Evidence that Basolateral But Not Apical Membrane Localization of Outwardly Rectifying Depolarization-Induced Cl− Channel in Airway Epithelia

The rat primary cultured-airway monolayer had been an excellent model for deciphering the ion channel after nystatin permeabilization of its basolateral or apical membrane (Hwang et al., 1996). After apical membrane permeabilization of rat primary cultured-airway monolayer, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive outwardly rectifying depolarization-induced Cl⁻ (BORDIC) currents were observed across the basolateral membrane in symmetrical NMG-Cl solution in this study. No significant Cl⁻ current induced by the application of voltage clamping was observed across the apical membrane in symmetrical NMG-Cl solution after basolateral membrane permeabilization. The halide permeability sequence for BORDIC current was Br⁻≒ I⁻ > Cl⁻. BORDIC current was not affected by basolaterally applied bumetanide (0.5 mm). Basolateral DIDS (0.2 mm) but not apical DIDS inhibited CFTR mediated short-circuit current (I _sc ) in an intact monolayer of rat airway epithelia, a T84 human colonal epithelial cell line, and a Calu-3 human airway epithelial cell line. This is the first report showing that depolarization induced Cl⁻ current is present on the basolateral membrane of airway epithelia. Received: 7 October 1999/Revised: 24 April 2000     

Cyclooxygenase-2, Not Microsomal Prostaglandin E Synthase-1, Is the Mechanism for Interleukin-1β-induced Prostaglandin E2 Production and Inhibition of Insulin Secretion in Pancreatic Islets

Arachidonic acid is converted to prostaglandin E(2) (PGE(2)) by a sequential enzymatic reaction performed by two isoenzyme groups, cyclooxygenases (COX-1 and COX-2) and terminal prostaglandin E synthases (cPGES, mPGES-1, and mPGES-2). mPGES-1 is widely considered to be the final enzyme regulating COX-2-dependent PGE(2) synthesis. These generalizations have been based in most part on experiments utilizing gene expression analyses of cell lines and tumor tissue. To assess the relevance of these generalizations to a native mammalian tissue, we used isolated human and rodent pancreatic islets to examine interleukin (IL)-1β-induced PGE(2) production, because PGE(2) has been shown to mediate IL-1β inhibition of islet function. Rat islets constitutively expressed mRNAs of COX-1, COX-2, cPGES, and mPGES-1. As expected, IL-1β increased mRNA levels for COX-2 and mPGES-1, but not for COX-1 or cPGES. Basal protein levels of COX-1, cPGES, and mPGES-2 were readily detected in whole cell extracts but were not regulated by IL-1β. IL-1β increased protein levels of COX-2, but unexpectedly mPGES-1 protein levels were low and unaffected. In microsomal extracts, mPGES-1 protein was barely detectable in rat islets but clearly present in human islets; however, in neither case did IL-1β increase mPGES-1 protein levels. To further assess the importance of mPGES-1 to IL-1β regulation of an islet physiologic response, glucose-stimulated insulin secretion was examined in isolated islets of WT and mPGES-1-deficient mice. IL-1β inhibited glucose-stimulated insulin secretion equally in both WT and mPGES-1(-/-) islets, indicating that COX-2, not mPGES-1, mediates IL-1β-induced PGE(2) production and subsequent inhibition of insulin secretion.     

Myogenic tone in mouse mesenteric arteries: evidence for P2Y receptor-mediated, Na+, K+, 2Cl− cotransport-dependent signaling

This study examines the action of agonists and antagonists of P2 receptors on mouse mesenteric artery contractions and the possible involvement of these signaling pathways in myogenic tone (MT) evoked by elevated intraluminal pressure. Both ATP and its non-hydrolyzed analog α,β-ATP triggered transient contractions that were sharply decreased in the presence of NF023, a potent antagonist of P2X₁ receptors. In contrast, UTP and UDP elicited sustained contractions which were suppressed by MRS2567, a selective antagonist of P2Y₆ receptors. Inhibition of Na⁺, K⁺, 2Cl⁻ cotransport (NKCC) with bumetanide led to attenuation of contractions in UTP- but not ATP-treated arteries. Both UTP-induced contractions and MT were suppressed by MRS2567 and bumetanide but were insensitive to NF023. These data implicate a P2Y₆-mediated, NKCC-dependent mechanism in MT of mesenteric arteries. The action of heightened intraluminal pressure on UTP release from mesenteric arteries and its role in the triggering of P2Y₆-mediated signaling should be examined further.