Retention of antidiuretic hormone-induced particle aggregates by luminal membranes separated from toad bladder epithelial cells.

Aggregates of intramembrane particles appear in the luminal membranes of renal collecting duct and amphibian bladder cells after stimulation by antidiuretic hormone (ADH). We undertook this freeze-fracture study to determine whether particle aggregates, once in place, remain in the luminal membrane of the amphibian bladder after the membrane is physically separated from the rest of the cell. We found that the aggregates do remain in high yield in isolated membranes stabilized with a bifunctional imidoester (DTBP) followed by fixation with glutaraldehyde, or unfixed but stabilized with DTBP. These findings support the view that the particles are intrinsic membrane components and that their organization in the form of aggregates does not depend on the presence of the intact cell. In addition, the availability of isolated membranes containing particle aggregates provides a starting point for the isolation of the water-conducting proteins.

Stabilization of vasopressin-induced membrane events by bifunctional imidoesters.

Vasopressin increases the water permeability of the luminal membrane of the toad bladder epithelial cell. This change in permeability correlates with the occurrence in luminal membranes of intramembrane particle aggregates, which may be the sites for transmembrane water flow. Withdrawal of vasopressin is ordinarily associated with a rapid reduction of water flow to baseline values and a simultaneous disappearance of the particle aggregates. The bifunctional imidoesters dithiobispropionimidate (DTBP) and dimethylsuberimidate (DMS), which cross-link amino groups in membrane proteins and lipids, slow the return of water flow to baseline after vasopressin withdrawal. Cross-linking is maximal at pH 10, and is reduced as pH is lowered. Freeze-fracture studies show persistence of luminal membrane particle aggregates in cross-linked bladders and a reduction in their frequency as water flow diminishes. Fusion of aggregate-containing cytoplasmic tubular membrane structures with the luminal membrane is also maintained by the imidoesters. Reductive cleavage of the central S-S bond of DTBP by beta-mercaptoethanol reverses cross-linking, permitting resumption of the rapid disappearance of the vasopressin effect. Bladders that have undergone DTBP cross-linking and beta-mercaptoethanol reduction respond to a second stimulation by vasopressin. Thus, the imidoesters provide a physiologic and reversible means of stabilizing normally rapid membrane events.

Activation energy for water diffusion across the toad bladder: evidence against the pore enlargement hypothesis.

The activation energy (E(A)) for the diffusion of water across the epithelial cell layer of the toad bladder was determined in the absence and presence of vasopressin. An experimental approach was employed which minimized the effects of unstirred layers and the thick supporting layer of the bladder on the measurement of water diffusion. E(A) in the absence of vasopressin was 11.7 +/-1.4 kcal.mole(-1); after vasopressin it was 10.6+/-1.1 kcal.mole(-1). The difference between the two values was not significant. The results are consistent with an increase in the number rather than the size of aqueous channels in the cell membrane, a finding which differs from the generally held view that the hormone increases the radius of pores in the membrane.

A saturable, vasopressin-sensitive carrier for urea and acetamide in the toad bladder epithelial cell.

The permeability of the toad bladder to a series of isotopically labeled nonelectrolytes was determined in the presence of 150 mM unlabeled acetamide. Under these conditions, overall bladder function was unimpaired, as shown by a normal response to vasopressin of short-circuit current and permeability coefficient of [(3)H]water,[(14)C]ethanol, and [(14)C]propionamide. The permeability of the bladder to isotopic acetamide and urea, however, was significantly depressed by unlabeled acetamide, in both the absence and presence of vasopressin. These experiments indicate a competition between unlabeled and isotopic species for binding sites, and show the existence of a saturable, vasopressin-sensitive carrier for urea and acetamide in the epithelial cell membrane.

Effect of phloretin on water and solute movement in the toad bladder.

