Na/H exchange and H-K ATPase increase distal tubule acidification in chronic alkalosis

We examined whether H(+)-ATPase, H(+)-K(+)-ATPase, and or Na+/H+ exchange mediates increased distal tubule acidification in animals with chronic metabolic alkalosis using pharmacological inhibitors of these H+ transporters in in vivo-perfused tubules of anesthetized rats. Chronic metabolic alkalosis was induced with furosemide followed by minimum electrolyte diet and HCO3 drinking water. The reduction in net HCO3 reabsorption was greater in distal tubules of alkalotic compared to control animals perfused with Schering 28080 to inhibit H(+)-K(+)-ATPase (-6.4 +/- 0.9 vs. -1.4 +/- 0.5 pmol/mm.min-1, P < 0.02) and with EIPA to inhibit Na+/H+ exchange (-11.1 +/- 1.7 vs. -6.6 +/- 0.9 pmol/mm.min-1, P < 0.01) but was similar in distal tubules of alkalotic and control animals perfused with bafilomycin to inhibit H(+)-ATPase. The greater reduction of distal tubule net HCO3 reabsorption in alkalotic compared to control animals induced by EIPA was eliminated by systemic infusion of the endothelin receptor antagonist bosentan (-4.6 +/- 0.7 vs. -4.4 +/- 0.7 pmol/mm.min-1, P = NS) but the greater reduction induced by Schering 28080 persisted. Urine endothelin-1 (ET-1) excretion was higher in animals with maintained alkalosis (164.5 +/- 23.7 vs. 76.6 +/- 10.8 fmol/day, P < 0.03), but decreased following KCl repletion to a value (86.7 +/- 10.0 fmol/day, P < 0.02 vs. respective before-KCl value) that was not different from that for KCl-repleted control animals (79.9 +/- 8.7 fmol/day, P = NS vs. KCl-repleted alkalotic animals). The data support that augmented distal tubule acidification in alkalotic animals is due to increased H(+)-K(+)-ATPase and Na+/H+ exchange activity, the latter stimulated by endogenous endothelins.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

Relationship between fluid transport and in situ inhibition of Na(+)-K+ adenosine triphosphatase in corneal endothelium

PURPOSE: To examine the relationship between the activity of the sodium pump of the corneal endothelium and corneal thickness. It was postulated that because inhibition pressure of the stroma decreases as thickness increases, a partially inhibited sodium pump would result in a new steady-state thickness of the cornea when reduced rates of fluid influx and efflux were equal. Measurements of physiologic behavior and biochemical activity were to be made in the same tissue and thus establish the relationship directly. METHODS: Rabbit corneas were superfused with a bicarbonate Ringer solution containing different concentrations of ouabain. Exposure to ouabain was either continuous for 4 hours or for an initial 10 minutes followed by ouabain-free superfusion. Thickness was measured, and, after superfusion, endothelium was removed from the corneas, sonicated, and assayed for Na(+)-K+ adenosine triphosphatase (ATPase) activity without further addition of ouabain to the assay medium. Thickness was also measured during superfusion with suboptimal concentrations of Na+ or HCO3- and with brefeldin A, an inhibitor of protein trafficking. RESULTS: Continuous exposure to ouabain caused corneas to swell, but no new steady-state thickness was reached. At low concentrations, swelling rates increased with time, as did the extent of inhibition of the Na(+)-K+ ATPase. With only a 10-minute exposure to ouabain, swelling rates with 10(-4) M to 10(-5) M decreased with the duration of ouabain-free superfusion. Similar swelling curves were obtained by reductions in Na+ or HCO3- concentrations in the superfusion medium, indicating that partial inhibition of the endothelial fluid transport processes, whether via the Na(+)-K+ ATPase or by suboptimal ionic conditions, led toward a new equilibrium thickness of the cornea. However, when superfusion was continued for more than 4 hours, the corneas exposed for 10 minutes to 3 x 10(-5) M or lower-concentration ouabain showed increasing Na(+)-K+ ATPase activity and began to thin, indicating a recovery of fluid transport capability. This recovery was blocked by addition of brefeldin A during the ouabain-free superfusion. CONCLUSIONS: Inhibition of Na(+)-K+ ATPase by low concentrations of ouabain increases with time. Temporary exposure to ouabain causes swelling at rates that decline with time as ouabain dissociates from enzyme sites. This dissociation, together with the turnover of Na(+)-K+ ATPase in the plasma membrane, can lead to recovery of normal thickness in ouabain-exposed corneas. Twenty percent of Na(+)-K+ ATPase in the endothelium is estimated to be intracellular, and about 20% of the activity can be inhibited without inducing swelling.     

