Na(+)-dependent transporters mediate HCO(3)(-) salvage across the luminal membrane of the main pancreatic duct

To study the roles of Na(+)-dependent H(+) transporters, we characterized H(+) efflux mechanisms in the pancreatic duct in wild-type, NHE2(-/-), and NHE3(-/-) mice. The pancreatic duct expresses NHE1 in the basolateral membrane, and NHE2 and NHE3 in the luminal membrane, but does not contain NHE4 or NHE5. Basolateral Na(+)-dependent H(+) efflux in the microperfused duct was inhibited by 1.5 microM of the amiloride analogue HOE 694, consistent with expression of NHE1, whereas the luminal activity required 50 microM HOE 694 for effective inhibition, suggesting that the efflux might be mediated by NHE2. However, disruption of NHE2 had no effect on luminal transport, while disruption of the NHE3 gene reduced luminal Na(+)-dependent H(+) efflux by approximately 45%. Notably, the remaining luminal Na(+)-dependent H(+) efflux in ducts from NHE3(-/-) mice was inhibited by 50 microM HOE 694. Hence, approximately 55% of luminal H(+) efflux (or HCO(3)(-) influx) in the pancreatic duct is mediated by a novel, HOE 694-sensitive, Na(+)-dependent mechanism. H(+) transport by NHE3 and the novel transporter is inhibited by cAMP, albeit to different extents. We propose that multiple Na(+)-dependent mechanisms in the luminal membrane of the pancreatic duct absorb Na(+) and HCO(3)(-) to produce a pancreatic juice that is poor in HCO(3)(-) and rich in Cl(-) during basal secretion. Inhibition of the transporters during stimulated secretion aids in producing the HCO(3)(-)-rich pancreatic juice.     

Inhibition of Rho-kinase reduces renal Na-H exchanger activity and causes natriuresis in rat

Rho-kinase regulates the actin cytoskeleton and therefore modulates transport. The role of Rho-kinase in Na-H exchanger (NHE) activity of rat proximal convoluted tubules (PCTs) was investigated using (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), a specific inhibitor of Rho-kinase. In spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats, apical and basolateral NHE activities were determined by measuring cell pH recovery following luminal NH4+ prepulse and basolateral sodium removal, respectively. Apical NHE activity was greater in 8 to 9 week old hypertensive SHR compared with WKY. Although Y-27632 suppressed pH(i) recovery in both strains, sensitivity was 50-fold higher in adult SHR. Y-27632 suppressed basolateral NHE in both strains with similar sensitivity. Apical NHE activity was not greater in 5-week-old, not yet hypertensive, SHR rats compared with WKY. In clearance studies, Na excretion was less in SHR than in WKY rats. Y-27632 increased Na excretion and fractional excretion Na in both strains but more so in SHR. (22)Na uptake of the brush border membrane vesicle taken from Y-27632-treated rats decreased more than that from vehicle-treated animals in both adult SHR and WKY. We conclude that apical NHE activity is increased in SHR PCT compared with controls and that inhibition of Rho-kinase reduces PCT NHE activities and causes natriuresis.     

Basolateral Na+/HCO3– cotransport activity is regulated by the dissociable Na+/H+ exchanger regulatory factor

