ATP-stimulated tetraethylammonium transport by rabbit renal brush border membrane vesicles.

These studies examined the ability of ATP to stimulate transport of the organic cation tetraethylammonium (TEA) into proximal tubular brush border membrane vesicles. ATP markedly enhanced TEA uptake for 1 h or more to values severalfold above those observed in the absence of ATP. The poorly hydrolyzable analogue of ATP, AMP-PNP (adenyl-5'-yl imidodiphosphate), reduced the effect of ATP but alone did not stimulate TEA uptake. GTP and ITP also stimulated TEA uptake, whereas other nucleotides did not. ATP-stimulated TEA uptake was saturable, temperature-dependent, and markedly reduced by the organic cations amiloride, quinidine, cimetidine, and verapamil, but only modestly reduced by the organic cations N'-methylnicotinamide and choline. Some inhibitors of other transport ATPases, including N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, and oligomycin, reduced the effect of ATP, whereas ouabain, vanadate, and azide did not. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid also reduced TEA uptake in the presence of ATP. Vinblastine, but not actinomycin D and colchicine (all inhibitors of P-glycoprotein-mediated transport), reduced TEA uptake. The reduction of TEA transport by amiloride and cimetidine was most consistent with competitive inhibition, whereas the inhibition produced by N-ethylmaleimide and vinblastine evidently was not. ATP also stimulated uptake of N'-methylnicotinamide but not that of vinblastine. These studies have identified a previously unrecognized process by which ATP hydrolysis may directly energize the reabsorption of organic cations from the renal tubule lumen.

Organic cation transport by rat hepatocyte basolateral membrane vesicles.

Hepatocyte basolateral membrane possesses transport systems for mediated uptake of organic cations, the first step in the subsequent biliary excretion and/or metabolism of these compounds. The purpose of these studies was to evaluate potential mechanisms for transport of this class of solutes across this membrane by measuring 3H-labeled tetraethylammonium ([3H]TEA) transport into rat hepatocyte basolateral membrane vesicles. [3H]TEA uptake was stimulated by an outwardly directed proton gradient consistent with TEA-proton exchange. Proton gradient-stimulated [3H]TEA uptake was inhibited by quinidine and by the combination of valinomycin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) but not by CCCP alone or by N1-methylnicotinamide (NMN). An outwardly directed TEA gradient also stimulated uptake of [3H]TEA with values at early time points exceeding those at equilibrium. This trans-stimulation or countertransport was saturable with an apparent Michaelis constant of 106 microM and maximal velocity of 434 pmol.mg-1.15 s-1. TEA countertransport was cis-inhibited by quinidine, cimetidine, and thiamine and by low temperature, but not by NMN. Thiamine was also capable of trans-stimulating [3H]TEA uptake. An outwardly directed potassium gradient enhanced and an inwardly directed potassium gradient reduced TEA countertransport but had no effect on [3H]TEA uptake occurring in the absence of other electrochemical driving forces. These studies indicate that there are at least two potential mechanisms in the hepatocyte basolateral membrane for transport of organic cations; organic cation-organic cation exchange (countertransport) and organic cation-proton exchange. Furthermore, the results are consistent with the existence of more than one transporter with different substrate affinities in each of these categories.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of in vitro metabolic acidosis on luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport in rabbit kidney proximal tubules.

The aim of this study was to evaluate the role of the kidney in mediating the signals involved in adaptive changes in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis. Proximal tubular suspensions were prepared from rabbit kidney cortex and incubated in acidic (A) or control (C) media (pH 6.9 vs 7.4, 5% CO2) for 2 h. Brush border membrane (BBM) and basolateral membrane (BLM) vesicles were isolated from the tubular suspensions and studied for the activity of Na+/H+ exchange and Na+:HCO3- cotransport. Influx of 1 mM 22Na at 10 s (pH6 7.5, pH(i) 6.0) into BBM vesicles was 68% higher in group A compared to group C. The increment in Na+ influx in the group A was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.1 mM in C vs 9.2 in A and Vmax of 31 nmol/mg protein per min in group C vs 57 in A. Influx of 1 mM 22Na at 10 s (pH0 7.5, pH(i) 6.0, 20% CO2, 80% N2) into BLM vesicles was 83% higher in the group A compared to C. The HCO3-dependent increment in 22Na uptake in group A was 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid sensitive, suggesting that Na+:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 11.4 mM in C vs 13.6 in A and Vmax of 35 nmol/mg protein per min in C vs 64 in A. The presence of cyclohexamide during incubation in A medium had no effect on the increments in 22Na uptake in group A. We conclude that the adaptive increase in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis is acute and mediated via direct signal(s) at the level of renal tubule.

