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J S Handler

Publications and source records attributed to J S Handler.

At least 55 records · Page 3Linked to original sources
PubMed1991

Renal Na-myo-inositol cotransporter mRNA expression in Xenopus oocytes: regulation by hypertonicity.

Canine renal cells in culture (MDCK cells) accumulate organic osmolytes, including myo-inositol (MI), in response to hypertonic stress. When medium tonicity is increased, intracellular concentration of MI rises because hypertonicity elicits increased uptake of MI via Na-MI cotransporter(s). To study the mechanism for this increase in cotransporter activity, poly(A)+ RNA isolated from MDCK cells maintained in hypertonic or isotonic medium was injected into Xenopus oocytes, and Na-dependent MI uptake was measured 3-5 days later. Poly(A)+ RNA from hypertonic cells induced clear expression of the cotransporter. In contrast, oocytes injected with poly(A)+ RNA isolated from MDCK cells maintained in isotonic medium exhibited cotransporter activity like oocytes injected with water. Upon size fractionation of RNA, peak activity appeared in a fraction that contained poly(A)+ RNA with median size of approximately 4 kilobases. Na-dependent MI uptake by poly(A)+ RNA-injected oocytes was inhibited by both phlorizin and phloretin. We suggest that hypertonicity-induced upregulation of the Na-MI cotransporter involves an increase in mRNA and synthesis of cotransporter protein(s).

Animals↗
PubMed1991

Myo-inositol and betaine transporters regulated by tonicity are basolateral in MDCK cells.

Myo-inositol and glycinebetaine are compatible osmolytes accumulated in the renal medulla and in MDCK cells cultured in hypertonic media. Both osmolytes are taken up by MDCK cells on Na-coupled transporters. The maximal velocity (Vmax) of both cotransporters is increased by culture in hypertonic medium. When hypertonic MDCK cells are shifted to isotonic medium there is a large transient efflux of osmolytes. To determine the polarity of the cotransporters and the transient efflux, we grew MDCK cells on a porous support to assay transport separately at their apical and basolateral surfaces. In hypertonic cells, basolateral uptake of both osmolytes was 1) more than 10-fold apical uptake, 2) greater than 96% Na dependent, 3) 25- (myo-inositol) and 16-fold (glycinebetaine) uptake in isotonic cells, reaching a maximum 24 h after the switch to hypertonic medium. When medium osmolarity was decreased from hypertonic to isotonic, myo-inositol uptake reversed to the isotonic level within 1 day; glycinebetaine uptake decreased more slowly. When medium osmolarity was decreased from hypertonic to isotonic, there was a large transient increase in basolateral efflux of both osmolytes.

Animals↗
PubMed1991

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.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

Apical membrane vesicles of ADH-stimulated toad bladder are highly water permeable.

Antidiuretic hormone (ADH) stimulation of the toad urinary bladder causes intracellular vesicles called aggrephores to fuse with the apical plasma membrane of granular cells. Aggrephore membranes contain particle aggregates. Particle aggregates are believed to be water channels that cause large increases in the water permeability (PF) of the granular cell apical membrane. Removal of ADH causes the retrieval of particle aggregate-containing apical membrane via endocytosis and a decline in PF. We have previously shown that fluid phase markers are sequestered in these particle aggregate-containing vesicles during retrieval of the apical membrane and that these vesicles can be recovered in cell homogenates. We have now loaded these vesicles with the self-quenching fluorophore carboxyfluorescein (CF) to measure and compare their PF with that of CF-loaded resealed human erythrocyte ghosts. The membranes of these retrieved vesicles have a very high water permeability. The minimum PF of 99% of these vesicles is 4.5 X 10(-2) cm/s. This PF is comparable with that of erythrocyte ghosts (5.4 X 10(-2) cm/s) measured under identical conditions. We conclude that these vesicles are highly permeable to water, and this is consistent with their postulated function of retrieving water channels that have been inserted into the apical membrane in response to ADH.

Animals↗
PubMed1989

Molecular cloning of cDNA coding for kidney aldose reductase. Regulation of specific mRNA accumulation by NaCl-mediated osmotic stress.

