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Demonstration of stereospecific opiate binding in the nervous tissue of the marine mollusc Mytilus edulis
440
Brain Research, 181 (1980) 440 445 ,!) Elsevier/North-Holland Biomedical Press
Demonstration of stereospecific opiate binding in the nervous tissue of the marine mollusc Mytilus edulis GEORGE B. STEFANO*, RICHARD M. KREAM and R. SUZANNE ZUKIN Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, N. Y. 10461 (U.S.A.) (Accepted September 6th. 1979) Key words:
opiate binding - - invertebrates - - D A L A - levallorphan
- FK 33-824 - - naloxone ....... dopamine
The pentapeptides methionine and leucine enkephalin have been isolated from brain and shown to act as agonists at opiate receptor sites3, 9, This discovery has stimulated numerous studies o f the pharmacology and cellular actions of these and other endogenous opiateslS, 16. The existence of biologically active small peptides in invertebrates is well documented2; such systems have in many cases provided valuable models for higher neural functioning. To date neither enkephalins nor opiate binding sites have been demonstrated in invertebrate tissue 8, Recently, Stefano and Catapane 13 showed that methionine and leucine enkephalin pharmacologically alter dopamine levels in the marine mollusc Mytilus edulis. The effect of the enkephatins was reversible by naloxone, a result which suggests involvement of an opiate receptor mechanism. The present investigation demonstrates the presence of opiate receptor sites in the nervous tissue of Mytilus edulis; these receptors are shown to have properties very similar t o those o f opiate receptors in the brain. This study is the first demonstration of opiate receptors in an invertebrate system, a potentially valuable model system in which to investigate the molecular mechanism of opiate action. Subtidal Mytilus edulis were harvested from the Long Island Sound at Northport, N.Y. For binding studies, pedal ganglia (Pg) from fresh Mytilus edulis were dissected on ice and pooled tissue from 15-30 animals (0.2 mg protein/ganglion) was homogenized in 50 vol. 0.32 sucrose pH 7.4 at 4 °C. The homogenate was centrifuged at 30,000 × g for 15 min, and the resulting pellet was washed twicein 50 m M Tris.Cl buffer, p H 7.4. Finally, the washed pellet or crude membrane fraction was resuspended in 200 vol. 50 mM Tris.Cl buffer pH 7.4 containing 0.1 ~ bovine serum albumin to supplement protein content. Binding analysis was carried out by a modification o f the method of Pert and Snyder v. Aliquots o f membrane suspension (0.5 ml, 0.25 mg protein) were incubated in triplicate at 4 °C for 90 min with [125I]FK 33-824 ('500 Ci/mmol) or [3H] naloxone at the indicated concentration in the presence of dextror* City University of New York, Medgar Evers College, Brooklyn, N.Y. 11225and East Coast Neuroscience Foundation, Inc., 113 Bayview Avenue, Northport, N.Y. 11768, U.S.A.
441 TABLE I Stereospecifie binding of opioid peptides and opiates to Mytilus edMis nervous tissue Incubation conditions and definition of sl~ecificbinding are as described in the text. Data are presented as mean :i- S.E.M. of 3 independent experiments; n 15 30 animals. Reagent
[t~q]FK 33-824 (2.0 >~ 10 !~M)
[aH]naloxone (1.0 > 10 s M)
Concentration
Stereospecifically botmd ligand (/inol/mg protein)
% Control binding
-33.5 _~ 5.0 10 s M 16.2 :L 3.0 FK 33-824 2 x 10 s M 12.8 5:4.2 Naloxone 12.0 1.5 × 10 1 M 58.8 KCI NaC1 1.5 x 10 1M 12.7 ::i: 1.2 NaClandMnCIz 1.5 × 10 tM,10 aM 63.6 ± 10.0 trypsin 1 mg/ml 2.3 :!: 0.5 Trypsin and trypsin inhibitor 1 mg/ml, 2 mg/ml 26.8 2.5
I00 48.3 38.2 175.5 37.9 189.9 6.9
--22.8 =t 4.2 naloxone 2 × 10 s M 9.2 ± 3.3 FK 33-824 10 s M 10.8 ~ 2.0 KC1 1.5 > 10 1 M 21.7 _q 2.0 NaCI 1.5 × 10 1 M 47.6 ± 5.0 NaClandMnC12 1.5 >, 10 1M,10 aM 45.4 ± 3.0 trypsin 1 mg/ml 4.2 5:1.5 trypsin and trypsin inhibitor I mg/ml,2mg/ml 20.5 ~ 1.2
100 40.2 47.2 95.2 208.8 199.1 18.4
80.0
90.0
