Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.     

Na+ and K+ transport at basolateral membranes of epithelial cells. III. Voltage independence of basolateral membrane Na+ efflux

Na+ efflux across basolateral membranes of isolated epithelia of frog skin was tested for voltage sensitivity. The intracellular Na+ transport pool was loaded with 24Na from the apical solution and the rate of isotope appearance in the basolateral solution (JNa23) was measured at timed intervals of 30 s. Basolateral membrane voltage was depolarized by either 50 mM K+, 5 mM Ba++, or 80 mM NH+4. Whereas within 30 s ouabain caused inhibition of JNa23, depolarization of Vb by 30-60 mV caused no significant change of JNa23. Thus, both pump-mediated and leak Na+ effluxes were voltage independent. Although the pumps are electrogenic, pump-mediated Na+ efflux is voltage independent, perhaps because of a nonlinear relationship between pump current and transmembrane voltage. Voltage independence of the leak Na+ efflux confirms a previous suggestion (Cox and Helman, 1983. American Journal of Physiology. 245:F312-F321) that basolateral membrane Na+ leak fluxes are electroneutral.     

Potassium dependence of sodium transport in frog skin

22Na+ and 42K+ fluxes across the basolateral membrane of the isolated epithelium of frog skin were investigated with regard to dependence on K+ in the basolateral solution. When K+ was removed from the basolateral solution (K+-free Ringer), there was a transient rise in short circuit current (Isc) that could be eliminated by pretreatment with ouabain. Concurrently, the apparent sodium efflux across the basolateral membrane (JNa*13) showed either no change or an immediate (1-2 min) small decrease (approximately equal to 10%) that was followed by a small transient increase. K+ fluxes showed either no change or a small decrease under these conditions. JNa*13 was partially ouabain sensitive during all of the above treatments. Furosemide partially inhibited both sodium and potassium flux after K+-free treatment. The pump, as defined by ouabain sensitivity of Na+ flux, continued to work even after 20 minutes of K+-free treatment. Pump activity may be maintained by potassium leaking from the cells that is recycled by the pump. However, the ouabain-sensitive transient rise in Isc after K+-free treatment cannot readily be explained by changes in either Na+ or K+ flux. A change in pump coupling ratio provides one explanation for these data.     

Role of Na+/H+ exchange in the control of intracellular pH and cell membrane conductances in frog skin epithelium

Ion-sensitive microelectrodes and current-voltage analysis were used to study intracellular pH (pHi) regulation and its effects on ionic conductances in the isolated epithelium of frog skin. We show that pHi recovery after an acid load is dependent on the operation of an amiloride-sensitive Na+/H+ exchanger localized at the basolateral cell membranes. The antiporter is not quiescent at physiological pHi (7.1-7.4) and, thus, contributes to the maintenance of steady state pHi. Moreover, intracellular sodium ion activity is also controlled in part by Na+ uptake via the exchanger. Intracellular acidification decreased transepithelial Na+ transport rate, apical Na+ permeability (PNa) and Na+ and K+ conductances. The recovery of these transport parameters after the removal of the acid load was found to be dependent on pHi regulation via Na+/H+ exchange. Conversely, variations in Na+ transport were accompanied by changes in pHi. Inhibition of Na+/K+ ATPase by ouabain produced covariant decreases in pHi and PNa, whereas increases in Na+ transport, occurring spontaneously or after aldosterone treatment, were highly correlated with intracellular alkalinization. We conclude that cytoplasmic H+ activity is regulated by a basolateral Na+/H+ exchanger and that transcellular coupling of ion flows at opposing cell membranes can be modulated by the pHi-regulating mechanism.     

