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

Abstract

1. Desensitization of Gs-coupled receptors, the beta 2-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 alpha 2A-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. 2. 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 alpha 2A-adrenoceptor desensitization was not specific for phenylethylamine (adrenaline) or imidazoline agonists (moxonidine). 3. 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. 4. In the absence of extracellular Ca2+ the endoplasmic reticulum Ca(2+)-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.5. 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.