Rapid desensitization of adrenaline‐ and neuropeptide Y‐stimulated Ca2+ mobilization in HEL‐cells

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Abstract
Desensitization of Gs‐coupled receptors, the β2‐adrenoceptor for example, involves rapid and slower components but little is known regarding the existence of rapid desensitization of Gi‐coupled receptors and its possible mechanisms. In HEL‐cells stimulation of α2A‐adrenoceptors by adrenaline or Y1‐like neuropeptide Y receptors by neuropeptide Y, transiently mobilizes Ca2+ from intracellular stores via a Gi‐protein. We have used this model to study the existence and possible mechanisms of rapid desensitization of a Gi‐mediated cellular response. Following stimulation by adrenaline or neuropeptide Y Ca2+ levels returned towards baseline a few minutes after agonist addition and were refractory to a second agonist exposure demonstrating rapid desensitization. Cross‐desensitization experiments with neuropeptide Y, adrenaline and moxonidine demonstrated the presence of homologous (both receptors) and heterologous desensitization (neuropeptide Y receptors only), and that the α2A‐adrenoceptor desensitization was not specific for phenylethylamine (adrenaline) or imidazoline agonists (moxonidine). The protein kinase C activator, phorbol ester, rapidly desensitized the hormonal Ca2+ responses and inhibitors of protein kinase C enhanced the hormonal responses inconsistently. The tyrosine kinase inhibitor, herbimycin, enhanced Ca2+ mobilization by adrenaline and neuropeptide Y, whereas the protein phosphatase inhibitor, okdadaic acid, did not affect Ca2+ mobilization or its desensitization. In the absence of extracellular Ca2+ the endoplasmic reticulum Ca2+‐ATPase inhibitor, thapsigargin, reduced hormone‐stimulated Ca2+ elevations, demonstrating that mobilization occurs from a thapsigargin‐sensitive pool in the endoplasmic reticulum. The inositol phosphate‐independent Ca2+ release modulator, ryanodine, significantly enhanced adrenaline‐ and neuropeptide Y‐stimulated Ca2+ elevations. Blockade of the endoplasmic reticulum Ca2+‐ATPase by thapsigargin in the presence of extracellular Ca2+ enhanced hormone‐stimulated Ca2+ increases, demonstrating the importance of this enzyme for the termination of the Ca2+ signal. It is concluded that adrenaline and neuropeptide Y‐stimulated Ca2+ mobilization in HEL‐cells occurs from a thapsigargin‐ and ryanodine‐sensitive store in the endoplasmic reticulum and desensitizes rapidly; this appears to involve multiple mechanisms including protein kinases, possibly acting on receptors, and Ca2+ release and sequestration mechanisms.