Initial Rate Of Na Research Articles

Structural and Functional Analysis of Transmembrane XI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na(+)/H(+) exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals by extruding an intracellular H(+) in exchange for one extracellular Na(+). We characterized structural and functional aspects of the critical transmembrane (TM) segment XI (residues 449-470) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM XI was mutated to cysteine in the background of the cysteine-less protein and the sensitivity to water-soluble sulfhydryl reactive compounds MTSET ((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES ((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues with at least moderate activity remaining. Of the residues tested, only proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The activity of the L465C mutant was almost completely eliminated, whereas that of the L457C and I461C mutants was partially affected. The structure of a peptide representing TM XI (residues Lys(447)-Lys(472)) was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. The structure consisted of helical regions between Asp(447)-Tyr(454) and Phe(460)-Lys(471) at the N and C termini of the peptide, respectively, connected by a region with poorly defined, irregular structure consisting of residues Gly(455)-Gly(459). TM XI of NHE1 had a structural similarity to TM XI of the Escherichia coli Na(+)/H(+) exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with residue Leu(465) contributing to the pore.

2-Aminoperimidine, a specific inhibitor of bacterial NhaA Na+/H+ antiporters

The diuretic drug amiloride and its numerous derivatives are competitive inhibitors of mammalian Na+/H+ antiporters and other eukaryotic antiporters. Most prokaryotic antiporters, including the major NhaA family of enterobacteria, are resistant to these compounds. We show that 2-aminoperimidine (AP), a guanidine-containing naphthalene derivative with some similarity to amiloride, acts as a specific inhibitor of NhaA from Escherichia coli. Similar concentrations (IC50 of 0.9 μM) inhibit the proton motive force dependent Na+(Li+)/H+ exchange reaction in inside-out sub-bacterial vesicles (at 10 mM NaCl, pH 8) as well as the initial rate of 22Na+/Na+ exchange mediated by pure NhaA in proteoliposomes. The inhibitor is specific to NhaA type antiporters, so AP is a new tool to study the mechanism and roles of NhaA antiporters of enterobacteria as well as the molecular basis of inhibition by an amiloride-like compound.

Rat proximal NHE3 adapts to chronic acid-base disorders but not to chronic changes in dietary NaCl intake.

In the proximal tubule, the apical Na(+)/H(+) exchanger identified as NHE3 mediates most NaCl and NaHCO(3) absorption. The purpose of this study was to analyze the long-term regulation of NHE3 during alkalosis induced by dietary NaHCO(3) loading and changes in NaCl intake. Sprague-Dawley rats exposed to a low-NaCl, high-NaCl, or NaHCO(3) diet for 6 days were studied. Renal cortical apical membrane vesicles (AMV) were prepared from treated and normal rats. Na(+)/H(+) exchange was assayed as the initial rate of (22)Na(+) uptake in the presence of an outward H(+) gradient. (22)Na(+) uptake measured in the presence of high-dose 5-(N-ethyl-N-isopropyl) amiloride was not different among models. Changes in NaCl intake did not affect NHE3 activity, whereas NaHCO(3) loading inhibited (22)Na(+) uptake by 30%. AMV NHE3 protein abundance assessed by Western blot analysis was unaffected during changes in NaCl intake. During NaHCO(3) loading, NHE3 protein abundance was decreased by 65%. We conclude that proximal NHE3 adapts to chronic metabolic acid-base disorders but not to changes in dietary NaCl intake.