Increased reabsorptive capacity after ureteral obstruction reduces the ability of glucose to inhibit phosphate reabsorption in rat kidney

BACKGROUND: Proximal tubular reabsorption of glucose (G), phosphate (Pi) and amino acids is energized by the transmembrane Na+ gradient, which explains why decreased concentration of one solute can enhance the transport of another. Accordingly, we postulated that the consistent increase in Pi reabsorption seen in the post-obstructed kidney (POK) could be caused, in part, by the low filtered load of glucose and reversed by glucose loading. METHODS: Renal function was examined before and after i.v. glucose loading in POKs (after release of 24 h of unilateral ureteral obstruction) and control kidneys (CK) of 10 adult rats. Brush-border membrane vesicle (BBMV) transports of Pi and glucose were assessed in POKs and CKs. RESULTS: In POKs GFR, urine flow and Na+ excretion were significantly reduced and tubular reabsorption of both Pi (T(P)/GFR) and glucose (TG/GFR) were significantly increased: T(P)/GFR, 2.0 +/- 0.2 vs 1.36 +/- 0.1; TmG/GFR, 23.4 +/- 1.7 vs 18.9 +/- 1.1 mmol/l. Glucose loading inhibited T(P)/GFR only in the CK. Initial Na+ gradient-dependent uptakes of D-glucose and Pi were similar in BBMVs from POK and CK. CONCLUSIONS: The increases in T(P)/GFR and TG/GFR seen in the POK do not result from decreased glucose delivery or from alterations in BBM Pi and glucose transporters. The reduced ability of glucose to inhibit Pi reabsorption in the POK results primarily from a generalized increase in proximal tubular reabsorption of Na+ and cotransported Pi and glucose. A specific rise in distal Pi transport capacity may be an additional adaptive response to the low filtered load of Pi in the POK. In addition, absent distal glucose reabsorption may further facilitate Pi reclamation at these sites.     

Na(+)-dependent and -independent Cl-/HCO-3 exchange mediate cellular HCO3- transport in cultured human intrahepatic bile duct cells

Biliary epithelial cells (cholangiocytes) modulate bile fluidity and alkalinity absorbing and/or secreting fluid and electrolytes, particularly HCO3- and Cl-. Mechanisms responsible for transepithelial H+/HCO3- secretion in human cholangiocytes are largely unknown. Human cholangiocytes isolated by enzymatic digestion and immunomagnetic purification from normal liver tissue obtained from reduced grafts used for pediatric liver transplantation were cultured in the presence of human hepatocyte growth factor. Maintenance of cholangiocyte phenotypic features was assessed using markers such as cytokeratin 19, gamma-glutamyltranspeptidase, vimentin, factor VIII-related antigen, desmin, epithelial membrane antigen (EMA), and human epithelial antigen (HEA) 125. Intracellular pH (pHi) transients were measured microfluorimetrically 2'7'-Bis(2-carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester (BCECF). In the absence of HCO3-, pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na(+)-dependent and amiloride-inhibitable. No Na(+)-independent recovery was recorded even after stimulation with agents raising intracellular cyclic adenosine monophosphate (cAMP) concentrations. In the presence of HCO3-, recovery from an intracellular acid load required Na+, but was only partly inhibited by amiloride. In these conditions H+ extrusion was inhibited by 4,4-diisothiocyan atostilben-2,2-disulfonic acid (DIDS) and by intracellular Cl- depletion. Acute removal of extracellular Cl induced a pHi alkalinization that was inhibited by DIDS. pHi recovery from an intracellular alkaline load (isohydric CO2 changes) was Cl(-)-dependent and DIDS-inhibitable. Administration of agents raising intracellular cAMP concentrations increased both Na(+)-dependent and Na(+)-independent Cl-/HCO-3 exchange activity. Stimulation of Cl-/HCO3- exchange activity was not prevented by the Cl- channel inhibitor 5'-nitro-2(2)-phenylpropyl-amino-benzoate(NPPB). In conclusion, human cholangiocytes possess two acid extruders (Na+/H+exchanger and Na(+)-dependent Cl-/HCO3- exchange) and an acid loader (Cl-/HCO3- exchange), whereas no evidence was found for cAMP activated H(+)-ATPase. Bicarbonate influx is thus mainly mediated by Na-dependent Cl-/HCO3- exchange, whereas Na+:HCO-3 cotransport is not active in the physiological range of pHi. Stimulation of Na(+)-independent Cl-/HCO3- exchanger by cAMP does not require activation of Cl- conductances. These mechanisms may underlay hormone-regulated biliary HCO3- secretion in the human biliary tree.     

