Biomedical subjects

S H Wright

Publications and source records attributed to S H Wright.

At least 37 records · Page 2Linked to original sources

PubMed1999

Real-time assessment of alpha-ketoglutarate effect on organic anion secretion in perfused rabbit proximal tubules.

To determine the quantitative roles of the basolateral and luminal Na(+)-dicarboxylate (Na-DC) cotransporters in establishing and maintaining the alpha-ketoglutarate (alphaKG) gradient required for renal tubular secretion of organic anions, we measured net steady-state transepithelial secretion of fluorescein (FL) in real time in isolated, perfused S2 segments of rabbit renal proximal tubules. Net "basal" FL secretion in the absence of exogenous alphaKG had a K(t) of approximately 4 microM and a maximal transepithelial secretion rate (J(max)) of approximately 380 fmol. min(-1). mm(-1) (where K(t) is the FL concentration that produces one-half the J(max)). It could be almost completely inhibited by basolateral p-aminohippurate (PAH). Selective inhibition of the basolateral Na-DC cotransporter indicated that recycling via this transporter of alphaKG that had been exchanged for FL supports approximately 25% of the "basal" FL secretion. Physiological alphaKG concentrations of 10 microM in the bath or 50 microM in the perfusate stimulated net secretion of FL by approximately 30 or approximately 20%, respectively. These data indicate that the basolateral Na-DC cotransporter supports approximately 42% of the net FL secretion. The luminal and basolateral effects of physiological concentrations of alphaKG were additive, indicating that the combined function of the luminal and basolateral Na-DC cotransporters can support approximately 50% of the net FL secretion. This apparently occurs by their establishing and maintaining approximately 50% of the outwardly directed alphaKG gradient that is responsible for driving basolateral FL/alphaKG exchange. The remaining approximately 50% would be maintained by metabolic production of alphaKG in the cells.

Animals↗

PubMed1998

Influence of substrate structure on turnover of the organic cation/H+ exchanger of the renal luminal membrane.

We examined the influence of organic cation (OC) structure on the rate of turnover of the OC/H+ exchanger in rabbit renal brush-border membrane vesicles (BBMV). The rate of efflux of [14C]tetraethylammonium ([14C]TEA) from BBMV, measured in the presence of an inwardly directed chemical gradient for test agent, provided an indirect measure of activity of the OC/H+(OC) exchanger. The trans-stimulation of [14C]TEA efflux from BBMV was a saturable function of increasing extravesicular concentration of both unlabeled TEA and tetramethylammonium (TMA), with an apparent Michaelis constant (Kt) for the interaction of these compounds with the OC/H+(OC) exchanger of 25 microM and 1 mM, respectively. The effect on [14C]TEA efflux of saturating extravesicular concentrations of a series of n-tetraalkylammonium compounds was examined. Whereas the short-chain compounds TMA and TEA markedly stimulated [14C]TEA efflux (by 830% and 690%, respectively), the long-chain compounds tetrapropylammonium and tetrabutylammonium were less effective, increasing efflux by only 40% and 120%, respectively. When the exchanger was saturated with tetrapentylammonium, mediated efflux of [14C]TEA was reduced. Increasing alkyl chain length was also correlated with an increase in the inhibitory effect (as measured by the apparent inhibition constant, Ki, or the IC50 value) that these compounds had against transport of [14C]TEA by the OC/H+(OC) exchanger; i.e., there was a correlation between decreasing IC50 and decreasing turnover of the OC/H+(OC) exchanger. This same correlation was observed for a broader set of test agents of diverse molecular structure, including a series of n-tetraalkylammonium and -phosphonium compounds and the OCs, choline, N1-methyl nicotinamide, 1-methyl-4-phenylpyridinium, and amiloride. Because high affinity of substrates for the OC/H+(OC) exchanger is correlated with increasing substrate hydrophobicity, we conclude that the interaction of hydrophobic OCs with the renal OC/H+(OC) exchanger results in the formation of a substrate-exchanger complex that has a comparatively low rate of turnover.

Animals↗

PubMed1998

Basolateral choline transport in isolated rabbit renal proximal tubules.

