Ethanolamine transport in human placental brush-border membrane vesicles.

Pathways for transport of ethanolamine by human placental epithelia were investigated by measurement of [3H]ethanolamine uptake in brush-border membrane vesicles isolated by divalent cation precipitation. The presence of a conductive uptake pathway for ethanolamine was suggested by the marked stimulation of ethanolamine uptake to levels exceeding equilibrium induced by an inside-negative potassium diffusion potential. Evidence to suggest conductive ethanolamine uptake resulted from a mediated transport process included 1) the concentration-dependent inhibition by choline; 2) trans-stimulation of choline and ethanolamine uptake by ethanolamine; and 3) substrate-specific inhibition by chemically related analogs. Transport of both choline and ethanolamine by a common facilitated diffusion mechanism is suggested by 1) trans-stimulation of choline uptake by ethanolamine; 2) mutual inhibition of conductive choline and ethanolamine uptake by ethanolamine and choline; 3) the effect of ethanolamine on the kinetics of conductive choline uptake; and 4) the rank order inhibition of choline and ethanolamine uptake by the same panel of chemical analogs. The present study identifies the presence of a facilitated diffusion mechanism as a brush-border membrane transport pathway for choline and ethanolamine accumulation by human placenta.

Molecular cloning and characterization of two novel human renal organic anion transporters (hOAT1 and hOAT3).

The cloned organic anion transporters from rat, mouse, and winter flounder (rOAT1, mOAT1, fROAT) mediate the coupled exchange of alpha-ketoglutarate with multiple organic anions, including p-aminohippurate (PAH). We have isolated two novel gene products from human kidney which bear significant homology to the known OATs and belong to the amphiphilic solute facilitator (ASF) family. The cDNAs, hOAT1 and hOAT3, encode for 550- and 568-amino-acid residue proteins, respectively. hOAT1 and hOAT3 mRNAs are expressed strongly in kidney and weakly in brain. Both genes map to chromosome 11 region q11.7. PAH uptake by Xenopus laevis oocytes injected with hOAT1 mRNA is increased 100-fold compared to water-injected oocytes. PAH uptake is chloride dependent and is not further increased by preincubation of oocytes in 5 mM glutarate. Uptake of PAH is inhibited by probenicid, alpha-ketoglutarate, bumetanide, furosemide, and losartan, but not by salicylate, urate, choline, amilioride, and hydrochlorothiazide.

Thiamine transport in human placental brush border membrane vesicles.

Pathways for transport of thiamine by the human placental epithelium were investigated using brush border membrane vesicles isolated by divalent cation precipitation. The presence of thiamine transport mechanisms mediating Na+-thiamine cotransport, proton/thiamine exchange and facilitated diffusion was assessed from [3H]-thiamine tracer flux measurements. The magnitude of intravesicular thiamine accumulation was unaffected by the imposition of an inwardly directed sodium gradient suggesting an absence of a mechanism mediating brush border membrane Na+-thiamine cotransport. Intravesicular thiamine accumulation was indistinguishable when measured in the presence and absence of conditions favoring the development of an inside-negative, potassium diffusion potential. The observed absence of conductive thiamine uptake suggests the absence of a mechanism mediating facilitated diffusion of thiamine in placental brush border membrane. The imposition of an inside-acid pH gradient was observed to induce concentrative accumulation of thiamine to levels exceeding equilibrium, suggesting the presence of a placental brush border membrane proton/thiamine exchange mechanism. Protonophore- induced dissipation of an imposed inside-acid pH gradient in the absence of membrane potential was observed to abolish concentrative accumulation of thiamine, suggesting a direct chemical coupling of protons and thiamine via a mediated exchange mechanism. Consistent with the functional properties expected for a mechanism mediating thiamine transport by organic cation exchange, the rate and magnitude of intravesicular [3H]-thiamine accumulation was increased when measured in the presence compared to the absence of an outwardly directed thiamine concentration gradient. Substrate specificity studies of the proton/thiamine exchange mechanism suggest that the amine at position four of the pyrimidine ring, but not the hydroxyethyl side chain or an unmodified thiazolium ring, is an important chemical determinant for interaction with the transporter substrate binding site(s). Substrate specificity studies further suggest the possible presence of three separate organic cation exchange mechanisms mediating transport of thiamine, guanidine and MIA across placental brush border membrane.

