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Department of Physiology, University of Sydney, NSW, Australia.
1. We investigated the mechanisms involved in the rise of myoplasmic calcium concentration ([Ca2+]i) when isolated rat ventricular myocytes were exposed to lactate. The intracellular pH (pHi) and [Ca2+]i were measured using the fluorescent indicators 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and fura-2, respectively. Cell shortening was used as a measure of contractile performance. 2. Exposure to 20 mM lactate at the normal extracellular pH (pHo 7.4) for 10 min caused the pHi to fall rapidly by 0.24 pH units and cell shortening was reduced. Thereafter, pHi partially recovered by 0.16 pH units, which was paralleled by a recovery of shortening. 3. Exposure to lactate at a reduced extracellular pH (pHo 6.4) induced a very large acidosis of 0.70 pH units and cell shortening was abolished. During maintained exposure to lactate the pHi remained constant and cell shortening did not recover. 4. Application of Na(+)-H+ exchanger inhibitors, amiloride or ethylisopropyl-amiloride (EIPA), abolished the recovery of pHi and shortening during maintained exposure to lactate at pHo 7.4 and caused an additional acidosis during maintained application of lactate at pHo 6.4. 5. Application of lactate at both the normal and reduced pHo resulted in a rapid, followed by a slower, rise in [Ca2+]i. The diastolic and systolic [Ca2+]i and the amplitude of the systolic rise in the [Ca2+]i (the Ca2+ transient) all increased in both the rapid and the slow phase. 6. When lactate was applied at pHo 7.4, in the presence of EIPA, the initial rise of [Ca2+]i still occurred but the slower increase was abolished. This suggests an involvement of the Na(+)-H+ exchanger in the slower rise of [Ca2+]i. 7. In conclusion, the Na(+)-H+ exchanger is an important regulator of pHi during a lactate-induced intracellular acidosis. The rise of [Ca2+]i involves at least two mechanisms: (i) a rapid component which may represent reduced myoplasmic Ca2+ buffering, impaired Ca2+ removal by the sarcoplasmic reticulum or a direct inhibitory effect of protons on the Na(+)-Ca2+ exchanger; (ii) a slower component linked to stimulation of Na(+)-H+ exchanger which causes an increased [Na+]i and stimulates the Na(+)-Ca2+ exchanger, resulting in an enhanced Ca2+ influx.
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