opioid receptors by sine and histidine
Hanissian, Silva Hrant, Ph.D.
The Ohio State University, 1988
UMI 300 N. Zeeb RA Ann Arbor, MI 48106 MODULATION OF BRAIN OPIOID RECEPTORS BY ZINC AND HISTIDINE
DISSERTATION
Presented in Partial Fulfillement of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University
by
Silva H. Hanissian, B.S.
*****
The Ohio State University
1988
Dissertation Committee:
Dr. Gopi Tejwani (Adviser)
Dr. Sarah Tjioe
Dr. John Enyeart
Dr. Hussein Abou-Issa Approved by
Adviser Department of Pharmacology TO MY FAMILY
ii ACKNOWLEDGEMENTS
I would like to give special thanks to my adviser,
Dr. Gopi Tejwani, for his constant and patient guidance throughout the length of this research, and more importantly for being an example of an honest and dedicated scientist. 1 would also like to thank him for his understanding, encouragement, friendship and support during my graduate work. I also thank the members of my reading committee, Drs. Sarah Tjioe, John Enyeart, and Hussein
Abou-Issa for their suggestions and comments.
I express my sincere gratitude to my family and friends who were an endless source of love and support.
Lastly, 1 am very thankful to Drs. Frank and Louise
Adler for their encouragement and constant interest in my progress.
Special thanks are extended to the American Health
Assistance Foundation for partially supporting this study.
iii VITA
October 20, I960 Bora - Beirut, Lebanon
August 1982 B.S., Memphis State University, Memphis Tennessee
1984-1988 Graduate Research Associate, Dept. Pharmacology, College of Medicine, The Ohio State University, Columbus, Ohio, USA
PUBLICATIONS
C . N. Robinson, J. L. Horton, D . O. Foshee, J . W. Jones, and S.H. Hanissian (1986) Comparison of substituent constants for correlation of nuclear magnetic resonance absorption of B-carbon atoms in ortho-substituted styrenes. J. Organic Chemistry. £1: 3535-3540.
A.A. Farooqui, G.A. Tejwani, C.D. Mahle, S.H. Hanissian, W.A. Taylor, and L.A. Horrocks (1987) Mono- and diacylglycerol lipases in spinal cord of lean and obese Zucker rats. Comp. Biochem. Phvsiol.. 87B (2): 341-344.
H.N. Bhargava, G.A. Matwyshyn, S.H. Hanissian, and G.A. Tejwani (1988) Opioid peptides in pituitary gland, brain regions and peripheral tissues of spontaneously hypertensive and Wistar-Kyoto normotensive rats. Brain Res.. 333-340.
S.H. Hanissian, G.A. Tejwani, C.D. Mahle, and J.A. Merola (1988) Effect of exercise on glycolytic enzymes of zucker fatty rats. Molec. Cell. Biochem.. 81: 177-186.
S.H. Hanissian and G.A. Tejwani (1988) Histidine abolishes the zinc inhibition of naloxone binding to opioid receptors in rat brain. Neuropharmacolocrv. in press.
iv G.A. Tejwani, K.P. Gudehithlu, S.H. Hanissian, I.E. Gienapp, c.c. Whitacre and W.B. Malarkey (1988) Naltrexone inhibits rat mammary tumorigenesis induced by stress and a carcinogen. Advances in Biosciences, in press.
G.A. Tejwani, K.P. Gudehithlu and S.H. Hanissian (1988) Role of endorphins in stress-facilitated mammary carcinogenesis. Teraoia del Comportamaento. Italy, in press.
S.H. Hanissian, G.A. Tejwani, C.D. Mahle, A. Derabach, B. Girten, and A.J. Merola (1986) Effects of exercise on muscle phosphofructokinase activity in Zucker rats. Fed. Proc. 4 5 . Abstract 4343.
C.D. Mahle, G.A. Tejwani, S.H. Hanissian, B. Girten, A. Dembach, and A.J. Merola (1986) Effect of long-term aerobic exercise on weight gain, food and water intake, heart rate and blood pressure in Zucker rats. Fed. Proc. 4 5 . Abstract 2686.
S.H. Hanissian, G.A. Tejwani, G.A. Matwyshyn, and H.N. Bhargava (1987) Concentration of endogenous opioid peptides in central and peripheral tissues of spontaneously hypertensive (SHR) rats. Fed. Proc. 46. Abstract 6669.
S.H. Hanissian and G.A. Tejwani (1987) Histidine abolishes zinc induced inhibition of naloxone binding to brain opioid receptors. The Pharmacologist 22111/ Abstract 35.
G.A. Tejwani, Z. Rossetti, C.D. Mahle, and S.H. Hanissian (1987) Effect of physical training and/or fatigue on catecholamine levels in lean and obese Zucker rats. Proc. Xth International Congress of Pharmacology. Abstract P741.
G.A. Tejwani, C.D. Mahle, and S.H. Hanissian (1987) Effect of physical training and/or fatigue on B-endorphin and met- enkephalin levels in lean and obese Zucker rats. Proc. Intl. Narcotics Res. Conference. Abstract P38.
K.P. Gudehithlu, S.H. Hanissian, G.A. Tejwani, I.E. Gienapp, and C.C. Whitacre (1988) Opiate antagonist inhibits rat mammary tumorigenesis induced by stress and DMBA. FASEB J.. 2(5). Abstract 4984.
G.A. Tejwani and S.H. Hanissian (1988) Regulation of delta opioid receptor binding by divalent cations. FASEB J .. 2(5). Abstract 4528.
S.H. Hanissian, H.M. Sharma, and G.A. Tejwani (1988) Effect of Maharishi Amrit Kalash (MAK) on brain opioid receptors. FASEB J, , 2X11/ Abstract 802. v FIELD OF STUDY
Major Field: Neuropharmacology Dr. Gopi Tejwani (Adviser).
vi TABLE OF CONTENTS
PAGE ACKNOWLEDGEMENTS . . iii
VITA...... iv
LIST OF TABLES...... ix
LIST OF FIGURES...... X
ABBREVIATIONS...... xv
SUMMARY...... xvi
CHAPTER PAGE
I. INTRODUCTION...... 1
History of opiates...... 1 Discovery of Opioid Receptors and Peptides.... 2 Distribution of Opioid Receptors in Animals... 5 Precursors of Opioid Peptides...... 6 Trace Elements in CNS Regulation...... 10 Zinc and Brain Development...... 13 Zinc and the Opioid System...... 16 Structure of the Opioid Receptors...... 17 Hypothesis and Specific Aims...... 21
II. MATERIALS AND METHODS...... 24
Reagents...... 24 Animals...... 25 Brain Membrane Preparation...... 26 Opioid Receptor Binding Assays...... 26 Scatchard Analysis...... 28 Protein Determination...... 30
III. RESULTS...... 35
Receptor Binding Studies Using the Opioid Receptor Antagonist Naloxone...... 35
vii CHAPTER PAGE
III. RESULTS...... 35 Effect of Zinc and/or Histidine on Various Subtypes of Opioid Receptors...... 54 Receptor Binding Studies Using the Mu-Receptor Agonist DAGO...... 54 Receptor Binding Studies Using the Delta-Receptor Agonist DSTLE...... 71 Receptor Binding Studies Using the Kappa-Receptor Agonist EKC...... 86 Receptor Binding Studies Using the Epsilon-Receptor Agonist Beta-Endorphin...... 93
IV. DISCUSSION...... 97 Physiological Implications...... 109
BIBLIOGRAPHY...... Ill
APPENDIX A ...... 119
viii LIST OF TABLES
Table Page
1. Properties and Functions of Opioid Receptors..... 4
2. The Concentrations of Several Trace Elements in Selected Brain Regions...... 12
3. Inhibition of [3H]-Naloxone Binding to Opioid Receptors of Rat Brain by Zinc Ions...... 41
4. [3H]-Naloxone Binding to Rat Brain Membranes: Summary of Equilibrium Binding Parameters Derived From Scatchard Plots...... 46
5* Effect of Divalent Cations on [3H]-Naloxone Binding to Rat Cortex Opioid Receptors...... 50
6 . Effect of Divalent Cations on [3H]-DAGO Binding to Rat Cortex Opioid Receptors...... 62
7. Effect of Divalent Cations on [3H]-DSTLE Binding to Rat Cortex Opioid Receptors...... 77
8 . Summary of Equilibrium Binding Parameters Derived From the Scatchard Plots of f3H]-Naloxone, [3H]-dago, [3H]-dstle, and [3H]-ekc in the Rat Cortex...... 100
ix LIST OF FIGURES
Figure Page
1. Diagramatic representation of structures of opioid peptide precursors...... 7
2. Distribution in rat brain of the three families of opioid peptides by immunocytochemistry...... 9
3. Saturation curves of [3H]-naloxone binding to rat brain opioid receptors using three different methods of brain membrane preparation...... 36
4. Equilibrium binding of [3H]-naloxone at 25°C to rat brain opioid receptors...... 36
5. Typical curves of [3H]-naloxone binding to rat cortex opioid receptors generated by direct binding assays...... 38
6 . Specific binding of [3H]-naloxone as a function of radiolabelled ligand concentration and time... 38
7. Inhibition of [3H]-naloxone binding to rat cortex opioid receptors by ZnCl2 ...... 39
8 . The effect of lmM histidine on the inhibition of [3H]-naloxone binding in the rat cortex by zinc ions...... 39
9. The effect of lmM histidine on the inhibition [3H]-naloxone binding in the rat midbrain by zinc ions...... 40
10. The effect of histidine on [3H]-naloxone binding to rat midbrain opioid receptors in the presence and absence of 25uM ZnCl2 ...... 40
11. The effect of histamine on [3H]-naloxone binding to rat cortex opioid receptors in the presence and absence of 25uM znCl2 ...... 43
x Figure Page 12. The effect of imidazole acetic acid on [3H]-naloxone binding to rat midbrain opioid receptors in the presence and absence Of 25uM ZnCl2 ...... 43
13. The effects of lmM citrate and lmM histamine on the zinc inhibition of [3H]-naloxone binding to rat cortex opioid receptors...... 44
14. Scatchard analysis of [3H]-naloxone binding to rat cortex opioid receptors in the presence and absence of 30uM ZnCl2 and/or lmM..histidine.... 45
15. Scatchard analysis of [3H]-naloxone binding to rat midbrain opioid receptors in the presence and absence of 30uM ZnCl2 and/or lmM histidine.... 45
16. Equilibrium binding of [3H]-naloxone in the presence and absence of 30uM ZnCl2 at 25°C...... 48
17. The inhibition of [3H]-naloxone binding to rat cortex opioid receptors by CuCl2 in the presence and absence of lmM histidine...... 48
18. The inhibition of [3H]-naloxone binding to rat cortex opioid receptors by HgCl2 in the presence and absence of lmM histidine...... 49
19. The inhibition of [3H]-naloxone binding to rat cortex opioid receptors by CdCl2 in the presence and absence of lmM histidine...... 49
20. The dual effect of CoCl2 on [3H]-naloxone binding to cortex opioid receptors is denoted by open squares. Histidine (lmM) was able to abolish the stimulatory effects of CoCl2, but not its inhibitory effects...... 52
21. The dual effect of NiCl2 on [3H]-naloxone binding to cortex opioid receptors in the presence and absence of lmM histidine...... 52
22. The effect of MnCl2 on [3H]-naloxone binding to rat cortex opioid receptors in the presence and absence of lmM histidine...... 53
23. The effect of MgCl2 on [3H]-naloxone binding to rat cortex opioid receptors in the presence and absence of 30uM ZnCl2 ...... 53
xi Figure Page
24. Typical curves of [3H]-DAGO binding to rat cortex opioid receptors generated by direct binding assays...... 55
25. Equilibrium binding of [3H]-DAGO in the rat cortex in the presence and absence of 30uM ZnCl2 ...... 55
26. The inhibition by ZnCl2 of [3H]-DAGO binding to rat cortex opioid receptors in the presence and absence of lmM histidine...... 56
27. The effect of histidine on [3H]-DAGO binding in the rat midbrain in the presence and absence of 30uM ZnCl2 ...... 56
28. The inhibition of [3H]-DAGO binding to rat cortex opioid receptors by HgCl2 in the presence and absence of lmM histidine...... 57
29. The inhibition of [3H]-0AGO binding to rat cortex opioid receptors by CuCl2 in the presence and absence of lmM histidine...... 57
30. The inhibition of [3H]-DAGO binding to rat cortex opioid receptors by CdCl2 in the presence and absence of lmM histidine...... 61
31. The effect of MgCl2 on [3H]-DAGO binding to rat cortex opioid receptors in the presence and absence of 30uM ZnCl2 ...... 61
32. Scatchard analysis of [3H]-DAGO binding to rat cortex opioid receptors in the presence and absence of 30uM ZnCl2 and/or lmM histidine...... 63
33. The effect of beta-mercaptoethanol on [3H]-DAG0 binding to cortex opioid receptors in the presence and absence of 30uM ZnCl2 ...... 65
34. The effect of dithiothreitol on [3H]-DAG0 binding to cortex opioid receptors in the presence and absence of 30uM ZnCl2 ...... 65
35. The inhibitory effects of dithiobisnitrobenzoic acid on [3H]-DAG0 binding to rat cortex opioid receptors in the presence and absence of 30uM ZnCl2 ...... 66
xii Figure Pago
36. The effects of bestatin and thiorphan on [3H]-DAGO binding to rat midbrain opioid receptors in the presence of 30uM ZnCl2 ...... 68
37. The effects of bestatin and thiorphan on [3H]-DAGO binding to rat hypothalamus opioid receptors in the presence of 30uM ZnCl2 ...... -...... 68
38. The effect of increasing concentrations of bestatin on [3H]-DAGO binding to rat hypothalamus opioid receptors...... 70
39. The effect of increasing concentrations of thiorphan on [3H]-DAGO binding to rat hypothalamus opioid receptors...... 70
40. Typical curves of [3H]-DSTLE binding to rat cortex opioid receptors generated by direct binding assays...... 72
41. Equilibrium binding of [3H]-DSTLB at 25°C to rat cortex opioid receptors...... 72
42. The inhibition of [3H]-DSTLE binding to rat cortex opioid receptors by ZnCl2 in the presence and absence of lmM histidine...... 73
43. The effect of histidine on [3H]-DSTLE binding to rat cortex opioid receptors in the presence and absence of lOOuM znCl2 ...... 73
44. The inhibition of [3H]-DSTLE binding to rat cortex opioid receptors by HgCl2 in the presence and absence of lmM histidine...... 74
45. The inhibition of [3H]-DSTLE binding to rat cortex opioid receptors by CuCl2 in the presence and absence of lmM histidine...... 74
46. The inhibition of [3H]-DSTLE binding to rat cortex opioid receptors by CdCl2 in the presence and absence of lmM histidine...... 76
47. The effect of MgCl2 on [3H]-DSTLE binding to rat cortex opioid receptors in the presences and absence of 100UM ZnCl2 ...... 78
48. The effect of MnCl2 on [3H]-DSTLE binding to rat cortex opioid receptors in the presencce and absence of lOOuM ZnCl2 ...... 78
xiii Figure Page
49. Scatchard analysis of [3H]-DSTLE binding to rat cortex opioid receptors in the presence and absence of lOOuM ZnCl2 and/or lmM histidine...... 81
50. The effect of beta-mercaptoethanol on [3H]-DSTLE binding to cortex opioid receptors in the presence and absence of 500UM ZnCl2 ...... 82
51. The effect of dithiothreitol on [3H]-DSTLE binding to cortex opioid receptors in the presence and absence of 30uM ZnCl2 ...... 82
52. The inhibitory effect of dithiobisnitrobenzoic acid on [3H]-DSTLE binding to rat cortex opioid receptors in the presence and absence of 500UM ZnCl2 ...... 83
53. The effects of bestatin and thiorphan on [3H]-DSTLE binding to rat cortex opioid receptors in the presence of 30uM ZnCl2 ...... 83
54. The stimulatory effects of MnCl?, MnCl2, and CaCl2 on C3H]-DADLE binding to dialyzea midbrain opioid receptors...... 85
55. The stimulatory effects of MnCl2, MnCl2, and CaC12 on [3H]-DADLE binding to dialyzed cortex opioid receptors...... 85
56. Equilibrium binding of [3H]-EKC to rat cortex opioid receptors at 0°C and.... 25°C...... 90
57. Typical curves of [3H]-EKC binding to rat cortex opioid receptors generated by direct binding assays...... 90
58. The inhibition of [3H]-EKC binding to rat cortex opioid receptors by ZnCl2 in the presence and absence of lmM histidine...... 91
59. The effect of histidine on [3H]-EKC binding to rat cortex opioid receptors in the presence and absence of 200uM ZnCl2 ...... 91
60. Scatchard analysis of [3H]-EKC binding to rat cortex opioid receptors in the presence and absence of 150uM ZnCl2 and/or lmM histidine 92
61. Typical curves representing the specific binding of [12SI]-beta-endorphin to rat cortex opioid receptors generated by direct binding assays.... 96 xiv Figure Page
62. Limiting slopes technique for determining scatchard plots...... 120
63. Scatchard plot curve peeling by the Rosenthal method...... 120
64. Comparison of the sensitivities of three methods of protein determination using bovine serum albumin as standard...... 121
xv abbreviations a n d s y m b o l s
ACTH Adrenocorticotrophic hormone
BCA Bicinchoninic acid
Bmax Maximum number of binding sites
B-ME B-Mercaptoethanol
BSA Bovine serum albumin
CLIP Corticotrophin-like intermediate lobe peptide
CNS Central nervous system
DADLE [Tyr-D-Ala-Gly-Phe-D-Leu] -enkephalin
DAGO [Tyr-D-Ala-Gly-Methyl-Phe-Glyol]-enkephalin
DPDPE [D-Pen-Gly-Gly-Phe-D-Pen]-enkephalin
DSTLE [Tyr-D-ser-Gly-Phe-Leu-Thr]-enkephalin
DTNB Dithiobisnitrobenzoic acid
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
EKC Ethylketocyclazocine
Hepes 4-(2-hydroxyethyl)-1-piperazine ethane sulphonate
IC50 The concentration of the cation which causes 50%
inhibition of ligand binding to the receptors
Kd Dissociation constant
MSH Melanocyte stimulating hormone
xv i SUMMARY
The effect of zinc and several trace elements was
studied on the binding of the opioid receptor antagonist
[3H]-naloxone and the agonists [3H]-DAGO, [3H]-DSTI*E, and
[3H]—EKC, specific for the mu, delta and kappa receptors, respectively, in several areas of the rat brain.
