Regional localization of renal Na(+)-H+ antiporter: response to respiratory acidosis.

The Na(+)-H+ antiporter of renal brush-border membranes has been well characterized and plays a role in adaptation to acidosis. Na(+)-H+ antiporter activity has been described in other renal regions, but its kinetics as well as its role in adaptation to acidosis are unclear. Thus we measured Na(+)-H+ antiporter activity in membrane vesicles of outer and inner stripes of outer medulla (OSOM and ISOM, respectively) and in plasma membranes from papilla and compared it to Na(+)-H+ antiporter activity of the cortex in control and hypercapnic rabbits. Chronic hypercapnia (induced by exposure to CO2 for 48 h) was associated with significantly higher PCO2 and plasma HCO3- and lower urine pH than controls. In control animals, magnitude of Vmax of amiloride-sensitive component of Na(+)-H+ antiporter (expressed as fluorescence units.300 micrograms protein-1.min-1) was 392.2 +/- 32 in cortex, 115 +/- 9.7 in OSOM, 66.1 +/- 9.4 in 15-25% (F1) fraction and 118.7 +/- 16.8 in 25-40% (F2) fraction of ISOM, respectively, and 79.3 +/- 5.2 in papilla. These values were significantly different from each other except between F1 and papilla and F2 and OSOM. The Km for Na, however, was not different, suggesting that the renal Na(+)-H+ antiporter is basically the same in different renal regions but displays different activity. Hypercapnia for 48 h increased significantly the amiloride-sensitive component of Na(+)-H+ antiporter by 60% in cortex, 43% in F1, and 29% in papilla but failed to alter Vmax in OSOM.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of H+ secretion by CO2 in turtle bladder: role of intracellular pH, exocytosis, and calcium.

We studied the interaction of intracellular pH, exocytosis, and cell calcium on the stimulation of H+ secretion by CO2 in turtle bladder. Intracellular pH was continuously monitored by the fluorescent dye 6-carboxyfluorescein and exocytosis was monitored by the release of mucosal fluorescein dextran. The initial stimulation of H+ secretion by 1 or 5% CO2 added to the serosal solution was accompanied by a similar and temporally related increase in exocytosis. Furthermore, a decrease in intracellular pH seems necessary for the early increase in H+ secretion and exocytosis. Because calcium plays an important role in exocytosis, we measured intracellular calcium in isolated cells with the fluorescent dye quin2. An increase in intracellular calcium (from 50 to 100 nM) was observed in isolated turtle bladder epithelial cells gassed with 5% CO2. To further evaluate the role of intracellular calcium on H+ secretion and exocytosis we utilized agents that alter cell calcium such as trifluoperazine and lanthanum. In the presence of CO2 these agents blocked partially the increase in H+ secretion and exocytosis but did not affect the decrease in intracellular H+. In conclusion, exocytosis, intracellular pH, and intracellular calcium play a key role in mediating CO2-stimulated H+ secretion in the turtle bladder.

Na-HCO3 cotransport and Na-H antiporter in chronic respiratory acidosis and alkalosis.

Renal acidification in renal proximal tubule is thought to be mediated by luminal Na-H antiporter and the HCO3- generated by this antiporter is removed from the cell by a basolateral Na-HCO3 cotransporter. To study the effect of respiratory acid-base disorders on these transport systems, we have measured the Na-HCO3 cotransport in basolateral membranes and Na-H antiporter in luminal membranes in control rabbits, rabbits exposed to 10% CO2 (chronic hypercapnia), and rabbits exposed to 10% O2-90% N2 (chronic hypocapnia). The Vmax of HCO3(-)-dependent 22Na uptake was significantly higher in chronic hypercapnia than controls (2.54 +/- 0.03 vs. 1.18 +/- 0.21 nmol.mg protein-1.3 s-1, P less than 0.001). Likewise, the Vmax of the Na-H antiporter was also increased compared with controls (924.9 +/- 42.1 vs. 549.1 +/- 62.8 fluorescence units (FU).300 micrograms protein-1.min-1). In chronic hypocapnia, the Vmax of Na-HCO3 cotransport was lower than controls (0.72 +/- 0.11 vs. 1.18 +/- 0.21 nmol.mg protein-1.3 s-1, P less than 0.05). There was no difference, however, in the Vmax of the Na-H antiporter between hypocapnia and control (524.2 +/- 24.3 vs. 549.1 +/- 62.8, FU.300 micrograms protein-1.min-1). The Vmaxs of the Na-HCO3 cotransport and of the Na-H antiporter in hypocapnic, control, and hypercapnic rabbits were linearly related (r = 0.81), suggesting a simultaneous adaptation of the two systems in respiratory acid-base disorders.(ABSTRACT TRUNCATED AT 250 WORDS)

Methyl isobutyl amiloride: a new probe to assess the number of Na-H antiporters.