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells

The present study aimed to clarify the existence of a Na+/Ca2+ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca2+ reabsorption in the kidney. Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca2+ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca2+ reabsorption. The vesicle preparation contained high, digitalis-sensitive (Na+ + K+)-ATPase activities indicating its origin from the basolateral portion of plasma membrane. The operation of a Na+/Ca2+ antiport device was excluded by the findings that steep Ca2+ gradients formed by ATP-dependent Ca2+ accumulation in the vesicles were not discharged by extravesicular Na+, and did not drive 45Ca2+ uptake into the vesicles via a Ca2+-45Ca2+ exchange. The ATP-dependent Ca2+ uptake into the vesicles became increasingly depressed with time by extravesicular Na+. This was not due to an impairment of the Ca2+ pump itself, but caused by Na+/Ca2+ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca2+ accumulation in the vesicles. Earlier observations on Na+-induced release of Ca2+ from vesicles pre-equilibrated with Ca2+, seemingly favoring the existence of a Na+/Ca2+ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na+/Ca2+ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca2+ reabsorption through tubule epithelium essentially depending on the operation of a Na+/Ca2+ antiport device.     

Autoradiographic and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog salivary gland (author's transl)

The distribution of Na pump sites (Na+-K+ ATPase) in the acinar cells of dog submandibular gland was demonstrated by light and electron microscopical radioautography of 3H-ouabain binding sites and electron microscopical ATPase cytochemistry. The grains of 3H-ouabain by light microscopical radioautography were localized to the basal parts of acini and/or the striated ducts, and a small quantity of the grains was also present on the luminal parts of acini. The grains of 3H-ouabain by electron microscopical radioautography and the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells, while slightly on the microvilli of the luminal plasma membranes. The present evidence that the distribution of ATPase on the acinar cells determined by the cytochemistry is well concomitant with that of 3H-ouabain binding sites on the acinar cells by the radioautography, suggests that the above mentioned ATPase is Na+-K+ ATPase, a Na pump. The relationship of the distribution of the Na+-K+ ATPase and the cation transport of the plasma membranes in the acinar cells of the dog submandibular gland are discussed.     

Cyclic AMP inhibits Na+/H+ exchange at the apical membrane of Necturus gallbladder epithelium

The effects of elevating intracellular cAMP levels on Na+ transport across the apical membrane of Necturus gallbladder epithelium were studied by intracellular and extracellular microelectrode techniques. Intracellular cAMP was raised by serosal addition of the phosphodiesterase inhibitor theophylline (3 mM) or mucosal addition of either 8-Br-cAMP (1 mM) or the adenylate cyclase activator forskolin (10 microM). During elevation of intracellular cAMP, intracellular Na+ activity (alpha Nai) and intracellular pH (pHi) decreased significantly. In addition, acidification of the mucosal solution, which contained either 100 or 10 mM Na+, was inhibited by approximately 50%. The inhibition was independent of the presence of Cl- in the bathing media. The rates of change of alpha Nai upon rapid alterations of mucosal [Na+] from 100 to 10 mM and from 10 to 100 mM were both decreased, and the rate of pHi recovery upon acid loading was also reduced by elevated cAMP levels. Inhibition was approximately 50% for all of these processes. These results indicate that cAMP inhibits apical membrane Na+/H+ exchange. The results of measurements of pHi recovery at 10 and 100 mM mucosal [Na+] and a kinetic analysis of recovery as a function of pHi suggest that the main or sole mechanism of the inhibitory effect of cAMP is a reduction in the maximal rate of acid extrusion. In conjunction with the increase in apical membrane electrodiffusional Cl- permeability, produced by cAMP, which causes a decrease in net Cl- entry (Petersen, K.-U., and L. Reuss, 1983, J. Gen. Physiol., 81:705), inhibition of Na+/H+ exchange contributes to the reduction of fluid absorption elicited by this agent. Similar mechanisms may account for the effects of cAMP in other epithelia with similar transport properties. It is also possible that inhibition of Na+/H+ exchange by cAMP plays a role in the regulation of pHi in other cell types.     