Cells generally respond to long-term hyperosmotic stress by accumulating nonperturbing organic osmolytes. Unlike bacteria, in which molecular mechanisms involved in the increased accumulation of osmolytes have been identified, those in multicellular organisms are virtually unknown. In mammals, during antidiuresis, cells of the renal inner medulla are exposed to high and variable extracellular NaCl. Under these conditions, the cells contain a high level of sorbitol and other osmolytes which help balance the high extracellular osmolality. PAP-HT25 is a continuous line of cells derived from rabbit renal inner medulla. When medium osmolality is increased by raising the NaCl concentration, these cells accumulate sorbitol. The sorbitol is synthesized from glucose in a reaction catalyzed by aldose reductase. When the medium is made hyperosmotic, aldose reductase activity increases because of a larger increase in the amount of enzyme. This increase is produced by the accelerated rate of synthesis of aldose reductase protein. The purpose of the present studies was to examine the mechanism of this increase in aldose reductase protein by measuring the relative abundance of aldose reductase mRNA. A cDNA clone coding for rabbit kidney aldose reductase was isolated. Antisense RNA probes transcribed from this clone hybridized specifically with a 1.5-1.6 kilobase mRNA in Northern blots. Cells grown chronically in hyperosmotic medium had a relative abundance of this specific mRNA which was six times that of cells grown in isoosmotic medium. When cells grown in isoosmotic medium were switched to hyperosmotic medium, the level of aldose reductase mRNA peaked (18-fold) at 18-24 h. The induction of aldose reductase mRNA by osmotic stress was reversible. Our finding of increased abundance of a specific mRNA in direct response to hyperosmotic stress represents the first report of such an effect in animals.

Aldehyde Reductase↗
PubMed1989

Adenosine-sensitive phosphoinositide turnover in a newly established renal cell line.

To aid in characterizing adenosine receptors in renal cells, primary cultures of rabbit cortical collecting tubule (RCCT) cells were infected with an adenovirus 12-simian virus 40 hybrid, resulting in a continuous cell line. The cells, designated RCCT-28A, retained their epithelial morphology and reacted with a monoclonal antibody specific for rabbit collecting tubule. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was stimulated by vasopressin (AVP), isoproterenol, prostaglandin E2 (PGE2), calcitonin, parathyroid hormone, and a potent adenosine A1- and A2-receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA). A more selective adenosine A1-receptor agonist, N6-cyclohexyl adenosine (CHA) inhibited basal and AVP-stimulated cAMP accumulation. Cytosolic free calcium was transiently elevated by bradykinin, PGE2, NECA, and CHA. To examine the mechanism by which adenosine analogues increase intracellular free calcium, phosphoinositide (PI) turnover was assessed in the 28A cells after labeling with myo-[3H]inositol. NECA and CHA increased [3H]inositol phosphate formation with an approximate half-maximal effective concentration of 0.1 microM for both analogues. The increase in PI turnover was blocked by the selective adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine and pretreatment of the 28A cells with pertussis toxin. These results suggest that adenosine analogues increase cytosolic free calcium by stimulating PI turnover.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Apical membrane endocytosis via coated pits is stimulated by removal of antidiuretic hormone from isolated, perfused rabbit cortical collecting tubule.

Antidiuretic hormone increases the water permeability of the cortical collecting tubule and causes the appearance of intramembrane particle aggregates in the apical plasma membrane of principal cells. Particle aggregates are located in apical membrane coated pits during stimulation of collecting ducts with ADH in situ. Removal of ADH causes a rapid decline in water permeability. We evaluated apical membrane retrieval associated with removal of ADH by studying the endocytosis of horseradish peroxidase (HRP) from an isotonic solution in the lumen. HRP uptake was quantified enzymatically and its intracellular distribution examined by electron microscopy. When tubules were perfused with HRP for 20 min in the absence of ADH, HRP uptake was 0.5 +/- 0.3 pg/min/micron tubule length (n = 6). The uptake of HRP in tubules exposed continuously to ADH during the 20-min HRP perfusion period was 1.3 +/- 0.8 pg/min/micron (n = 8). HRP uptake increased markedly to 3.2 +/- 1.1 pg/min/micron (n = 14), when the 20-min period of perfusion with HRP began immediately after removal of ADH from the peritubular bath. Endocytosis of HRP occurred in both principal and intercalated cells via apical membrane coated pits. We suggest that the rapid decline in cortical collecting duct water permeability which occurs following removal of ADH is mediated by retrieval of water permeable membrane via coated pits.

Animals↗
PubMed1988

Identification of specific apical membrane polypeptides associated with the antidiuretic hormone-elicited water permeability increase in the toad urinary bladder.

