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J S Handler

Publications and source records attributed to J S Handler.

At least 73 records · Page 4Linked to original sources
PubMed1986

Gangliosides modulate sodium transport in cultured toad kidney epithelia.

Cultured A6 epithelial cells from toad kidney form confluent monolayers with tight junctions separating the apical and basolateral membranes. These two membrane domains have distinct compositions and functions. Thus, sodium is actively transported across the epithelia from the apical to basolateral surface via amiloride-inhibitable sodium channels located in the apical membrane. Sodium transport is stimulated by vasopressin, cholera toxin, and 8-bromo-cAMP applied to the basolateral surface where the receptors, adenylate cyclase, and Na+/K+-ATPase are located. In a previous study (Spiegel, S., Blumenthal, R., Fishman, P.H., and Handler, J.S. (1985) Biochim. Biophys. Acta 821, 310-318), we demonstrated that exogenous gangliosides inserted into the apical membrane of A6 epithelia do not redistribute to the basolateral membrane. With the ability to vary selectively the ganglioside composition of the apical membrane, we examined the effects of gangliosides on sodium transport in A6 epithelia. When the apical surface of A6 epithelia were exposed to exogenous gangliosides, sodium transport in response to vasopressin, cholera toxin, and 8-bromo-cAMP was enhanced compared to epithelia not exposed to gangliosides. The effect was observed with bovine brain gangliosides, NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GD1a) and Gal beta-1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GM1), but not with the less complex ganglioside, Neu-Ac alpha 2----3Gal beta 1----4Glc beta 1----Cer (GM3). We examined A6 cells for endogenous gangliosides and found that, whereas GM3 was a major ganglioside, only trace amounts of GM1 and GD1a were present. Based on cell surface and metabolic labeling studies, these gangliosides were synthesized by the cells and were present on the apical as well as the basolateral surface. Bacterial sialidase, which hydrolyzes more complex gangliosides to GM1, was used to modify the endogenous gangliosides on the apical surface; after sialidase treatment, the epithelia were more responsive to vasopressin, cholera toxin, and 8-bromo-cAMP. Thus, gangliosides may be modulators of sodium channels present in the apical membrane of epithelial cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

Complete response to vasopressin requires epithelial organization in A6 cells in culture.

Arginine vasopressin (AVP) stimulates adenylate cyclase activity in A6 epithelia grown on filters but not in A6 epithelia grown on plastic culture dishes. When A6 cells are subcultured from culture dishes and seeded at high density on filters, stimulation of adenylate cyclase by AVP is not evident for approximately 3 days. Peak stimulation by AVP occurs between 5 and 10 days. The time course for development of responsiveness to AVP corresponds to the development of an ordered epithelium with polarized cells, tight junctions, and a high transepithelial resistance. A similar correlation is seen when mature filter-grown epithelia that respond to AVP are dissociated by chelation of calcium and subcultured as single cells onto another filter. Stimulation of adenylate cyclase is not evident until an epithelium with significant electrical resistance is reformed. Filters seeded at 1.6 X 10(6) cells/cm2 require 1-2 days to show AVP sensitivity and electrical resistance; filters seeded at 0.4 X 10(6) cells/cm2 require 4 days. Experiments to test the function of the subunits of adenylate cyclase with forskolin (catalytic subunit) and with cholera toxin (Ns, guanine nucleotide-binding subunit) indicate that they are functional during the entire time and that it is the receptor for AVP that is not functional until an ordered epithelium has been formed.

Adenylyl Cyclases↗
PubMed1986

Role of cell replication in regulation of Na-coupled hexose transport in LLC-PK1 epithelial cells.

The glucose concentration in growth medium has been shown to regulate the number of sodium-coupled glucose transporters in LLC-PK1 epithelial cells. Epithelia grown in high concentrations of glucose express fewer transporters than epithelia grown in low concentrations of glucose. In the present work, the effect of a dose of ionizing radiation sufficient to block the incorporation of thymidine was examined in order to gauge the importance of cell replication in the hexose transport regulatory process. The low rate of thymidine incorporation in the plateau phase was completely eliminated by ionizing radiation. Under conditions of irradiation that completely blocked thymidine incorporation, down-regulation, namely the loss of alpha-methylglucoside-concentrating capacity, brought about by switching the epithelium from low to high glucose-containing medium, is independent of the irradiation and therefore most likely is also independent of cell replication. In contrast, the up-regulatory phenomenon is strongly impaired by radiation. This impairment may be due to specific radiation impairment of gene expression necessary for the up-regulatory process. It is apparent from the dose-response data that up-regulation is not inhibited by irradiation in a simple manner and is not inhibited at the same radiation dose as cell replication.

