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V L Schuster

Publications and source records attributed to V L Schuster.

At least 37 records · Page 2Linked to original sources
PubMed1995

Identification and characterization of a prostaglandin transporter.

Carrier-mediated prostaglandin transport has been postulated to occur in many tissues. On the basis of sequence homology, the protein of unknown function encoded by the rat matrin F/G complementary DNA was predicted to be an organic anion transporter. Expression of the matrin F/G complementary DNA in HeLa cells or Xenopus oocytes conferred the property of specific transport of prostaglandins. The tissue distribution of matrin F/G messenger RNA and the sensitivity of matrin F/G-induced prostaglandin transport to inhibitors were similar to those of endogenous prostaglandin transport. The protein encoded by the matrin F/G complementary DNA is thus preferably called PGT because it is likely to function as a prostaglandin transporter.

Amino Acid Sequence↗
PubMed1993

A basolateral lactate/H+ co-transporter in Madin-Darby Canine Kidney (MDCK) cells.

Monolayers of Madin-Darby Canine Kidney (MDCK) cells grown on permeable filters generated lactate aerobically and accumulated it preferentially in the basolateral compartment, suggesting the presence of a lactate carrier. The mechanism of lactate transport across apical and basolateral membranes was examined by determining intracellular pH (pHi) microspectrofluorimetrically after addition of lactate to the extracellular solutions and by measuring uptake of [14C]lactate. Addition of 20 mM lactate to the apical compartment produced no change in pHi, whereas lactate added to the basolateral compartment rapidly and reversibly lowered pHi. Pyruvate produced similar results. Inhibitors of lactate/H+ co-transporters, alpha-cyano-4-hydroxycinnamate (CnCN) and quercetin, partially inhibited the fall in pHi produced by basolateral lactate. In contrast, the disulphonic stilbene. DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid) produced no inhibition at 0.5 mM. Kinetic analysis was performed by applying basolateral lactate at various concentrations and measuring the rate of entry (delta pHi/min) in the presence and absence of CnCN. Lactate flux was shown to occur by both non-ionic diffusion and a alpha-cyano-4-hydroxycinnamate-sensitive component (carrier). The latter has a Km of approximately 7 mM for the lactate anion. Propionate, but not formate, lowered pHi to the same degree as did equimolar lactate, but the propionate effect was not inhibited by CnCN. Influx of [14C]lactate was substantially greater across the basolateral membrane than across the apical membrane and occurred in the absence of Na+. We conclude that MDCK cells grown on permeable filters generate lactate aerobically and transport it across the basolateral membrane by way of a lactate/H+ cotransporter.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

A 45-kDa protein antigenically related to band 3 is selectively expressed in kidney mitochondria.

Anion exchange similar to that catalyzed by erythrocyte band 3 occurs across many nonerythroid cell membranes. To identify anion-exchange proteins structurally related to band 3, we immunoblotted rabbit kidney medullary membrane fractions with anti-band 3 antibodies. Immunoblots using antibodies to the cytoplasmic domain of band 3 revealed cross-reactive proteins in the plasma membrane fraction only. In contrast, two monoclonal antibodies against band 3 membrane domain labeled a 45-kDa protein; further immunoblotting and immunogold studies of membrane fractions and kidney sections using one of the anti-membrane domain antibodies showed that labeling was strongest in mitochondria of H(+)-secreting collecting duct cells. Tissue-to-tissue expression of the 45-kDa mitochondrial protein was variable: kidney medulla greater than heart greater than kidney cortex much greater than liver. We conclude that a 45-kDa protein with immunological cross-reactivity to the erythrocyte band 3 membrane domain is expressed in mitochondria in a highly cell-specific fashion and speculate that the protein may play a role in mitochondrial anion transport.

Animals↗
PubMed1991

Imaging of filter-grown epithelial cells: MDCK Na(+)-H+ exchanger is basolateral.

We report a simple method for growing epithelial cells on permeable supports and for imaging the cells from the apical side using an inverted microscope. Madin-Darby canine kidney (MDCK) cells were either seeded onto the conventional side of Millipore-CM filters or onto "inverted" filters. The peak transepithelial resistance of confluent monolayers was the same with either system. Cells on inverted filters that were stained with various dyes and imaged by epifluorescence exhibited more distinct intercellular spaces, cell margins, nuclei, and subapical vesicles. We also perfused both sides of inverted filters with HCO3/CO2-free saline and measured intracellular pH (pHi) using 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and digital imaging. The intrinsic buffer capacity of MDCK cells increased exponentially as pHi decreased. After an NH4Cl load, the H+ extrusion rate (JH+) in control saline was 2.42 +/- 0.62 mM/min. JH+ was completely blocked by 1 mM basolateral amiloride. In contrast, 1 mM apical amiloride had no effect. We conclude that 1) growth of epithelial cells on an inverted filter system is useful for the microspectrofluorimetric determination of pHi in single cells and for the imaging of apical/subapical structures, and 2) the Na(+)-H+ exchanger of MDCK cells is functionally polarized to the basolateral membrane.