It is generally believed that urea crosses the cell membrane through aqueous channels, and that its movement across the membrane is accelerated in the direction of net water flow (solvent drag effect). The present report presents evidence for a vasopressin-sensitive pathway for the movement of urea, other amides, and certain non-amides, which is independent of water flow. Phloretin, when present at 10(-4) M concentration in the medium bathing the luminal surface of the toad bladder, strongly inhibits the movement of urea, acetamide, and propionamide across the toad bladder, both in the absence and presence of vasopressin. The vasopressin-stimulated movement of formaldehyde and thiourea is also reduced. Osmotic water flow, on the other hand, is not affected; nor is the movement of ethanol and ethylene glycol, or the net transport of sodium. On the basis of these studies we would conclude that the movement of many, if not all, solutes across the cell membrane is independent of water flow, and that a vasopressin-sensitive carrier may be involved in the transport of certain solutes across the cell membrane.

Interaction of vasopressin and prostaglandins in the toad urinary bladder.

Prostaglandins are important modulators of the action of vasopressin. Others researchers have proposed that vasopressin stimulates prostaglandin synthesis, completing a negative feedback loop and thereby limiting vasopressin's antidiuretic effect. We have re-examined this question, using specific radioimmunoassay and thin-layer radiochromatography to determine prostaglandin synthesis by the toad bladder. Under control conditions, the bladder synthesizes prostaglandin (PG)E2 and thromboxane (TX)B2. There was no evidence for synthesis of PGE1 or PGF2 alpha by radioimmunoassay, or of other prostaglandins by radiochromatography. Furthermore, there was no evidence for metabolism of PGE2 by the bladder. Using a variety of protocols, in isolated epithelial cells as well as intact bladders, we were unable to detect any significant increase in PGE2 or TXB2 synthesis after stimulation with arginine vasopressin (AVP) or deamino-8-D-arginine vasopressin (DDAVP). Arachidonic acid, the specific precursor of prostaglandin synthesis, increased PGE2 synthesis twofold, and significantly inhibited AVP- and DDAVP-stimulated water flow by 60 and 75%, respectively. Naproxen and acetaminophen inhibited prostaglandin synthesis and enhanced water flow in response to AVP and DDAVP (44-54%). Our findings indicate that the toad bladder produces tow prostaglandins, PGE2 and TXB2, and that vasopressin does not alter their rate of synthesis. Because agents such as acetaminophen and naproxen inhibit prostaglandin synthesis and enhance vasopressin- and DDAVP-stimulated water flow, we suggest that it is the inhibitory effect of these agents on the hormone-independent rate of prostaglandin synthesis that is responsible for their enhancement of water flow. Furthermore, because AVP appears to increase prostaglandin synthesis by the intact kidney, we suggest that cells other than those of the collecting tubule are responsible for the increased prostaglandin production.

Selective inhibition of osmotic water flow by general anesthetics to toad urinary bladder.

Vasopressin increases the permeability of the total urinary bladder, an analogue of the mammalian renal collecting duct, to water and small solutes, especially the amide urea. We have observed that three general anesthetic agents of clinical importance, the gases methoxyflurane and halothane and the ultrashortacting barbiturate methohexital, reversibly inhibit vasopressin-stimulated water flow, but do not depress permeability to urea, or the the lipophilic solute diphenylhydantoin. In contrast to their effects in vasopressin-treated bladders, the anesthetics do not inhibit cyclic AMP-stimulated water flow, consistent with an effect on vasopressin-responsive adenylate cyclase. The selectivity of the anesthetic-induced depression of water flow suggests that separate adenylate cyclases and cyclic AMP pools may exist for control of water and urea permeabilities in to toad bladder. Furthermore, theophylline's usual stimulatory effect on water flow, but not its effect on urea permeability, was entirely abolished in methoxyflurane-treated bladders, suggesting that separate phosphodiesterases that control water and urea permeabilities are present as well. We conclude that the majority of water and urea transport takes place via separate pathways across the rate-limiting luminal membrane of the bladder cell, and that separate vasopressin-responsive cellular pools of cyclic AMP appear to control permeability to water and to urea.

Evidence for immunoglobulin Fc receptor-mediated prostaglandin2 and platelet-activating factor formation by cultured rat mesangial cells.

