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Stereospecificity of opiate-stimulated GTPase activity in brain
Neuropeptides5: 185-188, 1984
STEREOSPECIFICITY OF OPIAll3-STDaLbTED QTPASE ACTIVITYII BRAIN W. Hoss and P.H. Franklin, Center for Brain Research, Box 605, University of RochesterSchool of Medicine and Dentistry, Rochester,NY 14642 (reprint requeststo WH) Low Km GTP hydrolysis in rat brain is stimulated in a concentrationdependent manner by the opiate levorphanol but not by its inactive enantiomer dextrorphan. This finding demonstratesthe stereospecificityof and opioid-stimulatedGTPase in brain. The mu-selectiveligands levorphatiol DAGO are less potent and less efficaciousthan etorphineor DALA, suggesting that delta opioid receptors are regulated by the GTP binding protein Ni in brain. INTRODUCTION Opioids inhibit adenylate cyclase activity in brain (I-3) and in NGlOa15, neuroblastomax glioma hybrid cells (4) in a receptor-mediatedmanner. The potency, and the rank order of potency of opioids in the inhibition of adenylate cyclase in NGlO&15 cells correlatewell with receptor binding in this tissue (5). In contrast, with respect to opioid receptor binding in brain, the concentration-responsecurve of the inhibition of striatal adenylate cyclase by D-ala*-•ethionine-enkephalin (DAME) is shifted to the right (3). Morphine, moreover, is far less potent an inhibitorof striatal cyclase than DAME, displaying the reverse of the rank order of binding potency in brain (2,3). Receptor-mediatedinhibition of adenylate cyclase activity has an absolute requirement for GTP (61. The regulatory component, Ni, in the active GTP bound state, transduces the hormonal stimulus through the membrane to the catalyticsubunit. The result of the interactionamong GTP, Ni and the catalytic subunit is a decrease in the rate of cyclic-AMP accumulation. A low Km GTPase, which is associated with Ni in brain (71, terminatesthe couplingbetween hormone and adenylatecyclase. Opioid receptor-mediated stimulation of low Km GTPase has been demonstratedin NGlO&15 cells (5); in these cells, the potency of opioids for stimulatingGTPase is well correlatedwith their binding affinities and inhibition of adenylate cyclase. Recently,we have shown opioid-stimulated 185
GTPase that is reversible by naloxone in brain (8). We report here that opiates stereospecifically stimulatelow Km GTPase in rat brain. METHODS
Washed neural membranes were prepared from rat brain as described earlier (8). The assay for GTPase was a modification of the method of Cassel and Selinger (9) as described (8). RESULTS We had reported earlier that low Km GTP hydrolysis in rat brain was stimulated by the opioids etorphine and D-ala2-leucine-enkephalinamide (DALA) in a concentrationdependentmanner and that maximal DALA stimulation was naloxone-reversible (8). TABLE 1. Stimulationof low Km GTPase activityby opioids at a concentrationof 1 PM.
Opioid-Stimulated GTPase
Opioid
7.22 + 3.42 + 2.59 + -0.17 2
Etorphine Levorphanol DAGO Dextrorphan
o.isa
(p< o.ooots)b
0.46 (p< 0.005) 1.76 (p< 0.05) 0.64
a Means + SD of triplicatedeterminations,pmol*min'l*mg~l. b Student's t-test for the difference between GTP hydrolysis in the presenceand absence of opioid. In order to examine the stereoselectivity, the stimulationof GTPase by levorphanoland dextrorphanwere compared at a concentrationof 1 uM which produced the maximal stimulation. Table 1 shows that levorphanolhad about one-half the efficacy of etorphine and that dextrorphanwas inactive. The difference between GTP hydrolysis stimulatedby levorphanoland dextrorphan Dextrorphan was inactive in two was highly significant, p< 0.001. additional experiments over the concentrationrange of lO-'O to 10-5 M. Opioid-stimulated low Km GTPase is, therefore,stereospecificin brain. The mu-selectiveanonist D-ala2-N-Mephe4-gly-ol (DAGO)was also less efficacious than etorphiner The ED50 values of several opioids were determinedfrom concentrationresponse curves. As shown in Table 2, both levorphanoland DAGO are less potent than etorphineand DALA for stimulatingGTPase activityin brain.