Basolateral transport of tetraethylammonium by a clone of LLC-PK1 cells.

In these studies, a clone of cells derived from the porcine renal epithelial line LLC-PK1 grown on porous filters was used to evaluate basolateral uptake of the organic cation tetraethylammonium (TEA). (3H) TEA (1 microM) entered cells in a saturable and time-dependent manner achieving a steady-state value at 2 to 2.5 h. Uptake was reduced by hypothermia and the metabolic inhibitors sodium azide and iodoacetate. Several other organic cations in 1 mM concentrations inhibited the majority of TEA uptake. In lower concentrations, the inhibitory potency of these was: verapamil greater than cimetidine approximately amiloride approximately quinidine greater than procainamide approximately N1-methylnicotinamide. When sodium was replaced with potassium in the uptake medium, TEA uptake was also reduced consistent with electrogenic transport. However, uptake was reduced further by 1 mM cimetidine in the presence of both NaCl and KCl buffers. TEA uptake was not significantly different when the media pH was varied from 6.0 to 8.0. In addition, results of experiments in which intracellular pH was altered with NH4Cl were not consistent with the presence of organic cation/proton exchange. TEA/TEA exchange could not be demonstrated in experiments in which cells were preloaded with 1 mM nonradioactive TEA and uptake of (3H)TEA was measured or in which nonradioactive TEA in the external medium failed to enhance efflux from cells preloaded with (3H)TEA. These results indicate that the basolateral membrane of LLC-PKc10 cells has one or more transport processes for the mediated uptake of organic cations. However, the precise mechanism(s) involved in this transport remains to be elucidated.

A pH modifier site regulates activity of the Na+:HCO3- cotransporter in basolateral membranes of kidney proximal tubules.

HCO3- exit across the basolateral membrane of the kidney proximal tubule cell is mediated via an electrogenic Na+:HCO3- cotransporter. In these experiments, we have studied the effect of internal pH on the activity of the Na+:HCO3- cotransport system in basolateral membrane vesicles isolated from rabbit renal cortex. Equilibrium thermodynamics predicts that in the presence of constant intravesicular concentration of Na+, an increasing concentration of HCO3- will be associated with an increasing driving force for Na+:HCO3- cotransport across the vesicles. Our experimental approach was to preequilibrate the membrane vesicles with 1 mM 22Na+ at pHi 6.8-8.0 and known concentrations of HCO3-. The vesicles were diluted 1:100 into Na(+)-free solution at pH 7.4 and the net flux of 22Na+ was assayed over 5 s. The results demonstrate that the net flux of Na+ was significantly higher at pHi 7.2 than pHi 8.0 despite much higher [HCO3-] at pHi 8.0. This suggests that an internal pH-sensitive site regulates the activity of the Na+:HCO3- cotransporter. This modifier site inhibits the cotransporter at alkaline pH despite significant base concentration and is maximally functional around physiologic pH. The combination of modifier sites on the luminal Na+/H+ exchanger and the basolateral Na+:HCO3- cotransporter should help maintain intracellular pH in a narrow range with changes in extracellular pH.

Sulfhydryl groups are essential for organic cation exchange in rabbit renal basolateral membrane vesicles.

The effect of N-ethylmaleimide (NEM), an irreversible sulfhydryl modifying reagent, on the transport of organic cations in the renal basolateral membrane was examined. The studies were conducted examining the exchange of [3H]tetraethylammonium (TEA) for unlabeled TEA in basolateral membrane vesicles isolated from the outer cortex of rabbit kidneys. NEM inactivated TEA transport in a dose-dependent fashion with an IC50 value of 260 microM. The rate of TEA transport inactivation followed apparent pseudo-first-order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 4.0. The data imply that inactivation involves the binding of at least four molecules of NEM per active transport unit. This is most consistent with the presence of four sulfhydryl groups at this site. The substrate TEA displayed a dose-dependent enhancement of NEM inactivation, with 50% enhancement occurring at 365 microM TEA. Another organic cation, N1-methylnicotinamide, known to share a common transport mechanism with the TEA/TEA exchanger is also capable of increasing the reactivity of sulfhydryl groups to NEM. These results demonstrate that there are essential sulfhydryl groups for organic cation transport in the basolateral membrane. In addition, the capability of organic cations to alter the susceptibility to sulfhydryl modification suggests that these groups may have a dynamic role in the transport process.

