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Biomedical subjects

J S Handler

Publications and source records attributed to J S Handler.

At least 109 records · Page 6Linked to original sources
PubMed1981

Vasopressin-elicited refractoriness of the response to vasopressin in toad urinary bladder.

Incubation of the urinary bladder of Bufo marinus with high concentrations of vasopressin produces refractoriness to subsequent stimulation of water permeability by low concentrations of vasopressin. Development of refractoriness is directly dependent on concentration of vasopressin and duration of incubation with the hormone. Refractoriness develops in the absence of transepithelial water flow, is evident following a 2-h recovery period of incubation in hormone-free Ringer solution, and is reversed after prolonged incubation in hormone-free Ringer solution. Development and reversal of refractoriness is not altered by actinomycin D or cycloheximide. The steps at which refractoriness develops have been identified partially. Under different conditions, refractoriness involves: 1) reduced vasopressin-sensitive adenylate cyclase activity, 2) reduced epithelial cell cAMP accumulation in response to vasopressin the absence of demonstrable change in vasopressin-sensitive adenylate cyclase activity, cAMP phosphodiesterase activity, or loss of cAMP into the Ringer solution, and 3) refractoriness of water permeability response to exogenous cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Epithelial organization and hormone sensitivity of toad urinary bladder cells in culture.

Two continuous cell lines (TB-M and TB-6c) derived from epithelial cells of the toad urinary bladder form epithelia in culture that manifest hormone-sensitive transepithelial transport. Development of transepithelial electrical resistance (R) and transport rate (ISC) are dependent on time and density of cells seeded, but steady-state ISC and R are characteristic for each cell line and independent of seeding density. Some responses of intact toad bladder are preserved in culture, whereas others are altered or absent. Neither cell line responds to vasopressin. Analogues of cAMP increase sodium transport and urea permeability in both cell lines but do not affect water permeability. The intramembrane particle aggregates associated with the vasopressin- and cAMP-induced increase in water permeability of the intact bladder could not be detected in the cell lines. Aldosterone increases sodium transport in both cell lines, and the time course and concentration dependence of the response to aldosterone are similar to those of the intact bladder. The relative effect of a series of steroids on ISC reveals corticosterone to be a more potent mineralocorticoid in cultured cells than in the intact bladder.

Animals↗
PubMed1980

Insulin and serum increase the number of receptors for vasopressin in a kidney-derived line of cells grown in a defined medium.

A continuous strain of epithelial cells (LLC-PK1L) derived initially from pig kidney grows in a defined medium without serum or hormones. The epithelia from domes, a manifestation of transepithelial transport. The parent cell line, LLC-PK1, forms similar eithelia and has vasopressin-sensitive adenylate cyclase. In this study the binding of 3H-labeled [Lys8]vasopressin was used to examine the vasopressin receptors of LLC-PK1L cells. In the absence of serum, LLC-PK1L cells have receptors with a Kd (10 nM) for 3H-labeled [Lys8]vasopressin similar to that of the parent cell line and of membranes prepared from pig renal medulla, LLC-PK1L cells, however, have only 5% as many receptors as LLC-PK1 cells. The number of receptors and the vasopressin-sensitive adenylate cyclase activity are increased by growing LLC-PK1L cells in medium with 10% fetal bovine serum or with 10 microgram of insulin per ml. The combination of serum plus insulin has a greater effect than either alone. Serum and insulin do not change the apparent affinity of the receptors for vasopressin or the coupling between receptors and adenylate cyclase. Basal and NaF-stimulated adenylate cyclase activity are not affected. Several agents reported to induce receptors for hormones in other tissues have no effect on vasopressin receptors in LLC-PK1L cells.

Adenylyl Cyclases↗
PubMed1980

Studies of renal cell function using cell culture techniques.

