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

Membrane currents and contractions evoked by acetylcholine (ACh) in fr eshly dissociated canine tracheal myocytes were investigated using the nystatin perforated-patch recording technique. In cells held at -60 m V in the presence of nifedipine, ACh evoked inward current (I(ACh)) an d contraction. Caffeine mimicked the effects of ACh. I(ACh) and contra ctions could be evoked 3-4 min after removing external Ca2+ but were a bolished by prolonged exposure to Ca2+-free media. Both responses were restored within minutes of reintroduction of Ca2+, even though the ce lls were held at -60 mV in the presence of nifedipine. I(ACh) and ACh- evoked contractions were also reversibly abolished by continued exposu re to caffeine. Cyclopiazonic acid (CPA), a blocker of the sarcoplasmi c 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, depolariz ation of the membrane to +10 mV immediately before application of ACh led to a partial restoration of I(ACh). This restorative effect of dep olarization was potentiated by Bay K 8644 and antagonized by nifedipin e. In conclusion, I(ACh) and contractions in canine tracheal myocytes are mediated by Ca2+ released from an internal store that can be deple ted by prolonged removal of extracellular Ca2+, prolonged exposure to caffeine, or by blockade of the SR Ca2+-ATPase. At least two Ca2+ infl ux pathways appear to contribute to refilling of the internal store: o ne pathway that is not activated by depolarization or ACh and a second involving dihydropyridine-sensitive voltage-activated Ca2+ channels t hat may be in direct contact with the SR (i.e., conduct extracellular Ca2+ directly into the SR, bypassing the cytosol).