We measured the binding of [3H]-5-(N-methyl-N-isobutyl) amiloride (MIA) to purified rabbit renal brush border membranes. MIA binding was protein, temperature and time dependent with optimal binding at pH 8.0 or above. At low pH MIA binding was inhibited, suggesting competition between H+ ions and MIA for the MIA binding site. There was 70-80% specific binding which reached a plateau at 30 min and remained stable thereafter for 150 min. Scatchard analysis revealed one family of binding sites with Bmax of 3.4 +/- 0.4 pmoles/mg protein and Kd of 30.5 +/- 2.3 nM. MIA inhibited the Vmax of the Na-H antiporter (assessed by acridine orange quenching) in a dose dependent fashion with 100% inhibition at MIA concentration of 10(-3) M and this inhibition was greater than that of amiloride. We conclude that MIA, a potent inhibitor of the Na-H antiporter, displays a high percentage of specific binding to renal brush border membranes and can be used to assess the number of the Na-H antiporters.

Phorbol-ester is bound specifically to renal luminal membranes and stimulates the Na-H antiporter.

We characterized binding of [3H]-phorbol-12,13-dibutyrate (an activator of protein kinase C) to highly purified rabbit cortical renal luminal membranes and measured its effect on the kinetics of the Na-H antiporter. There was 95% specific binding to luminal membranes and this binding was time, temperature and pH dependent with optimal binding at 4 degrees C and pH 7.4. Scatchard analysis of the binding revealed Kd of 0.8 microM and Bmax of 19.4 pmoles/mg protein. Phorbol-12,13-dibutyrate stimulated the Vmax of the Na-H antiporter by 16.5% +/- 4.7 at 10(-6) M and by 19.9% +/- 5.4 at 10(-5) M. The protein kinase C inhibitor H7(1) prevented the observed stimulation. Thus, there is a correlation between phorbol ester binding and phorbol ester stimulation of the Na-H antiporter. These data demonstrate that protein kinase C plays a role in the stimulation of the Na-H antiporter in renal luminal membranes.

High affinity binding sites for epidermal growth factor (EGF) in renal membranes.

The kidney produces large quantities of EGF but the role of the kidney in binding and degradation of EGF is unknown. We studied 125I-EGF binding and degradation by highly purified cortical luminal and cortical basolateral membranes of rabbit renal cortex, and by medullary plasma membranes. Specific binding for 125I-EGF was found for the first time in cortical basolateral and medullary plasma membranes (60-80% of total binding) but not in cortical luminal membranes. There was little degradation (less than 4%) of EGF by any of the membranes. Scatchard analysis of 125I-EGF binding by cortical basolateral membranes revealed two distinct classes of binding sites: high and low affinity. The existence of high specific binding sites in cortical basolateral and in medullary plasma membranes suggests a physiologic role of EGF in the kidney.

Effect of agents that alter cell calcium and microfilaments on CO2 stimulated H+ secretion in the turtle bladder.

CO2 addition to the serosal solution of turtle bladder stimulates H+ secretion. This stimulation of H+ secretion is thought to be mediated, at least in part, by exocytosis of vesicles containing H+ pumps that are inserted in the plasma membranes. In other systems, exocytosis is regulated by cell calcium and microfilaments. We, therefore, investigated the role of cell calcium and microfilaments on CO2 stimulation of H+ secretion. Trifluperazine and calmidazolium, inhibitors of calmodulin, did not alter base line H+ secretion, but significantly inhibited CO2 stimulation of H+ secretion. Lanthanum, an agent that displaces membrane bound calcium, also significantly inhibited CO2 stimulated H+ secretion when added to the mucosal solution. Removal of serosal Na, a maneuver that presumably changes cytosolic calcium via inhibition of Na-Ca exchange, decreased both base line as well as CO2 stimulated H+ secretion. In bladders treated with the microfilament disrupting agent cytochalasin B, there was inhibition of both base line and CO2 stimulated H+ secretion. In summary, agents that alter cell calcium or the cytoskeleton, inhibit CO2 stimulated H+ secretion, a finding compatible with the suggestion that exocytosis plays a role in this phenomenon.

