INWARD CURRENT ACTIVATED BY CARBACHOL IN RAT INTESTINAL SMOOTH-MUSCLE CELLS

1 . Carbachol(0.1 mm or 10 mum)-evoked inward currents were studied with standard and perforated whole-cell patch clamp techniques in smooth muscle cells isolated from rat small intestine. The intracellular free Ca2+ concentration was monitored simultaneously with the fura-2 method. 2. With a K+-containing pipette solution, carbachol produced an inward current at - 60 mV and a large outward current at - 20 mV. 3. When NaCl was substituted for KCl in the external and pipette solutions, carbachol elicited inward currents at holding potentials more inside-negative than 0 mV. The reversal potential of the carbachol-induced current altered when external chloride (- 0.9 mV) was replaced by iodide (- 21.2 mV), thiocyanate (- 27.0 mV) and glutamate (18.2 mV). The carbachol-induced current at - 60 mV was slightly decreased by the replacement of external NaCl with Tris-Cl. 4. The carbachol-induced inward current at - 60 mV was accompanied by an increase in the intracellular concentration of free Ca2+. Both responses to carbachol were observed 2 min after exposure of the cells to a Ca2+-free solution containing 2 mm EGTA. 5. Intracellular application of heparin inhibited the inward current and Ca2+ transient responses to carbachol but not those to caffeine (10 mm). An inward current and Ca2+ transient were elicited after the patch membrane was ruptured at - 60 mV, using a patch pipette containing inositol 1,4,5-trisphosphate (InsP3). 6. It is concluded that the carbachol-induced inward current is due to increases in membrane Cl- and Na+ conductances. Ca2+ released from InsP3-sensitive stores may play a role in increasing both conductances.