Overexpression of non-functional LAT1/4F2hc in renal proximal tubular epithelial cells from the spontaneous hypertensive rat.

The present study examined the expression of type 1 L-amino acid transporter (LAT1) and its associated glycoprotein 4F2hc in freshly isolated renal proximal tubules and immortalized renal proximal tubular epithelial (PTE) cells from spontaneously hypertensive (SHR) and normotensive (WKY) rats. The study also examined the inward and outward transport of [(14)C]-L-leucine, the preferred substrate of LAT1. The abundance of LAT1 and 4F2hc was greater in SHR than in WKY, both in freshly isolated renal proximal tubules and immortalized renal proximal tubular cells. In the absence of extracellular Na(+) the BCH (2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid)-sensitive [(14)C]-L-leucine uptake in SHR PTE cells was approximately 50% that observed in WKY PTE cells (77+/-4 vs 164+/-7 pmol/mg protein). In the absence of extracellular Na(+) the affinity of the transporter for the substrate in WKY PTE cells was 7.7-fold that in SHR cells, as evidenced by lower K(0.5) values. Gene silencing with a LAT1 siRNA and a 4F2hc siRNA significantly reduced LAT1 and 4F2hc expression, which was accompanied by a marked reduction in Na(+)-independent [(14)C]-L-leucine uptake in both SHR and WKY PTE cells. The spontaneous and L-leucine-stimulated outward transfer of [(14)C]-L-leucine was Na(+)-independent in both SHR and WKY PTE cells. The spontaneous [(14)C]-L-leucine efflux was higher in WKY than in SHR PTE cells and the potency of L-leucine to stimulate [(14)C]-L-leucine efflux in WKY (EC(50) = 9 microM) was greater than in SHR PTE cells (EC(50) = 41 microM). It is concluded that the SHR kidney overexpress LAT1/4F2hc units which display low affinity for L-leucine transport.     

Functional regulation of Na+-dependent neutral amino acid transporter ASCT2 by S-nitrosothiols and nitric oxide in Caco-2 cells

We describe the regulation mechanisms of the Na(+)-dependent neutral amino acid transporter ASCT2 via nitric oxide (NO) in the human intestinal cell line, Caco-2. Exposure of Caco-2 cells to S-nitrosothiol, such as S-nitroso-N-acetyl-DL-penicillamine (SNAP) and S-nitrosoglutathione, and the NO-donor, NOC12, concentration- and time-dependently increased Na(+)-dependent alanine uptake. Kinetic analyses indicated that SNAP increases the maximal velocity (V(max)) of Na(+)-dependent alanine uptake in Caco-2 cells without affecting the Michaelis-Menten constant (K(t)). The stimulatory effect was partially eliminated by actinomycin D and cycloheximide. Increased Na(+)-dependent alanine uptake by SNAP was partially abolished by the NO scavengers, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide sodium salt (carboxy-PTIO) and N-(dithiocarboxy)sarcosine disodium salts (DTCS), as well as the NADPH oxidase inhibitor, diphenyleneiodonium. RT-PCR revealed that Caco-2 cells expressed the Na(+)-dependent neutral amino acid transporter ASCT2, but not the other Na(+)-dependent neutral amino acid transporters ATB(0,+) and B(0)AT1. These results suggested that functional up-regulation of ASCT2 by SNAP might be partially associated with an increase in the density of transporter protein via de novo synthesis.     

H2 O2 stimulates Cl- /HCO 3- exchanger activity through oxidation of thiol groups in immortalized SHR renal proximal tubular epithelial cells

