Cell volume regulated transporters of compatible osmolytes.

Virtually all cells respond to hypertonicity by accumulating certain small organic solutes (compatible osmolytes) that, in contrast to intracellular ions, do not perturb macromolecular function. Several important compatible osmolytes are accumulated by coupled transport. Transcription of genes encoding these cotransporters is increased by hypertonicity and a tonicity-responsive enhancer element has been identified. When cells return to an iso-osmotic environment, osmolytes are rapidly lost through a pathway that current evidence indicates may be a volume-sensitive chloride channel.

Monoclonal antibodies as probes of epithelial membrane polarization.

Monoclonal antibodies directed against antigens in the apical plasma membrane of the toad kidney epithelial cell line A6 were produced to probe the phenomena that underlie the genesis and maintenance of epithelial polarity. Two of these antibodies, 17D7 and 18C3, were selected for detailed study here. 17D7 is directed against a 23-kD peptide found on both the apical and basolateral surfaces of the A6 epithelium whereas 18C3 recognizes a lipid localized to the apical membrane only. This novel observation of an apically localized epithelial lipid species indicates the existence of a specific sorting and insertion process for this, and perhaps other, epithelial plasma membrane lipids. The antibody-antigen complexes formed by both these monoclonal antibodies are rapidly internalized by the A6 cells, but only the 18C3-antigen complex is recycled to the plasma membrane. In contrast to the apical localization of the free antigen, however, the 18C3-antigen complex is recycled to both the apical and basolateral surface of the epithelium, which indicates that monoclonal antibody binding interferes in some way with the normal sorting process for this apical lipid antigen.

Diseases and medical disabilities of enslaved Barbadians, from the seventeenth century to around 1838 part II.

The disease environment, health problems and causes of mortality of enslaved Barbadians are described. Data are derived mainly from documentary sources; also included are bio-archaeological data from analyses of skeletons recovered from Newton Plantation cemetery. Major topics include infectious diseases transmitted from person to person, as well as those contracted through water soil, and other environmental contaminations, and diseases transmitted by insects, parasites and other animals; nutritional diseases, including protein energy malnutrition, vitamin deficiencies, anaemia, and geophagy or "dirt eating"; dental pathologies, lead poisoning, alcoholism, traumas, and other disorders, including psychogenic death or illness caused by beliefs in witchcraft or sorcery.

Diseases and medical disabilities of enslaved Barbadians from the seventeenth century to around 1838. Part I.

The disease environment, health problems and causes of mortality of enslaved Barbadians are described. Data are derived mainly from documentary sources; also included are bio-archaeological data from analyses of skeletons recovered from Newton Plantation cemetery. Major topics include infectious diseases transmitted from person to person, as well as those contracted through water soil, and other environmental contaminations, and diseases transmitted by insects, parasites, and other animals; nutritional diseases, including protein energy malnutrition, vitamin deficiencies, anaemia, and geophagy or "dirt eating"; dental pathologies; and lead poisoning, alcoholism, traumas, and other disorders, including psychogenic death or illness caused by beliefs in witchcraft or sorcery.

Transepithelial water flow regulates apical membrane retrieval in antidiuretic hormone-stimulated toad urinary bladder.

Antidiuretic hormone (ADH) increases the osmotic water permeability (Posm) of toad urinary bladder. This increase is believed to be produced by fusion of intracellular vesicles called aggrephores with the granular cell apical plasma membrane. Aggrephores contain intramembrane particle aggregates postulated to be water channels. ADH-stimulated Posm is decreased by osmotic gradient exposure, which is termed flux inhibition. We studied flux inhibition by exposing ADH-stimulated bladders to various osmotic gradients. Osmotic water flow was initially proportional to the applied osmotic gradient, but Posm decreased with time. Ultrastructural and quantitative studies of endocytosis demonstrate that apical membrane retrieval was a direct function of the transepithelial osmotic gradient. Posm remained unchanged when apical membrane retrieval was blocked by incubation of bladders at 2 degrees C, or under low water-flow conditions. These effects were reversed by increases in temperature or the applied osmotic gradient. We conclude that apical membrane retrieval causes the phenomenon of flux inhibition.