In the renal proximal tubule, the activities of the basolateral Na(+)/HCO(3)(-) cotransporter (NBC) and the apical Na(+)/H(+) exchanger (NHE3) uniformly vary in parallel, suggesting that they are coordinately regulated. PKA-mediated inhibition of NHE3 is mediated by a PDZ motif-containing protein, the Na(+)/H(+) exchanger regulatory factor (NHE-RF). Given the common inhibition of these transporters after protein kinase A (PKA) activation, we sought to determine whether NHE-RF also plays a role in PKA-regulated NBC activity. Renal cortex immunoblot analysis using anti-peptide antibodies directed against rabbit NHE-RF demonstrated the presence of this regulatory factor in both brush-border membranes (BBMs) and basolateral membranes (BLMs). Using a reconstitution assay, we found that limited trypsin digestion of detergent solubilized rabbit renal BLM preparations resulted in NBC activity that was unaffected by PKA activation. Co-reconstitution of these trypsinized preparations with a recombinant protein corresponding to wild-type rabbit NHE-RF restored the inhibitory effect of PKA on NBC activity in a concentration-dependent manner. NBC activity was inhibited 60% by 10(-8)M NHE-RF; this effect was not observed in the absence of PKA. Reconstitution with heat-denatured NHE-RF also failed to attenuate NBC activity. To establish further a physiologic role for NHE-RF in NBC regulation, the renal epithelial cell line B-SC-1, which lacks detectable endogenous NHE-RF expression, was engineered to express stably an NHE-RF transgene. NHE-RF-expressing B-SC-1 cells (B-SC-RF) exhibited markedly lower basal levels of NBC activity than did wild-type controls. Inhibition of NBC activity in B-SC-RF cells was enhanced after 10 microM of forskolin treatment, consistent with a postulated role for NHE-RF in mediating the inhibition of NBC activity by PKA. These findings not only suggest NHE-RF involvement in PKA-regulated NBC activity, but also provide a unique molecular mechanism whereby basolateral NBC and apical NHE3 activities may be coordinately regulated in renal proximal tubule cells.     

Chronic hyperosmolality increases NHE3 activity in OKP cells.

This study investigated the effect of chronic hypertonicity on the OKP cell Na/H antiporter, encoded by Na/H exchanger 3 (NHE3). Chronic (48 h) increases in extracellular glucose, mannitol, or raffinose concentration caused a significant increase in Na/H antiporter activity, while increases in urea concentration were without effect. This effect was seen with changes in osmolality of only 20 mOsm/liter, a magnitude that is observed clinically in poorly controlled diabetes mellitus. Increases in mannitol concentration acutely inhibited and chronically stimulated Na/H antiporter activity. The increase in Na/H antiporter activity induced by hypertonic incubation was resistant to 10(-7) and 5 x 10(-6) M but inhibited by 10(-4) M ethylisopropyl amiloride, consistent with regulation of NHE3. In addition, hypertonicity increased total cellular and plasma membrane NHE3 protein abundance twofold, with only a small increase in NHE3 mRNA abundance. We conclude that chronic pathophysiologically relevant increases in tonicity lead to increases in NHE3 protein abundance and activity. This may be responsible for increased proximal tubule apical membrane Na/H antiporter activity in poorly controlled diabetes mellitus, which could then contribute to hypertension, glomerular hyperfiltration and diabetic nephropathy.     

Contributions of cellular leak pathways to net NaHCO3 and NaCl absorption.

Proton and formic acid permeabilities were measured in the in vivo microperfused rat proximal convoluted tubule by examining the effect on intracellular pH when [H] and/or [formic acid] were rapidly changed in the luminal or peritubular fluids. Apical and basolateral membrane H permeabilities were 0.52 +/- 0.07 and 0.67 +/- 0.18 cm/s, respectively. Using these permeabilities we calculate that proton backleak from the luminal fluid to cell does not contribute significantly to net proton secretion in the early proximal tubule, but may contribute in the late proximal tubule. Apical and basolateral membrane formic acid permeabilities measured at extracellular pH 6.62 were 4.6 +/- 0.5 X 10(-2) and 6.8 +/- 1.5 X 10(-2) cm/s, respectively. Control studies demonstrated that the formic acid permeabilities were not underestimated by either the simultaneous movement of formate into the cell or the efflux of formic acid across the opposite membrane. The measured apical membrane formic acid permeability is too small to support all of transcellular NaCl absorption in the rat by a mechanism that involves Na/H-Cl/formate transporters operating in parallel with formic acid nonionic diffusion.     

Endothelin-1/endothelin-B receptor–mediated increases in NHE3 activity in chronic metabolic acidosis