Cl(-) /HCO (3)(-) exchanger and Na(+) /H(+) exchanger 3 are the main transporters responsible for NaCl reabsorption in kidney proximal tubules (PT). It is well accepted that membrane exchangers can be regulated by reactive oxygen species (ROS). In the kidney, ROS are known to contribute to decreases in Na(+) excretion and consequently increase blood pressure. The present study investigated mechanisms by which H(2) O(2) -induced stimulation of Cl(-) /HCO (3)(-) exchanger activity is enhanced in proximal tubular epithelial (PTE) cells immortalized from spontaneously hypertensive rats (SHR) as compared to normotensive Wistar Kyoto (WKY). H(2) O(2) decreased K(m) values for Cl(-) /HCO (3)(-) exchanger activity in SHR PTE cells, but had no effect on the kinetic parameters in WKY cells. DTDP stimulated in a concentration-dependent manner Cl(-) /HCO (3)(-) exchanger activity in both cell lines, but SHR PTE cells were 2.4-fold more responsive to this oxidant. In contrast, thimerosal had no effect on exchanger activity in both cell lines. The effects of H(2) O(2) and DTDP upon the exchanger activity were blocked by DTT in WKY and SHR PTE cells. Similar to H(2) O(2), DTDP decreased K(m) values for Cl(-) /HCO (3)(-) exchanger activity in SHR PTE cells. Basal content of free thiol groups was higher in WKY PTE cells than in SHR. Upon H(2) O(2) treatment the free thiol groups decreased in both cell lines; however, this decrease was more pronounced in WKY cells. In conclusion, in SHR PTE cells H(2) O(2) stimulates Cl(-) /HCO (3)(-) exchanger activity via modification of thiol groups of intracellular and/or transmembrane protein. Furthermore, the thiol oxidation-dependent pathway also increases the HCO (3)(-) affinity in SHR PTE cells.     

The l-isomer-selective transport of aspartic acid is mediated by ASCT2 at the blood-brain barrier

Aspartic acid (Asp) undergoes l-isomer-selective efflux transport across the blood-brain barrier (BBB). This transport system appears to play an important role in regulating l- and d-Asp levels in the brain. The purpose of this study was to identify the responsible transporters and elucidate the mechanism for l-isomer-selective Asp transport at the BBB. The l-isomer-selective uptake of Asp by conditionally immortalized mouse brain capillary endothelial cells used as an in vitro model of the BBB took place in an Na+- and pH-dependent manner. This process was inhibited by system ASC substrates such as l-alanine and l-serine, suggesting that system ASC transporters, ASCT1 and ASCT2, are involved in the l-isomer selective transport. Indeed, l-Asp uptake by oocytes injected with either ASCT1 or ASCT2 cRNA took place in a similar manner to that in cultured BBB cells, whereas no significant d-Asp uptake occurred. Although both ASCT1 and ASCT2 mRNA were expressed in the cultured BBB cells, the expression of ASCT2 mRNA was 6.7-fold greater than that of ASCT1. Moreover, immunohistochemical analysis suggests that ASCT2 is localized at the abluminal side of the mouse BBB. These results suggest that ASCT2 plays a key role in l-isomer-selective Asp efflux transport at the BBB.     

Active transport of alanine by the neutral amino-acid exchanger ASCT1

ASCT1 protein is a member of the glutamate transporter superfamily, which shows system ASC selectivity and properties and has been characterized as a Na+-dependent neutral amino-acid exchanger. Here, by using ASCT1-expressing oocytes, the uptake of alanine and glutamate was measured to investigate ASCT1's ability to mediate a concentrative transport of alanine, ASCT1's sodium dependence, and the influence of pH on the mutual inhibition between alanine and glutamate. Alanine uptake was measured after 30 min incubation. Kinetic analysis of the Na+ dependence of alanine uptake showed an apparent K0.5 (affinity constant) value for Na+ of 23.1 +/- 4.3 mM (mean +/- SE). Concentration dependence of alanine uptake was tested at 100 and 1 mM Na+, with apparent K0.5 values of 0.16 +/- 0.04 and 1.8 +/- 0.4 mM, respectively, at pH 7.5, and 0.21 +/- 0.06 and 1.9 +/- 0.3 mM at pH 6. Vmax was not modified between 100 and 1 mM Na+ at either pH. ASCT1 actively transports alanine and accumulates it in the cytosol even when the Na+ concentration in the medium was as low as 1-3 mM. 22Na uptake studies revealed that Na+ transport was stimulated by the presence of alanine in the medium. Our results demonstrate that ASCT1 is able to mediate a concentrative transport of alanine, which is Na+-dependent but not coupled to the Na+ gradient.     

