The effect of intracellular pH on active shortening was studied in ventricular myocytes isolated enzymically from guinea-pig and rat (humanely killed by cervical dislocation). pHi was measured with carboxy-SNARF-1 (AM-loaded), and active shortening was recorded simultaneously using edge-detection (pacing at 1 Hz).
To quantify the steady-state pHi-shortening relationship, cells were superfused with various concentrations of ammonium (7.5-30 mM) or acetate (10-80 mM) at pH 7.4 and 37 °C (guinea-pig n = 365, rat n = 123). Sarcolemmal acid/base transport was largely suppressed by superfusing with Hepes-buffered solutions containing 30 µM cariporide (inhibits Na+-H+ exchange, NHE). Intracellular acidosis depressed contraction. The pHi-shortening relationship, evaluated for steady-state pHi and contraction, was sigmoidal with a Hill coefficient of 1.91 (guinea-pig) and 1.35 (rat), centred around resting pHi (for multicellular tissue, see Bountra & Vaughan-Jones, 1989).
To study the dynamic pHi-shortening relationship, shortening and pHi were measured during and after a 4 min exposure to 40 mM acetate. In order to establish the effect of sarcolemmal acid/base transport and intracellular buffering capacity, the protocol was executed in Hepes- or CO2/HCO3–-buffered medium, and in the presence of 10 µM S0859 (inhibits Na+-HCO3– co-transport, NBC; Ch’en & Vaughan-Jones, 2001) and/or 30 µM cariporide (guinea-pig n = 8-52, rat n = 7-25). Shortening displayed a clockwise hysteresis against pHi in the acid range (acetate addition) and in the subsequent alkaline range (acetate removal).
Acid hysteresis persisted after inhibition of NHE and NBC (guinea-pig, rat n = 7-18) or after imposition of CO2/HCO3–-buffered conditions (guinea-pig n = 38, rat n = 22). It may be caused by changes in pHi-sensitive Ca2+ fluxes at the sarcoplasmic reticulum and the sarcolemma exerting opposite effects on contraction, and with lesser or greater time delays, respectively.
Alkaline-hysteresis was large in Hepes-buffered superfusates. It was collapsed in the presence of NHE and NBC inhibitors (guinea-pig, rat n = 7-18). It was also collapsed in the presence of CO2/HCO3– buffer (guinea-pig n = 38, rat n = 22). Alkaline-hysteresis may be caused by a kinetic mismatch between changes of [Na+]i and pHi under these conditions, given that each of these parameters is a major modulator of contraction.
In conclusion, active shortening displays considerable pHi sensitivity, although the dependence during dynamic changes of pHi depends on the immediate pHi history of the myocyte.
This work was funded by the BHF, Wellcome Trust and ORS. We thank Dr H.-W. Kleeman of Aventis for kindly providing cariporide and S0859.