Tetraethylammonium transport by OK cells.

Mechanisms exist in renal proximal tubules for the mediated transepithelial secretion or reabsorption of endogenous and exogenous organic cations. In the studies presented here, the uptake of the organic cation tetraethylammonium (TEA) into confluent monolayers of opossum kidney cells was evaluated to determine if these cells might serve as an in vitro model of this transport pathway. 3H-TEA entered opossum kidney cells in a time-dependent manner. Uptake at early time points was saturable with an apparent Km of 59.1 +/- 11.2 microM and a Vmax of 1,292 +/- 210 fmol/micrograms of DNA. TEA uptake was inhibited in a dose-dependent manner by several other organic cations including amiloride, cimetidine, verapamil, procainamide, quinidine and N1-methylnicotinamide. With 1 mM concentrations of these compounds, uptake was virtually eliminated. However, another organic cation, N'-methylnicotinamide caused only minimal inhibition. TEA uptake was significantly reduced by sodium azide, suggesting dependence on oxidative phosphorylation. An alkaline medium pH enhanced TEA uptake, but, at the same pH, uptake was similar in the presence or absence of bicarbonate. When cellular pH was altered by ammonium chloride addition or removal, TEA uptake was not affected. Thus, organic cation/proton exchange, as has been demonstrated previously in apical membrane vesicles prepared from proximal tubules, is evidently not responsible for TEA uptake. Similarly, uptake does not appear to result from organic cation/organic cation exchange. These results indicate that the plasma membrane of opossum kidney cells contains a transport system(s) for the mediated uptake of organic cations and that these cells may be a useful mode for further study of renal epithelial transport of these solutes.

Potassium depletion increases luminal Na+/H+ exchange and basolateral Na+:CO3=:HCO3- cotransport in rat renal cortex.

Most HCO3- reabsorption in proximal tubules occurs via electroneutral Na+/H+ exchange in brush border membranes (BBMS) and electrogenic Na+:CO3=:HCO3- cotransport in basolateral membranes (BLMS). Since potassium depletion (KD) increases HCO3- reabsorption in proximal tubules, we evaluated these transport systems using BBM and BLM vesicles, respectively, from control (C) and KD rats. Feeding rats a potassium deficient diet for 3-4 wk resulted in lower plasma [K+] (2.94 mEq/liter, KD vs. 4.47 C), and higher arterial pH (7.51 KD vs. 7.39 C). KD rats gained less weight than C but had higher renal cortical weight. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0, 10% CO2, 90% N2) into BLM vesicles was 44% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was DIDS sensitive, suggesting that Na+:CO3=:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.2 mM in KD vs. 7.6 mM in C and Vmax of 278 nmol/min/mg protein in KD vs. 177 nmol/min/mg protein in C. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0) into BBM vesicles was 34% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 6.2 mM in KD vs. 7.1 mM in C and Vmax of 209 nmol/min/mg protein in KD vs. 144 nmol/min/mg protein in C. Uptakes of Na(+)-dependent [3H]glucose into BBM and [14C]succinate into BLM vesicles were not different in KD and C groups, suggesting that the Na+/H+ exchanger and Na+:CO3=:HCO3- cotransporter activities were specifically altered in KD. We conclude that adaptive increases in basolateral Na+:CO3=:HCO3- cotransport and luminal Na+H+ exchange are likely responsible for increased HCO3- reabsorption in proximal tubules of KD animals.

Mechanism of organic cation transport in rabbit renal basolateral membrane vesicles.