Physiological concentrations of zinc were inhibitory to the binding of naloxone, DAGO, and EKC, whereas delta receptors were insensitive to this inhibition. Copper, cadmium, and mercury also inhibited the binding of all the ligands studied to their receptors. Histidine was most effective in preventing the inhibitory effects of zinc and copper, whereas it was less effective on cadmium, and without any effect on the inhibition caused by mercury. Its metabolites histamine and imidazoleacetic acid, and also citrate were ineffective. Magnesium and manganese were stimulatory to opioid receptor binding, whereas cobalt and nickel had dual effects. Concentrations of zinc less than its IC5q totally prevented the stimulatory effects of magnesium and manganese on the mu and delta receptors on which zinc alone had no effects. The reducing reagents dithiothreitol and B- mercaptoethanol partially protected against zinc
xv ii inhibition, and the oxidizing reagent dithiobisnitrobenzoic acid even potentiated the inhibitory effects of zinc on
DSTLE and DAGO binding, although to different extents.
Thus, it appears that zinc exerts its inhibitory effects on opioid receptor binding mostly by oxidizing the SH- groups on these receptors since it was partially prevented by reducing reagents, but that this was not its only mechanism of action. These studies also show that the effect of zinc on the mu, delta and kappa opioid receptors, and on the binding of the opioid antagonist naloxone is different from that of the other inhibitory cations, and is not only by oxidizing the essential sulfhydryl groups on the receptors, but also by preventing the effects of stimulatory ions such as magnesium and manganese on these receptors. These results also show that there are structural differences between these three opioid receptor subtypes, and that the delta receptor probably does not have SH-groups essential for binding, or that these are not accessible for zinc binding.
xviii CHAPTER I
INTRODUCTION
History of Opiates
The history of the opium poppy began in antiquity.
Although the exact date of the first systematic cultivation
of this plant remains unknown, poppy seeds were found as
early as the Stone Age in the area of the lake dwellers
(1) . In Mesopotamia (4000-3000 B.C.), the poppy plant was
cultivated by the Sumarians to extract opium from it, who
referred to it as the "joy plant" (2) . The medicinal value of the poppy was first described in the Ebers Papyrus
(1600-1500 B.C.), and later in the works of Hippocrates
(463-377 B.C.), where various types and preparations of the plant were recommended for use as a hypnotic, catharrtic and narcotic (3) . In the early part of the nineteenth century, Sertumer in Germany separated meconic acid from opium, producing morphine from the alkaline base. Robiquet isolated narcotine in 1817 and codeine in 1832 in France.
By this time the use of opium and its products had become fashionable and widespread both in the United States and abroad.
1 2 Discovery of Opioid Receptors and Peptides
The modem era of opiate research is usually considered
to have begun in 1973 with the demonstration of
stereospecific opiate receptors (later termed "opioid"
receptors) in the central nervous system of animals and
humans. Evidence for the existence in animal brain of
stereospecific opioid binding came in 1973, simultaneously
from three laboratories in reports by Simon et al. (4),
Pert and Snyder (5), and Terenius (6 ). It was the discovery
of stereospecific opioid receptors and the finding that
they exist in every vertebrate that triggered the search
for an endogenous opiate-like factor and ligand for the
receptor. The first reports of such endogenous opioid
activity again came simultaneously from two laboratories.
John Hughes in Hans Kosterlitz's laboratory at Aberdeen,
Scotland (7) , and Lars Terenius and Wahlstrom in Uppsala,
Sweden (8 ), reported the presence of opioid activity in aqueous extracts of animal brain. In December 1975 Hughes et al. (7) reported the purification and characterization of two pentapeptides that had opioid activity from extracts of pig brain. These pentapeptides were named methionine- enkephalin and leucine-enkephalin by their discoverers.
Hughes et al. (7) also reported that the sequence of met- enkephalin was present in the structure of beta-1 ipotropin which was isolated much earlier from pituitary glands by C.
H. Li (9). Later, Cox et al. (10), and Bradbury et al. (11), Independently found potent opioid activity In the C- terminal fragment61”91 of beta-1ipotropin. The longer peptides derived from beta-lipotropin61”91 were named alpha61”76, beta61”91, and gamma 6 1 " 77 endorphin, respectively. Dynorphin was discovered by Avram Goldstein in 1979 from porcine pituitary (12).
The first evidence for the existence of multiple classes of opioid receptors was obtained by Martin and coworkers in
1976, in pharmacological experiments performed in chronic spinal dogs (13), where they showed that the pharmacological profiles of several types of opioids in neurophysiological and behavioral tests were sufficiently different to suggest the existence of three types of opioid receptors which Martin named mu, delta, and kappa. The existence of these receptors were later confirmed by the
Kosterlitz group (14), and by Woods and coworkers (15).
Lemaire et al. (16) demonstrated the presence of a receptor in rat vas deferens which seemed to be highly specific for beta-endorphin. It was named the epsilon receptor. There is now enough evidence to suggest that mu, delta, and kappa-receptors may be the sites of action for different opioid peptides (Table 1). The delta-receptor has the highest affinity for enkephalins. The question as to what is the endogenous ligand for the mu-receptors is presently difficult to answer. There is considerable TABLE 1. PROPERTIES AND FUNCTTCNS OF OPIOID RECEPTCRS
TYPS AAiiists Ml Morphine Cerebral cortesc- Analgesia Morphioeptin layers I and IV Hypothermia Leu-enkephalin m |y «i»in'i w T in-yMif rt-rry Met-entephalin Periaqueductal gray activity Lsvorphanol Hypothalamus Respiratory Etorphine Hialanus depression Guinea pig ileum Miosis Bradycardia
Delta Leu-enkephalin cerebral cortex- Analgesia Mst-enkephalln layers n , m , V Behavioral Limbic system effects Pontine nuclei Epileptic Substantia gelatinosa seizures House vas deferens
Kappa Dynorphin Hypothalamus Spinal Ketocyclazocine jwhw analgesia Ethyl- Guinea pig ileum Miosis ketocyclazocine House vas deferens Sedation Bremazocine
Epsilon Rat vas deferens Analgesia Beta-endorphin leukocytes iBxnune function Local blood flow
Sigma Phencyclidine Cortex Mydriasis Allylnormetazocina Hippocampus Respiratory stimulation Dysphoria Hallucinations Tachycardia
(Obtained from Ref. 19). 5
evidence suggesting that dynorphin is the endogenous ligand
for the kappa-opioid receptor.
Distribution of Opioid Receptors in Animals
Opioid receptors are distributed widely in the CNS
except the cerebellum. The regions that are rich in opiate
binding sites are in the limbic system and in all of the
areas that have been impl icated in pathways of pain
perception and modulation, including the substantia
gelatinosa of the dorsal spinal cord, the nucleus raphe
magnus, the medial thalamus and the periaqueductal and
periventricular grey regions. There is evidence for
differential distribution of opioid receptor subtypes in
the CNS. Thus, in the thalamus there seems to be a population of virtually pure mu-sites; the substantia nigra
seems to be also enriched with mu-receptors, whereas areas
rich in delta-receptors are the frontal cortex and the hippocampus. The highest density of kappa-sites is found in
layers V and VI of the cerebral cortex, and in the pyriform
cortex of the guinea pig. In human brain, kappa-binding
sites are found in high proportion in most regions except
the thalamus, with the highest level being in the
hypothalamus (15). Prggurgorg qt gglQld Pgptlflgg In the last few years several groups have elucidated
the biosynthetic origin of the opioid peptides. The presently known opioid peptides come from three different precursors: the beta-endorphin/ACTH precursor (known as proopiomelanocortin or POMC), the enkephalin precursor
(proenkephalin or proenkephalin A), and the dynorphin/neo- endorphin precursor (prodynorphin or proenkephalin B) (Fig.
1) . These precursors are biosynthetically and anatomically separate from each other, and are encoded by different genes. POMC has at its carboxyl terminal beta-endorphin and its precursor beta-1ipotropin which contains beta-KSH in some species. The mid-portion of this precursor contains
ACTH which is cleaved into alpha-MSH and CLIP in the intermediate lobe of the pituitary gland (18). At the amino terminus of POMC is gamma-MSH. Thus, POMC contains one opioid peptide (beta-endorphin), and three MSH-like peptides- alpha-MSH from ACTH, beta-MSH and gamma-MSH. Thus this precursor has a secretory role, and contains multiple di-basic cleavage sites for post-translational processing.
Proenkephalin obtained from adrenal medullary tissue codes for several active peptides (7) . For example, it codes for seven peptides with the [met]- or [leu]- enkephalin active core. Four of these peptides are met- enkephalin, two are carboxylterminal-extended Met- enkephalin-Arg6-Phe7 and -Arg6-Gly7-Leu®, and one copy of I n m «• m in m i« it* im m m w# m
A m i> m i ut precursor Si|Ml jusU aMSH 9-MSH ji- N'Tnm Ih I pfiUi a c o T HfH at Vi-MSH l-LFH
4ft* 4ft ft Pnprocaktpfcalia A
Sigui Met-cak Met-cak Met-rak Md-cak Ln«A Met-cak* ptptfai* AV-Gty’-Uu' Ari'-Phs* t t Peptide F rebuts
ft M. i Plcproeakepbalia > Si|aal Peptide
Fi^ncei Diairanullc represents lioa of structures of opioid peptide precursors. MS1I and cafcepheNa Prodynorphin, as sequenced and cloned from pituitary and brain, produces three main opioid peptides containing the leu-enkephalin sequence: alpha-neoendorphin, dynorphin A, and dynorphin B. Immunohistochemical studies of endogenous opioids suggest that the POMC-derivad peptides beta- lipotropin, beta-endorphin, ACTH and alpha-MSH are localized both in pituitary and brain (Fig. 2). The anterior and intermediate lobes of pituitary, as well as POMC neurons of the arcuate nucleus of the hypothalamus all synthesize POMC. However, the major site of POMC biosynthesis is in the pituitary gland. The existence of two separate neuronal populations immunoreactive for POMC peptides have been demonstrated. The major POMC neuronal population resides in the arcuate nucleus and periarcuate regions of the medial basal hypothalamus, with projections throughout the brain (20). A second, less extensive group of neurons occurs in the nucleus tractus solitarius. The enkephalins are localized in many neuronal systems in the brain, some forming local circuits and others with long tract projections. The biosynthetic source of met- enkephalin and leu-enkephalin is the proenkephalin precursor found in both the adrenal gland and the brain. Enkephalin-containing neuronal circuits have been described in medullary projections to the spinal cord, amygdaloid efferents in the stria terminalis, the nguro 2. Distribution ia rat brain of die tbrce Emilies of opioid pepdds by iramuno- cytochemistry. 0-END — jj-endorphm: ENK — [Leu]enkephalin; DVN — dynorphin A. Schematic pantsaginai representation with amygdaloid region shown separately below the doable Uses. Solid circles — neuronal pcnkarya: dots « fibers and terminals. Abbreviations: a — n. acmmbcm: abl“ amygdala basolateral m «"am ygdala. central tuaco™amyg dala. conical au am — amygdala, medial n_ aon “ anterior olfactory n^ ire * arcuate nu as—anterior thalamic nc bst» bed n. stria terminal is: eg ~cinguiate cortex: cp — caudate- putamentdg "dentate gyrucdh — dormi bore, spinal cord: dm " donomedial dm — dor sal tegmental a-: ent " ento rhinal cortex: fn — fasti gial a , cerebellum: fr — frontal cortex; gi —n. reticularis gigantocellulam: gp " globus pallidus: h “ habenula; hpc “ hippocampus: ic • inferior collieulus: ip ” interpeduncular tu Ic " locus cocruleuc Ig ** lamina glomeru- Io s l oMhctory bulb: lha * lateral hypothalamic area: Ire" lateral reticular rum * mammil lary mu; mv " mesencephalic tngeminal n j nts • n. tiactus solitarius: otu “olfactory tuber* detpag" periaqueductal graytpbn “ parabracbialmpgi " a. reticularis paragigamoceUularis: prr—pyriform cortex: pp “ perforant path: pvn — paraventricular iu pvt — periventricular thalamic tu: rd “ n. raphe dorsalis: rm ~ n. raphe magnnr s ~ subiculum: sc — superior coUi- caluc sac " substantia nigra, pan compacts: snr • substantia nigra, pars rebculao: son ” su- praopdcn.:spt " septum: sV * spinal trigeminal n.:vm — ventromedial n.:vp *■ ventral palli dum: vst " vestibular nn.: vu * ventral tegmental area. (Reprinted with permission from Watson hypothalamo-neurohypophyseal system, and the antorhinal- hippocampal system (21-23) (Fig. 2). Dynorphin A, dynorphin B (or rimorphin) and alpha- neoendorphin are present in neuronal systems that often parallel the distribution of the enkephalin systems in the brain and spinal cord (Fig. 2). Prodynorphin is the precursor of these peptides. Detailed immunohistochemical studies have described the wide central nervous system distribution of all three prodynorphin peptides (25,26). Physiological and clinical studies have shown that all these endogenous opioid families are heavily involved in systems that regulate the body's responses to stress. Endorphins regulate the perception of and the response to painful stimuli. In addition, endogenous opioid peptides are involved in stress-induced analgesia (27), cardio vascular control, regulation of the immune system, affective disorders, hormonal regulation, learning and memory, sexual behavior, locomotor activity, body temperature control, food intake, etc. (28). Trace Elements in CNS Regulation Since the discovery of opioid receptors in 1973, numerous investigations were conducted in which the effect of many monovalent and divalent cations on the binding of opioid peptides to their receptors was studied. However, there are virtually no reports about the effects of XI essential trace elements in general, and zinc ions in particular on the equilibrium binding of opioid peptides to the mu, delta, kappa, and epsilon opioid receptor subtypes in different areas of the rat brain. The term "trace elements" refers to a collective group of chemical elements present at low concentrations in biological cells. Some of these trace elements perform essential functions and thus must be obtained from the environment in adequate amounts to optimize cellular metabolism. The list of essential trace elements for animals is under constant debate, but includes the following 12 elements according to Nielsen: arsenic, chromium, cobalt, copper, iodine, iron, manganese, molybdenum, nickel, selenium, silicon, and zinc. Some evidence exists for the essentiality of eight additional elements: cadmium, tin, bromine, boron, fluorine, lead, lithium, and vanadium (29). Not all of the essential trace elements have known neurochemical functions. Those that have been shown to be required for the development and maintenance of the central nervous system include zinc, copper, cobalt, iron, manganese, selenium, and iodine. The concentrations of some of these essential trace elements both in human and rat brain areas are presented in Table 2. Of those trace elements known to influence neurochemical functions, zinc is clearly the most studied yet the least understood. TABUS 2. OONCHHRMTOW* OF HISTIDINE AND DIVALENT CATIONS IN PAT ERAm REGION CU2+ Zn2* Ml2* Mg2* HISTIDINE m e d u u a / p c n s 45 154 10.0 7.70 80 CEREBEIUUH 46 178 9.44 7.0 90 STRIATVJM 46 195 7.70 7.24 95 CORTEX 45 192 6.80 6.14 45 HIPFOCAMRJS 41 210 8.0 6.35 100 HYPOTHALAMUS 60 174 23.5 6.21 121 MIDERAIN 48 180 11.0 6.80 65 The total concentration of the divalent cations are obtained frcm Donaldson et al. (42). The values for histidine are frcn Taylor and Snyder (17). ^Concentrations are in uM, except for Mg2* cjcncentrations which are in nM. Values are expressed per gram wet weight. 