Angiotensin II stimulates both Na(+)-H+ exchange and Na+/HCO3- cotransport in the rabbit proximal tubule.

Angiotensin II (AII) is a potent stimulus for HCO3- reabsorption in the rat proximal tubule in vivo. To determine the ionic mechanism of increased HCO3- reabsorption, we have examined the effect of AII on luminal Na(+)-H+ exchange and basolateral Na+/HCO3- cotransport in perfused S1 proximal tubules isolated from superficial nephrons of the rabbit kidney. Transporter activity was assessed by removing Na+ from both luminal and basolateral (i.e., bath) solutions and determining the rate at which intracellular pH (pHi) increased after Na+ was returned to only the lumen or only the bath. pHi was measured with the pH-sensitive fluorescent dye 2', 7'-bis(2-carboxyethyl)-5(and 6)-carboxyfluorescein. We found that basolateral administration of 1 nM AII not only increased the rate of luminal Na(+)-H+ exchange approximately 3.5-fold but also increased the rate of basolateral Na+/HCO3- cotransport approximately 2.5-fold. 5-(N-Ethyl-N-isopropyl)amiloride (50 microM) blocked luminal Na(+)-H+ exchange before and after stimulation by AII but had no effect on basolateral Na+/HCO3- cotransport. Conversely, 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate (50 microM) blocked basolateral Na+/HCO3- cotransport before and after AII but had no effect on luminal Na(+)-H+ exchange. Our data thus indicate that, at least under the conditions of our assay, AII independently stimulates the transporters responsible for both the luminal and basolateral steps of transepithelial HCO3- reabsorption.     

Biochemical localization of hepatic surface-membrane Na+,K+-ATPase activity depends on membrane lipid fluidity.

Membrane proteins of transporting epithelia are often distributed between apical and basolateral surfaces to produce a functionally polarized cell. The distribution of Na+,K+-ATPase [ATP phosphohydrolase (Na+/K+-transporting), EC 3.6.1.37] between apical and basolateral membranes of hepatocytes has been controversial. Because Na+,K+-ATPase activity is fluidity dependent and the physiochemical properties of the apical membrane reduces its fluidity, we investigated whether altering membrane fluidity might uncover cryptic Na+,K+-ATPase in bile canalicular (apical) surface fractions free of detectable Na+,K+-ATPase and glucagon-stimulated adenylate cyclase activities. Apical fractions exhibited higher diphenylhexatriene-fluorescence polarization values when compared with sinusoidal (basolateral) membrane fractions. When 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) was added to each fraction, Na+,K+-ATPase, but not glucagon-stimulated adenylate cyclase activity, was activated in the apical fraction. In contrast, further activation of both enzymes was not seen in sinusoidal fractions. The A2C-induced increase in apical Na+,K+-ATPase approached 75% of the sinusoidal level. Parallel increases in apical Na+,K+-ATPase were produced by benzyl alcohol and Triton WR-1339. All three fluidizing agents decreased the order component of membrane fluidity. Na+,K+-ATPase activity in each subfraction was identically inhibited by the monoclonal antibody 9-A5, a specific inhibitor of this enzyme. These findings suggest that hepatic Na+,K+-ATPase is distributed in both surface membranes but functions more efficiently and, perhaps, specifically in the sinusoidal membranes because of their higher bulk lipid fluidity.     

Effect of inhibitors on sodium and chloride transport in brush border vesicles from human jejunum and ileum

Brush border vesicles were isolated from surgically resected pieces of human jejunum and ileum using a Mg2+/EGTA precipitation method. When compared to the homogenate, the final membrane preparation contained alkaline phosphatase at a 14 times higher concentration and almost no (Na++K+)-stimulated adenosine triphosphatase. An Na+/H+ antiport could be demonstrated in the jejunum by imposing a pH gradient between the interior and the outside of the vesicles (pHinside 5.2, pHoutside 7.2). In the presence of amiloride or harmaline, Na+/H+ antiport was inhibited by 60 +/- 5% (p less than 0.05) or 65 +/- 5% (p less than 0.05), respectively. In vesicles of human ileum we found an Na+/H+ antiport and in contrast to the jejunum a Cl-/OH- antiport could be demonstrated by imposing a pH gradient (pHinside 5.2, pHoutside 7.2). Besides this double-exchange mechanism for sodium and chloride, a Na+/Cl- cotransport and a Cl- conductive pathway could be detected in ileal brush border vesicles. In the presence of the anion transport inhibitors, furosemide, SITS and DIDS activities of Cl-/OH- antiport and Na+/Cl- cotransport were suppressed by 30 +/- 5% (p less than 0.05), 35 +/- 5% (p less than 0.05) and 40 +/- 5% (p less than 0.05), respectively. We conclude that absorption of sodium and chloride in the absence of organic solutes is mediated through different transport mechanisms at the luminal plasma membrane, which are in part subject to regulation by sodium and chloride transport inhibitors.     

