Recent advances in opiate receptor isolation and characterization have prompted a more molecular approach to study of cellular adaptation. The binding of opiate ligands to their respective receptor sites may stimulate membrane receptors which can communicate intracellularly to activate a number of major systems whose cumulative effects generate tolerance. This type of activation process may be similar in nature to the hormonal activation of membranal adenyl cyclase to increase the intracellular levels of cAMP. However, cellular adaptation to opiates may occur on an even more fundamental level requiring changes in a cellular constituent which serves to integrate broad areas of cellular activity.

 

CALCIUM AS A MODULATOR OF CELLULAR FUNCTION

 

Cell calcium functions in neurotransmitter activity and membrane stability and together with cyclic nucleotides, is thought to play a major role in regulating intracellular metabolism. By virtue of its obligatory involvement with cyclic nucleotides (13,14) and its role in neurotransmitter systems, Phillis (15,16) has suggested Ca++ may function as a primary and secondary messenger in the central nervous system. Table 1 outlines some of the areas of major involvement of Ca++, many of which are also directly or indirectly affected _by opiate treatment. Calcium activates tryptophan and tyrosine hydroxylase as well as adenyl and quanyl cyclase and phosphodiesterase activities (17). Calcium is also an obligatory requirement for excitation-contraction (23) and secretion coupling mechanisms (24) and neurotransmitter-receptor interactions (25,26). Its role in membrane stabilization, activation of neurotransmitter release (by functioning as a charge carrier), and preliminary association in the regulation of macromolecule synthesis (27-31) further support a role for cell constituent in cellular adaptation.

CALCIUM AND OPIATE ACTIONS

 

Pharmacological Studies 

 

Kakunago et al. (33) reported that intracisternal injections of Cat+ but not other ions Batt, Met, Sr++ or Zntt nor Nat or Kt antagonized opiate induced analgesia. Further EDTA or citrate antagonized Catt's ability to alter analgesia. This finding was more recently confirmed by Harris et al. (34) who demonstrated that Ca++ antagonism of opiate analgesia is sensitive to EGTA but not EDTA. These investigators also demonstrated that lauthanum, a well-known Ca++ antagonist (35) may have a neuroanatomical site of action similar to morphine in producing analgesia and in fact can substitute for morphine in a cross-tolerance situation in producing analgesia (36,37). These later findings support earlier work by Shikimi et al. (38) and his suggestion that the analgesic action of morphine may be due to the opiate's affect on Cat+ flux. While Ca++ was the only cation found to alter analgesia, Mg++ content was observed to increase after acute opiate treatment (39).

 

Shikimi et al. (40) demonstrated morphine decreased whole brain Ca++ in mice, an effect to which tolerance developed but was independent of analgesia tolerance. In accordance with this finding was the report by Marchand and Denis (41) of increases in urinary excretion of Ca++.

 

Studies in our laboratory have extended the work of Shikimi et al. (40) by demonstrating opiates in a dose dependent fashion cause Ca++ decreases in the brain in a fairly uniform manner (42). Many hypotheses regarding neurochemical basis for opiate tolerance and dependence regard primary mechanisms in terms of various neurotransmitters which may mediate the tolerance-dependence phenomena. Thus, it would be expected if a particular transmitter were involved, opiate effects may be seen predominately in that brain region where the neurotransmitter distribution is the greatest. Our data based upon assay of Ca++ loss in eight regional brain areas

(43) would indicate no predominant area of opiate induced Ca++ loss suggesting a lack of correlation between Ca++ depletion and opiate effects on any transmitter system. Kuhar et al.

(44) reported similar lack of correlation between opiate receptor binding and lessioned neurotransmitter regions. Further treatment with maximal Ca++ depleting doses of reserpine (5.0 mg/kg) and morphine (50 mg/kg) demonstrated additive Ca++ depletion indicating that morphine and reserpine sensitive Ca++ pools were in all likelihood distinct. In support of this finding Ross and Lynn (45) and Ross (46) showed reserpine pretreatment had no effect upon development of four hour and seven day tolerance to Ca++ depletion.

 

The depletion of Ca++ by morphine sulfate was also seen with the opiate congener levorphanol but not with the analgesically inactive (+) isomer dextrorphan. Naloxone effectively blocked the morphine induced decrease in Ca++ but did not prevent reserpine or pentobarbital from reducing calcium levels (47).