Antidiuretic hormone (ADH) increases the water permeability of the toad urinary bladder. The increase occurs in the apical plasma membrane of granular cells that line the urinary surface of the bladder and is produced by the insertion of water permeability units that have been identified by freeze-fracture electron microscopy as intramembrane particle aggregates. Under water-impermeable conditions, particle aggregates reside in intracellular vesicles called "aggrephores." In response to ADH, the aggrephores fuse with the apical plasma membrane and render it water permeable. When ADH is removed, intramembrane particle aggregates and aggrephores are retrieved from the apical membrane, and it returns to a water-impermeable state. To identify proteins involved in the water permeability response, we used lactoperoxidase/glucose oxidase to 125I-label external apical membrane proteins to compare control and ADH-treated bladders. Several polypeptides were consistently labeled in ADH-treated bladders and not in paired controls. After demonstrating that lactoperoxidase behaves as a fluid-phase marker and is sequestered in aggrephore-like vesicles when ADH is withdrawn, we used the technique of Mellman et al. [Mellman, I.S., Steinman, R. M., Unkeless, J. C. & Cohn, Z. A. (1980) J. Cell Biol. 86, 712-722] to label proteins endocytosed when water permeability declines after ADH is withdrawn to test whether the membrane proteins labeled in ADH-treated bladders behaved like particle aggregates. The internalized membranes contained polypeptides of the same molecular weights (55,000, 17,000-14,000, and 7,000) as those labeled on the apical surface of ADH-treated but not control bladders. These polypeptides are evidently involved in the ADH-stimulated water permeability response and may be components of particle aggregates.

Animals↗
PubMed1988

Dexamethasone accelerates differentiation of A6 epithelia and increases response to vasopressin.

When seeded heavily on a porous tissue culture dish, A6 cells, derived from the kidney of Xenopus laevis, form a highly differentiated epithelium within 4-6 days. When dexamethasone is added to the culture medium, morphological differentiation is completed by day 2, a time at which the control (untreated) is still a disorganized multilayer of cells. In addition to the morphologically evident monolayer of cuboidal cells, the accelerated differentiation is expressed as high transepithelial electrical resistance, short-circuit current, and adenylate cyclase response to vasopressin. When grown on impermeable plastic tissue culture dishes, A6 epithelia are less differentiated and do not respond to vasopressin. With the addition of dexamethasone at the time of seeding on a plastic tissue culture dish, vasopressin responsive adenylate cyclase activity is expressed, albeit at a slower rate than when grown on a porous surface. In addition, dexamethasone treatment of mature epithelia grown on a porous surface results, in hours, in an increase in the adenylate cyclase response to vasopressin. These results reveal two previously unrecognized interactions between adrenal steroid hormones and vasopressin, namely, accelerated differentiation and increased responsiveness of adenylate cyclase.

Animals↗
PubMed1988

Antidiuretic hormone moves membranes.

This review focuses on events at the apical plasma membrane of toad urinary bladder and mammalian collecting duct as their permeability to water changes in response to antidiuretic hormone (ADH) and to its withdrawal. The major marker of the permeability change is observed in freeze-fracture electron microscopy of the apical plasma membrane and consists of a dramatic increase in membrane particle aggregates and, in toad bladder but not in collecting duct, in fused vesicles (aggrephores) that contain particle aggregates in their limiting membranes. Withdrawal of ADH is accompanied by endocytosis at the apical membrane, reflecting retrieval of water-permeable, particle aggregate-containing membrane. Covalent labeling of the external surface of the apical membrane of toad bladder identifies specific proteins that are present in the apical membrane only during the response to ADH. Proteins of the same molecular weights are also present in the retrieved membrane when ADH is withdrawn. Several controversial areas are considered, including the extent of cell swelling as water flows across the epithelium from dilute apical solution to isotonic basal solution, whether only principal cells or principal cells and intercalated cells participate in the water permeability response of the collecting duct, the role of the cytoskeleton in the water permeability response, and the proposed second water permeability barrier that is affected by ADH, but not by adenosine 3',5'-cyclic monophosphate.

Animals↗
PubMed1987

Implications of tooth root hypercementosis in a Barbados slave skeletal collection.

A 17th- to 19th-century cemetery sample of 104 slaves from Newton Plantation (Barbados) shows uniquely high hypercementosis prevalence, as well as unexpectedly high and variable skeletal lead content. A variety of biological and archeological factors indicates that individuals with lower amounts of these anomalies (relative to age at death) were probably African-born, first-generation slaves. The hypercementosis is related to the progression of periodontal disease as assessed from alveolar bone. Although the hypercementosis is endemic in the Caribbean black population, it does not as yet have a clear explanation. We suggest the etiology might relate to chronic malnutrition involving periodic, seasonal rehabilitation.

