Calcium regulation during stimulus-secretion coupling: continuous measurement of intracellular calcium activities.

Accurate measurements of intracellular calcium activities in salivary gland epithelial cells of the insect Phormia regina were obtained with microelectrodes in which N,N'-di(11-ethoxycarbonyl)undecyl-N,N'-4,5-tetramethyl-3,6-dioxaoctane diacid diamide wsa incorporated in a liquid membrane system. When calibrated in solutions approximating the ionic concentration of the cell interior, these microelectrodes gave rapid stable responses that were linear functions of the logarithm of calcium activities and were not affected by potassium, sodium and magnesium. Continuous monitoring of calcium activities during serotonin-induced saliva release provided direct evidence of hormonal influence on transmembrane calcium movement and spontaneous regulation of intracellular calcium by stimulated cells.

Vanadate inhibition of ATP-dependent H+ transport in membrane vesicles from turtle bladder epithelial cells.

The ATP-dependent proton transport into vesicles of a mixed membrane fraction obtained from turtle bladder epithelial cells consists of at least two kinetically defined moieties: one, which is maximally inhibited by 25% with nanomolar levels of vanadate, but not inhibited at all with equimolar levels of N-ethylmaleimide, and another, which is maximally inhibited by 70% with micromolar levels of N-ethylmaleimide and by 25% with equimolar levels of vanadate. In contrast to the transport function, the associated enzymatic function (the ouabain-resistant ATPase activity) in these membranes, not inhibited by nanomolar levels of vanadate or N-ethylmaleimide, is maximally inhibited by 40% with micromolar levels of vanadate and by 13% with equimolar levels of N-ethylmaleimide. Independent of these kinetic differences between the enzyme and the transport functions, membranes containing the N-ethylmaleimide-sensitive proton transport function are electrophoretically separable from those containing the vanadate-sensitive transport function. For example, the kinetically defined, vanadate-sensitive proton transport function is recovered exclusively and kinetically identified in one of four electrophoretic membrane fractions, EF-II; while the N-ethylmaleimide-sensitive function is recovered in EF-III as well as in EF-II. Membranes of EF-IV, maximally enriched in ouabain-resistant ATPase activity, possess no proton transport function at all, even in the absence of N-ethylmaleimide or vanadate. Additional data under in vivo as well as under in vitro conditions are required to prove that the vanadate-sensitive proton transport in these vesicles is an in vitro manifestation of the mechanism responsible for generating the vanadate-sensitive luminal acidification process under in vivo conditions in the intact turtle bladder.

ATPase activity and ATP-dependent proton translocation in plasma membrane vesicles of turtle bladder epithelial cells.

ATP-induced quenching of fluorescence of acridine orange (a pH probe) or Oxonol V (a potential difference probe) is evoked in turtle bladder membrane vesicles in suspending media of appropriate ionic composition and is insensitive to oligomycin, valinomycin, and ouabain. These effects are ascribed to a membrane-bound, ouabain-resistant ATPase which mediates an active electrogenic proton transport.

The role of bicarbonate ions and of adenosine 3',5'-monophosphate (cAMP) in chloride transport by epithelial cells of bullfrog small intestine.

In an HCO3-free medium, isolated segments of bullfrog small intestine, stripped of their external muscle layers, displayed a small, serosal positive PD that did not, on the average, differ significantly from zero. Similarly, in this medium, the mean values of Isc and of net Na+ and Cl- absorption under short-circuit conditions did not differ significantly from zero. External HCO3- (25 mM) induced a highly significant serosal negative PD and Isc and a large net absorption of Cl-. Net Cl- absorption exceeded Isc, i.e., there was a significant net flux, JR, which was consistent with a net secretion of HCO3-. The ratio of the internal Cl-activity of the absorptive cells (alpha Cli) to its equilibrium value was larger in the presence than in the absence of HCO3-. In the presence of HCO3-, cAMP, added to the serosal medium, reversed the serosal negative PD and Isc, and inhibited, though it did not completely abolish, net Cl- absorption. JR was unchanged; tissue Cl- and alpha Cli were reduced, and tissue Na+ decreased and tissue K+ increased. When HCO3- and Cl- were removed from the bathing medium, the electrical response of the tissue to cAMP, though greatly attenuated, was not completely abolished. Under these conditions, cAMP induced a significant net Na+ absorption. A model for ion transport in the absorptive cells of the small intestine is proposed that is consistent with these findings.

ATP-dependent H+ transport by the turtle bladder: NBD-C1 preferentially inhibits the vanadate-insensitive component in isolated membranes.

We investigated the inhibitory effects of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) on ATP-dependent H+ accumulation by membrane vesicles prepared from the turtle urinary bladder epithelium. NBD-Cl at 30 microM was found to completely inhibit the vanadate-insensitive component of H+ transport, with half-maximal inhibition occurring at 4.2 to 5.4 microM. In contrast, the vanadate-inhibitable component was unaffected by 30 microM NBD-Cl. At high concentrations (300 microM), both components were fully inhibited. The results confirm the presence of two distinct H+ transport processes in turtle bladder membranes and identify selective inhibitors, NBD-Cl and vanadate, for each process.

Effects of valinomycin on vanadate-sensitive and vanadate-resistant H+ transport in vesicles from turtle bladder epithelium: evidence for a K+/H+ exchanger.