Choline can undergo both net secretion and net reabsorption by renal proximal tubules, but at physiological plasma levels net reabsorption occurs. During this process, choline enters the cells at the luminal side down an electrochemical gradient via a specific transporter with a high affinity for choline. It appeared likely that choline was then transported out of the cells against an electrochemical gradient at the basolateral membrane by countertransport for another organic cation. This possibility was examined by studying net transepithelial reabsorption and basolateral uptake and efflux of [14C]choline in isolated S2 segments of rabbit renal proximal tubules. Basolateral uptake, which was inhibited by other organic cations such as tetraethylammonium (TEA), appeared to occur by the standard organic cation transport pathway. However, the addition of TEA to the bathing medium not only failed to trans-stimulate net transepithelial reabsorption and basolateral efflux of [14C]choline but it actually inhibited transepithelial reabsorption by @60%. The results do not support the presence of a countertransport step for choline against an electrochemical gradient at the basolateral membrane. Instead, they suggest that choline crosses this membrane by some form of carrier-mediated diffusion even during the reabsorptive process.

Absorption↗

PubMed1998

Effect of alpha-ketoglutarate on organic anion transport in single rabbit renal proximal tubules.

The effect of exogenous alpha-ketoglutarate (alpha KG) and the peritubular Na(+)-dicarboxylate (Na-DC) cotransporter on organic anion/dicarboxylate (OA/DC) exchange in S2 segments of single, nonperfused rabbit proximal tubules was measured using 1 microM fluorescein (FL), a model OA, and epifluorescence microscopy. The effect of different transmembrane distributions of 10 microM alpha KG on peritubular FL uptake was measured at 37 degrees C using bicarbonate-buffered, nutrient-containing buffers, which are conditions similar to those found in vivo. Compared with FL uptake in the absence of exogenous alpha KG, preloading tubules with alpha KG (trans-configuration) or acute exposure to alpha KG (cis-configuration) increased FL uptake 62% and 54%, respectively, whereas a cis-trans-configuration of alpha KG increased FL uptake by 76%. The cis-stimulation of FL uptake by alpha KG was rapid, within 5-7 s. This stimulation was blocked 96% by simultaneous exposure to 2 mM Li+, indicating that stimulation of transport was secondary to the uptake of exogenous alpha KG. In the absence of exogenous alpha KG, selective inhibition of Na-DC cotransport using 2 mM Li+ or 1 mM methylsuccinate decreased FL uptake by 25% (effects that were reversible but not additive), suggesting that the Na-DC cotransporter recycles endogenous alpha KG that has left the cell in exchange for FL and that this activity supports approximately 25% of baseline activity of the OA/DC exchanger. With recycling of alpha KG accounting for approximately 25% of FL uptake and with accumulation of exogenous alpha KG accounting for another approximately 75% increase in FL uptake, Na-DC cotransport appears to directly support (25% + 75%)/175%, or approximately 57%, of total FL transport.

Animals↗

PubMed1998

Peritubular transport of ochratoxin A by single rabbit renal proximal tubules.

Epifluorescence microscopy was used to study peritubular transport of the fluorescent mycotoxin ochratoxin A (OTA) into single proximal tubule segments of the rabbit. Initial rates of OTA uptake into S2 segments were saturable and adequately described by Michaelis-Menten kinetics, with an apparent Km of 2.2+/-0.3 microM (SEM). Several lines of evidence indicated that peritubular uptake of OTA in S2 segments was effectively limited to the "classical" organic anion transporter. First, 5 mM p-aminohippurate (PAH) cis-inhibited the uptake of 1 microM OTA into tubules by 96%. Kinetic analysis of the inhibition of OTA uptake by PAH (100 microM to 5 mM) yielded an apparent Ki of 164 microM, similar to the 100 to 200 microM range of Km values previously reported for the peritubular uptake of PAH. Second, efflux of OTA from tubules was trans-stimulated 3.2-fold by the presence of 2.5 mM PAH in the uptake medium. Third, 100 microM alpha-ketoglutarate (alphaKG) trans-stimulated the uptake rate of 1 microM OTA by 1.8-fold. Fourth, besides PAH, other organic anions effectively cis-inhibited the uptake of 1 microM OTA into tubules (inhibitor, % inhibition): 1.5 mM alphaKG, 80%; 1 mM probenecid, 100%; 1 mM piroxicam, 100%; 1 mM octanoate, 100%. In contrast, 1.5 mM tetraethylammonium, an organic cation, blocked uptake of 1 microM OTA by only 7%. The inhibition of OTA uptake into S1 and S3 segments of the proximal tubule was qualitatively similar: 5 mM PAH cis-inhibited the uptake of 1 microM OTA by approximately 95% in both S1 and S3 segments. Thus, peritubular OTA uptake into all segments of the proximal tubule appears to be dominated by its interaction with the classical organic anion transporter. The high-affinity and relatively high capacity of this pathway for OTA suggest that peritubular uptake may be a significant avenue for the entry of this toxin into proximal tubule cells.

