Maximal Transport Capacity Research Articles

A Stereospecific myo-Inositol/D-chiro-Inositol Transporter in HepG2 Liver Cells: IDENTIFICATION WITH D-CHIRO-[3-3H]INOSITOL

D-chiro-Inositol is an epimer of myo-inositol that is found in certain mammalian glycosylphosphatidylinositol protein anchors and inositol phosphoglycans possessing insulin-like bioactivity. In order to generate a probe for metabolic studies, D-chiro-[3-3H]inositol was synthesized by selective reduction of D-chiro-3-inosose at pH 6.5 with sodium borotritide. D-chiro-[3-3H]Inositol was taken up by HepG2 human liver cells through a saturable and stereospecific pathway in which D-chiro-inositol and myo-inositol competed equally but L-chiro-inositol was not recognized. Dd-Glucose, but not L-glucose, competed for D-chiro-[3-3H]inositol uptake over glucose concentrations of 4-28 mM. Maximum transport capacity was 717 pmol/mg cell protein/3 h with a Km value of 348 microM. Uptake was reduced by 76% when sodium was eliminated from the medium and by 94% when the experiment was performed at 0 degrees C. The new myo/D-chiro-inositol transporter is distinct from the sodium-myo-inositol co-transporter found in many tissues and accounts for all of the saturable D-chiro-inositol uptake and for a portion of the saturable low affinity myo-inositol uptake in HepG2 cells. It may allow D-chiro-inositol to be used by cells in the presence of a relatively large amount of competing myo-inositol.

Phorbol Ester Activation of the Rat Vascular Myocyte Na + -H + Exchanger Isoform 1

Vascular myocytes from the spontaneously hypertensive rat (SHR) demonstrate elevated Na(+)-H(+) exchanger activity associated with increased cell proliferation and hyperresponsiveness to agonists such as phorbol esters. Since the Na(+)-H(+) exchanger isoform 1 (NHE-1) is stimulated by protein kinase C, we have investigated the effects of phorbol esters on NHE-1 activity and its phosphorylation in vascular myocytes of these rats. SHR cells demonstrated a larger alkalinization response to 12-O-tetradecanoylphorbol 13-acetate than Wistar-Kyoto rat (WKY) cells. Kinetic analyses indicated that whereas 12-O-tetradecanoylphorbol 13-acetate increased the maximal transport capacity of NHE-1 in both cell types, affinity for H+ was increased in WKY cells and cooperativity for H+ at the internal modifier site was reduced in SHR cells. In neither cell type was the subcellular distribution of NHE-1 altered by phorbol ester stimulation. NHE-1 phosphorylation was markedly reduced in WKY cells stimulated by the phorbol ester, an effect abolished by inhibition of protein kinase C. In contrast, NHE-1 phosphorylation in quiescent SHR cells was approximately double that of WKY cells and was reduced after phorbol ester treatment. Inhibition of protein kinase C in SHR cells led to a marked elevation of NHE-1 phosphorylation that was not associated with a change in the exchanger activity, but WKY cells exhibited a small, insignificant rise in NHE-1 phosphorylation. Thus, the kinetic responses of NHE-1 to phorbol esters in vascular myocytes of these rat strains are different, the changes in exchanger kinetics of SHR resembling those described in human hypertension. NHE-1 phosphorylation has an inverse relationship with protein kinase C activity. However, modulation of NHE-1 phosphorylation may not be associated with concurrent alterations in activity, indicating a role for non-phosphorylation-dependent mechanisms.

Lipopolysaccharide and tumor necrosis factor stimulate lung microvascular arginine uptake, a response attenuated by dexamethasone.