13 Zinc and Brain Development Many of the trace elements are needed for proper brain development (31); zinc displays perhaps the most dramatic effects, for when the supply is limited major congenital malformations of the brain are observed (32). In particular, zinc deficiency appears to affect rapidly * proliferating tissues, which renders the embryo and growing animals especially vulnerable to suboptimal zinc status (33) . In rats, severe maternal zinc deficiency during pregnancy results in fetal death and widespread teratogenesis where all organ systems are affected, and there is a high incidence of defects of the central nervous system (34,35). Even more impressive are the permanent adult behavior alterations that occur after a very brief period of zinc deprivation during perinatal development. These changes include impairment of long-term memory and perhaps learning and short-term memory as well (36). Since zinc is associated with approximately 100 zinc-containing and zinc-activated enzymes from various species (37), and since it is involved with the stability of biological membranes (38) and with the T-cell mediated immune response (39) , it is not surprising that zinc deficiency in adult animals presents a syndrome of remarkable complexity. With growing tissue, however, the effects are less diverse and seem to derive mainly from arrested cell growth, which in turn probably results from the established reguirement for 14 zinc during mitosis (40). Zinc is known to bs required by a number of enzymes involved in the processes of transcription and translation (41). Some workers consider the effect of zinc deficiency on RNA and/or protein synthesis (41) to be of great biochemical consequence in reducing the overall rate of cell division. However, the precise mode of action of zinc is still not known. With the exception of potassium, calcium, and magnesium, zinc is the most abundant intracellular metal (42). In general, concentrations in the grey matter are twice as high as those in white matter (4 3) . The highest concentrations of zinc in the brain are found in the hippocampus, followed by that in the cortex, striatum, and cerebellum (Table 2) . Zinc concentrations in rat brain are quite similar to that of the human brain. It has been reported that about 20% of the zinc in the brain is either free or bound to ligands of molecular weight less than 10,000 daltons (44). The remainder 80% of zinc is apparently chelated or bound to larger molecules. The ubiquity and essentiality of zinc in biology derives from its position in the periodic table of elements (45) . It is a group I IB element with a completed £ subshell and two additional $ . electrons. The chemically combined form is always the 2+ oxidation state, and there is no evidence that the metal undergoes oxidation or reduction in biologic reactions. Perhaps the most important characteristic of 15 zinc, at least from a biochemical point of view, is its ability to form complex ions. As a metal ion which possesses many of the complexation characteristics of a "transition” metal, zinc binds predominantly to ligands containing sulfur, nitrogen and, to a lesser extent, oxygen. Because of these properties, a large proportion of the zinc in organisms is bound to proteins. Fats, plasma filtrates and urine contain relatively low levels of zinc. Although zinc binds to many proteins, it may have a unique affinity for, and special function in, lipoproteins and proteins associated with biomembranes (46). Abnormal eating patterns and permanent behavioral changes of zinc-deficient rats have prompted many studies that have examined zinc and neurotransmitters. These studies were given further impetus by the observation of Hesse (47) that zinc-deficient rats express abnormal synaptic transmission when hippocampal mossy fiber axons are stimulated. The steady-state levels of brain norepinephrine, dopamine and serotonin are not greatly influenced by dietary zinc deficiency. Recent experiments have shown that chronic zinc deficiency in rats elevated hypothalamic norepinephrine (NE), whereas acute deficiency, while changing eating behavior, did not affect NE (48). Another study found lower levels of dynorphin, an endogenous opioid peptide, in hypothalamus but not in cerebral cortex (49) with zinc-deficiency. This might have 16 some relevance to the etiology of anorexia, since endogenous opioids have been shown to play a role In appetite regulation. Zinc and the Opioid System Several lines of evidence suggest that zinc might be involved in neurotransmitter homeostasis in addition to having effects on the steady-state levels of neurotransmitters. Stengaard-Pedersen et al. (50) have suggested that there might be a relationship between zinc and the enkephalins, and that zinc ions may be a physiologically important modulator of opioid receptor function in the hippocampal mossy fiber system. Their proposal was based on evidence that in guinea pigs, the staining pattern for enkephalin, based on immunocyto- chemistry, was congruent with that for zinc obtained by Timm's silver-sulfide staining, and that both were confined to the hippocampal mossy fiber pathway. Although the hippocampus contains relatively few opioid receptors and a low concentration of enkephalin, Stengaard-Pedersen et al. (50) point out that opioid peptides have been shown to have a profound effect on hippocampal pyramidal cells in culture, and on the electrical activity of hippocampal pyramidal neurons in vivo. Furthermore, Stengaard-Pedersen (51) has shown that zinc ions have the ability to inhibit the stereospecific binding of [3H]-enkephalinamide to 17 opioid receptors, possibly because of a zinc-thiol interaction. In addition, Baraidi et al. (52) have reported that zinc added in vitro inhibits [3H]-naloxone binding to rat brain membranes. Baraldi has also reported that administration of zinc to morphine-dependent-tolerant rata reduced the analgesic effect of morphine, and the naloxone- precipitated withdrawal syndrome (53). Interestingly, treating animals with morphine alone caused a decrease in zinc levels in their plasma and brain, and a decrease in ACTH levels in plasma, which is exactly the opposite effects produced by zinc supplementation alone. Thus, zinc is a very important divalent metal ion and an essential trace element with various effects on the CNS and on opioid receptors, as reported in a few preliminary experiments; therefore understanding its role in modulating the activity of different opioid receptor subtypes in various areas of the rat brain would be very valuable in that it will increase our knowledge about the mechanisms by which opioid receptors are regulated by zinc and other trace elements that are essential for normal growth and development. Structure of the Opioid Receptors Numerous studies were and still are being conducted in an attempt to try and purify the opioid receptor and study its structural components and the mechanisms of its 18 regulation. There has been considerable progress in the purification of active opioid binding sites. At least partial purification has been achieved in several laboratories (54-60). In every case the major purification step involved affinity chromatography, and 200-500 fold purification of the receptors was achieved. However, there is a lot of discrepancy about the molecular weight of the receptors and the number of its subunits, and different laboratories report different molecular weights and number of subunits for the opioid receptor. For example, Bidlack et al. (54) reported only three protein bands of molecular weight (M.W.) 43, 35, and 23 kdalton when they subjected their partially purified receptors to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) . Maneckjee et al. (59) achieved partial purification of mu opioid receptors, and upon electrophoresis of the purified protein on native PAGE, they obtained a band with a M.W. of 300-350 kdalton. Under denaturing conditions (SDS-PAGE), a major band of M.W. 94 kdalton and minor bands at M.W. 44 and 35 kdalton were seen. Fujioka et al. (60) used delta preferring ligands for purification of opioid receptors, and reported two major bands on SDS-PAGE, of M.W. 62 and 39 kdalton. The 62 kdalton may represent a subunit of the delta receptor, in good agreement with the crosslinked polypeptide of M.W. 58 kdalton of Klee et al. (61). The crosslinked delta binding protein reported by Howard et al. 19 (62) is somewhat smaller (53 kdalton), possibly reflecting differences in the purification and experimental conditions used. Purification of opioid binding proteins to apparent homogeneity has been reported to date from only two laboratories, one for an active binding subunit derived from mu receptors, the other for an affinity-labeled polypeptide derived from delta receptors. Gioannini et al. (63) have recently succeeded in the purification to virtual homogeneity from bovine striatal membranes of a polypeptide of H.H. 65 kdalton, which appears to be a binding component of mu receptors. By improvements in washing and elution procedures, the degree of purification that they achieved was about 77,000 fold. The other report of purification to apparent homogeneity comes from the laboratory of Klee (64) . This group has identified a binding subunit of delta opioid receptors from NG108-15 cells. This was a glycoprotein with a M.W. of 58 kdalton that had been affinity labelled with [3H]-fentanyl. The degree of purification that they achieved was 30,000 fold. However, the purified receptors from all of these laboratories were inactive, and therefore could not be used to study their regulation by different ions or compounds. Scientists appear to agree however, that the opioid receptor has essential sulfhydryl groups which have to be in the reduced form for the receptor to be active (66,67). 20 Another important feature about the opioid receptor is that it has essential histidine residues at the binding site which are necessary for the receptor to be active (68). If these histidine residues are altered by protein modifying reagents such as ethoxyformic anhydride or photooxidized in the presence of the anionic dye Rose Bengal, the opioid receptor loses its activity (6 8 ). As can be seen from these reports, it took about 12 years before significant progress was achieved in the purification of opioid receptors. This progress has been confined to the binding site portion of the receptors, and has not so far been extended to other components of the receptor complex. The reason for this was in large due to the difficulty of this task, especially since opioid receptors present a very small portion (about 0 .001%) of the total cell membrane proteins, and the remarkable sensitivity of opioid receptors to detergents, even those of the non-ionic variety (65) . However, with the two reports of purification to homogeneity of opioid binding proteins (63,64), there is hope that in the near future amino acid sequence of the receptors can be obtained which can then be used to prepare oligonucleotide probes for detection and isolation of cDNA complementary to receptor message from an appropriate cDNA library. This would permit complete sequencing of the cDNA and thus yield the total amino acid sequence of the receptor protein. Another area of research that has already begun to show results, but will now be greatly accelerated, is the production of antibodies to opioid receptor proteins (65). The availability of antibodies should further facilitate the distinction of opioid receptor types, speed up receptor purification, and provide the means for detailed immunohistochemical mapping of the various types of opioid receptors. Finally, reconstitution experiments of purified opioid binding sites and other receptor components (G proteins, adenylate cyclase) into artificial or natural membranes should aid in the elucidation of the steps triggered by receptor-ligand interaction and in our understanding of the functions of the endogenous opioid system. Hypothesis and Specific Alms In view of all these reports, our specific aims are: 1. To investigate the role of zinc ions, which are found in high concentrations in the brain and are known to oxidize sulfhydryl groups, and histidine, which is a good metal chelator, an endogenous compound and an essential part of the opioid receptor, in regulating opioid receptor activity in different areas of the rat brain. 2. Specifically, our goal is to observe if zinc and histidine have differential effects on the different subtypes of opioid receptors- mu, delta, kappa, and 22 •psilon-, in an attempt to explain whether these two endogenous compounds could detect and reveal any structural and regulatory differences between the different opioid receptor subtypes. 3. To study the effect of zinc and histidine on agonist and antagonist binding to the opioid receptors, to see if their binding is regulated differently, and if these compounds differentiate agonists from antagonists. 4. To compare the effect of zinc to that of other trace elements on opioid ligand binding to the different subtypes of opioid receptors. 5. To compare the effect of histidine on the zinc inhibition of opioid receptor binding to its effects on the inhibition by other trace elements. Therefore, we propose to perform a number of experiments using trace elements such as zinc, and histidine, in an attempt to answer the above questions. Thus, we will perform receptor binding studies using: 1. The opioid receptor antagonist naloxone. 2. The mu-receptor agonist DAGO. 3. The delta-receptor agonist DSTLE. 4. The kappa-receptor agonist EKC. 5. The epsilon-receptor agonist B-endorphin. The IC50 of the inhibitory ions, or the stimulatory effects of others will be measured, as well as their effects on the K0 and Bjnax of the opioid receptors in the presence and 23 absence of histidine. 6 . Reducing and oxidizing reagents will be used in the presence and absence of zinc, in an attempt to try and elucidate the mechanisms of action of this divalent cation. The differential effects of the above mentioned endogenous compounds on the different subtypes of opioid receptors may provide us a better understanding about their regulatory mechanisms, and perhaps give us a glimpse about any structural differences among opioid receptors. CHAPTER II MATERIALS AND METHODS Reagents The radioactive opioid ligands used in these studies were the following. [3H]-naloxone (specific activity 57.4 Ci/mmol); [3H]-DAGO (S.A. 30.3 Ci/mmol); [3H]-DSTLE (S.A. 32.1 Ci/mmol); [3H]-DADLE (S.A. 46.9 Ci/mmol); C3H]- dynorphin A 1 - 8 (S.A. 25.5 Ci/mmol); [3 H]- ethylketocyclazocine (S.A. 28.9 Ci/mmol), all obtained from NEN Research Products (Boston,MA). [3H]-DPDPE (S.A. 34 Ci/mmol), and [125I]-beta-endorphin (S.A. 2000 Ci/mmol) were both obtained from Amersham Corporation (Arlington Heights, IL) . The purity of all the radioactive materials was >96%. Hepes buffer was obtained from United States Biochemical Corp. (Cleveland, OH) . The highly purified metals such as ZnCl2, CuCl2, CdCl2, MgCl2, and HgCl2 were products of Sigma Chemical Company (St. Louis, MO) . MnCl2 and CoCl2 were obtained from Mallinckrodt (Paris, KY) . NiCl2 was a product of EM Science (Cherry Hill, N.J.). The liquid scintillation cocktail used was Scinti Verse E obtained from Fisher (Cincinnati, OH) . Bestatin, thiorphan, naloxone, histidine, histamine, imidazoleacetic acid, and 24 25 citrate were products of Sigma (St. Louis, MO) , and levorphanol was obtained from Hoffmann-LaRoche, Inc. (Nutley. N.J.). DAGO and DADLE were supplied by Bachem (Torrance, CA) , and bacitracin was purchased from Aldrich (Milwaukee, WI). Dialysis membrane with a molecular weight cutoff of 10,000 daltons was obtained from Sigma (St. Louis, MO) . Protein concentrations in brain samples were determined using the bicinchoninic acid method (73), and the reagents used were the following: bovine serum albumin as standard, sodium carbonate, sodium bicarbonate, sodium tartrate, and CUSO4 .5H 2O were obtained from Sigma (St. Louis, MO), and bicinchoninic acid was a product of Pierce (Rockford, IL). Whatman glass microfiber GF/B filters, and the cell harvester were both products of Brandel (Gaithersburg, MD). Animals Male Sprague Dawley rats (250-3OOg) were obtained from Zivic Miller Laboratories, Inc. (Allison Park, PA). Animals were housed in groups of four per cage and maintained on normal laboratory rat chow for at least 3 days before sacrificing them. They were exposed to 12 h light and dark cycles, a temperature of 72+2°F, and a humidity of 45+2%. 