The transports of [3H]tetraethylammonium (TEA), [3H]procainamide (PCA), and N1-[3H]methylnicotinamide (NMN) were studied in rabbit renal basolateral membrane vesicles (BLMVs) by use of a rapid filtration assay. All three compounds exhibited a similar uptake profile into the BLMVs and reached equilibrium by 1 h. In the presence of valinomycin, a K+ ionophore and K+ gradients (in to out), an inside-negative potential difference (PD) was generated that stimulated the uptake of TEA, PCA, and NMN by 1.9-, 1.9-, and 2.1-fold, respectively. The effect of PD could be blocked by the organic cation transport inhibitor mepiperphenidol. An inside-negative PD was also generated by a pH gradient (inside acidic). An overshoot of TEA uptake was produced, which was blocked by a valinomycin voltage clamp. Counterflow studies revealed that 1 mM TEA was capable of trans-stimulating 50 microM [3H]TEA uptake and producing a peak overshoot of nearly three times the equilibrium value, which was not abolished in the presence of a valinomycin voltage clamp or a gramicidin pH clamp. When an inside-negative PD was imposed on 1 mM TEA-loaded BLMVs, the uptake of [3H]TEA was 33% less. In contrast, neither NMN nor PCA produced a trans-stimulation of [3H]NMN or [3H]PCA transport, respectively. In addition, the effect of several organic cations on the TEA-TEA exchange mechanism was studied. Mepiperphenidol, PCA, choline, cimetidine, and NMN all demonstrated cis inhibition (82, 81, 58, 51, and 20%, respectively). Arginine, a basic amino acid, and probenecid, an organic anion transport inhibitor, had no effect. Choline was capable of trans-stimulating TEA uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Organic cation uptake by a cultured renal epithelium.

Several organic cations are actively transported by proximal renal tubules by mediated processes across both the apical and basolateral cell membranes. In order to evaluate this transport system in a cultured renal epithelium, uptake of 3H-tetraethylammonium (TEA) across the apical membrane was measured in LLCPK1 cells, a cell line with several characteristics of proximal tubules. 3H-TEA progressively entered these cells and reached a near-steady state by 30 min. Three-minute uptake was saturable with an apparent Vmax of 1,669 +/- 129 fmoles/micrograms DNA and apparent Km of 34.0 +/- 3.4 microM. 3H-TEA uptake was inhibited by an excess of nonradioactive TEA, other organic cations, sodium azide, and hypothermia. An alkaline external pH was associated with greater 3H-TEA uptake than an acid pH. However, efflux of 3H-TEA from cells was not appreciably affected by changes in external pH. Preincubation of cells in acid or alkaline media did not affect uptake. Alteration of cell pH by ammonium chloride addition or removal had little effect on 3H-TEA uptake. Finally, uptake of 3H-TEA was not accelerated by preloading cells with an excess of nonradioactive TEA. These results indicate that intact LLCPK1 cells possess a mechanism(s) in their apical membranes for the mediated transport of a prototypic organic cation. The mechanism(s) involved in this transport is uncertain. However, neither organic cation/proton nor organic cation/organic cation exchange appears to be the predominant process.

Protease effects on adenylate cyclase in potassium-depleted rabbit kidney.

Potassium depletion in rabbits induces a renal concentrating defect in vivo and decreased hydrosmotic response to arginine vasopressin (AVP) in isolated cortical collecting tubules (CCT) perfused in vitro. The molecular basis of the AVP resistance in potassium depletion was investigated by comparing AVP-responsive adenylate cyclase activities in CCT from potassium-depleted and control rabbits. Vasopressin-responsive enzyme activity was impaired in CCT dissected from kidneys of potassium-depleted rabbits but not when kidneys were treated with collagenase to improve microdissection conditions. Potassium depletion also depressed parathyroid hormone (PTH)-stimulated adenylate cyclase activity in proximal straight tubules (PST) dissected from untreated but not collagenase-treated kidneys. Commercially available collagenase, which also contains other proteolytic enzymes, increased AVP-sensitive adenylate cyclase activity in control CCT, and trypsin treatment of CCT dissected without collagenase abolished the decrease in AVP-sensitive activity induced by potassium depletion. Inclusion of trypsin inhibitor during collagenase treatment of kidneys lowered AVP response in CCT from potassium-depleted rabbits. These results demonstrate that potassium depletion impairs hormone-sensitive adenylate cyclase of CCT (and PST) by a protease-sensitive mechanism.

Effects of respiratory acidosis on HCO3- transport by rabbit collecting tubules.