Animals↗

PubMed1998

Peritubular transport of ochratoxin A in rabbit renal proximal tubules.

The transport of the nephrotoxic mycotoxin ochratoxin A across the renal peritubular membrane was examined in suspensions of rabbit renal proximal tubules. Ochratoxin A transport across the peritubular membrane was a high-affinity, low-capacity carrier-mediated process with a Jmax value of 0.12 +/- 0.4 nmol/mg of protein/min and a Km value of 1.4 +/- 0.1 microM. The apparent Michaelis constants for inhibition of [3H]para-aminohippurate (PAH) uptake by ochratoxin A inhibition was 1.5 microM, which is similar to the Km value for ochratoxin A uptake in tubule suspensions and suggests that ochratoxin A could be a substrate for the organic anion pathway. The capacity and affinity for peritubular ochratoxin A transport were 40-fold lower and > 100-fold greater, respectively, than those measured for the peritubular uptake of [3H]PAH in tubule suspensions. A concentration of 2.5 mM PAH, which reduced the uptake of [3H]PAH by 90%, reduced ochratoxin A uptake by only 40% to 50%, whereas probenecid concentrations of 0.6 to 2 mM reduced ochratoxin A accumulation in tubule suspensions up to approximately 80% to 90%. This probenecid-sensitive, PAH-insensitive uptake of ochratoxin A suggested that at least one mediated pathway other than the organic anion transporter was involved in the peritubular uptake of this mycotoxin. A 2 mM concentration of the fatty acid octanoate and 1.5 mM concentration of the nonsteroidal anti-inflammatory agent piroxicam were as effective as probenecid in blocking ochratoxin A uptake. The apparent Ki values for inhibition of ochratoxin A uptake by probenecid, piroxicam and octanoate were 30.5 +/- 7.9, 23.2 +/- 10.4 and 81.5 +/- 8.7 microM, respectively. The ability of octanoic acid to inhibit ochratoxin A transport to the same extent as probenecid and a greater extent than PAH suggests that a separate fatty acid transport pathway may be involved in the accumulation of ochratoxin A by suspensions of rabbit renal proximal tubules.

Animals↗

PubMed1998

Na-dependent transport of S-(1,2-dichlorovinyl)-L-cysteine by renal brush-border membrane vesicles.

Cytotoxicity after exposure to the nephrotoxicant S-(1, 2-dichloro-vinyl)-L-cysteine (DCVC) requires transport of this cysteine conjugate across the cell membrane. Although several basolateral transport pathways have been implicated in the uptake of this compound into renal proximal cells, the identity of the process or processes associated with transport across the luminal membrane is unclear. We used a preparation of luminal brush-border membrane vesicles to characterize the transport of [35S]DCVC in rabbit kidney. An inwardly directed Na-gradient stimulated the initial rate of DCVC uptake by 16-fold compared to uptake measured in the absence of Na+. The Na-dependent component of DCVC uptake was stimulated by imposition of an inside-negative electrical potential difference and was blocked by the presence of 5 mM unlabeled DCVC in the extravesicular solution. Transport of DCVC was adequately described by Michaelis-Menten kinetics with an apparent Kt of 0.5 mM. DCVC uptake was blocked by the presence in the extravesicular solution of 10 mM concentrations of phenylalanine, leucine and cysteine, but not by glycine, proline, lysine, taurine, N-acetyl DCVC, p-aminohippurate, lactate or succinate. Unlabeled DCVC inhibited uptake of [14C]phenylalanine by a mechanism that exerted a greater effect on the apparent Kt than on the Jmax of phenylalanine, implicating a possible competitive interaction between these compounds. The carrier-mediated permeability of DCVC (defined as the ratio of Jmax/Kt) in luminal brush border membranes was as large as or larger than that reported for a battery of other organic electrolytes, including several amino acids and organic anions. We conclude that luminal transport of DCVC in rabbit proximal cells is limited to a single Na-cotransport process that also handles phenylalanine.

