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Activation of phospholipase C (PLC) in neonatal rat cardiomyocytes (NCM) generates primarily inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) in response to rises in intracellular Ca(2+), or inositol 1,4-bisphosphate (Ins(1,4)P(2)) in response to norepinephrine (NE) (Matkovich, S. J. and Woodcock, E. A. (2000) J. Biol. Chem. 275, 10845-10850). To examine the PLC subtype mediating the alpha(1)-adrenergic receptor response, PLC-beta(1) and PLC-beta(3) were overexpressed in NCM using adenoviral infection (Ad-PLC-beta(1) NCM and Ad-PLC-beta(3) NCM, respectively) and PLC responses assessed from [(3)H]inositol phosphate (InsP) generation in the presence of 10 mm LiCl. The [(3)H]InsP response to NE (100 microm) was enhanced in Ad-PLC-beta(1) NCM relative to cells infected with blank virus (Ad-MX NCM), but was reduced in Ad-PLC-beta(3) NCM. In contrast, the [(3)H]InsP response to ATP (100 microm) was not elevated in Ad-PLC-beta(1) NCM, and was enhanced rather than diminished in Ad-PLC-beta(3) NCM, showing that effects of the two PLC-beta isoforms were specific for particular receptor types. PLC-delta(1) overexpression selectively reduced NE-induced [(3)H]InsP responses, without affecting the ATP stimulation. The reduced NE response was associated with a selective loss of PLC-beta(1) expression in Ad-PLC-delta(1) NCM. alpha(1)-Adrenergic receptor activation caused phosphorylation of PLC-beta(1) but not PLC-beta(3), whereas stimulation by ATP induced phosphorylation of PLC-beta(3) but not PLC-beta(1.) Taken together, these studies provide evidence that NE-stimulated InsP generation in NCM is primarily mediated by PLC-beta(1), despite the presence of both PLC-beta(1) and PLC-beta(3) isoforms.

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)P(3). However, in [(3)H]inositol-labeled rat neonatal cardiomyocytes (NCM) both initial and sustained [(3)H]InsP responses to alpha(1)-adrenergic receptor stimulation with norepinephrine (100 microM) were insensitive to the phosphatidylinositol 4,5-bisphosphate-binding agent neomycin (5 mM). Introduction of 300 microM unlabeled Ins(1,4, 5)P(3) into guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)-stimulated, permeabilized [(3)H]inositol-labeled NCM increased [(3)H]Ins(1,4,5)P(3) slightly but did not significantly reduce levels of its metabolites [(3)H]Ins(1,4)P(2) and [(3)H]Ins(4)P, suggesting that these [(3)H]InsPs are not formed principally from [(3)H]Ins(1,4,5)P(3). In contrast, the calcium ionophore A23187 (10 microM) provoked [(3)H]InsP responses in intact NCM which were sensitive to neomycin, and elevation of free calcium in permeabilized NCM led to [(3)H]InsP responses characterized by marked increases in [(3)H]Ins(1,4,5)P(3) (2.9 +/- 0.2% of total [(3)H]InsPs after 20 min of high Ca(2+) treatment in comparison to 0. 21 +/- 0.05% of total [(3)H]InsPs accumulated after 20 min of GTPgammaS stimulation). These data provide evidence that Ins(1,4, 5)P(3) generation is not a major contributor to G protein-coupled InsP responses in NCM, but that substantial Ins(1,4,5)P(3) generation occurs under conditions of Ca(2+) overload. Thus in NCM, Ca(2+)-induced Ins(1,4,5)P(3) generation has the potential to worsen Ca(2+) overload and thereby aggravate Ca(2+)-induced electrophysiological perturbations.