Histidyl residues at the active site of the Na/succinate cotransporter in rabbit renal brush borders

Summary Mono-, dicarboxylic acid-, andd-glucose transport were measured in brush border vesicles from renal cortex after treatment with reagents known to modify terminal amino, lysyl, -amino, guanidino, serine/threonine, histidyl, tyrosyl, tryptophanyl and carboxylic residues. All three sodium-coupled cotransport systems proved to possess sulfhydryl (and maybe tryptophanyl sulfhydryl, disulfide, thioether and tyrosyl) residues but not at the substrate site or at the allosteric cavity for the Na coion. Histidyl groups seem to be located in the active site of the dicarboxylic transporter in that the simultaneous presence of Na and succinate protects the transporter against the histidyl specific reagent diethylpyrocarbonate. Lithium, which specifically competes for sodium sites in the dicarboxylic acid transporter, substantially blocked the protective effect of Na and succinate. Hydroxylamine specifically reversed the covalent binding of diethylpyrocarbonate to the succinate binding site. The pH dependence of the Na/succinate cotransport is consistent with an involvement of histidyl and sulfhydryl residues. We conclude that a histidyl residue is at, or is close to, the active site of the dicarboxylate transporter in renal brush border membranes.     

Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles

Summary We have confirmed previous demonstrations of sodium gradient-stimulated transport ofl-alanine, phenylalanine, proline, and -alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these¹⁴C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability ofd-glucose to inhibit tracer uptake.l-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the¹⁴C-l-alanine uptake could be inhibited by proline and -alanine, while lysine and MeAIB were no more effective thand-glucose. Roughly 50% of the¹⁴C-phenylalanine uptake could be inhibited by proline and -alanine; lysine was as effective as proline and -alanine, and the effects of pairs of these amino acids at 50mm each were not cumulative. MeAIB was no more effective thand-glucose. We conclude that three pathways mediate the uptake of neutral,l, -amino acids. One system is inaccessible to lysine, proline, and -alanine. The second system carries a major fraction of thel-alanine flux; it is sensitive to proline and -alanine, but not to lysine. The third system carries half the¹⁴C-phenylalanine flux, and it is sensitive to proline, lysine, and -alanine. Since the neutral,l, -amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classicalA system, and since all of thel-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classicalASC system.l-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective thand-glucose in inhibiting lysine uptake, while proline and -alanine appear to inhibit a component of the lysine flux. We conclude that the¹⁴C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, -alanine, andl-alanine, and one which is inhibited byl-alanine and phenylalanine but inaccessible to proline, -alanine, and MeAIB. Fluxes of¹⁴C-proline and¹⁴C-MeAIB are completely inhibited byl-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit¹⁴C--alanine uptake. However, -alanine inhibits only 38% of the¹⁴C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports -alanine.     

Static and dynamic components of renal cortical brush border and basolateral membrane fluidity: Role of cholesterol

Summary Static polarization and differential polarized phase fluorimetry studies on rat renal cortical brush border (BBM) and basolateral membranes (BLM) were undertaken to determine the membrane components responsible for differences in BBM and BLM fluidity, whether these differences were due to the order or dynamic components of membrane fluidity and if a fluidity gradient existed within the bilayer. Surface membrane proteins rigidified both BBM and BLM fluidity. Neutral lipid extraction, on the other hand, caused a larger decrease in BBM than BLM fluorescence polarization (0.104vs. 0.60,P<0.01) using diphenyl hexatriene (DPH). Cholesterol addition to phospholipid fractions restored membrane fluidity to total lipid values in both BBM and BLM phospholipids. The response to cholesterol in the BBM was biphasic, while the BLM response was linear. Lateral mobility, quantitated using dipyrenylpropane, was similar in both BBM and BLM fractions at 35°C. BBM and BLM differed primarily in the order component of membrane fluidity as DPH-limiting anisotropy (r ) (0.212vs. 0.154,P<0.01) differed markedly between the two membrane fractions. The two membrane components also differed with respect to 2 and 12-anthroyloxy stearate (2-AS, 12-AS) probes, indicating a difference in the dynamic component of membrane fluidity may also be present. DPH and 12-As probes were also used to quantitate inner core membrane fluidity and showed the BBM was less fluid than the BLM for intact membranes, total lipid extracts and phospholipids. Results obtained using the surface membrane probes trimethylammonium-DPH (TMA-DPH) and 2-AS suggested a fluidity gradient existed in both BBM and BLM bilayers with the inner core being more fluid in both membranes. These data indicate cholesterol is in large part responsible for fluidity differences between BBM and BLM and that these membranes, while clearly differing in the order component of membrane fluidity, may also difer in the dynamic component as well.     

