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The inhibition of short-circuit current (Isc) in isolated frog skin and the induction of surface potentials in lipid bilayer membranes produced by the diuretic drug amiloride and a number of its chemical analogues was studied. The major conclusions of our study are: (a) The charged form of amiloride is the biologically active species. (b) Both the magnitude of Isc and the amiloride inhibitory effect are sensitive to the ionic milieu bathing the isolated skin, and these two features are modulated at separate and distinct regions on the transport site. (c) Amiloride is very specific in its inhibitory interaction with the Na+ transport site since slight structural modifications can result in significant changes in drug effectiveness. We found that substitutions at pyrazine ring position 5 greatly diminish drug activity, while changes at position 6 are less drastic. Alterations in the guanidinium moiety only diminish activity if the result is a change in the spatial orientation of the amino group carrying the positive charge. (d) Amiloride can bind to and alter the charge on membrane surfaces, but this action cannot explain its highly specific effects in biological systems.

Studies of phosphate transport in the proximal tubule have recently focused on interactions with cellular metabolism. The present studies demonstrate that two fatty acids, valerate and butyrate, and two tricarboxylic acid cycle intermediates, succinate and malate, stimulate net phosphate transport in the rabbit proximal tubule by 34-117%. Valerate had no effect on the total uptake of inorganic [32P]phosphate into suspensions of proximal tubules but did enhance the initial rate of influx. Net fluid transport was unaffected by these substrates although glucose absorption increased by 10-15% following the addition of either valerate or succinate. Since valerate, butyrate, and succinate are known to stimulate gluconeogenesis and respiration, we evaluated the role of gluconeogenesis in the stimulation of phosphate transport. The addition of 3-mercaptopicolinate (1 mM), an inhibitor of gluconeogenesis, did not alter phosphate transport, nor did it prevent the valerate-induced stimulation of phosphate transport. We conclude that valerate, butyrate, succinate, and malate enhance phosphate transport by the proximal convoluted tubule. This action appears to be unrelated to effects on gluconeogenesis and may be related to close links between phosphate transport and oxidative metabolism.

Various aspects of the interrelationship between ion transport and cellular metabolism were investigated using a suspension of rabbit cortical tubules that were mainly proximal in nature. Using the intact tubules, the compartmentation of K within the renal cell was studied by performing 42K uptake studies. The oxygen consumption (QO2) of the tubules was measured under similar conditions, as well as when the Na pump was stimulated by increasing Na+ entry with nystatin. In addition, the state 3 rate of respiration was measured when the mitochondria of digitonin-permeabilized tubules were stimulated by ADP. At 37 and 25 degrees C, a single-compartmental uptake of 42K was observed, which suggests that extracellular K+ communicates with a single compartment within the renal cell. Between 37 and 15 degrees C, the ouabain-sensitive QO2 and the initial 42K uptake rate were parallel in an Arrhenius-type plot, which indicated that active ion transport and oxidative phosphorylation remain tightly coupled within this temperature range. At all temperatures between 37 and 15 degrees C, nystatin stimulated the QO2, which demonstrates that the entry of Na+ into the renal cells was rate limiting for active Na+ transport throughout this temperature range. Between 37 and 20 degrees C, the nystatin-stimulated QO2 was nearly equal to the state 3 rate of respiration, which suggests that active ion transport may be limited by ATP availability under these conditions. At 15 degrees C, nystatin addition stimulated the QO2 well below the state 3 respiratory rate.

Oxygen (O2) consumption and net K+ uptake were measured simultaneously upon reintroduction of K+ into a K+-depleted suspension of renal tubules. The K+/O2 stoichiometries of 11.8 +/- 0.2 and 8.4 +/- 0.6 were obtained for reduced nicotinamide adenine dinucleotide- and flavoprotein-linked substrates, respectively. These values complement classical K+ to adenosine triphosphate (ATP) and ATP/O2 stoichiometries, thereby demonstrating a remarkably efficient coupling between the processes of Na+- and K+-dependent adenosinetriphosphatase-mediated ion transport and oxidative phosphorylation within the intact cell.