Ion exchangers mediating NaCl transport in the proximal tubule

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. We identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanism's of uphill Cl- entry across the apical membrane of proximal tubule cells. For steady state Cl- absorption to occur by these mechanisms, formate and oxalate must recycle from lumen to cell. Recycling of formate from lumen to cell may occur by H(+)-coupled formate transport and nonionic diffusion of formic acid in parallel with Na(+)-H+ exchange. Oxalate recycling from lumen to cell may take place by oxalate-sulfate exchange in parallel with Na(+)-sulfate cotransport. Cl- exit across the basolateral membrane is most likely mediated by Cl- channels. To identify the Na(+)-H+ exchanger (NHE) isoform(s) expressed on the brush border membrane of proximal tubule cells, we developed isoform-specific monoclonal and polyclonal antibodies. We found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the thin and thick limbs of the loop of Henle. NHE3 is also present in a population of subapical intracellular vesicles, suggesting possible regulation by membrane trafficking. The inhibitor sensitivity of Na(+)-H+ exchange in renal brush border vesicles indicates that it is mediated by NHE3 under baseline conditions and during up-regulation by metabolic acidosis. Increased apical membrane Na(+)-H+ exchange activity in response to metabolic acidosis and during renal maturation is associated with increased NHE3 protein expression. Finally, we found that the organic anion-dependent absorption of Cl- is markedly down-regulated in metabolic acidosis in parallel with the up-regulation of brush border membrane Na(+)-H+ exchange. Thus, differential regulation of apical membrane ion exchangers may provide a mechanism to regulate the relative rates of NaHCO3 and NaCl reabsorption.     

Biomarkers for malignancy in the columnar-lined esophagus

Toads of the genus Bufo are highly resistant to the toxic effects of digitalis glycosides, and the Na+,K(+)-ATPase of all toad tissues studied to date has been relatively insensitive to inhibition by digitalis and related compounds. In studies of brain microsomal preparations from two toad species, Bufo marinus and Bufo viridis, inhibition of ATPase activity and displacement of [3H]ouabain from Na+,K(+)-ATPase occurred over broad ranges of ouabain or bufalin concentrations, consistent with the possibility that more than one Na+,K(+)-ATPase isoform may be present in toad brain. The data could be fitted to one- or two-site models, both of which were consistent with the presence of Na+,K(+)-ATPase activity with high sensitivity to ouabain and bufalin. Ki (concentration capable of producing 50% inhibition of activity) values for ouabain in the one-site model were in the 0.2 to 3.7 microM range, whereas Ki1 values in the two-site model ranged from 0.085 to 0.85 microM, indicating that brain ATPase was at least three orders of magnitude more sensitive to ouabain than B. marinus bladder ATPase (Ki = 5940 microM). Ouabain was also an effective inhibitor of 86Rb+ uptake in B. marinus brain tissue slices (Ki = 3.1 microM in the one-site model; Ki1 = 0.03 microM in the two-site model). However, the relative contribution of the high ouabain-sensitivity site to the total activity was 17% in the transport assay as compared with 63% in the Na+,K(+)-ATPase enzymatic assay. We conclude that a highly ouabain-sensitive Na+,K(+)-ATPase activity is present and functional in toad brain but that its function may be partially inhibited in vivo.     

Effect of methyl isocyanate on rabbit cardiac Na+, K(+)-ATPase

The present study describes the effect of methyl isocyanate (MIC) on rabbit cardiac microsomal Na+, K(+)-ATPase. Addition of MIC in vitro resulted in dose-dependent inhibition of Na+, K(+)-ATPase, Mg(2+)-ATPase and K(+)-activated p-nitrophenyl phosphatase (K(+)-PNPPase). Activation of Na+, K(+)-ATPase by ATP in the presence of MIC showed a decrease in Vmax with no change in Km. Similarly, activation of K+ PNPPase by PNPP in the presence of MIC showed a decrease in Vmax with no change in Km. The circular dichroism spectral studies revealed that MIC interaction with Na+, K(+)-ATPase led to a conformation of the protein wherein the substrates Na+ and K+ were no longer able to bind at the Na(+)- and K(+)-activation sites. The data suggest that the inhibition of Na+, K(+)-ATPase was non-competitive and occurred by interference with the dephosphorylation of the enzyme-phosphoryl complex.     