Sulfate transport in human placental brush-border membrane vesicles.

Membrane transport pathways for transplacental transfer of sulfate were investigated by assessing the possible presence of a bicarbonate-coupled anion exchange mechanism for sulfate in the maternal facing membrane of human placental epithelial cells. The presence of a SO42-/HCO3- exchange mechanism was determined from 35SO42-tracer flux measurements in preparations of purified brush-border membrane vesicles. Under 10% CO2/90% N2 the imposition of an outwardly directed bicarbonate gradient (pH0 6/pHi 7.5) stimulated sulfate uptake to levels approximately 4-fold greater than observed at equilibrium. Maneuvers designed to offset the development of ion gradient-induced diffusion potentials (valinomycin, [K+]0 = [K+]i) significantly reduced bicarbonate gradient-induced sulfate uptake but concentrative accumulation of sulfate persisted. Early time point determinations performed in the presumed absence of membrane potential suggest the reduced level of bicarbonate gradient-induced sulfate uptake resulted from a more rapid dissipation of the imposed bicarbonate gradient. Concentrative accumulation of sulfate was not observed in the presence of a pH gradient alone under 100% N2. suggesting a preference of bicarbonate over hydroxyl ions as substrates for exchange. Static head determinations of opposing sulfate and bicarbonate gradients resulting in zero net flux of sulfate suggests the anion exchange mechanism mediates the electroneutral exchange of 2 bicarbonate or 1 carbonate for each sulfate. Sulfate uptake was increased with increasing intravesicular concentrations of carbonate at constant bicarbonate but was constant with increasing intravesicular concentrations of bicarbonate at constant carbonate suggesting carbonate as a substrate for anion exchange. The mechanism mediating bicarbonate gradient-induced sulfate uptake was sensitive to inhibition by stilbene derivatives, furosemide, bumetanide and probenecid. Substrate specificity studies suggest possible interactions of the anion exchange mechanism with salicylate, butyrate, thiosulfate, sulfite, selenate, chromate and oxalate. The results of this study provide evidence for the presence of a bicarbonate-coupled anion exchange mechanism as an electroneutral pathway for sulfate transport across the maternal-facing membrane of human placental epithelial cells.

Organic anion transport in rabbit renal basolateral membrane vesicles.

Pathways for p-aminohippurate (PAH) transport across the basolateral membrane of rabbit proximal tubule cells were investigated from studies of [3H] PAH uptake in membrane vesicles isolated by Percoll-density gradient centrifugation. The 10-s uptake of PAH was not significantly different when measured in the absence of cation gradients or in the presence of inwardly directed Na, Li, K or choline gradients that suggests the absence of a mechanism mediating Na-PAH cotransport. A probenicid-sensitive, trans-stimulation of [3H] PAH uptake was observed in the presence of an outward PAH gradient. PAH gradient-driven [3H] PAH uptake was cis-inhibited by glutarate, alpha-ketoglutarate, adipate and sebacate and outward gradients of alpha-ketoglutarate trans-stimulated probenicid-sensitive PAH uptake. A concentrative accumulation of PAH was measured in the presence of an inward Na gradient and the dicarboxylates glutarate or alpha-ketoglutarate. Compared to the absence of a pH gradient, an inside alkaline pH gradient induced an increased PAH uptake both in the presence and absence of CO2/HCO3. Inside-negative and inside-positive voltage differences were observed to stimulate and inhibit alpha-ketoglutarate gradient-driven PAH uptake, respectively. alpha-Ketoglutarate gradient-driven PAH uptake was progressively reduced in the presence of increasing penicillin concentration and an outward gradient of alpha-ketoglutarate induced an increased level of [14C] penicillin uptake. These results suggest the presence of a probenicid-sensitive organic anion exchange mechanism as a pathway for PAH and penicillin transport across the basolateral membrane of rabbit proximal tubule cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Choline transport in human placental brush-border membrane vesicles.