186
TABLE 2.
ED50 values
of opiolds
for
stimulating
2oa 41a 300b
Etorphine DALA Levorphanol DAGO a b
CTPase in brain.
5oob
Mean of three determinations. Value from a single determination.
DISCUSSION The stimulation of stereospecific in brain.
low Em GTPase by opioids was shown to be This, together with the concentration dependency
of the stimulation by several opioids and the naloxone reversibility, provides strong evidence for an opioid receptor-mediated effect. Although a definite conclusion cannot be drawn, the available data are compatible with the notion that opioid-stimulated GTPase is (at least) a delta reoeptor phenomenon in brain. The mu-selective ligands are both less potent and less effioacious than etorphine, whiah is nonselective , or DALA, which is a full agonist at delta receptors , suggesting that opiate-stimulated GTPase is not activated by mu opiold receptor in brain.
It is conceivable that GTP-binding proteins may link some classes of opioid receptors to effecters other than adenylate cyclase. A non-adenylate cyclase system regulated by GTP is light-activated phosphodlesteraae in retinal rod cells ( IO). In addition, thyrotropin releasing hormone (TBH) , which can stimulate prolactin release from pituitary-derived cell lines without affecting adenylate cyclase (111, stimulates low Km GTPase in membranes from these cells (12). examine additional opioids for their brain.
This work was supported 05232) to P.H.F.
in
part
It will, abilities
therefore, be of interest to to stimulate low Em GTPaae in
by a NIDA predoctoral
Fellowship
(DA-
REFERENCES 1.
Collier, H.O.J. and Roy, A.C. (1974). Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain homogenate. Nature 248: 24-27. 187
2.
Law, P.Y., Wu, J., Koehler, J.E. and Loh, H.H. (1981). Demonstration and characterizationof opiate inhibition of the striatal adenylate cyclase. Journal of Neurochemistry36: 1834-1846.
3.
Cooper, D.M.F., Londos, C., Gill, D.L. and Rodbell, M. (1982). Opiate receptor-mediatedinhibition of adenylate cyclase in rat striatal membranes. Journal of Neurochemistry38: 1164-1167.
4.
Sharma, S.K., Nirenberg,M. and Klee, W.A. (1975). Morphine receptors as regulators of adeny1.at.e cyclase activity. Proceedings of the NationalAcademy of Sciences (U.S.A.)72: 590-594.
5.
Koski, G. and Klee, W.A. (1981). Opiates inhibit adenylatecyclase by stimulating GTP hydrolysis. Proceedings of the National Academy of Sciences (U.S.A.)78: 4185-4189.
6.
Jakobs, K.H. (1979). Determinationof the turnoff reactions for the epinephrine-inhibitedhuman platelet adenylate cyclase. Molecular CellularEndocrinology16: 147-156.
7.
Enomoto, K. and Asakawa, T. (1984). GTPase activity associates with the inhibitory GTP-binding regulatory component of adenylate cyclase purified from rat brain. FEBS Letters 171: 233-239.
8.
Franklin, P.H. and Hess, W. (1984). Opiates stimulatelow Km GTPase in brain. Journal of Neurochemistry,in press.
9.
Cassel, D. and Selinger, 2. (1976). Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes. Biochimica et Biophysics Acta 452: 538-551.
10.
Stryer, L., Hurley, J.B. and Fung, B.K.-K. (1981). Transducin: an amplifier protein in vision. Trends in Biochemical Sciences 6: 245247.
11.
Gershengorn,M.C. (1982). Thyrotropinreleasing hormone. A review of mechanisms of acute stimulation of pituitary hormone release. Molecular and CellularBiochemistry45: 163-179.
12.
Hinkle, P.M. and Phillips,W.J. (1984). Thyrotropinreleasing hormone stimulatesGTP hydrolysis by membranes from GH4C1 rat pituitary tumor cells. Prooeedings of the National Academy of Sciences (U.S.A.), in press.