Alpha 2-adrenoceptors determine the response to nitric oxide inhibition in the rat glomerulus and proximal tubule

Arginine-derived nitric oxide exerts control over the processes of glomerular filtration and tubular reabsorption. The tonic influence of nitric oxide over both of these is eliminated by renal denervation. The hypothesis that the renal nerves function, in this regard, via the activation of alpha 2-adrenoceptors was tested by renal micropuncture. The physical determinants of glomerular filtration and proximal tubular reabsorption were assessed in Munich-Wistar rats before and during the administration of the nitric oxide synthase inhibitor NG-monomethyl L-arginine (L-NMMA). In one set of studies, the systemic infusion of the alpha 2-agonist B-HT 933 rendered nephron GFR, nephron plasma flow, and proximal reabsorption sensitive to reduction by L-NMMA after renal denervation. In a second set of studies, the infusion of the alpha 2 receptor antagonist, yohimbine, to rats with renal nerves intact was found to suppress the effects of L-NMMA on nephron plasma flow and proximal reabsorption. The effects of L-NMMA on nephron GFR and nephron plasma flow, afferent and efferent arteriolar resistances, and proximal reabsorption correlated with the level of underlying alpha 2-adrenergic activity. The activation of renal alpha 2-adrenoceptors increases the influence of arginine-derived nitric oxide in the glomerulus and proximal tubule.     

Peripheral nerve blocks improve analgesia after total knee replacement surgery

Using an in vitro cell system and Cs+ NMR techniques we were able to show that porcine aortic endothelial cells (PAEC) reduce their Na(+)-K(+)-ATPase activity upon an increase in intracellular cAMP. Reduction in the pump rate was due to phosphorylation of the alpha-subunit of the ATPase as shown by immunoprecipitation. Apart from a pump inhibiton using 8-Br-cAMP and IBMX, we were also able to show that changes in the Na(+)-K(+)-ATPase activity could be mediated by the adenosine-A2 and prostaglandin receptor agonists 5'-N-Ethylcarboxamidoadenosine and Iloprost, respectively. Parallel to a decrease in pump activity we also observed a decrease in intracellular Cs+, indicating opening of K+ channels.     

Short-term effect of epidermal growth factor (EGF) on sodium and glucose cotransport of isolated jejunal epithelial cells

This study was undertaken to assess the short-term effects of EGF on sodium and glucose uptake, glucose metabolism and Na+/K(+)-ATPase activity in isolated enterocytes of rats. Jejunal cells exposed to EGF had a significantly greater total uptake of sodium compared to controls after 6 min. Kinetic analysis of glucose transport across BBMV's demonstrated similar Km values but a significant increase of the Vmax in vesicles prepared from cells first exposed to EGF as compared to controls. EGF was also associated with a significant increase in glucose metabolism of jejunal enterocytes after 15 min. The activity of Na+/K(+)-ATPase increased in jejunal enterocytes exposed to EGF. The increase in Na+/K(+)-ATPase activity of the cells following EGF exposure was not accompanied by an increase in immunodetectable total or assembled Na+/K(+)-ATPase protein. EGF's effect on enzyme activity was abolished by removing NaCl from the incubation solution, and by preincubating the enterocytes with phlorizin prior to addition of EGF. Preincubation with amiloride did not inhibit the effect of EGF on Na+/K(+)-ATPase. The results confirm that EGF promotes uptake of both sodium and glucose by the jejunal mucosal cells, and suggest the effect of EGF on glucose and sodium is mediated through the brush-border membrane glucose-sodium transporter. The increase in Na+/K(+)-ATPase activity that occurs with EGF appears to be secondary to a rise in intracellular Na+ concentration. The short-term effects of EGF on glucose and sodium transport by the small intestine may have potential therapeutic implications.     