Animals↗

PubMed1998

Relation of cysteine conjugate nephrotoxicity to transport by the basolateral organic anion transport system in isolated S2 segments of rabbit proximal renal tubules.

We examined basolateral transport of the radiolabeled zwitterionic nephrotoxic cysteine S-conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), inhibition of such transport and the effects of inhibition of transport on the toxicity produced by DCVC in isolated S2 segments of rabbit proximal tubules. High concentrations of unlabeled DCVC itself and an unlabeled nontoxic cysteine S-conjugate, S-(2-benzothiazole)-L-cysteine cis-inhibited the basolateral uptake of radiolabeled DCVC by approximately 80 to 85%. High concentrations of para-aminohippurate, the prototype substrate for the basolateral organic anion transport system, and probenecid, a well-known inhibitor of basolateral organic anion transport, cis-inhibited the basolateral uptake of radiolabeled DCVC by approximately 70%, whereas a high concentration of L-phenylalanine had little effect. High concentrations of S-(2-benzothiazole)-L-cysteine and para-aminohippurate in the bathing medium with DCVC inhibited the loss of 86Rb (used as a K+ surrogate to measure toxicity) from S2 segments produced by DCVC alone to approximately the same extent as they inhibited uptake of DCVC. Under the same circumstances, probenecid completely inhibited 86Rb loss. These data indicate that in rabbit proximal renal S2 tubules basolateral entry of DCVC can occur to a major extent via the organic anion transport pathway and that inhibition of such entry can reduce toxicity to approximately the same extent that entry is reduced. They also suggest that probenecid provides additional protection from DCVC toxicity.

Animals↗

PubMed1997

Mast cell heterogeneity in the gastrointestinal tract: variable expression of mouse mast cell protease-1 (mMCP-1) in intraepithelial mucosal mast cells in nematode-infected and normal BALB/c mice.

Soluble granule chymases in rodent intestinal mucosal mast cells (IMMCs) may play an important role in altering epithelial permeability during immediate hypersensitivity reactions. Using a monoclonal antibody against the chymase mouse mast cell protease-1 (mMCP-1), we have shown that it is constitutively expressed in < or = 20% of esterase-positive (esterase+) IMMCs but not in esterase+ gastric mucosal mast cells (GMMCs) in normal BALB/c mice. Intestinal infection with mouse- or rat-adapted strains of Nippostrongylus brasiliensis resulted in IMMC hyperplasia with 100% of esterase+ IMMCs expressing mMCP-1. In contrast, there was a variable response in terms of numbers of GMMCs and of the proportion expressing mMCP-1. Esterase+ mast cells in the gastric submucosa, muscularis, ear pinna, lung parenchyma, major airway submucosa, and peritoneal cavity did not express mMCP-1. The few airway esterase+ mast cells expressing mMCP-1 were, like the great majority of IMMCs and GMMCs, located intraepithelially. In conclusion, mMCP-1 is predominantly expressed by intraepithelial mucosal mast cells but not in all sites; the immunological stimulus associated with intestinal nematodiasis substantially up-regulates mMCP-1 expression by mast cells in the jejunum but not in the stomach; IMMCs and GMMCs in BALB/c mice are phenotypically and possibly functionally distinct.

Animals↗

PubMed1996

NMDA receptor activation inhibits neuronal volume regulation after swelling induced by veratridine-stimulated Na+ influx in rat cortical cultures.

