CYTOSOLIC PH MEASUREMENTS IN SINGLE CARDIAC MYOCYTES USING CARBOXY-SEMINAPHTHORHODAFLUOR-1

This study examines the use of carboxy-seminaphthorhodafluor-1 (C-SNARF-1) as an indicator of cytosolic pH in isolated rat cardiac myocytes. The emission spectrum of C-SNARF-1 when excited at 530 nm contains two well-separated peaks at approximately 590 and 640 nm, corresponding to the acidic and basic forms of the indicator. This spectral feature allows the indicator to be used in the single excitation, dual emission ratio mode. When C-SNARF-1 is loaded into rat cardiac myocytes as the membrane permeant ester derivative, C-SNARF-1/AM, the indicator localizes within the cytosol with virtually no partitioning into the mitochondria. C-SNARF-1 does not load into isolated mitochondria in suspension. There was no evidence for the presence of nondeesterified C-SNARF-1 within the cells. C-SNARF-1 can be calibrated in situ using a technique that abolishes all transsarcolemmal pH gradients. A 0.7-unit shift in the apparent pK (pK(app) = pK - log10) between the in vitro calibration and the in situ calibration is consistent with a change in beta(I640 at pH 9/I640 at pH 5) in the cytosolic environment (beta(in situ)/beta(in vitro) = 0.21) and not a change in the true pK of the indicator. The contribution of cellular autofluorescence to the total signal can be made negligible. There is no effect of C-SNARF-1 on the contractile properties of rat cardiac myocytes. The resting cytosolic pH of myocytes maintained in a nonbicarbonate buffer of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) showed a large alkalotic shift compared with cells maintained in a bicarbonate buffer [7.48 +/- 0.17 (n = 35) in HEPES vs. 7.14 +/- 0.12 (n = 54) in bicarbonate buffer systems (mean +/- SD)]. C-SNARF-1 is well retained, and steady-state pH and transient changes in pH are easily monitored using a time-resolved fluorescence microscope system.