Performance of the lift ventricle in left ventricular obstructive cardiomyopathy

Whether volume expansion influences NaC1 reabsorption by the diluting segment of the nephron remains a matter of controversy. In the present studies this question has been examined in normal unanesthetized dogs, undergoing maximal water diuresis. Free water clearance (CH2O/GFR) has been used as the index of NaC1 reabsorption in the diluting segment. Three expressions have been employed for "distal delivery" of NaC1: a) V/GFR, designated as the "volume term"; b) (CNa/GFR + CH2O/GFR), the "sodium term;" and c) (CC1/GFR + CH2O/GFR), the "chloride term". The validity of these terms is discussed. Three techniques were used to increase distal delivery: 1) the administration of acetazolamide to dogs in which extracellular fluid (ECF) volume was not expanded (grop 1); 2) "moderate" volume expansion (group 2); and 3) "marked" volume expansion (group 3). CH2O/GFR increased progressively with rising values for "distal delivery" regardless of which term was used to calculate the latter. With all three delivery terms, differences in distal NaC1 reabsorption emerged between the two volume-expanded groups, though only with the "chloride" term did substantial differences also emerge between the nonexpanded group 1 dogs and both volume-expanded groups. In group 1, values for CH2O/GFR increased in close to a linear fashion up to distal delivery values equal to 24% of the volume of glomerular filtrate. However, at high rates of distal delivery the rate of rise of CH2O/GFR was less in group 2 than in group 1 and the depression of values was even greater in group 3. Within the limits of the techniques used, the data suggest that volume expansion inhibits fractional NaC1 reabsorption in the diluting segment of the nephron in a dose-related fashion. The "chloride" term was found to be superior to the "volume" and "sodium" terms in revealing these changes.     

A protocol to improve analgesia use in the accident and emergency department

In this experiment, intracellular K+ concentration ([K+]i) and ATPase activity of myocardiocytes were measured in early stage of burn injury. Comparing with control group, it was found that, 1. [K+]i were decreased after burn injury, [K+]i of 1st, 3rd, 8th and 24th hours were decreased to 96.2 +/- 1.3%, 85.8 +/- 1.3%, 65.9 +/- 1.0% and 73.7 +/- 1.1% of normal, respectively. 2. Cardiac sarcolemma total ATPase, Mg(2+)-ATPase and Na(+)-K(+)-ATPase activities were all reduced significantly at 8th hour after injury. These results suggest that, burn injury accelerates K+ efflux current, but inhibits K+ influx current, and the reduction of Na(+)-K(+)-ATPase activity is one reason of decrease of [K+]i after injury.     

The apical Cl/HCO3 exchanger of beta intercalated cells

There are two types of intercalated cells of the renal collecting duct; one secretes H+ and the other secretes HCO3-. The H(+)-secreting form has an apical vacuolar H(+)-ATPase and a basolateral Cl/HCO3 exchanger that cross-reacts with antibodies to band 3, the product of the AE1 gene. The HCO3(-)-secreting form has a basolateral vacuolar H(+)-ATPase and an apical Cl/HCO3 exchanger, whose identity has not been established previously. Apical membrane vesicles of beta intercalated cells purified from rabbit kidney cortex contain both an electroneutral Cl/HCO3 exchange activity and polypeptides that react with antibodies to band 3 on Western blots. Furthermore, both primary cultures of HCO3(-)-secreting intercalated cells and an immortalized cell line derived from these cells express AE1 and have an apical Cl/HCO3 exchanger. Apical membranes purified from these cells contain a 100-kDa polypeptide that cross-reacts with antibody to the cytoplasmic domain of band 3. These data suggest that the apical Cl/HCO3 exchanger of HCO3(-)-secreting intercalated cells is band 3.     

Intracellular pH in vascular smooth muscle: regulation by sodium-hydrogen exchange and multiple sodium dependent HCO3- mechanisms