Glucocorticoid modulates Na+/H+ exchange activity in vascular smooth muscle cells by nongenomic and genomic mechanisms

In vascular smooth muscle cells (VSMCs), Na+/H+ exchange (NHE) plays an important role in intracellular pH (pHi) regulation. The genomic effect of glucocorticoid (GC) on NHE activity has been suggested in VSMCs. However, the nongenomic and genomic effects of GC on NHE activity and the underlying intracellular signaling mechanisms have not yet been demonstrated in VSMCs. Also, it is not known whether there are specific surface-binding sites of GC to the plasma membrane of VSMCs. The effects of short (3 h)- and long (24 h)-term exposure to corticosterone (CORTI) on NHE activity were studied in cultured rat aortic VSMCs by using pHi measurement with the pH-sensitive fluorescent dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The NHE activity was calculated from the initial rate of Na+-dependent pHi recovery after the acid load. Short-term exposure of VSMCs to CORTI (10-6 mol/L) increased NHE activity, whereas long-term exposure to CORTI decreased it. The inhibitors of gene transcription (actinomycin D) and of protein synthesis (cycloheximide) did not affect the short-term effect of CORTI on NHE activity, but inhibited the long-term effect of CORTI on NHE activity. The cytosolic GC receptor (GR) antagonist (RU38486) inhibited both the short- and long-term effects of CORTI on NHE activity, but the cytosolic mineralocorticoid receptor antagonist (spironolactone) did not influence either the short- or long-term CORTI effects. Two protein kinase C (PKC) inhibitors (staurosporine A and calphostin C) and PKC down-regulation [24-h pre-exposure to phorbol 12-myristate 13-acetate (PMA)] inhibited both short- and long-term CORTI effects. Exposure to PMA for three hours mimicked the short-term CORTI effect. The short-term CORTI effect was inhibited by the disruptor of microtubule (colchicine), but not by the disruptor of filamentous-actin (cytochalasin B). The long-term exposure to CORTI decreased NHE (NHE-1) mRNA levels to 0.65 times the control level, whereas the short-term exposure to CORTI caused no effect. Scatchard analysis of [3H]CORTI surface binding to VSMCs showed a single class of CORTI binding sites with a Bmax of 876.2 fmol per mg of cell protein and a Kd of 12.2 nmol/L. RU38486 also inhibited [3H]CORTI surface binding to VSMCs. In VSMCs, NHE activity is stimulated by short-term exposure to CORTI, but is inhibited by long-term exposure to CORTI. The short-term stimulatory effect of CORTI on NHE activity is independent of gene transcription and protein synthesis, is mediated through the CORTI surface receptor, and occurs through a microtubule-dependent process. The long-term inhibitory effect of CORTI on NHE activity requires gene transcription and protein synthesis and occurs only through the cytosolic GR. The short- and long-term effects of CORTI on NHE activity occur via PKC activation. Therefore, CORTI differentially modulates NHE activity in VSMCs by nongenomic and genomic mechanisms.

Expression of monosaccharide transporters in the intestine of human diabetics

D-glucose and Dvgalactose are transported across the brush-border membrane, into enterocytes by the Na+Iglucose co-transporter (SGLTl). Fructose is transported from the intestinal lumen into the enterocytes by the fructose transporter (GLUTS). These monosaccharides exit the cell across the basolateral membrane by the monosaccharide transporter, GLUT2. It has been shown in rats with experimentally induced diabetes that the capacity of the small intestine to absorb D-glucose increases. This increase is mainly due to enhanced activity and abundance of SGLTl and GLUT2. No studies have been carried out to investigate if a similar situation occurs in the intestine of diabetic humans. We have assessed the expression of SGLTl, GLUTS, disaccharidases, and the structural proteins villin and l3-actin in the duodenum of healthy control individuals and patients with non-insulin dependent diabetes mellitus (NIDDM). Biopsy samples were removed, during endoscopy, from the second part of the duodenum of both control and diabetic subjects. Purified brush-border membrane vesicles (BBMV) were prepared from human biopsies using an established technique. A 4-fold increase in the abundance of SGLTI in the BBMV isolated from diabetic patients (85.2 ± 13.4 pmollmg protein, n=6) compared with controls (20.0 ± 5.6 pmollmg protein, n=6), was shown by quantitative western blotting. A similar increase in the initial rate of Na+-dependent Dsglucose transport in BBMV isolated from the intestine of diabetic patients compared with controls was also shown. The abundance of GLUTS, disaccharidases, villin and l3-actin were determined in the same BBMV by western blotting with appropriate antibodies. Our results indicate that levels of the fructose transporter, GLUTS, are also increased significantly in diabetics compared to controls. In contrast the abundance of lactase, sucrase, and the structural protein villin were only slightly elevated in diabetic patients. Total protein recoveries were equal in biopsies taken from both control and diabetic patients. We report here for the first time an increase in the capacity of the human intestine to absorb monosaccharides in NIDDM. This increase appears to be due to a combination of villus hypertrophy and a specific increase in the expression of SGLTl and GLUTS.

