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Cellular mechanisms underlying carbachol‐induced oscillations of calcium‐dependent membrane current in smooth muscle cells from mouse anococcygeus
Author(s) -
Wayman Christopher P,
McFadzean Ian,
Gibson Alan,
Tucker John F
Publication year - 1997
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0701279
Subject(s) - cyclopiazonic acid , carbachol , egta , chemistry , calcium , biophysics , patch clamp , extracellular , biochemistry , receptor , biology , organic chemistry
At a holding potential of −40 mV, carbachol (50 μ M ) 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 . 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 m M ), and by inclusion of 1 m M EGTA in the patch‐pipette filling solution. In calcium‐free extracellular medium (plus 1 m M 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 m M ). Inclusion of the inositol triphosphate (IP 3 ) 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 μ M ), prevented the activation of I oscil by carbachol. Caffeine (10 m M ) 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 μ M ) and a high (30 μ M ) concentration of ryanodine. Carbachol‐induced I oscil was abolished by the general calcium entry blocker SKF 96365 (10 μ M ) and by Cd 2+ (100 μ M ), but was unaffected by La 3+ (400 μ M ). As found previously, I DOC was also blocked by SKF 96365 and Cd 2+ , but not La 3+ ; the inhibition of I DOC preceded the abolition of I oscil by 27 s with SKF 96365 and by 30 s with Cd 2+ . Nifedipine (1 μ M ) 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%. 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.