Lipopolysaccharide (LPS), tumor necrosis factor-alpha, (TNF), and glucocorticoids can modulate endothelial nitric oxide (NO) production. L-Arginine is the exclusive precursor for NO biosynthesis, suggesting that NO generation and arginine transport are intimately linked. To further study this relationship, we examined the effects of LPS, TNF, and dexamethasone (DEX) on arginine uptake by rat lung microvascular endothelial cells. The transport of radiolabeled arginine was assayed in confluent cells grown in 24-well plates. The bulk (> 90%) of arginine transport was mediated by the Na(+)-independent carriers System y+ and System b0,+. Arginine transport was stimulated independently by LPS and TNF, a response first observed at 10 hours. Together, both agents exerted an additive effect on carrier-mediated uptake. The LPS- and TNF-induced increase in arginine transport activity was blocked by cycloheximide and actinomycin D, indicating the requirement for RNA and protein synthesis. The enhancement in transport activity was primarily due to an increase in Systems y+ maximal transport capacity (Vmax) with no change in transporter affinity and little change in System b0,+ activity. Treatment of cells with dexamethasone inhibited arginine transport activity in a time- and dose-dependent manner, an event that was abrogated by both actinomycin D and cycloheximide. The combination of DEX and LPS and TNF abrogated each other's antagonistic effects. These data indicate that LPS and TNF additively stimulate arginine transport in lung microvascular endothelial cells via a pathway that requires de novo protein synthesis (possibly of the transporter protein itself) and that this response is attenuated by DEX.

Insulin downregulates diabetic-enhanced intestinal glucose transport rapidly in ileum and slowly in jejunum.

The effect of insulin on intestinal sodium-dependent glucose transport and brush border membrane surface area was examined in male Sprague-Dawley rats following a 30-day period of nontreated streptozocin-induced diabetes. Nontreated diabetic rats were hyperglycemic and demonstrated increased jejunal and ileal Na-dependent glucose transport Jmax (maximal transport capacity) and Na-K ATPase activity compared with controls. Daily administration of insulin resulted in a steady decline in blood glucose levels over a period of 6 days. Jejunal Jmax was normalized after 2 days of insulin therapy, while ileal Jmax was normalized 12 h following a single insulin injection. The normalization of Na-K ATPase activity lagged behind Na-dependent glucose transport regulation by 24 h in both jejunum and ileum. Brush border surface area was increased in the ileum of diabetic rats as a result of an increase in microvillus height. Insulin treatment resulted in a decrease in ileal microvillus height to control values by 12 h, which correlated directly with the decrease in Na-dependent glucose transport. Insulin had no effect on jejunal brush border surface area. In conclusion, these findings indicate that the jejunum and ileum respond differentially to experimentally induced diabetes, and these regions also utilize different adaptive mechanisms to regulate Na-dependent glucose transport.

Molecular mechanisms of hepatic bile salt transport from sinusoidal blood into bile.

An increasingly complex picture has emerged in recent years regarding the bile salt transport polarity of hepatocytes. At the sinusoidal (or basolateral) plasma membrane two bile salt-transporting polypeptides have been cloned. The Na(+)-taurocholate-cotransporting polypeptide (Ntcp) can account for most, if not all, physiological properties of the Na(+)-dependent bile salt uptake function in mammalian hepatocytes. The cloned organic anion-transporting protein (Oatp1) can mediate Na(+)-independent transport of bile salts, sulfobromophthalein, estrogen conjugates, and a variety of other amphipathic cholephilic compounds. Hence, Oatp1 appears to correspond to the previously suggested basolateral multispecific bile sale transporter. Intracellular bile salt transport can be mediated by different pathways. Under basal bile salt flux conditions, conjugated trihydroxy bile salts bind to cytoplasmic binding proteins and reach the canalicular plasma membrane predominantly via cytoplasmic diffusion. More hydrophobic mono- and dihydroxy and high concentrations of trihydroxy bile salts associate with intracellular membrane-bound compartments, including transcytotic vesicles, endoplasmic reticulum (ER), and Golgi complex. A facilitated bile salt diffusion pathway has been demonstrated in the ER. The exact role of these and other (e.g., lysosomes, "tubulovesicular structures") organelles in overall vectorial transport of bile salts across hepatocytes is not yet known. Canalicular bile salt secretion is mediated by two ATP-dependent transport systems, one for monovalent bile salts and the second for divalent sulfated or glucuronidated bile salt conjugates. The latter is identical with the canalicular multispecific organic anion transporter, which also transports other divalent organic anions, such as glutathione S-conjugates. Potential dependent canalicular bile salt secretion has also been suggested to occur, but its exact mechanism and physiological significance remain unclear, since a potential driven bile salt uptake system has also been identified in the ER. Hypothetically, and similar to changes in cell volume, the intracellular potential could also play a role in the regulation of the number of bile salt carriers at the canalicular membrane and thereby indirectly influence the maximal canalicular bile salt transport capacity of hepatocytes.

