Heterogeneity of the Na(+)-H+ antiport systems in renal cells.

This study analyzes the differential characteristics of the Na(+)-H+ antiport systems observed in several epithelial and non-epithelial renal cell lines. Confluent monolayers of LLC-PK1A cells have a Na(+)-H+ antiport system located in the apical membrane of the cell. This system, however, is not expressed during cell proliferation or after incubation in the presence of different mitogenic agents. In contrast, confluent monolayers of MDCK4 express minimal Na(+)-H+ antiport activity in the confluent monolayer state but reach maximal antiport activity during cell proliferation or after activation of the cells by different mitogenic agents. Similar results were obtained with the renal fibroblastic cell line BHK. The system present in MDCK4 cells is localized in the basolateral membrane of the epithelial cell. In LLC-PK1A cells, an increase in the extracellular Na+ concentration produces a hyperbolic increase in the activity of the Na(+)-H+ antiporter. In MDCK4 and BHK cells, however, an increase in external Na+ produces a sigmoid activation of the system. Maximal activation of the system occur at a pHo 7.5 in LLC-PK1A cells and pHo 7.0 in MDCK4 cells. The Na(+)-H+ antiporter of LLC-PK1A cells is more sensitive to the inhibitory effect of amiloride (Ki 1.8 x 10(-7) M) than is the antiporter of MDCK4 cells (Ki 7.0 x 10(-6) M). Moreover, 5-(N-methyl-N-isobutyl)amiloride is the most effective inhibitor of Na(+)-H+ exchange in LLC-PK1A cells, but the least effective inhibitor in MDCK4 cells. Conversely, the analog, 5-(N,N-dimethyl)amiloride, is the most effective inhibitor of Na(+)-H+ exchange in MDCK4 cells, but is the least effective inhibitor in LLC-PK1A cells. These results support the hypothesis that Na(+)-H+ exchange observed in LLC-PK1A and other cell lines may represent the activity of different Na(+)-H+ antiporters.     

Hydrogen ion currents in rat alveolar epithelial cells.

Alveolar epithelial cells isolated from rats and maintained in primary culture were studied using the whole-cell configuration of the "patch-clamp" technique. After other ionic conductances were eliminated by replacing permeant ions with N-methyl-D-glucamine methanesulfonate, large voltage-activated hydrogen-selective currents were observed. Like H+ currents in snail neurons and axolotl oocytes, those in alveolar epithelium are activated by depolarization, deactivate upon repolarization, and are inhibited by Cd2+ and Zn2+. Activation of H+ currents is slower in alveolar epithelium than in other tissues, and often has a sigmoid time course. Activation occurs at more positive potentials when external pH is decreased. Saturation of the currents suggests that diffusion limitation may occur; increasing the pipette buffer concentration from 5 to 120 mM at a constant pH of 5.5 increased the maximum current density from 8.7 to 27.3 pA/pF, indicating that the current amplitude can be limited in 5 mM buffer solutions by the rate at which buffer molecules can supply H+ to the membrane. These data indicate that voltage-dependent H+ currents exist in mammalian cells.     

Actions of emigrated neutrophils on Na(+) and K(+) currents in rat ventricular myocytes

Interactions between neutrophils and the ventricular myocardium can contribute to tissue injury, contractile dysfunction and generation of arrhythmias in acute cardiac inflammation. Many of the molecular events responsible for neutrophil adhesion to ventricular myocytes are well defined; in contrast, the resulting electrophysiological effects and changes in excitation–contraction coupling have not been studied in detail. In the present experiments, rat ventricular myocytes were superfused with either circulating or emigrated neutrophils and whole-cell currents and action potential waveforms were recorded using the nystatin-perforated patch method. Almost immediately after adhering to ventricular myocytes, emigrated neutrophils caused a depolarization of the resting membrane potential and a marked prolongation of myocyte action potential. Voltage clamp experiments demonstrated that following neutrophil adhesion, there was (i) a slowing of the inactivation of a TTX-sensitive Na⁺ current, and (ii) a decrease in an inwardly rectifying K⁺ current.