Neurons and glia experience rapid fluctuations in transmembrane solute and water fluxes during normal brain activity. Cell volume must be regulated under these conditions to maintain optimal neural function. Almost nothing is known, however, about how brain cells respond to volume challenges induced by changes in transmembrane solute flux. As such, we characterized the volume-regulatory mechanisms of cultured cortical neurons swollen by veratridine-stimulated Na+ influx. Exposure of cortical neurons to 100 microM veratridine for 10-15 min caused a 1.8- to 2-fold increase in cell volume that persisted for at least 90 min. This volume increase was blocked by extracellular Na+ removal or by exposure to 5 microM tetrodotoxin, indicating that swelling is a result of Na+ entry via Na+ channels. Treatment of cells with veratridine together with various NMDA receptor antagonists had no effect on the magnitude of swelling. NMDA receptor antagonist-treated cells, however, underwent nearly complete volume recovery within 50-70 min after veratridine exposure. This recovery suggests that NMDA receptor activation disrupts neuronal osmoregulatory pathways. Volume regulation was blocked by Ba2+, quinidine, or 5-nitro-2-(3-phenylpropylamino) benzoic acid, indicating that swelling activates volume regulatory K+ and Cl- channels. Veratridine also caused a rapid, transient increase in intracellular Ca2+. Extracellular Ca2+ removal or intracellular Ca2+ chelation prevented or dramatically reduced veratridine-induced increases in intracellular Ca2+ and completely blocked volume recovery. These findings indicate that increases in Ca2+ during cell swelling induced by Na+ influx are required for activation of neuronal volume-regulatory pathways.

Animals↗

PubMed1996

Seroepidemiological survey of Taenia saginata cysticercosis in Kenya.

A sero-epidemiological study of Taenia saginata cysticercosis was carried out to determine the prevalence and distribution of the infection in three provinces of Kenya. Serum samples and meat inspection records were collected from cattle at slaughter at export and district abattoirs. Cattle origin and the presence of T. saginata cysticerci were noted as was the prevalence of other helminths such as Echinococcus granulosus and Fasciola gigantica. Serum samples were screened for circulating parasite antigen using a monoclonal antibody-based enzyme-linked immunosorbent assay (Ag-ELISA) and for ante-parasite antibody by indirect ELISA (Ab-ELISA). Eighty per cent of the sera were collected from cattle from the Rift Valley Province of Kenya. The prevalence of T. saginata cysticercosis and the other helminth infections varied between districts and was particularly high in Narok. Animal husbandry practices in arid areas such as Narok may be particularly conducive to transmission. The potential value of the Ag-ELISA for use in sero-epidemiological studies was verified by this study. It detected at least twice as many cases as T. saginata cysticercosis as meat inspection and, of the three methods investigated, was considered the most valuable.

Abattoirs↗

PubMed1996

Diagnosis of Taenia saginata cysticercosis in Kenyan cattle by antibody and antigen ELISA.

Sera from calves, either experimentally or naturally infected with Taenia saginata, were screened for an antibody response to T. saginata, and for parasite antigen, by enzyme-linked immunosorbent assays (ELISAs). An antibody response was detected by 3 weeks post infection (p.i.), rose to a peak at 10-12 weeks p.i., and was still in evidence 1 year p.i. Parasite antigen was first detected 4-7 weeks p.i. and persisted until the end of the experiment, over 1 year p.i. In the experimentally infected animals, cattle with 14 or more live cysticerci had detectable levels of parasite antigen in their sera at slaughter, while animals with live cyst burdens ranging from 0 to 4 were negative. Furthermore, levels of circulating antigen were positively correlated with live cysticercus burden in the experimental animals. In naturally infected cattle, 83% (5/6) of those with 30 or more live cysts, and 22% (5/23) of those with 1-29 live cysts, could be detected by the ELISA for parasite antigen, although no significant correlation between antigen level and live cyst burden could be detected. Antibody levels were not found to be associated with cyst burdens in either experimentally or naturally infected cattle. In slaughterhouse cattle, the antigen assay was almost three times as sensitive as meat inspection. However, there was no agreement between cattle found positive at meat inspection and those found positive by the antigen detection ELISA. One possible reason is that the ELISA only detects live cysts, while lesions left by dead cysts are more noticeable at meat inspection. The mouse monoclonal antibody-based antigen detection ELISA is of value for the diagnosis of naturally occurring, viable, T. saginata cysticercosis in live cattle and has an immediate application for field based epidemiological studies designed to determine prevalence.

Abattoirs↗

PubMed1996

Response of cell volume in Mytilus gill to acute salinity change.