Investigation of a Heat Pipe Array for Convective Cooling

A combined experimental and analytical investigation was conducted to evaluate a heat pipe convective cooling device consisting of sixteen small copper/water heat pipes mounted vertically in a 4 × 4 array which was 25.4 mm square. The analytical portion of the investigation focused on determination of the maximum heat transport capacity and the resistance of the individual heat pipes. The resistance of each heat pipe was found to be 2.51 K/Watt, or more than 3 times smaller than the resistance produced by a solid copper rod with the same dimensions. The maximum predicted heat rejection for the module was over 50 Watts, or a power density in excess of 7.75 Watts/cm2. In the experimental portion of the investigation, two different modules were tested. The first module utilized ten circular aluminum fins mounted on the condenser end of each heat pipe to enhance heat rejection, while the second contained only the sixteen copper/water heat pipes. The effects of flow velocity, input power, and base plate temperature on the overall thermal resistance and the heat rejection capacity were determined, as well as the pressure drop resulting from each module. The finned heat pipe array was found to have a lower overall thermal resistance and thus, a higher heat rejection capacity, but also resulted in a significantly larger pressure drop than the array without fins. The results of the heat pipe array experiments were also compared with experimental and empirical results obtained from flow over a flat plate 25.4 mm square.

Lithium Transport Pathways in Human, Chicken and Eel Erythrocytes

The contribution of four transport pathways to Li+ influx in human, chicken and eel erythrocytes was studied. All pathways were measured simultaneously in each kind of blood sample to avoid possible temporal variations in transport parameters. We found that: 1) Li+ influx via Na+-K+ pump increased 2-3 fold in the order human < eel < chicken; 2) by the countertransport exchange system lithium influx increases its variance between 15% and 35% when saline medium is replaced by choline Cl; 3) the anion exchange system (band 3 protein) shows very little interindividual variability on the lithium influx; 4) the lithium leak pathway, for human and eel red blood cells, is the major contribution,whereas it is negligible for chicken erythrocytes. It is concluded that a similar transport system exists in the red cell membranes of the three species which can transport lithium. However, the exchange system does not exhibit identical transport characteristics in the three species and shows a marked inter- and intra-species variability in maximum transport capacity and some differences in susceptibility towards the inhibitors.

Is dietary erucic acid hepatotoxic in pregnancy? An experimental study in rats and hamsters