Platelet and erythrocyte Mg2+, Ca2+, Na+, K+ and cell membrane adenosine triphosphatase activity in essential hypertension in blacks.

OBJECTIVE: To assess the relationship between intracellular Mg2+, Ca2+, Na+ and K+ and cell membrane adenosine triphosphatase (ATPase) activity in normotensive and hypertensive blacks. DESIGN: Intracellular cations and cell membrane ATPase activity were studied in black patients with untreated essential hypertension and age-, weight- and height-matched normotensive controls. Platelet, erythrocyte and serum Mg2+, Ca2+, Na+ and K+ levels as well as platelet and erythrocyte membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities were measured in all subjects. METHODS: Intracellular Na+ and K+ were measured by flame photometry and Mg+ and Ca+ by atomic absorption spectrophotometry. Cell membrane ATPase activity was determined by a colorimetric method. RESULTS: The hypertensive group consistently demonstrated depressed activity of each ATPase studied, with significantly lower serum Mg2+, serum K+, erythrocyte Mg2+ and platelet Mg2+ levels compared with the normotensive group. Platelet Na+ and Ca2+ and erythrocyte Ca2+ were significantly elevated in the hypertensive group. In the hypertensive group, mean arterial pressure (MAP) was inversely correlated with platelet and erythrocyte membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase. Serum Mg2+, serum Ca2+ and platelet Mg2+ were negatively correlated with MAP in the hypertensive group whilst erythrocyte and platelet Ca2+ were positively correlated. In the normotensive group, platelet Mg2+ and MAP were negatively, and erythrocyte Ca2+ and MAP, positively correlated. CONCLUSIONS: Black patients with essential hypertension have widespread depression of cell membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities with serum and intracellular Mg2+ depletion and cytosolic Na+ and Ca2+ overload, which may reflect an underlying membrane abnormality in essential hypertension. These cellular abnormalities may be related to the defective transport mechanisms that in turn may be aggravated by Mg2+ depletion.     

Erythrocyte membrane lipids and cationic transport systems in men.

OBJECTIVE: The relationship between erythrocyte membrane and plasma lipids and various transmembrane erythrocyte cationic fluxes was examined in 53 normal men. DESIGN: Different measurements of erythrocyte transport systems were obtained: Na(+)-Li+ countertransport activity; Na+, K+ cotransport activity; Na+, K(+)-ATPase pump activity and the ground membrane permeability for Na+ and K+ as well as the intra-erythrocyte Na+, K+ and Mg2+ concentrations. Plasma cholesterol, triglycerides, phospholipids, free fatty acids, low- and high-density lipoprotein cholesterol levels and the erythrocyte membrane contents of cholesterol, phospholipids and free fatty acids were obtained from fasting subjects. RESULTS: In single regression analysis the erythrocyte Na(+)-Li+ countertransport and Na+, K+ cotransport activities were negatively related to the erythrocyte membrane cholesterol, phospholipids and free fatty acids contents. The Na+, K(+)-ATPase pump activity as assessed by the ouabain-sensitive Na+ efflux was also inversely related to the membrane cholesterol and phospholipids contents. In multiple regression analysis the red blood cell Na(+)-Li+ countertransport activity was independently and negatively related to the membrane cholesterol and free fatty acids contents. CONCLUSION: Our data show that an elevated level of erythrocyte membrane lipids in normal men is accompanied by lower Na(+)-Li+ countertransport, Na+, K+ cotransport and Na+, K(+)-ATPase pump activities.     