Intestinal calcium transport in spontaneously hypertensive rats (SHR) and their genetically matched WKY rats

Calcium transport across the basolateral membranes of the enterocyte represents the active step in calcium translocation. This step occurs by two mechanisms, an ATP-dependent pump and a Ca2+/Na+ exchange process. These studies were designed to investigate these two processes in jejunal basolateral membrane vesicles (BLMV) of the spontaneously hypertensive rats (SHR) and their genetically matched controls, Wistar-Kyoto (WKY) rats. The ATP-dependent calcium uptake was stimulated several-fold compared with no ATP condition in both SHR and WKY, but no differences were noted between rate of calcium uptake in SHR and WKY. Kinetics of ATP-dependent calcium uptake at concentrations between 0.01 and 1.0 microM revealed a Vmax of 0.67 +/- 0.03 nmol/mg protein/20 sec and a Km of 0.2 +/- 0.03 microM in SHR and Vmax of 0.69 +/- 0.12 and a Km of 0.32 +/- 0.14 microM in WKY rats. Ca2+/Na+ exchange in jejunal BLMV of SHR and WKY was investigated in two ways. First, sodium was added to the incubation medium (cis-Na+). Second, Ca2+ efflux from BLMV was studied in the presence of extravesicular Na+ (trans-Na+). Both studies suggest a decreased exchange of calcium and Na+. Kinetic parameters of Na(+)-dependent Ca2+ uptake at concentrations between 0.01 and 1.0 microM exhibited Vmax of 0.05 +/- 0.01 nanmol/mg protein/5 sec and a Km of 0.21 +/- 0.13 microM in SHR and Vmax of 0.11 +/- 0.02 nanmol/mg protein/5 sec and a Km of 0.09 +/- 0.05 in WKY, respectively. These results confirm that the intestinal BLMV of SHR and WKY rats have two mechanisms for calcium extrusion, an ATP-dependent Ca2+ transport process and a Na+/Ca2+ exchange process. The ATP-dependent process appears to be functional in SHR; however, the Ca2+/Na+ exchange mechanism appears to have a marked decrease in its maximal capacity. These findings suggest that calcium extrusion via Ca2+/Na+ is impaired in the SHR, which may lead to an increase in intracellular calcium concentration. These findings may have relevance to the development of hypertension.     

Characterization of nucleoside transport systems in cultured rat epididymal epithelium

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.     

A phosphorylated phloretin derivative. Synthesis and effect on intestinal Na(+)-dependent phosphate absorption

2'-Phosphophloretin (2'-PP), a phosphorylated derivative of the plant chalcone, was synthesized. The effect of 2'-PP, on Na(+)-dependent phosphate uptake into intestinal brush-border membrane vesicles (BBMV) isolated from rabbit and rat duodenum and jejunum was examined. 2'-PP decreased Na(+)-dependent phosphate uptake into rabbit BBMV with an IC(50) of 55 nM and into rat BBMV with an IC(50) of 58 nM. 2'-PP did not affect Na(+)-dependent glucose, Na(+)-dependent sulfate, or Na(+)-dependent alanine uptake by rabbit intestinal BBMVs. 2'-PP inhibition of rabbit intestinal BBMV Na(+)-dependent phosphate uptake was sensitive to external phosphate concentration, suggesting that 2'-PP inhibition of Na(+)-dependent phosphate uptake was competitive with respect to phosphate. Binding of [(3)H]2'-PP to rabbit intestinal BBMV was examined. Binding of [(3)H]2'-PP was Na(+)-dependent with a K(0.5) for Na(+)(Na(+) concentration for 50% 2'-PP binding) of 30 mM. The apparent K(s) for Na(+)-dependent [(3)H]2'-PP binding to rabbit BBMVs was 58 nM in agreement with the IC(50) for 2'-PP inhibition of Na(+)-dependent phosphate uptake. These results indicate that 2'-PP bound to rabbit or rat intestinal BBMV Na(+)-phosphate cotransporter and inhibited Na(+)-dependent phosphate uptake. In rats treated with 2'-PP by daily gavage, the effect of 2'-PP on serum phosphate, serum glucose, and serum calcium was examined. In a concentration-dependent manner, 2'-PP reduced serum phosphate by 45% 1 wk after starting treatment. 2'-PP did not alter serum calcium or serum glucose. The apparent IC(50) for 2'-PP in vivo was 3 microM.     