 

Relationship of Ca++ to Development of Tolerance and  Dependence 

 

Many factors have been reported to influence the development of tolerance and a dependence on opiate drugs. Among the more prominent are those dealing with use of nucleic acid and protein synthesis inhibitors. Inhibitors of protein synthesis such as cycloheximide and puromycin have been shown to effectively alter development of analgesic tolerance (48). Tolerance to calcium depletion was observed by Shikimi et al. (40) and Ross (46) demonstrated that cycloheximide but not chloramphenicol effectively blocked tolerance to calcium depletion. These studies are summarized in Figure 1. These results together with the lack of effects of reserpine pretreatment suggest two important points. Cycloheximide has been reported to have a locus of action directed to interruption of nerve membrane synthesis (49,50) while chloramphenicol's action is directed toward mitochondrial synthesis. Based upon these findings, rapid tolerance to Ca++ depletion may be explained by changes in synthesis of nerve membrane protein. Secondly, lack of reserpine's effect upon the induction of Cat+ tolerance suggests the locus of action for tolerance development may reside at membrane sites other than those sensitive to neurotransmitter stimulation.

 

Subcellular Studies: Locus of Cat+ Depletion 

 

The earlier work of Shikimi et al. (40) and previous studies in our laboratory satisfy the criteria necessary to demonstrate calcium depletion as a specific opiate effect. However, it was of interest to us to further explore the locus of this calcium depletion with reference to specific subcellular calcium pools. If the calcium depletion is to be formally considered a specific effect of opiate treatment, one may expect that calcium levels would be altered in those sub-cellular fractions shown to preferentially bind opiate ligands. Pert and Snyder (51) initially reported that 3H-opiate ligands were predominently bound in vitro to crude mitochondrial fractions (P2) containing nerve endings, with less binding in microsomal (P3) fractions. Pert et al. (52) extended these observations by fractionating the crude mitochondrial fraction over sucrose gradients and demonstrating the majority of opiate binding to be associated with partially purified nerve ending fraction. More recent experiments, administering 3H-opiate ligands in vivo (53,54) have demonstrated association of the ligands with synaptic membrane fractions.

 

If the calcium decrease is the result of initial binding of the opiate agonist, causing displacement of Cat+, the locus of the calcium decrease may be confirmed by examining Ca++ oontent in subcellular fractions after acute in vivo treatment. Cardenas and Ross (55) have examined the Ca++ contents in 11 subcellular fractions obtained by Ficol-Sucrose gradients after acute opiate treatment. They report the locus of calcium depletion to be confined to the synaptic particulate fraction. A comparison of the subcellular distribution of opiate ligand binding (52) to calcium depletion may be seen in Table 2.

 

Crude separation of the subcellular fractions into P1 (nuclear), P2 (crude mitochondria), P3 (microsomal) and S (soluble) indicate a predominent loss of calcium from the P2 fraction. Similarly, but to a lesser extent, the predominant binding of opiate ligands (52%) occurs in this fraction. Sub-fractionation of the crude mitochondria (containing myelin, synaptic nerve endings and mitochondria) by sucrose (52) or Ficoll-Sucrose (55) reveals the predominant calcium loss occurs in the synaptosome particulate (73%), with a small but significant loss occuring in the myelin (20%). This locus of calcium depletion is similar to the distribution of opiate receptor binding and supports the original premise that the calcium decrease occurs via displacement of synaptosomal Ca++ after opiate binding.

 

In addition to calcium, magnesium as well plays an important role in function of biological membranes and serves as cofactor for many enzyme systems (56,57). However, Kakunaga et al. (32) were unable to antagonize opiate analgesia with intracisternal injections of magnesium. More recently, it has been demonstrated both Ca++ and Mg++ could inhibit binding of opiate ligands to the crude membrane homogenate (10,51,58). Subsequently, Nat and Mn++ were found to be the most effective in vitro modulators of opiate ligand binding (58-61). While Ca++ was found to be ineffective in differentiating agonist or antagonist binding.

 

Recent studies in our laboratory have examined the levels of Na+, K+ and Mg++ in subcellular fractions after a single dose of morphine. While calcium depletion was significant and confined to synaptic particulate fractions no changes in Na+, K+ or Mg++ were observed for any of the subcellular fractions (55)., It is difficult to resolve at this time whether or not any endogenous cation may be regulating the conformation of a drug receptor. However, Lee et al. (62) have recently suggested, based on comparisons of opiate ligand binding in Tris-HC1 vs. artificial buffers, that the presence of a combination of monovalent and divalent ions best contributes to affinity and accessibility of binding sites.

