Functional coupling of the calcium pump and glucose 6-phosphatase activity in liver microsomes: preliminary results

The addition of G-6-Pi to the incubation system for MgATP-dependent calcium transport in liver microsomes results in a marked stimulation of Ca2+ uptake. At physiological pH values (7.2-7.4), the G-6-Pi stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. In the system for the G-6-Pi-stimulated calcium uptake, G-6-Pi is actively hydrolyzed by the glucose 6-phosphatase activity of liver microsomes. Such an activity is not influenced by the concomitant calcium uptake. After the incubation of the system for the MgATP-dependent microsomal calcium transport in the presence of G-6-Pi, Pi and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose 6-phosphatase multicomponent system.     

Calcium sequestration activity in rat liver microsomes. Evidence for a cooperation of calcium transport with glucose-6-phosphatase

Mechanisms regulating the energy-dependent calcium sequestering activity of liver microsomes were studied. The possibility for a physiologic mechanism capable of entrapping the transported Ca2+ was investigated. It was found that the addition of glucose 6-phosphate to the incubation system for MgATP-dependent microsomal calcium transport results in a marked stimulation of Ca2+ uptake. The uptake at 30 min is about 50% of that obtained with oxalate when the incubation is carried out at pH 6.8, which is the pH optimum for oxalate-stimulated calcium uptake. However, at physiological pH values (7.2-7.4), the glucose 6-phosphate-stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. The Vmax of the glucose 6-phosphate-stimulated transport is 22.3 nmol of calcium/mg protein per min. The apparent Km for calcium calculated from total calcium concentrations is 31.9 microM. After the incubation of the system for MgATP-dependent microsomal calcium transport in the presence of glucose 6-phosphate, inorganic phosphorus and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. In the system for the glucose 6-phosphate-stimulated calcium uptake, glucose 6-phosphate is actively hydrolyzed by the glucose-6-phosphatase activity of liver microsomes. The latter activity is not influenced by concomitant calcium uptake. Calcium uptake is maximal when the concentration of glucose 6-phosphate in the system is 1-3 mM, which is much lower than that necessary to saturate glucose-6-phosphatase. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose-6-phosphatase multicomponent system. The physiological implications of such a cooperation are discussed.     

Characterization of Ni transport into brush border membrane vesicles (BBMVs) isolated from the kidney of the freshwater rainbow trout (Oncorhynchus mykiss)

The transport of nickel (Ni) across the renal brush border membrane of the rainbow trout was examined in vitro using brush border membrane vesicles (BBMVs). Both transmembrane transport of Ni into an osmotically active intravesicular space, and binding of Ni to the brush border membrane itself, were confirmed. Nickel (Ni) uptake fitted a two component kinetic model. Saturable, temperature-dependent transport dominated at lower Ni concentrations, with a moderate linear diffusive component of Ni transport apparent at higher Ni concentrations. An affinity constant (K_m) for Ni transport within the specifically described vesicular media was calculated as 17.9 ± 1.9 μM, the maximal rate of transport (J_max) was calculated as 108.3 ± 3.7 nmol mg protein⁻¹ min⁻¹, and the slope of the linear diffusive component was calculated as 0.049 ± 0.005 nmol mg protein⁻¹ min⁻¹ per μM of Ni. Efflux of Ni from BBMVs was fitted to an exponential decay curve with a half-time (T_1/2) of 125.2 ± 7.3 s. Ni uptake into renal BBMVs was inhibited by magnesium at a 100:1 Mg to Ni molar ratio, and by magnesium and calcium at a 1000:1 molar ratio. In the presence of histidine at a 100:1 histidine to Ni ratio, Ni uptake was almost completely abolished. At a 1:1 molar ratio, histidine inhibited Ni uptake by approximately 50%. Ni-histidine complexation was rapid, with a T_1/2 of 12.2 s describing the Ni-histidine equilibration time needed to inhibit Ni uptake into renal BBMVs by 50%. Characterization of Ni transport across cellular membranes is an important step in understanding both the processes underlying homeostatic regulation of Ni, and the toxicological implications of excessive Ni exposure in aquatic ecosystems.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 mediated calcium uptake in ATP depleted human red cells