Decreases in blood pH activate NHE3, the proximal tubular apical membrane Na/H antiporter. In cultured renal epithelial cells, activation of the endothelin-B (ET(B)) receptor increases NHE3 activity. To examine the role of the ET(B) receptor in the response to acidosis in vivo, the present studies examined ET(B) receptor-deficient mice, rescued from neonatal lethality by expression of a dopamine beta-hydroxylase promoter/ET(B) receptor transgene (Tg/Tg:ET(B)(-/-) mice). In proximal tubule suspensions from Tg/Tg:ET(B)(+/-) mice, 10(-8) M endothelin-1 (ET-1) increased NHE3 activity, but this treatment had no effect on tubules from Tg/Tg:ET(B)(-/-) mice. Acid ingestion for 7 days caused a greater decrease in blood HCO(3)(-) concentration in Tg/Tg:ET(B)(-/-) mice compared with Tg/Tg:ET(B)(+/+) and Tg/Tg:ET(B)(+/-) mice. Whereas acid ingestion increased apical membrane NHE3 by 42-46% in Tg/Tg:ET(B)(+/+) and Tg/Tg:ET(B)(+/-) mice, it had no effect on NHE3 in Tg/Tg:ET(B)(-/-) mice. In C57BL/6 mice, excess acid ingestion increased renal cortical preproET-1 mRNA expression 2.4-fold and decreased preproET-3 mRNA expression by 37%. On a control diet, Tg/Tg:ET(B)(-/-) mice had low rates of ammonium excretion, which could not be attributed to an inability to acidify the urine, as well as hypercitraturia, with increased titratable acid excretion. Acid ingestion increased ammonium excretion, citrate absorption, and titratable acid excretion to the same levels in Tg/Tg:ET(B)(-/-) and Tg/Tg:ET(B)(+/+) mice. In conclusion, metabolic acidosis increases ET-1 expression, which increases NHE3 activity via the ET(B) receptor.     

Hyposmolality stimulates apical membrane Na(+)/H(+) exchange and HCO(3)(-) absorption in renal thick ascending limb

The regulation of epithelial Na(+)/H(+) exchangers (NHEs) by hyposmolality is poorly understood. In the renal medullary thick ascending limb (MTAL), transepithelial bicarbonate (HCO(3)(-)) absorption is mediated by apical membrane Na(+)/H(+) exchange, attributable to NHE3. In the present study we examined the effects of hyposmolality on apical Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL of the rat. In MTAL perfused in vitro with 25 mM HCO(3)(-) solutions, decreasing osmolality in the lumen and bath by removal of either mannitol or sodium chloride significantly increased HCO(3)(-) absorption. The responses to lumen addition of the inhibitors ethylisopropyl amiloride, amiloride, or HOE 694 are consistent with hyposmotic stimulation of apical NHE3 activity and provide no evidence for a role for apical NHE2 in HCO(3)(-) absorption. Hyposmolality increased apical Na(+)/H(+) exchange activity over the pH(i) range 6.5-7.5 due to an increase in V(max). Pretreatment with either tyrosine kinase inhibitors or with the tyrosine phosphatase inhibitor molybdate completely blocked stimulation of HCO(3)(-) absorption by hyposmolality. These results demonstrate that hyposmolality increases HCO(3)(-) absorption in the MTAL through a novel stimulation of apical membrane Na(+)/H(+) exchange and provide the first evidence that NHE3 is regulated by hyposmotic stress. Stimulation of apical Na(+)/H(+) exchange activity in renal cells by a decrease in osmolality may contribute to such pathophysiological processes as urine acidification by diuretics, diuretic resistance, and renal sodium retention in edematous states.     

Role of the Na+/H+ antiporter in rat proximal tubule bicarbonate absorption.

Amiloride and the more potent amiloride analog, 5-(N-t-butyl) amiloride (t-butylamiloride), were used to examine the role of the Na+/H+ antiporter in bicarbonate absorption in the in vivo microperfused rat proximal convoluted tubule. Bicarbonate absorption was inhibited 29, 46, and 47% by 0.9 mM or 4.3 mM amiloride, or 1 mM t-butylamiloride, respectively. Sensitivity of the Na+/H+ antiporter to these compounds in vivo was examined using fluorescent measurements of intracellular pH with (2', 7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein (BCECF). Amiloride and t-butylamiloride were shown to be as potent against the antiporter in vivo as in brush border membrane vesicles. A model of proximal tubule bicarbonate absorption was used to correct for changes in the luminal profiles for pH and inhibitor concentration, and for changes in luminal flow rate in the various series. We conclude that the majority of apical membrane proton secretion involved in transepithelial bicarbonate absorption is mediated by the Na+-dependent, amiloride-sensitive Na+H+ antiporter. However, a second mechanism of proton secretion contributes significantly to bicarbonate absorption. This mechanism is Na+-independent and amiloride-insensitive.     