188
STEREOSPECIFICITY OF OPIAll3-STDaLbTED QTPASE ACTIVITYII BRAIN W. Hoss and P.H. Franklin, Center for Brain Research, Box 605, University of RochesterSchool of Medicine and Dentistry, Rochester,NY 14642 (reprint requeststo WH) Low Km GTP hydrolysis in rat brain is stimulated in a concentrationdependent manner by the opiate levorphanol but not by its inactive enantiomer dextrorphan. This finding demonstratesthe stereospecificityof and opioid-stimulatedGTPase in brain. The mu-selectiveligands levorphatiol DAGO are less potent and less efficaciousthan etorphineor DALA, suggesting that delta opioid receptors are regulated by the GTP binding protein Ni in brain. INTRODUCTION Opioids inhibit adenylate cyclase activity in brain (I-3) and in NGlOa15, neuroblastomax glioma hybrid cells (4) in a receptor-mediatedmanner. The potency, and the rank order of potency of opioids in the inhibition of adenylate cyclase in NGlO&15 cells correlatewell with receptor binding in this tissue (5). In contrast, with respect to opioid receptor binding in brain, the concentration-responsecurve of the inhibition of striatal adenylate cyclase by D-ala*-•ethionine-enkephalin (DAME) is shifted to the right (3). Morphine, moreover, is far less potent an inhibitorof striatal cyclase than DAME, displaying the reverse of the rank order of binding potency in brain (2,3). Receptor-mediatedinhibition of adenylate cyclase activity has an absolute requirement for GTP (61. The regulatory component, Ni, in the active GTP bound state, transduces the hormonal stimulus through the membrane to the catalyticsubunit. The result of the interactionamong GTP, Ni and the catalytic subunit is a decrease in the rate of cyclic-AMP accumulation. A low Km GTPase, which is associated with Ni in brain (71, terminatesthe couplingbetween hormone and adenylatecyclase. Opioid receptor-mediated stimulation of low Km GTPase has been demonstratedin NGlO&15 cells (5); in these cells, the potency of opioids for stimulatingGTPase is well correlatedwith their binding affinities and inhibition of adenylate cyclase. Recently,we have shown opioid-stimulated 185
GTPase that is reversible by naloxone in brain (8). We report here that opiates stereospecifically stimulatelow Km GTPase in rat brain. METHODS
Washed neural membranes were prepared from rat brain as described earlier (8). The assay for GTPase was a modification of the method of Cassel and Selinger (9) as described (8). RESULTS We had reported earlier that low Km GTP hydrolysis in rat brain was stimulated by the opioids etorphine and D-ala2-leucine-enkephalinamide (DALA) in a concentrationdependentmanner and that maximal DALA stimulation was naloxone-reversible (8). TABLE 1. Stimulationof low Km GTPase activityby opioids at a concentrationof 1 PM.
Opioid-Stimulated GTPase
Opioid
7.22 + 3.42 + 2.59 + -0.17 2
Etorphine Levorphanol DAGO Dextrorphan
o.isa
(p< o.ooots)b
0.46 (p< 0.005) 1.76 (p< 0.05) 0.64
a Means + SD of triplicatedeterminations,pmol*min'l*mg~l. b Student's t-test for the difference between GTP hydrolysis in the presenceand absence of opioid. In order to examine the stereoselectivity, the stimulationof GTPase by levorphanoland dextrorphanwere compared at a concentrationof 1 uM which produced the maximal stimulation. Table 1 shows that levorphanolhad about one-half the efficacy of etorphine and that dextrorphanwas inactive. The difference between GTP hydrolysis stimulatedby levorphanoland dextrorphan Dextrorphan was inactive in two was highly significant, p< 0.001. additional experiments over the concentrationrange of lO-'O to 10-5 M. Opioid-stimulated low Km GTPase is, therefore,stereospecificin brain. The mu-selectiveanonist D-ala2-N-Mephe4-gly-ol (DAGO)was also less efficacious than etorphiner The ED50 values of several opioids were determinedfrom concentrationresponse curves. As shown in Table 2, both levorphanoland DAGO are less potent than etorphineand DALA for stimulatingGTPase activityin brain.
