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0 proteins
Vol. 60, Nos. 13114, 199’7
1179
Abstracts
33 ANIMMUNOBUYT ASSAY FOR PTX ADP-RlBOSYLATlON OF BRAIN AND SPINAL CORT Gi/OPRCYTBINS N. W. DeL,app,D. E. Womer and H. E. Shannon. Eli LiIly ResearchLaboratories,
I&ulapolis,[email protected]. Pertussis toxin (PTX) blocks inhibitory G-proteins (Gi/O) coupled to muscarinic M2 and M4 receptors by ADP-ribosylation, and can be used to indicate participation of these subtypes it mediating physiological responses. PTX ADP-ribosyhttion of membranes in vitro is usuaII1 detent&d using [“PI NAD as substrate coupled with SDS-PAGE and autoradiographyto identilj labeled GVO.This study describes an alternativemethod. Membranes from rat brain and mouse spinal cord were solubilized by sonication in Tris buffet containing 1% sodium cholate. PTX was activated by incubation in 50 mM DTT. The ADP. ribosylation reaction (5Op.l)contained 25 mM Tris-HCl pH 8.0, 1 mM ATP, 100 pM GDP@)S, 1C mM thymidine. 1 mM EDTA, 12.5 mM D’IT, .025% SDS, 1.25 ugs activated PTX, 15 pM [‘I-T NAD (3 Cilmmole), and 20-100 pgs of suspended or solubihzed membraneprotein Reactions were carriedout for one hour at 30°C and were terminatedby heating at 100°C. G-proteins were separated by SDS-PAGE and were transferredto nitroceklose and immunoblottedwith an anti-Gi/Oantibody, Immunoreactivebands of 40 Mr were cut out, dissolved in scintihation fluid, and were counted fat 3H. Incorporation of [“H]-ADP-ribose into isolated Gil0 was 20-30 fold higher using solubilized versus suspended membranes.ADP-ribosylation of spinal cord membraneGil0 was reduced by 62% in samples taken from mice 7 days after an intrathecalinjection of 0.3 pgs/Kg PTX. ADP-ribosylatior of cholate-solubilired membranesusing [“I-IINAD coupled with scintihation counting of immunobloi bands provides a rapid,sensitive and quantitativemethod not requiringthe use of 32P.
34 THESTRlATALMUSCARLNIC
RECEPTOR INHIBITING DOPAMINE D,-ST~MULAT’ED CYCLASE ACTIVITY AS A TARGET FOR ANTICHOfJNERGfC ANTIPARKINSONDRUGS. ADIzNYLYL
P. Onali and M.C. Olianas, Section on Biochemical University of Cagliari, 09124 Cagliari, Italy.
Pharmacology,
Department
of Neurosciences,
Activation of striatal muscarinic receptors inhibits the stimulation of adenylyl cyclase by dopamine (DA) acting on Dr receptors. Recent studies have shown that full DA Dr receptor agonists can improve the motor disturbances in animal models of Parkinson’s disease. Thus, it is possible that the blockade of the muscarinic receptors inhibiting the striatal DA Dr receptor activity may contribute to the antiparkinsonian action of antimuscarinic drugs. In this report, we show that various antimuscarinic drugs, currently used in the treatment of Parkinson’s disease, potently antagonize the carbachol (CCh) inhibition of rat striatal Dr-stimulated adenylyl cyclase activity. Drug Ki values (nM) are: benztropine 2.4, biperiden 3.1, trihexyphenidyl5.0, procyclidine 8.2, ethopropazine 24.6, orphenadrine 41.5, diphenhydramine 164. There is a good correlation between the drug rank order of potencies and their clinical efficacies (r = 0.970; P = 0.0003). On the other hand, the drugs are weaker antagonists of the CCh stimulation of phosphoinositide hydrolysis in rat striatum, displaying the following Ki values (nM): berm&opine 48.8, biperiden 9.6, trihexyphenidyl 31, procyclidine 98, ethopropazine 175, orphenadrine 608, diphenhydramine 2260. These potencies poorly correlate with the clinical efficacies of the drugs (r = 0.776; P = 0.04). The data support the possibility that the antagonism of the muscarinic inhibition of striatal DA Dr receptor activity may be one of the mechanisms by which anticholinergic drugs exert their antiparkinsonian effect.