Taurocholate transport by basolateral plasma membrane vesicles isolated from human liver

Transport of taurocholate into the hepatocyte against unfavorable chemical and electrical gradients occurs via a sodium-dependent, carrier-mediated transport system. Although this cotransporter has been characterized in the rodent, it has not been demonstrated in man. Therefore, we utilized human liver, obtained via multiorgan donation but not used for transplantation, to prepare basolateral (sinusoidal) liver plasma membrane vesicles by a Percoll gradient method. Na+,K+-ATPase, a marker enzyme for the basolateral domain, was enriched 28.9-fold in the final membrane fraction compared with homogenate, whereas the bile canalicular membrane enzymes Mg++-ATPase and alkaline phosphatase were enriched only 3.4- and 6.4-fold, respectively. Marker enzyme activities for endoplasmic reticulum, lysosomes and mitochondria were not enriched compared with homogenate. Integrity of the membrane vesicles was confirmed by the demonstration of Na+-dependent concentrative uptake of the amino acid L-alanine (estimated intravesicular volume of 0.59 microliter per mg protein). An inwardly directed 100 mM Na+ gradient stimulated the initial rate of 2.5 microM taurocholate uptake and energized a transient 2-fold accumulation of the bile acid above equilibrium ("overshoot"). In contrast, uptake was slower and no overshoot occurred with a K+ gradient. A negative intravesicular potential, created by altering accompanying anions or by valinomycin-induced K+ diffusion potentials, did not enhance taurocholate uptake, suggesting an electroneutral cotransport mechanism. Chloride as the accompanying anion stimulated the initial rate of uptake compared with anions of lesser or greater lipid permeability. Na+-dependent taurocholate (4 microM) uptake was significantly inhibited by 250 microM cholate, taurocholate, glycocholate, taurochenodeoxycholate and bromsulfophthalein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Red cell membrane Na+ transport systems in hereditary spherocytosis: relevance to understanding the increased Na+ permeability

Red blood cells (RBCs) in hereditary spherocytosis (HS) show high sodium (Na+) and potassium (K+) movement across the membrane, resulting in dehydration. In general, these abnormal cation fluxes have been interpreted as "increased leaks" due to passive or electrodiffusional permeability of the RBC membrane. A study to elucidate the contribution of concomitant ouabain-resistant pathways (Na-K-2Cl cotransport and Na-Li countertransport) to abnormal Na+ permeability present in RBCs of subjects with HS has been undertaken. Accordingly, erythrocyte Na+ and K+ content and transmembrane cation movements via the Na-K pump, Na-K-2Cl cotransport, Na-Li countertransport, and Na+ passive diffusion, were measured in 25 non-splenectomized patients with HS and compared with the results obtained from the study of 11 patients with congenital non-spherocytic haemolytic anaemia (CNSHA) due to hereditary elliptocytosis (7 cases) and RBC enzyme defects (4 cases) and of 30 normal controls. Compared to the controls, patients with HS exhibited a highly significant (P<0.001) increase in all the Na+ transmembrane movements via passive diffusion (411+/-243 vs 105+/-40), Na-K pump (2615+/-970 vs 1874+/-359), Na-K-2Cl cotransport (males: 371+/-138 vs 190+/-42; females: 401+/-134 vs 104+/-44) and Na-Li countertransport (207+/-131 vs 98+/-41). This was associated with increased Na+ and decreased K+ content, resulting in a reduction of total cation (Na+ + K+) RBC concentration. Furthermore, significant correlations were also found between the patients' RBC cationic content and the mean corpuscular haemoglobin concentration (MCHC) (r=0.51, P<0.05) and between the Na+ passive leak and the haematocrit value (r=-0.44, P<0.05). In the patients with CNSHA, a less significant (P<0.01) increase of active (Na-K pump) and passive (leak) transmembrane permeability to Na+ was associated with normal transmembrane movements via Na-K-2Cl cotransport and Na-Li countertransport. The present study demonstrates that in HS, RBCs are characterized by a variable, but always significant increase of all the membrane transport systems leading to the extrusion of Na+, and that these abnormalities, regardless of their relation to membrane structural defects, may probably be valuable for the differential diagnosis between HS and other congenital defects of RBCs.     