The hypothesis that dietary erucic acid may contribute to the pathogenesis of intrahepatic cholestasis of pregnancy has been examined in pregnant rats and hamsters after prolonged feeding of diets containing 25% rapeseed oil rich in erucic acid (40% of fatty acids) or corn oil, without erucic acid. Both dietary oils were well tolerated, although weight gain was 17% to 20% less in animals receiving rapeseed oil. Rats and hamsters were studied on the last day of pregnancy and compared with age- and diet-matched nonpregnant animals. Histological examination showed no major morphologic abnormalities in liver, heart, kidneys, and adrenals. Similar microscopic deposits of fat were found in the livers and hearts of pregnant hamsters of both dietary groups. Chromatographic analysis of fatty acids in liver, heart, and kidney homogenates of hamsters and in isolated rat liver cells reflected the fatty acid composition of the dietary oils: oleic (18:1) and linoleic (18:2) acids were among the predominant fatty acids. Erucic acid was found in a higher proportion in the heart (14% by weight of total fatty acids) than in the liver (3%) and kidneys (3%) of animals fed rapeseed oil. Bile flow and biliary lipid composition was similar in rats and hamsters fed rapeseed or corn oil. Bile flow tended to be less in pregnant than in nonpregnant animals. Pregnant hamsters fed rapeseed oil tended to have the lowest bile flow. The lithogenic index of bile was slightly decreased in pregnant rats and increased in pregnant hamsters, although these proportional changes were similar for both diets. In all circumstances the lithogenic index remained below a value of 1. Biliary excretion of sulfobromophthalein (BSP) and maximal transport capacity of BSP into bile (BSP Tm) were similar in rats fed rapeseed or corn oil diets. In conclusion, no morphologic or hepatic functional abnormalities attributable to the diet could be detected in animals fed rapeseed oil with a high content of erucic acid, whereas pregnancy induced changes in bile secretion in rats and hamsters, irrespective of the diet administered. These observations are still insufficient to reject the possibility that rapeseed oil rich in erucic acid could be involved in the pathogenesis of intrahepatic cholestasis of pregnancy because dietary erucic acid could interact with a genetic metabolic predisposition to induce a disease that seems to be specific to humans.

Na + -H + Antiporter Phenotype, Abundance, and Phosphorylation of Immortalized Lymphoblasts From Humans With Hypertension

Previous studies have demonstrated an elevated Na(+)-H+ exchanger activity in various cell types from patients with essential hypertension. The phenotype of an increased maximal transport capacity is preserved in Epstein-Barr virus immortalized lymphoblasts from hypertensive patients. The mechanisms underlying this abnormality are unclear. In this study, we used lymphoblasts from hypertensive patients and normotensive control subjects with and without a family history of hypertension to determine (1) Na(+)-H+ exchanger activity using fluorometry with the pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, (2) Na(+)-H+ exchanger isoform 1 abundance with specific polyclonal antibodies, and (3) Na(+)-H+ exchanger phosphorylation by immunoprecipitation of the 32P-labeled transporter. Na(+)-H+ exchanger activity (in millimoles per liter per minute) measured when pHi was clamped at 6.0 was significantly higher in cells from hypertensive patients (18.8 +/- 0.6, P < .001) and those subjects with a family history of hypertension (16.4 +/- 0.6, P < .001) compared with normotensive control subjects (12.9 +/- 0.6). Exchanger abundance was identical in all three groups of subjects, indicating that increased activity in the hypertensive group was due to an elevated turnover number of the exchanger. Na(+)-H+ exchanger phosphorylation in quiescent cells was significantly elevated in cells from hypertensive patients (1.58 +/- 0.16, P < .001) compared with control subjects (1.00 +/- 0.07), and cells from normotensive subjects with a hypertensive family history showed intermediate values (1.23 +/- 0.14). Identical changes in Na(+)-H+ exchanger function and phosphorylation have been demonstrated in vascular smooth muscle cells from spontaneously hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Charge optimization for a triangular-shaped etched micro heat pipe

An analytical model considered to be the first analytical micro heat pipe model to predict the capillary limit of operation as well as the radius of curvature in the evaporator section and the optimal value of liquid charge was developed for a triangular-shaped, etched micro heat pipe. This model predicted optimal liquid prime values between 16-25% for various power levels and also indicated an optimal charge value which would not exceed 25%. Micro heat pipe arrays with liquid prime values ranging from 10 to 50% were used. The model was validated by comparing the predicted values of maximum heat transport capacity with experimental data at various liquid fill ratios. Although the model correctly predicted the trends associated with the variations in the fill ratio, significant differences were evident in the predicted and measured dryout level. 8 refs.

