pH regulation and response to AVP in A10 cells differ markedly in the presence vs. absence of CO2-HCO3-.
The fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to determine the effect of ambient CO2-HCO3- on the regulation of intracellular pH (pHi) and the pHi response to arginine vasopressin (AVP) in A10 vascular smooth muscle (VSM) cells. Steady-state pHi averaged 7.04 +/- 0.02 in the absence and 7.25 +/- 0.01 in the presence of CO2-HCO3-. In the absence of CO2-HCO3-, virtually all (greater than 96%) of the acid extrusion from acidification occurred by amiloride-sensitive Na(+)-H+ exchange. However, in the presence of CO2-HCO3-, acid extrusion after acidification occurred by both Na(+)-H+ exchange and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive Na(+)-dependent Cl(-)-HCO3- exchange. In CO2-HCO3(-)-containing media, amiloride-sensitive Na(+)-H+ exchange mediated 85% of acid extrusion at a pHi of 6.48, but the DIDS-sensitive acid extrusion mechanism (NA(+)-dependent Cl(-)-HCO3- exchange) was the dominant acid extrusion mechanism at a pHi of 6.94. Base exited A10 cells by a DIDS-sensitive process consistent with Na(+)-independent Cl(-)-HCO3- exchange. Both amiloride- and DIDS-sensitive processes regulated steady-state pHi in CO2-HCO3-. AVP (10(-7) M) alkalinized steady-state pHi in the absence of CO2-HCO3- (delta pHi = 0.08 +/- 0.01 pH units) by stimulating Na(+)-H+ exchange; however, AVP did not alter pHi of untreated cells in CO2-HCO3- (delta pHi = -0.01 +/- 0.01 pH units) because of concomitant stimulation of Na(+)-independent Cl(-)-HCO3-exchange. We conclude that the steady-state pHi, the mechanisms of pHi regulation, and the pHi response to AVP in A10 cells are critically influenced by the presence of extracellular CO2-HCO3-. Thus the potential contribution of pHi changes to VSM cell responses to vasoactive agents should be evaluated in the presence of CO2-HCO3-.
Kikeri
D
D
Harvard Center for the Study of Kidney Diseases, Harvard Medical School, Boston, Massachusetts.
Zeidel
M L
ML
Ballermann
B J
BJ
Brenner
B M
BM
Hebert
S C
SC
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism / Q000494:pharmacology*; D002245:Carbon Dioxide / Q000494:pharmacology*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D003470:Culture Media; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009131:Muscle, Smooth, Vascular; D014668:Vasotocin / Q000494:pharmacology*
D001639:Bicarbonates; D002712:Chlorides; D003470:Culture Media; D005452:Fluoresceins; D005456:Fluorescent Dyes; D002245:Carbon Dioxide; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D014668:Vasotocin