1179
Abstracts
33 ANIMMUNOBUYT ASSAY FOR PTX ADP-RlBOSYLATlON OF BRAIN AND SPINAL CORT Gi/OPRCYTBINS N. W. DeL,app,D. E. Womer and H. E. Shannon. Eli LiIly ResearchLaboratories,
I&ulapolis,[email protected]. Pertussis toxin (PTX) blocks inhibitory G-proteins (Gi/O) coupled to muscarinic M2 and M4 receptors by ADP-ribosylation, and can be used to indicate participation of these subtypes it mediating physiological responses. PTX ADP-ribosyhttion of membranes in vitro is usuaII1 detent&d using [“PI NAD as substrate coupled with SDS-PAGE and autoradiographyto identilj labeled GVO.This study describes an alternativemethod. Membranes from rat brain and mouse spinal cord were solubilized by sonication in Tris buffet containing 1% sodium cholate. PTX was activated by incubation in 50 mM DTT. The ADP. ribosylation reaction (5Op.l)contained 25 mM Tris-HCl pH 8.0, 1 mM ATP, 100 pM GDP@)S, 1C mM thymidine. 1 mM EDTA, 12.5 mM D’IT, .025% SDS, 1.25 ugs activated PTX, 15 pM [‘I-T NAD (3 Cilmmole), and 20-100 pgs of suspended or solubihzed membraneprotein Reactions were carriedout for one hour at 30°C and were terminatedby heating at 100°C. G-proteins were separated by SDS-PAGE and were transferredto nitroceklose and immunoblottedwith an anti-Gi/Oantibody, Immunoreactivebands of 40 Mr were cut out, dissolved in scintihation fluid, and were counted fat 3H. Incorporation of [“H]-ADP-ribose into isolated Gil0 was 20-30 fold higher using solubilized versus suspended membranes.ADP-ribosylation of spinal cord membraneGil0 was reduced by 62% in samples taken from mice 7 days after an intrathecalinjection of 0.3 pgs/Kg PTX. ADP-ribosylatior of cholate-solubilired membranesusing [“I-IINAD coupled with scintihation counting of immunobloi bands provides a rapid,sensitive and quantitativemethod not requiringthe use of 32P.
34 THESTRlATALMUSCARLNIC
RECEPTOR INHIBITING DOPAMINE D,-ST~MULAT’ED CYCLASE ACTIVITY AS A TARGET FOR ANTICHOfJNERGfC ANTIPARKINSONDRUGS. ADIzNYLYL
P. Onali and M.C. Olianas, Section on Biochemical University of Cagliari, 09124 Cagliari, Italy.
Pharmacology,
Department
of Neurosciences,
Activation of striatal muscarinic receptors inhibits the stimulation of adenylyl cyclase by dopamine (DA) acting on Dr receptors. Recent studies have shown that full DA Dr receptor agonists can improve the motor disturbances in animal models of Parkinson’s disease. Thus, it is possible that the blockade of the muscarinic receptors inhibiting the striatal DA Dr receptor activity may contribute to the antiparkinsonian action of antimuscarinic drugs. In this report, we show that various antimuscarinic drugs, currently used in the treatment of Parkinson’s disease, potently antagonize the carbachol (CCh) inhibition of rat striatal Dr-stimulated adenylyl cyclase activity. Drug Ki values (nM) are: benztropine 2.4, biperiden 3.1, trihexyphenidyl5.0, procyclidine 8.2, ethopropazine 24.6, orphenadrine 41.5, diphenhydramine 164. There is a good correlation between the drug rank order of potencies and their clinical efficacies (r = 0.970; P = 0.0003). On the other hand, the drugs are weaker antagonists of the CCh stimulation of phosphoinositide hydrolysis in rat striatum, displaying the following Ki values (nM): berm&opine 48.8, biperiden 9.6, trihexyphenidyl 31, procyclidine 98, ethopropazine 175, orphenadrine 608, diphenhydramine 2260. These potencies poorly correlate with the clinical efficacies of the drugs (r = 0.776; P = 0.04). The data support the possibility that the antagonism of the muscarinic inhibition of striatal DA Dr receptor activity may be one of the mechanisms by which anticholinergic drugs exert their antiparkinsonian effect.