Effect of metabolic or respiratory acidosis on rabbit renal medullary proton-ATPase.

Distal urinary acidification is thought to be mediated by an H+-ATPase sensitive to N-ethylmaleimide and dicyclohexyl-carbodiimide. We have studied the effect of chronic metabolic acidosis (NH4Cl for 3 days) or respiratory acidosis (inhalation of 10% CO2 for 2 days) on the H+-ATPase of plasma membranes prepared from the medulla. The enzymatic assay for the H+-ATPase was performed in the presence of ouabain and oligomycin and in the absence of Ca. H+-transport activity was assessed by the quenching of acridine orange in the presence of ATP. The 15-25% sucrose gradient fraction was enriched 40-fold in enzymatic activity over the homogenate, and 8-fold in enzymatic activity and 4-fold in H+-transport activity over the fluffy fraction (38,000 X g). Metabolic acidosis (pH less than 7.31) or chronic hypercapnia (PCO2 greater than 66 mmHg; 1 mmHg = 133.3 Pa) was induced for 2-3 days. Both groups showed the same enrichment factor in enzymatic and H+-transport assays as the control rabbits. Enzymatic and H+-transport activities, however, were not different between animals with respiratory acidosis and controls. Kinetic studies failed to disclose an increase in Vmax (673 vs. 702 mumol/(mg protein.min] or a decrease in Km (0.43 vs. 0.48 mM) in chronic hypercapnia as compared with controls. Metabolic acidosis also failed to increase H+-ATPase activity. These data demonstrate that the H+-ATPase of renal medulla does not display the expected increase in activity during acidosis. The role of this H+-ATPase in the adaptation to acidosis remains to be determined.

Na+-H+ antiporter in posthypercapnic state.

Posthypercapnic metabolic alkalosis has been attributed to decreased HCO3 excretion because of low glomerular filtration rate (GFR), volume contraction, or chloride depletion. We have previously shown that chronic hypercapnia enhances the Vmax of the Na+-H+ antiporter. We reasoned that an increased Vmax of the Na+-H+ antiporter could play a role in the maintenance of posthypercapnic metabolic alkalosis. To test this hypothesis, we measured the kinetics of the Na+-H+ antiporter by the dissipation of the quenching of acridine orange fluorescence in purified brush-border membrane obtained from posthypercapnic rabbits. The kinetic parameters were measured in controls and in rabbits that were exposed to hypercapnia for 48 h and then allowed to breathe room air for 3, 24, or 48 h. In luminal membranes prepared from posthypercapnic animals, the Vmax of the Na+-H+ antiporter was significantly increased after 3 and 24 h but not after 48 h compared with controls. The increase in Vmax was not different from that of hypercapnic animals. There was no difference in the Km of the Na+-H+ antiporter among these five groups. Amiloride inhibited the Vmax equally in membranes from control and posthypercapnic rabbits. Proton permeability was comparable among the groups. These data indicate that the increase in Vmax in posthypercapnic rabbits is mediated through the electroneutral Na+-H+ exchange and not through conductive H+ and Na+ pathway. Glucose uptake was not different in control and posthypercapnia, indicating a selective increase in Na+-H+ antiporter activity. At 3 and 24 h posthypercapnia, HCO3 concentration was higher than control.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic hypercapnia enhances Vmax of Na-H antiporter of renal brush-border membranes.

