Proximal tubule cells of the kidney contain, on their apical surface, an amiloride-sensitive Na/H antiporter that functions in Na reabsorption and proton secretion. We have investigated the localization of the antiporter in a cloned cell line of porcine renal origin, LLC-PKI/C14, which is often considered to be a useful model of the proximal tubule. Transport measurements were performed with differentiated monolayers grown on Nuclepore filters, permitting independent access to the apical and basolateral cell surfaces. In control experiments with LLC-PK,/Cl4 monolayers, three marker transport systems showed the expected polarity: 87% of ouabain-sensitive Rb uptake was at the basolateral surface, and 99% of Na-dependent a-methylglucoside transport and 93% of Na-dependent D-aspartate (L-glutamate) transport were at the apical surface. By contrast, the monolayers displayed significant Na/H antiporter activity (assayed as ethylisopropylamiloride-sensitive 22Na uptake) at both cell surfaces, with an apical uptake rate amounting to 44% and a basolateral rate amounting to 56% of the total. Significantly, the apical and basolateral antiporters could readily be distinguished from one another on the basis of ethylisopropylamiloride sensitivity. The apical system had an IC.5 of 13 FAM, close to that reported for kidney brush border vesicle preparations, whereas the basolateral system had an ICse of 44 nM, similar to values seen in undifferentiated LLC-PKI cells and other cultured cell lines.The PKE20 mutant, previously selected from LLC-PK,/Cl4on the basis of resistance to ethylisopropylamiloride, was found to overexpress the more resistant antiporter both during rapid growth and on its apical cell surface at confluence; normal amounts of the more sensitive antiporter were seen on the basolateral surface of confluent PKE20 cells. Taken together, these results suggest that there are two distinct forms of the Na/H antiporter, which are under separate genetic control.

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A synthetic analogue of ubiquinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, inhibits oxidation of succinate and NADH-linked substrates by rat liver mitochondria. Inhibition occurs both in the presence (state 3) and absence (state 4) of ADP. With isolated succinate-cytochrome c reductase complex from bovine heart mitochondria the quinone analogue inhibits succinate-cytochrome c reductase and ubiquinol-cytochrome c reductase activities but does not inhibit succinate-ubiquinone reductase activity. Inhibition of cytochrome c reductase activities is markedly dependent on pH in the range pH 7-8. At pH 7.0 inhibition occurs with an apparent Ki less than or equal to 1 x 10(-8) M, while at pH 8.0 the apparent Ki is more than an order of magnitude greater than this. Spectrophotometric titrations of 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole show a visibly detectable pKa at pH 6.5 attributable to ionization of the 6-hydroxy group. These results indicate that this quinone derivative is a highly specific and potent inhibitor of electron transfer in the b-c1 segment of the respiratory chain. Because of the structural analogy, it is likely that the mechanism of inhibition involves disruption of normal ubiquinone function. In addition, this inhibition depends on protonation of the ionizable hydroxy group of the inhibitory analogue or on protonation of a function group in the b-c1 segment.

The Ca2+-stimulated release of cortical vesicle (cortical granule) contents from the cell surface complex (CSC) of the sea urchin egg is an in vitro model for exocytosis. To gain insight into the molecular mechanism of exocytosis we investigated the sensitivity of this model to sulfhydryl modification and proteolytic digestion. Our findings include the following: (a) Proteolytic treatment with trypsin or pronase of CSC prepared from the eggs of StrongyIocentrotus purpuratus increased the free Ca 2+ concentration required to elicit exocytosis. Although a small increase in the Ca 2+ threshold was detected after mild proteolysis, high concentrations of trypsin (0.5 mg/ml) and prolonged incubation (3 h) were required to render the CSC unresponsive to high concentrations of Ca 2+ (0.5 raM). Despite the severity of the proteolytic digestions required to inactivate the CSC, the individual cortical vesicles remained intact, as gauged by the latency of ovoperoxidase, a cortical vesicle enzyme. (b) As previously shown (Haggerty, J. C., and R. C. Jackson, 1983, J. Biol. Chem. 258:1819-1825), cortical exocytosis can be blocked by sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). in this report we demonstrate that NEM inhibits by increasing the Ca 2+ threshold required for exocytosis. When CSC that had been completely inactivated by NEM modification was briefly digested, on ice, with a low concentration of trypsin (or several other proteases), exocytotic activity was restored. Although the Ca 2+ threshold of the reactivated CSC was slightly higher than that of untreated CSC, it was nearly identical to that of control CSC, which was trypsinized but not treated with NEM. We discuss the significance of these results with regard to the molecular mechanism of exocytosis.Cell surface complex (CSC) ~ prepared from the eggs of the sea urchin Strongylcocentrotus purpuratus is comprised of the vitelline layer, the plasma membrane and attached cortical secretory vesicles (CVs), also referred to as cortical granules (1). In response to physiologically relevant concentrations of free Ca 2+ the CSC undergoes a reaction that morphologically approximates exocytosis (2-4). This response can be followed microscopically (2-5), enzymatically (6, 7), and turbidimetAbbreviations used in this paper: CSC, cell surface complex; CV, cortical vesicle; DFP, diisopropylfluorophosphate; NEM, N-ethylmaleimide; PKME, buffer made with PIPES, KCI, MgCI2, and EGTA; SBT1, soybean trypsin inhibitor.rically (6,8). Recent investigations have shown that exocytosis in this in vitro model system can be inhibited by phenothiazine drugs (6,7,9) and by sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM; reference 6). Inhibition by sulfhydryl-modifying reagents suggested that proteinaceous elements of the CSC were required for cortical exocytosis and prompted us to investigate the effect of proteolytic digestion of CSC and NEM-inhibited CSC on cortical exocytosis. We show that cortical exocytosis can be completely inhibited by prolonged pro...