Cellular localization, membrane distribution, and possible function of guanylyl cyclases A and 1 in collecting ducts of rat

BACKGROUND: Natriuretic peptides regulate Na+ and H(2)O transport in the cortical collecting duct (CCD). We have shown that natriuretic peptides have no effect on ion conductances or water transport of principal cells (PC) even though a cGMP-regulated K+ channel is located in the basolateral membrane of these cells. METHODS: RT-PCR was used to screen for different guanylyl cyclases (GC) in CCD and to look for the expression of GC-1 and GC-A mRNA in CCD of male and female Wistar and Sprague-Dawley rats. Polyclonal antibodies were raised against the detected GC. BCECF was used to investigate the effects of ANP on intracellular pH in intercalated cells (IC). RESULTS: GC-A and GC-1 were detected. GC-A was immunolocalized in the luminal membrane of IC while GC-1 was mainly found in the luminal membrane of PC. GC-1 is expressed in Sprague-Dawley and Wistar rats except for male Sprague-Dawley rats, while GC-A is expressed in all strains. ANP (160 nM, n=11), urodilatin (140 nM, n=6), which had no effect in PC, significantly decreased pH(i) by 0.02+/-0.01 and 0.03 +/- 0.01 Units in IC, respectively. ANP as well as urodilatin and 8-Br-cGMP decreased the pH(i) recovery after acidification by 30 +/- 6% (n=12), 37 +/- 7% (n=8), and 19 +/- 3% (n=8), respectively. CONCLUSION: GC-A is located in the luminal membrane of IC of rat CCD and ANP acts through this receptor when regulating pH(i) via an inhibition of the Na+/H+-exchanger. PC do not possess GC-A. GC-1 seems to be the only GC in these cells of most rat strains tested and therefore, it could be responsible for the regulation of K+ channels in the basolateral membrane via cGMP-dependent protein kinase.     

Cellular mechanisms of acid secretion

Current ideas about the parietal cell are presented in the model shown in Figure 6; the cell is illustrated both in resting and stimulated states. The basal-lateral surface contains three major receptor classes mediated by either cyclic AMP responses or Ca2+ changes, Ca2+ channels and a Ca2+ pump, and a variety of ion transport pathways such as Na+:H+ and Cl-:HCO-3 exchange, a K+ conductance, a postulated NaKCl2 cotransport and the (Na+ + K+)ATPase. At rest, the tubulovesicles contain the (H+ + K+)ATPase. In the stimulated state, the apical (secretory canalicular) membrane contains a K+ and Cl- conductance as well as the (H+ + K+)ATPase. There is also a change in cytoskeletal arrangement associated with stimulation.     

Thyroid hormones increase Na+-H+ exchange activity in renal brush border membranes.

Na+-H+ exchange activity, i.e., amiloride-sensitive Na+ and H+ flux, in renal proximal tubule brush border (luminal) membrane vesicles was increased in the hyperthyroid rat and decreased in the hypothyroid rat, relative to the euthyroid animal. A positive correlation was found between Na+-H+ exchange activity and serum concentrations of thyroxine (T4) and triiodothyronine (T3). The thyroid status of the animal did not alter amiloride-insensitive Na+ uptake. The rate of passive pH gradient dissipation was higher in membrane vesicles from hyperthyroid rats compared to the rate in vesicles from hypothyroid animals, a result which would tend to limit the increase in Na+ uptake in vesicles from hyperthyroid animals. Na+-dependent phosphate uptake was increased in membrane vesicles from hyperthyroid rats; Na+-dependent D-glucose and L-proline uptakes were not changed by the thyroid status of the animal. The effect of thyroid hormones in increasing the uptake of Na+ in the brush border membrane vesicle is consistent with the action of the hormones in enhancing renal Na+ reabsorption. Further, the regulation of transtubular Na+ flux has now been shown to be concomitant with modulation of the entry of Na+ into the tubular cell across its luminal membrane, mediated by the exchange reaction, and with the previously reported control of the pumping of Na+ out of the cell across its basolateral membrane, mediated by the Na+,K+-ATPase.     

