Acute regulation of OAT3-mediated estrone sulfate transport in isolated rabbit renal proximal tubules.

We investigated the regulation of organic anion transport driven by the organic anion transporter 3 (OAT3), a multispecific OAT localized at the basolateral membrane of the renal proximal tubule. PMA, a PKC activator, inhibited uptake of estrone sulfate (ES), a prototypic substrate for OAT3, in a dose- and time-dependent manner. This inhibition was reduced by 100 nM bisindoylmaleimide I (BIM), a specific PKC inhibitor. The alpha(1)-adrenergic receptor agonist phenylephrine also inhibited ES uptake, and this effect was reduced by BIM. These results suggest that PKC activation downregulates OAT3-mediated organic anion transport. In contrast, epidermal growth factor (EGF) increased ES uptake following activation of MAPK. Exposure to PGE(2) or dibutyryl (db)-cAMP also enhanced ES uptake. Stimulation produced by PGE(2) and db-cAMP was prevented by the PKA inhibitor H-89, indicating that this stimulation required PKA activation. In addition, inhibition of cyclooxygenase 1 (COX1) (but not COX2) inhibited ES uptake. Furthermore, the stimulatory effect of EGF was eliminated by inhibition of either COX1 or PKA. These data suggest that EGF stimulates ES uptake by a process in which MAPK activation results in increased PGE(2) production that, in turn, activates PKA and subsequently stimulates ES uptake. Interestingly, EGF did not induce upregulation immediately following phenylephrine-induced downregulation; and phenylephrine did not induce downregulation immediately after EGF-induced upregulation. These data are the first to show the regulatory response of organic anion transport driven by OAT3 in intact renal proximal tubules.

Mucosal mast cells and nematode infection: strain-specific differences in mast cell precursor frequency revisited.

Mucosal mast cells (MMC) play an important role in the immune response against selected species of intestinal nematode. The kinetics with which different strains of inbred mice resolve infection with Trichinella spiralis correlates with their ability to mount MMC responses in the intestinal mucosa. Homologues of MMC that express and constitutively secrete abundant amounts of the granule chymase, mouse mast cell protease-1 (mMCP-1), can be generated in vitro from bone marrow cultures supplemented with interleukins-3 and -9, stem cell factor and transforming growth factor-beta1. Using the enhanced growth characteristics of these MMC homologues, a novel limiting dilution assay for mast cell precursor (MCp) frequency has been developed. The assay is highly specific, in that cultures containing mast cells are identified with mMCP-1 specific antibody, and almost three-fold more sensitive than previously published systems. MCp frequencies were compared in BALB/c and C57/BL10 strains of mice that, respectively, respond rapidly and slowly to infection with T. spiralis. MCp frequency (1/378 bone marrow cells) was significantly greater in BALB/c than C57/BL10 mice (frequency: 1/751). Similarly the rate of growth of MMC homologues and the production of mMCP-1 was significantly greater in BALB/c than in C57/BL10 bone marrow cultures.

Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues.

BACKGROUND: The mucosal mast cell (MMC) granule-specific beta-chymase, mouse mast cell protease-1 (mMCP-1), is released systemically into the bloodstream early in nematode infection before parasite-specific IgE responses develop and TGF-beta1 induces constitutive release of mMCP-1 by homologues of MMC in vitro. Intraepithelial MMC may also express the chemokine CCL2 (monocyte chemotactic protein-1) during nematode infection but the expression of this chemokine by MMC homologues has not been investigated. OBJECTIVE: To investigate the expression and to compare the mechanisms of constitutive release of the chymase, mMCP-1, and the chemokine, CCL2. METHODS: MMC homologues were generated by culturing bone marrow cells in the presence of TGF-beta1, IL-3, IL-9 and stem cell factor (SCF). The intracellular distribution of mMCP-1 and CCL2 was examined by confocal microscopy. The involvement of the Golgi complex and of protein synthesis in the constitutive release of mMCP-1 and CCL2 was investigated using the Golgi-disrupting agent brefeldin A and cycloheximide to block protein synthesis. Secreted analytes were quantified by ELISA. RESULTS: mMCP-1 colocalized with Golgi matrix protein 130 but was most abundant in the granules, whereas CCL2 was not found in the granules but appeared to be located uniquely in the Golgi complex. Extracellular release of mMCP-1 was significantly inhibited ( approximately 40%) by cycloheximide and by the Golgi-disrupting agent brefeldin A, indicating both continuous protein synthesis and transportation via the Golgi complex are required for optimal mMCP-1 secretion. A similar but more marked inhibitory effect with both compounds was demonstrated on the constitutive secretion of CCL2. CONCLUSION: The culture conditions that promote mMCP-1 expression and release by MMC homologues also promote the expression and release of CCL2. Constitutive release involves de novo protein synthesis and requires a functional Golgi complex, suggesting that similar mechanisms of extracellular secretion operate for both mediators.