Effect of adrenal steroid hormones on the response of the toad's urinary bladder to vasopressin.

This study was designed to examine the effect of adrenal steroid hormones on the response of the toad bladder to vasopressin. Aldosterone enhanced the short-circuit current response, the osmotic water flow response, and the urea permeability response to vasopressin. Since aldosterone also enhanced the short-circuit current response and the osmotic water flow response to adenosine 3',5'-monophosphate, the steroid effect on the bladder's response to vasopressin appears to be at a step beyond the stimulation of adenyl cyclase. Indirect evidence was obtained that the effect of adrenal steroid hormones on the osmotic water flow response to vasopressin is mediated by a different hormone-tissue interaction than that mediating the effect of adrenal steroid hormones on sodium transport. In experiments with three different pairs of mineralocorticoid and glucocorticoid analogues, the former had a greater effect on short-circuit current, the latter on the osmotic water flow response to vasopressin. In addition, the spirolactone SC-14266 markedly inhibited the short-circuit current effect of dexamethasone and had little or no inhibitory effect on the dexamethasone enhancement of the osmotic water flow response to vasopressin. Aldosterone and dexamethasone stimulate the oxidation by the bladder of glucose-6-(14)C and depress the rate of oxidation of glucose-1-(14)C compared with glucose-6-(14)C. SC-14266 inhibited the effect of dexamethasone on the oxidation of glucose-6-(14)C but did not alter the effect of the steroid on the rate of oxidation of glucose-1-(14)C compared with glucose-6-(14)C, suggesting that the latter is a glucocorticoid effect and the stimulation of glucose-6-(14)C oxidation a mineralocorticoid effect. Under conditions in which aldosterone has produced a marked enhancement of short-circuit current and the permeability response to vasopressin, the steroid had no detectable effect on cell water content or on cell sodium, potassium, or chloride.

Vasopressin-stimulated prostaglandin E biosynthesis in the toad urinary bladder. Effect of water flow.

Prostaglandin E biosynthesis and its effect on water permeability were investigated in the toad urinary bladder. Arginine vasopressin (1 mU/ml) increased prostaglandin E (PGE) biosynthesis from 0.5+/-0.1 to 5.0+/-0.4 pmol/min per hemibladder (mean +/-SEM, n= 8, P less than 0.001). Maximal vasopressin-stimulated PGE biosynthesis, 6.4+/-0.2 pmol/min per hemibladder, occurred at vasopressin concentrations in excess of 3 mU/ml. Half-maximal stimulation of PGE biosynthesis occurred at a vasopressin concentration of approximately 0.7 mU/ml, whereas half-maximal stimulation of water flow occurred at a vasopressin concentration of approximately 5 mU/ml. Vasopressin-stimulated PGE biosynthesis did not depend on water flow along an osmotic gradient or upon sodium transport. Thin-layer chromatographic analysis of the lipids released from hemibladders labeled with tritium-arachidonic acid revealed that vasopressin stimulates the release of arachidonic acid from intracellular lipid stores without affecting the percentage of free arachidonic acid converted to PGE. Neither cyclic AMP nor theophylline stimulated PGE biosynthesis although they mimic arginine vasopressin (AVP) in stimulating water permeability. Biosynthesis of PGE was inhibited by mepacrine, a phospholipase inhibitor, and by agents that inhibit arachidonic acid oxygenase. The inhibition of PGE biosynthesis resulted in augmented vasopressin- and theophylline-stimulated water flow, but had no effect on cyclic AMP-stimulated water flow. We interpret these results to mean that endogenous PGE inhibits basal and vasopressin-stimulated adenylate cyclase activity. In contrast to the effects of AVP on permeability and transport, AVP stimulates PGE biosynthesis by a mechanism that does not depend on an increase in cellular cyclic AMP levels. The water permeability response of the toad urinary bladder to vasopressin is inhibited by PGE synthesized by the bladder in response to vasopressin.

