Characterization of intracellular pH regulation in the guinea‐pig ventricular myocyte

Abstract

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 (β). The rate of pHi recovery from acid or base loads was used, in conjunction with the measurements of β, 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). The intracellular intrinsic buffering power (βi), 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 (βCO2) was consistent with buffering in a cell fully open to CO2. Because the full value of βCO2 develops slowly (2·5 min), it had to be measured under equilibrium conditions. The value of βCO2 increased monotonically with pHi. 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). The pHi dependence of flux through the transporters, in combination with the pHi and time dependence of intracellular buffering (βi+βCO2), 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.

Acidosis in models of cardiac ventricular myocytes

“…; Grabe & Oster 2001). In this section, we describe the elements of a dynamic model for pH buffering, proton transport and associated model components developed by Leem & VaughanJones (1998) and Leem et al (1999), and their integration with existing cell models to provide a simulation study of pH regulation in cardiac myocytes.…”

“…Typically, the proton buffering power in the myocyte is of the order of bZ20-90 mM per pH unit. This comprises (at least) two distinct intrinsic buffers (Leem et al 1999) where, from the Henderson-Hasselbalch equation, b i ðpH i Þ Z ln 10 !10 ðKpH i Þ 1 C ½B 1 !10 pK 1 ð1 C 10 pK 1 KpH i Þ 2 C ½B 2 !10 pK 2 ð1 C 10 pK 2 KpH i Þ 2 ! ; ð5:3Þ…”

“…The processes regulating pH have been modelled in a variety of tissue and cell types in which pH regulation plays an important role, including heart (Ch'en et al 1998; Leem et al 1999), nerve cells (Boron & De Weer 1976) and pancreatic ductal epithelium (Sohma et al 1996(Sohma et al , 2000, and in subcellular compartments, including intracellular organelles which maintain an acidic interior (such as lysosome, endocytic and secretory organelles, etc. ; Grabe & Oster 2001).…”

“…(b ) Acid transporters Leem et al (1999) have performed detailed and comprehensive measurements of transmembrane proton fluxes with varying intracellular pH under a variety of acid and base loading conditions in order to characterize the pH-dependence of the four transporter fluxes. Using a variety of pharmacological agents and ligand conditions, they were able to dissect out the contributions to net proton transport of the four individual proteins.…”

Pharmacological profile of KR-33028, a highly selective inhibitor of Na+/H+ exchanger

“…Intracellular pH, Na + , and Ca 2+ in the heart are regulated by several ion transporters, such as Na + -H + exchanger (NHE), Na-HCO 3 cotransporter (NBC), Na + -Ca 2+ exchanger (NCX), Cl-HCO 3 exchanger, and ATP-dependent H + pump (Leem et al, 1999). Of these transporters, the Na + -H + exchanger (NHE) is a dominant ion transporter in restoration of intracellular pH during ischemia and reperfusion and acts overloading intracellular Na + , which activates Na + -Ca 2+ exchanger in the reverse mode and leads to intracellular Ca 2+ overload in the heart (Karmazyn, 2001).…”