Transforming growth factor-beta1 mediates coexpression of the integrin subunit alphaE and the chymase mouse mast cell protease-1 during the early differentiation of bone marrow-derived mucosal mast cell homologues.

BACKGROUND: Mucosal mast cells (MMC) play a central role in gut hypersensitivities and inflammation. They are morphologically, biochemically and functionally distinct from their connective tissue counterparts. Massive hyperplasia of MMC occurs 7-10 days after intestinal infection with nematodes but it has never been possible to replicate this phenomenon in vitro. OBJECTIVE: (1) To determine whether mouse bone marrow-derived mast cells (mBMMC) grown in the presence of transforming growth factor (TGF)-beta1 could develop over the same time frame (7-10 days) as MMC in parasitized mice. (2) To compare the early expression of surface receptors (integrins alphaE and beta7, c-kit and FcepsilonR) with that of the MMC-specific granule chymase mouse mast cell protease-1 (mMCP-1). METHODS: Mouse bone marrow cells were cultured in the presence of IL-9, IL-3 and Stem Cell Factor (SCF) with or without TGF-beta1. mBMMC were quantified after toluidine blue or Leishmans' staining. Expression of MMC-specific mouse mast cell proteases was analysed by ELISA, immunohistochemistry and RT-PCR. Surface antigen expression was characterized by flow cytometry and confocal microscopy. RESULTS: TGF-beta1 promotes the development of abundant MMC-like mBMMC from bone marrow progenitor cells with kinetics, which closely parallel that seen in vivo. mRNA transcripts encoding mMCP-1 and -2 are readily detectable by day 4 ex vivo in cultures grown in the presence of TGF-beta1. Between 30 and 40% and 75-90% of the cells in these cultures on days 4 and 7, respectively, have typical mast cell morphology, are c-kit+, FcepsilonR+, integrin alphaEbeta7+, and express and secrete abundant mMCP-1. The integrin alphaE subunit is coexpressed with mMCP-1. CONCLUSION: The kinetics of mMCP-1+/alphaE+ mBMMC development, regulated by TGF-beta1, are consistent with that seen in vivo in the parasitized intestine. The normally down-regulatory functions of TGF-beta1 in haematopoiesis are superseded in this culture system by its ability to promote the early expression of alphaE and mMCP-1.

A novel tableted purgative for colonoscopic preparation: efficacy and safety comparisons with Colyte and Fleet Phospho-Soda.

BACKGROUND: Currently available aqueous purgatives used before colonoscopy are poorly tolerated. We designed a tableted sodium phosphate purge that we believe will yield much greater patient acceptance. METHODS: A total of 305 outpatients undergoing routine diagnostic colonoscopy were randomized to one of three preparation groups: Colyte (100 patients), Fleet Phospho-Soda (106 patients), or sodium phosphate tablets (99 patients). Endoscopists were blinded to the type of preparation administered and answered a questionnaire regarding preparation quality. Patients answered a questionnaire designed to analyze tolerability. Adverse events were closely followed and recorded. RESULTS: There were no significant differences in quality of preparation across the groups (80% excellent or good, 4% repreparation). Although hypocalcemia (4 of 71), hypokalemia (18 of 68), and hyperphosphatemia (39 of 69) were observed in patients receiving the tablets, no adverse events occurred. Patients preferred taking the tablets over Colyte and Fleet Phospho-Soda. CONCLUSION: The evaluation of a novel delivery system of a sodium phosphate purge is described. Intended for use before colonoscopy, it circumvents the poor taste and excessive volume of ingestion that are aversive to patients. The tableted purgative is equally effective, safe, and greatly preferred over the existing aqueous preparations. This may improve patient compliance with recommendations for screening colonoscopy.