Basolateral membrane Na+/H+ exchange enhances HCO3- absorption in rat medullary thick ascending limb: evidence for functional coupling between basolateral and apical membrane Na+/H+ exchangers.

The role of basolateral membrane Na+/H+ exchange in transepithelial HCO3- absorption (JHCO3) was examined in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. In Na(+)-free solutions, addition of Na+ to the bath resulted in a rapid, amiloride-sensitive increase in intracellular pH. In MTALs perfused and bathed with solutions containing 146 mM Na+ and 25 mM HCO3-, bath addition of amiloride (1 mM) or 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 50 microM) reversibly inhibited JHCO3 by 50%. Evidence that the inhibition of JHCO3 by bath amiloride was the result of inhibition of Na+/H+ exchange included the following: (i) the IC50 for amiloride was 5-10 microM, (ii) EIPA was a 50-fold more potent inhibitor than amiloride, (iii) the inhibition by bath amiloride was Na+ dependent, and (iv) significant inhibition was observed with EIPA as low as 0.1 microM. Fifty micromolar amiloride or 1 microM EIPA inhibited JHCO3 by 35% when added to the bath but had no effect when added to the tubule lumen, indicating that addition of amiloride to the bath did not directly inhibit apical membrane Na+/H+ exchange. In experiments in which apical Na+/H+ exchange was assessed from the initial rate of cell acidification following luminal EIPA addition, bath EIPA secondarily inhibited apical Na+/H+ exchange activity by 46%. These results demonstrate basolateral membrane Na+/H+ exchange enhances transepithelial HCO3- absorption in the MTAL. This effect appears to be the result of cross-talk in which an increase in basolateral membrane Na+/H+ exchange activity secondarily increases apical membrane Na+/H+ exchange activity.     

Effect of Ischemia—Reperfusion on Na+,K+-ATPase Expression in Human Liver Tissue Allograft: Image Analysis by Confocal Laser Scanning Microscopy

We have analyzed the effect of ischemia-reperfusion on expression of hepatic Na+,K+-ATPase on bile canalicular (BCM) and basolateral membranes (BLM) in human liver allografts using confocal laser scanning microscopy imaging. Na+, K+-ATPase, an integral membrane enzyme, plays a key role in the physiology and structure of hepatocytes, where it maintains the electrochemical gradients for Na+ and K+ across the cell membrane. The concentrations of these ions as well as their gradients regulate the active transport across the plasma membrane for bile acid and water from sinusoidal to canalicular membranes. In addition, Na+,K+-ATPase is also involved in cellular structure because of its close relationship with submembrane microfilaments and its implication in tight junction assembly. Therefore, Na+,K+-ATPase appears as an indicator of tissue viability and hepatic functionality during liver transplantation. Its localization and its function in BCM are still controversial. As in previous studies, we found an enzyme expression in both BLM and BCM. We show that ischemia induced a decrease in Na+,K+-ATPase expression only in BCM. This result could be explained by the differences in biochemical membrane environment between basolateral and bile canalicular Na+,K+-ATPase. Membrane lipid fluidity, which is more elevated in BLM than in BCM, could protect the enzyme during ischemia. After reperfusion, Na+,K+-ATPase expression was strongly decreased in both BCM and BLM. This alteration following reperfusion is probably due to multiple factors: direct alteration of the enzyme catalytic subunit and modification of its environment and membrane lipid fluidity by free radicals and changes in ATP levels and ionic distribution. This important decrease in Na+,K+-ATPase expression of both BLM and BCM could disturb not only hepatic secretory function but also cellular volume and structure during the postoperative period.     

