Abstract

Cyclic GMP relaxes swine tracheal smooth muscle. Relaxation occurs because of decreases in intracellular calcium concentration ([Ca⁺⁺]_i) that are thought to occur through hyperpolarization which inhibits calcium influx. Activation of K⁺ channels has been suggested as the underlying mechanism for the hyperpolarization. In the present study, the effects of 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP, a membrane-permeable analog of cyclic GMP) on acetylcholine (ACh)-induced increases in [Ca⁺⁺]_i were examined by laser scanning confocal microscopy in fluo 3-loaded single cells. Membrane potential and currents were measured by the perforated-configuration of patch-clamp method. 8-Bromo-cGMP (1 μM–0.1 mM) inhibited 0.1 μM ACh-induced oscillations in [Ca⁺⁺]_i in a concentration-dependent manner. Spontaneous changes in membrane potential were observed by the patch-clamp method. Acetylcholine (0.03 μM) did not affect the time-averaged mean potential. The spontaneous changes in membrane potential were reduced and the cells were depolarized by 0.1 μM ACh and to a greater degree by 1 μM ACh. This result is consistent with previous observations of ACh-induced depolarization in intact tissue. The application of 0.1 mM 8-Br-cGMP had no significant effects on spontaneous changes in membrane potential and did not induce changes in membrane potential in cells treated with 0.1 μM ACh. In voltage-clamped cells, ACh (0.1 μM) induced oscillations in calcium-activated K⁺ currents. 8-Bromo-cGMP (0.1 mM) inhibited these ACh-induced oscillations in currents, but had no significant effects on spontaneous changes in membrane current in unstimulated cells. These data indicate that 8-Br-cGMP inhibits ACh-induced increases in [Ca⁺⁺]_i by mechanisms other than regulation of membrane potential.