A novel function for transforming growth factor-beta1: upregulation of the expression and the IgE-independent extracellular release of a mucosal mast cell granule-specific beta-chymase, mouse mast cell protease-1.

Intestinal mucosal mast cells (IMMC) express granule neutral proteases that are regulated by T-cell-derived cytokines, including interleukin-3 (IL-3) and IL-9, and by stem cell factor (SCF). The IMMC-specific chymase, mouse mast cell protease-1 (mMCP-1), is released in substantial quantities into the blood stream during gastrointestinal allergic responses. We used cultured bone marrow-derived mast cells (mBMMC) to identify cytokines that regulate the expression and extracellular release of mMCP-1. When grown in IL-3-rich WEHI (15% vol/vol) and 50 ng/mL recombinant rat SCF (rrSCF) bone marrow cells supplemented with IL-9 (5 ng/mL) differentiated into mBMMC that expressed a maximum of less than 250 ng mMCP-1/10(6) cells and 189 ng mMCP-1/mL of culture supernatant. Supplementation of the same three cytokines with transforming growth factor-beta1 (TGF-beta1; 1 ng/mL) resulted in substantially enhanced expression (6 micrograms/10(6) mBMMC) and extracellular release (2 micrograms/mL of culture supernatant) of mMCP-1. The response to TGF-beta1 was dose-dependent, with maximal effect at 1 ng/mL, and was associated with immunohistochemical and ultrastructural changes in the secretory granules. IL-9-induced expression of mMCP-1 may be due to endogenously expressed TGF-beta1, because it was blocked by anti-TGF-beta antibodies. In conclusion, the expression and extracellular release of the IMMC-specific chymase, mMCP-1, is strictly regulated by TGF-beta1.

Influence of substrate structure on substrate binding to the renal organic cation/H+ exchanger.

The carrier-mediated exchange of H+ for organic cations ("OC/H+ exchange") is the active step in OC secretion in renal proximal tubules. Although hydrophobicity is known to be an important criterion for binding of substrates to this transporter, the degree to which steric parameters of substrate structure influence binding to the exchanger is unclear. We examined this issue by measuring the inhibition of OC/H+ exchange produced by a group of quaternary ammonium compounds which share a common structural motif: an N1-pyridinium residue. Activity of the OC/H+ exchanger was determined by measuring transport of [14C]tetraethylammonium (TEA) in brush-border membrane vesicles (BBMV) from rabbit renal cortex. Transport was measured in the presence of a pH gradient (pHin 6.0; pHout 7.5) to maximize TEA/H+ exchange. Apparent inhibitory constants (Ki values) for each test agent were measured. The test agents included 4-phenylpyridiniums and 3-phenylpyridiniums, quinoliniums and acridiniums. The planar structure of these compounds permits a direct test of whether the presence of planar hydrophobic mass in different orientations relative to the pyridinium motif exerts a systematic effect on substrate binding to the OC/H+ exchanger. The hydrophobicity of each group of compounds was systematically varied by addition of different substituents at the quaternary nitrogen. Whereas decreases in Ki proved to be proportional to hydrophobicity, the position of the phenyl-ring substituent(s) had no effect on substrate interaction with the exchanger. The results led to the development of a preliminary quantitative structure-activity relationship (QSAR) correlating substrate hydrophobicity and substrate binding to the OC/H+ exchanger. This QSAR was used to predict the binding of 1-methyl-4-phenylpyridinium (MPP+), (+) and (-)nicotine, (+) and (-)ephedrine, quinine and quinidine to the OC/H+ exchanger. Molecular graphics representation of the 3D structures of the test agents was used to develop a working model of a hydrophobic, planar receptor surface on the OC/H+ exchanger against which substrates are suggested to interact during binding. Development of the QSAR and receptor surface model open the way to quantitative tests of the specific physical and structural determinants of substrate selectivity by the renal OC/H+ exchanger.

pH and drug resistance. II. Turnover of acidic vesicles and resistance to weakly basic chemotherapeutic drugs.