Amiloride↗
PubMed1991

Axial heterogeneity of rabbit cortical collecting duct.

The rabbit cortical collecting duct (CCD) consists of three major cell types: principal cells transport K+, beta-intercalated cells absorb Cl-, and alpha-intercalated cells secrete H+. We used functional and histological methods to assess axial distribution of these cell types along rabbit CCD. In perfused CCDs, lumen-to-bath Rb+ rate coefficient (an index of principal cell K+ transport) was not different in tubules from outer cortex (1 mm from renal surface) compared with those from inner cortex (2 mm from renal surface), suggesting that principal cell function is homogeneous along the CCD. In contrast, Cl- rate coefficient (a measure of beta-intercalated cell function) was twice as high in CCDs from outer compared with inner cortex, suggesting heterogeneity of beta-intercalated cells along the CCD. To further investigate these regional differences, we fixed and embedded kidneys and identified three cell types in CCD cross sections using carbonic anhydrase staining and peanut lectin binding. Comparing tubule cross sections from outer with those from inner cortex, we found no axial difference in the fraction of cells that were either principal cells (64%) or total (lectin binding and nonlectin binding) intercalated cells (36%). However, the lectin-binding intercalated cell subset was significantly increased in outer compared with inner cortex. We conclude that there is not heterogeneity of principal cells along the rabbit CCD; however, beta-cell number and function are increased in outer CCD. Collecting duct heterogeneity begins within the cortical segment.

Absorption↗
PubMed1991

Colocalization of H(+)-ATPase and band 3 anion exchanger in rabbit collecting duct intercalated cells.

Two major types of intercalated cells (IC) have been previously defined in rabbit collecting duct: alpha-cells have a basolateral band 3-like anion exchanger and secrete H+, whereas beta-cells bind peanut agglutinin (PNA) apically and are believed to secrete HCO3-. To further define IC types, we double-labeled kidney sections with anti-H(+) -ATPase antibodies and with either an anti-band 3 antibody or PNA. We found four patterns of staining: 1) IC with apical H(+)-ATPase and basal band 3, a configuration consistent with ongoing H+ secretion, which prevailed in the inner stripe of outer medulla (OMCDi); 2) diffuse H(+)-ATPase labeling across the cell and basal band 3, which was most numerous in the outer stripe of outer medulla (OMCDo); 3) IC with "bright" apical peanut lectin, diffuse H(+)-ATPase, and no band 3, which was abundant in the cortical collecting duct (CCD) and probably represents HCO3(-)-secreting cells; and 4) "hybrid" cells with various staining combinations (e.g., apical lectin binding and apical H(+)-ATPase), which although they are uncommon, were seen in the CCD. Consistent with this immunocytochemical finding of hybrid cells, cell-sorting studies on isolated CCD IC showed that 6-18% of PNA-positive cells also stained positively for band 3. We conclude that 1) band 3-positive IC in the OMCD vary axially. Most OMCDi IC are probably active proton secretors, whereas up to one-half of OMCDo IC may be latent H+ secretors. 2) The diffuse H(+)-ATPase pattern in putative beta-cells differs from comparable results in the rat and is not consistent with a "reversed" alpha-cell. HCO3- secretion by beta-cells may be driven by an H+ extrusion mechanism other than the alpha-cell pump re-sorted to the basolateral membrane. 3) The possibility of hybrid cells that might combine alpha- and beta-cell transport proteins suggests a mechanism for functional reversal of collecting duct IC polarity.

Animals↗
PubMed1990

Organization of collecting duct intercalated cells.

Our understanding of the mechanisms by which the collecting duct transports HCO3 continues to evolve rapidly. The models put forth in Figure 1, though esthetically pleasing by virtue of their simplicity, will undoubtedly require modification as the above areas and others continue to be explored. It should be noted that a large percentage of the citations in this review emanate from colleagues of Dr. Donald Seldin who have been or currently are nephrologists at Southwestern Medical School in Dallas. The length of this list is testimony to the large number of investigators in the field of renal acid-base research who have been intellectually stimulated by their contact with Dr. Seldin.

