title: Intracellular pH in vascular smooth muscle: regulation by sodium-hydrogen exchange and multiple sodium dependent HCO3- mechanisms.
abstract: OBJECTIVE
  The aim was to determine the mechanisms, particularly bicarbonate dependent mechanisms, of intracellular pH (pHi) recovery from various acidoses in vascular smooth muscle and to explore the ATP dependency of the respective mechanisms.
  
  
  METHODS
  Experiments were conducted in rat aortic smooth muscle cells grown in primary culture and synchronised in a non-growing state by serum deprivation. pHi was measured in cells loaded with the pH sensitive fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF). Chloride efflux was studied by determination of the rate of efflux of 36Cl over 5 min. Cells were ATP depleted by substitution of glucose in the medium by 2-deoxyglucose. Acidoses were induced by CO2 influx and NH3 efflux techniques.
  
  
  RESULTS
  In the absence of HCO3-, the 5-(N-ethyl-N-isopropyl) amiloride (EIPA) sensitive Na+/H+ exchange accounted for the recovery from intracellular acidosis. In the presence of HCO3- ions the response to respiratory acidosis (CO2 influx) was predominantly via activation of Na+/H+ exchange and an EIPA sensitive Na+ and HCO3- dependent mechanism. A 4-acetamido-4'-isothiocyanostilbene-2',2'-sulphonic acids (SITS) sensitive Na+ dependent Cl-/HCO3- mechanism which is also sensitive to EIPA makes a small contribution during severe intracellular acidosis. Under such conditions HCO3- dependent mechanisms contributed about 40% to the overall pHi regulating capacity of vascular smooth muscle cells. However, under conditions which deplete cellular ATP these pHi regulating mechanisms account for virtually all of theses cells' ability to regulate pHi. The inability of Na+/H+ exchange to participate in pHi recovery under these circumstances, reduces the ability of vascular smooth muscle cells to recover pHi by approximately 50-60%. Chloride efflux was approximately linear over 5 min and was increased by 36% in the presence of extracellular HCO3-. Efflux in the presence of HCO3- was inhibited similarly by both SITS and EIPA.
  
  
  CONCLUSIONS
  At least three transporters contribute to recovery from acidosis in vascular smooth muscle: Na+/H+ exchange, an Na(+)-HCO3- cotransporter which is sensitive to EIPA, and an Na+ dependent HCO3-/Cl- exchange sensitive to both SITS and EIPA. The Na(+)-HCO3- cotransporter appears to be similar to that described in human vascular smooth muscle. When the Na+/H+ exchanger is attenuated by cellular ATP depletion, the alternative pathways, particularly the Na(+)-HCO3- cotransporter, ensure that substantial pHi regulatory capacity is maintained.
authors.affiliation: Baker Medical Research Institute, Prahran, Victoria, Australia.
authors.forename: P J
authors.initials: PJ
authors.lastname: Little
authors.forename: C B
authors.initials: CB
authors.lastname: Neylon
authors.forename: C A
authors.initials: CA
authors.lastname: Farrelly
authors.forename: P L
authors.initials: PL
authors.lastname: Weissberg
authors.forename: E J
authors.initials: EJ
authors.lastname: Cragoe
authors.forename: A
authors.initials: A
authors.lastname: Bobik
mesh_terms: D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000187:drug effects / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism*; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D027963:Chloride-Bicarbonate Antiporters; D006863:Hydrogen-Ion Concentration; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000378:metabolism*
chemical_list: D017920:Antiporters; D001639:Bicarbonates; D002352:Carrier Proteins; D027963:Chloride-Bicarbonate Antiporters; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000584:Amiloride; D000255:Adenosine Triphosphate; C039614:ethylisopropylamiloride