Kinetics of sodiumd-glucose cotransport in bovine intestinal brush border vesicles

Summary Brush border membrane vesicles (BBMV) purified from steer jejunum were used to study the kinetics of sodiumd-glucose cotransport under voltage clamped, zero-trans conditions. When the initial rate of glucose transport (J _gluc) was measured over a wide range of glucose concentrations ([S]=0.01–20mm), curvature of the Woolf-Augustinsson-Hofstee plots was seen, compatible with a diffusional and one major, high capacity (maximal transport rateJ _max=5.8–8.8 nmol/mg·min) saturable system. Further studies indicated that changes incis [Na] altered theK _t , but not theJ _max, suggesting the presence of a rapid-equilibrium, ordered bireactant system with sodium adding first.Trans sodium inhibitedJ _gluc hyperbolically. KCl-valinomycin diffusion potentials, inner membrane face positive, loweredJ _gluc, while potentials of the opposite polarity raiseJ _gluc. At low glucose concentrations ([S]<0.05mm), a second, minor, high affinity transport system was indicated. Further evidence for this second saturable system was provided by sodium activation curves, which were hyperbolic when [S]=0.5mm, but were sigmoidal when [S]=.0.01mm. Simultaneous fluxes of²²Na and [³H]glucose at 1mm glucose and 30mm NaCl yielded a cotransport-dependent flux ratio of 21 sodium/glucose, suggestive of 11 (Na/glucose) high capacity, low affinity system and a 31 (Na/glucose) high affinity, low capacity system. Kinetic experiments with rabbit jejunal brush borders revealed two major Na-dependent saturable systems. Extravesicular (cis) Na changed theK _t , but not theJ _max of the major system.     

Stimulation of intestinal Na+/d-glucose cotransport by monoclonal antibodies

Summary The small intestinal brush border membrane is endowed with a number of transport systems. Monoclonal antibodies were produced against integral membrane proteins and tested for their ability to bind to such membranes. For this purpose papain-digested, deoxycholate-extracted BBMVs from rabbit small intestine were used to immunize mice. Of the 765 hybridoma supernatants tested, 119 gave a significantly higher extent of binding to the crude antigen preparation as compared with the background. The monoclonal antibodies were also tested for their ability to influence the sodium-dependent uptake of solutes into intact BBMVs. Two monoclonal antibodies clearly showed stimulation of secondary actived-glucose transport, whereas sodium-dependent uptake ofl-alanine andl-proline was not affected. Hydrophobically labeled, i.e. intrinsic, membrane proteins of 175, 78 and 65 kilodaltons could be immunoprecipitated by both monoclonal antibodies, the 78 kDa band corresponding in all likelihood to the Na⁺/glucose cotransporter.     

Transport of p-aminohippuric acid,uric acid and glucose in highly purified rabbit renal brush border membranes

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na⁺ + K⁺)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%.Transport of d-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (E_A = 11.3–37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 μM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.     

Ketone body transport in renal brush border membrane vesicles

Ketone body uptake by renal brush border vesicles has been investigated. Ketone bodies enter into the brush border vesicles by a carrier-mediated process. The uptake is dependent on an Na⁺ gradient ([Na⁺]outside > [Na⁺]inside) and is electroneutral. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. A pH gradient (alkaline inside) also stimulates the ketone body uptake. Acetoacetate and 3-hydroxybutyrate share the same carrier as demonstrated by the accelerated exchange diffusion and mutual inhibitory effects.     

Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium

The alkaline phosphatases present on isolated brush border and basal lateral membranes of rat duodenal epitheilum were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6–9.8, similar substrate k_m's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin or papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000–150,000, and both enzymes show an R_f value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The V_max of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 μmoles/mg-h as opposed to 14 μmoles/mg-h. The differences in V_max are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.     

Ion permeability of rabbit intestinal brush border membrane vesicles

Summary The ion permeability of rabbit jejunal brush border membrane vesicles was studied by measuring unidirectional fluxes with radioactive tracers and bi-ionic diffusion potentials with the potential-sensitive fluorescent dye, diS–C₃-(5). Tracer measurements provide estimates of the absolute magnitudes of permeability coefficients, while fluorescence measurements provide estimates of relative and absolute ion permeabilities. The magnitudes of the permeability coefficients for Na⁺, K⁺, Rb⁺, and Br^– were approximately 5 nanoliters/(mg protein × sec) or 10^–5 cm/sec as determined by radioactive tracer measurements. The apparent selectivity sequence, relative to Na⁺, as determined by bi-ionic potential measurements was: F^–, isetheionate, gluconate, choline (<0.1)+(1.0)–(1.5)=NO ₃ ^– (1.5)–(2.3)+(2.4)+(2.5)+(2.6)+(3.9) 4 ^– +(12)–(40). The origin of this selectivity sequence and its relationship to the ion permeability of the brush border membrane in the intact epithelium are discussed.     

CAMP-dependent protein kinase inhibits proline transport across the rat renal tubular brush border membrane

Very little is known about the cellular mechanisms controlling renal tubular amino acid transport. cAMP-dependent protein kinase (cAK) modulates the activity of several ion channels and pumps in biological membranes. The direct influence of cAK on transmembrane amino acid transport has not been investigated. We studied the effect the cAK-mediated phosphorylation on Na+- and Cl–-linked proline transport across the rat renal brush border membrane (BBM). cAK bioassay and Western hybridization analysis using cAK subunit-specific antibodies demonstrated the presence of the enzyme in the BBM. Brush border membrane vesicles (BBMV) were phosphorylated using the hyposmotic shock technique. cAMP, by activating endogenous cAK,and exogenous, highly purified catalytic subunit of cAK inhibited NaCl-dependent proline transport by phosphorylated, lysed/resealed BBMV compared with control vesicles. The cAK-mediated inhibition of proline uptake was completely abolished when phosphorylation at the cytoplasmic (inner side) of the membrane was prevented by isosmotic, rather than hyposmotic, phosphorylation. The cAK-induced inhibition of proline transport was reversed by the specific cAK inhibitor peptide, PKl. These data suggest that cAMP-dependent protein kinase-mediated phosphorylation modulates Na+- and Cl–-linked proline transport across the tubular luminal membrane.     

Multiple transport pathways for neutral amino acids in rabbit jejunal brush border vesicles

Summary Amino acids enter rabbit jejunal brush border membrane vesicles via three major transport systems: (1) simple passive diffusion; (2) Na-independent carriers; and (3) Na-dependent carriers. The passive permeability sequence of amino acids is very similar to that observed in other studies involving natural and artificial membranes. Based on uptake kinetics and cross-inhibition profiles, at least two Na-independent and three Na-dependent carrier-mediated pathways exist. One Na-independent pathway, similar to the classical L system, favors neutral amino acids, while the other pathway favors dibasic amino acids such as lysine. One Na-dependent pathway primarily serves neutrall-amino acids including 2-amino-2-norbornanecarboxylic acid hemihydrate (BCH), but not -alanine or -methylaminoisobutyric acid (MeAIB). Another Na-dependent route favors phenylalanine and methionine, while the third pathway is selective for imino acids and MeAIB. Li is unable to substitute for Na in these systems. Cross-inhibition profiles indicated that none of the Na-dependent systems conform to classical A or ACS paradigms. Other notable features of jejunal brush border vesicles include (1) no -alanine carrier, and (2) no major proline/glycine interactions.     