These studies were performed to determine whether in vitro elevation of bath PCO2 with associated reduction in pH (acute respiratory acidosis) affected bicarbonate transport by isolated perfused rabbit cortical collecting tubules (CCT) and collecting tubules from the outer (OMCTos) and inner (OMCTis) stripes of the outer medulla. When the PCO2 was elevated and pH reduced from approximately 7.4 to 7.0 the rate of total CO2 absorption increased to 252% of that observed at pH 7.4 in CCT, 146% in OMCTos, and 150% in OMCTis. In OMCTis, pretreatment with colchicine inhibited the stimulation of total CO2 absorption associated with respiratory acidosis, whereas lumicolchicine did not. Similar inhibition was observed in the presence of maptam and a low calcium concentration and in the presence of a calmodulin inhibitor. No differences were observed in apical or basolateral membrane morphometry of principal or intercalated cells between control tubules and those subjected to respiratory acidosis. The results indicate that acute respiratory acidosis stimulates acidification by the rabbit distal nephron in vitro through a process(es) that, at least in OMCTis, evidently involves the cell cytoskeleton and changes in cell calcium and calmodulin activities.

Prostaglandins and the urinary concentrating defect in potassium-depleted rabbits.

Urinary prostaglandin E (UPGE) excretion increased significantly after 1 and 2 wk of potassium depletion (KD) in female New Zealand White rabbits on ad libitum water intake [UPGE control, 21.3 +/- 4.6 ng PGE/mg creatinine; 1 wk KD, 40.4 +/- 6.1 ng PGE/mg creatinine (P less than 0.01); 2 wk KD, 31.9 +/- 14.9 ng PGE/mg creatinine (P less than 0.05)]. In vivo prostaglandin inhibition with indomethacin or meclofenamate significantly increased urinary osmolality after 12 h of dehydration and exogenous vasopressin (1.25 U) from 794 +/- 59 to 1,163 +/- 113 mosmol/kgH2O (P less than 0.01). In vitro prostaglandin inhibition with indomethacin or meclofenamate corrected the antidiuretic hormone (ADH) unresponsiveness of isolated perfused cortical collecting tubules (CCTs) from KD rabbits. Furthermore, preincubation with pertussis toxin, an agent that inactivates the guanine nucleotide inhibitory (Ni) subunit of adenylate cyclase, normalized the ADH response of KD CCTs, suggesting that prostaglandins may attenuate ADH action on the CCT through activation of Ni and contribute to the urinary concentrating defect associated with KD.

Bicarbonate transport in collecting tubules from outer stripe of outer medulla of rabbit kidneys.

The purpose of this study is to characterize the features of bicarbonate (total CO2) transport in isolated perfused collecting tubules obtained from the outer stripe of the outer medulla (OMCTos) of rabbit kidneys. Under control conditions (25 mM HCO3- in the perfusate and bath), all OMCTos studied absorbed total CO2 at a mean rate of 8.61 +/- 0.44 pmol.mm-1.min-1. Ouabain (10(-4) M in the bath) did not affect the rate of total CO2 absorption (JtCO2). Addition of the diethylstilbene 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a concentration of 10(-4) M or replacement of bath chloride by gluconate reduced JtCO2 by approximately 50%, whereas replacement of luminal chloride increased JtCO2 by 40%. The carbonic anhydrase inhibitors acetazolamide and ethoxyzolamide in concentrations of 10(-4) M had little effect on JtCO2. In a concentration of 10(-3) M, acetazolamide reduced JtCO2 by only 31%. OMCTos obtained from rabbits with ammonium chloride-induced metabolic acidosis did not have increased rates of total CO2 absorption compared with the control, but treatment of animals with mineralocorticoids increased JtCO2. These results indicate that OMCTos are capable of significant bicarbonate absorption in vitro. This absorption 1) is independent of sodium transport, 2) appears to require, at least in large part, HCO3- or OH- -Cl- exchange across the basolateral cell membrane of acid-secreting cells, 3) is much more resistant to inhibition by carbonic anhydrase inhibitors than reported previously for other rabbit nephron segments, and 4) is stimulated by prior mineralocorticoid treatment of animals but not by prior metabolic acidosis in vivo.

Cimetidine transport in rabbit renal cortical brush-border membrane vesicles.