 

While the sodium model (58,59) is useful for in vitro discrimination of opiate ligand binding, the presence of 100 mM Nat prevents physiologic uptake/release and binding of Ca++ to synaptic membranes (63-67). An alternative explanation for regulation of opiate ligand binding offered by Cardenas and Ross (55), views the opiate receptor in a Ca++ associated conformation. The binding of morphine induces Ca++ displacement shifting the membrane to a Ca++-dissociated state which may be reversed by naloxone (47).

 

ADAPTATION TO SUBCELLULAR LOSS OF CALCIUM

 

As stated earlier, the binding of opiate ligands to their receptor sites is extremely sensitive to inorganic ions (51, 59). Calcium and magnesium, as well as sodium and lithium, but not potassium, have been reported to alter ligand binding (10,58). If cellular adaptation at a membrane level is involved in the induction of tolerance to Ca++ depletion, it would appear that repeated administration of opiates may cause changes in one or more of the monovalent or divalent ions. Figure 2 illustrates the effects of tolerance and dependence upon calcium contents in synaptic particulate material. Twenty-four hours after morphine pellet implant Cat+ levels are significantly lower than control. Following seventy-two hours, calcium levels are significantly increased by 57% over control. The administration of naloxone to morphine pelleted mice reversed the elevated calcium to control levels.

 

 

Table 3 outlines the results of similar studies which measured Nat, K+ and Met in synaptosomal particulate from morphine pelleted mice. These studies indicate that Ca++ contents are slightly lowered at twenty-four hours. No changes are reported for Nat, Kt or Met. At seventy-two hours, magnesium was increased 13%. No changes were observed for Nat or K+. Naloxone induced withdrawal response produced a return to control of Met levels while having no effect on the monovalent ions.

The slight but significant changes in magnesium observed at seventy-two hours are best described as indirect when compared to changes in particulate calcium, since no changes in Mg++ are seen after acute opiate treatment (thirty minutes) (55) nor twenty-four hours after pellet implant. Pharmacological intervention using Mg++ to inhibit opiate analgesia was also without effect (32,34). These results would suggest that calcium content of the synaptic particulate significantly increases during the chronic exposure to morphine through pellet implant.

 

Means represent separate determinations from three separate experiments using three pooled mice brains in each experiment. Synaptosomal particulate was prepared according to methods outlined by Cardenas and Ross (1976). Cation contents were determined using the wet ash technique of Hanig et al. (1972). Pellets containing morphine base were implanted as described by Way et al. (1969). Groups of mice were sacrificed at twenty-four or seventy-two hours + ten minutes after 2 mg/kg naloxone. Cation contents were unaffected by the number of jumps the mice made at five, ten or twenty minutes (unpublished observation, Jones, D.J. and Ross, D.H., 1975). Control animals received lactose pellets + 2 mg/kg naloxone at seventy-two hours.

 

A further insight into this increase in synaptic calcium content may be had by comparing the endogenous Cat+ content with Ca45 binding capacity of the membranes during various treatments with opiate drugs. In this way, the possible formation of new calcium binding sites may be detected by observing the relationship between Cal"/Ca". Table 4 illustrates the Ca40/Ca45 associations with the synaptic particulate fractions. Naloxone effectively reverses the increase in Ca" content after pellet implant to control. Acute opiate treatment produces decreases in Ca" content, but an increase in Ca45 binding capacity.

 

* SPM fractions were obtained by methodology outlined in reference 55. NLX (4 mg/kg) administered at seventy-two hours and animals sacrificed ten minutes later. Preliminary experiments indicated jumping activity at five, ten or twenty minutes was independent of Ca++ content or binding.

 

These findings would suggest that acute exposure to opiates produces a membrane depleted state allowing more Ca" to bind. Chronic exposure to opiates causes adaptation by the membrane in such a manner as to provide for new calcium binding sites. The increased Ca" content during chronic treatment would thereby reduce the membrane capacity to bind Ca".