The vanadate-sensitive component of the ATP-dependent H+ gradient formed in isolated vesicles from a urinary epithelium was abolished by valinomycin omission. This suggests that vanadate-sensitive H+ transport has an absolute requirement for intravesicular K+ and that the transport may be due to a K+/H+ exchanger. Sensitivity to the inhibitor SCH28080 supports this conclusion. On the other hand, valinomycin affects the initial velocity of vanadate-resistant transport without altering its maximum gradient. This is consistent with the development of a membrane potential consequent to electrogenic uniport H+ transport.

Physiological role for vanadate-inhibitable active H+ transport: a new model for distal urinary acidification.

We have shown elsewhere that membranes isolated from the turtle bladder epithelium have both vanadate-resistant (VR) and vanadate-sensitive (VS) components of ATP-dependent H+ transport. VR appears to be due to a vacuolar-type H+ uniport while VS has properties of an H/K exchanger. In the present study we used bladders from chronically alkalotic turtles, which do not acidify urine, and found them to yield membranes which retain VR but lack VS transport. Thus VS occurs only in membranes from bladders secreting acid, implying that VS H+ transport (1) plays a physiological role in the secretion and (2) is a functional marker for the apical membrane of the acid-secreting cells. These results suggest a new model for distal urinary acidification.

Bafilomycin A1 at nanomolar concentrations saturably inhibits a portion of turtle bladder acidification current.

An earlier report indicated that acid secretion in turtle urinary bladder is driven by an unusual vacuolar H+-ATPase and that the ATPase accounts for essentially all acid secreted. These results, however, are difficult to reconcile with the acid transporters currently ascribed to the renal collecting duct. Here, we re-examine the effect of bafilomycin A1, an inhibitor of vacuolar (V-type) H+-ATPases, on acid secretion by intact isolated bladders from Pseudemys scripta turtles. Serosal-side bafilomycin had no effect on the transepithelial acidification current (AC). In the mucosal solution, bafilomycin inhibited the AC, with inhibition developing over the range 0.1-10 nmoll(-1), with a sigmoidal dose-response curve, and an IC50 of 0.47 nmoll(-1). At saturation, approximately 70% of H+ secretion was inhibited. The remaining 30% could be abolished by 30 micromoll(-1) Sch-28080, which is a level that in other systems is known to inhibit H+/K+-ATPase transport activity specifically and essentially completely. When the order of addition was reversed (Sch-28080 first), there was no change in the magnitude of the effect produced by either inhibitor, and the two together again eliminated the AC. The data indicate that baseline acid secretion in intact bladders is due (i) in part to a highly bafilomycin-sensitive process, with sensitivity typical of vacuolar H+ ATPases; and (ii) in part to a more bafilomycin-resistant process that is sensitive to Sch-28080.

Determination of the membrane potential of cultured mammalian Schwann cells and its sensitivity to potassium using a thiocarbocyanine fluorescent dye.

The membrane potential of cultured rat sciatic nerve Schwann cells was determined with conventional microelectrode and voltage-sensitive fluorescent dye, Di-S-C3(5), optical techniques. The value for membrane potential obtained with microelectrodes was -42.1 +/- 4.7 mV (n = 8). Using optically determined fluorescent intensity changes caused by changes in external potassium ion concentration, in the presence or absence of valinomycin (null point method), the membrane potential was estimated at -45.7 +/- 6.2 mV (n = 7); with a gramicidin and valinomycin double ionophore method it was -52.2 +/- 9.1 (n = 4). The membrane potential of Schwann cells was found to be potassium sensitive at and above the physiological range of [K+] at 27.5 mV/10x delta[K+], which is approximately half the Nernstian value. This result suggests that other ion permeabilities strongly influence the resting membrane potential of cultured Schwann cells. Since Na+ had little effect on the membrane potential, it is concluded that Cl- is a likely candidate for the other permeant ionic species. The optical method has been shown to be a useful tool for the systematic study of the membrane potential of Schwann cells in culture and for the characterization of its ionic basis and regulation.

Electrophysiologic study of rabbit proximal tubular cell monolayers in primary culture.

Primary cultures of renal cortical cells prepared by selective sieves have been found to display some characteristics of renal proximal tubular epithelium but their site of origin has not been confirmed by electrophysiologic studies. Cells were cultured in a defined medium on collagen gels. Confluency was approached after 7-10 days but gels were found to have zero transepithelial resistance unless they were allowed to contract spontaneously. With the appearance of a nonzero resistance, there was a change in morphology to a more columnar cell with better developed microvilli. These structural features were particularly prominent in clusters of proliferating cells observed on and around remnants of original tubules embedded in the gel. In noncontracted cultures there was no focal cell clustering and cells were squamous-like with rudimentary microvilli, similar in appearance to cells grown on plastic culture dishes. Measurements made in contracted monolayers yielded an average transepithelial resistance of 6.5 omega cm2, a spontaneous transepithelial potential difference of +0.9 mV, measured with respect to the serosa, and an apical membrane potential of -75 mV when cells were bathed in 0.4 mM K and -49 mV when cells were bathed in 4 mM K media. Mucosal protamine (50 micrograms/ml) increased transepithelial resistance by 22%, suggesting that the epithelial cell tight junctions were responsive to external stimuli. Monolayers were anion selective, giving a dilution potential (lumen-directed NaCl gradient) of -2.6 mV with respect to the serosa. These experiments show that primary culture of rabbit renal cortical cells separated by differential sieves displays electrophysiologic and morphologic characteristics of a proximal renal tubular epithelium. Confluency and attainment of differentiated morphology and function are promoted when monolayer cells are not bound to an unyielding substrate.