Black or African American↗
PubMed1987

Isolation and characterization of specialized regions of toad urinary bladder apical plasma membrane involved in the water permeability response to antidiuretic hormone.

Antidiuretic hormone (ADH) increases the apical (external facing) membrane water permeability of granular cells that line the toad urinary bladder. In response to ADH, cytoplasmic vesicles called aggrephores fuse with the apical plasma membrane and insert particle aggregates which are visualized by freeze-fracture electron microscopy. Aggrephores contain particle aggregates within their limiting membranes. It is generally accepted that particle aggregates are or are related to water channels. High rates of transepithelial water flow during ADH stimulation and subsequent hormone removal decrease water permeability and cause the endocytosis of apical membrane and aggrephores which retrieve particle aggregates. We loaded the particle aggregate-rich endocytic vesicles with horseradish peroxidase (HRP) during ADH stimulation and removal. Epithelial cells were isolated and homogenized, and a subcellular fraction was enriched for sequestered HRP obtained. The HRP-enriched membrane fraction was subjected to a density shifting maneuver (Courtoy et al., J. Cell Biol. 98:870, 1984), which yielded a purified membrane fraction containing vesicles with entrapped HRP. The density shifted vesicles were composed of approximately 20 proteins including prominent species of 55, 17 and 7 kD. Proteins of these molecular weights appear on the apical surface of ADH-stimulated bladders, but not the apical surface of control bladders. Therefore, we believe these density shifted vesicles contain proteins involved in the ADH-stimulated water permeability response, possibly components of particle aggregates and/or water channels.

Animals↗
PubMed1987

Isolation, growth, and characterization of a gluconeogenic strain of renal cells.

LLC-PK1 cells, derived from pig kidney, retain several properties of the proximal tubule, but are incapable of gluconeogenesis, due to the lack of fructose-1,6-bisphosphatase (FBPase) [Am. J. Physiol. 248 (Cell Physiol. 17): C181-185, 1985]. Cells incapable of gluconeogenesis require a hexose, pentose, or nucleoside to provide ribose-5-phosphate for RNA biosynthesis. To induce or select cells that express FBPase activity, we cultured LLC-PK1 cells in glucose-free medium. We obtained cells (designated LLC-PK1-FBPase+) that express FBPase activity and are capable of growing in the complete absence of sugars or nucleosides. The cells have apical membrane enzyme activities that differ from those of wildtype cells. Tests of metabolic flow through the gluconeogenic pathway, using 3-mercaptopicolinic acid, a specific inhibitor of phosphoenolpyruvate carboxykinase, confirmed that the cells are gluconeogenic. LLC-PK1-FBPase+ cells grown in medium containing 5 mM glucose for five weekly passages continued to express FBPase activity and apical membrane enzyme activities characteristic of the FBPase+ strain. When switched back to glucose-free medium, they proliferated well. The strain appears to be stable. It should provide a model for studying the relationship between gluconeogenesis and other proximal tubule functions. An incidental finding is that in both strains, the activity of lactate dehydrogenase varied directly with the concentration of glucose in the growth medium, indicating that the expression of lactate dehydrogenase may be regulated by glucose or a metabolite of glucose.

Animals↗
PubMed1987

Precursors of ribose 5-phosphate suppress expression of glucose-regulated proteins in LLC-PK1 cells.

Withdrawal of glucose from the medium bathing mammalian cells in culture results in cessation of growth and induces the synthesis of two stress proteins (Mr approximately 94-100 kDa and 78-80 kDa) that have been termed glucose-regulated proteins (GRPs). In LLC-PK1 cells, proteins of the same molecular weights, assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography, are fully induced by 24 h in glucose-free medium. The GRPs from LLC-PK1 cells cross-react with antibodies to GRPs of nonpolar cells, confirming their identification. Since glucose is not essential for energy production in cultured cells, but is essential for ribose 5-phosphate and nucleotide biosynthesis (Wice et al. J. Biol. Chem. 256: 7812-7819, 1981), we tested the effect of several precursors of ribose 5-phosphate on the induction of GRPs. The addition of 25 mM fructose or galactose to glucose-free medium suppressed the induction of GRPs. The pyrimidine ribonucleosides uridine and cytidine also suppressed GRP synthesis, ribose and the purine ribonucleosides guanosine and adenosine suppressed partially. The results, coupled with indirect evidence in the literature, lead to the suggestion that cell ribose 5-phosphate or a related metabolite regulates the expression of GRPs.

Adenosine↗