Regulatory volume increase after secretory volume decrease in colonic epithelial cells under muscarinic stimulation

To address the question of whether colonic secretory cells change their volume in response to carbachol (CCh) stimulation and, if so, the mechanisms involved therein, we used two-photon laser scanning microscopy to measure the volume of individual epithelial cells in the fundus region of crypts isolated from the guinea-pig distal colon. We also measured the volume of human colonic epithelial T84 cells using an electronic sizing technique. Both types of colonocytes responded to stimulation by CCh with shrinkage and then underwent a regulatory volume increase (RVI), even during continued stimulation by CCh. The secretory volume decrease (SVD) induced by CCh was antagonized by atropine, BAPTA loading and niflumic acid, a blocker of Ca²⁺-activated Cl ^- channels. An increase in the intracellular free [Ca²⁺] was observed with fura-2 during these volume responses to CCh. Removal of all Na⁺ or K⁺ or of most of the Cl^- from the extracellular solution abolished the RVI, but not the preceding SVD. The RVI, but not the preceding SVD, was abolished by bumetanide, a blocker of the Na ⁺-K⁺-2Cl^- cotransporter. We conclude that guinea-pig crypt colonocytes and human T84 cells exhibit a cytosolic Ca ²⁺-dependent SVD and undergo a subsequent RVI that is dependent on the operation of Na⁺-K⁺-2Cl^- cotransporters.