Ionic basis of methacholine-induced shrinkage of dissociated eccrine clear cells

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Abstract
The goal of the present study was to elucidate the ionic mechanisms by which cholinergic stimulation induces cell shrinkage in eccrine clear cells. Dissociated Rhesus monkey eccrine sweat clear cells were prepared by collagenase digestion of freshly isolated secretory coils and immobilized on a glass slide in a perfusion chamber at 30°C. The cell was visualized by light microscopy with differential interference contract (DIC) and was recorded with a video system (15,000× total magnification). The cell volume was calculated from the maximal cross section of the cell. Methacholine (MCh)-induced cell shrinkage, which was as much as 30% of resting cell volume, was dose dependent and pharmacologically specific. MCh-induced cell shrinkage was persistent in some cells but tended to partially wane with time in others. MCh-induced cell shrinkage was dependent on the chemical potential gradient for KCl, i.e., increasing [K] in the bath ([K] o ) from 5 to 120mm caused MCh to induce cell swelling, whereas removing [Cl] o at 120mm K partially restored the MCh-induced cell shrinkage. The interpolated null [K] o (medium [K] where the cell volume did not change by MCh) of 71mm agreed with the predicted [K] o,null. MCh-induced cell shrinkage was inhibited completely by 1mm quinidine (K-channel blocker) and partially by 1mm diphenylamine-2-carboxylic acid (DPC, a Cl-channel blocker), but not by 0.1mm ouabain or 0.1mm bumetanide, suggesting that MCh-induced cell shrinkage may be due to activation of both K and Cl channels with the resultant net KCl efflux down the chemical potential gradient. That Ca/calmodulin may be involved in cholinergic regulation of Cl and K channels is suggested because 10 μm ionomycin also induced cell shrinkage, MCh failed to induce cell shrinkage in a Ca-free medium after the endogenous Ca store was depleted, and (6-aminohexyl)-5-chlorol-naphthalenesulfonamide (W-7, a putative inhibitor of calmodulin) also inhibited MCh-induced cell shrinkage in a reversible manner.

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