Chronic hypercapnia is associated with increased proximal HCO3 reabsorption that is thought to be mediated by a Na-H antiporter. We hypothesized that chronic hypercapnia would be associated either with increased Vmax or with decreased Km of the Na-H antiporter. To test this hypothesis we made rabbits hypercapnic for 48 h by exposure to 10% CO2. In both control and hypercapnic animals, cortical luminal membranes were enriched over the homogenate 16-fold in alkaline phosphatase and 10-fold in maltase activity. The kinetic activity of the Na-H antiporter was measured by the dissipation of the quenching of acridine orange by addition of different Na concentrations. Chronic hypercapnic rabbits had significantly higher Vmax of the Na-H antiporter of luminal membranes than controls (593 +/- 81 vs. 252 +/- 40 arbitrary fluorescence units X min-1 X 300 micrograms protein-1, P less than 0.01). The Km, however, was not different between control and hypercapnic rabbits. 22Na uptake in presence of an outwardly directed pH gradient was significantly higher in vesicles from hypercapnic rabbits than controls. Amiloride inhibited the Na-H antiporter (as assessed by acridine orange quenching or 22Na uptake) to the same degree in membranes from both control and hypercapnic rabbits, suggesting that the increase in Vmax is mediated by the electroneutral component of the Na-H antiporter. In addition, under voltage clamp conditions by K and valinomycin the Vmax was still increased in membranes from hypercapnic animals, again suggesting that the increase in Vmax is mediated by the electroneutral component of the Na-H antiporter. The uptake of D-[3H]glucose by luminal membranes was not different between control and hypercapnic rabbits, indicating a specific enhancement of the Na-H antiporter. Acute hypercapnia (4 h) failed to increase the Vmax of the Na-H antiporter despite comparable increase in PCO2. Thus chronic hypercapnia, but not acute hypercapnia, induces a selective and specific increase in the Vmax of Na-H antiporter, and this may mediate the adaptation to chronic hypercapnia.(ABSTRACT TRUNCATED AT 250 WORDS)

Anion exchanger is present in both luminal and basolateral renal membranes.

Binding of the anion-exchange inhibitor 3H2-labeled 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3H2]DIDS of 5.5 microM and 19.3 microM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolateral and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 35SO4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward-directed Cl gradient, OH gradient or HCO3 gradient than in the absence of these gradients. There was an early anion-dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion-dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HCO3-dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 microM. The similar values of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward-directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na-dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well-known Na-dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport.

Renal basolateral membrane Na-Ca exchange is electrogenic.

A Na-Ca exchange system is present in highly purified basolateral renal tubular membranes. In the present study we examined the electrogenicity of the system by measuring 45Ca uptake in the presence of favorable electric gradient created by K and valinomycin, by measuring the uptake of the lipophilic cation methytriphenylphosphonium (MTPP+) and by utilizing the voltage-sensitive dye DiS-C3(5). In the presence of an inward directed K gradient, 45Ca uptake in basolateral vesicles loaded with Na and suspended in K was significantly higher in the presence than in the absence of valinomycin. Under conditions favoring the operation of Na-Ca exchange system (i.e. outward directed Na gradient and in presence of external calcium) MTPP+ uptake was significantly higher in the presence than in the absence of a Na. Na-dependent Ca uptake was associated with a decrease in the fluorescence of the voltage-sensitive dye DiS-C3(5), indicating hyperpolarization. Conversely, Na-dependent Ca extrusion was associated with an increase in the fluorescence of the dye indicating depolarization. The fluorescence changes seemed to be specific for Na and Ca since they could not be reproduced when Li replaced Na or when Mg replaced Ca. These data demonstrate that the Na-Ca exchange system in renal basolateral membranes is electrogenic with stoichiometry greater than 2:1 Na:Ca ratio.

Intracellular pH of the turtle bladder assessed with fluorescent probes.

Intracellular pH of the turtle bladder was measured with fluorescent probe 6-carboxyfluorescein (6-CF) diacetate. In isolated cells this probe provides reliable, reproducible and fast measurements of intracellular pH. The probe was mainly located in the cytosol and thus the values of intracellular pH mainly reflect cytosolic pH. The values of intracellular pH obtained with 6-CF were very similar to those measured with 14C-methylamine and 'null point' technique. The 6-CF technique was capable of detecting small changes in intracellular pH induced by acetazolamide. The intracellular pH of the mitochondrial-rich and granular cell fraction was not different. In addition to assessing intracellular pH of isolated cells, it was possible to monitor the intracellular pH of whole bladders continuously with 6-CF. Addition of CO2 to serosal solution decreased intracellular pH while perfusion with NH3 increased intracellular pH. Thus, 6-CF provides reliable and accurate measurements of intracellular pH in isolated cells and in whole bladders. This technique is capable of detecting rapid changes in intracellular pH and provides continuous monitoring of intracellular pH and thus should allow correlation of changes in urinary acidification with intracellular H+ concentration.

Relationship of K and ammonia transport by the turtle bladder.