Animals↗
PubMed1986

Activators of protein kinase C inhibit sodium transport in A6 epithelia.

To evaluate the role of protein kinase C in sodium transport via amiloride-sensitive sodium channels, we studied the effect of activators of protein kinase C on short-circuit current in epithelia formed by A6 cells in culture. In A6 epithelia, short-circuit current is equivalent to net sodium transport and is blocked by low concentrations of amiloride added to the solution bathing the apical surface. After any of four different activators of protein kinase C [phorbol 12,13-dibutyrate (20 ng/ml), phorbol 12-myristate 13-acetate (20 ng/ml), 1-oleoyl-2-acetylglycerol (50 micrograms/ml), and mezerein (10 ng/ml)] was added to the solution bathing the apical surface, short-circuit current fell, and electrical resistance rose. Nystatin added to the apical solution reversed the inhibition of short-circuit current, indicating that activators of protein kinase C inhibited transport at amiloride-sensitive sodium channels in the apical plasma membrane. Under some conditions, the activators also stimulated amiloride-insensitive short-circuit current. The ion transport represented by the amiloride-insensitive short-circuit current appears to be the result of basal to apical transport of chloride, but this has not been established conclusively.

Amiloride↗
PubMed1986

Porous-bottom dishes for culture of polarized cells.

Porous-bottom dishes offer several advantages for growing and studying epithelia in culture. Many epithelia differentiate more on porous surfaces than on plastic tissue culture dishes. In addition, separate solutions can be maintained on each side of the epithelium and can be sampled easily for studies of transport and other polarized functions. We describe the fabrication of dishes with a cellulose ester filter, a collagen-coated polycarbonate filter, or a collagen membrane forming the surface for cell attachment at the bottom of the dish.

Animals↗
PubMed1986

Fluorescent markers to study membrane retrieval in antidiuretic hormone-treated toad urinary bladder.

Antidiuretic hormone (ADH) stimulation of toad urinary bladder causes fusion of intracellular vesicles called aggrephores with the apical plasma membrane of granular cells. Aggrephores contain intramembrane particle aggregates whose appearance in the apical membrane is believed to produce a large increase in its water permeability. ADH removal (ADH washout) is thought to cause the retrieval of aggrephores into granular cell cytoplasm. We studied granular cell uptake of dextran and horseradish peroxidase conjugated with fluorescein, rhodamine, or both during ADH washout. Granular cell uptake of fluorescent dextran was dependent on prior exposure to ADH, a linear function of dextran concentration, and increased by a transepithelial osmotic gradient. Immediately after removal of ADH, granular cell fluorescence was finely dispersed and located near the apical surface. Subsequently, it coalesced into larger bodies. This change was most apparent when a single bladder was subjected to two cycles of ADH stimulation and removal using a dextran containing a different fluorophore for each cycle. The ultrastructural correlate for these fluorescent patterns was identified using rhodamine-labeled horseradish peroxidase. Electron microscopy showed that after detachment from the apical membrane, label was initially in tubular-shaped vesicles near the apical surface. Later, these vesicles clustered near multivesicular bodies and transferred their label to these structures. These tubular vesicles closely resemble the morphology of aggrephores visualized by freeze-fracture electron microscopy. We conclude that these fluorescent compounds can be used as markers for the luminal contents of membrane retrieved during ADH washout and allow detailed study of its intracellular processing.

Animals↗
PubMed1986

Adenosine 3',5'-cyclic monophosphate stimulates chloride secretion in A6 epithelia.

Basal and aldosterone-stimulated short-circuit current (Isc) of A6 epithelia are known to be equivalent to net apical to basal Na flux and are completely inhibited by 0.05 mM amiloride added to the solution bathing the apical surface of the epithelium. In the absence of amiloride, the Isc stimulated by adenosine 3',5'-cyclic monophosphate (cAMP) is also equivalent to net apical to basal Na flux. However, amiloride does not completely inhibit the cAMP-stimulated Isc. In this study, the cAMP-stimulated, amiloride-insensitive Isc was characterized, using vasopressin or forskolin to raise cell cAMP. After basal Isc is inhibited by amiloride, forskolin stimulates Isc, conductance, and bidirectional 36Cl flux. Stimulation of Isc depends on the presence of both Na and Cl; stimulation of conductance depends on the presence of Cl. 36Cl flux studies showed that the cAMP-stimulated, amiloride-insensitive Isc is equivalent to net Cl flux. It is inhibited by ouabain and by furosemide or bumetanide added to the solution bathing the basal surface of the epithelium. In view of the effect of cAMP in some other epithelia, we suggest that cAMP activates apical membrane Cl channels that are in series with a Na-K-Cl cotransporter in the basolateral plasma membrane.