Cellular NH4+/K+ transport pathways in mouse medullary thick limb of Henle. Regulation by intracellular pH

Fluorescence and electrophysiological methods were used to determine the effects of intracellular pH (pHi) on cellular NH4+/K+ transport pathways in the renal medullary thick ascending limb of Henle (MTAL) from CD1 mice. Studies were performed in suspensions of MTAL tubules (S-MTAL) and in isolated, perfused MTAL segments (IP-MTAL). Steady-state pHi measured using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) averaged 7.42 +/- 0.02 (mean +/- SE) in S-MTAL and 7.26 +/- 0.04 in IP-MTAL. The intrinsic cellular buffering power of MTAL cells was 29.7 +/- 2.4 mM/pHi unit at pHi values between 7.0 and 7.6, but below a pHi of 7.0 the intrinsic buffering power increased linearly to approximately 50 mM/pHi unit at pHi 6.5. In IP-MTAL, NH4+ entered cells across apical membranes via both Ba(2+)-sensitive pathway and furosemide-sensitive Na+:K+(NH4+):2Cl- cotransport mechanisms. The K0.5 and maximal rate for combined apical entry were 0.5 mM and 83.3 mM/min, respectively. The apical Ba(2+)-sensitive cell conductance in IP-MTAL (Gc), which reflects the apical K+ conductance, was sensitive to pHi over a pHi range of 6.0-7.4 with an apparent K0.5 at pHi approximately 6.7. The rate of cellular NH4+ influx in IP-MTAL due to the apical Ba(2+)-sensitive NH4+ transport pathway was sensitive to reduction in cytosolic pH whether pHi was changed by acidifying the basolateral medium or by inhibition of the apical Na+:H+ exchanger with amiloride at a constant pHo of 7.4. The pHi sensitivities of Gc and apical, Ba(2+)-sensitive NH4+ influx in IP-MTAL were virtually identical. The pHi sensitivity of the Ba(2+)-sensitive NH4+ influx in S-MTAL when exposed to (apical+basolateral) NH4Cl was greater than that observed in IP-MTAL where NH4Cl was added only to apical membranes, suggesting an additional effect of intracellular NH4+/NH3 on NH4+ influx. NH4+ entry via apical Na+:K+ (NH4+):2Cl- cotransport in IP-MTAL was somewhat more sensitive to reductions in pHi than the Ba(2+)-sensitive NH4+ influx pathway; NH4+ entry decreased by 52.9 +/- 13.4% on reducing pHi from 7.31 +/- 0.17 to 6.82 +/- 0.14. These results suggest that pHi may provide a negative feedback signal for regulating the rate of apical NH4+ entry, and hence transcellular NH4+ transport, in the MTAL. A model incorporating these results is proposed which illustrates the role of both pHi and basolateral/intracellular NH4+/NH3 in regulating the rate of transcellular N H4+ transport in the MTAL.     

Models of the Na-K pump in cardiac muscle predict the wrong intracellular Na+ activity

The Na-K pump in cardiac Purkinje strands has been carefully studied with voltage clamp and Na+-selective microelectrodes. In three of these studies both the rate of change of intracellular Na+ activity, a(Nai), after pump blockade, and the time constant of reduction of a(Nai) after an Na+ load were measured. These two parameters can be employed with a formalism relating pump activity to a(Nai) in order to predict the a(Nai) in the steady state. Several formalisms were tested: (a) a first-order dependence on a(Nai); (b) a model based on the assumption of a single, saturable, Na+-binding site that must be occupied for transport to occur; (c) a model based on n equivalent, saturable, Na+ binding sites per pump molecule all of which must be occupied for transport to occur. The first two models predicted an a(Nai) that is far below the value of about 6 mM that is experimentally obtained. The third model would work for n greater than or equal to 4. These results suggest that either the cardiac Na-K pump is not well described by available Na-K pump models for n less than 4 or that the measured Na+ influx rate, extrusion rate or a(Nai) are in error.     

