CHOLINERGIC REGULATION OF VOLTAGE-DEPENDENT CA2-MUSCLE CELLS( CHANNELS IN PORCINE TRACHEAL SMOOTH)

To investigate cholinergic regulation of voltage-dependent Ca2+ channe ls (VDCs) in airway smooth muscle, we measured inward currents through VDCs in enzymatically dispersed porcine tracheal smooth muscle cells using conventional (10 mM Ca2+ as charge carrier) and nystatin-perfora ted (5 mM Ba2+ as charge carrier) whole cell patch clamp techniques. C arbachol (CCh) had significant and dose-dependent inhibitory effects o n inward currents (12% with 10(-7) M and 42% with 10(-6) M) in perfora ted whole cell clamp experiments, but had no effect on currents in con ventional whole cell experiments. CCh also shifted the steady-state in activation curve to more negative potentials. Further experiments test ed the hypothesis that CCh inhibits VDCs in part by the activation of protein kinase C (PKC). Phorbol 12,13-diacetate, an exogenous PKC acti vator, inhibited currents through VDCs, and calphostin C, a specific P KC inhibitor, antagonized the inhibitory effect of CCh. Furthermore, i ntracellular exposure to the activating PKC fragment 530-558, using a pipette perfusion technique, also inhibited currents through VDCs. We conclude that cholinergic receptor stimulation can inhibit inward Ca2 currents through VDCs of porcine tracheal smooth muscle and that this effect may be mediated in part by activation of PKC.