Resistance to chemotherapeutic agents is a major cause of treatment failure in patients with cancer. The primary mechanism leading to a multidrug-resistant phenotype is assumed to be plasma-membrane localized overexpression of drug efflux transporters, such as P-glycoprotein (P-gp). However, acidic intracellular organelles can also participate in resistance to chemotherapeutic drugs. In this study, we investigated, both experimentally and theoretically, the effect of acidic vesicle turnover on drug resistance. We have developed a general model to account for multiple mechanisms of resistance to weakly basic organic cations, e.g. anthracyclines and Vinca alkaloids. The model predicts that lower cytosolic concentrations of drugs can be achieved through a combination of high endosomal turnover rates, a low endosomal pH, and an alkaline-inside pH gradient between cytosol and the extracellular fluid. Measured values for these parameters have been inserted into the model. Computations using conservative values of all parameters indicate that turnover of acidic vesicles can be an important contributor to the drug-resistant phenotype, especially if vesicles contain an active uptake system, such as H+/cation exchange. Even conservative estimates of organic cation-proton antiport activity would be sufficient to make endosomal drug extrusion a potent mechanism of resistance to weakly basic drugs. The effectiveness of such a drug export mechanism would be comparable to drug extrusion via drug pumps such as P-gp. Thus, turnover of acidic vesicles can be an important factor in chemoresistance, especially in cells that do not overexpress plasma membrane-bound drug pumps like P-glycoprotein.

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.

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.

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.

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.

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.

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.

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.

Kinetics of interactions of para-aminohippurate, probenecid, cysteine conjugates and N-acetyl cysteine conjugates with basolateral organic anion transporter in isolated rabbit proximal renal tubules.

Kinetics of the first 15 s of para-aminohippurate (PAH) uptake across the basolateral membrane of single isolated S2 segments of rabbit proximal renal tubules and the effects of probenecid and cysteine conjugates on them were determined. For PAH uptake in control tubules, Kt (the concentration of PAH at 1/2 Jmax) was about 110 microM and Jmax (maximal rate of PAH transport) was about 6.5 pmol min-1 nl-1. In tubules preloaded with alpha-ketoglutarate (alpha-KG), thereby stimulating PAH/alpha-KG countertransport, Jmax doubled with little change in Kt. Probenecid cis-inhibited PAH uptake with an apparent Ki of about 13 to 15 microM whether or not the tubules were preloaded with alpha-KG. High probenecid concentrations cis-inhibited PAH uptake by > 98%, indicating that essentially all movement of PAH across the basolateral membrane is carrier mediated. Zwitterionic nephrotoxic cysteine conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and nontoxic cysteine conjugate, S-(2-benzothiazole)-L-cysteine (BTC) cis-inhibited PAH uptake (apparent Ki: approximately 86 microM for DCVC; approximately 37 microM for BTC) at least as effectively as their negatively charged N-acetyl derivatives (NAC-DCVC and NAC-BTC) (apparent Ki: approximately 310 microM for NAC-DCVC; approximately 35 microM for NAC-BTC). The inhibition by both DCVC and NAC-DCVC was competitive in nature. NAC-DCVC also cis-inhibited net transepithelial secretion of PAH by isolated, perfused S2 segments. The presence of DCVC and NAC-DCVC, as well as PAH itself, in the bathing medium trans-stimulated the 15 s efflux of PAH across the basolateral membrane of single S2 segments with oil-filled lumens. These data indicate that these cysteine conjugates and their N-acetyl derivatives, not only interact competitively with the PAH transporter, but are transported by it.

Short-term cell volume regulation in Mytilus californianus gill.

Long-term acclimation of Mytilus californianus to 60% artificial sea water (585 mosmol l-1; ASW) led to a 30-40% decrease in the taurine (53.5-36.9 mumol g-1 wet mass) and betaine (44.8-26.2 mumol g-1 wet mass) content of gill tissue, compared with that of control animals held in 100% ASW (980 mosmol l-1). The K+ content of gills did not change following long-term acclimation to reduced salinity. In contrast, losses of all three solutes during a brief (60 min) exposure to 60% ASW were less than or equal to 15%. Nevertheless, the swelling of gill cells that occurred after acute exposure to 60% ASW was followed by a return towards the control volume. Direct optical measurement of single gill filaments confirmed that, during an acute exposure to reduced salinity, ciliated lateral cells increased in cell height (volume) and then underwent a regulatory volume decrease (RVD) with a half-time of approximately 10 min. This short-term RVD was completely inhibited by exposure to 1 mmol l-1 quinidine, a K+ channel blocker, but only when the drug was applied to the basolateral aspect of the gill epithelium. Application of 1 mumol l-1 valinomycin relieved the inhibition by quinidine of the gill RVD. However, addition of valinomycin did not accelerate the rate of RVD observed in the absence of quinidine. These results indicate that long-term acclimation of Mytilus californianus gill in dilute sea water involves primarily losses of taurine and betaine, whereas short-term regulation of cell volume may involve an electrically conductive loss of intracellular K+ and a counter ion.