 

FUNCTIONAL IMPLICATION OF ALTERED Ca++ METABOLISM

 

The increased Ca++ content and decreased Ca" binding capacity of synaptic particulate fractions during chronic morphine exposure would suggest some adaptive response is occuring in the membrane. Collier (7) has presented a theory for the genesis of tolerance and physical dependence which postulates an increase in membrane receptors for opiates during tolerance. However, recent research has failed to provide any direct evidence for an increase in narcotic receptors (9-12) during tolerance development. An alternative to Collier's hypothesis would consider activation of a membrane receptor which was biochemically linked to intracellular processes. Modification of membrane response could then activate an intracellular messenger system, leading to adaptation by the cell.

 

From a functional aspect, increased synaptosomal Ca++ content may be correlated with recent reports of increased neurotransmitter turnover and possible enzyme induction during development of opiate tolerance. Acute morphine treatment has been reported to increase dopamine turnover in striatal areas (68,69). This effect was found to be dose dependent and stereospecific (70,71) and tolerance was shown to develop (69) which paralleled pharmacological tolerance. Induction of tyrosine hydroxylase activity has also been reported (72) and with selective changes occurring in the rat striatum and hypothalamus. Since this enzyme is known to be rate limiting for the biosynthesis of dopamine and norepinephrine, it is of interest to note the recent reports of activation of tyrosine hydroxylase activity by physiological Ca"- concentrations. Morgenroth et al. (18) have demonstrated that physiological concentrations of Ca++ in the range of 50 11M significantly stimulate this enzyme in central noradrenergic neurons, while EGTA was found to activate the enzyme from dopaminergic neurons (73).

 

This apparent modulation of enzyme activity by the presence or absence of Ca++ may have special relevance considering the known capacity of norepinephrine, dopamine and Ca++ to activate adenyl cyclase. Conceivably, the action of opiates may indirectly, by increased transmitter turnover, stimulate adenyl cyclase. In addition, alteration in membrane Ca++ by opiates may directly affect the activity of adenyl cyclase. Recent evidence suggests that cyclic nucleotides may play a predominant role in mode of action of narcotics (74). Opiates may also act directly upon the cyclase system as suggested by the work of Klee et al. (75) and Sharma et al. (76). The binding of opiates may, through alteration of membrane Ca++ activate a messenger system which may in turn activate, at the intracellular level, mechanisms for transport of cations, neurotransmitters and enzyme activities, or more importantly, directly regulate various cellular functions such as phosphorylation of nuclear and synaptic membrane protein.

 

SUMMARY

 

The functional relationship of Ca++ binding to cellular response is of great importance in understanding how the cell responds to its surrounding environment. Areas for future research regarding interaction of narcotics with calcium metabolism must include studies on Cat+ binding protein regulation of cyclic nucleotides. The low concentrations of Ca++ required for activation of adenyl cyclase and cyclic nucleotide phosphodiesterase (19,22) provide a basis for the suggestion that their activities in vivo are regulated by variations in intracellular Ca++ content.

 

Cellular adapation to opiates appears to include selective changes in membrane Ca++ receptors, to accommodate the cell to continued exposure to opiates. A wide variety of cellular responses to opiates, such as enzyme regulation and phosphorylation of specific proteins may therefore be regulated through Cat+ binding protein control of second messenger systems.

 

REFERENCES

 

1. Axelrod, J.: The enzymatic N-demethylation of narcotic drugs. J. Pharm. exp. Ther. 117:322-330 (1956).

2. Cochin, J. and Axelrod, J.: Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine. J. Pharm. exp. Ther. 125:105-110 (1959).

3. Dole, V.P.: Biochemistry of addiction. Ann. Rev. Biochem. 39:821-840 (1970).

4. Hug, C.C.: Characteristics and theories related to acute and chronic tolerance development, CRC Chemical and Biological Aspects of Drug Dependence. Edited by Mule, S.J. and Brill, H., CRC Press, Cleveland, p. 307-359 (1972).

5. Clouet, D.H. and Iwatsubo, K.: Mechanisms of tolerance to and dependence on narcotic analgesic drugs. Ann. Rev. Pharm. 15:49-71 (1975).

6. Shuster, L.: Repression and de-repression of enzyme synthesis as a possible explanation of some aspects of drug action. Nature 189:314-315 (1961).

7. Collier, H.O.J.: Tolerance, physical dependence and receptors (A theory of the genesis of tolerance and physical dependence through drug induced changes in the number of receptors). Adv. Drug Res. 3:171-188 (1966).

8. Goldstein, A. and Goldstein, D.B.: Enzyme expansion theory of drug tolerance and physical dependence, The Addictive States. Edited by Wikler, A., Williams and Wilkins, Baltimore (1968).

