Dependence of Na+/H+ antiport activation in cultured rat aortic smooth muscle on calmodulin, calcium, and ATP. Evidence for the involvement of calmodulin-dependent kinases.
The role of Ca2+/calmodulin-dependent processes in the activation of the Na+/H+ antiport of primary cultures of rat aortic smooth muscle was studied using 22Na+ uptake and measurement of intracellular pH (pHi) with the fluorescent pH dye 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein. Antiport activation following exposure to serum and by the induction of an intracellular acidosis could be markedly attenuated by calmodulin antagonists. Ionomycin also transiently elevated pHi and 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ influx, effects consistent with activation of the antiport; these effects were abolished in cells exposed to calmodulin antagonists or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. Activation of the antiport following intracellular acidosis was markedly affected by cellular ATP depletion. A comparison of the abilities of control and 2-deoxy-D-glucose-treated cells to increase 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ influx in response to graded acidifications indicated that attenuation of Na+/H+ antiport activity was due to both a shift of its pHi dependence and to a reduction in maximal activity. The results suggest that the Na+/H+ antiport of rat aortic smooth muscle is dependent on Ca2+/calmodulin-dependent processes, presumably phosphorylation, which influences its activity by modulating (i) an intracellular proton dependent regulatory mechanism (allosteric site) and (ii) the maximum activity of the antiport.
Little
P J
PJ
Clinical Research Unit, Alfred Hospital, Prahran, Australia.
Weissberg
P L
PL
Cragoe
E J
EJ
Bobik
A
A
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism*; D003847:Deoxyglucose / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D017923:Sodium-Hydrogen Exchangers
D002147:Calmodulin; D002352:Carrier Proteins; D004987:Ethers; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; C039465:5-dimethylamiloride; D015759:Ionomycin; D000584:Amiloride; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D011494:Protein Kinases; D002118:Calcium