Aldosterone activates Na+/H+ exchange in vascular smooth muscle cells by nongenomic and genomic mechanisms

In vascular smooth muscle cells (VSMCs), Na+/H+ exchange (NHE) plays an important role in intracellular pH (pHi) regulation. Recently, nongenomic effect of aldosterone (ALDO) on NHE activity has been suggested in VSMCs. However, the nongenomic and genomic effects of ALDO on NHE and the intracellular signaling mechanisms for these effects have not fully been determined in VSMCs. The effects of short- (3 hr) and long- (24 hr) term exposure to ALDO on NHE activity were examined in cultured VSMCs from rat thoracic aortae by using single-cell pHi measurement with the pH-sensitive dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The NHE activity was calculated from the initial rate of Na+-dependent pHi recovery after acid load. The NHE activity significantly increased after short- and long-term exposure of VSMCs to ALDO (10(-6) M). The inhibitors of gene transcription (actinomycin D) and of protein synthesis (cycloheximide) had no effect on the short-term ALDO effect, but inhibited the long-term ALDO effect. The antagonists of the mineralocorticoid receptor (MR) (spironolactone) and of the glucocorticoid receptor (GR) (RU38486) caused no effect on the short-term ALDO effect, but inhibited the long-term ALDO effect. Two protein kinase C (PKC) inhibitors (staurosporine A and calphostin C) and PKC down-regulation (24 hr pre-exposure to phobol 12-myristate 13-acetate, PMA) inhibited both the short- and long-term ALDO effects. Exposure of VSMCs to PMA for 3 hours mimicked the short-term effect of ALDO on NHE activity. ALDO significantly increased PKC activity in VSMCs. The short-term ALDO effect was inhibited by disruptors of microtubule (colchicine) and of filamentous-actin (cytochalasin B). Long-term exposure of ALDO caused a threefold increase in NHE (NHE-1) mRNA levels. The short-term effect of ALDO on NHE activity is not mediated through either MR or GR, occurs independent of gene transcription and protein synthesis, and occurs through a mechanism involving the structural elements of cytoskeleton. The long-term effect of ALDO on NHE activity occurs through both MR and GR and requires gene transcription and protein synthesis. Both short- and long-term effects of ALDO are mediated through PKC activation. Therefore, ALDO activates NHE by nongenomic and genomic mechanisms in VSMCs.

Na +-H + exchange and intracellular pH regulation in colonocytes from the chick

The involvement of Na +-H + exchange in chicken colonocyte homeostasis was investigated. Colonocyte pH (pH i) was measured with 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The proton ionophore FCCP reduced basal pH i, indicating that cytosolic [H +] is not at electrochemical equilibrium across the membrane. External Na + removal decreased pH i and subsequent addition of Na + returns pH i towards its control value. The rate of pH i recovery from an acid load was Na +-dependent ( K m for Na +, 24 mM) and inhibited by EIPA (IC 50, 0.18 μM). The initial rate of Na +-dependent cell alkalization increased as the pH i decreased from 7.2 to 6.6 (Hill coefficient, 1.88). Radioisotope flux studies revealed that an outwardly directed proton gradient transiently stimulated Na + uptake into BBMV isolated from the chick colon. EIPA and amiloride inhibited pH gradient-driven Na + uptake (IC 50 of 4 μM and 32 μM, respectively). The K m for Na + of pH gradient-driven Na + uptake was 6.8 mM. The Hill coefficient of the relationship between the initial rate of pH-driven Na + uptake and the intravesicular pH was 0.70. It is concluded that a Na +-H + exchanger is involved in pH i homeostasis in chicken colonocytes and that these cells possess at least two types of Na +-H + antiporters with different sensitivity to EIPA and different kinetic parameters.