Intracellular Na+ regulation in cardiac myocytes

Intracellular [Na+] ([Na+]i) is regulated in cardiac myocytes by a balance of Na+ influx and efflux mechanisms. In the normal cell there is a large steady state electrochemical gradient favoring Na+ influx. This potential energy is used by numerous transport mechanisms, including Na+ channels and transporters which couple Na+ influx to either co- or counter-transport of other ions and solutes. Six sarcolemmal Na+ influx pathways are discussed in relatively quantitative terms: Na+ channels, Na+/Ca2+ exchange, Na+/H+ exchange, Na+/Mg2+ exchange, Na+/HCO3- cotransport and Na+/K+/2Cl- cotransport. Under normal conditions Na+/Ca2+ exchange and Na+ channels are the dominant Na+ influx pathways, but other transporters may become increasingly important during altered conditions (e.g. acidosis or cell volume stress). Mitochondria also exhibit Na+/Ca2+ antiporter and Na+/H+ exchange activity that are important in mitochondrial function. These coupled fluxes of Na+ with Ca2+, H+ and HCO3- make the detailed understanding of [Na+]i regulation pivotal to the understanding of both cardiac excitation-contraction coupling and pH regulation. The Na+/K+-ATPase is the main route for Na+ extrusion from cells and [Na+]i is a primary regulator under physiological conditions. [Na+]i is higher in rat than rabbit ventricular myocytes and the reason appears to be higher Na+ influx in rat with a consequent rise in Na+/K+-ATPase activity (rather than lower Na+/K+-ATPase function in rat). This has direct functional consequences. There may also be subcellular [Na+]i gradients locally in ventricular myocytes and this may also have important functional implications. Thus, the balance of Na+ fluxes in heart cells may be complex, but myocyte Na+ regulation is functionally important and merits focused attention as in this issue.     

Reconstitution of an inwardly rectifying potassium channel from the basolateral membranes of Necturus enterocytes into planar lipid bilayers.

Basolateral membrane vesicles from Necturus enterocytes, highly (greater than 20-fold) enriched in Na+,K+-ATPase, were reconstituted into planar lipid bilayers. The principal channel activity observed is selective for K+ over Na+ and Cl-. This K+ channel is blocked by Ba2+ and Leiurus quinquestriatus venom but is not affected by Ca2+ over the range of 10(-3) to less than 10(-7) M and is not inhibited by charybdotoxin. L. quinquestriatus venom also markedly reduces the conductance of the basolateral membrane of intact villus cells of Necturus small intestine. The open-time probability (Po) of the channel displays a voltage-dependence characteristic of an "inward rectifier"; i.e., the channel inactivates when the basolateral membrane is depolarized and Po increases with increasing hyperpolarization of that barrier. Assuming that similar properties prevail under physiological conditions, this characteristic could provide, in part, an explanation for the parallelism between Na+-pump and K+-leak activities of the basolateral membrane observed in this epithelium. Thus, an increase in rheogenic Na+-pump activity at the basolateral membrane would hyperpolarize that barrier and, in turn, increase the open time of this K+ channel.     

KCNQ1-dependent transport in renal and gastrointestinal epithelia

Mutations in the gene encoding for the K+ channel alpha-subunit KCNQ1 have been associated with long QT syndrome and deafness. Besides heart and inner ear epithelial cells, KCNQ1 is expressed in a variety of epithelial cells including renal proximal tubule and gastrointestinal tract epithelial cells. At these sites, cellular K+ ions exit through KCNQ1 channel complexes, which may serve to recycle K+ or to maintain cell membrane potential and thus the driving force for electrogenic transepithelial transport, e.g., Na+/glucose cotransport. Employing pharmacologic inhibition and gene knockout, the present study demonstrates the importance of KCNQ1 K+ channel complexes for the maintenance of the driving force for proximal tubular and intestinal Na+ absorption, gastric acid secretion, and cAMP-induced jejunal Cl- secretion. In the kidney, KCNQ1 appears dispensable under basal conditions because of limited substrate delivery for electrogenic Na+ reabsorption to KCNQ1-expressing mid to late proximal tubule. During conditions of increased substrate load, however, luminal KCNQ1 serves to repolarize the proximal tubule and stabilize the driving force for Na+ reabsorption. In mice lacking functional KCNQ1, impaired intestinal absorption is associated with reduced serum vitamin B12 concentrations, mild macrocytic anemia, and fecal loss of Na+ and K+, the latter affecting K+ homeostasis.     

Membrane ion transport in erythrocytes of salt hypertensive Dahl rats and their F2 hybrids: the importance of cholesterol.