phan (10 -5 M) or levorphanol (10 -.5 M). Free ligand was separated from m e m b r a n e b o u n d 12'SI-labeled ligand by filtration under reduced pressure through G F / B glass fiber filters ( W h a t m a n ) . The filters were rapidly washed with 2.5 ml aliquots of 50 m M Tris.Cl, p H 7.4 (4 °C). Filters were then counted in a Packard g a m m a counter for b o u n d [12~I]FK 33-824 or transferred to aquasol-toluene (2:1) a n d assayed by liquid scintillation spectrometry for b o u n d [all] naloxone (Intertechnique A B A L SL 40). Stereospecific b i n d i n g is defined as b i n d i n g in the presence of 10-.5 M dextrorphan m i n u s b i n d i n g in the presence of 10-2 M levorphanol. Protein concentration was determined by the method of Lowry et al. 6. Displacement analysis was as previously described4, iv. [125I]FK 33-824 (500 Ci/mmol) was prepared using soluble lactoperoxidase and carrier-free N a 12,5I as previously described 4. [aH]Naloxone (16.2 C i / m m o l ) w a s obtained from New England Nuclear. Met-enkephalin, D-ala"-met-enkephalin ( D A L A i and F K 33-824 were obtained from Peninsula Laboratories. In pharmacological experiments, drugs (0.1 ml) at the indicated concentrations were administered within 2 h of harvesting to the intact animal by topical application to the pedal ganglia (Pg). The Pg of 4 animals (equaling 1 N) were excised 60 rain after drug application, pooled, a n d assayed for d o p a m i n e spectrofluorometrically 12. The
442 data obtained from replicate assay of pooled tissues were statistically analyzed by a one-tailed Student's t-test. In order to determine whether the pharmacological effects of opioid peptides on dopamine levels in Mytilus edulis might be mediated through opiate receptor sites, binding of radiotabeled opiates to Pg homogenates was examined. Both [le51]FK 33824 (an enkephalin analog of enhanced stability) and [3H]naloxone exhibit stereospecific binding (Table I). Binding of [12'5I]FK 33-824 is saturable with respect to radiolabeled ligand concentration (data not shown); maximal binding occurs at 10-s M peptide to approximately 2.5 × 10 -1:~ tool sites/rag protein. Half-maximal binding of [125I]FK 33-824 occurs at 4.5 × 10 -~ M ligand; at this concentration specific binding constitutes approximately 50~o of total binding at 4 C . Displacement of [125I]FK 33-824 by non-radioactive peptide or by naloxone is half-maximal at about 1.0 / 10-8 M. Similarly, binding of [ZH]naloxone to this tissue is saturable (data not shown): maximal binding occurs at 5.0 10-8 M (to the same number of sites} and halfmaximal binding, at 2.0 10-s M. The IC~0 for displacement of [3HI naloxone by the non-radioactive narcotic or by FK 33-824 is also about 1.0 - I0 s M. Thus. binding of opiates to Mytilus edulis Pg is specific, saturable, reversible, and of high affinity. The density of opiate binding sites in this tissue is very similar to that found in rat brain striatum TM. By contrast, specific binding of radiolabeled opiates to non-neuronal tissue from Mytilus edulis as. for example, mantle, is essentially negligible t. 57~, total binding to this tissue). This result suggests that opiate binding sites are restricted to neuronal tissue. The effects of various salts and protein reagents on opmte binding to Mytilus edulis Pg were also investigated (Table I). The binding of [t'~5I]FK 33-824is markedly inhibited by sodium: this inhibition is reversed by manganese. B2ycontrast, the binding of [3H]naloxone is enhanced 2-fold in the presence of high sodium and is relatively unaffected by manganese. These differential effects of ions on opiate agonist and antagonist binding are very similar to those observed for opiate binding to rat brain homogenates ~1. Trypsin abolished almost completely specific binding of both ligands (Table 1). Pretreatment of tissue with soybean trypsin inhibitor reversed the trypsin inhibition of opiate binding. These findings indicate that the opiate binding sites in Mytilus edulis are probably proteinaceous. In order to determine if specific opiate binding in Mytilus edulis correlates with enkephalin action in vivo. the ability of these same enkephalin derivatives and other opiates to alter dopamine levels in Mytilus edulis was examined (Table II). Dopamine levels at 30 and 90 min were at control levels (Table I). FK 33-824 (10 -5 M), and D A L A (10 -5 Mj, another enkephatin analog of enhanced stability, each produced a 5 0 ~ increase (P < 0.001) in endogenous dopamine levels within 60 min of the last drug application (Table II). (At 30 and 90 rain dopamine levels in the presence or absence of enkephalin were the same within experimental error.) In previous studies, it was shown that 4.0 > 10-5 M methionine enkephalin was required to produce similar increases in dopamine levels T:3. The concentrations of these drugs required to elicit half-maximal stimulation of dopamine levels were between 1 , 10- ~i M. [aZTI]FK 33-