Na+/H+ antiporter (NHE-1 isoform) in cultured vascular smooth muscle from the spontaneously hypertensive rat

An increase in Na+/H+ antiporter activity may be involved in hyperproliferation of vascular smooth muscle cells (VSMC) and possibly in the vascular hyperplasia characteristic of hypertension. The present study was designed to examine cell proliferation, Na+/H+ exchange activity, and mRNA levels of the NHE-1 isoform of the Na+/H+ antiporter in cultured aortic VSMC derived from the spontaneously hypertensive rat (SHR) and from normotensive controls, the Wistar/Kyoto rat (WKY). VSMC derived from the SHR grown in early (2 to 6), but not in later (7 to 10) sub-passages, exhibited an increase in [3H]-thymidine incorporation and shorter doubling times as compared to those derived from WKY rats. Na+/H+ exchange activity assayed in the nominal absence of HCO3-/CO2, as the rate of intracellular pH (pHi) recovery after cell acidification was significantly higher in cells from SHR than in those from WKY rats when cells were studied in early sub-passages, but not in cells studied in later sub-passages. In cells grown in early sub-passage, Na+/H+ exchange activity assessed as the initial rate of Na+i accumulation following acute cell acidification was also significantly higher in SHR than WKY cells both in the nominal absence (10.22 +/- 1.15 and 6.98 +/- 1.17 mmol Na+i/90 seconds, P < 0.05, respectively) and in the presence of HCO3-/CO2 (9.94 +/- 1.02 and 5.59 +/- 0.86 mmol Na+/90 seconds, P < 0.01, respectively). There were no detectable differences in the level of steady-state Na+/H+ antiporter (NHE-1) mRNA between VSMC from SHR and WKY rats. Our findings indicate that Na+/H+ exchange activity is increased in cultured aortic VSMC derived from SHR as compared to those derived from WKY rats. The higher functional activity of the Na+/H+ antiporter in VSMC from the SHR is due to a post-transcriptional event(s) and may be related to enhanced growth in culture.

Sodium transport in erythrocytes of the mollusc Anadara broughtonii: Evidence for inhibition by quinine

Sodium transport through the molluscan erythrocyte membrane was examined using 22Na as a tracer. Incubation of the red cells in standard saline resulted in a rapid 22Na uptake reaching steady state concentration (about 21.5 mmol/l cells) in the first 60 min. A similar pattern in the time course of 22Na uptake was seen in the erythrocytes incubated in mantle fluid. The average value of unidirectional Na + influx, measured as a 5-min 22Na uptake, was 7.76 ± 0.36 mmol/1 cells/5 min or 93 ± 4.3 mmol/1 cells/hr. The initial rate of Na + influx increased in a saturable fashion as a function of external Na + concentration with apparent AT., of 380±12mM and V max of 14.3 ± 2.4 mmol/1 cells/5 min. Amiloride (1 mM), furosemide (1 mM), and DIDS (0.1 mM) had no effect on either initial Na + influx (5 min 22Na uptake) or equilibrium Na + concentration (60 min and 120min 22Na uptake) in the molluscan red cells exposed to standard saline. Quinine (1 mM) caused a significant fall in the initial Na + influx (by 48%) and in 60-min 22Na uptake (by 32%) as compared with control levels. In the presence of 0.1 mM ouabain, 22Na uptake into the red cells was enhanced by an average 27% and 44% during 60 min and 120 min of cell incubation, respectively. The ouabain-sensitive Na + accumulation in the red cells reflected a contribution of the Na, K-pump to Na + transport and the mean value was 5.6 ± 1.0 mmol/1 cells/hr.

Na+/H+ exchanger-2 is an O-linked but not an N-linked sialoglycoprotein.