The relationship between K and ammonia transport was investigated in the turtle bladder. At serosal pH 6.4, ammonia transport is preferentially from serosa to mucosa and is, at least in part, mediated by NH4+ transport. Since K and NH4+ share similar features such as permeability and stimulation of Na-K-ATPase, we studied the interaction of transport of these ions by the turtle bladder. Removal of K from the mucosal solution inhibited partially ammonia transport from serosa to mucosa and the inhibition was reversible by restoration of K. In contrast, removal of serosal K failed to inhibit ammonia transport. Since NH4+ can replace K in the activation of Na-K-ATPase in turtle bladder plasma membrane fraction with similar K, we examined the effect of ouabain on ammonia transport. Ouabain added to the serosal solution failed to inhibit ammonia transport thus, suggesting that the Na-K-ATPase is not required for ammonia entry into the cell. Methylammonium (a competitive inhibitor of NH4+ transport in other systems) decreased both ammonia transport and the observed increase in short circuit current elicited by NH4Cl addition to the serosal solution. This finding suggests that NH4+ and methylammonium are transported through a common pathway in the serosal side. Since the permeability of the serosal side to K and NH4+ is similar, we evaluated the effect of serosal depolarization and the effect of barium, an inhibitor of K channels, on ammonia transport. Serosal depolarization inhibited ammonia transport but barium did not affect ammonia flux.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma membrane proton ATPase from human kidney.

Distal urinary acidification is thought to be mediated by a proton ATPase (H+-ATPase). We isolated a plasma membrane fraction from human kidney cortex and medulla which contained H+-ATPase activity. In both the cortex and medulla the plasma membrane fraction was enriched in alkaline phosphatase, maltase, Na+,K+-ATPase and devoid of mitochondrial and lysosomal contamination. In the presence of oligomycin (to inhibit mitochondrial ATPase) in the presence of ouabain (to inhibit Na+,K+-ATPase) and in the absence of Ca (to inhibit Ca2+-ATPase) this plasma membrane fraction showed ATPase activity which was sensitive to dicyclohexylcarbodiimide and N-ethylmaleimide. This ATPase activity was also inhibited by vanadate, 4,4'-diisothiocyano-2,2'-disulfonic stilbene and ZnSO4. In the presence of ATP, but not GTP or UTP, the plasma membrane fraction of both cortex and medulla was capable of quenching of acridine orange fluorescence, which could be dissipated by nigericin indicating acidification of the interior of the vesicles. The acidification was not affected by presence of oligomycin or ouabain indicating that it was not due to mitochondrial ATPase or Na+,K+-ATPase, respectively. Dicyclohexylcarbodiimide and N-ethylmaleimide completely abolished the acidification by this plasma membrane fraction. In the presence of valinomycin and an outward-directed K gradient, there was increased quenching of acridine orange, indicating that the H+-ATPase is electrogenic. Acidification was not altered by replacement of Na by K, but was critically dependent on the presence of chloride. In summary, the plasma membrane fraction of the human kidney cortex and medulla contains a H+-ATPase, which is similar to the H+-ATPase described in other species, and we postulate that this H+-ATPase may be involved in urinary acidification.

Na-Ca exchange in renal tubular basolateral membranes.

We investigated the Na-dependent Ca transport in purified bovine luminal and basolateral renal tubular membranes. Na-dependent Ca uptake was observed in basolateral but not in luminal kidney tubular membranes. 45Ca uptake in basolateral membrane vesicles loaded with Na and suspended in K buffer was significantly greater than that observed in vesicles loaded with Na and suspended in Na buffer. The Na ionophore ETH-227 inhibited Na-dependent Ca uptake indicating that the Ca uptake was dependent on Na gradient. The Ca taken up in the presence of Na gradient could be released by the Ca ionophore A-23187 suggesting that Ca was accumulated inside the vesicles. In vesicles loaded with 45Ca, addition of Na to the media promoted Ca efflux. Methyl triphenylphosponium uptake and Ca uptake were significantly higher in the presence of a Na gradient as compared to those observed in the presence of other monovalent cation gradients, indicating the specificity for Na gradient and arguing against a calcium-activated sodium conductance pathway. The Na-dependent Ca uptake varied with intravesicular Na concentration with an apparent Km of 20-40 mM. The Km for Ca of the Na-dependent Ca uptake was 50 microM and the Vmax was 0.2 nmol/mg protein/min. The Na-dependent Ca uptake was inhibited by LaCl3, tetracaine and verapamil, unaffected by quinidine and amiloride, and slightly stimulated by chymotrypsin. These data demonstrate that the Na-dependent Ca transport by renal basolateral membranes is mediated by a Na-Ca exchange system and not by a calcium-activated sodium conductance pathway.