Transport of univalent cations across the erythrocyte membrane of hypertensive rats of various ages

In the erythrocytes incubated at low temperature (3-6 degrees C), the uptake of Li+ in 6- and 16-week old spontaneously hypertensive rats (SHR) was significantly higher than in the normotensive rats (WKY) of the same age. During the incubation of cells at 37 degrees C no difference occurred in either ouabain-sensitive or ouabain-resistant fluxes of Rb+, Na+ and Li+ between the 16-week old SHR and the WKY. K+ efflux from the erythrocytes at 3 degrees C was consistently stimulated after addition to the incubation medium of 1 mmol/l Ca2+. The value of Ca2+-dependent K+-transport was significantly elevated in 16-week old SHR than in the WKY, but there was no difference in 6-week old rats. Propranolol-induced Ca2+-dependent K+ efflux from the cells at 22 degrees C was markedly higher in 6- and 16-week old SHR as compared with the WKY. The results provide a further evidence in favor of the hypothesis on the existence of a "membrane defect" in red blood cells in the SHR.     

Comparative changes in the blood-brain barrier and cerebral infarction of SHR and WKY rats

Hypertension is involved in the exacerbation of stroke. It is unclear how blood-brain barrier (BBB) tight-junction (TJ) and ion transporter proteins critical for maintaining brain homeostasis contribute to cerebral infarction during hypertension development. In the present study, we investigated cerebral infarct volume following permanent 4-h middle cerebral artery occlusion (MCAO) and characterized the expression of BBB TJ and ion transporter proteins in brain microvessels of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats at 5 wk (prehypertension), 10 wk (early-stage hypertension), and 15 wk (later-stage hypertension) of age. Hypertensive SHR show increased infarct volume following MCAO compared with WKY control rats. BBB TJ and ion transporter proteins, known to contribute to edema and fluid volume changes in the brain, show differential protein expression patterns during hypertension development. Western blot analysis of TJ protein zonula occludens-2 (ZO-2) showed decreased expression, while ion transporter, Na(+)/H(+) exchanger 1 (NHE-1), was markedly increased in hypertensive SHR. Expression of TJ proteins ZO-1, occludin, actin, claudin-5, and Na(+)-K(+)-2Cl(-) cotransporter remain unaffected in SHR compared with control. Selective inhibition of NHE-1 using dimethylamiloride significantly attenuated ischemia-induced infarct volume in hypertensive SHR following MCAO, suggesting a novel role for NHE-1 in the brain in the regulation of ischemia-induced infarct volume in SHR.     

Na(+)-coupled transport of L-carnitine via high-affinity carnitine transporter OCTN2 and its subcellular localization in kidney

The mechanism of Na(+)-dependent transport of L-carnitine via the carnitine/organic cation transporter OCTN2 and the subcellular localization of OCTN2 in kidney were studied. Using plasma membrane vesicles prepared from HEK293 cells that were stably transfected with human OCTN2, transport of L-carnitine via human OCTN2 was characterized. Uptake of L-[(3)H]carnitine by the OCTN2-expressing membrane vesicles was significantly increased in the presence of an inwardly directed Na(+) gradient, with an overshoot, while such transient uphill transport was not observed in membrane vesicles from cells that were mock transfected with expression vector pcDNA3 alone. The uptake of L-[(3)H]carnitine was specifically dependent on Na(+) and the osmolarity effect showed that Na(+) significantly influenced the transport rather than the binding. Changes of inorganic anions in the extravesicular medium and of membrane potential by valinomycin altered the initial uptake activity of L-carnitine by OCTN2. In addition, the fluxes of L-carnitine and Na(+) were coupled with 1:1 stoichiometry. Accordingly, it was clarified that Na(+) is coupled with flux of L-carnitine and the flux is an electrogenic process. Furthermore, OCTN2 was localized on the apical membrane of renal tubular epithelial cells. These results clarified that OCTN2 is important for the concentrative reabsorption of L-carnitine after glomerular filtration in the kidney.     

