Ca(2+)-activated but not G protein-mediated inositol phosphate responses in rat neonatal cardiomyocytes involve inositol 1,4, 5-trisphosphate generation.
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.
Matkovich
S J
SJ
Cellular Biochemistry Laboratory, Baker Medical Research Institute, Melbourne 8008, Victoria, Australia.
Woodcock
E A
EA
D000818:Animals; D000831:Animals, Newborn; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000502:physiology*; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis*; D007295:Inositol Phosphates / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D009355:Neomycin / Q000494:pharmacology; D009638:Norepinephrine / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D010738:Type C Phospholipases / Q000502:physiology
D007295:Inositol Phosphates; D000001:Calcimycin; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D009355:Neomycin; D002118:Calcium; D009638:Norepinephrine