The relative role of various Ca2+ transport systems in the regulation of Ca2+ cytosolic free Ca2+ concentration was evaluated using rabbit renal proximal tubules. Intracellular compartmentation was evaluated through Ca2+ releases induced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), A23187, and ruthenium red (RR) alone and in combination. In a Ca2+-free solution after 1 h of incubation, FCCP released 43 +/- 4%, A23187 54 +/- 3%, and RR 29 +/- 5% of total cellular Ca2+; in addition, RR inhibited the rate of FCCP-induced release, confirming its mitochondrial origin. In 1 mM Ca2+, the releases were 57 +/- 9%, 70 +/- 5%, and 34 +/- 10%, respectively. Therefore, the mitochondrial Ca2+ content is 20-60 nmol/mg of mitochondrial protein, sufficiently large to effectively buffer cell Ca2+. To evaluate the role of the plasma membrane Na:Ca exchanger, 10(-4) M ouabain was added and caused a slight decline in total cell Ca2+ content and no change in ionized Ca2+ measured by the null-point method, suggesting that the plasmalemmal Na+:Ca2+ exchanger does not play an important role in Ca2+ extrusion. Cytosolic free Ca2+ increased when 100 mM sodium was replaced with equimolar choline or tetramethylammonium. However, tetramethylammonium replacement released 55% of the mitochondrial Ca2+ content by increasing mitochondrial Ca2+ efflux without affecting the Ca2+ influx pathway. These results suggest that Na+ replacements in this tissue increase ionized Ca2+ by increasing mitochondrial Ca2+ efflux and not by inhibition of Na+:Ca2+ exchange at the plasma membrane.

At pH 5.0, the electrical potential (delta psi, interior negative) across the plasma membrane of Staphylococcus aureus exhibits a minimum of -85 to -90 mV; the pH gradient (delta pH, interior alkaline) across the membrane approximates a maximum of about -100 mV. Under these conditions, uptake of the aminoglycoside gentamicin is negligible, and viability of the organism is not impaired by the antibiotic. In contrast, at pH 7.5, at which delta psi is about -130 mV and delta pH is 0, gentamicin uptake is observed and the drug markedly decreases viability. Dramatically, when the ionophore nigericin is added at pH 5.0, gentamicin uptake is induced, there is a striking decrease in viability, and the effect is associated with an increase in delta psi at the expense of delta pH. Consistently, valinomycin, which dissipates delta psi in the presence of potassium, abolishes gentamicin uptake and killing. In addition, from pH 5.0 to pH 7.5, there is a direct relationship between the magnitude of delta psi and both gentamicin uptake and its bactericidal effect. However, a threshold delta psi of -75 to -90 mV is apparently necessary to initiate uptake and killing. These observations provide a strong indication that delta psi plays a critical role in the uptake and antibacterial action of gentamicin and suggest that nigericin-like ionophores may be clinically useful in synergy with aminoglycosides.

The kinetics of oxygen consumption and (Na+,K+)-ATPase-mediated K+ transport was examined by reintroducing K+ into a K+-depleted suspension of renal tubules. In the presence of the substrates glucose, lactate, and alanine, a K+/O2 ratio of 10.4 +/- 0.2 was obtained, and the apparent K1/2 for K+ transport with respect to external K+ concentration was 0.9 mM. Supplementation of the substrates with the short chain fatty acid, butyric, had a 3-fold effect on the kinetic parameters examined: 1) the quantity of (Na+,K+)-ATPase-mediated ion transport per oxygen consumed fell by 17 +/- 2%; 2) the maximum rate of K+ transport increased by nearly 50%; and 3) the apparent K1/2 for transport with respect to external K+ concentration rose to 1.5 mM. These results indicate that despite decreasing the quantity of ATP produced per oxygen consumed, short chain fatty acids are able to increase the overall production of ATP during periods of high metabolic demand. The coupling between the two major metabolic processes of the renal cell, (Na+,K+)-ATPase-mediated ion transport and mitochondrial oxidative phosphorylation, is addressed in the context of these findings.