The A23187 induced calcium uptake in ATP depleted cells was determined at pH 6.9 in the presence of trifluoperazine (TFP, 0.30 mM), compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). Apart from verapamil the drugs all increased the maximum rate of ionophore-mediated calcium flux by 50-60 per cent. After the ionophore addition some time elapsed before the calcium flux attained the maximum value, and this time dependence could be interpreted as a slow uptake of A23187 into the membrane: five seconds after the addition of A23187 half of the added ionophore was able to transport calcium through the membrane. The effect of pH on the ionophore-mediated calcium uptake was determined in the absence and presence of TFP. At pH 7.4 the maximum rate of calcium flux in the absence of TFP was two to three times higher than that at pH 6.9 and TFP increased the uptake rate by 98 per cent.     

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10–100 μM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 μM. In contrast, concentrations of dibutyryl cyclic AMP between 200 μM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 μM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 μM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent either treatment alone. It is conclude that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process.     

Cellobiose uptake by the cellulolytic ruminal anaerobe Fibrobacter (Bacteroides) succinogenes

Cellobiose transport by the cellulolytic ruminal anaerobe Fibrobacter (Bacteroides) succinogenes was measured using randomly tritiated cellobiose. When assayed at the same concentration (1 mM), total cellobiose uptake was one-fourth to one-third that of total glucose uptake. The abilities of F. succinogenes to transport cellobiose or glucose were not affected by the sugar on which the cells were grown. Aspects of the simultaneous transport of [14C(U)]glucose and [3H(G)]cellobiose, the failure of high concentrations of cold glucose to compete with hypothetical [3H(G)]glucose (derived externally from [3H(G)]cellobiose), and differential metal-ion stimulation of cellobiose transport indicate a cellobiose permease, rather than cellobiase plus glucose permease, was responsible for cellobiose transport. Glucose (10-fold molar excess) partially inhibited cellobiose transport. This was enhanced by prior incubation of the cells with glucose, suggesting subsequent metabolism of the glucose was responsible for the inhibition. Compounds interfering with electron transport or maintenance of transmembrane ion gradients inhibited cellobiose uptake, indicating that active transport rather than a phosphoenolpyruvate:phosphotransferase system catalyzed cellobiose transport. Na+, but not Li+, stimulated cellobiose transport.     

Reassessment of insulin effects on the Vmax and Km values of hexose transport in isolated rat epididymal adipocytes

Effects of insulin on the kinetic parameters of hexose transport in rat epididymal adipocytes were re-examined. The transport activity was assessed by measuring the rate of uptake of 3-O-[3H]methyl-D-glucose (MeGlc) under equilibrium exchange and zero-trans conditions. The incubation was carried out at 37 degrees C in an infant incubator. During the incubation, the cell suspension (25%, v/v, in a total volume of 48 microliter) was mechanically swirled at a rate of 600 rpm (r = 2 mm). The swirling facilitated the rapid uptake of MeGlc without stimulating the basal transport activity by "mechanical agitation". The basal and insulin-treated cells were incubated under identical conditions, except for the length of the incubation period. The incubation was terminated by the addition of 350 microliters of 1 mM phloretin, which inhibited transport in approximately 0.06 s. The time course of MeGlc uptake was consistent with the view that the process was a multiple-phase reaction. The initial phase of the reaction was completed when the intracellular distribution space of MeGlc was approximately 1% of the total cell volume. Insulin (10 nM) increased the Vmax value of MeGlc uptake 16-fold in equilibrium exchange experiments and 18-fold in zero-trans experiments. At the same time, the hormone decreased the Km value of MeGlc uptake from 11.7 to 5.4 mM in equilibrium exchange experiments and from 9.7 to 4.8 mM in zero-trans experiments. It is concluded that the major effect of insulin on MeGlc uptake is to increase the Vmax value, but the hormone has the additional effect of lowering the apparent Km value.     

