Upon transferring confluent monolayers of Madin-Darby canine kidney (MDCK) cells from a low-Ca2+ medium (1-5 microM) to one with 1.8 mM Ca2+ (Ca switch), tight junctions (TJs) assemble and seal, and transepithelial electrical resistance (TER) develops in 4-5 h, presumably through exocytotic fusion that incorporates junctional components to the surface membrane. In the present work we test this possibility and observe 1) that the Ca switch raises the cytosolic concentration of this ion; 2) that it also increases the membrane area by 22%; 3) that chloroquine, a drug which prevents exocytosis, blocks both the increase of surface membrane and the sealing of TJs; and 4) that if monolayers are not permanently switched to 1.8 mM Ca2+, but are subject to a 15-min pulse, cytosolic free Ca2+ concentration [( Ca2+]c) transiently increases but returns to low values (14 +/- 11 nM) and TER does not develop. Comparisons of the time course of TJ sealing with levels of [Ca2+]c, as well as the relationship between these parameters and extracellular Ca2+ levels, suggest that this ion may act from the extracellular side or in a narrow intracellular domain in the close vicinity of the plasma membrane.

Essential hypertension, a rise in blood pressure of undetermined cause, includes 90% of all hypertensive cases and is a highly important public health challenge that remains, however, a major modifiable cause of morbidity and mortality. This review emphasizes that, from an evolutionary point of view, we are adapted to ingest and excrete <1 g of sodium (2.5 g of salt) per day and that essential hypertension develops when the kidneys become unable to excrete the amount of sodium ingested, unless blood pressure is increased. The renal-mean arterial pressure set-point model is briefly described to explain that a shift of the pressure natriuresis relationship toward abnormally high pressure levels is a pathophysiological characteristic of essential hypertension. Evidence indicating that this anomaly in the pressure natriuresis relationship arises from a sympathetic nervous system dysfunction is briefly formulated, and the most widely accepted pathophysiologic proposal to explain the development of this sympathetic dysfunction is described, with commentaries about novel action mechanisms of some drugs currently used in essential hypertension treatment.

Patch-clamp techniques were used to study a K channel in the cell membrane of MDCK cells. This cell line derives from the kidney of a normal dog, presumably from the distal nephron, a region involved in potassium secretion. The cells were cultured in confluent monolayers and approached from the apical side. The K channel we describe is Ca2+ and voltage activated, has a conductance of 221 +/- 7 pS, and can be inhibited by 10 mM tetraethylammonium and by 1 mM quinidine, but not by 4-aminopyridine, nor by 1 mM Ba2+ added to the outer side. Using the whole-cell configuration, we find that most of the cationic conductance of the membrane is constituted by a K-specific one (maximum K conductance 32.1 +/- 3.9 nS vs. a leak conductance of 1.01 +/- 0.17 nS). Comparisons of the maximum K conductance with that of a single K channel indicates that an MDCK cell has an average of 145 such channels. The membrane capacity is 24.5 +/- 1.4 pF.

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Madin-Darby canine kidney (MDCK) cells plated at confluence and incubated for 20 h in low (5 microM) Ca2+ have no tight junctions (TJs), and their Na+-K+-ATPase is randomly distributed over the surface. On transfer to normal Ca2+ levels (1.8 mM) ("Ca2+ switch"), TJs and transepithelial resistance develop quickly, trapping a considerable fraction (35%) of the surface Na+-K+-ATPase on the apical (incorrect) side. This misplaced enzyme is subsequently removed from this region or inactivated, demonstrating that polarization proceeds despite TJs. Simultaneously, the amount of Na+-K+-ATPase on the basolateral side increases in a higher proportion (125%), than could be accounted for by relocation of the misplaced apical enzyme. This incorporation is prevented by cycloheximide, ammonium chloride, primaquine, or chloroquine, suggesting that Na+-K+-ATPase originates in an intracellular pool and that its surface insertion requires synthesis of new enzyme or of a protein factor, since it is carried to the surface membrane through a mechanism of exocytosis. In summary, asymmetric distribution of ion pumps depends 1) on polarized insertion of Na+-K+-ATPase as well as 2) on removal or inactivation of misplaced enzyme.