Relationship between dietary Na+ intake, aldosterone and colonic amiloride-sensitive Na+ transport

The effect of changes in dietary Na+ intake on plasma aldosterone levels and electrogenic amiloride-sensitive Na+ transport (Iamil) was studied in the rat distal colon. Five groups of rats were fed on diets containing different amounts of Na+. Estimated Na+ intake ranged from about 400-80,000 mu equiv Na+/kg body weight (BW) per d. Both variables investigated, Iamil and plasma aldosterone, depended non-linearly on Na+ intake. Reduction of the daily Na+ intake increased plasma aldosterone levels and if these levels reached the value 200 pg/ml or more then Iamil was induced. The corresponding Na+ intake was 1300 mu equiv Na+/kg BW per d. Iamil was not observed at lower aldosterone levels and higher Na+ intakes. Aldosterone infusion for 7 d produced similar changes in Iamil compared with dietary Na(+)-depleted animals and made the estimation of maximum transport capacity of Iamil possible. We conclude that Iamil operates only if Na+ intake decreases below minimal Na+ requirement in growing rats and that the maximum transport capacity of this pathway is reached only after very severe Na+ deprivation.

Aldosterone modulates sodium kinetics of Na,K-ATPase containing an alpha 1 subunit in A6 kidney cell epithelia.

Short-term aldosterone (10(-6) M, 2.5 h) induces in A6-C1 cell epithelia an increase in Na transport, which is due to the in situ activation of the apical Na channel and, presumably, the basolateral Na pump (Na,K-ATPase). We have now directly measured the effect of aldosterone on the transport activity of endogenous Na pumps and hybrid Na pumps containing an exogenous alpha 1 subunit by measuring the pump current (Ip) across epithelia apically permeabilized with amphotericin B. Aldosterone (2.5 h) had no significant early effect on the maximal Ip, nor on the Na concentration required for half-maximal activation. In contrast, it increased the Ip at physiological intracellular Na concentrations (1.7-fold at 5 mM Na). This effect was blocked by the protein synthesis inhibitor cycloheximide. Hybrid pumps containing the transfected cardiotonic steroid-resistant alpha 1 subunit of Bufo marinus were also stimulated by aldosterone (2.5 h). A long aldosterone treatment (4 days) increased the maximal Ip produced by the endogenous pumps 1.5 to 2.1-fold. In conclusion, aldosterone acts on Na pumps containing an alpha 1 subunit in two ways. During its early phase of action it stimulates their transport activity by increasing their apparent Na affinity at physiological intracellular Na concentrations. In the long term it produces an increase in the maximal transport capacity, which corresponds to the known increase in the number of Na pumps.

Secretin prevents taurocholate-induced intrahepatic cholestasis in the rat

Secretin is known to stimulate the flow of bicarbonate-rich bile from the bile-duct epithelium, but has no effect on hepatocytes. To investigate the effects of secretin on bile production during intrahepatic cholestasis, we infused secretin into rats with taurocholate-induced cholestasis. Secretin was given at 0.25 and 0.50 units.min-1.kg-1 to Wistar rats that simultaneously received a continuous infusion of taurocholic acid at above its maximum hepatic transport capacity to produce cholestasis. When taurocholic acid was infused at doses of 1.4 and 1.6 mumol.min-1.100 g b.w.-1, bile volume decreased in control rats. In contrast, the simultaneous infusion of secretin significantly increased bile flow and the biliary excretion of bile acids and bicarbonate. The serum taurocholic acid level at the end of the experiment was significantly lower in the secretin-treated groups than in the control group. These findings indicate that secretin prevents taurocholate-induced cholestasis and may enhance the biliary excretion of bile acids.