26 Brain Membrana Preparation Membrane preparations from different brain areas of adult male Sprague Dawley rats (250-300g) sacrificed by decapitation were prepared according to the method of Wood (69) with slight modifications. Briefly, the brain was dissected into the following areas, and immediately frozen in liquid nitrogen: hippocampus, striatum, cortex, hypothalamus, midbrain, medulla/pons, and cerebellum. Brain parts were pooled from the rats, and homogenized in 20mM Hepes buffer, pH 7.5 (lOOmg wet weight/ml), and the homogenate centrifuged at 49,000 x g for 15 min at 4°C. The supernatant was discarded and the pellet homogenized in its original volume of buffer. This step was repeated and the homogenized pellet was incubated at 37°c for 40 min to allow any endogenous opioid peptides to dissociate from their receptors and be degraded by proteolytic enzymes. The suspension was finally centrifuged at 49,000 x g for 15 min at 4°C, and the pellet homogenized in 22 ml of buffer/250mg original tissue wet weight. This homogenate represented our working membrane preparation from each brain region. Opioid Receptor Binding Assays Assays using membrane suspensions (1 ml, consisting of about 1 mg protein) from the specific brain regions described above were performed in duplicates. [3H]-naloxone (0.5nM), [3H]-DAG0 (InM), [3H]-DSTLE (InM) , [3H]-EKC (2nM), 27 or [125I]-beta-endorphin (280 pM) were incubated with various concentrations of cations and/or lmM histidine for lh at 25°C. The suspensions were then rapidly filtered on Whatman GF/B filters using a Brandel cell harvester to trap the labelled membranes, and followed by 3x3 ml washes with ice cold Hepes buffer (20mM, pH 7.5). When using [3H]- ligands, the filters were placed in vials containing liquid scintillation cocktail, and the radioactivity counted using a Beckman liquid scintillation counter. When [125I]-beta- endorphin was used, the filters were placed in polypropylene tubes and counted in a Gamma counter. Specific binding of the radioactive ligands to the opioid receptors was determined as the difference between the amount bound in the absence and presence of 1 uM levorphanol (70,71) (specific binding * total binding- non-specific binding). When [3H]-EKC was used, 100 nM DAGO and DADLE were included in the assay mixture to suppress the binding of this ligand to mu and delta receptors. When using [12 5I]-beta-endorphin in the binding studies,0 . 2ml of membrane preparation was used per tube in the presence and absence of 3 uM naloxone which was included to determine non-specific binding. The binding of [125I]-beta- endorphin was also performed in the presence of lOOnM each of DAGO and DADLE, or lOOnM DAGO alone to determine the extent to which beta-endorphin binds to mu and delta receptors. For saturation studies, [3H]-naloxone was used 28 over a concentration range of 0-5 nM, [3H]-DAGO and [3H]- DSTLE over a range of 0-20 nM, that of [3H]-EKC was O-lOnM, and [125 ] -beta-endorphin from 0-400 pM. All incubations were carried out for l h at 25°C. For each brain area studied, [3H]-naloxone binding in the presence and absence of ZnCl2 and/or histidine was conducted by using membrane suspensions that were dialyzed for 3 h at 4°C against 2x20 volumes of 20 mM Hepes buffer, pH 7.5. This was performed to ensure the removal of all free ions and small peptides from the membrane preparations that were not removed during the washing steps and could otherwise affect the assay. To determine the effects of histidine, histamine, imidazoleacetic acid, and citrate on the binding of opioid ligands to their receptors, these compounds were used over a concentration range of 0-5mM. Scatchard Analysis The radioactive opioid ligands were used over the same concentration range as that described for the saturation experiments. Binding was carried out using 1ml of the working membrane suspensions (0 .2ml when using [125I]-beta- endorphin) , in the presence and absence of 30uM ZnCl2 and/or lmM histidine. When using [3H]-DSTLE, lOOuM ZnCl2 was included in the assay mixture, and 150uM of ZnCl2 was * included when [3H]-EKC was the ligand. The tubes were 29 incubated for Ih at 25°C. Binding was terminated by rapid filtration as described above. Specific binding is determined as the difference between the amount of radioactivity bound in the absence and presence of luM levorphanol, or 3uM naloxone when using [125I]-beta- endorphin. In addition, DAGO and DADXJ2 (lOOnM each) were included in the assay mixture when [3H]-EKC or [125I]-beta- endorphin were the ligands. The IC50 values of the cations were determined on the IBM PC computer using the GraphPad program and non-linear regression. Equilibrium binding parameters such as Kq and Bw,av were determined using the limiting slopes technique, followed by curve peeling by the Rosenthal method described in reference (72). Please refer to Figs. 62 and 63 in Appendix A for better understanding of this method. Briefly, in the limiting slopes technique, the slopes and intercepts of the straight-line segments approximating the tangents to the curve as the Scatchard plot intersects the axes can give us considerable information (see Fig. 62, Appendix A). In the case of two classes of sites, if the non-specific binding has been exactly determined, these slopes and intercepts (1^, S^, I2, and S2) enable us to calculate K^, R^, K2 and R2 (where K= the association constant, and R- B ^ x ) . These calculations are easily done by a calculator. This is then followed by the graphic curve peeling method described in 30 Fig. €3 of Appendix A, which gives us very good estimates of the equilibrium binding parameters. Protein Determination Protein concentrations in the brain samples were determined using the bicinchoninic acid (BCA) method developed by a group from the Pierce Chemical Co. in Rockford, Illinois (73). The principle of this assay is based upon the property of proteins to react with alkaline copper II to produce copper I. The BCA protein Assay Reagent reacts with copper I to form an intense purple color which is measured at 562 nm. BCA, the key component in the BCA Protein Assay Reagent, forms alkali metal salts which are soluble in water due to the polar carboxylic acid groups. The purple reaction product, formed by the interaction of two molecules of BCA with one cuprous ion, is also water-soluble enabling the spectrophotometric measurement of an aqueous protein solution. Protein + Cu2 + ------> BCA - Cu+ complex The BCA Protein Assay Reagents are the following: Reagent A - consists of an aqueous solution of 1% BCA-Na2, 2% NaC0 3 .H2O, 0.16% Na2 tartrate, 0.4% NaOH and 0.95% NaHCC>3 . The pH is adjusted to 11.25 with NaOH. Reagent B - consists of 4% CuS04 .5H2O in deionized water. Reagents A and B are stable indefinitely at room temperature. The BCA Protein Assay Working Reagent (WR) is 31 prepared by mixing 50 parts Reagent A to 1 part Reagent B. To run the assay, mix 1 volume of sample or bovine serum albumin standard with 20 volumes of WR (usually 50ul of sample or standard with 1ml of HR). incubate at 60°C for 30 min. Cool all the tubes to room temperature before reading the absorbance of purple color at 562 nm. Bovine serum albumin is used as standard in the range of lug-50ug/50ul aliquot. The advantages of using the BCA method for determining protein concentrations in samples over other methods are many. The method of Lowry et al. (74) for protein determination relies on the Folin-Ciocalteau reagent to enhance the sensitivity of the biuret reaction. The instability of this reagent in an alkaline medium demands precise timing of both reagent addition and mixing with sample in order to obtain accurate results. Non-ionic detergents as well as some buffer salts used at concentrations useful for protein solubilization can interfere by forming insoluble precipitates with the Folin- Ciocalteau reagent. It seems apparent that most of the difficulties associated with the Lowry method are due to the peculiarities of the detection reagent used. The coomassie blue method of Bradford (75) is based on the observation that Coomassie Brilliant Blue G-250 exists in two different color forms, red and blue. The red form is converted to the blue form upon binding of the dye to protein. The protein-dye complex has a high extinction coefficient thus leading to high sensitivity in measurement of the protein. The binding of the dye to protein is a very rapid process (approximately 2 min), and the protein-dye complex remains dispersed in solution for a relatively long time (about 1 hour) until the absorbance is measured at 595nm, thus making the procedure quite rapid without critical timing of the assay. This method for determining protein concentrations in samples has several advantages over Lowry's method, the most important ones being that Lowry's procedure is subject to interference by several cations, EDTA, different buffers, thiol reagents, and carbohydrates, whereas all of these do not interfere with Bradford's method. In addition, in Lowry's method precise timing of the assay is critical, whereas in the coomassie blue method it is not. However, Bradford's method for determining protein concentrations has also its drawbacks which led us to use the BCA method for our studies. One of the major disadvantages of Bradford's method is the time consuming procedure of protein reagent preparation using the Coomassie Brilliant Blue G-250 dye which must be dissolved in 95% ethanol and phosphoric acid, and then filtered on Whatman filter papers for hours before it is ready to use. The commercially available reagent is very costly, therefore we prepared our own reagent in the lab when we were using this procedure. In addition, these 33 solutions have to be prepared fresh whenever needed, since they are not stable for more than a few days, even when refrigerated. Another major disadvantage of this method is that it is not as sensitive as the BCA method, and can not detect protein concentrations less than Sug/ml in samples. Lastly, since the protein-dye complex is stable for only one hour, only about 30 samples can be prepared and analyzed at a time, which is not convenient when hundreds of samples are to be analyzed. This is not a problem with the BCA method which allows analysis of hundreds of samples without regulating reaction time. Thus, it is clear that the BCA method for protein determination has many advantages over Lowry* s and Bradford's methods. The reagents used in the BCA method are stable indefinitely at room temperature, and therefore do not need to be prepared fresh on the day the assay is performed, in addition, the BCA reagent has excellent stability at elevated temperatures, which has opened up the possibility of working out several different incubation protocols. The temperatures at which this protein assay can be carried out are room temperature, 37°C, and 60°C. This type of protocol flexibility allows the investigator to "fine tune" the sensitivity needed for a particular assay. It is noteworthy to point out that the room temperature incubations can be extended overnight, allowing the preparation of samples in the late afternoon and measuring of absorbances the following morning. This makes running the assay very relaxed, especially since the incubation timings are not critical. The BCA assay is also more tolerant to compounds that interfere with the Lowry method. Of particular importance is the tolerance of the BCA method to detergents. Since detergents are frequently used for solubilizing proteins and their complete removal prior to assaying with the Lowry method is often difficult, this becomes a significant advantage of the BCA technique. Also noteworthy is the fact that denaturing reagents (4M guanidine-HCl or 3M urea) are fairly well tolerated by the BCA method, while they interfere with the Lowry's method. Thus, it is clear that the BCA method is superior to the Lowry's method for protein determination. Briefly, the BCA technique offers manipulative simplifications, more tolerance toward interfering substances, greater working reagent stability, increased sensitivity, and greater protocol flexibility when compared to the standard Lowry or Bradford's Coomassie Brilliant Blue G-250 assays. For a comparison of the sensitivities of these three protocols of protein determination, please refer to Fig. 64 in Appendix CHAPTER III RESULTS Receptor Binding Studies Uaing ttlfl Opioid Receptor Antagonist Naloxone The saturation curves for [3H]-naloxone binding to rat brain opioid receptors using three different methods of membrane preparation are presented in Fig. 3. The first method used, as indicated in Fig. 3, is essentially the same as that described in the Materials and Methods Section, except that the buffer used included lOmM EDTA, and the whole brain membrane preparations were preincubated for 15 min at 3 0°C. It was not appropriate to use this method in our binding asays since EDTA was present in the buffer which would chelate the ions that were of interest to us. The second method that was used (Fig. 3) included no EDTA, had a 40 min preincubation at 37°C of the membrane preparations (as described in the Materials and Methods section) and was the method of choice. It was subsequently used to prepare brain membranes throughout all of our studies. Finally, the third method involved no preincubation step during the brain membrane preparation, which is essential to allow any endogenous opioid peptides 35 1 “ 1500 £ I 1200 m LJ 900 u_ £ GOO 2 x 300 o —- c i z i ao as 1.0 1.5 3H-NAL0X0NE CnM3 Figure 3. Saturation curvts of [3H]-naloxone binding to rat brain opioid receptors using three different methods of brain membrane preparation. Method 1- Preincubation of the brain membranes was carried on for 15 min at 30°C, and the assay buffer included lOmM EDTA. Method 2- Is described in detail in the Materials and Methods section, included no EDTA, and the p re incubation of the brain membrane preparations was for 40 min at 3 7 ° c . Method 3 - Is similar to Method 2, but did not include a preincubation step. 2 1500 2 1200 900 L u 600 300 i o r 120 INCUBATION TIME (minutes) Figure 4. Equilibrium binding of [3H]-naloxone (O.SnM) at 25®C to rat brain opioid receptors. See the Materials and Methods Section for details. 37 to dissociate from their receptors and be degraded by proteolytic enzymes. This step is necessary, as seen in Fig. 3, to obtain maximum binding of our radiolabeled ligand, since the free opioid receptors (Fig. 3, method 2) show a much higher specific binding of naloxone than the receptors which were not subjected to preincubation (Fig. 3, method 3). Hence the second method, which is described in detail in the previous Chapter, was used in all of the subsequent experiments. The equilibrium binding of the opioid receptor antagonist naloxone is shown in Fig. 4, which shows [ 3H] - naloxone binding reaching equilibrium after 30 minutes of incubation at 25°C. Fig. 5 depicts [3H]-naloxone binding reaching saturation with about 2nM of the radiolabelled ligand. The specific binding of naloxone both as a function of radiolabelled ligand concentration and time is presented in Fig. 6 . The inhibition of [3H]-naloxone binding to rat cortex opioid receptors by zinc ions is shown in Fig. 7. Fig. 8 shows that ImM histidine is capable of preventing the zinc inhibition by increasing the IC5 Q of zinc. The dose- dependent effect of zinc was also observed in other brain areas studied such as the midbrain (Fig. 9) , striatum and hippocampus (Table 3) . Table 3 shows the IC50 of zinc in the presence and absence of ImM histidine in the brain areas described above. Table 3 also shows the IC5 Q of zinc 38 6000 u 5000 X% 2 0 0 0 * o — i £ 1 0 0 0 * i 3= 3H-NAL0X0NE CnMl Figure 5. Typical curve* of [3H]-naloxone binding to rat cortex opioid receptors generated by direct binding assays. Open circles denote total binding; closed circles denote specific binding calculated as the difference between total binding and non-specific binding which is represented by closed triangles. c (V o faL 0 7 ■ O ■ u - 0 3 LU XE o _ j 3H-NALDXONE CnMl Figure 6 . Specific binding of t3H]-naloxone as a function of radiolabelled ligand concentration and time. 39 2 2 looo as w E 7 5 0 u *2 s i - m 1 § 25Q ? * a 0 ■6 A 3 ZnCl? CM] Figure 7. Inhibition of [3H]-naloxone binding to rat cortex opioid receptors by ZnCl2 • to 1000 m o 750 500- 250 10"7 10“« 10*5 IQ*4 lO*30 ZnCl2 CM] Figure 8. The inhibition of [3H]-naloxone binding to rat cortex opioid receptors by ZnCl2 in the presence (o} and absence (e) of ImM histidine. 1200 40 2 CD 900 C_) 600 £ 300 l (*= 0 0 Figure 9. The inhibition of [3H]-naloxone binding to rat midbrain opioid receptors by ZnCl2 in the presence (e) and absence (o) of ImM histidine. 