Pathways for transport of choline by human placental epithelia were investigated using brush border membrane vesicles isolated by divalent cation precipitation. The presence of choline transport mechanisms mediating Na(+)-choline cotransport, choline/H+ exchange and facilitated diffusion were assessed from [3H]choline tracer flux measurements. The rate and magnitude of intravesicular choline accumulation was unaffected by the imposition of an inwardly directed Na+ gradient suggesting an absence of a mechanism mediating brush border membrane Na(+)-choline cotransport. The imposition of inside-acid or inside-alkaline pH gradients was observed to have no significant effect on choline uptake suggesting choline is not a substrate for placental epithelial organic cation/H+ exchange. Conditions favoring the development of an inside-negative K+ diffusion potential was observed to induce a concentrative accumulation of choline to levels exceeding equilibrium suggesting the presence of a conductive uptake pathway for choline in placental brush border membrane. Evidence to suggest conductive choline uptake resulted from a mediated transport process includes a demonstration of the counterflow phenomena, the concentration-dependent inhibition by hemicholinium-3 (IC50 approximately equal to 100 microM) and the saturable rate of conductive choline uptake (Km approximately equal to 300 microM, Vmax approximately equal to 30 nmol/mg per min). Substrate specificity studies of the mechanism mediating conductive choline uptake suggest the interaction of choline with the transport protein occurs at a minimum of two sites: a site of negativity with the positively charged nitrogen group and a site of hydrogen bonding to the primary alcohol. Several commonly prescribed pharmaceuticals known to cross the placental barrier including imipramine, verapamil, propranolol, quinine, flurazepam, amiloride and ritodrin were observed to inhibit conductive choline uptake suggesting an interaction with the mechanism mediating conductive choline transport. Conductive choline uptake was unaffected by the presence of the basic amino acids lysine, arginine and histidine; the neurotransmitters serotonin, dopamine and histamine and the vitamins thiamine and carnitine which suggests the mechanism mediating conductive choline transport is not a pathway for placental uptake of these compounds.

Uric acid transport in rat renal basolateral membrane vesicles.

Pathways for urate transport across the basolateral membrane of rat proximal tubule cells were investigated using membrane vesicles isolated from rat renal cortex. The presence of an anion exchange mechanism(s) operative in the mode of alpha-keto-glutarate/urate; Cl-/urate and OH-(HCO3-)/urate as well as a mediated conductive mechanism was assessed from tracer flux measurements. In the presence of an inwardly directed Na+ gradient an alpha-ketoglutarate dependent concentrative accumulation of PAH but not urate was observed suggesting an absence of the mediated exchange of alpha-ketoglutarate for urate. The imposition of an outwardly directed Cl- gradient stimulated urate uptake in the absence but not the presence of conditions designed to minimize membrane potential development suggesting an indirect electrostatic coupling of urate uptake to a Cl- gradient-induced diffusion potential. Conditions favoring the development of an inside-positive K+ diffusion potential was observed to induce an inhibitor-sensitive, concentrative accumulation of urate in the absence of Cl-. The stimulation of urate uptake measured in the presence of an inside-alkaline pH gradient was not of sufficient magnitude to suggest the apparent conductive urate uptake was secondary to a membrane voltage induced, inside alkaline pH gradient and the operation of an OH-(HCO3-)/urate exchanger. The evidence obtained from the present investigation suggests rat basolateral membrane urate transport occurs by a mediated, conductive mechanism and is not coupled to Cl-, alpha-ketoglutarate or HCO3-.(ABSTRACT TRUNCATED AT 250 WORDS)

Human placental brush-border membrane Na(+)-pantothenate cotransport.