Mechanism of neomycin stimulation of d-glucose uptake in rabbit intestinal brush border membrane

In order to study the effect of the antibiotic neomycin on the intestinal epithelium, d-glucose was used as a probe molecule and its transport into rabbit brush border membrane vesicles was measured by a rapid filtration method. Treatment of the epithelium with neomycin sulfate prior to the preparation of the brush border membrane enhanced the d-glucose uptake, whereas neutral N-acetylated neomycin did not. This action of neomycin was related to its polycationic character and not to its bactericidal action. No significant difference could be demonstrated between the protein content or disaccharidase-specific activities of the brush border fractions from treated or non-treated intestines. Electrophoretic protein patterns of SDS-solubilized membrane were not significantly different after neomycin treatment. To gain more information on the mechanism involved in the stimulation of d-glucose transport, experiments were conducted on phosphatidyl glycerol artificial membranes and the results compared with those obtained with brush border membrane. At a concentration of 10^?7 M, neomycin decreased the nonactin-induced K⁺ conductance by a factor of approx. 100. The membrane conductance was linearly dependent on the neomycin concentration and the conductance in 10^?2 M KCl was 10 times that in 10^?3 M KCl. The valence of neomycin was estimated, from the slope of these curves, to be between 6 and 4. In contrast, acetylated neomycin had no effect on the nonactin-induced K⁺ membrane conductance. Therefore, the effect of neomycin on artificial membrane is related to its 4 to 6 positive charges. It is proposed that the stimulation of sugar transport in brush border membrane is related to screening of the membrane negative charges by the positively-charged neomycin. Accumulation of anions at the membrane surface then occurs and their diffusion into the intravesicular space would increase the transmembrane potential which, in turn, stimulates the entry of d-glucose.     

Interactions between biliary lipid micelles and intestinal brush border membranes investigated by 1H and 31P nuclear magnetic resonance

The effect of taurocholate and lecithincholesterol-taurocholate mixed micelles on the structure of isolated intestinal brush border membranes was investigated by nuclear magnetic resonance (NMR). Rabbit brush border membranes isolated by a Mg²⁺ precipitation step were chosen for this study because of their stability and integrity as revealed by ³¹P NMR. Incubation of taurocholate with the brush border membranes does not induce significant solubilization of these membranes even when the taurocholate/phospholipid ratio reaches 3.0 ¹H NMR studies indicate that taurocholate is included in the membrane bilayer at low concentration (3 mM). However this biliary salt produces a size diminution of the vesicles when its concentration increases. Incorporation of lecithin or lecithin-cholesterol in micelles of taurocholate and subsequent incubation with brush border membranes lead simultaneously to a decrease in the ³¹P NMR isotropic/bilayer line ratio, and to an increase in . These results indicate a protective effect of these compounds against lytic damage of taurocholate. Futhermore the equilibrium distribution of lecithin between mixed micelles and the membrane bilayer is strongly in favour of complete integration of micellar components in the bilayer. These data suggest that uptake of lipids from the micellar phase by isolated brush border membranes involves an interaction of the micelles with membranes followed by a fusion process.     

Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with glycoconjugates from rabbit intestinal brush border membranes: Relationship with ABH blood group determinants

The capacity of cholera toxin (CT) and type I heat-labile enterotoxin produced by Escherichia coli isolated from human intestine (LTh) to interact with glycoconjugates bearing ABH blood group determinants from rabbit intestinal brush border membranes (BBM) was studied. On the basis of the type of intestinal compounds related to the human ABH blood group antigens, rabbits were classified as AB or H. Toxin binding to the intestinal glycolipids and glycoproteins depends on the blood group determinant borne by the glycoconjugate and on the analyzed toxin. LTh was capable of interacting preferentially with several blood group A- and B-active BBM glycolipids compared to those isolated from animals lacking these antigens (H rabbits). Also, LTh preferably bound to several BBM glycoproteins from AB rabbit intestines compared to those from H ones. One of these glycoproteins, the sucrase-isomaltase complex (EC 3.2.1.48-10) isolated from AB and H rabbits showed the same differential LTh binding. Conversely, CT practically did not recognize either blood group A-, B-, or H-active glycolipids and glycoproteins. These results may be relevant for carrying out in vivo experiments in rabbits in order to disclose the role of ABH active-glycoconjugates in the secretory response induced by LTh in rabbit intestine.     