443
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444
824 (10 -a M) also produced an increase in dopamine, although tile maxtmal level ol dopamine achieved was not as great as that observed for FK 33-824 or t)-ala '~, metenkephalin. The opiate antagonists naloxone and levallorphan at concentrations as high as 8 × 10-a M did not alter dopamine levels. In addition, the active opiate dextrorphan (8 X 10.5 M) produced no change in these levels+ Prior treatment of tissue with naloxone or levallorphan (4 ." 10+ ~' M) completely inhibited the pharmacological effects of met- and D-ala2-met-enkephalin, FK 33-824, and [1271]FK 33-824. The presence of stereospecific, high affinity opiate binding sites in neuronal tissue of the invertebrate Mytilus edulis has been demonstrated. The binding affinities and sensitivities to salts observed in this tissue suggest that binding of opiates may be to receptor sites similar to those found in rat brain. The finding that the opioid peptide [t25I]FK 33-824 binds with an affinity approximately 10-fold greater than that found for the naloxone may indicate that the mollusc binding sites are analogous to enkephalin-type + opiate receptors described for brain tissue and neuroblas~oma cetlsS, 10. The ability of FK 33-824 and DAKA to raise dopamine levels in Mytilus edulis by approximately 50% was also demonstrated. The concentrations of these highly stable opioid peptides required to elicit this response are lower than that previously found la for methionine enkephalin. This result suggests that the naturally occurring opioid ligand of mammalian brain may be susceptible to proteolytic digestion in invertebrate tissue as well. The finding that dopamine, cAM P, and c G M P levels in the bivalve Anodonta cygnea are also altered by exogenously applied methionine enkephalin or morphine suggests that opiate binding sites may be more generally found in invertebrate neuronal tissue t4. The finding of opiate receptors in invertebrate nervous tissue may have important implications for the evolutionary development of brain opiate receptors. The finding of a population of receptors selective for the shorter opioid peptides suggests that the 'early' opiate receptor was a homogeneous one. Thus. the '6 ~ or enkephalin-receptor may have been the prototypic receptor from which the "it' and +K' types have evolved. In addition, the presence in invertebrate tissue of specific binding sites for enkephalins suggests a physiological role for the opiate receptor that transcends the mammalian pain pathways. The recent finding of enkephalin in the earthworm 1 complements our study and provides further support for the role of the endorphin system in more primitive nervous tissue. Finally, the opiate receptors of the mollusc Mytihts edulis provide a valuable model system in which to investigate the detailed ionic mechanisms associated with opiate action. The advantages of the invertebrate ganglion are 3-fold. First. the neurons of such systems may be large relative to those of brain. They thus provide the possibility of readily visualizing the neuronal substructure. Second. this system provides the potential for intracellular recording with more than one microelectrode inserted into a single cell. Such an experimental design would enable a detailed investigation of the exact ionic changes that accompany opiate receptor binding. Finally, the invertebrate nervous tissue provides a simple nervous system in which to monitor precisely the cellular and electrical effects of specific chemical additions, tt may thus be possible, by elucidating the mechanisms of opiate action in the
445 invertebrate system, to further the understanding of fundamental aspects of opiate receptor functioning in higher organisms including man. We would like to thank Endo Laboratories for generously providing samples of naloxone and Hoffman LaRoche for providing levallorphan and dextrorphan. This work was supported in part by NIH Grants DA 01843 (to R.S.Z.) RR 08171 (to G.B.S.), East Coast Neuroscience Foundation and the Division of Research Resources.