186
TABLE 2.
ED50 values
of opiolds
for
stimulating
2oa 41a 300b
Etorphine DALA Levorphanol DAGO a b
CTPase in brain.
5oob
Mean of three determinations. Value from a single determination.
DISCUSSION The stimulation of stereospecific in brain.
low Em GTPase by opioids was shown to be This, together with the concentration dependency
of the stimulation by several opioids and the naloxone reversibility, provides strong evidence for an opioid receptor-mediated effect. Although a definite conclusion cannot be drawn, the available data are compatible with the notion that opioid-stimulated GTPase is (at least) a delta reoeptor phenomenon in brain. The mu-selective ligands are both less potent and less effioacious than etorphine, whiah is nonselective , or DALA, which is a full agonist at delta receptors , suggesting that opiate-stimulated GTPase is not activated by mu opiold receptor in brain.
It is conceivable that GTP-binding proteins may link some classes of opioid receptors to effecters other than adenylate cyclase. A non-adenylate cyclase system regulated by GTP is light-activated phosphodlesteraae in retinal rod cells ( IO). In addition, thyrotropin releasing hormone (TBH) , which can stimulate prolactin release from pituitary-derived cell lines without affecting adenylate cyclase (111, stimulates low Km GTPase in membranes from these cells (12). examine additional opioids for their brain.
This work was supported 05232) to P.H.F.
in
part
It will, abilities
therefore, be of interest to to stimulate low Em GTPaae in
by a NIDA predoctoral
Fellowship
(DA-
REFERENCES 1.
Collier, H.O.J. and Roy, A.C. (1974). Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain homogenate. Nature 248: 24-27. 187
2.
Law, P.Y., Wu, J., Koehler, J.E. and Loh, H.H. (1981). Demonstration and characterizationof opiate inhibition of the striatal adenylate cyclase. Journal of Neurochemistry36: 1834-1846.
3.
Cooper, D.M.F., Londos, C., Gill, D.L. and Rodbell, M. (1982). Opiate receptor-mediatedinhibition of adenylate cyclase in rat striatal membranes. Journal of Neurochemistry38: 1164-1167.
4.
Sharma, S.K., Nirenberg,M. and Klee, W.A. (1975). Morphine receptors as regulators of adeny1.at.e cyclase activity. Proceedings of the NationalAcademy of Sciences (U.S.A.)72: 590-594.
5.
Koski, G. and Klee, W.A. (1981). Opiates inhibit adenylatecyclase by stimulating GTP hydrolysis. Proceedings of the National Academy of Sciences (U.S.A.)78: 4185-4189.
6.
Jakobs, K.H. (1979). Determinationof the turnoff reactions for the epinephrine-inhibitedhuman platelet adenylate cyclase. Molecular CellularEndocrinology16: 147-156.
7.
Enomoto, K. and Asakawa, T. (1984). GTPase activity associates with the inhibitory GTP-binding regulatory component of adenylate cyclase purified from rat brain. FEBS Letters 171: 233-239.
8.
Franklin, P.H. and Hess, W. (1984). Opiates stimulatelow Km GTPase in brain. Journal of Neurochemistry,in press.
9.
Cassel, D. and Selinger, 2. (1976). Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes. Biochimica et Biophysics Acta 452: 538-551.
10.
Stryer, L., Hurley, J.B. and Fung, B.K.-K. (1981). Transducin: an amplifier protein in vision. Trends in Biochemical Sciences 6: 245247.
11.
Gershengorn,M.C. (1982). Thyrotropinreleasing hormone. A review of mechanisms of acute stimulation of pituitary hormone release. Molecular and CellularBiochemistry45: 163-179.
12.
Hinkle, P.M. and Phillips,W.J. (1984). Thyrotropinreleasing hormone stimulatesGTP hydrolysis by membranes from GH4C1 rat pituitary tumor cells. Prooeedings of the National Academy of Sciences (U.S.A.), in press.
188