EMPTYING AND REFILLING OF CA2+ STORE IN TRACHEAL MYOCYTES AS INDICATED BY ACH-EVOKED CURRENTS AND CONTRACTION

Membrane currents and contractions evoked by acetylcholine (ACh) in freshly dissociated canine tracheal myocytes were investigated using the nystatin perforated-patch recording technique. In cells held at -60 mV in the presence of nifedipine, ACh evoked inward current (I(ACh)) and contraction. Caffeine mimicked the effects of ACh. I(ACh) and contractions could be evoked 3-4 min after removing external Ca2+ but were abolished by prolonged exposure to Ca2+-free media. Both responses were restored within minutes of reintroduction of Ca2+, even though the cells were held at -60 mV in the presence of nifedipine. I(ACh) and ACh-evoked contractions were also reversibly abolished by continued exposure to caffeine. Cyclopiazonic acid (CPA), a blocker of the sarcoplasmic reticulum (SR) Ca2+-ATPase, reduced I(ACh) by >95% within 15 min but had little or no effect on the contractile responses evoked by ACh. I(ACh) was restored after washout of CPA even though cells were held at -60 mV. After depleting the Ca2+ store with the use of CPA, depolarization of the membrane to +10 mV immediately before application of ACh led to a partial restoration of I(ACh). This restorative effect of depolarization was potentiated by Bay K 8644 and antagonized by nifedipine. In conclusion, I(ACh) and contractions in canine tracheal myocytes are mediated by Ca2+ released from an internal store that can be depleted by prolonged removal of extracellular Ca2+, prolonged exposure to caffeine, or by blockade of the SR Ca2+-ATPase. At least two Ca2+ influx pathways appear to contribute to refilling of the internal store: one pathway that is not activated by depolarization or ACh and a second involving dihydropyridine-sensitive voltage-activated Ca2+ channels that may be in direct contact with the SR (i.e., conduct extracellular Ca2+ directly into the SR, bypassing the cytosol).