Intracellular pH regulation of CA1 neurons in Na+/H+ isoform 1 mutant mice

To understand the role of Na(+)/H(+) exchanger 1 (NHE1) in intracellular pH (pH(i)) regulation and neuronal function, we took advantage of natural knockout mice lacking NHE1, the most ubiquitously and densely expressed NHE isoform in the central nervous system (CNS). CA1 neurons from both wild-type (WT) and NHE1 mutant mice were studied by continuous monitoring of pH(i), using the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. In the nominal absence of CO(2)/HCO(3)(-), steady-state pH(i) was higher in WT neurons than in mutant neurons. Using the NH(4)Cl prepulse technique, we also show that H(+) flux in WT neurons was much greater than in mutant neurons. The recovery from acid load was blocked in WT neurons, but not in mutant neurons, by removal of Na(+) from the extracellular solution or by using 100 microM 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate (HOE 694) in HEPES buffer. Surprisingly, in the presence of CO(2)/HCO(3)(-), the difference in H(+) flux between WT and mutant mice was even more exaggerated, with a difference of more than 250 microM/s between them at pH 6.6. H(+) flux in CO(2)/HCO(3)(-) was responsive to diisothiocyanato-stilbene-2, 2'-disulfonate (DIDS) in the WT but not in the mutant. We conclude that (a) the absence of NHE1 in the mutant neurons tended to cause lower steady-state pH(i) and, perhaps more importantly, markedly reduced the rate of recovery from an acid load; and (b) this difference in the rate of recovery between mutant and WT neurons was surprisingly larger in the presence, rather than in the absence, of HCO(3)(-), indicating that the presence of NHE1 is essential for the regulation and/or functional expression of both HCO(3)(-)-dependent and -independent transporters in neurons.     

Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule.

The apical transport processes responsible for proton secretion were studied in the isolated perfused rabbit S3 proximal tubule. Intracellular pH (pHi) was measured with the pH dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady state pHi in S3 tubules in nominally HCO3(-)-free solutions was 7.08 +/- 0.03. Removal of Na+ (lumen) caused a decrease in pHi of 0.34 +/- 0.06 pH/min. The decrease in pHi was inhibited 62% by 1 mM amiloride (lumen) and was unaffected by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (lumen) and Cl- removal (lumen, bath). After a brief exposure to 20 mM NH4Cl, pHi fell by approximately 0.7 and recovered at a rate of 0.89 +/- 0.15 pH/min in the nominal absence of Na+, HCO3-, organic anions, and SO4(2-) (lumen, bath). 1 mM N,N'-dicyclohexylcarbodiimide (lumen), 1 mM N-ethylmaleimide (lumen), 0.5 mM colchicine (bath), and 0.5 mM iodoacetic acid (lumen, bath) inhibited the Na+-independent pHi recovery rate by 73%, 55%, 77%, and 86%, respectively, whereas 1 mM KCN (lumen, bath) did not inhibit pHi recovery. Reduction of intracellular, but not extracellular chloride, also decreased the Na+-independent pHi recovery rate. In conclusion, the S3 proximal tubule has an apical Na+/H+ antiporter with a Michaelis constant for Na+ of 29 mM and a maximum velocity of 0.47 pH/min. S3 tubules also possess a plasma membrane H+-ATPase that can regulate pHi, has a requirement for intracellular chloride, and utilizes ATP derived primarily from glycolysis.     

Mechanism of apical and basolateral Na(+)-independent Cl-/base exchange in the rabbit superficial proximal straight tubule.

