Intracellular Ca2+ requirement for activation of the Na+/H+ exchanger in vascular smooth muscle cells.
The role for intracellular Ca2+ in modulating activity of the Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. Na+/H+ exchange was activated by four distinct stimuli: 1) phorbol 12-myristate 13-acetate, 2) thrombin, 3) cell shrinkage, and 4) intracellular acid loading. [Ca2+]i was independently varied between 40 and 200 nM by varying the bathing Ca2+ from 10 nM to 5.0 mM. Thrombin-induced intracellular Ca2+ transients were blocked with bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). In the absence of stimulators of Na+/H+ exchange, varying [Ca2+]i above or below the basal level of 140 nM did not activate Na+/H+ exchange spontaneously. However, varying [Ca2+]i did affect stimulus-induced activation of Na+/H+ exchange. Activation of the exchanger by phorbol 12-myristate 13-acetate was blunted by reduced intracellular Ca2+ (half-maximal activity at 50-90 nM [Ca2+]i), consistent with a Ca2+ requirement for protein kinase C (Ca2+/phospholipid-dependent enzyme). Activation of the exchanger by thrombin in protein kinase C-depleted cells was also sensitive to reduced intracellular Ca2+ (half-maximal activity at 90-140 nM [Ca2+]i) and was increased 40% by raising [Ca2+]i to 200 nM. Activation of the exchanger by cell shrinkage or intracellular acid loads was not significantly affected over the range of [Ca2+]i tested. Thus, altered [Ca2+]i does not itself affect Na+/H+ exchange activity in vascular smooth muscle but instead modulates activation of the transporter by particular stimuli.
Mitsuhashi
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Division of Nephrology, University of California, San Francisco 94143.
Ives
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D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017923:Sodium-Hydrogen Exchangers; D013917:Thrombin / Q000494:pharmacology
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D011493:Protein Kinase C; D013917:Thrombin; D002118:Calcium