l Alumets, J., Hakanson, R., Sundler, F. and Thorell, J., Neuronal localisation of immunoreactive enkephalin and /3-endorphin in the earthworm, Nature (Lond.), 279 (1979) 805 806. 2 Frontali, N. and Gainer, H., Peptides in invertebrate nervous systems. In H. Gainer (Ed.), Peptides in Neurobiology, Plenum Press, N.Y., 1977 pp. 259 285. 3 Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A. and Morris, H. R., Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature (Lond.), 258 (1975), 577 579. 4 Kream, R. M. and Zukin, R. S., Binding characteristics of a potent enkephalin analog, Biochem. biophys. Res. Comnum., in press. 5 Lord, J. A. H., Waterfield, A. A., Hughes, J. and Kosterlitz, H. W., Endogenous opioid peptides, multiple agonists and receptors, Nature (Lond.), 267 (1977) 495 499. 6 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265 275. 7 Pert, C. B. and Snyder, S. H., Properties of opiate-receptor binding in rat brain, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 2243-2247. 8 Pert, C. B., Aposhian, D. and Snyder, S. H., Phylogenetic distribution of opiate receptor binding, Brain Research, 75 (1974) 356 361. 9 Simantov, R. and Snyder, S. H., Morphine-like peptide in mammalian isolation, structure elucidation, and interactions with the opiate receptor, Proc. nat. A cad. Sci. (Wash.), 70 (1976) 2515-2519. 10 Simon, E. J. and Hiller, J. M., The opiate receptors, Ann. Rev. Pharmacol. Toxicol., 18 (1978) 371-394. 11 Simon, E. J., Hiller, J. M., Groth, J. and Edelman, I., Further properties of stereo-specific opiate binding in rat brain: on the nature of the sodium effect, J. Pharmacol. exp. Ther., 192 (1975) 531-537. 12 Stefano, G. B. and Catapane, E. J., The effects of temperature acclimation on monoamine metabolism, J. Pharmacol. exp. Ther., 203 (1977) 449-456. 13 Stefano, G. B. and Catapane, E. J., Enkephalins increase dopamine levels in the CNS of a marine mollusc, Life Sci., 24 (1979) 1617-1622. 14 Stefano, G. B. and Hiripi, L., Methionine enkephalin and morphine alter moneamine and cyclic nucleotide levels in the cerebral ganglia of the freshwater bivalve Anodonta o'gnea, Life Sci., in press. 15 Waterfield, A. A., Smokcum, R. W. J., Hughes, J., Kosterlitz, H. W. and Henderson, G., In vitro pharmacology of the opioid peptides, enkephalin and endorphins, Europ. J. Phormaeol., 43 (1977) 107-116. 16 Zieglgfinsberger, W. and Fry, J. P., Actions of enkephalin on cortical and striatal neurones of naive and morphine tolerant/dependent rats. In H. W. Kosterlitz (Ed.), Opiates and Endogenous Opioid Peptides, North-Holland, Amsterdam, 1976, 231 238. 17 Zukin, R. S. and Kream, R. M., Chemical crosslinking ofa solublized enkephalin macromolecular complex, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 1593-1597.