Acid-Base Equilibrium↗
PubMed1989

Reduction in sensitivity to Cl- channel blockers by HCO3- -CO2 in rabbit cortical collecting duct.

We examined the ability of HCO3- -CO2 to modify the potency of Cl- channel blockers in the renal cortical collecting duct (CCD) for the following two reasons. 1) From a practical point of view, there is, to our knowledge, no information regarding the effect of the HCO3- -CO2 buffer system on the potency of Cl- channel blockers. 2) We showed in the companion manuscript [Am. J. Physiol. 257 (Cell Physiol. 26): C94-C101, 1989] that HCO3- -CO2 stimulates transepithelial anion exchange in the CCD. Based on precedent in the literature, we postulated that HCO3- stimulates the basolateral membrane Cl- conductance. Here, we demonstrate that several Cl- channel blockers can reduce CCD Cl- self exchange when the solutions are buffered in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Concentrations of blockers producing 80% inhibition in HEPES, pH 7.4, produced only 20% inhibition in 25 mM HCO3- -CO2, pH 7.4. The ability of HCO3- -CO2 to reduce blocker potency had an IC50 of only 2 mM. We also examined interactions of HCO3- -CO2 and blockers with regard to the principal cell basolateral Cl- conductance. Blockers did not alter the Rb+ flux, a marker of K+ transport, but did reduce transepithelial conductance (GT), i.e., the blockers inhibited the principal cell basolateral Cl- conductance. As was the case with intercalated cell anion exchange, GT measurements indicated that HCO3- -CO2 impaired the ability of Cl- channel blockers to inhibit the principal cell Cl- conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1989

Stimulation of Cl- self exchange by intracellular HCO3- in rabbit cortical collecting duct.

In rabbit cortical collecting duct, Cl- self exchange accounts for most of the transepithelial Cl- tracer rate coefficient, KCl (nm/s); a small fraction is effected by Cl--HCO3- exchange and Cl- diffusion. We previously reported that changing from a CO2-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) bath to a 5% CO2-25 mM HCO3- bath stimulates Cl- self exchange. Here, we examine in further detail the individual components of the CO2-HCO3- system that stimulate KCl. Addition of 0.5% CO2 to a HEPES bath (final pH = 7.24) stimulated KCl by 70 +/- 19 nm/s, a delta KCl comparable to that induced by 1% CO2 (pH 7.12), 6% CO2 (pH 6.6), or 6% CO2-25 mM HCO3- (pH 7.4). The roles of intracellular pH (pHi) and HCO3- concentration were examined by clamping pHi using high K+ and nigericin. Increasing pHi from 6.9 to 7.6 in solutions without exogenous CO2 or HCO3- increased KCl by 71 +/- 17 nm/s. These results suggest that pHi might regulate anion exchange. However, during such a pHi-shift experiment, metabolically derived CO2 produces a concomitant change in intracellular HCO3- concentration [( HCO3-]i). To determine whether an increase in [HCO3-]i could stimulate Cl- self exchange, we replaced HEPES with 6% CO2-5 mM HCO3- isohydrically (pHi clamped at 6.9). With this increase in [HCO3-]i at constant pHi, KCl increased by 51 +/- 10 nm/s. These maneuvers had negligible effects on Cl- diffusion and Cl--HCO3- exchange. These experiments demonstrate that increases in cell [HCO3-] (or perhaps CO2) can stimulate transepithelial anion exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

K+ and Rb+ transport by the rabbit CCD: Rb+ reduces K+ conductance and Na+ transport.

We compared transport of K+ and Rb+ across the rabbit cortical collecting duct to gain insight into the mechanisms of K+ secretion. Passive tracer fluxes, active secretory rates, electrophysiological behavior, and the ability of each ion to support Na+-K+-ATPase activity were determined. When active transport was inhibited by amiloride, K+ permeability was twice the Rb+ permeability. Transepithelial conductance (GT) was half as great in solutions where 5 mM Rb+ replaced 5 mM K+. When 4 mM Ba2+ was added to the lumen, both Rb+ and K+ permeability fell to values not different from that expected for paracellular diffusion. The relationship between Ba2+-induced changes in the K+ and Rb+ permeabilities and in the simultaneously measured GT provides strong evidence that K+ transport across the apical membrane is largely, if not exclusively, conductive. We also determined that net K+ secretion is greater than net Rb+ secretion (when each is the abundant ion). The reasons for this difference probably involve several steps in the K+ secretory process and include the following: 1) reduced ATPase activity in the presence of Rb+ (approximately 80%) compared with K+, 2) reduction of Na+ absorption, and 3) partial blockade of the apical (and perhaps basolateral) K+ conductance. Although there were quantitative differences between K+ and Rb+ transport, we found no evidence suggesting that these ions are transported by different mechanisms.