2500 | 2000 u 1500 u_ a looo LU S 500* X o _! •X Z I 31 ffl 10-* HISTIDINE 00 Figure 10. The effect of histidine on [3H]-naloxone binding to rat midbrain opioid receptors in the presence (e) and absence (o) of 25uM ZnClj- TABUS 3. INHIBITION OP (^-NAIOSXIE BINDING TO OPIOID RECEPTORS OF FAT ERAIN BY ZINC ICNS iCtjQ of zn2+ (i*!) UNDIALYZED DIALYZED -HistidineIU a +Histidine" 1 J _U-Histidine 4 1 J I +Histidine" Itll, CORTEX 6014.5 320111 (5.3) 3614 220130 (6 .1) MIDBRAIN 42113 116118 (2 .8 ) 3113 177140 (5.7) STRIATUM 36116 9814.5 (2.7) 44114 262144 (6.0) HIPPOCAMPUS 56111 139120 (2.5) 4018.5 244138 (6.1) Numbers in parentheses represent the fold increase in zinc IC50 in the presence of ImM histidine. The values for zinc IC50 reported here are the MEAN ± S.D. of at least two experiments performed in duplicate. Student t-test was used to compare the 1050*8 of the group -histidine to that of +histidine; *P<0.001 compared to the -histidine group. ICso= The concentration of zinc which causes 50% inhibition of [3H]-naloxone binding. 42 in those same areas but using membrane preparations that were dialyzed for 3 h at 4°C. Pig. 10 depicts that b histidine concentrations up to 5mM have no effect on [3H]-naloxone binding in the rat midbrain. However, when 25uM zinc was included in the assay mixture with various concentrations of histidine, histidine concentrations greater than lOOuM were able to prevent the zinc inhibition. Histidine metabolites such as histamine and imidazoleacetic acid, and also citrate were without effects on the zinc inhibition of [3H]-naloxone binding (Figs. Il ls). In fact, citrate by itself was inhibitory to the binding of [3H]-naloxone to opioid receptors (Fig. 13). Scatchard analysis revealed a single high affinity binding site for [3H]-naloxone (Figs. 14 and 15). In undialyzed membrane preparations, zinc ions increased the Kd of the opioid receptors for [3H]-naloxone by 2 and 3.5 fold in the cortex and midbrain membranes, respectively (Table 4; Figs. 14 and 15). Histidine alone had no significant effect on the Kq in cortex and midbrain (Table 4) . When histidine and zinc were used together, histidine decreased the zinc-induced increase in the significantly in the cortex, and to a lesser extent in the midbrain. was not affected in either tissue upon the addition of ImM histidine and/or 30uM zinc (Table 4; Figs. 14 and 15), since analysis of variance followed by Scheffe's test did not reveal any statistically significant differences. When “ 1500 £ S 1200 m u. goo £ 600 UJ s 300 —Is zI 5= lO " 4 HISTAMINE CM! Figure 11. The effect of histamine on [3H]-naloxone binding to rat cortex opioid receptors. 2500 % 2000 £ “ 1500 U_ B 1000 LU 500 ■ X5 o 3* i W 10*5 10- IMI0AZ0LE ACETIC ACID CW Figure 12. The effect of imidazole acetic acid on [3H]- naloxone binding to rat midbrain opioid receptors in the presence (e) and absence (o) of 25uM ZnCl2. to rat cortex opioid receptors. opioid cortex rat to iue 3 Te fet o ctae IM () n histamine and (o) (ImM) citrate of effects The 13. Figure IM () n h zn ihbto o [ of inhibition zinc the on (♦) (ImM) ^-NALOXONE SPECIFIC BINDING (CPM) 1200 1500 300 600 900 - With] ^4 Hlstoeine 3 ]nlxn binding H]-naloxone With1 aH Citrate 3 " 0 1 2 - 0 1 1 - 0 1 44 0.15 • Control O With IsM Histidine 3 . 0.12 * With 30sM ZnCl2 _c ♦ With ZnCl* ♦ 5 0.09 LL. £ Histidine £ h. 0.06 '•i. M 0.03 0.00 0 25 50 75 100 125 B0UN0 (fmol/mg protein) Figure 14. Scatchard analysis of [3H]-naloxone binding bo rat cortex opioid receptors in the presence and absence of 30uM ZnCl2 and/or ImM histidine. 0.10 Control ^ aoe o With IsM Histidina ♦ With 30^H ZnCl2 S 0.06 ♦ With ZnCl2 Histidina S ’ 0.04 15 30 45 60 75 B0UN0 (fmol/mg protein) Figure 15. Scatchard analysis of [3H]-naloxone binding to rat midbrain opioid receptors in the presence and absence of 30uM ZnCl2 and/or ImM histidine. TABLE 4. [3 H)-NAK»OONE BOOING ID BAT BRAIN MEMBRANES: SlfMARY OF EQUILIBRIUM BINDING PARAMETERS DERIVED EBON SCATCHARD PLOTS OONIHDL HISTIDINEZINC ZINC + HISTIDINE 1. CORTEX Kq 0.87+0.10 0.82+0.065 1.78+0.075* 1,10+0.21** fiiHY 85+2.0 124+17.0 111+14.5 119+15.0 2.00KIEX-DIALYZED Kq 1.70+0.12 1.14+0.08 2.91+0.48* 1.80+0.54 B ^ 108+23.0 69+18.0 99+8.1 91+11.0 3. MIDBRAIN Kq 0.93+0.20 1.06+0.06 3.21+0.71* 2.90+0.36 B ^ 89+21.0 89+15.0 71+4.5 97+2.5 4. MIDERAIN-DIALyZED Kq 1.73+0.78 2.20+1.40 4.0+0.86* 3.20+0.60 B^v 78+21.0 80+31.0 75+20.0 87+12.0 The values reported are the MEAN + S.D. of at least two experiments performed in duplicate. Kq = Dissociation constant, in rM. B ^ y = Maximal runber of binding sites, in tnol/wq protein. Zinc = 30 uM; Histidine = 1 nM. Statistical analysis was performed using two-way analysis of variance followed by Scheffe's multiple ocnparison test. *P<0.05 vs. control; **P<0.05 vs. zinc. 47 the cortex and midbrain membrane preparations ware dialyzed, the effect of histidine and/or zinc on the Kq and Bgmx of the opioid receptors was quite similar to those of the undialyzed membranes (Table 4). Since zinc decreased the affinity of the opioid receptors for naloxone, we performed additional experiments to see whether zinc affected the equilibrium binding of [3H]-naloxone to opioid receptors. Fig. 16 shows the time course of [3H]-naloxone binding to rat cortex opioid receptors in the presence and absence of 30uM zinc. This data indicates that naloxone reaches equilibrium after 15 min of incubation in the presence of zinc, compared to 30 min in the absence of zinc, and therefore it is not necessary to prolong the incubation period when zinc is present in the assay mixture. The effects of several other cations such as Hg2+, Cu2+, Cd2+, Co2+, Ni2+, Mg2+, and Mn2+ on [3H]-naloxone binding were also studied in the presence and absence of histidine. The IC50 values of some of these ions which were inhibitory to naloxone binding are reported in Table 5 in the presence and absence of ImM histidine. We observed that histidine was capable of increasing the IC50 of Cu2+ by 2.7 fold, Cd2+ by 1.3 fold, but did not increase that of Hg2+. In the same tissue preparations, histidine increased the IC5 Q of zinc by 5.3 fold. Figs. 17, 18 and 19 show the inhibitory effects of Cu2+, Cd2+, and Hg2+, respectively, in the presence and absence of ImM histidine. 48 1S00 1200 900 s 600 300 0 30 60 90 120 TIME OF INCUBATION C sinutas) Figure 16. Equilibrium binding of [3H]-naloxone in the rat cortex in presence (o) and absence (e) of 30uM ZnCl2* eoo OD 600 Ss S 400 200 - 4 10“* 10 * C u C l , CM3 Figure 17. The inhibition of [3H]-naloxone binding to rat cortex opioid receptors by CUCI2 in the presence (o) and absence (e) of ImM histidine. 1000 49 i£ m 750 c_> u_ u 500 £ 250 Figure 18. The inhibition of [3H]-naloxone binding to rat cortex opioid receptors by HgCl2 in the presence (e) and absence (o) of ImM histidine. L3 800 z £ 600 uS. 400 200 10- 10-* CdCl2 CM3 Figure 19. The inhibition of [3H]-naloxone binding to rat cortex opioid receptors by CdCl2 in the presence (e) and absence (o) of ImM histidine. TABLE 5. EFFECT OF DIVALBfT CMTCNS CN [^-NAIflXCNE BINDING TO RAT OQRZEX OPIOID RECEPTORS IC5 0 (*0 £ 2 K m ______~ HISTIDINE______+ HISTIDINE Zn2* 60114 320111* (5,3) CU2+ 2412.0 6512,4*(2.7) Cd2+ 2011.0 2515.0 (1.3) Hg2+ 1313.5 811.3 (0.6) ICcn=» The concentration of the cation which causes 50% inhibition of 3 H-nalawxie binding. Nunfcers in parentheses represent the fold increase in IC5 0 in the presence of lirM histidine. The values for the IC^'s reported here are the MEAN 1 S.D. of 2-4 experiments performed in duplicate. Student t-test was used to oonpare the ICgo's of the groqp -histidine to that of -fhistidine; *P<0.001 oonpared to the -histidine grcup. The effectiveness of these cations in inhibiting [ 3H] - naloxone binding to rat cortex opioid receptors was of the following order: Hg2*>Cd2*,Cu2*>Zn2*. Histidine prevented the inhibitory effects of these ions by increasing their IC50 in the following order: Zn2+>Cu2+>Cd2+>Hg2+. Pig. 20 depicts the effect of Co2* on the binding of [3H]-naloxone to cortex opioid receptors in the presence and absence of ImM histidine. As seen in this figure, Co2* has a dual effect on naloxone binding, first stimulating it at concentrations between 5-5 OuM, and then inhibiting it at concentrations greater than lOOuM. Histidine abolished the stimulatory action, and was without any effect on the inhibitory action of Co2+. The effect of Ni2* on naloxone binding to cortex opioid receptors is shown in Fig. 21. Ni2* stimulated naloxone binding at concentrations between 50-500uM, and inhibited it at higher concentrations. Histidine did not abolish either effect of Ni2*, but overall decreased the specific binding of naloxone. The action of Mn2* on naloxone binding is presented in Fig. 22, which shows the stimulatory effect of Mn2* at concentrations greater than lOOuM. Histidine prevented this effect to a certain extent. Fig. 23 depicts the slight stimulatory effect of Mg2* on naloxone binding, which was completely abolished by the addition of 30uM zinc to the assay mixture. This shows that 3 OuM zinc is able to overcome the effect of millimolar concentrations of Mg2*. 52 2500 i£ CD 2000 U 1500 1000 With IsM Histidina 500 l s Figure 20. The dual effect of CoCl2 on [3H]-naloxone binding to cortex opioid receptors (o). Histidine (ImM) was able to abolish the stimulatory effects of CoCl2, but not its inhibitory effects (#). 2000 so 1500 CJ a 500 z 1 With IsM Histidina £ CHI Figure 21. The dual effect of NiCl2 on [3H]-naloxone binding to cortex opioid receptors in the presence (e) and absence (o) of ImM histidine. 53 1000 £ | CO 750 o u £ 10- MnCl m Figure 22. The effect of MnCl2 on [3H]-naloxone binding to rat cortex opioid receptors in the presence (o) and absence (e) of ImM histidine. 1200 900 ■ 600 300 lQ-= 1Q-* 10-3 10*z MgCl2 Z H 1 Figure 23. The effect of MgCl2 on [3H]-naloxone binding to rat cortex opioid receptors in the presence (o) and absence (e) of 3OuM ZnCl2. 54 EFFECT OF ZINC AND/OR HISTIDINE ON VARIOUS SUBTYPES OF OPIOID RECEPTORS 1. Receptor Binding Studies Using the Mu-Receptor Agonist DAGO (TD-Al A N-METHYL-PHEa. GLYOI^I -ENKEPHALIN) The saturation curve for [3H]-DAGO binding to rat cortex opioid receptors is presented in Fig. 24, which shows that saturation is reached with about lOnM DAGO. The time-course for [3H]—DAGO binding to cortex opioid receptors in the presence and absence of 3OuM zinc is presented in Fig. 25. It shows that the binding of [3H]-DAGO reaches equilibrium after 1 hour incubation at 25°C, whereas in the presence of zinc, equilibrium is reached after about 90 minutes of incubation. Zinc inhibition of [3H]-DAGO binding in the presence and absence of ImM histidine is presented in Fig. 26. It shows that the IC5 Q of zinc is about 37uM, and that histidine increases it to about 25OuM. Fig. 27 shows that concentrations of histidine between lOOuM and 500uM are able to prevent the zinc inhibition of [3H]-DAG0 binding to the midbrain, whereas histidine concentrations greater than ImM are inhibitory. The inhibition of [3H]-DAG0 binding by Hg2+ in the presence and absence of histidine is shown in Fig. 28. Histidine was not able to prevent the inhibition of DAGO binding by Hg2+. Fig. 29 represents the inhibition of DAGO binding by Cu2+, and shows that histidine is capable of preventing the inhibitory effect of Cu2+. The 55 5000 §- 4000 3000 S 2000 ■oj 1000 Figure 24. Typical curves of [3H]-DAGO binding to rat cortex opioid receptors generated by direct binding assays. Open circles denote total binding; closed circles denote specific binding calculated as the difference between total binding and non-specific binding which is represented by closed triangles. 1800 L3 Z i 1500 CD C_J 1200 U. CJ S. 900 U1 u OL CD O 600 (j •< ai 300 z CD 0 30 60 90 120 TIME OF INCUBATION (m inutes) Figure 25. Equilibrium binding of [3H]-DAGO (InM) in the rat cortex in presence (o) and absence (e) of 3OuM Zncl2 * iua 6 Ta niiin y Zncl by inhibition Tha 26. Figura a crac pod aatr i ta rcna a ad absanca and (a) pracanca tha in racaptors opioid cortaacrat o o UM histidina. UnM of(o) ’’H-OAGO SPECIFIC BINDING ^I-DAGO SPECIFIC BINOING (cpa) (cpa) 1200 U> o» to i\> ut Ul to O a o D o o a CD o x to M—t •—«C3 Z [ of2 m 3 *—• ]DG bnig to binding H]-DAGO o o 0* Ma o Ul o \ 57 1500 00 <_> 1000 3 8- 500 H g C l2 CM] Figure 28. The inhibition of [3H]-DAGO binding to rat cortex opioid receptors by HgCl2 in the presence (o) and absence (e) of ImM histidine. 03 5 1500 2 m u_ 1000 u S. UJ u Q_ cn 500 oi C uC l Figure 29. The inhibition of [3H]-DAGO binding to rat cortex opioid receptors by CuCl2 in the presence (o) and absence (e) of ImM histidine. 58 inhibitory effect of Cd2+ is presented in Fig. 30, which shows that histidine increases the IC50 of Cd2+ from about 20uM to about 25uM. Thus, the inhibitory effects of these cations on [3H]-DAG0 binding to cortex opioid receptors is of the following order: Hg2+>Cd2+ ,Cu2+>Zn2+. Histidine prevented their inhibitory effects by increasing their IC50 in the following order: Zn2+>Cu2+>Cd2+>Hg2+. Table 6 represents the IC5q of these different cations on [3H]-DAGO binding in the presence and absence of ImM histidine. Since naloxone is an opioid receptor antagonist with about fifteen times higher affinity for the mu receptors than for the delta or kappa receptors, we compared the effects of 2inc, copper, cadmium, and mercury, in the presence and absence of histidine, on the binding of [3H]-naloxone to that of [3H]-DAGO in the rat cortex. As seen in Tables 5 and 6 , these ions had the same order of inhibiton on [3H]- DAGO binding as that of [3H]-naloxone. Histidine increased their IC50 to the same extent for both ligands. This suggests that these cations do not differentiate mu receptor agonists from antagonists, since they affected their binding in a similar fashion, and to the same extent. Fig. 31 depicts the stimulatory effect of Mg2+ on [3H]- DAGO binding to cortex opioid receptors; this effect is completely prevented when 30uM zinc is included in the assay mixture with increasing concentrations of Mg2+. Thus, as with naloxone binding, 30uM zinc was capable of 59 preventing the stimulatory effect of millimolar concentrations of Mg2+. Scatchard analysis of [3H]-DAGO binding to the rat cortex opioid receptors revealed a curvilinear scatchard plot which could be resolved into a high and a low affinity binding site (Fig. 32A). The high affinity site had a KD of 0.40 nM and a Bq^x of 30 fmol/mg protein. The low affinity site had a KD of 1.94 nM and a Bj,a x of 110 fmol/mg protein. In the presence of ImM histidine (Fig. 32B), the high affinity site had a KD of 0.35 nM, and a B ^ y of 30 fmol/mg protein, whereas the low affinity site had a KD of 3.70 nM, and a B^y of 140 fmol/mg protein. Fig. 32C shows the scatchard plot of [3H]-DAGO binding to rat cortex opioid receptors in the presence of 30uM ZnCl2, where the high affinity site has a KD of 2.0 nM, and a Bmax of 30 fmol/mg protein, and a low affinity site with a Kq of 4.35 nM, and a B»ax of 150 fmol/mg protein. Lastly, in the presence of histidine (ImM) and ZnCl2 (30uM) together, the Kq for the high affinity site was 1.40 nM, and the B ^ y was 30 fmol/mg protein; the low affinity site had a Kq of 3.40 nM, and a B^x of 135 fmol/mg protein. These experiments were repeated at least three times, and the experimental error was less than 10%. Thus the results of the scatchard analysis show that: a) histidine had no effect on the Kq and Bnax of the high affinity binding site, whereas zinc increased the KD without affecting the B^ax? when histidine was present with zinc, it decreased the KD for the high affinity site compared to zinc alone, but did not restore it to control levels, and the was unaffected; b) histidine and zinc both alone and together increased the K0 for the low affinity site; the 3m »v was increased in the presence of histidine or zinc, and was slightly decreased when the two were included in the assay mixture together. These results show that both histidine and zinc have similar effects on the low affinity binding sites of the mu receptors. Histidine alone has no effect on the high affinity site, and decreases the zinc-induced increase in the Kd, although not completely. These observations extend the results depicted in Fig. 27, which shows that histidine concentrations greater than 500uM which are inhibitory to [3H]-DAGO binding to opioid receptors also affect the KD and BJnax of the low affinity sites of the mu receptors by increasing them. On the other hand, Fig. 10 shows that histidine concentrations up to 5mM that have no effect on [3H] -naloxone binding, have also no effect on the Kq and Bmax of °pi°id receptors for naloxone (Table 4). Opioid receptors have been reported to have essential sulfhydryl groups which must be in the reduced form for the receptor to be active (66,67). Since divalent metal ions such as Cu2+ and Zn2+ are also oxidizing agents, we believe that these metals inhibit the binding of DAGO to the opioid receptors by oxidizing the essential thiol groups on the 1500 61 2 i 1200 m CJ 900 oS. 600 (-3 ■< a i 300- JF 10-? 