Cimetidine is an organic cation and commonly prescribed drug that is eliminated primarily by proximal renal tubular secretion. The present studies evaluated cimetidine transport in rabbit renal cortical brush-border membrane vesicles (BBMV). [3H]Cimetidine uptake varied inversely with media osmolarity and was stimulated with uphill transport above equilibrium values (overshoot) produced by an initial proton gradient directed from the vesicle interior outwardly. Uphill transport occurred earlier and was of greater magnitude at 25 degrees C than at 5 degrees C. pH-stimulated [3H]cimetidine uptake was inhibited by excess nonradiolabeled cimetidine, quinidine, and procainamide but was affected little by probenecid. Tetraethylammonium inhibited cimetidine uptake in the presence and absence of an initial proton gradient, indicating that nonionic diffusion and simple diffusion cannot totally account for cimetidine transport in BBMV. The protonophore carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP) inhibited pH-stimulated cimetidine uptake but had no effect on uptake occurring in the absence of an initial pH gradient. Preloading BBMV with an excess of procainamide enhanced cimetidine uptake. However, in the presence of FCCP, the combination of FCCP and valinomycin, or nigericin the effect of preloading with procainamide was diminished, suggesting that the apparent countertransport of cimetidine produced by procainamide was indirect and due to generation of a transvesicular proton gradient. These results are consistent with the hypothesis that cimetidine is transported across BBMV by organic cation-proton exchange.

Effect of potassium depletion and protein intake in vivo on renal tubular bicarbonate transport in vitro.

These studies evaluated the effect of decreased dietary potassium and increased dietary protein content in rabbits on systemic acid-base parameters, urine pH, and segmental renal tubular bicarbonate handling in vitro. Animals fed a potassium-deficient high-protein diet (KD) for 2 wk developed metabolic acidosis (arterial blood pH 7.31 vs. 7.43) and excreted a more acid urine than control animals fed a diet with normal amounts of potassium and protein; whereas, animals fed the same potassium deficient diet to which supplemental potassium was added (KD + K+) excreted an even more acid urine and had a blood pH of 7.36. In superficial proximal convoluted and straight tubules, there were no differences in rates of fluid and total CO2 absorption between control and KD tubules. Cortical collecting tubules obtained from KD and KD + K+ animals absorbed, and control tubules secreted total CO2 in vitro. With an ambient potassium concentration of 2.5 mM, collecting tubules obtained from the inner stripe of the outer medulla of KD animals absorbed significantly less total CO2 than control tubules. The same tendency was observed in collecting tubules from the outer stripe of the outer medulla. Plasma aldosterone levels fell with development of potassium depletion. Administration of deoxycorticosterone for 1 day to KD animals was associated with a lower urine pH and higher arterial blood pH than in untreated KD animals and with increased total CO2 absorption by both cortical collecting tubules and tubules from the inner stripe of the outer medulla.(ABSTRACT TRUNCATED AT 250 WORDS)

Procainamide transport in rabbit renal cortical brush border membrane vesicles.

Previous studies in canine and rat renal cortical brush border membrane vesicles (BBMV) and some results in isolated perfused rabbit proximal tubules indicate that organic cations may be transported across the apical cell membrane by an organic cation/proton exchange process. To determine more directly whether organic cations are transported across the apical cell membrane of rabbit proximal tubules, [3H]procainamide uptake in BBMV was studied. Procainamide uptake was linearly related to the inverse of the media osmolarity, indicating uptake into an intravesicular space. A proton gradient directed from vesicle interior outwardly stimulated and an opposite gradient inhibited procainamide uptake. pH-stimulated uptake was inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) and was also reduced by an inwardly directed sodium gradient. pH-stimulated procainamide uptake was inhibited by other organic cations including the quarternary ammonium ion tetramethylammonium, indicating that the effect of proton gradients was not due to changes in nonionic diffusion. pH-stimulated procainamide uptake at 10 s was saturable with an apparent Km of 5.4 X 10(-4) M and Vmax of 4.7 X 10(-10) mol X mg protein-1. Uptake of [3H]procainamide was enhanced when BBMV were preloaded with nonradioactive procainamide but this was prevented by FCCP and valinomycin. Finally, an outwardly directed potassium gradient in the presence of valinomycin failed to significantly stimulate procainamide uptake. These results are consistent with a mechanism of secretion that involves electroneutral exchange of procainamide for protons across the apical cell membrane of rabbit proximal tubules.

In vivo and in vitro studies of urinary concentrating ability in potassium-depleted rabbits.