A polyclonal antibody (Ab597) was produced in rabbit against a fusion protein of glutathione-S-transferase and the last 87 amino acids of the Na+/H+ exchanger isoform, NHE2. By Western blotting, Ab597 recognized proteins of 75 and 85 kDa in PS120/NHE2 membranes (PS120 cells stably transfected with NHE2), and this antibody did not cross-react with NHE1 and NHE3. When Ab597 was used to immunocytochemically stain PS120/NHE2 cells, permeabilization of the cells was required for staining, confirming the putative membrane topology of NHE2 that the C-terminus is cytoplasmic. NHE1 is N-glycosylated. NHE2 was predicted to be N-glycosylated as it contains one potential N-linked glycosylation site (N350VS), which is conserved among NHE1, NHE3, and NHE4. However, NHE2 was resistant to peptide:N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) digestion, suggesting that NHE2 is not N-glycosylated. In contrast, neuraminidase shifted the mobility of the 85 kDa NHE2 protein in PS120/NHE2 membranes into an 81 kDa band, and O-glycanase further shifted the mobility of the neuraminidase-treated 81 kDa protein to 75 kDa. Incubation of PS120/NHE2 cells with benzyl N-acetyl-alpha-D-galactosaminide (Bz alpha GalNAc), an O-glycosylation inhibitor, decreased the size of the 85 kDa protein to 81 kDa. This treatment had no effect on the initial rate of Na+/H+ exchange of PS120/NHE2 cells. The 75 kDa protein was not affected by the glycosidase treatment of PS120/NHE2 membranes or the Bz alpha GalNAc treatment of PS120/NHE2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular pH regulation in cecal epithelial cells from the chick

Intracellular pH (pH i) regulation has been investigated in cells isolated from the proximal ceca of the chicken. pH i was measured with the pH-sensitive dye, 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein in nominally HCO 3 −-free solutions. Under resting conditions the pH i was 7.08. Removal of extracellular Na + decreased pH i by approx. 0.24 pH units and the subsequent addition of Na + increased pH i towards the control value. This Na +-dependent pH i recovery was inhibited by 5-( N-ethyl - N-isopropyl)amiloride (EIPA). Following an intracellular acidification, by abrupt withdrawal of NH 4CI, pH i alkalinized in the nominally absence of Na +. Rotenone, N-ethyleimide, N, N′-dicyclohexylcarbodiimide, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, iodoacetic acid and SCH 28080 inhibited the Na +-independent pH i recovery rate by 82, 82, 67, 74, 77 and 50%, respectively. Bafilomycin A 1 was without effect. Na +-independent cell alkalization was stimulated by external K +. In the presence of N-ethylmaleimide addition of Na + induced a rapid pH i recovery. The initial rate of this recovery exhibited first-order dependence on Na + concentration and it was inhibited by EIPA. The initial rate of Na +-dependent cell alkalization increased with a Hill coefficient greater than one when pH i was reduced from 7.2 to 6.2. The ‘set point’ for the exchange is appox. 7.5. These studies demonstrate that in cecal epithelial cells exist at least two mechanisms for proton secretion: a Na +-H + exchanger and a Na +-independent proton transport system.

Glucocorticoids stimulate Na+/H+ antiporter in OKP cells.

Previous studies have demonstrated that systemic administration of glucocorticoids stimulates proximal tubule acidification in part by increasing Na+/H+ antiporter activity; however, these studies could not exclude the possibility that changes in Na+/H+ antiporter activity were secondary to glucocorticoid-induced hemodynamic changes. The present study examined the effect of dexamethasone on Na+/H+ antiporter activity in quiescent OKP cells. Na+/H+ antiporter activity was assayed as the initial rate of Na(+)-dependent pH recovery from an acid load. Intracellular pH was measured using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Dexamethasone produced a dose- and time-dependent stimulation of Na+/H+ antiporter activity in OKP cells. Dexamethasone produced a 24% stimulation in Na+/H+ antiporter activity at 10(-9) M and an approximately 40% stimulation of Na+/H+ antiporter activity at both 10(-8) and 10(-6) M. The effect of 10(-6) M dexamethasone was seen within 4 h of incubation and was due to an increase in maximal velocity (Vmax, 3.03 vs. 1.79 pH units/min) with no change in the affinity constant for sodium (KNa, 47.2 vs. 42.0 mM). The stimulatory effect of dexamethasone on Na+/H+ antiporter activity was blocked by cycloheximide and was not observed with 10(-8) M aldosterone. These data demonstrate a direct effect of glucocorticoids to stimulate Na+/H+ antiporter activity in OKP cells.

