Phorbol myristate acetate induces endocytosis as well as exocytosis and hydroosmosis in toad urinary bladder

The induction of the hydroosmotic response in the toad urinary bladder is considered to be associated with membrane addition mediated by exocytosis at the affected luminal membrane and reversed by endocytic retrieval at that surface. The permeability, exocytosis and endocytosis are initiated by antidiuretic hormone (ADH) receptor interaction on the basolateral membrane. In other hormone responsive systems, phorbol ester (phorbol myristate acetate, PMA), a tumor promoter, has been implicated in the regulation of various transport processes through the activation of protein kinase C and cytoskeletal protein phosphorylation. We found that addition of 10(-6) M PMA to the mucosa induces an hydroosmotic response which is gradual and which reaches a maximum within 60 min, equal to about 1/3 the maximal ADH response. Morphologically, PMA causes rapid exocytosis of the granules, endocytosis of horseradish peroxidase from the mucosal medium into tubules and multivesicular bodies and elongation of apical microvilli. Controls treated with mucosal 0.1% dimethylsulfoxide (DMSO) or an inactive PMA isomer on the mucosal surface, or PMA on the serosal surface lack the hydroosmotic, exocytic, endocytic and cytoskeletal changes. Addition of serosal ADH to PMA-treated bladders results in a precocious hydroosmotic and exocytic ADH response, but a lowering of the maximal response. Also pretreatment of bladders with PMA prevented the ADH-induced increase in transepithelial potential difference. Thus, apical events mediating the PMA hydroosmotic response are correlated with exo- and endocytosis and elongation of apical microvilli.     

Intracellular calcium as a modulator of transepithelial permeability to water in frog urinary bladder

The divalent cation ionophore A 23187 was used to evaluate the action of intracellular calcium on net transepithelial water movement across the isolated frog urinary bladder. Incubation with the ionophore increases the net basal water flux in a dose-dependent fashion but independent of the extracellular calcium concentration. Bladders pretreated with A 23187 and exposed thereafter to an increase in calcium concentration exhibit a water permeability that under certain conditions can be comparable to that achieved with antidiuretic hormone (ADH). Lowering the serosal calcium at the peak of the hydrosmotic responses to both ADH and A 23187 inhibited the maintenance of the net water flux. The action of a supramaximal dose of ADH is blunted in bladders pretreated with A 23187, while the hydrosmotic effects of a submaximal dose are enhanced when the ionophore is added together with the hormone. The results show that an increase in transepithelial water movement can be triggered by calcium and that serosal calcium is needed to sustain the response. This hydrosmotic response may be dependent upon the rate at which intracellular calcium concentrations change and on the absolute concentration attained. It is suggested that calcium is involved in the action of ADH on water permeability and may act as a modulator of the hydrosmotic response.     

Evaluation by capacitance measurements of antidiuretic hormone induced membrane area changes in toad bladder

A technique for estimating effective transepithelial capacitance in vitro was used to investigate changes in epithelial cell membrane area in response to antidiuretic hormone (ADH) exposure in toad bladder. The results indicate that transepithelial capacitance increases by about 30% within 30 min after serosal ADH addition and decreases with ADH removal. This capacitance change is not blocked by amiloride and occurs whether or not there is a transepithelial osmotic gradient. It is blocked by methohexital, a drug which specifically inhibits the hydro-osmotic response of toad bladder to ADH. We conclude that the hydro-osmotic response of toad bladder to ADH is accompanied by addition of membrane to the plasmalemma of epithelial cells. This new membrane may contain channels that are permeable to water. Stimulation of Na⁺ transport by ADH is not related to membrane area changes, but appears to reflect activation of Na⁺ channels already present in the cell membrane before ADH challenge.     