Membrane transport pathways for transplacental transfer of the water-soluble vitamin pantothenate were investigated by assessing the possible presence of a Na(+)-pantothenate cotransport mechanism in the maternal facing membrane of human placental epithelial cells. The presence of Na(+)-pantothenate cotransport was determined from radiolabeled tracer flux measurements of pantothenate uptake using preparations of purified brush-border membrane vesicles. Compared with other cations the imposition of an inward Na+ gradient stimulated vesicle uptake of pantothenate to levels approximately 40-fold greater than those observed at equilibrium. The observed stimulation of pantothenate uptake was not the result of indirect electrostatic coupling to an inside positive Na+ diffusion potential. In the absence of Na+ and pantothenate concentration gradients an inside negative voltage difference induced a Na(+)-dependent net influx of pantothenate, suggesting the presence of an electrogenic Na(+)-pantothenate cotransport mechanism. The effect of biotin on the kinetics of Na(+)-dependent pantothenate uptake and the effect of pantothenate on the kinetics of Na(+)-dependent biotin uptake suggested that placental absorption of biotin and pantothenate from the maternal circulation occurs by a common Na+ cotransport mechanism in apical brush-border membrane.

Human placental brush-border membrane Na(+)-biotin cotransport.

Membrane transport pathways for transplacental transfer of the water-soluble vitamin biotin were investigated by assessing the possible presence of a Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells. The presence of Na(+)-biotin cotransport was determined from radiolabeled tracer flux measurements of biotin uptake using preparations of purified brush-border membrane vesicles. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of biotin to levels approximately 25-fold greater than those observed at equilibrium. The voltage sensitivity of Na+ gradient-driven biotin uptake suggested Na(+)-biotin cotransport is electrogenic occurring with net transfer of positive charge. A kinetic analysis of the activation of biotin uptake by increasing Na+ was most consistent with an interaction of Na+ at 2 sites in the transport protein. Static head determinations used to identify the magnitude of opposing driving forces bringing flux through the cotransport mechanism to equilibrium indicated a coupling ratio of 2 Na+ per biotin. Substrate specificity studies using chemical analogues of biotin suggested both the terminal carboxylic acid of the valeric acid side chain and a second nucleus of anionic charge were important determinants for substrate interaction with the cotransport protein. Initial rate determinations of biotin uptake indicate biotin interacts with a single saturable site (Km = 21 microM) with a maximal transport rate of 4.5 nmol/mg/min. The results of this study provide evidence for an electrogenic Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells.

Effect of chronic acid loading on rat renal basolateral membrane bicarbonate transport.

The effect of chronic acid loading on the activity of luminal membrane Na(+)-H+ exchange and basolateral membrane Na+/HCO3- cotransport and Cl(-)-HCO3- exchange was investigated using membrane vesicles isolated from rat renal cortex. Na(+)-H exchange activity was increased approx. 50% in brush-border membranes isolated from acidemic compared to control kidneys. Na+/HCO3- cotransport and Cl(-)-HCO3- exchange activity was increased approx. 45% and 100%, respectively, in basolateral membranes isolated from acidemic kidneys. The increased Na+/HCO3- cotransport activity resulted from an increased apparent maximal rate of transport (Vmax) with no change in affinity (Km) for Na+. In contrast to acid/base transport activities chronic acid loading had no effect on the activity of basolateral membrane Na+/dicarboxylate cotransport. These results suggest proximal tubule cells coordinately increased luminal and basolateral membrane acid/base transport activities to accommodate an adaptive increase in the capacity for transcellular bicarbonate reabsorption.

C1/HCO3 exchange in human placental brush border membrane vesicles.