The possibility of Fc-dependent uptake of IgG immune complexes was examined in subcultured rat mesangial cells free of monocytes. 195Au-labeled colloidal gold particles were coated either with BSA only or with BSA followed by rabbit anti-BSA-IgG or the F(ab')2 fragment of the IgG. Mesangial cells preferentially took up 195Au particles covered with BSA-anti-BSA-IgG over those covered with BSA or the F(ab')2 fragment. This uptake was a time-dependent and saturable process inhibitable by sodium azide or cytochalasin B. Using phase-contrast microscopy in the light reflectance mode, it was established that essentially all mesangial cells took up IgG-coated gold particles. By electron microscopy the process was shown to consist of vesicular uptake with delivery to endosomes. Mesangial binding-uptake of the IgG-covered particles was associated with stimulation of PGE2 synthesis and production of platelet-activating factor, a lipid mediator of inflammation. To characterize the potential Fc receptor for IgG we used the rosetting technique with sheep red blood cells coated with IgG subclass-specific mouse monoclonal antibodies. 50% of mesangial cells exhibited rosetting with red cells coated with mouse IgG2a, whereas negligible rosetting was observed with IgG2b or IgG1. Competition experiments confirmed the specificity of IgG2a binding. We conclude that cultured rat mesangial cells exhibit specific receptors for IgG and that occupancy of Fc receptors results in endocytosis and is associated with generation of PGE2 and platelet-activating factor. These observations may be of significance for immune-mediated glomerular diseases.

Relationship of aggregated intramembranous particles to water permeability in vasopressin-treated toad urinary bladder.

It has been previously demonstrated with freeze-fracture electron microscopy that vasopressin induces specific structural alterations of the luminal membrane of granular cells from toad urinary bladder in a dose-dependent fashion. These alterations consist of aggregated intramembranous particles and are observed both in the presence and absence of an osmotic gradient. We examined the effect of methohexital, a selective inhibitor of vasopressin-stimulated water flow, and the effect of phloretin, a selective inhibitor of urea permeability, on the structure of the granular cell luminal membrane. Methohexital treatment of the vasopressin-stimulated toad bladder reduced both the osmotic water flow and vasopressin-induced alterations of membrane structure to the same extent. Phloretin reduced urea permeability but not water flow or particle aggregation. Since neither agent affects vasopressin-stimulated sodium movement, these findings indicate that the phenomenon of particle aggregation is specifically related to vasopressin-induced water permeability and not to changes in urea or sodium permeability.

A new proposal for the action of vasopressin, based on studies of a complex synthetic membrane.

The total osmotic flow of water across cell membranes generally exceeds diffusional flow measured with labeled water. The ratio of osmotic to diffusional flow has been widely used as a basis for the calculation of the radius of pores in the membrane, assuming Poiseuille flow of water through the pores. An important assumption underlying this calculation is that both osmotic and diffusional flow are rate-limited by the same barrier in the membrane. Studies employing a complex synthetic membrane show, however, that osmotic flow can be limited by one barrier (thin, dense barrier), and the rate of diffusion of isotopic water by a second (thick, porous) barrier in series with the first. Calculation of a pore radius is meaningless under these conditions, greatly overestimating the size of the pores determining osmotic flow. On the basis of these results, the estimation of pore radius in biological membranes is reassessed. It is proposed that vasopressin acts by greatly increasing the rate of diffusion of water across an outer barrier of the membrane, with little or no accompanying increase in pore size.

The surface charge of isolated toad bladder epithelial cells. Mobility, effect of pH and divalent ions.

The surface charge of epithelial cells isolated from the toad bladder has been determined by the microscope method of cell electrophoresis. The cells possess a net negative charge, and a net surface charge density of 3.6 x 10(4) electronic charges per square micron at pH 7.3. Estimates of net surface charge over the alkaline pH range indicate (a) that an average distance of the order of 40 A separates the negatively charged groups, and (b) that amino as well as acid groups are present at the electrophoretic surface of shear. A significant increase in mobility following cyanate treatment of the cells suggests that a large proportion of the amino groups are the epsilon-amino groups of lysine. In view of the known effects of calcium and other divalent ions on cell permeability and cell adhesion, the extent of binding of calcium and magnesium to the cell surface was determined by the electrophoretic technique. Mobility was significantly decreased in the presence of calcium or magnesium, indicating that these ions are bound by surface groups. When the pH was lowered from 7.3 to 5.2, calcium binding was markedly decreased, an observation consistent with competition between calcium and hydrogen ions for a common receptor site.