One cytotoxic effect of neutrophils appears to be initiated by enhanced Na⁺ entry into the myocytes. Thus, manoeuvres that precluded activation of Na⁺ channels, for example holding the membrane potential at −80 mV, significantly increased the time to cell death or prevented contracture entirely. A mathematical model for the action potential of rat ventricular myocytes has been modified and then utilized to integrate these findings. These simulations demonstrate the marked effects of (50-fold) slowing of the inactivation of 2–4% of the available Na⁺ channels on action potential duration and the corresponding intracellular Ca²⁺ transient. In ongoing studies using this combination of approaches, are providing significant new insights into some of the fundamental processes that modulate myocyte damage in acute inflammation.     

State-dependent inactivation of K+ currents in rat type II alveolar epithelial cells

Inactivation of K+ channels responsible for delayed rectification in rat type II alveolar epithelial cells was studied in Ringer, 160 mM K-Ringer, and 20 mM Ca-Ringer. Inactivation is slower and less complete when the extracellular K+ concentration is increased from 4.5 to 160 mM. Inactivation is faster and more complete when the extracellular Ca2+ concentration is increased from 2 to 20 mM. Several observations suggest that inactivation is state-dependent. In each of these solutions depolarization to potentials near threshold results in slow and partial inactivation, whereas depolarization to potentials at which the K+ conductance, gK, is fully activated results in maximal inactivation, suggesting that open channels inactivate more readily than closed channels. The time constant of current inactivation during depolarizing pulses is clearly voltage-dependent only at potentials where activation is incomplete, a result consistent with coupling of inactivation to activation. Additional evidence for state-dependent inactivation includes cumulative inactivation and nonmonotonic from inactivation. A model like that proposed by C.M. Armstrong (1969. J. Gen. Physiol. 54: 553-575) for K+ channel block by internal quaternary ammonium ions accounts for most of these properties. The fundamental assumptions are: (a) inactivation is strictly coupled to activation (channels must open before inactivating, and recovery from inactivation requires passage through the open state); (b) the rate of inactivation is voltage-independent. Experimental data support this coupled model over models in which inactivation of closed channels is more rapid than that of open channels (e.g., Aldrich, R.W. 1981. Biophys. J. 36:519-532). No inactivation results from repeated depolarizing pulses that are too brief to open K+ channels. Inactivation is proportional to the total time that channels are open during both a depolarizing pulse and the tail current upon repolarization; repolarizing to more negative potentials at which the tail current decays faster results in less inactivation. Implications of the coupled model are discussed, as well as additional states needed to explain some details of inactivation kinetics.     

Na(+)-K(+)-ATPase activity in alveolar epithelial cells increases with cyclic stretch

Na(+)-K(+)-ATPase pumps (Na(+) pumps) in the alveolar epithelium create a transepithelial Na(+) gradient crucial to keeping fluid from the pulmonary air space. We hypothesized that alveolar epithelial stretch stimulates Na(+) pump trafficking to the basolateral membrane (BLM) and, thereby, increases overall Na(+) pump activity. Alveolar type II cells were isolated from Sprague-Dawley rats and seeded onto elastic membranes coated with fibronectin or 5-day-conditioned extracellular matrix. After 2 days in culture, cells were uniformly stretched for 1 h in a custom-made device. Na(+) pump activity was subsequently assessed by ouabain-inhibitable uptake of (86)Rb(+), a K(+) tracer, and BLM Na(+) pump abundance was measured. In support of our hypothesis, cells increased Na(+) pump activity in a "dose-dependent" manner when stretched to 12, 25, or 37% change in surface area (DeltaSA), and cells stretched to 25% DeltaSA more than doubled Na(+) pump abundance in the BLM. Cells on 5-day matrix tolerated higher strain than cells on fibronectin before the onset of Na(+) pump upregulation. Treatment with Gd(3+), a stretch-activated channel blocker, amiloride, a Na(+) channel blocker, or both reduced but did not abolish stretch-induced effects. Sustained tonic stretch, unlike cyclic stretch, elicited no significant Na(+) pump response.     

