Characterization of intracellular pH regulation in the guinea-pig ventricular myocyte.
1. Intracellular pH was recorded fluorimetrically by using carboxy-SNARF-1, AM-loaded into superfused ventricular myocytes isolated from guinea-pig heart. Intracellular acid and base loads were induced experimentally and the changes of pHi used to estimate intracellular buffering power (beta). The rate of pHi recovery from acid or base loads was used, in conjunction with the measurements of beta, to estimate sarcolemmal transporter fluxes of acid equivalents. A combination of ion substitution and pharmacological inhibitors was used to dissect acid effluxes carried on Na+-H+ exchange (NHE) and Na+-HCO3- cotransport (NBC), and acid influxes carried on Cl--HCO3- exchange (AE) and Cl--OH- exchange (CHE). 2. The intracellular intrinsic buffering power (betai), estimated under CO2/HCO3--free conditions, varied inversely with pHi in a manner consistent with two principal intracellular buffers of differing concentration and pK. In CO2/HCO3--buffered conditions, intracellular buffering was roughly doubled. The size of the CO2-dependent component (betaCO2) was consistent with buffering in a cell fully open to CO2. Because the full value of betaCO2 develops slowly (2.5 min), it had to be measured under equilibrium conditions. The value of betaCO2 increased monotonically with pHi. 3. In 5 % CO2/HCO3--buffered conditions (pHo 7.40), acid extrusion on NHE and NBC increased as pHi was reduced, with the greater increase occurring through NHE at pHi < 6.90. Acid influx on AE and CHE increased as pHi was raised, with the greater increase occurring through AE at pHi > 7.15. At resting pHi (7.04-7.07), all four carriers were activated equally, albeit at a low rate (about 0.15 mM min-1). 4. The pHi dependence of flux through the transporters, in combination with the pHi and time dependence of intracellular buffering (betai + betaCO2), was used to predict mathematically the recovery of pHi following an intracellular acid or base load. Under several conditions the mathematical predictions compared well with experimental recordings, suggesting that the model of dual acid influx and acid efflux transporters is sufficient to account for pHi regulation in the cardiac cell. Key properties of the pHi control system are discussed.
Leem
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University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK.
Lagadic-Gossmann
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Vaughan-Jones
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D000818:Animals; D001578:Benzopyrans; D001639:Bicarbonates / Q000378:metabolism; D002021:Buffers; D002245:Carbon Dioxide / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport; D008955:Models, Cardiovascular; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D012508:Sarcolemma / Q000378:metabolism; D012964:Sodium / Q000378:metabolism
D001578:Benzopyrans; D001639:Bicarbonates; D002021:Buffers; D002712:Chlorides; D005456:Fluorescent Dyes; C063917:SNARF dye; D002245:Carbon Dioxide; D012964:Sodium