Transport of 2-aminoisobutyric acid in mesophyll protoplasts of Pisum sativum L. Application of silicone oil layer centrifugation

Uptake of 2-amino [1-¹⁴C]isobutyric acid by pea mesophyll protoplasts was investigated using silicone oil layer centrifugation. Uptake was expressed on the basis of the ³H₂O-space of the protoplast pellet. The ³H₂O-space can be used as a measure of protoplast volume, taking into consideration that about 10% of it is extracellular space. At concentrations in the range 10 μM–10 mM, the uptake of 2-aminoisobutyric acid (Aib) was linear with time for at least 1 h. At an external concentration of 10 μM, up to a 10-fold accumulation of Aib in the protoplasts was observed during 1 h of incubation. The concentration-dependence of the uptake rate conforms to the sum of a Michaelis-Menten term and a linear term. Large differences in uptake rates were found for different preparations of protoplasts, especially at low concentrations of Aib. This could be attributed to differences in the activity of the saturable component. Both transport components were strongly inhibited by 10 μM CCCP, even when transport was apparently downhill.     

Effect of verapamil and sulfhydryl reagents on calcium transport in bovine spermatozoa

Calcium uptake by intact bovine epididymal spermatozoa is not affected by low concentrations (up to 0.75 mM) of the calcium transport blocker verapamil. Under these conditions, calcium transport into sperm mitochondria is highly inhibited. At higher verapamil concentrations (1.0, 1.5 mM), calcium transport into intact sperm is also inhibited, and this inhibition cannot be relieved by disrupting the plasma membrane with filipin. Calcium uptake into intact sperm is highly inhibited by mersalyl and this inhibitory effect can be completely relieved when the plasma membrane is disrupted by filipin. This effect of mersalyl is not dependent on the presence of phosphate in the incubation medium. Phosphate itself, up to 2 mM, enhances calcium uptake into the cells; this effect decreases at higher concentrations and is depressed 57% at 10 mM phosphate. This inhibitory effect of high phosphate concentration can be blocked by mersalyl. It is suggested that the calcium carrier itself and not a phosphate carrier of the plasma membrane is inhibited by mersalyl. It is possible that there is a symporter for calcium and phosphate in the plasma membrane of bovine spermatozoa.     

Na+ and pH dependence of 5-methyltetrahydrofolic acid and methotrexate transport in freshly isolated hepatocytes

The dependence of the high-affinity transport systems for 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) and methotrexate on sodium ions and on pH was examined in freshly isolated rat hepatocytes. Previous studies indicated that transport of these folate derivatives was sodium-dependent. Experiments to determine the Km for sodium of 5-CH3-H4PteGlu transport showed no dependence on extracellular sodium. However, uptake was sodium-dependent when hepatocytes were preincubated for 30 min in sodium-free medium, a treatment which resulted in an increase in the transmembrane pH gradient (delta pH = pH out-pH in) and a decrease in the uptake of 5-CH3-H4PteGlu. Uptake of methotrexate displayed a linear dependence on extracellular sodium ions. Uptake of 5-CH3-H4PteGlu increased linearly as the transmembrane pH gradient decreased; i.e., as the medium became more acid with respect to the cytosol. Lineweaver-Burk and Scatchard plots of 5-CH3-H4PteGlu uptake indicated an apparent Km for H+ of about 24 nM, equivalent to a pH of 7.6. Hill-plots suggested a stoichiometry of 1:1 for the interaction of protons with the 5-CH3-H4PteGlu transport system. Both the Km and Vmax for 5-CH3-H4PteGlu transport were increased at pH 5.5 compared to pH 7.4, suggesting that extracellular protons increased the number of and/or the activity of the membrane carrier. In contrast, methotrexate transport was maximal at pH 7 where the transmembrane pH gradient was zero. These results suggest the possibility that 5-CH3-H4PteGlu may be cotransported along with H+ ions in hepatocytes, although they do not rule out a 'catalytic coupling' whereby protons interact with the carrier to stimulate substrate flux without concomitant H+ transport.     