Binding and degradation of125I-insulin by isolated rat renal brush border membranes: Evidence for low affinity,high capacity insulin recognition sites

Summary The kidney plays a major role in the handling of circulating insulin in the blood, primarily via reuptake of filtered insulin at the luminal brush border membrane.¹²⁵I-insulin associated with rat renal brush border membrane vesicles (BBV) in a time-and temperature-dependent manner accompanied by degradation of the hormone to trichloroacetic acid (TCA)-soluble fragments. Both association and degradation of¹²⁵I-insulin were linearly proportional to membrane protein concentration with virtually all of the degradative activity being membrane assoicated. Insulin, proinsulin and desoctapeptide insulin all inhibited the association and degradation of¹²⁵I-insulin by BBV, but these processes were not appreciably afected by the insulin-like growth factors IGF-I and IGF-II or by cytochromec and lysozyme, low molecular weight, filterable, proteins, which are known to be reabsorbed in the renal tubules by luminal endocytosis. When the interaction of¹²⁵I-insulin with BBV was studied at various medium osmolarities (300–1100 mosm) to alter intravesicular space, association of the ligand with the vesicles was unaffected, but degradation of the ligand by the vesicles decreased progressively with increasing medium osmolarity. Therefore, association of¹²⁵I-insulin to BBV represented binding of the ligand to the membrane surface and not uptake of the hormone or its degradation products into the vesicles. Attempts to crosslink¹²⁵I-insulin to a high-affinity insulin receptor using the bifunctional reagent disuccinimidyl suberate revealed only trace amounts of an¹²⁵I-insulin-receptor complex in brush border membrane vesicles in contrast to intact renal tubules where this complex was readily observed. Both binding and degradation of¹²⁵I-insulin by brush border membranes did not reach saturation even at concentrations of insulin approaching 10^–5 m. These results indicate the presence of low-affinity, high-capacity binding sites for¹²⁵I-insulin on renal brush border membranes which can clearly distinguish insulin from the insulin-like growth factors and other low molecular weight proteins and polypeptides, but which do not differentiate insulin from its analogues ad do the biological receptors for the hormone. The properties and location of these binding sites make them attractive candidates for the sites at which insulin is reabsorbed in the renal tubule.     

Ca2+ dependency of Na+ transport by rabbit renal brush border membrane

Summary Intracellular Ca²⁺ has been suggested to play an important role in the regulation of epithelial Na⁺ transport. Previous studies showed that preincubation of toad urinary bladder, a tight epithelium, in Ca²⁺-free medium enhanced Na⁺ uptake by the subsequently isolated apical membrane vesicles, suggesting the downregulation of Na⁺ entry across the apical membrane by intracellular Ca²⁺. In the present study, we have examined the effect of Ca²⁺-free preincubation on apical membrane Na⁺ transport in a leaky epithelium, i.e., brush border membrane (BBM) of rabbit renal proximal tubule. In contrast to toad urinary bladder, it was found that BBM vesicles derived from proximal tubules incubated in 1mm Ca²⁺ medium exhibited higher Na⁺ uptake than those derived from proximal tubules incubated in Ca²⁺-free EGTA medium. Such effect of Ca²⁺ in the preincubation medium was temperature dependent and could not be replaced by another divalent cation, Ba²⁺ (1mm). Ca²⁺ in the preincubation medium did not affect Na⁺-dependent BBM glucose uptake, and its effect on BBM Na⁺ uptake was pH gradient dependent and amiloride (10^–3 m) sensitive, suggesting the involvement of Na⁺/H⁺ antiport system. Addition of verapamil (10^–4 m) to 1mm Ca²⁺ preincubation medium abolished while ionomycin (10^–6 m) potentiated the effect of Ca²⁺ to increase BBM Na⁺ uptake, suggesting that the effect of Ca²⁺ in the preincubation medium is likely to be mediated by Ca²⁺-dependent cellular pathways and not due to a direct effect of extracellular Ca²⁺ on BBM. Neither the proximal tubule content of cAMP nor the inhibitory effect of 8, bromo-cAMP (0.1mm) on BBM Na⁺ uptake was affected by the presence of Ca²⁺ in the preincubation medium, suggesting that Ca²⁺ in the preincubation medium did not increase BBM Na⁺ uptake by removing the inhibitory effect of cAMP. Addition of calmodulin inhibitor, trifluoperazine (10^–4 m) to 1mm Ca²⁺ preincubation medium did not prevent the increase in BBM Na⁺ uptake. The effect of Ca²⁺ was, however, abolished when protein kinase C in the proximal tubule was downregulated by prolonged (24 hr) incubation with phorbol 12-myristate 13-acetate (10^–6 m). In summary, these results show the Ca²⁺ dependency of Na⁺ transport by renal BBM, possibly through stimulation of Na⁺/H⁺ exchanger by protein kinase C.     