Membrane transport pathways for transplacental transfer of CO2/HCO3 were investigated by assessing the possible presence of a Cl/HCO3 exchange mechanism in the maternal-facing membrane of human placental epithelial cells. Cl/HCO3 exchange was tested for in preparations of purified brush border membrane vesicles by 36Cl tracer flux measurements and determinations of acridine orange fluorescence changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake to levels approximately 2-fold greater than observed at equilibrium. Maneuvers designed to offset the development of ion gradient-induced diffusion potentials (valinomycin, Ko = Ki) significantly reduced HCO3- gradient-driven Cl- uptake but concentrative accumulation of Cl- persisted. Early time point determinations performed in the presumed absence of membrane potential suggests the reduced level of HCO3- gradient-driven Cl- uptake resulted from a more rapid dissipation of the HCO3- concentration gradient. Concentrative accumulation of Cl- was not observed in the presence of a pH gradient alone under 100% N2, suggesting a preference of HCO3- over OH- as a substrate for transport. As monitored by acridine orange fluorescence the Cl- gradient-dependent collapse of an imposed pH gradient (pHo 8.5/pHi 6) was accelerated in the presence of CO2/HCO3 when compared with its absence, indicating coupling of HCO3- influx to Cl- efflux. Increasing concentrations of the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid were observed to cause a stepwise reduction in HCO3- gradient-driven Cl- uptake (I50 approximately 25 microM) further suggesting the presence of a Cl/HCO3 exchange mechanism. The results of this study provide evidence for a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive Cl/HCO3 exchange mechanism in the maternal-facing membrane of human placental epithelial cells. The identification of an ion-coupled HCO3- transport pathway in placental epithelia may suggest functional roles in mediating transplacental transfer of CO2 as well as maintenance of fetal acid/base balance.

Dicarboxylate transport in human placental brush-border membrane vesicles.

Pathways for transport of dicarboxylic acid metabolites by human placental epithelia were investigated using apical membrane vesicles isolated by divalent cation precipitation. The presence of Na+/dicarboxylate cotransport was assessed directly by [14C]succinate tracer flux measurements and indirectly by fluorescence determinations of voltage sensitive dye responses. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of succinate achieving levels approximately 5-fold greater than those observed at equilibrium. The increased succinate uptake was specific for Na+ as no stimulation was observed in the presence of Li+, K+ or choline+ gradients. In addition to concentrative accumulation of succinate, a direct coupling of Na+/succinate cotransport was suggested by the absence of a sizeable conductive pathway for succinate uptake and decreased succinate uptake levels associated with a more rapid decay of an imposed Na+ gradient. Na+ gradient-driven succinate uptake was not the result of parallel Na+/H+ and succinate/OH- exchange activities but was reduced by the Na+-coupled inhibitor harmaline. The voltage sensitivity of Na+ gradient-driven succinate uptake suggests Na+/succinate cotransport is electrogenic occurring with net transfer of positive charge. Substrate-specificity studies suggest the tricarboxylic acid cycle intermediates as candidates for transport by the Na+-coupled pathway. Decreasing pH increased the citrate-induced inhibition of succinate uptake suggesting divalent citrate as the preferred substrate for transport. Initial rate determinations of succinate uptake indicate succinate interacts with a single saturable site (Km 33 microM) with a maximal transport rate of 0.5 nmol/mg per min.

Proton-coupled organic cation transport in renal brush-border membrane vesicles.

We had previously proposed that organic cations are transported across the brush-border membrane in the canine kidney by a H+ exchange (or antiport) system (Holohan, P.D. and Ross, C.R. (1981) J. Pharmacol. Exp. Ther. 216, 294-298). In the present report, we demonstrate that in brush-border membrane vesicles the transport of organic cations is chemically coupled to the countertransport of protons, by showing that the uphill or concentrative transport of a prototypic organic cation, N1-methylnicotinamide (NMN), is chemically coupled to the flow of protons down their chemical gradient. In a reciprocal manner, the concentrative transport of protons is coupled to the counterflow of organic cations down their concentration gradient. The transport of organic cations is monitored by measuring [3H]NMN while the transport of protons is monitored by measuring changes in acridine orange absorbance. The functional significance of the coupling is that a proton gradient lowers the Km and increases the Vmax for NMN transport.

Cl(-)-HCO3- exchange in rat renal basolateral membrane vesicles.