Cystine loading induces Fanconi's syndrome in rats: in vivo and vesicle studies

To better understand the link between lysosomal cystine accumulation and the renal impairment seen in cystinosis, we have studied the effect of cystine loading in vivo, on renal function of rats, and in brush-border membrane vesicles (BBMV) prepared from the kidney cortex of the treated rats. Intraperitoneal injection of cystine dimethyl ester (CDME) (400 mumol, twice a day, for 5 days) led to an increased urine volume and excretion of glucose, phosphate, and protein. Kinetic analysis of alpha-methylglucoside initial flux in BBMV showed reduction in maximal transport capacity (Vmax, from 10.1 +/- 1.3 to 8.5 +/- 0.7 nmol.min-1.mg protein-1; P < 0.01) with no change in Michaelis constant (Km, 4.80 +/- 0.08 and 4.90 +/- 0.05 mM). The number of phlorizin binding sites declined (from 6.5 +/- 0.7 to 4.1 +/- 0.4 pmol/mg protein; P < 0.01) with no significant change in the affinity for phlorizin (0.64 +/- 0.08 and 0.59 +/- 0.06 microM). In the cortex homogenate, cystine concentration, which was undetectable in controls, increased to 0.97 +/- 0.09 nmol 1/2 cystine/mg protein. Two hours after CDME administration, ATP content declined to approximately 50% of control values. This decline was transient, and ATP content was recovered to control values 5 h after CDME administration. The treatment did not affect ouabain-sensitive adenosinetriphosphatase activity (40.0 +/- 3.9 and 38.6 +/- 4.7 nmol Pi.mg protein-1.min-1) or the number and affinity of ouabain binding sites (Bmax = 1.48 +/- 0.25 and 1.44 +/- 0.18 pmol/mg, and Kd = 0.68 +/- 0.09 and 0.72 +/- 0.12 microM, respectively). (ABSTRACT TRUNCATED AT 250 WORDS)

L-thyroxine inhibits intestinal phosphate absorption in developing rabbits

Little is known about effects of l-thyroxine on phosphate absorption. To characterize effects of l-thyroxine on intestinal phosphate transport, four week old suckling rabbits were injected with l-thyroxine. 24 hours following injection, phosphate transport was studied in brush border membrane vesicles. Vesicles from l-thyroxine-treated and control animals demonstrated sodium-phosphate cotransport however uptake was significantly greater in controls than in treated animals. Controls demonstrated an “overshoot” in that peak phosphate uptake was nearly three-fold the equilibrium value whereas minimal overshoot was observed in treated animals. V max and K m for sodium-dependent phosphate transport were 230.4±6.4 pmol·mg protein −1·10s −1 and 0.034±0.05 mmol/L respectively for controls, and 97.7±4.4 pmol·mg protein −1·10s −1 and 0.023±0.006 mmol/L for treated animals. While phosphate uptake was greater in controls than in treated animals, maximal stimulation of phosphate uptake occurred between 75 and 100 mmol/L sodium in both groups. Uptake was half-maximally stimulated by 36.3±13.0 mmol/L sodium in controls, and 41.4±10.0 mmol/L sodium in treated animals. These results indicate that within 24 hours, l-thyroxine suppresses intestinal sodium-coupled phosphate transport in four week old suckling rabbits. This is due to decreased maximal transport capacity without changes in transporter affinity for phosphate or sodium.

Oral Vanadate Reduces Na+-Dependent Glucose Transport in Rat Small Intestine

The effects of oral vanadate supplementation on intestinal morphometry and glucose transport were examined in STZ-induced diabetic and age-matched control male Sprague-Dawley rats. Animals received 0.1 mg/ml vanadium pentoxide in their drinking water over 14 days. Vanadate reduced intestinal glucose maximal transport capacity in both diabetic and control animals. In jejunum tissue, this decrease in glucose absorption was a direct consequence of downregulation of the glucose carrier and was not related to changes in mucosal morphometry. In the ileum tissue of control animals, the vanadate-induced decrease in glucose maximal transport capacity occurred in conjunction with an increase in carrier affinity and mucosal morphometric measurements. In the ileum tissue of diabetic animals, the vanadate-induced decrease in glucose maximal transport capacity occurred with a decrease in mucosal morphometric measurements. Na(+)-K(+)-adenosine triphosphatase activity was affected by vanadate only in diabetic animals. These results demonstrate that oral vanadate supplementation results in downregulation of the small intestinal sodium-dependent glucose carrier in both diabetic and nondiabetic rats. Furthermore, the vanadate effect may be occurring at the cellular level.