Sodium-dependent antiporters in choroid plexus epithelial cultures from rabbit.

The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na(+)-dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO3- plus 95% O2/5% CO2. Intracellular pH (pHi) was determined from the steady-state distribution of [14C]benzoate. Recovery after acidification with NH4Cl was rapid (t1/2 = 5 min) and was dependent on external Na+ (EC50 = 12 mM). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na+/H+ antiport, blocked 80% of recovery when [Na+] was 5 mM with IC50 values of 100 nM. However, neither drug blocked recovery in normal [Na+]. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of Cl-/HCO3- antiports, blocked recovery of pHi in a dose-related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na+ channel, nor with dimethylbenzamil, an inhibitor of Na+/Ca2+ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH-clamped with nigericin and KCl, the initial rate of 22Na+ uptake was very rapid (227 pmol/micrograms of DNA/min at pH 6.2), was dependent on external [Na+] with an EC50 value of 8 mM, and was inversely related to the pH of the medium. The maximal inhibition of 22Na+ uptake by hexamethyleneamiloride was 60% with an IC50 value of 76 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal Na+/H+ Exchanger Activity is Up-Regulated by Bowel Resection in the Weanling Rat

Increased Na+/H+ exchanger activity is associated with cellular hyperplasia. Cellular hyperplasia is an adaptive response to small-intestinal resection. Therefore, we hypothesized that the small-intestinal Na+/H+ exchanger activity increases in response to small-intestinal resection. Twenty-one-d-old, male Sprague-Dawley rats were randomly divided to receive either a 70% small intestinal resection (n = 59), or a mid-small intestinal transection (n = 49). Seven d postoperatively, the animals were killed and the Na+/H+ exchanger activity of the intestinal remnants was studied by a well validated brush border membrane vesicle technique. The initial rate of Na+ uptake in the presence of an outwardly directed pH gradient and the Vmax of the amiloride-sensitive Na+ uptake were significantly increased (p < 0.01 and p < 0.001, respectively) in the resection as compared with the transection remnants and to a greater magnitude in the distal as compared with the proximal remnants. Km values were not significantly different. The amiloride-sensitive Na+ uptake in the setting of various intravesicular pH was significantly greater (p < 0.001) in the distal resection as compared with the distal transection remnants, with points of enhanced Na+/H+ exchanger activity of intravesicular pH 6.62 and 6.87, respectively. The presence and activation of the Na+/H+ exchanger's internal modifier site was confirmed by demonstrating the effect of intravesicular pH on Na+ efflux. The present study demonstrates an up-regulation of intestinal Na+/H+ exchange activity in a small-bowel resection model in the weanling rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-potassium pump and sodium-calcium exchange in adult and neonatal canine cardiac sarcolemma

The purpose of this study was to determine whether the Na(+)-K+ pump and the Na(+)-Ca2+ exchanger, two systems thought to be important in excitation-contraction coupling in the heart, change during postnatal development. In adult and neonatal canine cardiac sarcolemmal preparations, Na(+)-K(+)-adenosine-triphosphatase (ATPase) activity and specific [3H]ouabain binding were found to be higher in the adult compared with the neonate, although Na(+)-K(+)-ATPase turnover numbers were not significantly different. Furthermore, ouabain association and dissociation rate constants, examined under a variety of conditions, were the same for both the neonate and adult preparations. These results suggest that the number of pump units per milligram sarcolemmal protein changes during postnatal development. In contrast, the initial rate of Na(+)-dependent-45Ca2+ influx, an index of Na(+)-Ca2+ exchange activity, was not significantly different in the two age groups. Additionally, the sensitivity of the exchanger to extravesicular Ca2+ and K+ was the same. In summary, during postnatal development sarcolemmal Na(+)-K(+)-ATPase and [3H]ouabain binding increase from neonate to adult, whereas Na(+)-Ca2+ exchange activity remains the same. This may result in a greater importance of Ca2+ flux through the Na(+)-Ca2+ exchange system in the neonate compared with the adult.