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles

The sodium-dependent entry of proline and glycine into rat renal brushborder membrane vesicles was examined. The high K_m system for proline shows no sodium dependence. The low K_m system for glycine entry is strictly dependent on a Na⁺ gradient but shows no evidence of the carrier system having any affinity for Na⁺. The low K_m system for proline and high K_m system for glycine transport appear to be shared. Both systems are stimulated by a Na⁺ gradient and appear to have an affinity for the Na⁺. The effect of decreasing the Na⁺ concentration in the ionic gradient is to alter the K_m for amino acid entry and, at low Na⁺ concentrations, to inhibit the V for glycine entry.     

Parathyroid hormone inhibition of phosphate transport in renal brush border vesicles from phosphate-depleted dogs

Dietary phosphate (P_i) restriction increases renal P_i reabsorption and induces resistance to the phosphaturic action of parathyroid hormone. Na⁺-gradient-stimulated P_i transport in membrane vesicles isolated from the renal brush border of experimental animals has been shown to parallel changes in renal P_i reabsorption induced by dietary P_i restriction and in vivo administration of parathyroid hormone. Dietary P_i restriction has been shown to markedly inhibit the phosphaturic response to parathyroid hormone in rats and dogs. Parathyroid hormone has been reported not to decrease the Na⁺-gradient-stimulated transport of P_i in brush border membrane vesicles isolated from dietary P_i restricted rats unless the rats were administered an acute P_i load prior to killing, however, thyroparathyroidectomy of rats fed a low P_i diet has been reported to increase Na⁺-gradient-stimulated P_i transport. Using the dietary P_i restricted dog, we demonstrated no significant decrease in renal reabsorption of P_i in response to parathyroid hormone administration. However, significant decreases in P_i transport in brush border membrane vesicles isolated from the kidneys of dietary P_i restricted dogs were observed in response to in vivo parathyroid hormone administration. These data demonstrate that the resistance to the phosphaturic action of parathyroid hormone observed in vivo does not include resistance to the inhibitory effect of parathyroid hormone on P_i transport in brush border membrane vesicles. Thus, the data suggest that parathyroid hormone continues to alter P_i transport characteristics of the brush border membrane in states of P_i depletion despite the resistance to parathyroid hormone seen in vivo.     

Parathyroid hormone-induced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells

Summary In order to examine the possibility of parathyroid hormone-mediated ultrastructural rearrangements in target epithelium, isolated canine renal proximal tubular cells were grown on a collagen-coated semipermeable membrane in a defined medium. Scanning and transmission electron microscopy of these monolayers revealed abundant microvilli. Exposure of the proximal tubular cells to parathyroid hormone resulted in a biphasic changes involving: (1) dramatic shortening and rarefaction of microvilli within 1 min; and (2) recovery of microvillar topography after 5 min. A similar shortening of microvilli was observed following exposure to ionomycin, whereas incubation with cyclic AMP resulted in an elongation of microvilli. Parathyroid hormone stimulated cyclic AMP production and increased cytoplasmic free calcium concentration in cultured proximal tubular cells. Pretreatment of cells with a calmodulin inhibitor abolished the effect of parathyroid hormone on brush border topography. Shortening of microvilli was associated with a disappearance of microvillar core filaments. Staining of F-actin with fluoresceinphalloidin showed that parathyroid hormone resulted in fragmentation of stress fibers. It is concluded that parathyroid hormoneinduced cell activation involves cytoplasmic-free calcium, calmodulin, and the cytoskeleton.