Characterisation and cloning of a Na(+)-dependent broad-specificity neutral amino acid transporter from NBL-1 cells: a novel member of the ASC/B(0) transporter family

Na(+)-dependent neutral amino acid transport into the bovine renal epithelial cell line NBL-1 is catalysed by a broad-specificity transporter originally termed System B(0). This transporter is shown to differ in specificity from the B(0) transporter cloned from JAR cells [J. Biol. Chem. 271 (1996) 18657] in that it interacts much more strongly with phenylalanine. Using probes designed to conserved transmembrane regions of the ASC/B(0) transporter family we have isolated a cDNA encoding the NBL-1 cell System B(0) transporter. When expressed in Xenopus oocytes the clone catalysed Na(+)-dependent alanine uptake which was inhibited by glutamine, leucine and phenylalanine. However, the clone did not catalyse Na(+)-dependent phenylalanine transport, again as in NBL-1 cells. The clone encoded a protein of 539 amino acids; the predicted transmembrane domains were almost identical in sequence to those of the other members of the B(0)/ASC transporter family. Comparison of the sequences of NBL-1 and JAR cell transporters showed some differences near the N-terminus, C-terminus and in the loop between helices 3 and 4. The NBL-1 B(0) transporter is not the same as the renal brush border membrane transporter since it does not transport phenylalanine. Differences in specificity in this protein family arise from relatively small differences in amino acid sequence.     

Ultrastructural and functional changes in the jejunal epithelium of spontaneously hypertensive rats

Ultrastructural studies on the epithelium, sugar transport and immunocytochemistry of Na+-glucose cotransporter (SGLT1) were carried out in the jejunum of Spontaneously Hypertensive Rats (SHR) and their normotensive genetic control, Wistar-Kyoto (WKY) rats. Electron microscopy studies showed a regular brush-border membrane in the jejunal enterocytes of WKY rats, with colloidal gold particles, representing SGLTI, localized at the microvilli of the absorptive epithelial cells. However, a patchy loss of microvilli was detected in the jejunal sections from SHR, with no presence of colloidal gold particles, indicating the absence of the SGLT1 protein. Most adjacent microvilli were normal in size like those found in WKY rats, and SGLT1 labeling was observed. All these changes were accompanied by a reduction in Na+-dependent D-glucose and D-galactose uptakes in the jejunal BBMVs isolated from SHR, when compared to WKY rats. We conclude that ultrastructural changes were paralleled by modifications in the sugar transport and in the localization of SGLT1 in the jejunal epithelium of SHR.     

Nucleoside transport in L1210 murine leukemia cells. Evidence for three transporters

L1210 murine leukemia cells have two nucleoside transport activities that differ in their sensitivity to nitrobenzylmercaptopurine riboside (NBMPR). This study re-examines NBMPR-insensitive nucleoside transport in these cells and finds that it is mediated by two components, one Na(+)-dependent and the other Na(+)-independent. A mutant selected previously for loss of NBMPR-insensitive transport lacks only the Na(+)-independent activity. When NBMPR is used to block efflux via the NBMPR-sensitive transporter, uptake of formycin B (a nonmetabolized analog of inosine) is concentrative in both the parental and mutant cells, but the intracellular concentration of the nucleoside is 5-fold lower in the parental cells. Decreased accumulation of formycin B in the parental cells is due to efflux of the nucleoside via the NBMPR-insensitive, Na(+)-independent transporter that the mutant lacks. The Na(+)-dependent transporter appears to accept most purine, but not pyrimidine, nucleosides as substrates. Two exceptions are uridine, a good substrate, and 7-deazaadenosine, a poor substrate. In contrast, all of the nucleosides tested are substrates for the Na(+)-independent transporter. We conclude that L1210 cells have three distinct nucleoside transporters and that the specificity of the Na(+)-dependent transporter is similar to that of one of the two Na(+)-dependent nucleoside transporters seen in mouse intestinal epithelial cells.     

Expression of rat liver Na+/L-alanine co-transport in Xenopus laevis oocytes. Effect of glucagon in vivo.