Pathways for HCO3- transport across the basolateral membrane were investigated using membrane vesicles isolated from rat renal cortex. The presence of Cl(-)-HCO3- exchange was assessed directly by 36Cl- tracer flux measurements and indirectly by determinations of acridine orange absorbance changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake compared to Cl- uptake under 100% N2 in the presence of a pH gradient alone. Mediated exchange of Cl- for HCO3- was suggested by the HCO3- gradient-induced concentrative accumulation of intravesicular Cl-. Maneuvers designed to offset the development of ion-gradient-induced diffusion potentials had no significant effect on the magnitude of HCO3- gradient-driven Cl- uptake further suggesting chemical as opposed to electrical Cl(-)-HCO3- exchange coupling. Although basolateral membrane vesicle Cl- uptake was observed to be voltage sensitive, the DIDS insensitivity of the Cl- conductive pathway served to distinguish this mode of Cl- translocation from HCO3- gradient-driven Cl- uptake. No evidence for K+/Cl- cotransport was obtained. As determined by acridine orange absorbance measurements in the presence of an imposed pH gradient (pHo 7.5/pHi 6), a HCO3- dependent increase in the rate of intravesicular alkalinization was observed in response to an outwardly directed Cl- concentration gradient. The basolateral membrane vesicle origin of the observed Cl(-)-HCO3- exchange activity was verified by experiments performed with purified brush-border membrane vesicles. In contrast to our previous observations of the effect of Cl- on HCO3- gradient-driven Na+ uptake suggesting a basolateral membrane Na+-HCO3- for Cl- exchange mechanism, no effect of Na+ on Cl-HCO3- exchange was observed in the present study.

HCO3- transport in basolateral membrane vesicles isolated from rat renal cortex.

The mechanism of HCO3- translocation across the proximal tubule basolateral membrane was investigated by testing for Na+-HCO3- cotransport using isolated membrane vesicles purified from rat renal cortex. As indicated by 22Na+ uptake, imposing an inwardly directed HCO3- concentration gradient induced the transient concentrative accumulation of intravesicular Na+. The stimulation of basolateral membrane vesicle Na+ uptake was specifically HCO3(-)-dependent as only basolateral membrane-independent Na+ uptake was stimulated by an imposed hydroxyl gradient in the absence of HCO3-. No evidence for Na+-HCO3- cotransport was detected in brush border membrane vesicles. Charging the vesicle interior positive stimulated net intravesicular Na+ accumulation in the absence of other driving forces via a HCO3(-)-dependent pathway indicating the flow of negative charge accompanies the Na+-HCO3- cotransport event. Among the anion transport inhibitors tested, 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid demonstrated the strongest inhibitor potency at 1 mM. The Na+-coupled transport inhibitor harmaline also markedly inhibited HCO3- gradient-driven Na+ influx. A role for carbonic anhydrase in the mechanism of Na+-HCO3- cotransport is suggested by the modest inhibition of HCO3- gradient driven Na+ influx caused by acetazolamide. The imposition of Cl- concentration gradients had a marked effect on HCO3- gradient-driven Na+ influx which was furosemide-sensitive and consistent with the operation of a Na+-HCO3- for Cl- exchange mechanism. The results of this study provide evidence for an electrogenic Na+-HCO3- cotransporter in basolateral but not microvillar membrane vesicles isolated from rat kidney cortex. The possible existence of an additional basolateral membrane HCO3(-)-translocating pathway mediating Na+-HCO3- for Cl- exchange is suggested.

Na+/HCO3-co-transport in basolateral membrane vesicles isolated from rabbit renal cortex.