Brain Research, 181 (1980) 440 445 ,!) Elsevier/North-Holland Biomedical Press
Demonstration of stereospecific opiate binding in the nervous tissue of the marine mollusc Mytilus edulis GEORGE B. STEFANO*, RICHARD M. KREAM and R. SUZANNE ZUKIN Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, N. Y. 10461 (U.S.A.) (Accepted September 6th. 1979) Key words:
opiate binding - - invertebrates - - D A L A - levallorphan
- FK 33-824 - - naloxone ....... dopamine
The pentapeptides methionine and leucine enkephalin have been isolated from brain and shown to act as agonists at opiate receptor sites3, 9, This discovery has stimulated numerous studies o f the pharmacology and cellular actions of these and other endogenous opiateslS, 16. The existence of biologically active small peptides in invertebrates is well documented2; such systems have in many cases provided valuable models for higher neural functioning. To date neither enkephalins nor opiate binding sites have been demonstrated in invertebrate tissue 8, Recently, Stefano and Catapane 13 showed that methionine and leucine enkephalin pharmacologically alter dopamine levels in the marine mollusc Mytilus edulis. The effect of the enkephatins was reversible by naloxone, a result which suggests involvement of an opiate receptor mechanism. The present investigation demonstrates the presence of opiate receptor sites in the nervous tissue of Mytilus edulis; these receptors are shown to have properties very similar t o those o f opiate receptors in the brain. This study is the first demonstration of opiate receptors in an invertebrate system, a potentially valuable model system in which to investigate the molecular mechanism of opiate action. Subtidal Mytilus edulis were harvested from the Long Island Sound at Northport, N.Y. For binding studies, pedal ganglia (Pg) from fresh Mytilus edulis were dissected on ice and pooled tissue from 15-30 animals (0.2 mg protein/ganglion) was homogenized in 50 vol. 0.32 sucrose pH 7.4 at 4 °C. The homogenate was centrifuged at 30,000 × g for 15 min, and the resulting pellet was washed twicein 50 m M Tris.Cl buffer, p H 7.4. Finally, the washed pellet or crude membrane fraction was resuspended in 200 vol. 50 mM Tris.Cl buffer pH 7.4 containing 0.1 ~ bovine serum albumin to supplement protein content. Binding analysis was carried out by a modification o f the method of Pert and Snyder v. Aliquots o f membrane suspension (0.5 ml, 0.25 mg protein) were incubated in triplicate at 4 °C for 90 min with [125I]FK 33-824 ('500 Ci/mmol) or [3H] naloxone at the indicated concentration in the presence of dextror* City University of New York, Medgar Evers College, Brooklyn, N.Y. 11225and East Coast Neuroscience Foundation, Inc., 113 Bayview Avenue, Northport, N.Y. 11768, U.S.A.
441 TABLE I Stereospecifie binding of opioid peptides and opiates to Mytilus edMis nervous tissue Incubation conditions and definition of sl~ecificbinding are as described in the text. Data are presented as mean :i- S.E.M. of 3 independent experiments; n 15 30 animals. Reagent
[t~q]FK 33-824 (2.0 >~ 10 !~M)
[aH]naloxone (1.0 > 10 s M)
Concentration
Stereospecifically botmd ligand (/inol/mg protein)
% Control binding
-33.5 _~ 5.0 10 s M 16.2 :L 3.0 FK 33-824 2 x 10 s M 12.8 5:4.2 Naloxone 12.0 1.5 × 10 1 M 58.8 KCI NaC1 1.5 x 10 1M 12.7 ::i: 1.2 NaClandMnCIz 1.5 × 10 tM,10 aM 63.6 ± 10.0 trypsin 1 mg/ml 2.3 :!: 0.5 Trypsin and trypsin inhibitor 1 mg/ml, 2 mg/ml 26.8 2.5
I00 48.3 38.2 175.5 37.9 189.9 6.9
--22.8 =t 4.2 naloxone 2 × 10 s M 9.2 ± 3.3 FK 33-824 10 s M 10.8 ~ 2.0 KC1 1.5 > 10 1 M 21.7 _q 2.0 NaCI 1.5 × 10 1 M 47.6 ± 5.0 NaClandMnC12 1.5 >, 10 1M,10 aM 45.4 ± 3.0 trypsin 1 mg/ml 4.2 5:1.5 trypsin and trypsin inhibitor I mg/ml,2mg/ml 20.5 ~ 1.2
100 40.2 47.2 95.2 208.8 199.1 18.4
80.0
90.0