The response of gill cell volume in Mytilus californianus and Mytilus trossolus (=edulis) to acute changes in salinity was assessed using three independent indicators: optical measurement of lateral cell height, measurement of intracellular water content using radiolabeled tracers and measurement of the contents of the major osmolytes of the gills. Optical measurements indicated significant variation in the response of individual lateral cells of M. californianus to acute low-salinity shock. Lateral cell height increased by approximately 20% shortly after abrupt exposure to 60% artificial sea water (ASW). Following this initial swelling, we estimate that a substantial regulatory volume decrease (RVD) was present in 25% of the trials. More commonly, however, an RVD was either absent or minimal: cell height remained elevated for at least 1 h, then returned to the control height when gills were re-exposed to 100% ASW. Changes in the combined water space of all cells in the gill, measured as the difference between total water space and extracellular space ([14C]polyethylene glycol space), indicated that cell volume regulation in the gill as an organ was also absent or minimal. Cell water space was 2.16 ml g-1 dry mass in isolated gills of M. californianus acclimated to 100% sea water in the laboratory and increased to 2.83 ml g-1 dry mass after a 6 min exposure to 60% ASW. Cell water space was still 2.81 ml g-1 dry mass after 1 h in 60% ASW and returned to 2.06 ml g-1 dry mass upon re-exposure to 100% ASW. Consistent with these observations, the gill contents of the principal cytoplasmic osmolytes (taurine, betaine and K+) were unchanged (approximately 450, 250 and 230 mu mol g-1 dry mass, respectively) following exposure of gills from 100% ASW-acclimated mussels to 60% ASW. A decrease in cell water space to 2.66 ml g-1 dry mass after 4 weeks of acclimation to 60% ASW corresponded with a 37% decrease in betaine content; taurine and K+ contents were unchanged. The changes in water space and solute content of gills from freshly collected M. californianus and M. trossolus were also consistent with the absence of volume regulation; cell water space remained elevated for at least 1 h after low-salinity exposure, and solute contents were unchanged after this period. We calculated the potential energetic cost of cell volume regulation for mussels exposed to 12 h of sinusoidal fluctuations between 100% and 50% sea water; solute uptake for full volume regulation in all tissues would cost a minimum of approximately 30% of the standard metabolic rate during the period of salinity increase. The routine absence of substantial cell volume regulation in Mytilus gill may reflect the potentially high energetic cost of volume regulation in the face of the large and frequent salinity fluctuations that are regularly encountered by estuarine bivalves.

Animals↗

PubMed1996

Salinity change and cell volume: the response of tissues from the estuarine mussel Geukensia demissa.

The response of cell volume to changes in external salinity was assessed in four tissues (gill, mantle, hemolymph cells and ventricle) of the estuarine mussel Geukensia demissa by using one or more of the following three indicators of cell volume response: changes in cell dimensions, cell water space and cell solute content. All three techniques indicated that short-term volume regulation was generally absent from gill tissue. Lateral cell height in gills, measured using differential interference contrast (DIC) microscopy, increased by approximately 20% after an abrupt exposure to reduced salinity (60% artificial sea water, ASW). There was significant variability in the observance of a regulatory volume decrease (RVD) subsequent to the initial swelling; cells remained swollen for 1 h after low-salinity exposure in two-thirds of the trials, while there was a return of cell volume towards control values in the remaining one-third of the trials. Lateral cell height increased linearly when salinity was gradually decreased from 100 to 60% ASW over 135 min. Cell height then returned to control values when the salinity was abruptly returned to 100% ASW, indicating that an RVD was not elicited by a slow change in salinity of the type normally encountered by estuarine mussels. Cumulative cell water space in gills increased by 47% after exposure to 60% ASW and the cells remained swollen for at least 4 h, returning to control values when gills were returned to 100% ASW. Consistent with the overall lack of an RVD, there was only a small decrease (approximately 5%) in cumulative osmolyte content (primarily taurine, betaine and K+) after 4 h in 60% ASW. Decreases in both cell water space and osmolyte content after 3 weeks of acclimation to 60% ASW indicated a long-term RVD of approximately 60%. Individual cells in the mantle epithelium also generally lacked an RVD in response to lowered salinity. Both abrupt and gradual decreases in salinity caused an increase in mantle cell height to a maximum of 25-30%, and cell height returned to the control height when salinity was abruptly returned to 100% ASW. Corresponding with the lack of an RVD in individual mantle cells, there was no change in solute content of the mantle tissue after 4 h of exposure to low salinity. The response of the volume of spherical hemolymph cells to 1 h of abrupt exposure to low salinity, calculated from measured cell diameters, likewise indicated that an RVD is generally lacking in these hemolymph cells. In the ventricle, however, there was a significant decrease in amino acid and betaine content after 4 h of exposure to low salinity, suggesting tissue-specific variability in the cellular response to salinity change. The consistent lack of a short-term RVD in many tissues may serve to avoid large energetic expenditures associated with repeated volume regulation in the face of the frequent, short-term changes in salinity encountered by estuarine mussels.