Studies on Na+ and H+ translocation through the Fo part of the Na(+)-translocating F1Fo ATPase from Propionigenium modestum: discovery of a membrane potential dependent step.

The purified ATPase of Propionigenium modestum (F1Fo) was incorporated into liposomes, and the F1 part was dissociated. The Fo-liposomes catalyzed proton uptake in response to a potassium diffusion potential (inside negative). Proton translocation was abolished by rebinding F1 to the Fo-liposomes or after incubation with the c-subunit-specific inhibitor dicyclohexylcarbodiimide (DCCD). Proton uptake was also sensitive to the presence of external Na+ or Li+ ions and was completely abolished at 2 mM NaCl or 150 mM LiCl, respectively. However, the same concentrations of these salts in the internal volume of the Fo-liposomes were without effect, suggesting that the cation binding site is not accessible from both sides of the membrane simultaneously. An open channel-type of transport through Fo from P. modestum is therefore excluded. The Fo-liposomes also catalyzed Na+ influx or efflux in response to a K+ diffusion potential that was negative on the inside or outside, respectively. These Na+ fluxes could not be created, however, by delta pNa+ of about 60-180 mV. The initial rate of Na+ uptake depended strongly on the size of the membrane potential with no significant conductivity below -40 mV, followed by a proportional increase up to about -115 mV. In the absence of a membrane potential, the Fo-liposomes catalyzed 22Na+ counterflow against a 28-fold concentration gradient. Uptake of 22Na+ into Fo-liposomes against delta pNa+ (counterflow) was completely prevented by imposing an inside-positive potassium diffusion potential of 90 mV. The catalysis of 22Na+ counterflow by Fo from P. modestum is a clear indication of a carrier (transporter)-type mechanism and excludes a channel mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature-dependent effects of cholesterol on sodium transport through lipid membranes by an ionizable mobile carrier

Temperature-jump relaxation experiments on Na + transport by (221)C 10-cryptand were carried out in order to study the influence of cholesterol and its temperature-dependence on ion transport through thin lipid membranes. The experiments were performed on large, negatively charged unilamellar vesicles (LUV) prepared from mixtures of dioleoylphosphatidylcholine, phosphatidic acid and cholesterol (mole fractions 0–0.43), at various temperatures and carrier concentrations. The initial rates of Na + transport and the apparent rate constants of its translocation by (221)C 10 increased with the carrier concentration and the temperature. The incorporation of cholesterol into the membranes significantly reduced the carrier concentration- and temperature-dependence of these two parameters. The apparent energy required to activate the transport decreased significantly with increasing carrier concentrations at any given cholesterol molar fraction, and increased significantly with the cholesterol molar fraction at any given carrier concentration. Our interpretation of the action of cholesterol on this transport system is based on the assumption that the binding cavity of cryptands is likely to be located towards the aqueous side of the dipole layer. The results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes, and of the interactions occurring between an ionizable mobile carrier and the membrane.

Presence of [formula omitted] exchange activity and its role in regulation of intracellular calcium concentration in bovine adrenal chromaffin cells

