title: Intracellular acidification associated with changes in free cytosolic calcium. Evidence for Ca2+/H+ exchange via a plasma membrane Ca(2+)-ATPase in vascular smooth muscle cells.
abstract: The purpose of this study was to define the mechanism whereby agonists that increase free cytosolic calcium (Cai2+) affect intracellular pH (pHi) in smooth muscle. Rat aortic vascular smooth muscle cells grown on coverslips were loaded with BCECF/AM or fura-2/AM for continuous monitoring of pHi or Cai2+, respectively, in a HCO3-/CO2- containing medium. Recovery from rapid increases in Cai2+ produced by 1 microM angiotensin (Ang) II (delta Cai2+ -229 +/- 43 nM) or 1 microM ionomycin (delta Cai2+ -148 +/- 19 nM) was accompanied by a fall in pHi (delta pHi, -0.064 +/- 0.0085 P < 0.01, and -0.05 +/- 0.012 pH units, P < 0.01, respectively). Neither the fall in pHi nor the rise in Cai2+ elicited by Ang II was prevented by pretreatment with agents which block the action of this agonist on pHi via the stimulation of the Cl/HCo3 exchangers (DIDS, 50 microM) or the Na+/H+ antiporter (EIPA, 50 microM). In the presence of DIDS and EIPA, Ang II produced a fall in pHi (delta pHi, -0.050 +/- 0.014, P < 0.01) and a rise in Cai2+ (delta Ca2+ 252 +/- 157 nM, P < 0.01). That the change in pHi was secondary to changes in Cai2+ was inferred from the finding that, when the rise in Cai2+ elicited by Ang II was prevented by preincubation with a Ca2+ buffer, BAPTA (60 microM), the fall in pHi was abolished as well (delta pHi, 0.0014 +/- 0.0046). The pHi fall produced by Ang II and ionomycin was prevented by cadmium at a very low concentration (20 nM) which is known to inhibit plasma membrane Ca(2+)-ATPase activity (delta pHi -0.002 +/- 0.0006 and -0.0016 pH units, respectively). Cadmium also blunted Cai2+ recovery after Ang II and ionomycin. These findings suggest that the fall in pHi produced by these agents is due to H+ entry coupled to Ca2+ extrusion via the plasma membrane Ca(2+)-ATPase. Our results indicate that agonists that increase Cai2+ cause intracellular acidification as a result of Ca2+/H+ exchange across the plasma membrane. This process appears to be mediated by a plasma membrane Ca(2+)-ATPase which, in the process of extruding Ca2+ from the cell, brings in [H+] and thus acidifies the cell.
authors.affiliation: Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Daugirdas
authors.forename: J
authors.initials: J
authors.lastname: Arrieta
authors.forename: M
authors.initials: M
authors.lastname: Ye
authors.forename: G
authors.initials: G
authors.lastname: Flores
authors.forename: D C
authors.initials: DC
authors.lastname: Battle
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000143:Acids / Q000378:metabolism*; D000468:Alkalies / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology; D001692:Biological Transport; D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D011522:Protons; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
chemical_list: D000143:Acids; D000468:Alkalies; D011522:Protons; D002104:Cadmium; D000804:Angiotensin II; D004533:Egtazic Acid; D015759:Ionomycin; D000584:Amiloride; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium; C039614:ethylisopropylamiloride