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 β-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 β-adrenoceptors (adenylate cyclase) and angiotensin II (protein kinase C). The results show that a 60-min preincubation with N-ethylmaleimide (3 μM) abolished the facilitatory effect of isoprenaline (0.1 μM) and angiotensin II (0.1 μM) on the stimulation-induced release of noradrenaline and reduced the inhibitory action of neuropeptide Y (0.3 μM). N-ethylmaleimide had no effect on the stimulatory action of either phorbol dibutyrate (0.01, 0.1 μM), forskolin (10 μM), or a combination of 8-bromo adenosine-3′,5′-monophosphate (90 μM) and 3-isobutyl-l-methylxanthine (100 μM). However, at a higher concentration (10 μM), N-ethylmaleimide reduced the facilitatory effect of phorbol dibutyrate (0.1 μM) and the combination of 8-bromo adenosine-3′,5′-monophosphate (90 μM) and 3-isobutyl-l-methylxanthine (100 μM). This suggests that N-ethylmaleimide at 3 μM but not 10 μM was selective for receptor-mediated modulation of noradrenaline release without directly affecting the adenylate cyclase (forskolin, 8-bromo adenosine-3′,5′-monophosphate + 3-isobutyl-l-methylxanthine) or protein kinase C (phorbol dibutyrate) transduction pathways. In atria from mice pretreated with pertussis toxin (1.5 μg/mouse), N-ethylmaleimide preincubation (1 and 3 μM) resulted in a more pronounced reduction of the inhibitory action of neuropeptide Y (0.3 μM). 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 β-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 μM) resulted in non-specific effects on signal transduction mechanisms in the present experimental conditions. © 1990.