Transport of tricarballylate by intestinal brush-border membrane vesicles from steers.

Tricarballylic acid is a non-metabolizable rumen bacterial fermentation product of the naturally occurring tricarboxylic acid trans-aconitic acid. The aim of the present study was to investigate intestinal absorption of tricarballylate using brush-border membrane vesicles (BBMVs) isolated from the proximal jejunum of steers by a Ca2+ precipitation method with subsequent differential centrifugation. Transport of tricarballylate was investigated indirectly (influence of tricarballylate on the uptake of 14C-labelled citrate) as well as directly (uptake of 3H-labelled tricarballylate). Citrate as well as tricarballylate uptake (at a concentration of 0.05 mmol l-1) was strongly stimulated by an inwardly directed initial Na+ gradient. Furthermore, transport of both tricarboxylates under Na+ gradient conditions was clearly enhanced by lowering the extravesicular pH from 7.8 to 5.6. The imposition of an inwardly directed H+ gradient (pH(out)/pH(in) = 5.6/7.8) further enhanced the intravesicular accumulation of citrate as well as of tricarballylate compared with pH(out)/pH(in) = 5.6/5.6. Unequivocal evidence for a common transport site for tricarballylate and citrate was obtained from 'cis-inhibition' and 'trans-stimulation' of Na(+)-dependent citrate uptake by tricarballylate. In further experiments the influence of different substances on the uptake of 3H-labelled tricarballylate was evaluated. Unlabelled tricarballylate, citrate, succinate as well as trans- and cis-aconitate significantly inhibited the accumulation of 3H-labelled tricarballylate by BBMVs. Tricarballylate uptake as a function of the tricarballylate concentration revealed a Na(+)-dependent saturable component (apparent kinetic parameters: maximal transport capacity (Vmax) = 119 pmol (mg protein)-1 (3s)-1; affinity constant (Km) = 0.097 mmol l-1) and a Na(+)-independent diffusional component (diffusion constant: 169 nl (mg protein)-1 (3s)-1). It is concluded that tricarballylate and citrate are transported across the intestinal brush-border membrane by a common, Na(+)-dependent transport mechanism. The stimulatory influence of a low extravesicular pH most probably indicates that the protonated forms of tricarboxylates are better transported than the trivalent species.

Epithelial ion transport in the ileal J-pouch after proctocolectomy in the rat.

In ulcerative colitis and polyposis coli, creation of an ileal pouch with ileoanal anastomosis after proctocolectomy has become a common surgical method. The aim of our study was to characterize the adaptation of the epithelial ion transport function in the pouch by using electrophysiologic techniques. Proctocolectomy and ileoanal anastomosis was performed in rats either with (pouch) or without (control) creation of an ileal J-pouch. To characterize the epithelial barrier function, impedance analysis was performed 6 months after surgery. Epithelial resistance was 29 +/- 2 omega.cm2 in controls and was unchanged in the pouch (28 +/- 4 omega.cm2; NS). In contrast, subepithelial resistance increased from 33 +/- 3 omega.cm2 to 54 +/- 5 omega.cm2 (P < 0.01) owing to work hypertrophy of the muscle layers in the pouch. To detect changes in net ion transport, ion tracer fluxes were measured in the Ussing chamber. In control ileum, absorptive net Na and Cl fluxes of similar magnitude were present, indicating that electroneutral NaCl absorption was the predominant ion transport system. Neither Isc nor net Na and Cl fluxes were significantly altered in the pouch. Glucose-coupled Na absorption was measured as the 3-o-methyl-glucose-induced increase in Isc. Km remained unaltered, while Vmax decreased from 7.5 +/- 2.1 mu eq.h-1 cm-2 in controls to 1.7 +/- 0.8 mu eq.h-1 cm-2 (P < 0.05) in the pouch. Then, maximal transport capacity for electrogenic Cl secretion was measured as the Cl-induced increase in Isc blockable by serosal bumetanide (in the presence of theophylline and prostaglandin E1).(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of L-alanine transport systems in brush-border membrane vesicles from rat placenta during late gestation.