9. Klee, W.A. and Streaty, R.A.: Narcotic receptor sites in morphine dependent rats. Nature 248:61-63 (1974).

10. Hitzeman, R.J., Hitzeman, B.A. and Loh, H.H.: Binding of 3H-naloxone in the mouse brain: Effects of ions and tolerance development. Life Sci. 14:2393-2404 (1974).

11. Pert, C.B. and Snyder, S.H.: Opiate receptor: Demonstration in nervous tissue. Science 179:1011-1014 (1973).

12. Hollt, V., Dum, J., Blasig, J., Schubert, P. and Herz, A.: Comparison of in vivo and in vitro parameters of opiate receptor binding in naive and tolerant/dependent rodents. Life Sci. 16:1823-1828 (1975).

13. Rasmussen, H.: Cell communcation, calcium ion, and cyclic adenosine monophosphate. Science 170:404-412 (1970).

14. Rasmussen, H., Goodman, D.B.P. and Tennenhouse, A.: The role of cyclic AMP and calcium in cell activation. CRC Critical Rev. in Biochem. 1:95-148 (1972).

15. Phillis, J.W., Lake, N. and Yarbrough, G.: Calcium mediation of the inhibitory effects of biogenic amines on cerebral cortical neurones. Brain Res. 53:465-469 (1973).

16. Phillis, J.W.: The role of calcium in the central effects of biogenic amines. Life Sci. 14:1189-2101 (1974).

17. Knapp, S., Mandell, A.J. and Bullard, W.P.: Calcium activation of brain tryptophan hydroxylase. Life Sci. 16: 1583-1594 (1975).

18. Morgenroth, V.H., Boodle-Biher, M.C. and Roth, R.H.: Activation of tyrosine hydroxylase from central noradrenergic neurons by calcium. Mol. Pharm. 11:427-435 (1975).

19. Brostrom, C.O., Huang, Y.C., Breckenridge, B.Mcl. and Wolff, D.J.: Identification of a calcium binding protein as a calcium dependent regulator of brain adenylate cyclase. Proc. Nat. Acad. Sci. 72:64-68 (1975).

20. Ferrendelli, J.A., Kinscherf, D.A. and Chang, M.M.: Regulation of levels of guanosine cyclic 3',5' monophosphate in the central nervous system: Effects of depolarizing agents. Mol. Pharm. 9:445-454 (1973).

21. Olson, D.R., Kon, C. and Breckenridge, B.Mcl.: Calcium ion effects on guanylate cyclase of brain. Life Sci. 18: 935-940 (1976).

22. Kakiuchi, S., Yamazaki, R., Teshima, Y., Uenshi, K. and Miyamoto, E.: Multiple cyclic nucleotide phosphodiesterase activities from rat tissues and occurrence of a Ca++ + Mg++ ion dependent phosphodiesterase and its protein activator. Biochem. J. 146:109-120 (1975).

23. Somlyo, A.V. and Somlyo, A.P.: Vascular smooth muscle I. Normal structure, pathology, biochemistry, and biophysics. Pharma. Rev. 20:197-272 (1968).

24. Douglas, W.W.: Stimulus-secretion coupling: The concept and clues from chromaffin and other cells. Brit. J. Pharm. 34:451-474 (1968).

25. Triggle, D.J.: Neurotransmitter-Receptor Interactions. Academic Press, New York and London (1971).

26. Triggle, D.J.: Effects of calcium on excitable membranes and neurotransmitter action. Prog. Surf. Mem. Sci. 5: 267-331 (1972).

27. Seeman, P.: The membrane actions of anesthetics and tranquilizers. Pharm. Rev. 24:583-655 (1972).

28. Katz, B. and Miledi, R.: The effect of calcium on acetylcholine release from motor nerve terminals. Proc. Roy. Soc. Biol. 161:496-503 (1965).

29. Roy, A.K.: Inhibition of the alanine T-RNA amino acylation by Ca++. Biochem. Biophys. Acta 246:349-352 (1971).

30. Rao, K.N., de Smit, M., Howells, A.J. and Bygrove, F.L.: Inhibition of Ca++ of TRNA amino-acylation in preparation of rat liver. Febs. Letters 41:185-188 (1974).