The Effect of Calcium on Non-heme Iron Uptake,Efflux, and Transport in Intestinal-like Epithelial Cells (Caco-2 Cells)

It has been suggested that calcium inhibits the absorption of dietary iron by directly affecting enterocytes. However, it is not clear if this effect is due to a decreased uptake of iron or its efflux from enterocytes. We studied the effect of calcium on the uptake, efflux, and net absorption of non-heme iron using the intestinal-like epithelial cell line Caco-2 as an in vitro model. Caco-2 cells were incubated for 60 min in a buffer supplemented with non-heme iron (as sulfate) and calcium to achieve calcium to iron molar ratios ranging from 50:1 to 1,000:1. The uptake, efflux, and net absorption of non-heme iron were calculated by following a radioisotope tracer of ⁵⁵Fe that had been added to the buffer. Administration of calcium and iron at molar ratios between 500 and 1,000:1 increased the uptake of non-heme iron and decreased efflux. Calcium did not have an effect on the net absorption of non-heme iron. At typical supplementary doses for calcium and non-heme iron, calcium may not have an effect on the absorption of non-heme iron. The effect of higher calcium to iron molar ratios on the efflux of non-heme iron may be large enough to explain results from human studies.     

Interaction and transport of poly(L-lysine) dendrigrafts through liposomal and cellular membranes: the role of generation and surface functionalization

Two generations of poly(l-lysine) dendrigrafts (DGLs) were studied with regard to their ability to interact with and translocate through liposomal and cellular membranes. Partial guanidinylation of the surface amino groups of the starting dendrigrafts afforded the guanidinylated derivatives whose membrane translocation properties were also assessed. Mixed liposomes, consisting of dihexadecyl phosphate, phosphatidylcholine, and cholesterol, were employed as model membranes, while A549 human lung carcinoma cells were used for cellular uptake studies. At high surface group/liposomal phosphate molar ratios and depending on the structure of the DGL, the interaction led to aggregation. Dendrigraft liposomal internalization was achieved, however, at low molar ratios. Thus translocation of the second generation dendrigrafts was rather limited at 25 degrees C, which, however, was enhanced when the bilayer was in the liquid-crystalline phase. In contrast, third-generation counterparts exhibited minor translocational ability. Furthermore, the introduction of a guanidinium group to dendrigrafts was found to enhance their transport through liposomal membranes. On the other hand, cellular uptake by A549 cells was monitored up to 3 h incubation time via fluorescence registration employing fluorescein-labeled dendrigrafts. The efficiency of dendrigraft internalization was enhanced by the presence of the guanidinium groups, while DGLs were preferentially localized in the nucleus and nuclear membrane, as revealed by fluorescence microscopy.     

A Thiamine/H+ Antiport Mechanism for Thiamine Entry into Brush Border Membrane Vesicles from Rat Small Intestine

