Ca2+-activated but not G protein-mediated inositol phosphate responses in rat neonatal cardiomyocytes involve inositol 1,4,5-trisphosphate generation

Abstract

Inositol phosphate (InsP) responses to receptor activation are assumed to involve phospholipase C cleavage of phosphatidylinositol 4,5-bisphosphate to generate Ins(1,4,5)P3. However, in [3H]inositol-labeled rat neonatal cardiomyocytes (NCM) both initial and sustained [3H]InsP responses to α1- adrenergic receptor stimulation with norepinephrine (100 μM) were insensitive to the phosphatidylinositol 4,5-bisphosphate-binding agent neomycin (5 mM). Introduction of 300 μM unlabeled Ins(1,4,5)P3 into guanosine 5'-3-O-(thio)triphosphate (GTPγS)-stimulated, permeabilized [3H]inositol-labeled NCM increased [3H]Ins(1,4,5)P3 slightly but did not significantly reduce levels of its metabolites [3H]Ins(1,4)P2 and [3H]Ins(4)P, suggesting that these [3H]InsPs are not formed principally from [3H]Ins(1,4,5)P3. In contrast, the calcium ionophore A23187 (10 μM) provoked [3H]InsP responses in intact NCM which were sensitive to neomycin, and elevation of free calcium in permeabilized NCM led to [3H]InsP responses characterized by marked increases in [3H]Ins(1,4,5)P3 (2.9 ± 0.2% of total [3H]InsPs after 20 min of high Ca2+ treatment in comparison to 0.21 ± 0.05% of total [3H] InsPs accumulated after 20 min of GTPγS stimulation). These data provide evidence that Ins(1,4,5)P3 generation is not a major contributor to G protein-coupled InsP responses in NCM, but that substantial Ins(1,4,5)P3 generation occurs under conditions of Ca2+ overload. Thus in NCM, Ca2+-induced Ins(1,4,5)P3 generation has the potential to worsen Ca2+ overload and thereby aggravate Ca2+-induced electrophysiological perturbations.