Inhibition of vasopressin-stimulated prostaglandin E biosynthesis by chlorpropamide in the toad urinary bladder. Mechanism of enhancement of vasopressin-stimulated water flow.

Chlorpropamide is known to enhance the water permeability response of the toad urinary bladder to vasopressin and to theophylline. In other studies, we have shown that prostaglandin E synthesis by the toad bladder inhibits the water permeability response to arginine vasopressin and to theophylline. In this study, the effect of chlorpropamide on vasopressin-, theophylline-, and cyclic AMP-stimulated water flow and on prostaglandin E biosynthesis was investigated in the toad urinary bladder in vitro. Chlorpropamide inhibited prostaglandin E biosynthesis during vasopressin-, theophylline- and cyclic AMP-stimulated water flow. Tolbutamide and glyburide, two other sulfonylurea compounds, also enhanced vasopressin-stimulated water flow and inhibited vasopressin-stimulated prostaglandin E biosynthesis. We conclude that the mechanism of enhancement on vasopressin-stimulated water flow by the sulfonylureas is the inhibition of prostaglandin E biosynthesis.

Taurine behaves as an osmolyte in Madin-Darby canine kidney cells. Protection by polarized, regulated transport of taurine.

Using a clonal growth assay, we demonstrated that taurine, a nonperturbing osmolyte accumulated in kidney medulla, brain, and some other tissues of hypertonic experimental animals can function as a nonperturbing osmolyte in Madin-Darby canine kidney (MDCK) cells. The taurine content of hypertonic MDCK cells is twice that of isotonic MDCK cells (isotonic 160 nmol/mg protein; hypertonic 320 nmol/mg protein). Therefore we studied taurine transport in MDCK cells grown on porous supports and then studied the effect of hypertonicity which is known to elicit increased uptake of some other nonperturbing osmolytes by MDCK cells. Basal uptake exceeded apical uptake, with Km and Vmax of 56 microM and 933 pmol/min.mg protein on the basal surface and 10 microM and 50 pmol/min.mg protein on the apical surface. On both surfaces, virtually all taurine uptake was Na+ and Cl- dependent. 24 h after cells were shifted to hypertonic medium (500 mosmol/kg), taurine uptake doubled on the basolateral surface without change on the apical surface. The response to hypertonicity was the result of an increase in Vmax without change in Km. There was no change in taurine efflux when cells were shifted from isotonic to hypertonic medium. When cells adapted to hypertonic medium were shifted to isotonic medium, a large transient basolateral efflux of taurine occurred within 10 min. We conclude that taurine can function as a nonperturbing osmolyte in MDCK cells and that tonicity-regulated taurine transport is a basolateral function in MDCK cells.

Medium tonicity regulates expression of the Na(+)- and Cl(-)-dependent betaine transporter in Madin-Darby canine kidney cells by increasing transcription of the transporter gene.

Betaine is one of the major compatible osmolytes accumulated by kidney derived Madin-Darby canine kidney cells cultured in hypertonic medium. Betaine is accumulated by Na(+)- and Cl(-)-dependent uptake from the medium. To gain insight into the mechanism by which hypertonicity evokes an increase in the Vmax of the betaine transporter in Madin-Darby canine kidney cells, we measured the relative abundance of mRNA for the transporter in cells shifted to a hypertonic medium and found parallel increases in mRNA abundance and cotransporter activity. The increase in mRNA levels preceded the increase in transporter activity slightly. Transcription of the gene for the transporter rose rapidly and to the same relative extent as mRNA abundance in cells shifted to hypertonic medium, indicating that transcription of the gene for the cotransporter plays a major role in regulating the accumulation of betaine in response to hypertonicity.

Identification of the second tonicity-responsive enhancer for the betaine transporter (BGT1) gene.