Adaptation, Physiological↗

PubMed1995

Paraquat2+/H+ exchange in isolated renal brush-border membrane vesicles.

The mechanism(s) by which paraquat (1,1'-dimethyl-4,4'-bipyridinium), a divalent organic cation (OC) and proximal tubule nephrotoxicant, crosses renal cell membranes is unclear. The structurally-related monovalent OC, 1-methyl-4-phenylpyridinium (MPP+), crosses the renal brush border via OC/H+ exchange using the same pathway by which tetraethylammonium (TEA) is transported. We examined whether paraquat shares the TEA(MPP+)/H+ exchanger by examining 14C-paraquat transport in rabbit renal BBMV. Compared to a pH equilibrium condition (pH 7.5in:7.5o), an H-gradient (pH 6in:7.5o) stimulated the 5 s and 60 s uptakes of 230 microM paraquat by 51% and 108%, respectively, and this stimulation was blocked by both 20 mM unlabeled paraquat and TEA. Pre-loading BBMV with 2 mM unlabeled TEA (under conditions of pH equilibrium) stimulated by 3-fold the 60 s uptake of 120 microM paraquat and by 5 min produced a transient intravesicular accumulation of paraquat that exceeded equilibrium (2 h) uptake by 45%. The presence of 200 microM paraquat in the extravesicular solution competitively inhibited H-gradient-stimulated transport of 14C-TEA in renal BBMV, increasing the apparent Kt for TEA transport from 169 microM to 379 microM, without significantly influencing the Jmax (16.0 vs. 15.4 nmol mg-1 min-1). The calculated Ki for paraquat (presumably equal to its Kt for transport) after transport was between 160 and 220 microM (depending upon the method of estimation). Significantly, the Kt for MPP+/H exchange is 12 microM, suggesting that the affinity of the exchanger is profoundly influenced by the presence on paraquat of a second positive charge. We conclude that renal transport of paraquat involves the OC/H+ exchanger of proximal cell luminal membranes and that this pathway may play a role in the renal secretion of polyvalent organic cations.

1-Methyl-4-phenylpyridinium↗

PubMed1995

Tetrapentylammonium (TPeA): slowly dissociating inhibitor of the renal peritubular organic cation transporter.

The efflux of tetraethylammonium (TEA) from suspensions of rabbit renal proximal tubules is completely blocked by 500 microM tetrapentylammonium (TPeA) in the extracellular medium. The basis of this trans-inhibition of TEA transport by TPeA was examined in tubule suspensions. At TPeA concentrations < 10 microM, efflux of TEA was reduced by approximately 50%, whereas at concentrations > 10 microM, TPeA reduced efflux an additional 50% to produce a near complete block of TEA efflux. Increasing concentrations of TPeA from 0-500 microM were found to produce a biphasic, concentration-dependent trans-inhibition of TEA efflux from tubule suspensions suggesting that TPeA may block efflux by binding to both a high and low affinity TPeA binding site. The trans-inhibition of TEA efflux by TPeA at low concentrations (< 10 microM) may result from a slow carrier turnover when TPeA is bound to the carrier site. To determine whether the inhibitory effectiveness of TPeA was also associated with its slow dissociation from the carrier site, the effect of a 10 s preincubation with 1 microM TPeA on TEA uptake was examined. The uptake of TEA by tubules preincubated for 10 s with TPeA was reduced by approximately 30-50% compared to control tubules not preincubated with TPeA. A 10 s preincubation with 150 microM unlabeled TEA had no effect on TEA uptake compared to control tubules not preincubated with TEA. When the 10 s preincubation with 1 microM TPeA was followed by a 10 min recovery period, TEA uptake returned to control levels, indicating that the prolonged inhibition was reversible. This prolonged inhibition of TEA uptake after a 10 s preincubation with 1 microM TPeA, as suspected, may arise from a slow dissociation of TPeA from the OC transporter following a rapid association to the binding site. TPeA inhibition of TEA uptake into tubules was competitive in nature with a Ki of 1 microM. The ability of TEA to compete with TPeA for binding to the carrier suggests that the binding of TPeA to the carrier can be displaced by large concentrations of TEA. These observations suggest that the interactions of TPeA, and perhaps similarly large hydrophobic OCs, with the OC transporter are complex.