Amiloride↗
PubMed1986

Neurohypophysial peptide potencies in cultured anuran epithelia (A6).

To characterize the V2 receptor (for antidiuretic hormone), we have studied the effect of a number of neurohypophysial hormone analogues on cyclic AMP (cAMP) accumulation and short-circuit current in cultured epithelia formed by A6 cells. A6 is the designation of a continuous cell line derived from the kidney of Xenopus laevis. The order of potency for stimulating cAMP accumulation and short-circuit current in A6 epithelia is like that for stimulating water permeability in toad urinary bladder. As anticipated, arginine vasotocin (AVT), the antidiuretic hormone of Amphibia, is more potent than arginine vasopressin (AVP), the antidiuretic hormone of most mammals. The two hormones differ only in the third amino acid (Phe-3 in AVP is a substitution for Ile-3 in AVT). However, there are a number of striking differences in the responsiveness of these amphibian V2 receptors and mammalian V2 receptors to changes in the 7th, 8th, and 9th amino acids where AVT and AVP are identical. 1) Substitution of Lys-8 for Arg-8 in AVP results in marked loss of potency in Amphibia, whereas there is only modest loss of potency in mammals. 2) Desglycinamide AVP is nearly as potent as AVP in Amphibia, whereas it is inactive in mammals. 2) Tocinoic acid, lacking amino acids 7, 8, and 9, has activity in Amphibia, but pressinoic acid, lacking the same three amino acids, is inactive.

Amino Acids↗
PubMed1986

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.

Animals↗
PubMed1985

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.

Animals↗
PubMed1985

Regulation of expression of the sodium-coupled hexose transporter in cultured LLC-PK1 epithelia.

A variety of techniques have been used to study the sodium-coupled hexose transporter in epithelia formed by LLC-PK1 cells. The expression of the transporter is affected by the density and age of the culture and by the concentration of glucose in the growth medium. Sodium-coupled hexose transport appears as the epithelium becomes confluent and increases further as the epithelium matures. The increased transport is associated with increased transport in apical plasma membrane vesicles. Epithelia grown in medium containing 5 mM glucose express more transporters than epithelia grown in medium containing 25 mM glucose. The increase in transport is not the result of an extracellular signal that is generated as a consequence of the concentration of glucose. The response to different hexoses that are or are not transported on the carrier indicates that it is the metabolism of glucose that acts as the signal for expression of more or fewer transporters. The results are compared to similar studies of the effects of substrate concentration on expression of transporters in cultured fibroblasts and the intestines in situ.

Animals↗
PubMed1985

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.

Animals↗
PubMed1985

Adenosine stimulates sodium transport in kidney A6 epithelia in culture.

The effects of adenosine receptor agonists and antagonists were examined in epithelia formed in culture by A6 cells, a continuous cell line derived from Xenopus laevis kidney. A6 epithelia have a high electrical resistance and a short-circuit current that is equal to net sodium flux from mucosal to serosal surface. Adenosine, 2-chloroadenosine, 5'-(N-ethyl)carboxamidoadenosine, and N6-(L-2-phenylisopropyl) adenosine produced concentration-dependent increases in short-circuit current. Stimulation of short-circuit current by 2-chloroadenosine occurred at concentrations of 0.05 microM and above, with half-maximal stimulation occurring at 0.3 microM. 5'-(N-ethyl)carboxamidoadenosine was more potent than N6-(L-2-phenylisopropyl)adenosine, the usual order of potency for activation of stimulatory adenosine receptors. Theophylline (100 microM), an adenosine receptor antagonist, reduced the short-circuit current response to adenosine and 2-chloroadenosine by 85-90%. Amiloride, an agent that inhibits both basal and adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated short-circuit current in A6 epithelia, completely and reversibly inhibited short-circuit current stimulated by 2-chloroadenosine. Adenosine and 2-chloroadenosine stimulated adenylate cyclase activity in a crude membrane preparation from A6 cells. Stimulation by adenosine was blocked by adenosine deaminase. 2-Chloroadenosine increased cell cAMP accumulation in intact epithelia. The results provide evidence that adenosine and adenosine receptor agonists stimulate adenylate cyclase and active sodium transport in an epithelial cell line of renal origin.

2-Chloroadenosine↗