When certain cells are exposed to a hypertonic solution, transcription of the BGT1 gene is markedly increased. The ensuing rise in betaine transport leads to cellular accumulation of betaine that protects the cells from the stress of hypertonicity. We have previously identified a tonicity-responsive enhancer (TonE1) in the 5' flanking region of the BGT1 gene. It was recognized, however, that full activation of transcription requires additional sequence upstream from the TonE1. Now we report that there is another TonE (named TonE2) 72 base pairs upstream from the TonE1. TonE1 and TonE2 act synergistically to stimulate transcription of BGT1 in response to hypertonicity.

Gangliosides do not move from apical to basolateral plasma membrane in cultured epithelial cells.

Both qualitative and quantitative approaches were used to ascertain whether gangliosides, incorporated into the apical plasma membrane of cultured epithelial cells from kidney of toad (A6) and dog (MDCK), were able to redistribute past the tight junctions to the basolateral membrane. The apical surfaces of confluent epithelia were exposed to rhodaminyl gangliosides and the distribution of the inserted gangliosides was assessed qualitatively by fluorescence microscopy. All of the fluorescence was confined to the apical surface for at least 1 h after the fluorescent gangliosides had become incorporated; none appeared on the basolateral surface. These observations were confirmed by incubating the cells with anti-rhodamine antibodies and 125I-labeled protein A. In order to quantitate further the ganglioside distribution, binding assays were performed using 125I-labeled cholera toxin, which binds specifically to ganglioside GM1. Exogenous GM1 added to the apical membrane was not detected on the basolateral membrane 4 h after its incorporation even though there was extensive disappearance of the inserted ganglioside, presumably through endocytosis. To directly examine the behaviour of endogenous gangliosides, the apical surface of the epithelial cells was exposed to bacterial neuraminidase, which hydrolyzes more complex gangliosides to GM1. The cells exhibited a 10-fold increase in binding of cholera toxin to their apical surface, but no increase in binding to their basolateral surface. Thus, no cellular pathways for movement from apical to basolateral plasma membrane appear to be available for implanted or endogenous gangliosides.

Transepithelial transport of vinblastine by kidney-derived cell lines. Application of a new kinetic model to estimate in situ Km of the pump.

We present a new transport model that may be useful for many kinds of transepithelial transport experiments. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. We apply the model to studies of a multidrug efflux pump, P-glycoprotein, which is normally located in the apical plasma membrane of certain transporting epithelia such as kidney proximal tubule cells. To determine the functional properties of this multidrug transporter in an epithelium, we studied the transepithelial transport of the chemotherapeutic drug, vinblastine, in epithelia formed by the kidney cell lines MDCK, LLC-PK1, and OK. We have previously shown that basal to apical flux of 100 nM vinblastine was about five times higher than apical to basal flux in MDCK epithelia, indicating that there is a net transepithelial transport of vinblastine across MDCK epithelia. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer in a concentration-dependent manner, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. According to the transport model the apical membrane pump has Michaelis-Menten kinetics with an apparent Km = 1.1 microM. Net basal to apical transport of vinblastine was also observed in LLC-PK1 cells and OK cells which are other kidney-derived cell lines. The order of potency of the transport is LLC-PK1 greater than MDCK greater than OK cells. The organic cation transporter is not involved in this vinblastine transport because vinblastine transport in MDCK cells was not affected by 3 mM tetramethyl- or tetraethylammonium. Inhibitors of vinblastine transport in MDCK cells was not affected by potency, were verapamil greater than vincristine greater than actinomycin D greater than daunomycin. The transport pattern we observed is that predicted to result from the function of the multidrug transporter in the apical plasma membrane.

Transport in cultured epithelia.

Recent progress in studying epithelia in culture indicates that techniques already available and applied to naturally occurring epithelia can be applied effectively to cultured epithelia. Studies using this approach are summarized as well as new information the studies have yielded regarding the mechanism of action of aldosterone and the regulation of expression of the sodium-coupled hexose transporter. In addition, special features of cultured epithelia should lead to new approaches to understanding epithelial biology as well as epithelial transport.