Effect of Calcium and Other Divalent Cations on Intracellular pH Regulation of Frog Skeletal Muscle
1. We examined, in frog semitendinosus muscle, the effect of calcium release, induced by depolarization or caffeine, on intracellular pH (pHi) recovery from an acid load applied at least 40 min later. We also studied the effect of external Ca and other divalent cations on recovery. We used pH-sensitive micro-electrodes; the external pH (pH0) was always 7·35.
2. In fibres depolarized by 50 mM-K, constant [K] x [Cl] in the presence of 1 mM-tetracaine (which blocks Ca release), the rate of pHi recovery from 5% C02-induced acidification was 0·15±0·02 ΔpHi h-1 (n = 7), whereas in depolarized fibres that had never been exposed to the drug, the rate of recovery was 0·27±0·01 ΔpHi h-1 (n = 5). Yet, when Ca release was not blocked and the depolarized fibres were exposed to tetracaine shortly before C02 exposure, a similar slow rate of 0·14±0·03 ΔpHi h-1 (n = 7) was observed. When Ca release was blocked by tetracaine, but the drug washed out before recovery, the rate was again 0·27 ±0·02 ΔpHi h-1 (n = 6).
3. In fibres first depolarized to about —23 mV in 50 mM-K, constant [K] x [Cl] (recovery of 0·23±0·034 ΔpHi h-1, n = 6), and then repolarized to —79 mV in 2·5 mM-K, the slow rate of recovery was the same (0·03 ± 0·02 ΔpHi h-1) as that in fibres without a history of depolarization and thus of Ca release.
4. In fibres depolarized to —50 mV (15 mM-K, constant Cl) and then exposed to caffeine (4 mM) which releases Ca from intracellular stores, the recovery was the same (0·07 ±0·03 ΔpHi h-1, n = 5) as in depolarized fibres not exposed to caffeine (0·09±0·01 ΔpHi h-1, n = 5).
5. We conclude that in frog muscle transient Ca release induced by either depolarization or caffeine does not affect the rate of subsequent pHi recovery. Tetracaine reversibly inhibits pHi recovery, but this inhibition is not due to its blocking of Ca release.
6. Recovery from C02-induced acidification of fibres depolarized to —21 mV in 50 mM-K, constant Cl was halved, from 0·31 ±0·04 ΔpHi h-1 (n = 10) to 0·15±0·01 ΔpHi h-1 (n = 13), when external Ca was raised from 4 to 10 mM. There was no further reduction in recovery at 14 mM-Ca (0·15±0·044 ΔpHi h-1, n = 4). Lowering Ca from 4 to 1 mM did not affect recovery (0·32±0·04 ΔpHi h-1, n = 8). The removal of both Ca and Mg (either with or without 2 mM-EDTA) also did not affect recovery (0·31 ±0·03 ΔpHi h-1, n = 7).
7. Ba at 4, 10 or 20 mM reduced recovery from C02 acidification to 0·19±0·01 (n = 5), 0·16±0·01 (n = 6) and 0·13±0·03 ΔpHi h-1 (n = 5), respectively. Mg at 8 mM or Sr at 6 mM had no effect on recovery, but 20 mM-Mg or Sr reduced recovery to 0·23±0·02 ΔpHi h-1 (n = 6), respectively. Ni or Cd, each at 1 mM, reduced recovery to 0·21 ±0·01 ΔpHi h-1 (n = 6) and 0·12±0·02 ΔpHi h-1 (n = 7), respectively.
8. The reduced recovery from C02-induced acidification in 10 mM-Ca was not affected by 1 mM-amiloride (0·15±0·01 ΔpHi h-1, n = 8), even though we previously found that amiloride reduced the higher recovery rate observed in 4 mM-Ca by about 50%. 4-acetamido-4' -isothiocyanostilbene-2,2' -disulphonic acid (SITS) (0·1 mM) nearly abolished the recovery in 10 mM-Ca (0·05±0·02 ΔpHi h-1, n = 11). We conclude that elevated external Ca preferentially inhibits Na-H exchange.
9. In agreement with this conclusion, 10 mM-Ca abolished recovery in fibres acidified by pre-pulsing with NH4Cl, where recovery was shown to be due almost entirely to Na-H exchange. Cd had a similar effect. The Ca and Cd inhibitions could be partially reversed. Raising Mg to 8 mM was ineffective. La (1 mM) reduced recovery to 0·04±0·02 ΔpHi h-1 (n = 5); this effect also was partially reversible.
10. These observations on the effect of multivalent cations in the bathing solution are compatible with an external inhibitory cation binding site on the Na-H exchanger of frog skeletal muscle, with binding order La = Cd > Ni > Ba > Ca > Sr ≥ Mg. This sequence suggests an anionic site of weak field strength.
Putnam, R. W.,
& Roos, A.
(1986). Effect of Calcium and Other Divalent Cations on Intracellular pH Regulation of Frog Skeletal Muscle. The Journal of Physiology, 381, 221-239.