Partial purification and reconstitution of renal basolateral Na+-Ca2+ exchanger into liposomes.

The Na+-Ca2+ exchange system in renal tubular basolateral membranes was partially purified and incorporated into liposomes. Solubilization of basolateral membranes with 1% cholic acid in the presence of 2.5% soybean phospholipids and proteolytic treatment with Pronase (20 micrograms/ml) as described (Wakabayashi, S., and Goshima, K. (1982) Biochim. Biophys. Acta 693, 125-133) allowed partial purification and reconstitution of the Na+-Ca2+ exchange system into liposomes. The Na+-dependent Ca2+ uptake in the reconstituted liposomes was 25 times higher than the Na+-dependent Ca2+ uptake in the native basolateral membranes. Eadie-Hofstee analysis of the Na+-dependent Ca2+ uptake revealed a Vmax of 201 pmol of Ca2+/mg of protein/45 s and a Km for Ca2+ of 2.7 microM. The stoichiometry (n) of the Na+-Ca2+ exchange system was determined from the Na+ gradient which opposes constant membrane potential so that no net Ca2+ transport occurs. In the presence of constant negative membrane potential, the value for n was 3.09 +/- 0.22, and in the presence of constant positive membrane potential, the value for n was 2.89 +/- 0.2. Thus, the stoichiometry of the renal Na+-Ca2+ exchange system is approximately 3Na+:1Ca2+.

High-affinity calcium binding sites in luminal and basolateral renal membranes.

We evaluated Ca binding by highly purified luminal (L) and basolateral (BL) tubular membranes prepared from beef kidney. Ca binding was measured by using 45Ca and a rapid-filtration technique. After Ca uptake reached equilibrium, the vesicles were lysed and the amount of 45Ca retained in the membranes was considered the bound Ca. Ca binding in both membranes accounted for approx. 80% of total Ca uptake. Analysis of binding data by Scatchard plot revealed the presence of two distinct types of binding sites in both L and BL membranes. The high-affinity binding sites showed a similar affinity constant of 10(-5)M for both L and BL membranes, but the maximum number of binding sites was 0.75 and 1.6 nmol/mg protein, respectively. In contrast, the low-affinity binding sites were similar regarding affinity constant and maximum number of binding sites in the two membranes. In L and BL membranes, high-affinity binding sites were selective for Ca, as high concentrations of divalent cations were required to inhibit Ca binding. In both membranes Ca binding was inhibited by ruthenium red, LaCl3, and detergents, and it was stimulated by calmodulin inhibitors (trifluoperazine, calmidazolium), ionophore A-23187, and ATP. These results demonstrate that L and BL membranes possess high-affinity binding sites with different capacities but similar characteristics as regards affinity constant and stimulation and inhibition of binding. The data further demonstrate that most of Ca uptake by these membranes represents binding.

Glucagon degradation by luminal and basolateral rabbit tubular membranes.

Glucagon is avidly degraded by the kidney, but the relative contribution of the luminal and basolateral tubular membranes to this process is unknown. We studied 125I-glucagon degradation by purified luminal (L) and basolateral (BL) tubular membranes prepared from rabbit kidney cortex, which showed enrichment vs. homogenate of marker enzyme activities (Na-K-ATPase for BL and maltase for L) of 10- and 14-fold, respectively. Renal homogenates and both tubular membrane fractions degraded glucagon avidly without reaching saturation even at pharmacologic concentration (10(-5) M) of the hormone. At physiologic concentration (3 x 10(-11) M) BL membranes degraded substantial amounts of glucagon (8.1 +/- 0.9 pg . micrograms protein-1 . h-1) even though at lesser rates (P less than 0.001) than the luminal fraction (33.3 +/- 1.9 pg . micrograms protein-1 . h-1). Competition experiments suggested that glucagon-degrading activity in both fractions includes both specific and nonspecific components, and the potency of different enzyme inhibitors to decelerate glucagon degradation was strikingly similar in the two membrane preparations. Glucagon degradation differed in several important aspects from the manner in which tubular membranes catabolize insulin, including absolute degradation rates and relative degrading capacity of the membranes vs. homogenates, both being substantially higher for glucagon. These results provide direct evidence that the renal metabolism of glucagon also involves its degradation by peritubular cell membranes.