The excretion of sulfobromophthalein (BSP) is determined by hepatic uptake (storage capacity) and secretion (transport maximum). We calculated the BSP excretion data in six rabbits by measuring the rate of rise of BSP at various infusion rates. The relative storage capacity was determined to be 0.34 +/- 0.13 mg.mg-1.dl-1.kg-1. The transport maximum was calculated to be 0.91 +/- 0.12 mg.min-1.kg-1. These results compare closely with previously reported results using more direct measurements that require anesthesia and surgical cannulation of the biliary tree. Repeated studies were easily performed. Our method provides a reliable and simple technique to study the effects of various conditions or substances on BSP excretion in conscious, unanesthetized rabbits.

The intracellular ATP, ADP, AMP, and orthophosphate (Pi) levels were measured in the midgut of Manduca sexta. The nucleotide levels were identical in tissues either "fresh" frozen or equilibrate in regular (32 mM) K or low (8 mM) K solutions. The calculated [ATP]/[ADP][Pi]ratio was approximately 300 M-1, which is low compared to other tissues. Given the ability of this ratio to control the respiratory rate, it is speculated that this low value may cause the maximal uncontrolled respiration normally observed in the midgut. The kinetics to anoxia of active transport (Isc) and the redox level of the mitochondrial cytochromes were measured simultaneously in the midgut. The cytochromes became reduced with a time constant of 0.75 +/- 0.15 min, whereas that for Isc inhibition was 2.1 +/- 0.15 min after a delay of 0.25 min. The difference between these two kinetic rates indicates that an intermediate form of energy exists in this tissue to energize active K transport. Measurements of ATP levels during the transition to anoxia indicate that its decay kinetics are sufficiently slow for ATP to be the immediate energy source for active transport in this tissue.

S-(1,2,3,4,4-Pentachloro-1,3-butadienyl)-L-cysteine (PCBC) has been identified as the penultimate compound responsible for hexachlorobutadiene-induced nephrotoxicity. The primary goal of these studies was to determine the mechanism of PCBC-induced toxicity in rabbit renal proximal tubules by examining the early changes in tubular physiology. PCBC (20-500 microM) induced a specific sequence of toxic events. Following 15 min of exposure, 200 microM PCBC increased basal (25%) and ouabain-insensitive (78%) respiration. This was followed by a decrease in basal (46%), nystatin-stimulated (54%), and ouabain-insensitive (21%) respiration and a decrease in glutathione content (79%). Finally, there was a decrease in cell viability as measured by a decrease in LDH retention at 60 min. Direct probing of mitochondrial function revealed that the initial increase in respiration resulted from the uncoupling of oxidative phosphorylation, while the late changes in respiration appeared to result from gross mitochondrial damage characterized by inhibited state 3 respiration, inhibited cytochrome c-cytochrome oxidase, and inhibited electron transport. Studies utilizing tubules with decreased glutathione content revealed that glutathione plays little if any role in the early events of PCBC-induced toxicity. These results suggest that PCBC-induced mitochondrial dysfunction may initiate the renal proximal tubule injury.

Several metabolic aspects of primary active transport have been explored in this communication. One emphasized theme entailed the need to investigate the properties of the mitochondria and the active transport systems within the intact cell. Several methodological and conceptual approaches were described that permitted such an analysis. The answers provided were sometimes qualitative or quantitative. Qualitative information was provided regarding the cytosolic signal linking active transport with respiration, suggesting that the cytosolic ADP concentration was an important element in that link. The intact renal cell was found to work normally at 50 to 60% of its maximal respiratory capacity, indicating that sufficient reserve capacity was present for increased metabolic demands. Several examples were described in which a combination of qO2 measurements and/or optical techniques were used to differentiate between effects of agents which act primarily on transport or metabolic events. Finally, the control of transport by metabolism was discussed, primarily emphasizing the role of ATP and Pi. One of the overall conclusions from these studies is that, in general, the mitochondria and the transport systems seem to display similar properties in the intact cell as they do in isolated form. However, uncertainties concerning the cellular microenvironment surrounding the mitochondria and the plasma membrane transporters have produced some interesting surprises concerning their function in the intact cell. More quantitative information on the energy compartmentation of the renal cell would be helpful to clarify numerous aspects of metabolic function.

