Prejunctional beta-adrenoceptors, angiotensin II and neuropeptide Y receptors on sympathetic nerves in mouse atria are linked to N-ethylmaleimide-susceptible G-proteins.
We used the alkylating agent N-ethylmaleimide in order to investigate G-proteins linked to release-modulating prejunctional receptors of sympathetic nerves in mouse atria incubated with [3H]-noradrenaline. The receptors tested were facilitatory beta-adrenoceptors and angiotensin II receptors and inhibitory neuropeptide Y receptors. In order to evaluate the specificity of the N-ethylmaleimide treatment, we tested N-ethylmaleimide against the second messenger pathways that are linked to beta-adrenoceptors (adenylate cyclase) and angiotensin II (protein kinase C). The results show that a 60-min preincubation with N-ethylmaleimide (3 microM) abolished the facilitatory effect of isoprenaline (0.1 microM) and angiotensin II (0.1 microM) on the stimulation-induced release of noradrenaline and reduced the inhibitory action of neuropeptide Y (0.3 microM). N-ethylmaleimide had no effect on the stimulatory action of either phorbol dibutyrate (0.01, 0.1 microM), forskolin (10 microM), or a combination of 8-bromo adenosine-3'5'-monophosphate (90 microM) and 3-isobutyl-1-methylxanthine (100 microM). However, at a higher concentration (10 microM), N-ethylmaleimide reduced the facilitatory effect of phorbol dibutyrate (0.1 microM) and the combination of 8-bromo adenosine-3',5'-monophosphate (90 microM) and 3-isobutyl-1-methylxanthine (100 microM). This suggests that N-ethylmaleimide at 3 microM but not 10 microM was selective for receptor-mediated modulation of noradrenaline release without directly affecting the adenylate cyclase (forskolin, 8-bromo adenosine-3',5'-monophosphate + 3-isobutyl-1-methylxanthine) or protein kinase C (phorbol dibutyrate) transduction pathways. In atria from mice pretreated with pertussis toxin (1.5 micrograms/mouse), N-ethylmaleimide preincubation (1 and 3 microM) resulted in a more pronounced reduction of the inhibitory action of neuropeptide Y (0.3 microM). The nature of this interaction is unclear. Since N-ethylmaleimide has been shown in other studies to inactivate G-proteins, the inhibitory effect of N-ethylmaleimide on prejunctional beta-adrenoceptors, angiotensin II receptors and neuropeptide Y receptors of sympathetic nerves may suggest that G-proteins are involved with these receptors, although other effects of N-ethylmaleimide on the receptor coupling processes cannot be ruled out. Moreover, it appears that the concentration of N-ethylmaleimide used is critical since a higher concentration (10 microM) resulted in non-specific effects on signal transduction mechanisms in the present experimental conditions.
Foucart
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S
Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia.
Murphy
T V
TV
Majewski
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D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology*; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006325:Heart Atria; D006329:Heart Conduction System / Q000378:metabolism*; D051379:Mice; D009200:Myocardial Contraction / Q000187:drug effects; D009478:Neuropeptide Y / Q000378:metabolism / Q000494:pharmacology; D037342:Pertussis Toxin; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D011945:Receptors, Angiotensin / Q000378:metabolism; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D013564:Sympathetic Nervous System / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D009478:Neuropeptide Y; D011943:Receptors, Adrenergic, beta; D011945:Receptors, Angiotensin; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter; D010566:Virulence Factors, Bordetella; D000804:Angiotensin II; D015240:Phorbol 12,13-Dibutyrate; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D005033:Ethylmaleimide