Actin depolymerization in the cyclic AMP-stimulated toad bladder epithelial cell, determined by the DNAse method.

Previous studies with the rhodamine phalloidin binding assay have shown that antidiuretic hormone and 8-Br-cAMP rapidly depolymerize F-actin in toad bladder epithelial cells. We have extended these studies with the DNAse inhibition assay and have found that in isolated epithelial cell suspensions, G-actin increases from 37 to 56% of total actin following 8-br-cAMP stimulation. The G-actin concentration in the epithelial cell greatly exceeds its critical concentration, indicating the requirement for a G-actin sequestering protein or proteins in this system.

Morphological aspects of the action of ADH.

Early studies employing biophysical techniques provided a model for ADH-induced water flow in which the number of small water-conducting channels in the outer facing membrane is increased by the hormone. With the development of new concepts and techniques in cell biology, the problem of ADH action now centers on organelle movement, fusion, endocytosis and vesicular traffic with the cell. In this review, endocytosis and vesicular traffic are discussed, and their application to the action of ADH is considered.

Neonatal myasthenia gravis: specific advantages of repetitive stimulation over edrophonium testing.

A premature infant with neonatal myasthenia gravis is presented to illustrate the utility of electrodiagnosis. The patient, born to a mother with myasthenia gravis, suffered additional problems, including hypoxia and subependymal hemorrhage which potentially contributed to hypotonia and poor respiratory effort, thus complicating the diagnosis. Standard testing with edrophonium originally was negative which cast doubt on the diagnosis; however, a repetitive motor nerve stimulation test demonstrated a significant decremental response which was consistent with neonatal myasthenia gravis. This decremental response was corrected following intravenous infusion of edrophonium. In the newborn with suspected myasthenia gravis, repetitive motor nerve stimulation may be a more reliable diagnostic procedure than the more frequently recommended pharmacologic tests. Use of this electrodiagnostic procedure in combination with pharmacologic testing may improve diagnostic accuracy in the premature infant and lead to earlier treatment.

Evidence that the heads of ADH-sensitive aggrephores are clathrin-coated vesicles: implications for aggrephore structure and function.

Antidiuretic hormone (ADH) induces the fusion of long tubular organelles (aggrephores) with the luminal membrane of the receptor cell, and the delivery of particle aggregates to the membrane. Water flow is believed to take place through the particles. Nothing is known about the origin of the particle aggregates, their incorporation into the aggrephores, or the possible relationship of the aggrephores to the vesicular traffic that takes place in the epithelial cell. In the present studies of the ADH-sensitive epithelial cells of the toad urinary bladder, we have found that the spherical heads of the aggrephores appear to be clathrin-coated vesicles. We propose that vesicles originating from sites such as the Golgi or the luminal membrane may be engaged in aggrephore assembly, the resupply of particle aggregates to the aggrephores, and/or the removal of aggregates, and that the aggrephores may be central points in the pattern of vesicular traffic in the cell.

Alteration of luminal membrane structure by antidiuretic hormone.

The final step in the action of antidiuretic hormone (ADH) is the insertion of aggregates of water-conducting particles into the luminal membrane of the receptor cell. The aggregates arise from cytoplasmic tubular structures that fuse with the luminal membrane. This review presents a number of questions about this process, along with current attempts to answer them. The following topics are addressed: 1) the exact role of the cytoskeleton in promoting tubular fusion, 2) the nature of the translocation process leading to fusion, 3) the point in the sequence at which ADH enters, 4) the composition and structure of the water-conducting particles. Some of the answers require a more complete understanding of the structure of the apical portion of ADH-sensitive cells; recent morphological studies are therefore included.