The placental uptake of L-alanine was studied by using purified brush-border membrane vesicles from rat trophoblasts. Saturation curves were carried out at 37 degrees C in buffers containing 100 mM (zero-trans)-NaSCN, -NaCl, -KSCN, -KCl, or -N-methyl-D-glucamine gluconate. The uncorrected uptake results were fitted by non-linear regression analysis to an equation involving one diffusional component either one or two saturable Michaelian transport terms. In the presence of NaCl, two distinct L-alanine transport systems were distinguished, named respectively System 1 (S-1; Vm1 about 760 pmol/s per mg of protein; KT1 = 0.5 mM) and System 2 (S-2; Vm2 about 1700 pmol/s per mg; KT2 = 9 mM). In contrast, in the presence of K+ (KCl = KSCN) or in the absence of any alkali-metal ions (N-methyl-D-glucamine gluconate), only one saturable system was apparent, which we identify as S-2. When Na+ is present, S-1, but not S-2, appears to be rheogenic, since its maximal transport capacity significantly increases in the presence of an inside-negative membrane potential, created either by replacing Cl- with the permeant anion thiocyanate (NaSCN > NaCl) or by applying an appropriate K+ gradient and valinomycin. alpha-(Methylamino)isobutyrate (methyl-AIB) appears to be a substrate of S-1, but not of S-2. For reasons that remain to be explained, however, methyl-AIB inhibits S-2. We conclude that S-1 represents a truly Na(+)-dependent mechanism, where Na+ behaves as an obligatory activator, whereas S-2 cannot discriminate between Na+ and K+, although its activity is higher in the presence of alkali-metal ions than in their absence (Na+ = K+ > N-methyl-D-glucammonium ion). S-2 appears to be fully developed 2 days before birth, whereas S-1 undergoes a capacity-type activation between days 19.5 and 21.5 of gestation, i.e. its apparent Vmax. nearly doubles, whereas its KT remains constant.

Intracellular pH and Na+/H+ antiport activity of cultured skin fibroblasts from diabetics

Increased leucocyte Na+/H+ antiport activity has previously been demonstrated in both hypertensive subjects and Type 1 diabetic patients with nephropathy and may indicate a predisposition to hypertension in such diabetic patients. We have studied intracellular pH and Na+/H+ antiport activity in cultured skin fibroblasts from diabetic patients with and without nephropathy, together with non-diabetic controls to assess if such differences persisted in cultured cells. Fibroblasts from diabetic patients with nephropathy were significantly more alkaline [median (range): 6.90 (6.82 to 7.07)] compared to both normoalbuminuric diabetic patients [6.81 (6.75 to 6.89)] or normal controls [6.82 (6.77 to 6.93)] (P < 0.001 for both). This was associated with a raised Na+/H+ antiport activity in cells from patients with nephropathy when intracellular pH (pHi) was clamped to pH 6.5, without any differences in the maximal transport capacity of the antiport at pHi 6.2. Using both intracellular pH and Na+/H+ antiport activity at pHi 6.5, patients with nephropathy were separated from uncomplicated subjects with a sensitivity of 92% and a specificity of 100%. In conclusion, the raised Na+/H+ antiport activity in cells from patients with diabetic nephropathy persists despite passaging in vitro, thus indicating a heritable component, and results mainly from an increased apparent affinity of the antiport for intracellular H+.