10-8 I0-s CdCl2 00 Figure 30. The inhibition of [3H]-DAGO binding in the rat cortex by CdCl2 in the presence (o) and absence (e) of ImM histidine. 2000 L3 Z 1500 03I (_> £ S- 1000 L U o 5? | 5 0 0 01 z tn 0 10*5 10“4 10*3 10-2 10"1 H g C lj [M3 Figure 31. The effect of HgCl2 on [3H]-DAGO binding to rat cortex opioid receptors in the presence (o) and absence (e) of 30uM ZnCl2. TABLE 6. EFFECT OF DIVAlfNT CATICNS ON (3H]-Cft30 BINDING TO RAT OOKIEX OPIOID RECEPTORS ICgQ (pM) Zn2* 37 ± 7.0 250 ± 25,0* (6 .8 ) CU2+ 23 ± 2.5 130 + 12.5* (5.7) ca2+ 20 ± 1.0 25 + 0.10* (1.3) Hg2+ 9 ± 1.0 7 + 0.60 (0.8) IC5 Q = The ooncentraticn of the cation which causes 50% inhibition of [3H)-DM30 binding. Numbers in parentheses represent the fold increase in IC5 0 in the presence of luM histidine. The ic^ 0 values reported here are the MEAN + S.D. of 2-4 experiments performed in duplicate. Student t-test was used to ccnpare the IC^g's of the group -histidine to that of +histidine; *P< 0.005 ocnpared to the -histidine group. cortex opioid receptors. A. Control; B. with ImM histidine; ImM with B. [ ZnCl Control; of A. 3OuM With C. receptors. anlaysis opioid Scatchard cortex 32. Figure B/F (fed/eg protein/pH) B/F (feol/eg protoin/pN) 5 10 5 20 250 200 150 protein) (feol/eg 100 BOUND 50 0 5 10 150 100 protein) 50 (fnol/ng BOUND P —! 0 3 !- a— _____190 _____190 Kca- 2.0 nM 2.0 Kca- K q *- -110 4.39 nM 4.39 2 tmml/mq * D. With 30uM ZnCl 30uM With * D. fmol/mg protaln protein fMl/Mg 02 03 0 BOUND (frol/eg protein) (frol/eg BOUND 3 B W N D (fool/eg protein) (fool/eg D N W B 5 10 5 20 250 200 150 100 50 0 ]DG bnig o rat to binding H]-DAGO 2 and ImM histidine. ImM and W 3.40 nM 3.40 W "30 0 3 l" Kb*- JlnM 70 Kb*- 140 140 fmol/mg protein fmol/ protaln 64 mu-receptors and decreasing their affinity for DAGO. Histidine is able to prevent their effects by chelating these metal ions. In view of all this, we decided to look at the effect of several known reducing agents and an oxidizing agent on [3H]-DAGO binding in the presence and absence of 30uM zinc. Fig. 33 shows that the reducing agent beta-mercaptoethanol (B-ME) has no effect on [3H]-DAGO binding in the rat cortex in the absence of zinc at concentrations up to 5mM, after which it has inhibitory effects. In the presence of 30uM zinc, beta-mercaptoethanol had no effects on the zinc inhibition of DAGO binding (Fig. 33) . Another reducing agent, dithiothreitol (DTT), had no effects on [3H]-DAGO binding to rat cortex mu-receptors by itself, but increased the binding in the presence of 30uM zinc by about 50% (Fig. 34). Fig. 35 represents the action of the oxidizing reagent dithiobisnitrobenzoic acid (DTNB) on [3H]-DAG0 binding in the rat cortex. In the absence of zinc, DTNB decreased the binding of DAGO in a dose- dependent manner and at a concentration of 2mM the binding decreased by 75%. In the presence of 30uM zinc, DTNB potentiated the inhibition of DAGO binding induced by zinc. The studies using these sulfhydryl reagents extend previous findings about the presence of essential SH-groups on the opioid receptors. Thus, we demonstrated that DTT was able to prevent the inhibitory effects of zinc by about 50% at a concentration of lOmM, whereas B-ME which is a weaker 65 2 1500 2 1250 1000 C_) 750 500- L3 at 10“ 4 IQ"3 0-MERCAPTOETHANOL CM! Figure 33. The effect of beta-mercaptoethanol on [3H]-DAGO binding to cortex opioid receptors in the presence (o) and absence (e) of 30uM ZnC12. 2000 2 2 1500 ca LJ CJ- X o 1000 LU 500 Q l 3= 10-* 10-3 DITHIOTHREITOL EMI Figure 34. The effect of dithiothreitol on [3H]-DAGO binding to rat cortex opioid receptors in presence (o) and absence (e) of 30uM ZnC12. 66 «■ 2000 £ s 1500 u u 1000 81 O U 500 O I DITHI0BISNITR0BENZ0IC ACID CHI Figure 35. The inhibitory effects of dithiobisnitrobenzoic acid on £3H]-DAGO binding to rat cortex opioid receptors in the presence (o) and absence (e) of 30uM ZnCl^. reducing reagent than DTT could not. However, DTT did not completely reverse the effect of zinc on [3H]-DAGO binding, which shows that the inhibitory effect of zinc is probably by two mechanisms: by oxidizing SH-groups on the mu- receptors essential for binding, and by competing with stimulatory ions such as Mg2+ and preventing their binding to cation binding sites on the opioid receptor, and preventing their effects. The oxidizing reagent DTNB inhibited [3H]-DAGO binding to opioid receptors, and this effect was potentiated in the presence of 30uM zinc. This again shows that zinc can cause inhibition of opioid receptor binding by another mechanism besides oxidizing SH- groups on the receptor. The enkephalinase inhibitor thiorphan, and the amino- peptidase inhibitor bestatin (76) have been reported to have analgesic effects when administered to animals (77,78). These enkephalin and endorphin-degrading enzymes are zinc-metalloenzymes. Bestatin and thiorphan inhibit the activity of these enzymes by complexing the zinc ions present at their active site through the thiol groups present in these inhibitors. Since these peptidase inhibitors exert their effects by complexing the zinc ions present in these enzymes, we wanted to see whether they were able to complex zinc ions in vitro and reverse their inhibitory effects on [3H]-DAGO binding, which could also explain their analgesic activity. Thus, Figs. 36 and 37 68 1 8 0 0 2 2 1 5 0 0 1200 9 0 0 6 0 0 - LS «F 10*e 10-7 10-« io-3 jq-4 kj-3 DRUG CHI Figure 36. The effects of bestatin (e) and thiorphan (o) on [ H]—DAGO binding to rat midbrain opioid receptors in the presence of 30uM ZnCI2. The specific binding of [3H]-DAGO in the absence of zinc and drugs (e), and in the absence of drugs only (♦) are denoted on the figure. g 1000 2 s 7 5 0 o a§ X W 5 0 0 5? 3 4 •..... o g 2 5 0 r i= 0 it ■ * * -* 0 lO"10 10-® 1Q-® IQ"7 10-* DRUG DO Figure 37. The effects of bestatin (e) and thiorphan (o) on [3H]-DAGO binding to rat hypothalamus opioid receptors in the presence of 30uM ZnCl2. The specific binding of [3H]- DAGO in the absence of zinc and drugs (a), and in the absence of drugs only (a) are denoted on the figure. 69 represent the effects of increasing concentrations of bestatin and thiorphan on the zinc inhibition of [3H]-DAGO binding in the midbrain and hypothalamus, respectively. In both tissues thiorphan and bestatin did not have any significant effects on the zinc inhibition of [3H]-DAGO binding. Bestatin and thiorphan, when used alone, had no effect on [3H]-DAGO binding over a concentration range of lOOnM-lOOuM (Figs. 38 and 39). From these studies bestatin and thiorphan do not appear to be able to complex free zinc ions in the brain. Thus, it appears that the analgesic effect of these enzyme inhibitors is not because of the removal of free zinc ions present in the brain, and therefore preventing their inhibitory effect, but presumably because they inhibit the enzymes that degrade the endogenous opioid peptides, and thus increase their concentration in the brain. 70 £ IQOO* § s 750 \3 500*- n n 250 I SF iir7 io~* i0“s io~4 BESTATIN 00 Figure 38. The effect, of increasing concentrations of bestatin on [ 3H]-DAGO binding to rat hypothalamic opioid receptors. 1250 £ £ 1000 m u li_ 750 L u U 500* I 250* SF 10-a THIORPHAN DO Figure 39. The effect of increasing concentrations of thiorphan on [3H]-DAGO binding to hypothalamic opioid receptors. 71 Z* BflS-SPtor Binding Studies Using tbs Delta-Receptor Agonist DSTLE ( fTYR-D-SER-GLY-PHE-LEU-THRI-ENKEPHALIN) The saturation curve of [3H]-DSTLE binding in the rat cortex is presented in Fig. 40, which shows that saturation is reached with about 6nM [3H]-DSTLE after one hour incubation at 25°C. The equilibrium binding of [3H]-DSTLE is shown in Fig. 41 which reveals that about 90% of maximum binding of [3H]-DSTLE is achieved after one hour incubation at 25°C. The zinc inhibition curve of [3H]-DSTLE binding to rat cortex opioid receptors is presented in Fig. 42, which also displays the effect of ImM histidine on the zinc inhibition of DSTLE binding. As depicted in Fig. 42, it can be seen that zinc is not as effective in inhibiting [3H]- DSTLE binding as it was on [3H]-DAGO or [3H]-naloxone binding. The IC5 Q of zinc on [3H]-DSTLE binding is about 550uM and 620uM in the absence and presence of histidine, respectively. Fig. 43 shows that histidine does not have significant effects on the zinc inhibition of [3H]-DSTLE binding in the rat cortex, but that histidine itself is inhibitory at concentrations greater than 500uM. The inhibition of [3H]-DSTLE binding in the cortex by Hg2+ in the presence and absence of ImM histidine is displayed in Fig. 44. The IC50 of Hg2+ is about 14uM, and is not affected significantly by histidine. Fig. 45 shows the inhibition of [3H]-DSTLE binding to cortex opioid receptors by Cu2+, in the presence and absence of ImM histidine. The 72 3500 3000- » 1500 1000 500 Figure 40. Typical curves of [3H] -DSTLE binding to rat cortex opioid receptors generated by direct binding assays. Open circles denote total binding; closed circles denote specific binding calculated as the difference between total binding and non-specific binding which is represented by closed triangles. 1000 7 5 0 500 Si to 2 5 0 o i m 0 3 0 6 0 90 12Q TIME OF INCUBATION (m inutas) Figure 41. Equilibrium binding of [3H]-DSTLE (InM) at 25°C to rat cortex opioid receptors. 73 1000 £ s 7 5 0 t 5 0 0 2 5 0 gi ir 10"® 10"® r4 ZnCl2 CM3 Figura 42. Tha inhibition of [3H]-DSTLE binding to rat cortax opioid racaptors by ZnCl2 in tha prasanca (o) and abaanca (a) of ImM histidina. 1000 7 5 0 LJ S.u 5 0 0 - & in 2 5 0 oi 5= itr4 HISTIDINE [MI Figura 43. Tha affact of histidina on [3H] -DSTLE binding to rat cortax opioid racaptors in tha prasanca (o) and absanea (a) of lOOuM ZnCl2- 74 6 0 0 1 4 0 0 u 200 cn IQ*7 10”8 10-5 10“4 1Q“3 H gC lz 0 0 Figure 44. The inhibition of [3H]-DSTLE binding to rat cortex opioid receptors by HgCl2 in the presence (o) and absence (e) of liBH histidine. 6 0 0 * CJ §. *00 o 8* cn ot 1 0 * 7 10“8 10_s 10“4 10"3 C u C l2 CM3 Figure 45. The inhibition of [3H]-DSTLE binding to rat cortex opioid receptors by CuCl2 in the presence (o) and absence (e) of ImM histidine. IC5o o f Cu2+ is about 33uM, and histidina inreases it to about 85uM. The inhibitory action of Cd2+ in the presence and absence of ImM histidina is depicted in Fig. 46, which reveals that histidine increases the IC50 of Cd2+ from 58uM to about 130uM. Thus, the inhibitory effect of these cations on [3H]-DSTLE binding is of the following order: Hg2+>Cu2+>Cd2+>Zn2+. Histidine prevents the effects of these ions by increasing their IC50 as follows: Cu2+>Cd2+>Zn2+>Hg2+. The IC5Q values of these cations in the presence and absence of ImM histidine is depicted in Table 7. The stimulatory effect of Mg2+ on [ 3H] -DSTLE binding (Fig. 47) was significantly decreased in the presence of lOOum Zn2+ which by itself had no significant effect on [3H]-DSTLE binding. Fig. 48 shows that lOOuM Zn2+ completely prevented the stimulatory effects of Mn2+ on [3H]-DSTLE binding in the cortex. From these experiments we can deduce that perhaps the delta-opioid receptors either do not contain SH-groups, or that these are not accessible to zinc which is the reason that physiological concentrations of zinc ions could not inhibit delta receptor binding. However, the studies with Mg2+ and Mn2+ which were both stimulatory to the delta opioid receptor binding, also confirm and extend our previous findings with DAGO that zinc ions compete with these stimualtory ions, prevent them from binding to the cation binding sites on the opioid receptors to exert their effects, and bind to iue 6 Te niiin f [ CdCl of by receptors inhibition absence opioid The cortex 46. Figure ^-OSTLE SPECIFIC BINDING (e) o £ 1000 250 500 750 of ImM histidine. ImM of 07 Q6 1- Q* 1Q-3 1Q-* 10-S 1Q-6 10-7 dl CM] CdCl2 2 n Ue rsne o and (o) TABLE 7. EFFECT OF DTVAIflfT CATIONS ON [^l-DSTIE BINDING TO BAT OORIEX OPIOID RBCEPTCRS IC5 0 (*<> CATION -HISTIDINE -tfgSTIDINE Zrr+ 550 + 45 620 ± 70 (1.1) CU2+ 33+2.5 85 + 10* (2.6) Cd2+ 58 + 2.5 130 + 5.0*(2.2) Hg2 14 + 2.5 12 ± 1.5 (0.9) IC5 0 » The concentration of the cation which causes 50% inhibition of [3 H]-DGTI£ binding. Numbers in parentheses represent the fold increase in IC5 Q in the presence of ImM histidine. The IC5 0 values reported here are the MEAN ± S.D. of 2-4 experiments performed in dtplicate. Student t-fcest was used to ocnpare the IC^q's of the group -histidine to that of +histidine; *Pc 0.005 ocnpared to the -histidine group. 78 1500 s 1200 900 I u 8* 600 tn 300- ai nz I0“s 10"4 10*3 10’z HgCl2 DO Figure 47. The effect of MgCl2 on [3H]-DSTLE binding to rat cortex opioid receptors in the presence (o) and absence (e) of lOOuM ZnCl2• L9 1500 63 1200 m 900 oc l 600 tn ai 3= 10“s 10’ 4 10"3 I0*2 MnClj D O Figure 48. The effect of MnCl2 on [3H]-DSTLE binding to rat cortex opioid receptors in the presence (o) and absence (e) of lOOuM ZnCl2. 79 these sites to cause inhibition of the opioid receptors. The scatchard analysis of [3H]-DSTLE binding to rat cortex opioid receptors in the presence and absence of XOOuM ZnCl2 and/or ImM histidine is depicted in Fig. 49. The high affinity site has a KD of 0.09 nM and a Bgiax of 20 fmol/mg protein. The low affinity site had a KD of 0.82 nM and a B ^x of 80 fmol/mg protein. In the presence of ImM histidine (Fig. 49B), the high affinity site was not detected, whereas the Kq of the low affinity site was increased to 1.80 nM, and the B ^ x to 135 fmol/mg protein. Fig. 49C shows the scatchard plot of [3H]-DSTLE binding to rat cortex opioid receptors in the presence of lOOuM ZnCl2, where the high affinity site is also undetected, and the Kq of the low affinity site was increased to 3.10 nM, and the ®max to 132 fmol/mg protein. Lastly, in the presence of histidine (ImM) and ZnCl2 (100UM) together (49D), the high affinity site was also undetected; histidine was not capable of preventing the effect of Zn2+, and the low affinity site had a Kq of 2.80 nM and a B ^ x of 150 fmol/mg protein. Thus the results of the scatchard analysis show that: a) histidine and zinc both alone and together abolished the high affinity binding site of [3H]-DSTLE in the rat cortex; and b) histidine and zinc both alone and together increased the Kq and the Bmax for the low affinity binding site of [3H]-DSTLE. This again is in agreement with Fig. 43, where histidine is shown not to be effective in 80 preventing the zinc inhibition, but is itself inhibitory at concentrations greater than 500uM. The reducing reagents beta-mercaptoethanol (B-ME) and dithiothreitol (DTT) by themselves had no effect on [3H]- DSTLE binding in the cortex at concentrations betveen 0- lOmM, but at a 5mM concentration were capable of preventing the inhibitory action of 500uM Zn2+ as depicted in Figs. 50 and 51, respectively. The oxidizing reagent DTNB inhibited the specific binding of [3H]-DSTLE by 65% at a concentration of 2mM (Fig. 52). When 500uM Zn2+ was included in the assay mixture with increasing concentrations of DTNB, the inhibitory effects of both compounds together was potentiated, and the specific binding of [3H]-DSTLE in the rat cortex was decreased by more than 80% in the presence of 500uM Zn2+ and 2mM DTNB (Fig. 52) . From these studies we can conclude that the inhibitory effect of 500uM zinc on [3H]-DSTLE binding is a) by oxidizing SH-groups since it was prevented by 5mM DTT and B-ME, and b) by another mechanism since it potentiated the inhibitory effects of DTNB. Fig. 53 shows that in the presence of 30uM Zn2+, bestatin and thiorphan have no effects on [3H]-DSTLE binding in the cortex, and inhibit it by about 20% at concentrations greater than 400uM. DADLE is not a very specific delta receptor agonist, and has considerable affinity to the mu receptors. We used this C. With lOOuM ZnCl2 ; D. With lOOuM ZnCl2 and lmM histidine. lmM and ZnCl2 histidine; lmM lOOuM With ; With D. B. ZnCl2 Control; lOOuM A. With C. receptors. opioid cortex iue 9 Sacad nlss f 3]DTEbnigt rat to binding [3H]-DSTLE of analysis Scatchard 49. Figure B/F (faol/ag protoin/pH) B/F (faol/ag proteln/pM) .12 .06 • .06 .01 .02 .04 .03 • .03 .09* .03 ■ BOUND (faol/ag protein) (faol/ag BOUND 5 10 150 100 50 0 protein) (faol/ag BOUND 3 6 9 120 90 60 30 0 v * » \ * - 3 3 -1 | a \ \ \ \ Kei- a O nM DO a - i e K I \ \ g « / l o « f 0 * - 1 . . U n l a t o r p • K 1 O / j l/n o M ^ SO - — U praUin i U a r p 1 \ \ ' faol/ag faol/ag / K q • \ • \ • m - 0 nM 10 . 