Protons as substitutes for sodium and potassium in the sodium pump reaction

The role of protons as substitutes for Na+ and/or K+ in the sodium pump reaction was examined using inside-out membrane vesicles derived from human red cells. Na+-like effects of protons suggested previously (Blostein, R. (1985) J. Biol. Chem. 260, 829-833) were substantiated by the following observations: (i) in the absence of extravesicular (cytoplasmic) Na+, an increase in cytoplasmic [H+] increased both strophanthidin-sensitive ATP hydrolysis (nu) and the steady-state level of phosphoenzyme, EP, and (ii) as [H+] is increased, the Na+/ATP coupling ratio is decreased. K+-like effects of protons were evidenced in the following results: (i) an increase in nu, decrease in EP, and hence increase in EP turnover (nu/EP) occur when intravesicular (extracellular) [H+] is increased; (ii) an increase in the rate of Na+ influx into K+(Rb+)-free inside-out vesicles and (iii) a decrease in Rb+/ATP coupling occur when [H+] is increased. Direct evidence for H+ being translocated in place of cytoplasmic Na+ and extracellular K+ was obtained by monitoring pH changes using fluorescein isothiocyanate-dextran-filled vesicles derived from 4',4-diisothiocyano-2',2-stilbene disulfonate-treated cells. With the initial pHi = pHo = pH 6.2, a strophanthidin-sensitive decrease in pHi was observed following addition of ATP provided the vesicles contained K+. This pH gradient was abolished following addition of Na+. With alkali cation-free inside-out vesicles, a strophanthidin-sensitive increase in pH was observed upon addition of both ATP and Na+. The foregoing changes in pHi were not affected by the addition of tetrabutylammonium to dissipate any membrane potential and were not observed at pH 6.8. These ATP-dependent cardiac glycoside-sensitive proton movements indicate Na,K-ATPase mediated Na+/H+ exchange in the absence of extracellular K+ as well as H+/K+ exchange in the absence of cytoplasmic Na+.     

Effect of cell sodium on Na+/K(+)-ATPase-dependent sodium efflux in cortical collecting tubule of rabbits under different aldosterone status

Aldosterone increased the tubular volume in cortical collecting tubules (CCD) of rabbit kidney. It modulated the rate of cell sodium accumulation, under condition of ATPase inhibition (4 degrees C, in the absence of K+). In contrast, the relationship between Na+/K(+)-ATPase-dependent Na+ extrusion rate and intracellular Na+ concentration (Nai+) was similar in control, adrenalectomized, and aldosterone-treated adrenalectomized animals: Na+ extrusion rate increased with Nai+, up to 70 mM Nai+, and then plateaued. This indicates that aldosterone does not modify the characteristics of Nai(+)-dependent Na+ extrusion rate by the Na+/K(+)-ATPase pump in CCD.     

Transport energetics of the Na+ pump in Aplysia californica gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na+ transporter (Na+ pump). Increased activity of the Na+ pump, coupled to luminal Na+/AIB symporter activity and basolateral membrane depolarization, changed the Na+ transport energetics across the basolateral membrane to a greater extent than the change in Na+ transport energetics across the luminal membrane.     

Cytosolic pH regulation in osteoblasts. Interaction of Na+ and H+ with the extracellular and intracellular faces of the Na+/H+ exchanger

The interaction of Na and H ions with the extracellular and intracellular sites of the Na+/H+ exchanger of the osteosarcoma cell line UMR-106 was investigated. Na ions interact with a single, saturable extracellular transport site. H+ and amiloride appear to compete with Na+ for binding to this site. The apparent affinity for extracellular Na+ (Nao+) and amiloride was independent of intracellular H+ (Hi+), Nai+, or an outwardly directed H+ gradient. The interaction of H+ with the intracellular face of the exchanger had a sigmoidal characteristic with a Hill coefficient of approximately 2. The apparent affinity for Hi+ was independent of Nao+ between 25 and 140 mM. The apparent affinity for Hi+, but not the number of intracellular sites, increased with the increase in the outwardly directed H+ gradient across the membrane. Nai+/Ho+ exchange (reverse mode) is an electroneutral process with a Na+/H+ stoichiometry of 1. The dependence of Nai+/Ho+ exchange on Nai+ was sigmoidal, with a Hill coefficient of 2.16. Nai+ competes with Hi+ for binding to at least the transport site. The apparent affinity for Nai+ decreased with the increase in the outwardly directed H+ gradient. High Ho+ inhibited exchange activity in the reverse mode. We conclude that intracellular Na+ and H+ can activate the exchanger. The exchanger has two separate and asymmetric extracellular and intracellular transport sites. The relative apparent affinities of the internal transport site for Na+ and H+ are determined by the direction and magnitude of the H+ gradient across the membrane. Kinetic characterization of the exchanger suggests that Na+/H+ exchange is compatible with a simultaneous transport model, although a ping-pong transport model could not be excluded.     