31. Ross, D.H. and Villaneuva, R.P.: Unpublished observations (1976).

32. Kakunaga, T., Kaneto, H. and Hano, K.: Pharmacologic stuides on analgesics - VII. Significance of the calcium ion in morphine analgesia. J. Pharm. exp. Ther. 153:134141 (1966).

33. Kaneto, H.: Inorganic ions: The role of calcium, Narcotic Drugs: Biochemical Pharmacology. Edited by Clouet, D.H., Plenum Press, New York, p. 300-309 (1971).

34. Harris, R.A., Loh, H.H. and Way, E.L.: Effects of divalent cations, cotian chelators and an ianophore on morphine analgesia and tolerance. J. Pharm. exp. Ther. 195:488 (1975).

35. Weiss, G.B.: Cellular pharmacology of lanthanum. Ann. Rev. Pharm. 14:343-354 (1974).

36. Harris, R.A., Loh, H.H. and Way, E.L.: Antinociceptive effects of lanthanum and cerium in non-tolerant and morphine tolerant-dependent animals. J. Pharm. exp. Ther. 196:288 (1976).

37. Harris, R.A., Iwamoto, E.T., Loh, H.H. and Way, E.L.: Analgesic effects of lanthanum cross tolerance with morphine. Brain Res. 100:221 (1975).

38. Shikimi, T., Kaneto, H. and Hano, K.: Effect of morphine on the liberation of acetylcholine from the mouse cerebral cortical slices in relation to the calcium concentration in the medium. Jap. J. Pharm. 17:136-137 (1967).

39. Shikimi, T. and Kaneto, H.: Unpublished observations.

40. Shikimi, T., Kaneto, H. and Kano, K.: Narcotic Drugs: Biochemical Pharmacology. Edited by Clouet, D.H., Plenum Press, New York, p. 303 (1971).

41. Marchand, D. and Denis, G.: Diuretic effect of chronic morphine treatment in rats. J. Pharm. exp. Ther. 162: 331-337 (1968).

42. Ross, D.H., Medina, M.A. and Cardenas, H.L.: Morphine and ethanol: Selective depletion of regional brain calcium. Science 186:63-65 (1974).

43. Cardenas, H.L. and Ross, D.H.: Morphine-induced depletion of calcium in discrete regions of rat brain. J. Neurochem. 24:487-493 (1975).

IONS, OPIATES AND CELLULAR ADAPTATION 279

44. Kuhar, M.J., Pert, C.B. and Snyder, S.H.: Regional distribution of opiate receptor binding in monkey and human brain. Nature 245:447-450 (1973).

45. Ross, D.H. and Lynn, S.C.: Characterization of acute tolerance to morphine using reserpine and cycloheximide. Biochem. Pharmacol. 24:1135-1137 (1975).

46. Ross, D.H.: Tolerance to morphine induced calcium depletion. Br. J. Pharm. 55:431-437 (1975).

47. Ross, D.H., Lynn, S.C. and Cardenas, H.L.: Selective control of calcium levels by naloxone. Life Sci. 18:789796 (1976).

48. Cox, B.M. and Osman, 0.: Inhibition of the development of tolerance to morphine in rats by drugs which inhibit rebonucleic acid or protein synthesis. Br. J. Pharmac. 38:157-170 (1970).

49. Morgan, I.G. and Austin, L.: Synaptosomal protein synthesis in a cell-free system. J. Neurochem. 15:41-51 (1968).

50. Barondes, S.H.: Synaptic macromolecules. Ann. Rev. Biochem. 43:147-168 (1974).

51. Pert, C.B. and Snyder, S.H.: Properties of opiate receptor binding in rat brain. Proc. Nat. Acad. Sci. 70:22432247 (1973).

52. Pert, C.B., Snowman, A.M. and Snyder, S.H.: Localization of opiate receptor binding in synaptic membranes of rat brain. Brain Res. 70:184-188 (1974).

53. Clouet, D.H. and Williams, N.: Localization in brain particulate fractions of narcotic analgesic drugs administered intracisternally to rats. Biochem. Pharm. 22: 1283-1293 (1973).

54. Mule, S.J., Casella, G. and Clouet, D.H.: Localization of narcotic analgesics in synaptic membranes of rat brain. Res. Commun. Chem. Pathol. Pharmacol. 9:55-77 (1974).

55. Cardenas, H.L. and Ross, D.H.: Calcium depletion of synaptosomes after morphine treatment. Br. J. Pharm. 57:521-526 (1976).