The possible association of salt hypertension and altered lipid metabolism with abnormalities of particular systems transporting sodium and potassium has been studied in erythrocytes of Dahl rats and their F2 hybrids fed a high-salt diet since weaning. Our attention was paid to the Na(+)-K+ pump, Na(+)-K+ cotransport and especially to passive membrane permeability for Na+ and Rb+ (Na+ and Rb+ leak), because the Na+ leak was found to be dependent on the genotype, age and salt intake of Dahl rats, whereas the Rb+ leak was suggested to be a potential marker of salt sensitivity in Dahl and Sabra rats. Young male Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats kept on a low-salt (0.3% NaCl) or high-salt diet (8% NaCl) were used for the progenitor study. The subsequent genetic study was based on 135 young male SS/Jr x SR/Jr F2 hybrids fed a high-salt diet since weaning. Ouabain (5 mmol/l) and bumetanide (10 micromol/l) were used to distinguish the contribution of the Na(+)-K+ pump, Na(+)-K+ cotransport and passive membrane permeability to measured net Na+ fluxes and unidirectional Rb+ (K+) movements. Compared to normotensive SR/Jr animals, salt-loaded SS/Jr rats had higher blood pressure (BP), elevated erythrocyte Na+ content, and increased Na+ and Rb+ leaks together with enhanced Na+ and Rb+ transport mediated by the Na(+)-K+ pump and Na(+)-K+ cotransport system. Salt hypertensive Dahl rats were also characterized by elevated plasma levels of total cholesterol and triglycerides, which were positively associated with BP of F2 hybrids (r=0.27 and 0.24, p< 0.01). In F2 hybrids, mean arterial pressure correlated significantly with erythrocyte Na+ content (r=0.24, p<0.01) and ouabain-sensitive Na+ extrusion, but not with the passive membrane permeability for Na+ or Rb+ (r=-0.02 and 0.06, not significant). Both of the above-mentioned significant associations could partially be ascribed to the dependence of erythrocyte Na+ content and ouabain-sensitive Na+ extrusion on plasma cholesterol (r=0.18 and 0.21, p<0.05). Our results support the idea that abnormal lipid metabolism and/or altered Na+,K(+)-ATPase function play an important role in the pathogenesis of salt hypertension in salt-sensitive Dahl rats.     

Studies on the specificity of the effects of oxygen metabolites on cardiac sodium pump

Isolated myocytes of rat heart, and sealed sarcolemmal vesicles of bovine heart, were used to examine the selectivity of the effects of partially reduced oxygen species (generated by a mixture of xanthine and xanthine oxidase) on cardiac sodium pump and several other ion transporters of the plasma membrane. When myocytes were exposed to xanthine plus xanthine oxidase, there were time-dependent inhibitions of ouabain-sensitive 86Rb+ uptake and (Na+ + K+)-ATPase activity that could be prevented by allopurinol, or by catalase and superoxide dismutase; suggesting the involvements of H2O2 or oxygen free radicals in the inhibition of the pump. This inhibition preceded any significant decrease in cellular ATP or in the number of viable cells. While ouabain increased 45Ca2+ uptake by myocytes as expected, exposure to xanthine plus xanthine oxidase decreased 45Ca2+ uptake; suggesting that the Na+, Ca2(+)-exchanger of the intact myocytes is also inhibited by oxygen metabolites. Simultaneous inhibitions of the pump, the Na+, Ca2(+)-exchange, the Na+, H(+)-exchange, and the Na+, Pi-cotransport activities also occurred in sarcolemmal vesicles that were treated with xanthine plus xanthine oxidase. These findings indicate that inactivations of the sodium pump and other sarcolemmal ion carriers are early events in the oxidant-induced damage to the cardiomyocyte. In the rat heart myocytes, a fraction of (Na+ + K+)-ATPase that seems to be more sensitive to ouabain, was inactivated more rapidly upon exposure of myocytes to xanthine plus xanthine oxidase; raising the possibility of the existence of different pump populations with different sensitivities to extracellularly generated oxygen metabolites.     

Effect of CO2 on rat colonic Na absorption: studies with nystatin.