Basolateral Na(+)-H+ antiporter. Mechanisms of electroneutral and conductive ion transport

The basolateral Na-H antiporter of the turtle colon exhibits both conductive and electroneutral Na+ transport (Post and Dawson. 1992. American Journal of Physiology. 262:C1089-C1094). To explore the mechanism of antiporter-mediated current flow, we compared the conditions necessary to evoke conduction and exchange, and determined the kinetics of activation for both processes. Outward (cell to extracellular fluid) but not inward (extracellular fluid to cell) Na+ or Li+ gradients promoted antiporter-mediated Na+ or Li+ currents, whereas an outwardly directed proton gradient drove inward Na+ or Li+ currents. Proton gradient-driven, "counterflow" current is strong evidence for an exchange stoichiometry of > 1 Na+ or Li+ per proton. Consistent with this notion, outward Na+ and Li+ currents generated by outward Na+ or Li+ gradients displayed sigmoidal activation kinetics. Antiporter-mediated proton currents were never observed, suggesting that only a single proton was transported per turnover of the antiporter. In contrast to Na+ conduction, Na+ exchange was driven by either outwardly or inwardly directed Na+, Li+, or H+ gradients, and the activation of Na+/Na+ exchange was consistent with Michaelis-Menten kinetics (K1/2 = 5 mM). Raising the extracellular fluid Na+ or Li+ concentration, but not extracellular fluid proton concentration, inhibited antiporter-mediated conduction and activated Na+ exchange. These results are consistent with a model for the Na-H antiporter in which the binding of Na+ or Li+ to a high-affinity site gives rise to one-for-one cation exchange, but the binding of Na+ or Li+ ions to other, lower-affinity sites can give rise to a nonunity, cation exchange stoichiometry and, hence, the net translocation of charge. The relative proportion of conductive and nonconductive events is determined by the magnitude and orientation of the substrate gradient and by the serosal concentration of Na+ or Li+.     

Mechanical stretching of alveolar epithelial cells increases Na(+)-K(+)-ATPase activity.

Alveolar epithelial cells effect edema clearance by transporting Na(+) and liquid out of the air spaces. Active Na(+) transport by the basolaterally located Na(+)-K(+)-ATPase is an important contributor to lung edema clearance. Because alveoli undergo cyclic stretch in vivo, we investigated the role of cyclic stretch in the regulation of Na(+)-K(+)-ATPase activity in alveolar epithelial cells. Using the Flexercell Strain Unit, we exposed a cell line of murine lung epithelial cells (MLE-12) to cyclic stretch (30 cycles/min). After 15 min of stretch (10% mean strain), there was no change in Na(+)-K(+)-ATPase activity, as assessed by (86)Rb(+) uptake. By 30 min and after 60 min, Na(+)-K(+)-ATPase activity was significantly increased. When cells were treated with amiloride to block amiloride-sensitive Na(+) entry into cells or when cells were treated with gadolinium to block stretch-activated, nonselective cation channels, there was no stimulation of Na(+)-K(+)-ATPase activity by cyclic stretch. Conversely, cells exposed to Nystatin, which increases Na(+) entry into cells, demonstrated increased Na(+)-K(+)-ATPase activity. The changes in Na(+)-K(+)-ATPase activity were paralleled by increased Na(+)-K(+)-ATPase protein in the basolateral membrane of MLE-12 cells. Thus, in MLE-12 cells, short-term cyclic stretch stimulates Na(+)-K(+)-ATPase activity, most likely by increasing intracellular Na(+) and by recruitment of Na(+)-K(+)-ATPase subunits from intracellular pools to the basolateral membrane.     

Chemical modifications of the Na+-H+ antiport in Escherichia coli membrane vesicles

The effects of chemical modifications of the Na+-H+ antiport in Escherichia coli have been analyzed by studying the resulting variations of the energy-dependent, downhill Na+ efflux from membrane vesicles. The histidyl reagent diethylpyrocarbonate (EtO)2C2O3 prevents the activation of the Na+ efflux mechanism by delta microH+ or its components. Inactivation of the antiporter by (EtO)2C2O3 is completely reversed by hydroxylamine. The data suggest that histidine residues are involved in the molecular mechanism of the Na+-H+ antiport. In contrast, no conclusive evidence suggesting participation of carboxylic, tyrosine or sulfhydryl residues in the Na+-H+ exchange reaction has been obtained.