The factors responsible for the urinary concentrating defect associated with the potassium-depleted (KD) state are uncertain. The present studies were designed to, first, determine whether a urinary concentrating defect exists in potassium-depleted rabbits and, second, to use the technique of in vitro perfusion to evaluate directly the antidiuretic hormone (ADH) responsiveness of cortical collecting tubules (CCT) in this setting. Feeding female New Zealand White rabbits a potassium-deficient diet for 2 wk caused a significant fall in plasma potassium levels in both the ad-libitum and controlled water intake groups (P less than 0.001). Muscle potassium content after 2 wk of potassium restriction fell from 45.6 +/- 0.9 to 29.0 +/- 1.2 meq/100 g fat-free dry solids (P less than 0.001). Renal papillary sodium content fell significantly from a control value of 234.6 +/- 8.0 to 182.46 +/- 10.0 meq/kg H2O after 2 wk of potassium restriction. Maximal urinary osmolality measured after 12 h of dehydration and 1.25 U pitressin IM was significantly decreased in rabbits after 2 wk of potassium restriction in both the ad-libitum and controlled water intake groups (P less than 0.001). The relationship between plasma potassium concentration and maximum urinary osmolality was significantly correlated in both the ad-libitum and controlled water intake groups, r = 0.73 and 0.68 (P less than 0.001), respectively. In addition, refeeding KD rabbits with normal chow for 1 wk resulted in normalization of both plasma potassium levels and urinary concentrating ability. CCT from control and KD rabbits were perfused in vitro at 25 degrees C. The hydraulic conductivity coefficient, Lp, was significantly reduced at all doses of ADH tested in tubules from KD rabbits when compared with control tubules. In addition, the maximal hydraulic conductivity in tubules from KD rabbits when tested with 200 microU/ml ADH at 37.5 degrees C was only 23% of control values (P less than 0.05). Furthermore, this reduced ADH responsiveness persisted when the bath potassium was elevated from 5 to 20 mM. The reflection coefficient for NaCl when compared with raffinose was 0.91 in tubules from KD animals. Thus, these data suggest that the ADH-resistant urinary concentrating defect associated with potassium depletion is due, at least in part, to a diminished responsiveness of the CCT to ADH. Therefore, further studies were designed to investigate the cellular steps involved in this abnormal response. There was no difference in the 8-para-chlorophenylthio cyclic AMP induced hydroosmotic response between CCT from KD and control rabbits. Since the cAMP-induced hydroosmotic response was similar between KD and control CCT, experiments were performed to evaluate the contribution of phosphodiesterase (PDIE) activity by using the potent PDIE inhibitor isobutylmethylxanthine (10(-4) and 10(-3)M) in the presence of ADH (200 U/ml). Although Lp was increased by PDIE inhibition in CCT from both control and KD animals, the overall hydroosmotic response in CCT from KD rabbits was still significantly reduced when compared with controls. The final experiments used forskolin to evaluate further the adenylate cyclase complex. The resulting hydroosmotic response in CCT from KD rabbits was almost identical to that obtained in controls. In conclusion, these data suggest that the decreased responsiveness of CCT from KD rabbits to ADH involves a step at or proximal to the stimulation of the catalytic subunit of adenylate cyclase, and that PDIE activity makes no contribution to this abnormal hydroosmotic response.

Further studies of organic base secretion by rabbit proximal tubules.

The aim of the present studies was to further characterize the mechanisms responsible for the secretion of the organic base procainamide by measuring [3H] procainamide transport in superficial (SF) and juxtamedullary (JM) S2 segments of rabbit proximal tubules perfused in vitro. When perfusate pH was changed from 7.4 to 6.2, procainamide secretion increased by 227% in SF and by 159% in JM tubules. In the presence of ouabain the acidic perfusate caused only a minimal increase in procainamide secretion. When the perfusate pH was increased, procainamide secretion was reduced. Acetazolamide, 10(-5) M in the bath, reduced procainamide secretion in both SF and JM tubules. In SF tubules amiloride, 10(-4) M in the perfusate, inhibited procainamide secretion, whereas nonradioactive procainamide, quinidine, and cimetidine did not. When ambient sodium was reduced to 10 mM, the rate of procainamide secretion was low and was not affected by amiloride in the perfusate or acetazolamide in the bath. Amiloride in the bath caused a dose-related inhibition of procainamide secretion. The rate of procainamide secretion by cortical collecting tubules was very low and was not affected by acetazolamide. The mechanisms of renal organic base secretion are complex. Some of the present data are consistent with a mechanism involving organic base-proton exchange across the apical cell membrane. However, other data are not consistent with this being the major mechanism involved.