Poly(A)+ RNA (mRNA) isolated from rat liver was injected into Xenopus laevis oocytes, and expression of Na+/L-alanine transport was assayed by measuring Na(+)-dependent uptake of L-[3H]alanine. Expression of Na+/L-alanine transport was detected 3-7 days after mRNA injection, and was due to an increment of the Na(+)-dependent component. After injection of 40 ng of total mRNA, Na(+)-dependent uptake of L-alanine was 2.5-fold higher than in water-injected oocytes. In contrast with Na+/L-alanine transport by water-injected oocytes, expressed Na+/L-alanine transport was inhibited by N-methylaminoisobutyric acid, was inhibited by an extracellular pH of 6.5 and was saturated at approx. 1 mM-L-alanine. After sucrose-density-gradient fractionation, highest expression of Na+/L-alanine uptake was observed with mRNA of 1.9-2.5 kb in length. Compared with mRNA isolated from control rats, mRNA isolated from glucagon-treated rats showed a approx. 2-fold higher expression of Na+/L-alanine transport. The results demonstrate that both liver Na+/L-alanine transport systems (A and ASC) can be expressed in X. laevis oocytes. Furthermore, the data obtained with mRNA isolated from glucagon-treated rats suggest that glucagon regulates liver Na+/L-alanine transport (at least in part) via the availability of the corresponding mRNA.     

Sodium-dependent, concentrative nucleoside transport in Walker 256 rat carcinosarcoma cells

Nucleoside transport in Walker 256 cells was reexamined using formycin B, a nonmetabolized analog of inosine. In the presence of dipyridamole to inhibit the equilibrative (facilitated diffusion) transporter previously described in these cells, the initial rate of uptake of 1 microM formycin B was 10-fold greater in Na(+)-containing medium than in Na(+)-free medium. In the presence of Na+ and dipyridamole the intracellular concentration of formycin B exceeded that in the medium within one min and was 6-fold greater than that of the medium by 5 min. Na(+)-dependent transport of formycin B was inhibited by low concentrations of inosine, but not thymidine. Furthermore, Na(+)-dependent transport of uridine, but not thymidine, was apparent in the presence of dipyridamole. These data indicate that Walker 256 cells have, in addition to the previously described equilibrative transporter, a concentrative nucleoside transporter. The specificity of this transporter appears to correspond to one of the two Na(+)-dependent transporters previously described in mouse intestinal epithelial cells.     

Intestinal brush border calcium uptake in spontaneously hypertensive rats and their genetically matched WKY rats

The current studies were designed to characterize calcium transport by intestinal brush border membrane in the spontaneously hypertensive rat (SHR) and normotensive control, the Wistar-Kyoto (WKY) rat. The biochemical and functional purity of the intestinal brush border membranes in SHR and WKY rats was validated by marker enzymes and the ability to transiently transport D-glucose in the presence of Na+ gradient. Calcium transport into duodenal and jejunal vesicles represented a minor binding component and transmembrane movement as evident by initial rate studies, A23187 studies, and lanthanum displacement experiments. Initial rate and time course of calcium uptake was lower in SHR compared with WKY rats. Kinetic analysis of calcium uptake by the jejunum (total uptake minus binding component) showed a Vmax of 6.98 +/- 0.2 and 1.8 +/- 0.2 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.001), whereas Km values were 0.76 +/- 0.04 and 0.87 +/- 0.1 mM for WKY rats and SHR, respectively. Similar kinetic analysis of calcium uptake by the duodenal segments showed a Vmax of 10.3 +/- 0.8 and 2.8 +/- 0.2 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.01). Km values were 0.7 +/- 0.2 and 0.3 +/- 0.06 mM (P greater than 0.05). Vmax of calcium uptake in the 2-week-old rats (prehypertensive period) was 6.0 +/- 0.3 and 3.53 +/- 0.3 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.001), whereas Km values were 0.60 +/- 0.07 and 0.5 +/- 0.01 mM, respectively. These results suggest that calcium binding and uptake by duodenal and jejunal intestinal brush border membranes of SHR is significantly decreased compared with WKY rats. The decrease in transmembrane calcium uptake is secondary to decrease in Vmax and is present before the appearance of hypertension, implying a genetically determined defect in calcium uptake in intestinal brush border membranes of the SHR.