Cellular mechanisms underlying carbachol-induced oscillations of calcium-dependent membrane current in smooth muscle cells from mouse anococcygeus

Abstract

1. At a holding potential of −40 mV, carbachol (50 μM) produced a complex pattern of inward currents in single smooth muscle cells freshly isolated from the mouse anococcygeus. Membrane currents were monitored by the whole-cell configuration of the patch-clamp technique. Previous work has identified the first, transient component as a calcium-activated chloride current (I_Cl(Ca)) and the second sustained component as a store depletion-operated non-selective cation current (I_DOC). The object of the present study was to examine the cellular mechanisms underlying the third component, a series of inward current oscillations (I_oscil) superimposed on I_DOC.

2. Carbachol-induced I_oscil (amplitude 97±11 pA; frequency 0.26±0.02 Hz) was inhibited by the chloride channel blocker anthracene-9-carboxylic acid (A-9-C; 1 mM), and by inclusion of 1 mM EGTA in the patch-pipette filling solution.

3. In calcium-free extracellular medium (plus 1 mM EGTA), carbachol produced an initial burst of oscillatory current which lasted 94 s before decaying to zero; I_oscil could be restored by re-admission of calcium. The frequency, but not the amplitude, of I_oscil increased with increasing concentrations of extracellular calcium (0.5–10 mM).

4. Inclusion of the inositol triphosphate (IP₃) receptor antagonist heparin (5 mg ml⁻¹) in the patch-pipette filling solution, or pretreatment of cells with the sarcoplasmic reticulum (SR) calcium ATPase inhibitor cyclopiazonic acid (CPA; 10 μM), prevented the activation of I_oscil by carbachol. Caffeine (10 mM) activated both I_Cl(Ca) and I_DOC and prevented the induction of I_oscil by carbachol. Caffeine and CPA also abolished I_oscil in the presence of carbachol, as did both a low (3 μM) and a high (30 μM) concentration of ryanodine.

5. Carbachol-induced I_oscil was abolished by the general calcium entry blocker SKF 96365 (10 μM) and by Cd²⁺ (100 μM), but was unaffected by La³⁺ (400 μM). As found previously, I_DOC was also blocked by SKF 96365 and Cd²⁺, but not La³⁺; the inhibition of I_DOC preceded the abolition of I_oscil by 27 s with SKF 96365 and by 30 s with Cd²⁺. Nifedipine (1 μM) produced a partial inhibition of the carbachol-induced I_oscil frequency at holding potentials of −20 mV and −60 mV and, in addition, reduced I_DOC at −60 mV by 18%.

6. It is concluded that carbachol-induced inward current oscillations in mouse anococcygeus cells are due to a calcium-activated chloride current, and reflect oscillatory changes in cytoplasmic calcium ion concentration. These calcium oscillations are derived primarily from the SR stores, but entry of calcium into the cell is necessary for store replenishment and maintenance of the oscillations. Capacitative calcium entry (via I_DOC) appears to be important not only for sustained contraction of this tissue, but also as a route for re-filling of the SR and, therefore, represents an important target for the development of novel and selective drugs.

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