Animals↗

PubMed1995

Structure and interaction of inhibitors with the TEA/H+ exchanger of rabbit renal brush border membranes.

The renal secretion of organic cations (OCs) involves a carrier-mediated exchange of OC for H+ in the luminal membrane of proximal cells. To assess the influence of chemical structure on the interaction of potential substrates with this process we examined the effect of a series of quaternary ammonium compounds on the transport of the OC tetraethylammonium (TEA) in a preparation of isolated renal brush-border membrane vesicles. Apparent inhibitory potency varied over a factor of 10(4), as expressed in inhibitor coefficients (KiTEA) whose approximate values ranged from 0.5 microM to 5 mM. The poorest inhibitors of TEA/H+ exchange were those molecules with carboxyl or hydroxyl residues, whereas the addition of methylene groups to a parent molecule tended to increase inhibitory potency. A plot of apparent KiTEA versus calculated octanol:water partition coefficient (expressed in terms of a relative lipophilicity factor) showed a clear correlation between these two parameters, although there was considerable variability between apparent lipophilicity and KiTEA for molecules with very different parent structures. For select groups of molecules with similar parent structures (e.g., the n-tetraalkylammoniums or the 4-phenylpyridinium, 3-phenylpyridinium, and quinolinium compounds) the correlation between calculated lipophilicity and apparent KiTEA was more marked. However, even within these groups of closely related parent structures, there appeared to be subtle, but systematic, variations in inhibitory potency that may have been related to the influence of steric factors on the binding of inhibitors to the TEA/H+ exchanger. We conclude that the lipophilic nature of a quaternary ammonium compound represents the predominant factor in the binding to and subsequent inhibition of, luminal TEA/H+ exchange. Specific steric factors may influence the binding of substrate to the exchanger, but play a secondary role in this interaction.

Acridines↗

PubMed1995

Basolateral transport of taurine in epithelial cells of isolated, perfused Mytilus californianus gills.

We found that the basolateral surface of the gill epithelium of the marine mussel Mytilus californianus possesses a carrier-mediated process capable of concentrating taurine within epithelial cells. We used retrograde perfusion of gill sections to demonstrate the kinetics, specificity and ion-dependence of taurine transport. [3H]taurine was concentrated relative to a space marker ([14C]mannitol); this accumulation was blocked by the inclusion of 10 mmol l-1 unlabeled taurine in the perfusate. The drop in [3H]taurine uptake at increasing concentrations of unlabeled taurine was fitted to Michaelis-Menten kinetics and indicated a basolateral process with a taurine concentration at which transport is half-maximal (Kt) of 35.3 mumol l-1 and a maximal flux (Jmax) of 0.35 mumol g-1 wet mass h-1. Taurine accumulation on the apical surface had a higher affinity (Kt = 9.5 mumol l-1) and a higher maximum rate of transport (Jmax = 1.23 mumol g-1 h-1). Basolateral transport was inhibited by inclusion in the perfusate of 1 mmol l-1 of another beta-amino acid (beta-alanine), but not by inclusion of alpha-alanine, glutamic acid or betaine. The dependence of basolateral taurine transport on Na+ (when replaced with N-methyl-D-glucamine) was sigmoidal with an apparent Hill coefficient of 2.3, indicating that more than one Na+ is necessary for the transport of each taurine molecule. Complete substitution of Cl- in bathing media reduced taurine accumulation by 90% and 70% on the apical and basolateral surfaces, respectively. Taurine accumulation on both surfaces was reduced by only 20% when Cl- was reduced from 496 to 73 mmol l-1, suggesting that taurine uptake is not significantly influenced by the changes in Cl- concentration accompanying the salinity fluctuations normally encountered by mussels. We estimate that the various Na+ and Cl- gradients naturally encountered by epithelial cells are capable of providing ample energy to maintain a high intracellular concentration of taurine. We suggest that the ability of epithelial cells to accumulate taurine across the basolateral surface from the hemolymph plays a significant role in the intracellular regulation of this important osmolyte and may effect osmolality-dependent changes in the intracellular concentration of taurine.

Amino Acids↗