Seroepidemiological study of Taenia saginata cysticercosis in Swaziland.

A cross-sectional study of Taenia saginata cysticercosis in Swaziland using a serodiagnostic ELISA for parasite antigen is described. The seroprevalence and the levels of parasite antigen were compared in the sera of cattle from different geographical localities, and from areas of high or low population density. Cattle from the Lowveldt region, which has a hot and dry climate relative to the other areas investigated, exhibited significantly higher serum antigen levels. Seroprevalence was also higher in the Lowveldt but this difference was not found to be significant. Within the Lowveldt, antigen levels were found to be slightly elevated in cattle from more highly populated areas. It is suggested that either human behaviour and/or practices in animal husbandry, or increased susceptibility of cattle to reinfection at certain times of the year, may enhance transmission in the Lowveldt since climatic conditions in this region are not conducive to transmission.

Betaine transport in rabbit renal brush-border membrane vesicles.

Transport of the organic osmolyte betaine was characterized in brush-border membrane vesicles (BBMV) isolated from rabbit renal cortex. Inwardly directed gradients of either Na+ or H+ supported concentrative uptake in a manner consistent with the presence of parallel Na(+)-betaine and H(+)-betaine cotransport processes. Concentrative uptake occurred in the presence of membrane potential alone, indicating that betaine transport is electrogenic. Accumulation of betaine was not dependent on chloride in the medium. Whereas L-proline inhibited both the H(+)- and Na(+)-sensitive components of betaine transport, glycine blocked the H(+)-sensitive pathway and had little effect on Na(+)-sensitive betaine transport. Both pathways were adequately described by Michaelis-Menten kinetics. Under Na(+)-gradient conditions (pH equilibrium), the maximal rate of total betaine transport (Jmax) = 50.8 +/- 13.3 nmol.mg-1.min-1 and the concentration of total betaine producing half-maximal uptake (Kt) = 4.1 +/- 0.5 mM. Under H(+)-gradient conditions (Na+ free), Jmax = 102.5 +/- 10.5 nmol.mg-1.min-1 and Kt = 2.8 +/- 0.3 mM. Imposition of both Na+ and H+ gradients increased Jmax (142 +/- 25.5 nmol.mg-1.min-1) to a level significantly greater than that noted in the presence of a Na+ gradient alone. We conclude that betaine transport in renal BBMV involves two distinct transport pathways that are differentiated on the basis of sensitivity to either Na+ or H+ and by their specificity to proline and glycine.

Betaine transport in the gill of a marine mussel, Mytilus californianus.

The integumental epithelium of Mytilus californianus gill accumulates amino acids directly from seawater against chemical gradients exceeding 10(7) to 1. In the present report, we confirm the presence of betaine in Mytilus tissue and identify a transport process in the gill for this organic osmolyte. Betaine content of gill tissue from animals acclimated to 100% seawater (980 mosM) was 45 mmol/kg wet wt, similar to that of taurine (53 mmol/kg). The kinetics of betaine uptake were adequately described by the Michaelis-Menten equation, with a Kt of 6 microM and Jmax of 7 mumol.g-1.h-1. Betaine transport was inhibited by L-proline and related structural analogues, and by alanine. L-Proline transport, which involves both high- and low-affinity pathways, was partially inhibited by betaine. The low-affinity proline pathway transports lysine. Betaine and L-lysine showed no reciprocal inhibitory interactions. This pattern of structural specificity suggested that betaine transport in the gill is confined to the alanine-proline pathway. Uptake of 0.5 microM betaine into gills was inhibited by 97% when Na+ in seawater was replaced with Li+. Activation of betaine transport in the gill was a near-linear function of the external Na+ concentration up through 100% artificial seawater (ASW, 425 mM Na+). Acute exposure of the gill to 60% ASW inhibited betaine uptake by 83%. Maintenance of normal osmotic concentration (980 mosM, by addition of mannitol to 60% ASW) reduced inhibition to 31%, similar to the level predicted from the availability of Na+.(ABSTRACT TRUNCATED AT 250 WORDS)