Outwardly oriented H⁺ gradients greatly enhanced thiamine transport rate in brush border membrane vesicles from duodenal and jejunal mucosa of adult Wistar rats. At a gradient pH_in5:pH_out7.5, thiamine uptake showed an overshoot, which at 15 sec was three times as large as the uptake observed in the absence of the gradient. Under the same conditions, the binding component of uptake accounted for only 10–13% of intravesicular transport. At the same gradient, the K _m and J _max values of the saturable component of the thiamine uptake curve after a 6 sec incubation time were 6.2 ± 1.4 μm and 14.9 ± 3 pmol · mg⁻¹ protein · 6 sec⁻¹ respectively. These values were about 3 and 5 times higher, respectively, than those recorded in the absence of H⁺ gradient. The saturable component of the thiamine antiport had a stoichiometric thiamine: H⁺ ratio of 1:1 and was inhibited by thiamine analogues, guanidine, guanidine derivatives, inhibitors of the guanidine/H⁺ antiport, and imipramine. Conversely, the guanidine/H⁺ antiport was inhibited by unlabeled thiamine and thiamine analogues; omeprazole caused an approximately fourfold increase in thiamine transport rate. In the absence of H⁺ gradient, changes in transmembrane electrical potential did not affect thiamine uptake. At equilibrium, the percentage membrane-bound thiamine taken up was positively correlated with the pH of the incubation medium, and increased from about 10% at pH 5 to 99% at pH 9. Received: 17 July 1997/Revised: 16 September 1997     

Calcium transport in intact human erthrocytes

Intact human erythrocytes can be readily loaded with calcium by incubation in hypersomotic media at alkaline pH. Erythrocyte calcium content increases from 15-20 to 120-150 nmol/g hemoglobin after incubation for 2 h at 20 degree C in a 400 mosmol/kg, pH 7.8 solution containing 100 mM sodium chloride, 90 mM tetramethylammonium chloride, 1 mM potassium chloride, and 10 mM calcium chloride. Calcium uptake is a time-dependent process that is associated with an augmented efflux of potassium. The ATP content in these cells remains at more than 60% of normal and is not affected by calcium. Calcium uptake is influenced by the cationic composition of the external media. The response to potassium is diphasic. With increasing potassium concentrations, the net accumulation of calcium initially increases, becoming maximal at 1 mM potassium, then diminishes, falling below basal levels at concentrations above 3 mM potassium. Ouabain inhibits the stimulatory effect of low concentrations of potassium. The inhibitory effects of higher concentrations of potassium are ouabain insensitive and independent of the external calcium concentration. Sodium also inhibits calcium uptake but this inhibition can be modified by altering the external concentration of calcium. The effux of calcium from loaded erythrocytes is not significantly altered by changes in osmolality, medium ion composition, or ouabain. It is concluded that hypertonicity increases the net uptake of calcium by increasing the influx of calcium and that some part of the sodium potassium transport system is involved in this influx process.     

The administration of lipopolysaccharide, in vivo, induces alteration in L-leucine intestinal absorption

The objective of the present study was to determine the alterations in L-leucine intestinal uptake by intravenous administration of Lipopolysaccharide (LPS), which is a constituent of gram negative bacterial, causative agent of sepsis. The amino acid absorption in LPS treated rabbits was reduced compared to the control animals. The LPS effect on the amino acid uptake was due to an inhibition of the Na+-dependent system of transport, through both reduction of the apparent capacity transport (Vmax) and diminution of the Na+/K-ATPase activity. The results have also shown that the LPS decreases the mucosal to serosal transepithelial flux and the transport across brush border membrane vesicles of L-leucine. The study of possible intracellular mechanisms implicated in the LPS effect, showed that the second messengers calcium, protein kinase C and c-AMP did not play any role in this effect. However, the absence of ion chloride in the incubation medium removes the LPS inhibition and the intracellular tissue water was affected by the LPS treatment. Therefore, the inhibition in the L-leucine intestinal absorption, by intravenous administration of LPS, could be mainly produced by the secretagogue action of this endotoxin on the gut.     