An increase in ambient CO2 tension from 3% to 11% augments colonic Na absorption in the rat. The membrane site of action of CO2 was examined by measuring colonic Na absorption in the Ussing chamber when nystatin was used to permeabilize the luminal (apical) membrane. The equal rates of ouabain-sensitive Na absorption at 3% and 11% CO2 in the presence of nystatin and at 11% CO2 in its absence suggested that CO2 acted at the luminal membrane. This finding was also observed at a submaximal rate of Na absorption (produced by lowering bathing solution Na from 140 to 27 mmol/L) and in a Cl-free solution (to prevent cell swelling). The basolateral membrane was indeed rate limiting for Na absorption in the presence of nystatin, because methylprednisolone (3 mg/kg SC for 3 days to increase sodium-potassium--stimulated adenosine triphosphatase activity) increased Na absorption measured in the presence of nystatin and because CO2 increased absorption in steroid-treated rats in the absence of nystatin. These results validate the protocol and confirm the luminal site of action of CO2 and nystatin on colonic Na absorption.     

Polarized Na+/H+ exchange function is pliable in response to transepithelial gradients of propionate.

Short-chain fatty acids are produced at high concentration in the colonic lumen and stimulate electroneutral Na+ absorption by activating apical Na+/H+ exchange in colonocytes. We used an epithelial cell line derived from a human colon carcinoma (HT29-18-C1) to study activation of apical and basolateral Na+/H+ exchange by a short-chain fatty acid, propionate. Confluent cell monolayers on membrane filters were loaded with 2',7'-bis(2-carboxyethyl)-5 (and 6)-carboxyfluorescein (a fluorescent pH indicator) and intracellular pH was monitored with a digital fluorescence imaging microscope. Cells acidified by transient exposure to NH4Cl demonstrated both apical and basolateral Na+/H+ exchange. In this condition, apical Na+/H+ exchange was 50% of the total Na+/H+ exchange activity. Similar results were obtained when cells were bilaterally perfused with apical and basolateral propionate in an isosmotic medium (130 mM propionate at each membrane surface). However, apical Na+/H+ exchange was a significantly larger fraction (76%) of the total Na+/H+ exchange activity when cells were acidified by exposure to apical propionate alone. Conversely, in cells acidified by basolateral propionate alone, apical Na+/H+ exchange was 21% of the total Na+/H+ exchange activity. The change in relative activity was observed in individual cells which expressed both apical and basolateral Na+/H+ exchange and occurred rapidly (within 7 min). In the presence of transepithelial propionate gradients, all Na(+)-dependent alkalinization was sensitive to 3 microM 5-(N-ethyl-N-isopropyl)amiloride, a potent Na+/H+ exchange inhibitor. These results suggest that transepithelial gradients of short-chain fatty acids, which occur in vivo, can cause preferential activation of apical Na+/H+ exchange.     

Mechanisms of absorption of amino acids and oligopeptides. Control and implications in human diet therapy

The mechanisms involved in the absorption of amino acids and oligopeptides are reviewed regarding their implications in human feedings. Brush border and basolateral membranes are crossed by amino acids and di-tripeptides by passive (facilitated or simple diffusion) or active (Na+ or H+ co-transporters) pathways. Active Na(+)-dependent system occurs mainly at brush border and simple diffusion at basolateral, both membranes have the passive facilitated transport. Free-amino acids use either passive or active transport systems whereas di-tripeptides do mainly active (H+ co-transporter). Brush border have distinctive transport system for amino acids and di-tripeptides. The former occurs mainly by active Na+ dependently whereas the later is active H(+)-dependent with little affinity for tetra or higher peptides. Free amino acids are transported at different speed by saturable, competitive carriers with specificity for basic, acidic or neutral amino acids. Di and tripeptides have at least two carriers both electrogenic and H(+)-dependent. The basolateral membrane transport of amino acids is mostly by facilitated diffusion while for di-tripeptides it is an active anion exchange associated process. The main regulation of amino acids and di-tripeptide transport is the presence o substrate at the mucosal membrane with higher the substrate higher the absorption. Di and tripeptides are more efficiently absorbed than free amino acids which in turns are better absorbed than oligopeptides. So di-tripeptides result in better N-retention and is particularly useful in cases of lower intestinal absorption capacity. The non-absorbed peptides are digested and fermented by colonic bacteria resulting short-chain fatty acids, dicarboxylic acids, phenolic compounds and ammonia. Short-chain fatty acid provides energy for colonocytes and bacteria and the ammonia not fixed by bacteria returns to the liver for ureagenesis.