Recent studies suggest that the major pathway for exit of HCO3- across the basolateral membrane of the proximal tubule cell is electrogenic Na+/HCO3- co-transport. We therefore evaluated the possible presence of Na+/HCO3- co-transport in basolateral membrane vesicles isolated from the rabbit renal cortex. Imposing an inward HCO3- gradient induced the transient uphill accumulation of Na+, and imposing an outward Na+ gradient caused HCO3- -dependent generation of an inside-acid pH gradient as monitored by quenching of acridine orange fluorescence, findings consistent with the presence of Na+/HCO3- co-transport. In the absence of other driving forces, generating an inside-positive membrane potential by imposing an inward K+ gradient in the presence of valinomycin caused net Na+ uptake via a HCO3- -dependent pathway, indicating that Na+/HCO3- co-transport is electrogenic and associated with a flow of negative charge. Imposing transmembrane Cl- gradients did not appreciably affect HCO3- gradient-stimulated Na+ influx, suggesting that Na+/HCO3- co-transport is not Cl- -dependent. The rate of HCO3- gradient-stimulated Na+ influx was a simple, saturable function of the Na+ concentration (Km = 9.7 mM, Vmax = 160 nmol/min/mg of protein), was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (I50 = 100 microM), but was inhibited less than 10% by up to 1 mM amiloride. We could not demonstrate a HCO3- -dependent or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive component of Na+ influx in microvillus membrane vesicles. This study thus indicates the presence of a transport system mediating electrogenic Na+/HCO3- co-transport in basolateral, but not luminal, membrane vesicles isolated from the rabbit renal cortex. Analogous to the use of renal microvillus membrane vesicles to study Na+/H+ exchange, renal basolateral membrane vesicles may be a useful model system for examining the kinetics and possible regulation of Na+/HCO3- co-transport.

Mechanism of renin release in exercising dog.

Exercise is associated with an increase in plasma renin activity (PRA). The purpose of this study was to determine the role of the prostaglandin (PG) and adrenergic pathways in the renin release with exercise in the dog. One group of animals (n = 4) was exercised under control untreated and indomethacin- and meclofenamate- (2 mg/kg) treated conditions. A 155% increase in PRA was not influenced by PG inhibition. In a second group (n = 7) PRA was 1.22 +/- 0.32, 3.29 +/- 1.59, 6.28 +/- 2.85, and 5.30 +/- 2.00 ng ANG I X ml-1 X h-1 at rest and during light, moderate, and heavy exercise, respectively. Guanethidine treatment (15 mg/kg) decreased mean PRA by 41, 50, 70, and 73% at rest and during the three levels of exercise, respectively. In a third group (n = 5) control exercise runs were repeated after metoprolol treatment. Selective beta 1-blockade completely abolished the increment in PRA observed with exercise. These data demonstrate that the elevation of PRA during exercise in the dog is mediated by increased sympathetic nerve activity involving beta 1-receptors and that it is not dependent on alterations in PG synthesis.

Effect of K+ and H+ on sodium/citrate cotransport in renal brush-border vesicles.

The uptake of citrate by renal brush-border vesicles, prepared according to the method of Vannier, occurs by Na+-linked cotransport. It is 'positive rheogenic', i.e., stimulated by an (inside) negative, and inhibited by an (inside) positive electrical potential. The question arises whether, besides Na+, other ions (e.g., K+ and H+) participate in the cotransport. As to K+, neither an inward nor an outward directed K+ gradient has a significant effect on the citrate movement, but at equal concentrations of K+ inside and outside, equilibrium exchange of citrate, and to a smaller extent, the Na+-linked net uptake of citrate, are significantly stimulated. This observation is consistent with a hypothetical model in which K+ acts by accelerating both the empty and the fully loaded translocator. As to H+, citrate uptake is also stimulated by decreasing extravesicular pH, an effect previously attributed to protonization of the citrate anion in the assumption that the resulting secondary citrate anion is more acceptable to the translocator site. It was found, however, that the pH effect is still apparent if the concentration of the secondary citrate is kept constant by adjusting the total citrate concentration. This is taken as an argument against the above assumption and as being consistent with H+-linked cotransport. After the overshoot peak citrate exits slowly, and even after several hours does not attain equilibrium distribution, presumably owing to trapping by vesicular calcium.