Cell culture, a powerful tool for the study of cell biology, offers advantages for the study of renal cell function. Epithelial cells derived from a variety of organs, including the kidney, form oriented epithelial sheets in culture that have many structural characteristics (microvilli, tight junctions) of epithelia in situ. There is evidence of transepithelial transport of salt and water by cells of two lines (MDCK and LLC-PK1) derived from mammalian kidney. LLC-PK1 cells may also manifest the glucose transport system of the proximal tubule. Cells of both lines have adenylate cyclase activity sensitive to hormones. Two lines of cells derived from toad urinary bladder form epithelia with a high transepithelial resistance and transport sodium actively from apical to basolateral surface. The rate of sodium transport in both lines is stimulated by cyclic AMP and by aldosterone. There are important differences in the characteristics of the response of the two lines to aldosterone as well as in their sensitivity to inhibition of sodium transport by amiloride. These differences may lead to new insights regarding the molecular events in the response to aldosterone and in the inhibitory action of amiloride. Cultures of kidney cells have also been used effectively to study the biosynthesis of the hormonal derivative of vitamin D and to study prostaglandin production. In addition, cell culture is ideally suited for study of the developmental biology of the kidney.

Aldosterone↗
PubMed1979

Toad urinary bladder epithelial cells in culture: maintenance of epithelial structure, sodium transport, and response to hormones.

Epithelial cells from the toad urinary bladder have been grown in continuous culture. Many of the cells resemble the granular cell type of the urinary bladder. They form an epithelium with typical tight junctions and gap junctions. The transport properties of two cell lines have been examined. When cells of the line designated TB-M or of line TB-6c are grown on collagen-coated nucleopore filters, epithelia are formed that have transepithelial potential differences of 40 and 20 mV, resistances of 5000 and 10,000 omega-cm2, and short-circuit currents (ISC) of 8.5 and 2.5 muA/cm2, respectively. Net mucosa to serosa sodium transport accounts for all of ISC in line TB-M and for 70% of ISC in line TB-6c. Vasopressin, which stimulates adenylate cylase and ISC in the intact bladder, has no effect on the cells in culture. Cyclic AMP stimulates ISC and lowers resistance in both lines. Aldosterone stimulates ISC in both lines. This is accompanied by a fall in resistance in line TB-M and no change in resistance in line TB-6c. Amiloride inhibits ISC in TB-M cells under basal conditions and after stimulation by aldosterone. In line TB-6c amiloride has no effect under basal conditions but lowers ISC of aldosterone-treated cells to the basal level. Thus, the cells have retained the ability to form oriented, high-resistance epithelial membranes that manifest hormone-sensitive transepithelial sodium transport.

Aldosterone↗
PubMed1977

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.

Adenylyl Cyclases↗
PubMed1977

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.

Animals↗
PubMed1976

Study of enzymes regulating vasopressin-stimulated cyclic AMP metabolism in separated mitochondria-rich and granular epithelial cells of toad urinary bladder.

The epithelial cells of the toad urinary bladder are morphologically heterogenous. In order to relate the effect of vasopressin on cyclic AMP metabolism to cell type, the epithelial cells were separated by the density gradient technique of Scott, Sapirstein and Yoder (Science 184:797, 1974). The separation was verified by electron-microscopy and by observing that the band of cells enriched in mitochondria-rich cells was enriched in carbonic anhydrase activity compared to the band of granular cells. A large portion of cells collected from the gradient was considered to be nonviable, precluding further study of their function as intact cells. Vasopressin-stimulated adenylate cyclase activity in homogenates of granular cells was simular to that in homogenates of mitochondria-rich cells. Cyclic nucleotide phosphodiesterase activity was also similar in the two types of cell. Thus, the enzymes known to be involved in cyclic AMP metabolism in response to vasopressin appear to be located in both major cell types.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Release of cyclic AMP by toad urinary bladder.

Cyclic AMP accumulates in the Ringer solution bathing the toad urinary bladder in vitro. At least 4 times more cyclic AMP is released into the solution bathing the serosal surface than into the solution bathing the mucosal surface. Most of the cyclic AMP originates in the epithelial cells rather than the stroma. Vasopressin increased the content of cyclic AMP in the epithelial cells and increases the amount of cyclic AMP in the Ringer solution. Since there is not an increase in medium cyclic AMP when cell cyclic AMP levels are increased by theophylline, it is suggested that theophylline may reduce the permeability of the cell membrane to cyclic AMP. Finally, it is demonstrated that 10 mM NaF increase the amount of cyclic AMP in the epithelial cells and in the solution bathing the bladder, but block the effect of vasopressin on water permeability, presumably at a step subsequent to the formation of cyclic AMP.

Animals↗