OBJECTIVES: The aim was to determine the mechanisms, particularly bicarbonate dependent mechanisms, of intracellular pH (pHi) recovery from various acidoses in vascular smooth muscle and to explore the ATP dependency of the respective mechanisms. METHODS: Experiments were conducted in rat aortic smooth muscle cells grown in primary culture and synchronised in a non-growing state by serum deprivation. pHi was measured in cells loaded with the pH sensitive fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF). Chloride efflux was studied by determination of the rate of efflux of 36Cl over 5 min. Cells were ATP depleted by substitution of glucose in the medium by 2-deoxyglucose. Acidoses were induced by CO2 influx and NH3 efflux techniques. RESULTS: In the absence of HCO3-, the 5-(N-ethyl-N-isopropyl) amiloride (EIPA) sensitive Na+/H+ exchange accounted for the recovery from intracellular acidosis. In the presence of HCO3- ions the response to respiratory acidosis (CO2 influx) was predominantly via activation of Na+/H+ exchange and an EIPA sensitive Na+ and HCO3- dependent mechanism. A 4-acetamido-4'-isothiocyanostilbene-2',2'-sulphonic acids (SITS) sensitive Na+ dependent Cl-/HCO3- mechanism which is also sensitive to EIPA makes a small contribution during severe intracellular acidosis. Under such conditions HCO3- dependent mechanisms contributed about 40% to the overall pHi regulating capacity of vascular smooth muscle cells. However, under conditions which deplete cellular ATP these pHi regulating mechanisms account for virtually all of theses cells' ability to regulate pHi. The inability of Na+/H+ exchange to participate in pHi recovery under these circumstances, reduces the ability of vascular smooth muscle cells to recover pHi by approximately 50-60%. Chloride efflux was approximately linear over 5 min and was increased by 36% in the presence of extracellular HCO3-. Efflux in the presence of HCO3- was inhibited similarly by both SITS and EIPA. CONCLUSIONS: At least three transporters contribute to recovery from acidosis in vascular smooth muscle: Na+/H+ exchange, an Na(+)-HCO3- cotransporter which is sensitive to EIPA, and an Na+ dependent HCO3-/Cl- exchange sensitive to both SITS and EIPA. The Na(+)-HCO3- cotransporter appears to be similar to that described in human vascular smooth muscle. When the Na+/H+ exchanger is attenuated by cellular ATP depletion, the alternative pathways, particularly the Na(+)-HCO3- cotransporter, ensure that substantial pHi regulatory capacity is maintained.     

Potential anxiolytic properties of R-(+)-8-OSO2CF3-PAT, a 5-HT 1A receptor agonist

The inhibitory effect of 2% ethanol (400 mM) in the incubation medium on several characteristics of the Na(+)-ATPase of basolateral plasma membranes from rat kidney proximal tubular cells was investigated. Ethanol did not change the Km of the enzyme for Mg2+, ATP or Na+; it did not change either the optimal pH or temperature values of the incubation medium for the enzyme to act and finally, it did not affect the apparent energy of activation of the enzyme. It was also found that 2% ethanol produced stronger inhibition of the ATPase when it is in an activated or stimulated state, than when it is working at its lower basal level. The presented results can be explained by assuming that 2% ethanol in the incubation medium inhibits Na(+)-ATPase activity by affecting the enzyme structure as well as its activating mechanism.     

Calcium-activated potassium conductances on cultured nontransformed dog pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDECs) mediate the pancreatic secretion of fluid and electrolytes. Membrane K+ channels on these cells regulate intracellular K+ concentration; in combination with the Na+/H+ antiport and Na+,K+ adenosine triphosphatase (ATPase), they may also mediate serosal H+ secretion, balancing luminal HCO3- secretion. We describe the K+ conductances on well-differentiated and functional nontransformed cultured dog PDECs. Through 86Rb+ efflux studies, we demonstrated Ca(2+)-activated K+ channels that were stimulated by A23187, thapsigargin, and 1-ethyl-2-benzimidazolinone, but not forskolin. These conductances also were localized on the basolateral membrane because 86Rb+ efflux was directed toward the serosal compartment. Of the K+ channel blockers, BaCl2, charybdotoxin, clotrimazole, and quinidine, but not 4-aminopyridine, apamin, tetraethylammonium, or iberiotoxin, inhibited 86Rb+ efflux. This efflux was not inhibited by amiloride, ouabain, and bumetanide, inhibitors of the Na+/H+ antiport, the Na+,K(+)-ATPase pump, and the Na+,K+,2Cl- cotransporter, respectively. When apically permeabilized PDEC monolayers were mounted in Ussing chambers with a luminal-to-serosal K+ gradient, A23187 and 1-ethyl-2-benzimidazolinone stimulated a charybdotoxin-sensitive short-circuit current (Isc) increase. Characterization of K+ channels on these cultured PDECs, along with previous identification of Cl- channels (1), further supports the importance of these cells as models for pancreatic duct secretion.     