phan (10 -5 M) or levorphanol (10 -.5 M). Free ligand was separated from m e m b r a n e b o u n d 12'SI-labeled ligand by filtration under reduced pressure through G F / B glass fiber filters ( W h a t m a n ) . The filters were rapidly washed with 2.5 ml aliquots of 50 m M Tris.Cl, p H 7.4 (4 °C). Filters were then counted in a Packard g a m m a counter for b o u n d [12~I]FK 33-824 or transferred to aquasol-toluene (2:1) a n d assayed by liquid scintillation spectrometry for b o u n d [all] naloxone (Intertechnique A B A L SL 40). Stereospecific b i n d i n g is defined as b i n d i n g in the presence of 10-.5 M dextrorphan m i n u s b i n d i n g in the presence of 10-2 M levorphanol. Protein concentration was determined by the method of Lowry et al. 6. Displacement analysis was as previously described4, iv. [125I]FK 33-824 (500 Ci/mmol) was prepared using soluble lactoperoxidase and carrier-free N a 12,5I as previously described 4. [aH]Naloxone (16.2 C i / m m o l ) w a s obtained from New England Nuclear. Met-enkephalin, D-ala"-met-enkephalin ( D A L A i and F K 33-824 were obtained from Peninsula Laboratories. In pharmacological experiments, drugs (0.1 ml) at the indicated concentrations were administered within 2 h of harvesting to the intact animal by topical application to the pedal ganglia (Pg). The Pg of 4 animals (equaling 1 N) were excised 60 rain after drug application, pooled, a n d assayed for d o p a m i n e spectrofluorometrically 12. The
442 data obtained from replicate assay of pooled tissues were statistically analyzed by a one-tailed Student's t-test. In order to determine whether the pharmacological effects of opioid peptides on dopamine levels in Mytilus edulis might be mediated through opiate receptor sites, binding of radiotabeled opiates to Pg homogenates was examined. Both [le51]FK 33824 (an enkephalin analog of enhanced stability) and [3H]naloxone exhibit stereospecific binding (Table I). Binding of [12'5I]FK 33-824 is saturable with respect to radiolabeled ligand concentration (data not shown); maximal binding occurs at 10-s M peptide to approximately 2.5 × 10 -1:~ tool sites/rag protein. Half-maximal binding of [125I]FK 33-824 occurs at 4.5 × 10 -~ M ligand; at this concentration specific binding constitutes approximately 50~o of total binding at 4 C . Displacement of [125I]FK 33-824 by non-radioactive peptide or by naloxone is half-maximal at about 1.0 / 10-8 M. Similarly, binding of [ZH]naloxone to this tissue is saturable (data not shown): maximal binding occurs at 5.0 10-8 M (to the same number of sites} and halfmaximal binding, at 2.0 10-s M. The IC~0 for displacement of [3HI naloxone by the non-radioactive narcotic or by FK 33-824 is also about 1.0 - I0 s M. Thus. binding of opiates to Mytilus edulis Pg is specific, saturable, reversible, and of high affinity. The density of opiate binding sites in this tissue is very similar to that found in rat brain striatum TM. By contrast, specific binding of radiolabeled opiates to non-neuronal tissue from Mytilus edulis as. for example, mantle, is essentially negligible t. 57~, total binding to this tissue). This result suggests that opiate binding sites are restricted to neuronal tissue. The effects of various salts and protein reagents on opmte binding to Mytilus edulis Pg were also investigated (Table I). The binding of [t'~5I]FK 33-824is markedly inhibited by sodium: this inhibition is reversed by manganese. B2ycontrast, the binding of [3H]naloxone is enhanced 2-fold in the presence of high sodium and is relatively unaffected by manganese. These differential effects of ions on opiate agonist and antagonist binding are very similar to those observed for opiate binding to rat brain homogenates ~1. Trypsin abolished almost completely specific binding of both ligands (Table 1). Pretreatment of tissue with soybean trypsin inhibitor reversed the trypsin inhibition of opiate binding. These findings indicate that the opiate binding sites in Mytilus edulis are probably proteinaceous. In order to determine if specific opiate binding in Mytilus edulis correlates with enkephalin action in vivo. the ability of these same enkephalin derivatives and other opiates to alter dopamine levels in Mytilus edulis was examined (Table II). Dopamine levels at 30 and 90 min were at control levels (Table I). FK 33-824 (10 -5 M), and D A L A (10 -5 Mj, another enkephatin analog of enhanced stability, each produced a 5 0 ~ increase (P < 0.001) in endogenous dopamine levels within 60 min of the last drug application (Table II). (At 30 and 90 rain dopamine levels in the presence or absence of enkephalin were the same within experimental error.) In previous studies, it was shown that 4.0 > 10-5 M methionine enkephalin was required to produce similar increases in dopamine levels T:3. The concentrations of these drugs required to elicit half-maximal stimulation of dopamine levels were between 1 , 10- ~i M. [aZTI]FK 33-
