Richard M. Hays

THE EFFECT OF NEUROHYPOPHYSEAL HORMONES ON THE PERMEABILITY OF THE TOAD BLADDER TO UREA

With the introduction of the countercurrent hypothesis (1) to explain renal concentrating ability there has been renewed interest in the role of urea in renal tubular function. Recent studies (2-5) suggest that urea makes a major contribution to the urinary concentrating mechanism. If this is the case, one might expect important differences in the permeability of the renal tubule to urea in the presence or absence of antidiuretic hormone. Further, such movement of urea across the tubule might be passive, or effected by means of an active transport system, such as has been described for the bull frog kidney (6-8). The difficulty of exploring such problems in the intact mammalian kidney need not be emphasized. It would be desirable to use a simpler system, in which the movement of urea across living cells could be measured directly. The studies of Ussing, Zerahn, Koefoed-Johnsen, and Andersen (9-12) have demonstrated the usefulness of isolated, surviving amphibian membranes as models for the study of active transport and other membrane phenomena. In the studies reported below we have used the toad bladder, a tissue that resembles the mammalian renal tubule in several respects (13).

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.

EFFECTS OF CHRONIC HYPERCAPNIA ON ELECTROLYTE AND ACID-BASE EQUILIBRIUM. II. RECOVERY, WITH SPECIAL REFERENCE TO THE INFLUENCE OF CHLORIDE INTAKE

Previous studies in both the rat (1, 2) and the dog (3) have demonstrated that chronic respiratory acidosis induces profound alterations in electrolyte balance, but little is known concerning the changes that occur when the stimulus of hypercapnia is withdrawn. The present experiments were undertaken in order to define the process of recovery, and to determine how it is influenced by the availability of sodium and chloride in the diet. For this purpose, balance studies were carried out in dogs returned to room air after a long period of exposure to a high CO2 atmosphere.

The role of water diffusion in the action of vasopressin.

SummaryVasopressin produces a large increase in the osmotic flow of water across the toad bladder, with little apparent change in the diffusion rate of tritiated water. This discrepancy between osmotic and diffusional net flow is the basis of the pore theory of vasopressin action. The present studies show that there is in fact a large (at least 10-fold) increase in water diffusion subsequent to addition of vasopressin, which is masked by unstirred layers and by the resistance offered to diffusion by the thick layer of connective tissue and muscle supporting the bladder epithelial cells. An even higher diffusion rate would be anticipated with the complete elimination of unstirred layers, and of barriers to diffusion remaining within the epithelial layer itself. An alternative to the pore hypothesis is considered, in which vasopressin acts solely by increasing the diffusion rate of water across the luminal membrane of the epithelial cell.

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.

THE PROBLEM OF CLINICAL VASOPRESSIN RESISTANCE: IN VITRO STUDIES

Excerpt In recent years, there has been considerable progress in our understanding of the role of antidiuretic hormone in the renal concentrating mechanism. Studies by Hargitay and Kuhn,1Wirz,2Gott...

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.

The effect of vasopressin on the cytoskeleton of the epithelial cell

Vasopressin (AVP) promotes the fusion of vesicles containing water channels with the apical membrane of receptor cells in the amphibian bladder and mammalian kidney. Fusion is accompanied by depolymerization of the actin cytoskeleton. In this review, we present the evidence for actin depolymerization by AVP in the whole cell, and the application of confocal microscopy and immunogold electron microscopy in localizing depolymerization to the apical region of the receptor cell.

Membrane pathways for water and solutes in the toad bladder: I. Independent activation of water and urea transport.

SummaryVasopressin activates a number of transport systems in the toad bladder, including the systems for water, urea, sodium, and other small solutes. Evidence from experiments with selective inhibitors indicates that these transport systems are to a large extent functionally independent. In the present study, we show that the transport systems can be separately activated. Low concentrations of vasopressin (1 mU/ml) activate urea transport with virtually no effect on water transport. This selective effect is due in part to the relatively greater inhibitory action of endogenous prostaglandins on water transport. Low concentrations of 8-bromoadenosine cyclic AMP, on the other hand, activate water, but not urea transport. In additional experiments, we found that varying the ratio of exogenous cyclic AMP to theophylline activated water or urea transport selectively. These studies support the concept of independently controlled systems for water and solute transport, and provide a basis for the study of individual luminal membrane pathways for water and solutes in the accompanying paper.

The Movement of Water Across Vasopressin-Sensitive Epithelia

Publisher Summary This chapter presents the evidence for the pore enlargement hypothesis and reviews the recent studies that provide a basis for an alternative view of water movement across vasopressin-sensitive epithelia and cell membranes in general. It was proposed that vasopressin enlarges pores in the membrane—permitting an increase of Poiseuille flow—with only a small accompanying increase in the diffusion rate of labeled water. Extraneous layers of a vasopressin-sensitive epithelium—the urinary bladder of the toad—retard the diffusion rate of tritiated water to a significant extent. When this retarding effect is taken into account, a number of conclusions can be drawn about the effects of vasopressin on the structure of the luminal membrane of the epithelial cell. First, the diffusion rate of water increases dramatically following vasopressin, possibly enough to account fully for the increase in water flow. Second, the activation energy for diffusion remains high following hormone treatment. This indicates that water is moving through the membrane in a highly bonded state and that there may be no change in the physical properties of the aqueous pathway. Finally, it cannot be said with certainty whether the membrane sites are channels or simply points in the membrane through which individual water molecules can move.