The present study was undertaken to determine the magnitude and mechanism of base transport via the apical and basolateral Na(+)-independent Cl-/base exchangers in rabbit isolated perfused superficial S2 proximal tubules. The results demonstrate that there is an apical Na(+)-independent Cl-/base exchanger on both membranes. HCO3- fails to stimulate apical Cl-/base exchange in contrast to the basolateral exchanger. Inhibition of endogenous HCO3- production does not alter the rate of apical Cl-/base exchange in Hepes-buffered solutions. Both exchangers are inhibited by H2DIDS and furosemide; however, the basolateral anion exchanger is more sensitive to these inhibitors. The results indicate that the apical and basolateral Cl-/base exchangers differ in their transport properties and are able to transport base equivalents in the absence of formate. The formate concentration in rabbit arterial serum is approximately 6 microM and in vitro tubule formate production is < 0.6 pmol/min per mm. Formate in the micromolar range stimulates Jv in a dose-dependent manner in the absence of a transepithelial Na+ and Cl- gradient and without a measurable effect on Cl(-)-induced equivalent base flux. Apical formic acid recycling cannot be an important component of any cell model, which accounts for formic acid stimulation of transcellular NaCl transport in the rabbit superficial S2 proximal tubule. We propose that transcellular NaCl transport in this nephron segment is mediated by an apical Na+/H+ exchanger in parallel with a Cl-/OH- exchanger and that the secreted H+ and OH- ions form H2O in the tubule lumen.     

H+-ATPase activity in selective disruption of H+-K+-ATPase alpha 1 gene of mice under normal and K-depleted conditions

The outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis by two luminal proton ATPases, H(+)-ATPase and H(+)-K(+)-ATPase (HKA), both of which are in the intercalated cells (ICs) of OMCD. We showed previously that HKAalpha1 (gastric H(+)-K(+)-ATPase) activity is the essential H(+)-K(+)-ATPase activity under normal conditions, and that HKAalpha2 (colonic H(+)-K(+)-ATPase) is induced and mediates increased proton-secretion under K-depleted conditions. To better understand the role of H(+)-ATPase (potassium-independent) in acid secretion and the relationship between H(+)-ATPase and a specific HKA isoform, we examined H(+)-ATPase activity in the H(+)-K(+)-ATPasealpha1 knockout (KO) mice under normal and K-depleted conditions. Mice were fed a potassium-free diet and studied after 7 days. Segments of the OMCD were perfused in vitro, and intracellular pH (pH(i)) was measured by ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. The isolated OMCD tubules obtained from mice fed a potassium-free diet were examined by fluorescent immunocytochemistry with an antibody to the 31-kDa subunit of H(+)-ATPase (E-11) and were compared with those obtained from a normal diet. In the absence of Na(+) and K(+), the H(+)-ATPase-mediate pH(i) recovery rates were 6.7 +/- 1.1 x 10(-4) units/s (n = 7 ICs) in wild-type (WT) mice and increased to 8.7 +/- 1.8 x 10(-4) (P < 0.05; n = 6) in HKAalpha1 KO mice. K-independent proton transport activity was significantly inhibited by the H(+)-ATPase inhibitor bafilomycin A(1) (BAF, 10 nM) with luminal applied in both WT and KO mice. Comparison of the results indicated upregulation of BAF-sensitive H(+)-ATPase activity in KO mice. To determine the intracellular localization of H(+)-ATPase in the intercalated cells of OMCD, we dissected the OMCD and performed fluorescent immunocytochemistry with the H(+)-ATPase antibody in the WT and KO mice. In the WT mice, on normal diet, H(+)-ATPase staining distributed diffusely throughout the intercalated cells and was slightly polarized to the apical plasma membrane in the KO mice, consistent with increase in the H(+)-ATPase-mediate pH(i) recovery in the KO mice. One week of a potassium-free diet resulted in a significant increase in the degree of H(+)-ATPase polarization at the apical plasma membrane in both WT and KO mice. Hypokalemia stimulates H(+)-ATPase in the intercalated cells of OMCD of both WT and KO mice. The enhanced activity of H(+)-ATPase plays an important role in compensatory proton secretion in the HKAalpha1 KO mice under normal conditions.     

Regulation and intracellular localization of the epithelial isoforms of the Na+/H+ exchangers NHE2 and NHE3