56. Williams, R.J.P.: The biochemistry of sodium, potassium, magnesium and calcium. Q. Rev. Lond. Chem. Soc. 24:331365 (1970).

57. Madiera, V.M. and Antunes-Madiera, M.C.: Interaction of Ca+2 and Mg+2 with synaptic plasma membranes. Biochem. Biophys. Acta 323:396-407 (1973).

58. Pert, C.B. and Snyder, S.H.: Opiate receptor binding of agonists and antagonists affected differentially by sodium. Mot. Pharm. 10:868-879 (1974).

59. Simon, E.J., Hiller, J.M. and Edelman, I.: Stereo-specific binding of the potent narcotic analgesic [3H] etorphine to rat brain homogenate. Proc. Nat'l. Acad.Sci. 70:1947-1949 (1973).

60. Simon, E.J., Holler, J.M., Groth, J. and Edelman, I.: Further properties of stereospecific opiate binding sites in rat brain: On the nature of the sodium effect. J. Pharm. exp. Ther. 192:531-537 (1975).

61. Pasternak, G.W., Snowman, A.M. and Snyder, S.H.: Selective enhancement of (3H] opiate agonist binding by divalent cations. Mol. Pharm. 11:735-744 (1975).

62. Lee, C.Y., Akera, T., Stolman, S. and Brody, T.M.: Saturable binding of dihydromorphine and naloxone to rat brain tissue in vitro. J. Pharm. exp. Ther. 194:583-592 (1975).

63. Stahl, W.L. and Swanson, P.D.: Uptake of calcium by sub-cellular fractions isolated from ouabian treated cerebral tissues. J. Neurochem. 16:1553-1564 (1969).

64. Stahl, W.L. and Swanson, P.D.: Movements of calcium and other cations in isolated cerebral tissue. J. Neurochem. 18:415-428 (1971).

65. Swanson, P.D., Anderson, L. and Stahl, W.L.: Uptake of calcium ions by synaptosomes from rat brain. Biochim. Biophys. Acta 356:174-183 (1974).

66. Yoshida, H. and Ichida, S.: Effects of Na+ on Catt-uptake of the snyaptic plasma membrane. Life Sci. 15:685693 (1974).

67. Ichida, S., Hata, F., Matsuda, T. and Yashida, H.: Effects of Na+ and other monovalent cations on Ca++ efflux from synaptosomes. Jap. J. Pharmacol. 26:31-37 (1976).

68. Clouet, D.H. and Ratner, M.: Catecholamine biosynthesis in brains of rats treated with morphine. Science 168: 854-856 (1970).

69. Gauchy, C., Agid, Y., Glowinski, J. and Cheramy, A.: Acute effects of morphine on dopamine synthesis and release and tyrosine metabolism in the rat striatum. Eur. J. Pharmacol. 22:311-319 (1973).

70. Smith, C.B., Sheldon, M.I., Bednarczyk, J.H., Villarreal, J.E.: Morphine induced increases in the incorporation of 14C-tyrosine into 14C-dopamine and 14C-norepinephrine in the mouse brain: Antagonism by naloxone and tolerance. J. Pharm. exp. Ther. 180:547-557 (1972).

71. Gessa, G.L., Vargin, L., Biggio, G. and Togliamonte, A.: Effect of methadone on brain dopamine metabolism, Frontiers in Catecho1amine Research. Edited by Usdin, E. and Snyder, S.H., Pergamon, New York, p. 1011-1014 (1973).

72. Reis, D.J., Hess, P. and Azmitia, E.C.: Changes in enzymes subserving catecholamine metabolism in morphine tolerance and withdrawal in rat. Brain Res. 20:309-315 (1970).

73. Morgenroth, V.H., Boodle-Bicher, M.C. and Roth, R.H.: Dopaminergic neurons: Activation of tyrosine hydroxylase

by a calcium chelator. Mot. Pharm. 12:41-48 (1976).

74. Collier, H.O.J. and Roy, A.C.: Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain homogenate. Nature 248:24-27 (1974).

75. Klee, W.A., Sharma, S.H. and Nirenberg, M.: Opiate receptors as regulators of adenyl cyclase. Life Sci. 16: 1869-1874 (1975).

76. Sharma, S.H., Klee, W.A. and Nirenberg, M.: Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance. Proc. Nat. Acad. Sci. 72:3092-3096 (1975).