Energization of the sugar transport mechanism in the plasmalemma of isolated mesophyll protoplasts

The mechanism of 3-O-methyl-d-glucose transport through the plasmalemma has been investigated in protoplasts isolated from the mesophyll of Pisum sativum L. var. Dan.Analysis of the fluxes after 50 minutes of uptake showed that the gradual decrease in slope of the net uptake curve with time was not due to any decline in uptake capacity; it represented the approach to flux equilibrium of a small compartment of the protoplast, probably the cytoplasm.The energy of activation for initial flux into this compartment was 20 kilocalories per mole between 17 and 27 C. Very high discrimination was shown with regard to sugar isomers. Light strongly promoted flux (by a factor of 2.5 in the case of methyl glucose). Initial flux showed sharply contrasting inhibitor sensitivity in the light and the dark. Light uptake was sensitive to the proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP), but stable for at least the first 10 minutes to the ATPase inhibitors quercetin, rutin, and diethylstilbestrol, as well as to arsenate. Dark uptake, on the other hand, was stable to CCCP but was immediately depressed by quercetin, rutin, diethylstilbestrol, and arsenate.Protoplasts which received a light pretreatment before incubation in the dark took up methyl glucose at the accelerated light rate for the first 7 minutes. Moreover, the light pretreatment sensitized subsequent initial dark uptake to CCCP, and conferred on it the stability to ATPase inhibitors and arsenate characteristic of light uptake. After about 7 minutes the characteristic inhibitor responses of dark uptake were resumed.It is proposed that more than one mode of energy-coupling for sugar transport may operate in these protoplasts.     

Mechanism and Regulation of Isoleucine Excretion in Corynebacterium glutamicum

Whole cells of Corynebacterium glutamicum were loaded with high cytoplasmic l-isoleucine concentrations, and isoleucine excretion from these cells was studied in terms of mechanism and regulation. The transmembrane isoleucine flux could be differentiated into carrier-mediated uptake, carrier-mediated excretion, and diffusion. After discrimination from the other transmembrane solute movements, the outward-directed flux, which was due to the activity of the isoleucine excretion carrier, was characterized with respect to its energy dependence and its regulation at the level of expression. Isoleucine excretion was shown to function as a secondary transport process, driven by the membrane potential and coupled to the movement of protons, presumably with a stoichiometry of 2:1 (H(sup+)/isoleucine). Of a variety of putative transport substrates, only leucine was able to compete for isoleucine at the cis (cytosolic) side of the export carrier. Cytoplasmic isoleucine concentrations higher than 20 mM induce the activity of the isoleucine excretion system. This effect is specific for isoleucine and is inhibited by the presence of chloramphenicol. Apart from leucine, other amino acids and related amino acid analogs are not able to induce isoleucine excretion. The complex pattern of regulation of the isoleucine excretion system at the level of activity and expression is shown to be related to the pattern of regulation of the isoleucine uptake system in C. glutamicum in terms of physiological significance.     

Voltage-sensitive calcium flux promoted by vesicles in an isolated cardiac sarcolemma preparation

Summary The effect of membrane potential on the vesicular uptake of calcium in an isolated cardiac sarcolemma preparation from canine ventricle was evaluated. Membrane potentials were developed by the establishment of potassium gradients across the vesicular membranes. In the presence of valinomycin, the fluorescence changes of the voltage sensitive dye, diS-C₃-(5) were consistent with the development of potassium equilibrium potentials. Using EGTA to remove endogenous calcium from the preparation and to maintain a low intravesicular calcium concentration over time, the uptake of calcium was linear from 5 to 100 sec, in the absence of sodium, at both –98 and –1 mV. The rate of calcium uptake (calcium influx) was approximately twofold greater at –1 mV than at –98 mV, and prepolarization of the membrane potential to –98 mV did not enhance calcium influx upon subsequent depolarization to –1 mV. Hence, calcium influx was voltage-sensitive but not depolarization-induced and did not inactivate with time. Furthermore, the calcium influx was not inhibited by the organic calcium antagonists, which suggests that this flux did not occur via the transient calcium channel. Evaluation of calcium influx over a wide range of membrane potentials produced a profile consistent with the hypothesis that calcium entered the vesicles through the pathway responsible for the persistent inward current observed in voltage-clamped isolated myocytes. A model was proposed to account for these results.     

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.