The Na+/H+ exchangers (NHE) are a ubiquitous family of membrane proteins that catalyze the counter-transport of extracellular Na+ for intracellular H+ and are important for intracellular pH and cell volume regulation. The major epithelial isoforms, NHE2 and NHE3, are thought to have more specialized roles in regulating Na+ and water absorption and are differentially expressed in epithelial tissues. NHE2 and NHE3 not only differ with respect to their response to various endogenous and exogenous factors but exhibit different intracellular localization as well. NHE2 is primarily located at the plasma membrane, whereas NHE3 is mostly sequestered in an intracellular compartment corresponding to the recycling endosome. Furthermore, NHE3 is localized to the apical pole, whereas polar localization of NHE2 has been controversial. The author has recently localized NHE2 to the apical membrane of a renal epithelial cell line and identified a 45-residue-long region of the cytosolic domain (corresponding to residues 731-777 of the rat NHE2) to be critical for apical targeting. Although SH3 domains of various proteins were found to bind to this and a more carboxy-terminal proline-rich region in vitro, the functional significance of these interactions appears inconsequential. Deletion of both proline-rich regions did not affect Na+/H+ exchange nor its response to hypertonicity and metabolic depletion. However, loss of residues 731-777, which bound specifically in vitro to the SH3 domain of the cytoskeletal protein, alpha-spectrin, mistargets NHE2 to the basolateral surface.     

Handling of digoxin and ouabain by renal tubular cells (LLC-PK1).

Digoxin is known to be secreted by renal tubular cells, but the mechanisms are still not fully understood. In this study, we examined renal tubular cell handling of digoxin and ouabain using LLC-PK1 cells, a model of proximal renal tubular cells. The cells were used in suspension for binding experiments and in monolayers on permeable filters for transport studies. The specific binding of digoxin to the cells, presumably to the ouabain binding site (i.e., membrane Na+,K(+)-ATPase), were characterized by Kd of 2.6 x 10(-7) M and Bmax (total number of specific binding sites) of 1.6 x 10(6)/cell. Kd and Bmax of ouabain binding were 1.3 x 10(-7) M and 1.9 x 10(6)/cell, respectively. In transport experiments, digoxin showed significantly higher flux than ouabain from the basolateral to the apical side across the cell monolayers. Importantly, this secretory transport was not inhibited by ouabain concentrations sufficient to block membrane Na+,K(+)-ATPase and to displace digoxin from the binding site on the enzyme (i.e., 10(-6) to 10(-4) M ouabain). However, the digoxin secretion was decreased by low temperature or excess digoxin in a concentration-dependent manner. These data suggest that digoxin undergoes unidirectional transport in favor of secretion, which does not involve its binding to the ouabain binding sites on membrane Na+,K(+)-ATPase.     

Indomethacin and sodium retention in the rat: role of inhibition of prostaglandin E2 synthesis.

1. To further explore the Na(+)-retaining effect of indomethacin along the whole length of the nephron, the Na(+)-K(+)-ATPase activity of isolated tubules from indomethacin-pretreated rats was compared with that of tubules isolated from intact rats and exposed directly to prostaglandin E2. 2. Indomethacin increased Na(+)-K(+)-ATPase activity in the proximal convoluted tubule (+24%, P < 0.001 versus control), proximal straight tubule (+75%, P < 0.001 versus control), medullary thick ascending limb (+68%, P < 0.001 versus control), cortical thick ascending limb (+7%, not significant) and cortical collecting duct (+18%, P < 0.025 versus control). In contrast, in the distal convoluted tubule indomethacin decreased Na(+)-K(+)-ATPase activity by -42% (P < 0.001 versus control). 3. Indomethacin also strongly increased Na(+)-K(+)-ATPase activity in the cortical collecting duct of adrenalectomized rats. 4. In isolated tubules from control rats, prostaglandin E2 reduced Na(+)-K(+)-ATPase activity in the proximal convoluted tubule (-33%, P < 0.05), proximal straight tubule (-60%, P < 0.001), medullary thick ascending limb (-43%, P < 0.001), cortical thick ascending limb (-25%, P < 0.001) and cortical collecting duct (-45%, P < 0.001) and in the distal convoluted tubule, prostaglandin E2 increased Na(+)-K(+)-ATPase activity (+32%, P < 0.05). 5. That these changes in Na(+)-K(+)-ATPase activity in indomethacin-pretreated rats and prostaglandin E2-treated controls are similar in magnitude but occur in opposite directions suggests that the response to indomethacin is mediated by inhibition of prostaglandin E2 synthesis in the nephron. In the cortical collecting duct the effect of indomethacin is aldosterone-independent.     

Dissociation and redistribution of Na+,K(+)-ATPase from its surface membrane actin cytoskeletal complex during cellular ATP depletion.