The steady-state transport kinetics of the interaction between external sodium and the diuretic drug, amiloride, was studied in isolated anuran skin epithelia. We also investigated the effect of calcium on the amiloride-induced inhibition of short-circuit current (Isc) in these epithelial preparations. The major conclusions of this study are: (a) amiloride is a noncompetitive inhibitor of Na entry in bullfrog and grassfrog skin, but displays mixed inhibition in R. temporaria and the toad. A hypothesis which states that the interaction sites for amiloride and Na on the putative entry protein are spatially distinct in all of these species is proposed. (b) The stoichiometry of interaction between amiloride and the Na entry mechanism is not necessarily one-to-one. (c) The external Ca requirement for the inhibitory effect of amiloride is not absolute. Amiloride, at all concentrations, is equally effective in inhibiting Isc of bullfrog skin independently from the presence or absence of external Ca.

The midgut of Hyalophora cecropia actively transports potassium from hemolymph to lumen and the energy for this process appears to be intimately linked to oxidative metabolism. In the present investigation, we monitored concurrently the rate of active transport and the redox levels of the components of the respiratory chain in the intact tissue under a variety of experimental conditions. Approximately equal concentrations of cytochromes a3, a, c and b-557 were found. Other investigators (Pappenheimer, Jr, A.M. and Williams, C.M. (1954) J. Biol. Chem. 209, 915, Shappirio, D.G. and Williams, C.M. (1957) Proc. R. Soc. Lond. Ser. B 147, 233 and Chance, B. and Pappenheimer, Jr, A.M. (1957) J. Biol, Chem, 209, 931) have indentified cytochrome b-557 with b5 and found that it exists primarily in an extramitochondrial location. Steady-state experiments demonstrated that all these cytochromes were approximately 50% reduced while active transport proceeded at a high rate in regular cecropia Ringer containing 32 mM KCl. When the potassium concentration was reduced, the active transport decreased and all the cytochromes became more oxidized. Addition of 1 mM cyanide inhibited active transport by 90% and caused a 100% reduction of all cytochromes. Redox state and short circuit current (Isc) kinetics measured as the tissue was made anoxic showed that all the respiratory enzymes, except cytochrome b-557, became fully reduced at a faster rate than the rate of inhibition of the Isc. The rate of cytochrome b-557 reduction followed kinetically the Isc. These observations are interpreted in a scheme where cytochrome b-557 (possibly b5) branches off cytochrome c from the conventional resporatory chain, utilizing cytochrome a3 as the terminal oxidase for both branches. Cytochrome b-557 may be involved in providing a direct link between oxidative metabolism and active transport in the midgut of the silkworm.

Active Na transport across frog skin was separated from passive Na movement utilizing urea influx as a measure of passive (shunt) permeability. In this manner, the response of the overall active Na transport system to an applied potential was determined over a range from +200 mV to -100 mV. Active Na transport displays saturation as a function of applied potential, and both the level of saturation and the potential at which it is achieved are functions of the Na concentration in the external solution. The saturation with potential appears to involve a different step in the transport process than the saturation of Na flux as a function of external Na concentration. The observations can be qualitatively described by either a one-barrier or two-barrier model of the Na transport system.

Properties of the shunt pathway (a pathway in parallel to the Na transport system) in frog skin have been examined. The permeability of this shunt to urea increases markedly when the skin is depolarized to -100 mv (inside negative) but hyperpolarization to +100 mv produces no change in urea permeability compared to short-circuit conditions. The permeability increase at depolarizing potentials is dependent on the external solute concentration and is considerably reduced by the presence of external Ca. Neither urea permeability nor its response to changes in potential difference are affected by complete inhibition of Na transport by ouabain. In ouabain-poisoned skins, movements of Na, K, Cl, and mannitol through the shunt change in parallel with urea movements. Ion fluxes under these conditions and their response to potential can be described by the constant field equation. The selectivity of the shunt is in the order Cl > urea > K > Na > mannitol and this order does not appear to be affected by the absolute magnitude of the shunt permeability. Arguments are presented suggesting that the pathway is mainly between cells and that its permeability may be affected by cell swelling.