Intracellular calcium and alpha 1b-adrenoceptor phosphorylation
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Published on: May 7, 2013
J A García-Sáinz1, A Mendoza-Mendoza, J Vázquez-Prado
1Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F., Mexico. agarcia@ifisiol.unam.mx
Background:
Desensitization of G protein-coupled receptors is associated with receptor phosphorylation. Two groups of kinases seem to participate in such receptor phosphorylation, i.e., second messenger-activated protein kinases and G protein-coupled receptor kinases. Calcium seems to play a role in the phosphorylation of some G protein-coupled receptors. The role of calcium in alpha 1b-adrenoceptor phosphorylation has not been critically assessed.
Methods:
Rat-1 fibroblasts stably expressing the hamster alpha 1b-adrenergic receptor were used. To study receptor phosphorylation cells metabolically labeled with [32P]Pi were lysed and the receptor immunoprecipitated using a polyclonal antibody generated against the receptor carboxyl terminal decapeptide. Intracellular calcium was determined by using Fura-2 fluorescence.
Results:
Norepinephrine, endothelin-1, and lysophosphatidic acid increased intracellular calcium concentration. All these agents and phorbol myristate acetate (PMA) induce alpha 1b-adrenoceptor phosphorylation. The intracellular chelator, BAPTA, abolished the increase in intracellular calcium induced by the previously mentioned agents but did not affect the receptor phosphorylation induced by norepinephrine, PMA, or lysophosphatidic acid. Under these conditions, receptor phosphorylation induced by endothelin was slightly but consistently decreased. Thapsigargin increased intracellular calcium concentration but was unable to induce alpha 1b-adrenoceptor phosphorylation and decreased PMA-induced receptor phosphorylation. No increase in receptor phosphorylation was observed when calcium ionophores were used.
Conclusions:
Our data indicate that an increase in [Ca2+]i is not sufficient to induce alpha 1b-adrenoceptor phosphorylation and that buffering of [Ca2+]i does not alter the receptor phosphorylation induced by norepinephrine, lysophosphatidic acid, and PMA. A marginal role of calcium in the alpha 1b-adrenoceptor phosphorylation induced by endothelin-1 cannot be discarded.
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