Na+,K(+)-ATPase of human placenta during gestational hypertension: a biochemical-biophysical study

1. Na+,K(+)-ATPase is the membrane enzyme catalysing the active transport of Na+ and K+ across the plasma membrane of animal cells. A reduced activity of Na+,K(+)-ATPase has been described in gestational hypertension in a variety of cell types, in agreement with the hypothesis that gestational hypertension can induce membrane transport modifications similar to those reported for essential hypertension. The causes of the reduced Na+,K(+)-ATPase activity are still debated. 2. The aim of the present work was to investigate the molecular mechanism of the reduced enzymic activity in gestational hypertension using as a model Na+,K(+)-ATPase purified from human placenta. Na+,K(+)-ATPase obtained from term placentas of eight healthy pregnant women and eight age-matched women with gestational hypertension was purified as previously described. 3. We observed in gestational hypertension: (i) a significant increase in the activation energies above transition temperature; (ii) a significant decrease in the fluorescence polarization of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (i.e. increased fluidity) and an increase in the mean lifetime (modified hydrophobicity); (iii) a lower Kq, suggesting an enzymic structural modification; and (iv) an increased mean lifetime and rotational relaxation time of pyrene isothiocyanate, indicating a modified ATP binding site.     

Structure-function study on gastric proton pump

H+, K(+)-ATPase is a proton pump responsible for gastric acid secretion. It actively transport proton and K+ coupled with the hydrolysis of ATP, resulting in the formulation of a 10(6) fold proton gradient across the plasma membrane of parietal cells. The pump belongs to a family of P-type ATPases which include the Na+ pump (Na+, K(+)-ATPase) and the Ca2+ pump (Ca(2+)-ATPase). This review focuses on the structure-function relationship of this proton pump by using functional antibodies, specific inhibitor(s), a fluorescent reagent and site-directed mutants. First we prepared monoclonal antibodies which modified the functions of the H+, K(+)-ATPase . One of the antibodies, HK2032 inhibited the H+, K(+)-ATPase activity and the chloride conductance in gastric vesicles opened by S-S cross-linking, suggesting that the chloride pathway is in the H+, K(+)-ATPase molecule, and that the H+, K(+)-ATPase is a multi-functional molecule. Other antibody, HK4001 inhibited the H+, K(+)-ATPase activity by inhibiting its phosphorylation step. By using this antibody we found an H+, K(+)-ATPase isoform in the rabbit distal colon. Second we found that scopadulcic acid B, a main ingredient of Paraguayan traditional herb, is an inhibitor specific for the H+, K(+)-ATPase. This compound inhibited the H+, K(+)-ATPase activity by stabilizing the K(+)-form of the enzyme. Third we studied the conformational changes of the H+, K(+)-ATPase by observing the fluorescence of FITC-labeled enzyme. H+, K(+)-ATPase did not utilize acetylphosphate instead the ATP as an energy source of active transport, suggesting that the energy transduction system is not common among P-type ATPases. Finally we constructed a functional expression system of the H+, K(+)-ATPase in human kidney cells. By using this functional expression system in combination with site-directed mutagenesis, we studied the significance of amino acid residues in the catalytic centers (a phosphorylation site and an ATP binding site) and the putative cation binding sites. We newly found the sites determining the affinity for cations.     

Double immunofluorescence staining of Fos and Jun in the hypothalamus of the rat

Epidermal growth factor (EGF) triggers rapid and delayed effects on gluconeogenesis, cytosolic (lactate/pyruvate ratio) and mitochondrial (3-hydroxybutyrate/acetoacetate ratio) redox states (Soler, C. and Soley, M., Biochem. J., 294 (1993) 865-872). This study attempts to determine whether the mechanism by which EGF modulates any of these parameters is dependent on the regulation of Na+/H+ exchange and/or Na+/K(+)-ATPase activities. The Na+/H+ exchange was inhibited by either amiloride or the analogue 5-(N,N-hexamethylene)amiloride (HMA), and the Na+/K(+)-ATPase activity was inhibited by ouabain. The delayed EGF inhibition of gluconeogenesis, increase of the lactate/pyruvate ratio and decrease in the 3-hydroxybutyrate/acetoacetate ratio were unaltered in the presence of amiloride, HMA or ouabain. The rapid EGF stimulation of gluconeogenesis was also observed in the presence of HMA or ouabain. Although Na+/H+ exchange and/or Na+/K(+)-ATPase are regulated by EGF, our results indicate that these activities are not required for the effects of EGF on gluconeogenesis and/or cytosolic and mitochondrial redox state.