INHIBITORY EFFECT OF MUSCARINIC RECEPTOR ACTIVATION ON CA2+ CHANNEL CURRENT IN SMOOTH-MUSCLE CELLS OF GUINEA-PIG ILEUM

1. The effect of muscarinic receptor stimulation on voltage-gated calcium channel currents was examined in whole-cell voltage-clamped smooth muscle cells of the guinea-pig ileum. 2. In cells voltage clamped at -60 mV and in which calcium channel currents (I-Ca) were elicited repeatedly by depolarizing pulses (25 ms duration, 0.25 Hz frequency) to 0 mV, carbachol (CCh, 10 mu M) induced an inward current (I-CCh) and suppressed I-Ca, in a biphasic manner; an initial transient component was followed by a more sustained one. 3. A calcium channel current (I-Ba), when Ba2+ was used as a charge carrier, was also suppressed by CCh in a biphasic manner, as with I-Ca. The sustained phase of the I-Ba suppression was significantly smaller than that of the I-Ca suppression, suggesting that Ca2+ entry exerts a potentiating effect on the current suppression. 4. CCh had little or no effect on calcium channel currents (I-Ca and I-Ba) in cells dialysed with a pipette solution containing EGTA (20 mM). 5. Inclusion of GDP-beta-X (1 mM) in the pipette solution abolished I-CCh and the suppression of I-Ba. With GTP-gamma-S (10 mu M) in the pipette, the sustained phase of the I-Ba suppression remained almost unchanged even after removal of CCh. 6. Pretreatment with 2 mu g ml(-1) pertussis toxin (PTX), which abolished I-CCh, did not change noticeably the initial transient and sustained phases of I-Ba suppression. 7. Neomycin (100 mu M) or heparin (5 mg ml(-1)) in the pipette each abolished the initial transient component of I-CCh as well as the initial transient phase of I-Ba suppression. 8. The biphasic effect of CCB on I-Ba was observed in the presence of either staurosporine (1 mu M) or 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (100 mu M). Phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate (up to 10 mu M) had no inhibitory effect on I-Ca and I-Ba. 9. The results suggest that stimulation of the muscarinic receptor causes a biphasic suppression of the voltage-gated calcium channel currents through a PTX-insensitive G protein in guinea-pig ileal smooth muscle cells. The initial transient phase may be brought about by the release of Ca2+ from internal storage sites, and the sustained phase by a Ca2+-dependent mechanism which is independent of the phosphatidylinositol pathway.