443
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824 (10 -a M) also produced an increase in dopamine, although tile maxtmal level ol dopamine achieved was not as great as that observed for FK 33-824 or t)-ala '~, metenkephalin. The opiate antagonists naloxone and levallorphan at concentrations as high as 8 × 10-a M did not alter dopamine levels. In addition, the active opiate dextrorphan (8 X 10.5 M) produced no change in these levels+ Prior treatment of tissue with naloxone or levallorphan (4 ." 10+ ~' M) completely inhibited the pharmacological effects of met- and D-ala2-met-enkephalin, FK 33-824, and [1271]FK 33-824. The presence of stereospecific, high affinity opiate binding sites in neuronal tissue of the invertebrate Mytilus edulis has been demonstrated. The binding affinities and sensitivities to salts observed in this tissue suggest that binding of opiates may be to receptor sites similar to those found in rat brain. The finding that the opioid peptide [t25I]FK 33-824 binds with an affinity approximately 10-fold greater than that found for the naloxone may indicate that the mollusc binding sites are analogous to enkephalin-type + opiate receptors described for brain tissue and neuroblas~oma cetlsS, 10. The ability of FK 33-824 and DAKA to raise dopamine levels in Mytilus edulis by approximately 50% was also demonstrated. The concentrations of these highly stable opioid peptides required to elicit this response are lower than that previously found la for methionine enkephalin. This result suggests that the naturally occurring opioid ligand of mammalian brain may be susceptible to proteolytic digestion in invertebrate tissue as well. The finding that dopamine, cAM P, and c G M P levels in the bivalve Anodonta cygnea are also altered by exogenously applied methionine enkephalin or morphine suggests that opiate binding sites may be more generally found in invertebrate neuronal tissue t4. The finding of opiate receptors in invertebrate nervous tissue may have important implications for the evolutionary development of brain opiate receptors. The finding of a population of receptors selective for the shorter opioid peptides suggests that the 'early' opiate receptor was a homogeneous one. Thus. the '6 ~ or enkephalin-receptor may have been the prototypic receptor from which the "it' and +K' types have evolved. In addition, the presence in invertebrate tissue of specific binding sites for enkephalins suggests a physiological role for the opiate receptor that transcends the mammalian pain pathways. The recent finding of enkephalin in the earthworm 1 complements our study and provides further support for the role of the endorphin system in more primitive nervous tissue. Finally, the opiate receptors of the mollusc Mytihts edulis provide a valuable model system in which to investigate the detailed ionic mechanisms associated with opiate action. The advantages of the invertebrate ganglion are 3-fold. First. the neurons of such systems may be large relative to those of brain. They thus provide the possibility of readily visualizing the neuronal substructure. Second. this system provides the potential for intracellular recording with more than one microelectrode inserted into a single cell. Such an experimental design would enable a detailed investigation of the exact ionic changes that accompany opiate receptor binding. Finally, the invertebrate nervous tissue provides a simple nervous system in which to monitor precisely the cellular and electrical effects of specific chemical additions, tt may thus be possible, by elucidating the mechanisms of opiate action in the
445 invertebrate system, to further the understanding of fundamental aspects of opiate receptor functioning in higher organisms including man. We would like to thank Endo Laboratories for generously providing samples of naloxone and Hoffman LaRoche for providing levallorphan and dextrorphan. This work was supported in part by NIH Grants DA 01843 (to R.S.Z.) RR 08171 (to G.B.S.), East Coast Neuroscience Foundation and the Division of Research Resources.
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