Acidosis increases resting cytosolic [Ca2+], (Cai) of myocardial preparations; however, neither the Ca2+ sources for the increase in Cai nor the effect of
acidosis on mitochondrial free [Ca2+], (Cam) have been characterized. In this study cytosolic pH (pHi) was monitored in adult rat left ventricular myocytes loaded with the acetoxymethyl
ester (AM form) of
SNARF-1. A stable decrease in the pHi of 0.52 +/- 0.05 U (n = 16) was obtained by switching from a
bicarbonate buffer equilibrated with 5% CO2 to a
buffer equilibrated with 20% CO2. Electrical stimulation at either 0.5 or 1.5 Hz had no effect on pHi in 5% CO2, nor did it affect the magnitude of pHi decrease in response to hypercarbic
acidosis. Cai was measured in myocytes loaded with
indo-1/free
acid and Cam was monitored in cells loaded with
indo-1/AM after quenching cytosolic
indo-1 fluorescence with
MnCl2. In quiescent intact myocytes bathed in 1.5 mM [Ca2+], hypercarbia increased Cai from 130 +/- 5 to 221 +/- 13 nM. However, when
acidosis was effected in electrically stimulated myocytes, diastolic Cai increased more than resting Cai in quiescent myocytes, and during pacing at 1.5 Hz diastolic Cai was higher (285 +/- 17 nM) than at 0.5 Hz (245 +/- 18 nM; P < 0.05). The magnitude of Cai increase in quiescent myocytes was not affected either by sarcoplasmic reticulum (SR) Ca2+ depletion with
ryanodine or by SR Ca2+ depletion and concomitant superfusion with a Ca(2+)-free
buffer. In unstimulated intact myocytes hypercarbia increased Cam from 95 +/- 12 to 147 +/- 19 nM and this response was not modified either by
ryanodine and a Ca(2+)-free
buffer or by 50 microM
ruthenium red in order to block the mitochondrial uniporter. In mitochondrial
suspensions loaded either with
BCECF/AM or
indo-1/AM,
acidosis produced by
lactic acid addition decreased both intra- and extramitochondrial pH and increased Cam. Studies of mitochondrial
suspensions bathed in
indo-1/free
acid-containing
solution showed an increase in extramitochondrial Ca2+ after the addition of
lactic acid. Thus, in quiescent myocytes, cytoplasmic and intramitochondrial
buffers, rather than transsarcolemmal Ca2+ influx or SR Ca2+ release, are the likely Ca2+ sources for the increase in Cai and Cam, respectively; additionally, Ca2+ efflux from the mitochondria may contribute to the raise in Cai. In contrast, in response to
acidosis, diastolic Cai in electrically stimulated myocytes increases more than resting Cai in quiescent cells; this suggests that during pacing, net cell Ca2+ gain contributes to enhance diastolic Cai.