Tetracyclines reduce Na+/K+ pump capacity in Calu-3 human airway cells.

We studied the effect of tetracyclines on the Na+/K+ pump activity in Calu-3, a human airway cell line. To estimate Na+/K+ pump capacity on the basolateral membrane, an ouabain-sensitive component of the short-circuit current (Isc) was measured in the presence of nystatin, an ionophore of Na+. The application of ouabain (1 mM) to the basolateral solution completely inhibited the Isc generated by adding nystatin (50 microM) to the apical solution. Tetracycline (TC), minocycline (MC), or demethylchlortetracycline (DC) at 0.5 mM applied to the apical but not to the basolateral solution also decreased the nystatin-induced Isc. Neither phlorizin- nor diphenylamine-2-carboxylic acid-sensitive Isc was affected by TC, MC, or DC. These results indicate that tetracyclines may permeate only through the apical membrane with the result that the Na+/K+ pump's capacity for Na+ extrusion should be suppressed without a decrease in Cl- transport.     

Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder

Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.     

Intracellular sodium affects ouabain interaction with the Na/K pump in cultured chick cardiac myocytes

Whether a given dose of ouabain will produce inotropic or toxic effects depends on factors that affect the apparent affinity (K0.5) of the Na/K pump for ouabain. To accurately resolve these factors, especially the effect of intracellular Na concentration (Nai), we have applied three complementary techniques for measuring the K0.5 for ouabain in cultured embryonic chick cardiac myocytes. Under control conditions with 5.4 mM Ko, the value of the K0.5 for ouabain was 20.6 +/- 1.2, 12.3 +/- 1.7, and 6.6 +/- 0.4 microM, measured by voltage-clamp, Na-selective microelectrode, and equilibrium [3H]ouabain-binding techniques, respectively. A significant difference in the three techniques was the time of exposure to ouabain (30 s-30 min). Since increased duration of exposure to ouabain would increase Nai, monensin was used to raise Nai to investigate what effect Nai might have on the apparent affinity of block by ouabain. Monensin enhanced the rise in Na content induced by 1 microM ouabain. In the presence of 1 microM [3H]ouabain, total binding was found to be a saturating function of Na content. Using the voltage-clamp method, we found that the value of the K0.5 for ouabain was lowered by nearly an order of magnitude in the presence of 3 microM monensin to 2.4 +/- 0.2 microM and the magnitude of the Na/K pump current was increased about threefold. Modeling the Na/K pump as a cyclic sequence of states with a single state having high affinity for ouabain shows that changes in Nai alone are sufficient to cause a 10-fold change in K0.5. These results suggest that Nai reduces the value of the apparent affinity of the Na/K pump for ouabain in 5.4 mM Ko by increasing its turnover rate, thus increasing the availability of the conformation of the Na/K pump that binds ouabain with high affinity.     

Voltage dependence of partial reactions of the Na+/K+ pump: predictions from microscopic models

A theoretical treatment of the voltage dependence of electroneutral Na+-Na+ and K+-K+ exchange mediated by the Na+/K+ pump is given. The analysis is based on the Post-Albers reaction scheme in which the overall transport process is described as a sequence of conformational transitions and ion-binding and ion-release steps. The voltage dependence of the exchange rate is determined by a set of 'dielectric coefficients' reflecting the magnitude of charge translocations associated with individual reaction steps. Charge movement may result from conformational changes of the transport protein and/or from migration of ions in an access channel connecting the binding sites with the aqueous medium. It is shown that valuable mechanistic information may be obtained by studying the voltage dependence of transport rates at different (saturating and nonsaturating) ion concentrations.     