3 - nM 2 8 . 0 ein te o r p • CD «*. ■N. LL. . i l 1 0 . = 2 0 . 5 -S. 5 0 . - 1 CD . h 8 c .04 a w A O 3 0 . P &L 4 0 * o a 2 0 . .06 6 0 . 0 0 BOUND (faol/ag protoin) (faol/ag BOUND 5 10 150 100 50 0 BOUND (faol/ag protein) (faol/ag BOUND 5 10 150 100 50 0 ^ H I O -IS H— ^ v 0 \^ \ • ■135 feel/eg protein feel/eg ■135 w a a \ Ko-1.80 r* -.0 rM o-Z.60 K \ a ^ / l fac v \ ein te o r p 82 £ i 750 m S. 500 uj 250 tn ai 0 0-MERCAPTOETHANGL CM3 Figure 50. The effect of beta-mercaptoethanol on [3H]-DSTLE binding to rat cortex opioid receptors in the presence (o) and absence (e) of 500uM ZnCl2. 1000 750 u £ 500 LU '•Wo 8* LJ 250- tn Ql 3= 10-' DITHI0THREIT0L CM3 Figure 51. The effect of dithiothreitol on [3H]-DSTLE binding to rat cortex opioid receptors in the presence (o) and absence (e) of 500uM ZnCl2- 83 1000 7 5 0 us_ 5 0 0 2 5 0 S 0 OITHIQ0ISN1TROBEN2OIC AGIO CM] Figure 52. The inhibitory effect of dithiobisnitrobenzoic acid on [3H]-DSTLE binding to rat cotex opioid receptors in the presence (o) and absence (e) of SOOuM ZnCl2. 1000 s ffi u 7 5 0 3. o 5 0 0 8* tn 2 5 0 a 10"7 10“* 10~s 10- DRUG CM] Figure 53. The effects of bestatin (o) and thiorphan (#) on [3H]-DSTLE binding to the rat cortex opioid receptors in the presence of 30uM ZnCl2. The specific binding of [3H]- DSTLE in the absence of zinc and drugs (■) , and in the absence of drugs only (♦) are denoted on the figure. 84 ligand in a few experiments to study the effect of Ca2+, Mn2+, and Mg2+ on the binding of this agonist to the delta and mu receptors. Figs. 54 and 55 show the stimulatory effects of these ions on [3H]-DADLE binding to dialyzed rat midbrain and cortex membrane preparations, respectively. In the cortex, the stimulatory effect of these ions was of the increasing order: Mn2+ > Mg2+ > Ca2+ (Fig. 55) . In the midbrain, the order was Ca2+ > Mn2+ > Mg2+ (Fig. 54). we used [3H]-DPDPE, which has been reported to be a very specific delta-receptor agonist (81) , in several experiments to compare the effects of the cations studied on different types of ligands. We were not successful in obtaining any data when using [3H]-DPDPE since we could not gat any specific binding using this ligand. 85 2 1000 5 750 2 . u *' o 500 250- ai *F IQ-3 10-4 10-3 10-2 10*1 OIVALENT CATION CM3 Figure 54. Th« stimulatory effacts of MnCl2 (o), MgCl2 (*)# and CaCl2 (•) on [3H]-DADLE binding to dialyzed cortex opioid receptors. 1200 a u 900 u . u8 cn a 300 lO"5 ID-4 ID"3 10-2 10"1 OIVALENT CATION CMI Figure 55. The stimulatory effects of MnCl2 (o), MgCl? (*)* and CaCl2 (•) on [3H]-DADI£ binding to dialyzed midbrain opioid receptors. 86 3. Receptor Binding Studies Using fchs Kappa-Receptor Agonists Dvnorphin and EKC (Ethvlketocvclazocinel The opioid receptor agonist [3H]-dynorphin A1”® was used to label the kappa opioid receptors in the rat cortex. Since dynorphin A is a peptide and can undergo rapid degradation by endogenous peptidases, bestatin and thiorphan were included in the assay mixture to inhibit the action of these peptidases. In addition, we included lOOnM of each DAGO and DADLE in the assay mixture, since dynorphin has some affinity to the mu and delta opioid receptors, and thus it is necessary to saturate these receptors by their own high affinity ligands in order to obtain dynorphin binding only to the kappa receptors. When bestatin and thiorphan were ommitted, even a 15 min incubation period at 25°C was sufficient to cause complete degradation of the radioactive dynorphin, whereas 20uM thiorphan and 30uM bestatin prevented its degradation. However, binding studies using this radioactive opioid peptide gave us a lot of problems, the most notable one being very high non-specific binding which was not acceptable to continue our studies using this ligand. Therefore, we continued our investigations using [3H]- ethylketocyclazocine (EKC), which is also a kappa receptor agonist, but is not a peptide and therefore would not be degraded by peptidases. However, EKC has also some affinity towards the mu and delta opioid receptors, which makes it 87 necessary to suppress its binding to these two receptors by including lOOnM of each DAGO and DADLE in all of our experiments when using [3H]-EKC as the ligand. The equilibrium binding of [3H]-EKC to rat cortex opioid receptors both at 0°C and 25°C is represented in Fig. 56. When the incubation of cortex membranes and [3H]-EKC is carried on at 0°c, equilibrium is reached after a one hour incubation period, whereas a 30 min incubation is enough to reach equilibrium when the assay is conducted at 25°C. Fig. 57 shows the total, non-specific, and specific binding curves of [3H]-EKC in the rat cortex. As seen in this figure, saturation is reached with 8nM of [3H]-EKC. The inhibition of [3H]-EKC binding to rat cortex kappa receptors by zinc ions is depicted in Fig. 58, which shows that the IC50 of zinc in inhibiting [3H]-EKC binding is about 150uM. Histidine (lmM) increased the IC50 of zinc by 4.2 fold to 625uM. Histidine alone had no effect on [3H]- EKC binding in the rat cortex up to a lmM concentration after which it decreased it slightly, as seen in Fig. 59. It was also capable of reversing the inhibitory effect of 200uM zinc on the binding of [3H]-EKC in a dose-dependent manner, and at a dose of lmM it completely prevented it. However at higher concentrations of histidine, the effect of zinc was again inhibitory. The scatchard analysis of [3H]-EKC binding to rat cortex opioid receptors reveals a high and a low affinity binding site (Fig. 60). The high affinity site has a KD of 1.43 nM and a Bj„ax of 25 fmol/mg protein. The low affinity site has a Kq of 5 nM and a Bmax of 145 fmol/mg protein. Histidine did not affect the KD and ^ of both the high and low affinity sites of the kappa receptors, and in the presence of lmM histidine (Fig. 60B) , the high affinity site has a Kd of 1.41 nM, and a of 30 nM, whereas the low affinity site has a KD of 5.60 nM, and a Bmv of 155 fmol/mg protein. Fig. 60C shows the scatchard plot of [3H]- EKC binding to rat cortex opioid receptors in the presence of 150uM ZnCl2 , where the high affinity site is not detected, and the low affinity site has a KD of 8.5 nM, and a Bmax of 175 fmol/mg protein. Lastly, in the presence of histidine (lmM) and ZnCl2 (150uM) together (Fig. 60D), the high affinity site is also undetected, whereas the low affinity site has a KD of 5.10 nM, and a B^y of 150 fmol/mg protein, suggesting that histidine prevented the effect of zinc on the low affinity sites of the kappa receptors completely. Thus the results of the scatchard analysis show that: a) histidine has no significant effects on the K d and the of the high affinity binding site of [3H]-EKC, whereas zinc destroyed the high affinity site, and increased the Kq of the low affinity site by 1.7 fold, without changing the number of low affinity binding sites dramatically; and b) when histidine and zinc are used together, the high affinity binding site is still 89 undetected, but histidine restored the KD and Bn,ax of the low affinity site to control levels. 90 1000 2 2 750 5* 500 £ 250 i OF 0 30 60 90 120 INCUBATION TIME Cm in ) Figure 56. Equilibrium binding of [3H]-EKC to rat cortex opioid receptors at 0°C (o) and 25°C ( e ) . 3500 3000 2500 2000 c_> ac 1500- LU I 3F 1000 - 500- aH-EKC CnMl Figure 57. Typical curves of [3H]-EKC binding to rat cortex opioid receptors generated by direct binding assays. Open circles denote total binding; closed circles denote specific binding calculated as the difference between total binding and non-specific (*) binding. 91 1000 9 9 750 500 250 0 10-* 10“s 10"* 10"* ZnClz CM3 Figure 58. Tli* inhibition of £3H]-£KC binding to rat cortex opioid receptors by ZnCl2 in ths presence (o) and absence (e) of liaM histidine. 1000 750 es —L J O8- 500 U 250 10"4 HISTIDINE 00 Figure 59. The effect of histidine on [3H]-EKC binding to rat cortex opioid receptors in the presence (o) and absence (e) of 200UM ZnCl2- Ka,- 1.41 nM 29 faol/ag V w |. 30 faol/ag protaln protaln Kbb- 5. BO nM L .02 *v * mi* 199 faol/ag protaln 5 .01 0 50 100 150 200 0 50 100' 150 200 250 BOUND (fsol/ag protein) BOUND (faol/ag protein) C 0 Kq - 5. 10 i * .0150 Kb - B. S nM BUwi-275 faol/ag protaln -1SD faol/ag protaln > .0125 5 .020 g^.oiool- .0075 - .0050 ■ J -010 ■ ™ .0025 - .005 - 0 50 100 150 0 50 100 150 200 BOUND (faol/ag protein) BOUND (faol/ag protein) Figure 60. Scatchard analysis of [3H]-EKC binding to rat cortex opioid receptors. A. Control; B. With lmM histidine; C. With 150uM ZnCl2 ; D. With ISOuM ZnCl2 and lmM histidine. 93 4_. RgCQPtor binding studies using the ap s i 1 on- receptor agonist 6-endorohin Attempts were made to study the binding of the opioid peptide [125I ]-fl-endorphin to rat cortex opioid receptors in the presence and absence of ZnCl2 and/or histidine. The major drawback in performing binding assays using this radioactive opioid peptide was the extremely high non specific binding obtained, especially in the presence of zinc (>90%), which made these experiments impossible to replicate. We performed a large number of experiments using [125I]-5-endorphin in an attempt to show the presence of its receptors in the rat brain. We carried out the experiments in the presence and absence of the protease inhibitor bacitracin (50ug/ml brain membranes) which has been reported to prevent the degradation of 5-endorphin. However, bacitracin itself decreased the specific binding of 5-endorphin by more than 70%. This was in agreement with several investigators who reported the same phenomenon (82) . Therefore, we eliminated bacitracin from subsequent experiments. To determine non-specific binding, we tried both naloxone and levorphanol (3uM each), and obtained higher specific binding in the presence of naloxone, and hence it was included in the remaining experiments for determining non-specific binding. Experiments were performed to look at the saturation of epsilon receptors by [^^5i ]-6-endorphin. This was not achieved because of several reasons. At high concentrations of this radioactive peptide the nbn-specific binding was extremely high, and thus accurate results could not be obtained. In addition, extremely high radioactivity was needed in order to perform the experiments necessary to reach saturation, since the specific activity of the compound was very high. Thus, in order to obtain scatchard plots to study the affinity of this ligand for the epsilon receptors, we performed experiments using a fixed amount of radioactivity in the presence of increasing concentrations of cold 0-endorphin. We again failed to obtain any results using this method, since the non-specific binding was very high (>90%), and the radioactivity in the presence of increasing concentrations of cold 0-endorphin increased instead of decreasing, even after bacitracin was included in the assay mixture to prevent the degradation of the peptide. In addition, in all of these experiments, the filters were soaked in a solution containing Hepes buffer (20mM, pH 7.5), containing 0.4% BSA and 0.01% polylysine for 30min. This procedure has been reported to decrease the non specific binding dramatically (83) ; however, we performed a number of experiments using this technique, and did not see any improvement in reducing the non-specific binding. 0-endorphin has been reported to have some affinity toward the mu and delta opioid receptors (84). Hence experiments were performed where [125I ]-6 -endorphin was either used alone, or in the presence of lOOnM DAGO, or in the presence of lOOnM each of DAGO and DADLE to suppress the binding of this peptide to the mu and delta receptors. The only data that we obtained using radioactive 5- endorphin, which were reproducible are reported in Fig. 61. It shows that [125I]-5-endorphin binds to rat cortex opioid receptors, although saturation is not reached since it is used only up to 400pM. It also shows that when DAGO and DADLE are present in the incubation mixture to suppress its binding to the mu and delta receptors, the binding of fl- endorphin is decreased by about 75%, suggesting that only 25% of the binding obtained when using 6 -endorphin alone is to its own receptors, whereas 75% is to the mu and delta receptors. In addition, when DAGO alone was included in the assay mixture with [125I]-B-endorphin, the binding of the radioactive peptide decreased by about 60%, suggesting that about 40% of 5-endorphin binds to the delta and epsilon receptors, and 60% to the mu-receptors. From these experiments we can conclude that 60% of 5-endorphin binds to mu receptors, 25% to the epsilon receptors, and 15% to the delta receptors. 96 LD z r . 15000 o 2£ 5 12500 <_J u - C-) 10000 ci- c n E Q. u 7500 □_ o n 5000 2500 i ur CM 1251-^-ENDORPHIN (pM) Fig. 61. Typical curvas rapresanting tha spacific binding of 112 5I]-bata-andorphin to rat cortaac opioid racaptors ganarated by diract binding assays. Closad circlas danota spacific binding using [125I]-bata-andorphin alona; opan circlas danota spacific binding of [125I]-bata-andorphin in tha prasanca of cold DAGO and DADLE (lOOnM aach), vharaas closad trianglas raprasant tha spacific binding of this paptida in tha prasanca of cold DAGO (100 nM) . CHAPTER IV DISCUSSION The effect of essential trace elements on brain opioid receptors have been less thoroughly studied than other ions. The presence of these trace elements in animals is crucial, since they must be obtained from the environment in adequate amounts to optimize cellular metabolism. Some of the trace elements are required for the development and maintenance of the central nervous system; therefore we studied the regulation of opioid receptors in the rat brain by some of these ions. In addition, there is a relative lack of information about opioid receptor modulation by essential trace elements in the literature, and thus we decided to gain more knowledge about the regulation of various subtypes of opioid receptors in different areas of the brain using zinc, copper, cadmium, nickel, cobalt, magnesium, manganese, and also mercury. However, of all these ions, we focussed our attention mainly on zinc, since its deficiency displays the most dramatic effects in animals, for when the supply is limited, major congenital malformations of the CNS are observed (32,36). Zinc ions inhibited the binding of the opioid antagonist 97 98 naloxone, and the specific agonists for the mu, delta, and kappa receptors to different extents, suggesting some structural differences between different opioid receptor subtypes. The IC5Q of zinc for different subtypes of opioid receptors in the rat cortex was of the following order: delta> kappa> mu with the zinc IC50's being 550uM: 150uM: 37uM. Histidine increased them to 620uM: 625uM: 250uM, that is by 1.1 : 4.2 : 6.8 fold. The concentration of z inc ions in the rat cortex is about 192u M, and that of histidine is about 45uM (Table 2). Thus, at physiological concentrations of zinc, the mu and kappa receptors could be under zinc inhibition, whereas the delta receptors are not. Histidine prevented the effect of zinc significantly on the mu and kappa receptors, but not on the delta receptors, even though the concentration of histidine used was greater than physiological levels. Thus, if zinc is exerting its effects by inhibiting essential SH- groups on the opioid receptors as reported (51,67), our studies show that the mu, kappa, and delta receptors are sensitive to zinc to different extents, which could reflect a differential presence of sulfhydryl groups on these receptors and their accessibility to zinc ions, and thus structural differences among these receptor subtypes. The effects of zinc and histidine on the binding of the opioid antagonist naloxone in the cortex revealed an IC50 of about 60uM for zinc, which was increased by 5.3 fold to 99 320uM by histidine. Naloxone is a non-specific opioid antagonist and shows a decreasing order of potency of approximately 1: 15: 16 for mu: delta: kappa receptors (79). Thus, the IC50 of zinc on naloxone binding reflects its binding to all three receptor subtypes, and is closer to that of the mu receptors, probably due to higher affinity of naloxone for the mu receptors. Scatchard analysis using (3H]-DAGO, [3H]-DSTLE, (3H]-EKC and [3H] -naloxone in the presence and absence of IC50 concentrations of zinc and/or lmM histidine revealed that the binding of the agonists DAGO, DSTLE and EKC displayed curvilinear scatchard plots, whereas that of naloxone was linear. A summary of the equilibrium binding parameters derived from the scatchard plots of these ligands is presented in Table 8 . Zinc increased the Kq of both the high and low affinity sites of mu receptors for DAGO, having no effect on the Bmax of the high affinity site, but increasing that of the low affinity site, zinc abolished the high affinity binding sites of the delta and kappa receptors, and increased the Kq of their low affinity sites by about 3 .8 and 1 .7 fold for the delta and kappa receptors respectively, and also increased their number of low affinity binding sites. Histidine (lmM) had no effect on the Kq and B ^ x of the high affinity sites of the mu and kappa receptors, but abolished the high affinity sites of the delta receptors. It also increased the Kq and Bmax 100 TABLE S. SUMMARY OF EQUILIBRIUM BINDING PARAMETERS DERIVED FROM THE SCATCHARD PLOTS OF [3H]-NALOXONE, [3H]-DAGO, [3H]-DSTLE, AMD [3H]-EKC IN THE RAT CORTEX MU DELTA KAPPA ALL LIGANDS USED DAGO DSTLE EKC NALOXONE 1. CONTROL K0X 0.40+0.08 0.09+0.05 1.43+0.10 0.87+0.10 ®maxl 30+10.0 20±4.10 25*5.10 85+2.0 *02 1.94+0.20 0.82±0.20 5.0*0.09 *max2 110+14.0 80+8.20 145±2.40 2. HISTIDINE K0 i 0.35+0.025 1.41+0.05 0.82*0.065 Bmaxi 30+10.0 30+8.0 124±17.0 *D2 3.70+0.75 1.80*0.30*'** 5.