Establishment and maintenance of a polar distribution of Na+,K(+)-ATPase is essential for efficient Na+ reabsorption by proximal tubule cells and is dependent upon the formation of a metabolically stable, detergent-insoluble complex of Na+,K(+)-ATPase with the actin membrane cytoskeleton. The present studies show that cellular ATP depletion results in a rapid duration-dependent dissociation of Na+,K(+)-ATPase from the actin cytoskeleton and redistribution of Na+,K(+)-ATPase to the apical membrane. During ATP depletion, total cellular Na+,K(+)-ATPase activity was unaltered, but the Triton-X-100-insoluble fraction (cytoskeleton associated) of Na+,K(+)-ATPase activity decreased (P less than 0.01), with a corresponding increase in the detergent-soluble fraction of Na+,K(+)-ATPase (P less than 0.01). Indirect immunofluorescent studies of cells with depleted ATP revealed a redistribution of Na+,K(+)-ATPase from the basolateral membrane into the apical membrane and throughout the cytoplasm. ATP depletion also resulted in the redistribution of F-actin from a primarily cortical concentration to a perinuclear location. There was also a rapid, duration-dependent conversion of monomeric G-actin to F-actin starting during the first 5 min of ATP depletion. Taken together, these data suggest that ATP depletion causes profound alterations in cell polarity by inducing major changes in the actin cytoskeletal architecture.     

Impairment of sodium pump and Na/H exchanger in erythrocytes from non-insulin dependent diabetes mellitus patients: effect of tea catechins

BACKGROUND: Tea catechins (EGCG, EGC, ECG and EC) possess many important biological properties. We evaluated the effect of tea catechins on erythrocyte membrane Na(+)/K(+)-ATPase and sodium/hydrogen exchanger (NHE) activity in normal (control) and NIDDM subjects. METHODS: Erythrocyte membrane Na(+)/K(+)-ATPase and NHE activity were determined in normal and non-insulin dependent diabetes mellitus (NIDDM) patients. In vitro effect of tea catechins was studied by incubating membrane/intact erythrocytes in assay medium prior to Na(+)/K(+)-ATPase/NHE activity determination. RESULTS: A 24.2% decrease in the activity of Na(+)/K(+)-ATPase (p<0.001) and 39.37% increase in activity of NHE (p<0.02) were observed in NIDDM subjects compared to normal. Tea catechins inhibited the activity of Na(+)/K(+)-ATPase and NHE in both normal and NIDDM erythrocytes, the effect was concentration-dependent. The inhibitory effect of EGCG and ECG at micromolar concentrations was greater compared to EGC and EC on Na(+)/K(+)-ATPase. On NHE the inhibition of tea catechins was in the order: EC>EGC>ECG>EGCG at concentrations up to 10 micromol/l. CONCLUSIONS: This data may help to explain the anti-carcinogenic and cardioprotective effects of tea catechins. The effect of tea catechins on Na(+)/K(+)-ATPase and NHE may be explained due to a direct effect of these compounds on plasma membrane leading to a change in membrane fluidity.     

Human renal organic anion transporters: characteristics and contributions to drug and drug metabolite excretion

The kidney is a key organ for promoting the excretion of drugs and drug metabolites. One of the mechanisms by which the kidney promotes excretion is via active secretion. Secretion of drugs and their metabolites from blood to luminal fluid in the nephron is a protein-mediated process that normally involves either the direct or indirect expenditure of energy. Renal transporters for organic anions are located in the proximal tubule segment of the nephron. The primary transporters of organic anions on the basolateral membrane (BLM) of proximal tubule cells are members of the organic anion transporter (OAT) family (mainly OAT1 and OAT3). The sulfate-anion antiporter 1 (SAT-1; hsat-1) may also contribute to organic anion transport at the basolateral membrane. On the apical membrane, the multi-drug resistance-associated protein 2 (MRP2) is an important transport protein to complete the secretion process. However, there are several transport proteins on the basolateral and apical membranes of proximal tubule cells in human kidneys that have not been fully characterized and whose role in the secretion of organic anions remains to be determined. This review will primarily focus on the human renal basolateral and apical membrane transporters for organic anions that may play a role in the excretion of drugs and drug metabolites.