Charge translocation by the Na+/K+ pump under Na+/Na+ exchange conditions: intracellular Na+ dependence

The effect of intracellular (i) and extracellular (o) Na+ on pre-steady-state transient current associated with Na+/Na+ exchange by the Na+/K+ pump was investigated in the vegetal pole of Xenopus oocytes. Current records in response to 40-ms voltage pulses from -180 to +100 mV in the absence of external Na+ were subtracted from current records obtained under Na+/Na+ exchange conditions. Na+-sensitive transient current and dihydroouabain-sensitive current were equivalent. The quantity of charge moved (Q) and the relaxation rate coefficient (ktot) of the slow component of the Nao+-sensitive transient current were measured for steps to various voltages (V). The data were analyzed using a four-state kinetic model describing the Na+ binding, occlusion, conformational change, and release steps of the transport cycle. The apparent valence of the Q vs. V relationship was near 1.0 for all experimental conditions. When extracellular Na+ was halved, the midpoint voltage of the charge distribution (Vq) shifted -25.3+/-0.4 mV, which can be accounted for by the presence of an extracellular ion-well having a dielectric distance delta=0.69+/-0.01. The effect of changes of Nai+ on Nao+-sensitive transient current was investigated. The midpoint voltage (Vq) of the charge distribution curve was not affected over the Nao+ concentration range 3.13-50 mM. As Nai+ was decreased, the amount of charge measured and its relaxation rate coefficient decreased with an apparent Km of 3.2+/-0.2 mM. The effects of lowering Nai+ on pre-steady-state transient current can be accounted for by decreasing the charge available to participate in the fast extracellular Na+ release steps, by a slowly equilibrating (phosphorylation/occlusion) step intervening between intracellular Na+ binding and extracellular Na+ release.     

Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder

The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na+ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal 22Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na+ flux was a linear function of mucosal Na+ concentration. Ouabain-sensitive Na+ flux and ouabain-sensitive, Na+-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na+ with the basolateral transport process is assumed to involve the interaction of some number of Na+ ions, n, with a maximal flux, MMAX, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value for MMAX of 287.8 pM cm-2 sec-1 with an intracellular Na concentration of 2.0 mM Na+ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na+-induced current, we calculate a Na+ to K+ coupling ratio of 1.40 +/- 0.07 for the transport process.     

Quantification of Ca(2+)-ATPases in porcine duodenum. Effects of 1,25(OH)2D3 deficiency.

Previous studies have identified a calmodulin-stimulated ATP-dependent Ca2+ pump as the major Ca2+ efflux pathway in enterocytes. Here, we developed methods to quantify the number of Ca2+ pumps in basolateral and intracellular membranes from porcine duodenum. By the use of a pig strain with a genetic defect in renal 1 alpha-hydroxylase, we were able to investigate the influence of 1,25(OH)2D3-deficiency on the number of Ca(2+)-ATPases in porcine duodenum. The amount of Ca(2+)-ATPase in isolated basolateral membranes was 5.5 +/- 0.7 micrograms/mg protein, while the Vmax of ATP-dependent Ca2+ transport into inside-out resealed basolateral membrane vesicles was 2.6 +/- 0.4 nmol/mg protein per min. From these data we estimated roughly about 95 x 10(3) plasma membrane Ca2+ pump sites per enterocyte. In addition, the amount of intracellular Ca(2+)-ATPase in microsomal fractions was 0.41 +/- 0.02 microgram/mg protein. Comparison of these parameters between control and rachitic animals showed that Ca2+ pump capacities in both basolateral membranes and microsomal fractions of porcine duodenum are not influenced by 1,25(OH)2D3-deficiency. In conclusion, stimulatory effects of 1,25(OH)2D3 on intestinal Ca2+ transport most likely result from specific effects on apical influx and facilitation of cytosolic Ca2+ diffusion by Ca(2+)-binding proteins and not from an increase in Ca2+ pumping capacity in basolateral membranes.