Absorption↗
PubMed1988

Absence of transepithelial anion exchange by rabbit OMCD: evidence against reversal of cell polarity.

In the rabbit cortical collecting duct (CCD), Cl tracer crosses the epithelium predominantly via an anion exchange system that operates in either a Cl-Cl or Cl-HCO3 exchange mode. In the present study, we used the 36Cl lumen-to-bath rate coefficient (KCl, nm/s), a sensitive measurement of CCD transepithelial anion transport, to investigate the nature of Cl transport in the medullary collecting duct dissected from inner stripe, outer medulla (OMCD). The KCl in OMCD perfused and bathed in HCO3-Ringer solution was low (46.2 +/- 8.5 nm/s) and similar to that value observed in the CCD when anion exchange is inhibited and Cl permeates the epithelium by diffusion. Unlike KCl in CCD, KCl in OMCD was not stimulated by adenosine 3',5'-cyclic monophosphate (cAMP). OMCD KCl was not altered by bath Cl and/or HCO3 removal, demonstrating the absence of transepithelial Cl-Cl and Cl-HCO3 exchange. To test the hypothesis that metabolic alkalosis could reverse the polarity of intercalated cells and thus induce an apical Cl-HCO3 exchanger in H+-secreting OMCD cells, we measured KCl in OMCD from rabbits made alkalotic by deoxycorticosterone and furosemide. Although the base-line KCl was slightly higher than in OMCD from control rabbits, the value was still far lower than the KCl under comparable conditions in CCD. Moreover, KCl in OMCD from alkalotic rabbits was unchanged by cAMP, or by sequential removal of bath HCO3 and Cl. Immunocytochemistry using peanut lectin and a monoclonal antibody to-erythrocyte band 3 failed to reveal any evidence for alkalosis-induced reversal of either CCD or OMCD intercalated cell polarity.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkalosis↗
PubMed1988

Characteristics of the paracellular pathway of rabbit cortical collecting duct.

The nature of the paracellular pathway of the rabbit cortical collecting duct (CCD) was examined under conditions designed to eliminate all cellular ion transport. Transcellular conductive pathways were blocked by addition of amiloride and Ba2+ to the perfusate. Cl self-exchange was eliminated by removing Cl from the bath solution, and HCO3 transport was eliminated by omitting HCO3 and CO2 from the solutions. The residual transepithelial conductance (GT) and radioisotopic tracer flux under these conditions probably occur via the paracellular pathway. The GT measured in nontransporting CCD bathed in NaCl solutions was 1.1-1.2 mS/cm2. When Na or Cl was replaced by a less mobile ion, the GT decreased by an amount commensurate with the decrease in solution conductivity. The Na-to-Cl permeability ratio determined by NaCl dilution voltages ranged from 0.55 to 0.82. An independent estimate of paracellular selectivity was obtained by comparing the lumen-to-bath tracer rate coefficients for Na (kNa) and Cl (kCl). The ratio kNa:kCl was 0.75. These observations suggest that the paracellular pathway displays a Na:Cl permselectivity not substantially different from the ratio of their mobilities in water (0.65). Additional experiments demonstrated that the summed partial ionic conductances of Na and Cl calculated from the tracer fluxes were in close agreement with the measured GT. We conclude that the paracellular conductance of CCD is nonselective in character and approximately 1-2 mS/cm2 in magnitude.

Animals↗
PubMed1988

Inhibition of cortical collecting tubule chloride transport by organic acids.

Cl self-exchange by the rabbit cortical collecting tubule (CCT) occurs via an apical anion exchanger in series with a basolateral Cl conductance. We studied the effects of organic acids on CCT Cl self-exchange. We found no evidence for transport of acid anions by the self-exchange system. Rather, Cl self-exchange was inhibited by a variety of organic acids. The degree of inhibition correlated with the chloroform/water partition coefficient and was enhanced by lowering pH, indicating inhibition by the lipid-soluble, protonated species. Inhibition by the representative acid iso-butyrate was dose-dependent and showed sidedness (basolateral greater than apical). Iso-butyrate also reversibly reduced transepithelial conductance without altering K permeability, suggesting inhibition of the principal cell basolateral Cl conductance. Because small organic compounds with similar lipid solubilities but no carboxyl group had no effect, both the carboxyl group and the lipid-solubility of organic acids appear to be important. The results are consistent with blockade of chloride channels by organic acids.

1-Butanol↗