The presence of a Na + Ca 2+ exchanger in bovine adrenal chromaffin cells was demonstrated by measuring the efflux of 45Ca 2+ which had been preloaded into cells by a brief depolarization. The efflux of 45Ca 2+ was dependent on extracellular Na + (Na + o); 45Ca 2+ efflux was significantly decreased by replacing Na + o with N-methylglucamine (NMG), or Li +. Replacement of Na + o by NMG increased the resting intracellular Ca 2+ concentration ([Ca 2+] i) of freshly isolated chromaffin cells. This could be reversed by adding Na +, suggesting that Na + Ca 2+ exchanger activity was involved in maintaining [Ca 2+] i at its resting level. The initial rate of Na +-dependent [Ca 2+] i recovery after Ca 2+ loading by depolarization was dependent on the level of [Ca 2+] i. There was an apparent linear relationship between the activity of the Na + Ca 2+ exchanger and [Ca 2+] i both in the presence and absence of Na + o. When cells were treated with other stimuli, including 10 μM DMPP or 40 mM caffeine, the ability of the stimulated cells to decrease [Ca 2+] i was significantly reduced upon replacing Na + o with NMG. Our data show that the Na + Ca 2+ exchanger is one of the major pathways for regulating [Ca 2+] i in chromaffin cells in both resting and stimulated states.

PH-dependent heterogeneity of acidic amino acid transport in rabbit jejunal brush border membrane vesicles.

Initial rates of Na(+)-dependent L-glutamic and D-aspartic acid uptake were determined at various substrate concentrations using a fast sampling, rapid filtration apparatus, and the resulting data were analyzed by nonlinear computer fitting to various transport models. At pH 6.0, L-glutamic acid transport was best accounted for by the presence of both high (Km = 61 microM) and low (Km = 7.0 mM) affinity pathways, whereas D-aspartic acid transport was restricted to a single high affinity route (Km = 80 microM). Excess D-aspartic acid and L-phenylalanine served to isolate L-glutamic acid flux through the remaining low and high affinity systems, respectively. Inhibition studies of other amino acids and analogs allowed us to identify the high affinity pathway as the X-AG system and the low affinity one as the intestinal NBB system. The pH dependences of the high and low affinity pathways of L-glutamic acid transport also allowed us to establish some relationship between the NBB and the more classical ASC system. Finally, these studies also revealed a heterotropic activation of the intestinal X-AG transport system by all neutral amino acids but glycine through an apparent activation of Vmax.

Improved stability of rabbit and rat intestinal brush border membrane vesicles using phospholipase inhibitors

The initial rates of Na +-dependent d-aspartate and d-glucose uptakes were shown to decline from the time of resuspension of brush border membrane vesicles isolated from rabbit and rat jejunum by standard divalent cation precipitation procedures. The former were however more stable than the latter and followed quite closely the decrease in the intravesicular volume, thus suggeting that the loss of transport activity may involve both nonspecific opening of the vesicles and either direct or indirect specific inactivation of the transporters. Uptake rates for both substrates did tend to stabilize at 6–24 h from resuspension, however this final ‘next day’ uptake activity was too low to be of practical use in kinetic studies. Freeezing aliquots of rabbit jejunal vesicles in liquid N 2 until the time of assay resulted in complete stabilization of d-glucose uptake. A modified homogenate buffer designed to inhibit a broad spectrum of phospholipase activities resulted in a partial stabilization of glucose transport by rabbit jejunal vesicles with, on average, an over 6-fold enrichment in the ‘next day’ stable specific activity of uptake as compared to unfrozen vesicles. The modified homogenate buffer also improved the stability and the ‘next day’ specific activities of d-glucose uptake in rat jejunal brush border vesicles and d-aspartic acid uptake in rabbit jejunal vesicles. It also completely stabilized the intravesicular volume in the latter preparation. An evaluation of the kinetic parameters of Na +-dependent d-glucose transport in rabbit vesicles prepared from either the standard homogenate media and frozen in liquid N 2 or the modified media and allowed to stabilizeovernight, revealed a single transport system with a K m of 0.31–0.32 mM as the best model to fit the data. As such the modifications to the homogenate media do not appear to effect the functional properties of d-glucose transport in the membrane. While being less efficient in stabilizing the vesicles than the rapid freezing protocol, it is shown that the modified homogenate should however be preferred when dealing with slowly permeant ions like choline since it provides in this case the only alternative to reliable measurement of uptake rates across a stable and equilibrated vesicle preparation.