«0±0.17** ®max2 140+2.50 135+4.10* 155±4.10 3. ZINC *D1 2.0+0.20* 1.78±0.075* Bb s x I 30+9.50 111*14.50 *D2 4.35+0.23* 3.10+0.25* 8.50±0.80* *00X2 150+12.50 132*11.10* 175±12.0 4. HISTIDINE + ZINC Kd i 1.40+0.13* 1.10+0.21** *maxl 30+5.0 118+15 *D2 3.40±0.33* 2.80+0.60* 5.10*0.05** Bmax2 135±7.50 150*2.10* 150*1.25 Tha values reported are MEAN±S.D. from 2-3 experiments performed in duplicate. Xq i ** Th* dissociation constant of tbs high affinity binding sits, in nM; Kq 3* Tha dissociation constant of tha low affinity binding sits, In nM. Maximal numbar of high affinity binding sitas, and Maximal numbar of low affinity binding sitas, wbaro tha B-»y valuos ara expressed in fmol/mg protain. Histidine— lmM; Zinc* 30uM with naloxona and DAGO, lOOuM with DSTLE, and 150uM with EKC. Statistical analysis was parforaad using Student t-tast. *P<0.05 vs. control; **P<0.05 vs. zinc. the low affinity sites of the delta and mu receptors, and had no effect on those of the kappa receptors. Lastly, when histidine and zinc were used together, the high affinity sites of the delta and kappa receptors were still undetectable, but histidine restored the Kjj and Bn,,v of the low affinity site of kappa receptors to control values, with no significant effects on those of the delta receptors. As to the mu receptors, histidine decreased the Kq of both sites in presence of zinc, but did not return them to control levels. It also restored the B^ y for the low affinity site of mu receptors to normal levels. The agonists DAGO, DSTLE and EKC (in presence of DAGO and DADLE to suppress mu and delta sites) have been reported to be very specific to the mu, delta, and kappa receptors, respectively. Therefore their binding to more than one site either reflects that they could bind to other opioid receptor subtypes at higher concentrations as reported previously (86), or that this is due to binding to subtypes of these receptors (mu^, mu2, kappakappa2, etc.) that have been recently reported (80). As to naloxone binding, zinc increased the KD significantly, and histidine restored it to control values. To date, there are only two reports about the effect of zinc on opioid receptor binding. The first one was reported by Stengaard-Pedersen (51) , who showed that zinc inhibited enkephalinamide binding to rat brain mu receptors, and 102 increased the Kq while decreasing tha B ^ y of the receptors at concentrations greater than lmM. The second study was conducted by Baraldi et al. (52), who showed that zinc decreased the affinity of [3H]-naloxone binding to rat brain membranes, without any effect on the number of binding sites. These results are in agreement with ours. We found that zinc decreased the Kq of naloxone, but did not affect the number of binding sites; we found no decrease in the Bmax of mu receptors when we used DAGO as the ligand, although we obtained results similar to those of Stengaard- Pedersen (51) for the Kq. No other information is available in the literature about the effect of zinc ions on the KD and Botax of opioid receptors, especially the delta and kappa subtypes. Several experiments were performed regarding the inhibition of the binding of naloxone, DAGO, and DSTLE by other trace elements. They revealed that Cu2+ and Cd2+ inhibited the binding of these ligands in the following order: DAGO, naloxone> DSTLE, and the inhibition by Hg2+ was DAGO DSTLE, naloxone. Histidine had no effect on the inhibition of the binding of these ligands by Hg2+, but increased the IC50 of Cu2+ as follows: DAG0> DSTLE> naloxone, and of Cd2+: DSTLE> DAGO, naloxone. Thus copper, cadmium, and mercury were found to be more potent inhibitors of opioid receptor binding than zinc, with the following order for DAGO and naloxone: Hg2+ >Cd2 + , Cu2+>Zn2+, whereas that for DSTLE was essentially the same except that Cu2+ was almost twice as potent as Cd2+. The affinity of histidine for divalent metal ions and its ability for chelate formation decreases in the following order: Hg2+>Cu2+>Ni2+>Zn2+>Pb2+>Co2+>Cd2+>Mn2+>Mg2+ (85). However, histidine was most effective in preventing the inhibitory effects of zinc and copper, and to a much lesser extent that of cadmium and mercury. There are no reports in the literature about the effect of mercury on opioid receptors, and we do not know why histidine was not effective in preventing its inhibitory effects in our experiments. It is possible that mercury binds very strongly to membrane proteins, and hence histidine is not capable of complexing with it. Cobalt and nickel had dual actions on [3H]-naloxone binding, first stimulating it at low concentrations, then inhibiting it at higher levels (Figs. 20 and 21). Histidine decreased the effects of Ni2+, and prevented the stimulatory action of Co2+. Pasternak et al. (87) reported that nickel ions slightly increased the binding of [3H]-naloxone to rat brain homogenates at a concentration of 2 0uM, and inhibited it at higher concentrations. We observed a similar pattern when using naloxone, but the stimulatory effects of nickel were obtained at concentrations between 5 0 - 5 0 0 U M , and higher amounts were inhibitory. This discrepancy could be due to differences in our experimental conditions, and also 104 because we used cortex membranes in our study, whereas they used whole brain homogenates. Magnesium was stimulatory to naloxone, DAGO and DSTLE binding, and the inclusion of zinc completely prevented its stimulatory effects on naloxone and DAGO binding. Zinc was capable of significantly reducing the effect of Mg2+ on delta receptors at a concentration at which zinc by itself had no effect on DSTLE binding. Mn2+ had also stimulatory effects on naloxone and DSTLE binding; histidine decreased the effect of Mn2+ on naloxone binding to a certain extent, whereas zinc completely prevented the effects of up to SmM manganese on DSTLE binding. At concentrations of Mn2+ greater than 5mM, the addition of zinc made Mn2+ inhibitory on DSTLE binding. This again shows us that zinc is acting by another mechanism besides oxidizing SH-groups, since physiological concentrations of Zn2+ had no effect on DSTLE binding, but prevented the effects of stimulatory ions such as Mg2+ and Mn2+, possibly by competing with them for the divalent cation binding sites on the receptors, and preventing their binding there. Paterson et al. (88) reported that Mn2+ and Mg2+ were stimulatory to naloxone, DAGO, DPDPE, and DADLE binding at concentrations up to lmM, whereas they were inhibitory to dynorphin binding. These results agree with our report on these two ions. Histidine residues have been shown to be essential for opioid receptor activity, for when inactivated or altered, 105 tha receptors lose their activity (68). Histidine is also an endogenous compound, and the precursor of the neurotransmitter histamine which along with its metabolite » imidazoleacetic acid had no effects on opioid receptor binding. Thus, these studies show that histidine, besides being a precursor for histamine, is also a modulator of opioid receptor activity in the brain by a) complexing the inhibitory ions Zn2+, Cu2+, and Cd2+; b) histidine had no effect on the KD and B ^ x of the high affinity sites of the mu and kappa receptors, but abolished the high affinity site of the delta receptors; c) histidine by itself increased the Kq and Bmax of the low affinity sites of mu and delta receptors, with no effect on those of the kappa receptors; d) histidine prevented the effect of zinc completely on the mu and kappa receptors, but did not restore the Kq of the low affinity site of mu receptors to control levels, whereas it restored that of the kappa receptors, and had no effect on the zinc inhibition of delta receptor binding. The effects of histidine, as we mentioned before, were obtained using a non-physiolgical concentration of this compound. However, as we can see in Figs. 43, and 59, physiological concentraions of histidine prevented the effects of IC50 concentrations of zinc on kappa receptor binding, whereas it had no effect on the zinc inhibition of delta receptors. Fig. 27 shows that physiological levels of histidine were not very effective 106 in preventing the inhibitory effect of zinc on the mu receptors. However, it is possible that physiological concentrations of zinc in the cortex might be higher than the one used in this experiment, and hence histidine might be more effective if higher amounts of zinc were present, as shown in the kappa receptor studies. Opioid receptors have been reported to have essential sulfhydryl groups which must be in the reduced form for the receptors to be active (66,67). Since divalent metal ions such as copper and zinc are also oxidizing agents, they could inhibit the binding of opioid ligands by oxidizing the essential thiol groups on the opioid receptors and decreasing their affinity for their ligands. Histidine is a good metal chelator (85), and could prevent the effects of divalent metals by chelating them. This led us to try several reducing reagents and an oxidizing agent to look at their effects on [3H]-DAG0 and [3H]-DSTLE binding in the presence and absence of zinc. Thus, we observed that the reducing reagent B-ME by itself decreased the binding of DAGO in the cortex at concentrations greater than 5mM. In the presence of 30uM zinc, B-ME had no effects on the zinc inhibition (Fig. 33). However, B-ME had no effect on DSTLE binding, and at concentrations greater than 5mM it prevented the inhibitory action of 500uM zinc (Fig. 50) . The very high and non-physiological concentrations of zinc ions required to get inhibition of DSTLE binding suggests that essential SH-groups are not present on the delta receptors. The fact that the reducing reagent B-ME prevented the inhibition by zinc of DSTLE binding suggests that B-ME could be complexing zinc itself, since it has SH- groups capable of binding zinc ions. On the other hand, the reducing reagent DTT had no effects on [3H]-DAGO and [3H]- DSTLE binding by itself, but increased their binding in the presence of zinc by 50% (Fig. 34) and 71% (Fig. 51), respectively. In this case, DTT is probably preventing the effect of zinc on the mu receptors by keeping their essential SH-groups in the reduced form, but may also bind zinc directly since this ion was used at a concentration of 500uM. However, DTT did not prevent the effect of zinc on these receptors completely, suggesting that an additional mechanism exists for zinc inhibition. The oxidizing reagent DTNB decreased the binding of DAGO and DSTLE by itself, and in the presence of zinc (30uM for DAGO and 500uM for DSTLE), their inhibitory effects were potentiated (Figs. 35 and 52) . These studies show that the inhibitory effects of zinc involve more than oxidizing the SH-groups on the opioid receptors, since its effects were not completely reversed by reducing reagents, and were potentiated by the oxidizing reagent DTNB. This also supports our previous hypothesis that the inhibitory effects of zinc are at least partially due to prevention of the effects of stimulatory ions such as magnesium and manganese. 108 The peptidase inhibitors bestatin and thiorphan were used to see if they had any effects on [3H]-DAGO and [3H]- DSTLE binding in the presence and absence of zinc. They have been shown to complex with zinc ions present on the metalloenzymes through thiol groups present in these inhibitors, thus inhibiting the activity of these enzymes. Bestatin and thiorphan have also been shown to have analgesic effects when injected into animals (77,78) and this is believed to be by inhibiting the activity of enkephalinase and other amino-peptidases, and indirectly increasing the concentration of enkephalins and endorphins in the brain. Bestatin and thiorphan showed no signifcant effects on the action of 30uM zinc on [3H]-DAGO and [3H]- DSTLE binding in the rat midbrain (Fig. 36), hypothalamus (Fig. 37), and cortex (Fig. 53). They also had no effect on the binding of [3H]-DAG0 when used alone at various concentrations (Figs. 38 and 39). Thus, their effects are probably only due to the inhibition of these enzymes, and not by binding free zinc present in the brain and preventing its inhibitory effects on opioid receptors. Thus, our studies reveal that essential trace elements are important modulators of opioid receptor activity in the CNS, and possibly in the periphery, and that their deficiency or excess may have important effects on the opioid system. In addition, our studies show that there are important structural differences among the different opioid 109 receptor subtypes, and that not all of them may have SH- groups essential for binding, or that they are inaccessible to zinc, since zinc ions were not capable of inhibiting the binding of DSTLE to the delta receptors at physiological concentrations. Also, zinc ions prevent the stimulatory effects of Mg2+, Mn2+, Co2+, and Ni2+ by preventing their action on the divalent cation sites of the opioid receptors, suggesting that zinc has a dual action, first oxidizing SH-groups essential for binding on the mu and kappa receptors, and by competing with stimulatory ions and preventing their effects. This latter effect is what we believe is occurring with zinc on DSTLE binding to delta receptors. Lastly, we showed that histidine may be an endogenous chelator of divalent cations in the brain, which also has important effects on opioid receptors, either directly by decreasing the affinity of the low affinity binding sites of the mu and delta receptors and by abolishing the high affinity site of the delta receptors, or indirectly by complexing these ions and preventing their inhibitory effects on the opioid receptors. Physiological implications Opioid peptides have been shown to decrease immune function, whereas zinc supplementation increases it (89, 90) . Thus, it is possible that zinc supplementation can increase immune function by preventing the effects of 110 opioid peptides on their receptors. mi receptors are involved in opiate addiction, and when stimulated can cause analgesia, respiratory depression, miosis, bradycardia, etc. As we have demonstrated, under normal physiological concentrations of zinc, mu receptors are partially inhibited by zinc. However in zinc deficiency, these receptors may not be inhibited to the same extent because of a lack of zinc, and therefore it may be easier for morphine to bind to opioid receptors and produce addiction. In such a situation opiate addiction would be facilitated. Thus, zinc supplementation may prevent opiate addiction and decrease the severity of withdrawal symptoms in addicts. In fact, this has been demonstrated to be true in morphine-dependent rats, where zinc supplementation reduced the analgesic effects of morphine, and the naloxone-precipitated withdrawal symptoms (53). 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(e) denotes Lowry's method for protein determination where the absorbance is read at 540nm. Bradford's method (e) using Brilliant Blue G is carried out using 3 . ml of the dye diluted 1:5 in deionized water, and the absorbance of the samples read at 595nm, between 10-30 min after the addition of the dye.