Chloride-induced increment in short-circuiting current of the turtle bladder. Effects of in-vivo acid-base state

Evidence for the participation of conductive and non-conductive (exchange) transmembrane anion pathways in the luminal acidification, alkalinization, and chloride-reabsorptive functions of the turtle bladder is provided from the pattern of Cl- -induced changes in transepithelial electrical parameters of isolated urinary bladders from three groups of donor turtles: control or post-absorptive turtles (those killed 5 days after feeding); acidotic turtles (NH4Cl-loaded); and alkalotic turtles (NaHCO3-loaded). The predominance of each of the three aforementioned transport functions as well as the response to Cl- -addition is altered by the in-vivo electrolyte balance of the turtle. In post-absorptive bladders, which are poised for acidification and Cl- reabsorption, the mucosal and serosal addition of Cl- to Na+-free, (HCO3- + CO2)-containing media increases the negative short-circuiting current (Isc). In acidotic bladders, which are poised for acidification but not Cl- reabsorption, mucosal Cl- addition has no effect on this Isc whereas serosal Cl- addition increases the negative Isc in a manner identical to that observed in the post-absorptive bladders. Alkalotic bladders do not possess an acidification function but instead are poised for Cl- reabsorption and cAMP-dependent electrogenic alkali secretion (positive Isc). In these bladders, serosal Cl- addition is without effect while mucosal Cl- addition produces transient changes in this positive Isc. It is found that these results can be replicated by a model of the turtle bladder in which transmembrane Cl- and HCO3- conductive and exchange paths mediate transepithelial acidification, alkalinization and Cl- reabsorption.     

Evidence of basolateral water permeability regulation in amphibian urinary bladder

It is well known that arginine vasopressin (AVP) produces up to a 40-fold increase (0.1 to 4,0 μL/min·cm²) in net water flux across the amphibian urinary bladder under an osmotic gradient (mucosal side 10% hypotonic). No AVP effect is observed when the gradient is in the opposite direction (serosal hypotonic). Similar asymmetrical behavior to osmotic gradients occurs in the frog corneal epithelium. This rectification phenomenon has not been satisfactorily explained. We measured net water fluxes in bladder sacs and confirmed that AVP has no effect when the serosal bath is hypotonic. We reasoned that the ‘abnormal’ serosal osmolarity was inducing changes in membrane water permeability, the very parameter being measured. Thus, we studied the effect of solution osmolarity on diffusional water flow (J_dw) across the frog bladder using ³H₂O. As expected, AVP doubled J_dw (in either direction from 12 to 21 μL/min·cm²) when the serosal solution was iso-osmolar regardless of mucosal osmolarity. However, in the AVP-stimulated bladders, hypo-osmolarity of the serosal solution reduced J_dw by 42%, an effect that was reversible when normal osmolarity was re-established. Amphotericin B (instead of AVP) was used to irreversibly increase the permeability to water of the apical membrane. Under these conditions, basolateral hypotonicity also reversibly decreased J_dw by 32%, suggesting the basolateral membrane as the site where permeability is reduced. SEM and TEM of the tissue shows extreme swelling when it was exposed to serosal hypotonicity with or without AVP and typical surface morphology changes following hormone stimulation. We conclude that this swelling may initiate a signaling mechanism that reduces basolateral water permeability. These findings constitute evidence of basolateral water channel permeability regulation, which can also contribute to cell volume regulation.     

Cellular lithium and transepithelial transport across toad urinary bladder

Summary Toad urinary bladders were exposed on either their mucosal or serosal surfaces, or on both surfaces, to medium in which sodium was replaced completely by lithium. With mucosal lithium Ringer's, serosal sodium Ringer's, short-circuit current (SCC) declined by about 50 percent over the first 60 min and was then maintained over a further 180 min. Cellular lithium content was comparable to the sodium transport pool. With lithium Ringer's serosa, SCC was abolished over 60 to 120 min whether the mucosal cation was sodium or lithium. Measurements of cellular ionic composition revealed that the epithelial cells gained lithium from both the mucosal and serosal media. With lithium Ringer's mucosa and serosa, cells lost potassium and gained lithium and a little chloride and water, but these changes in cellular ions could not account for the current flow across the tissue under these conditions, which must, therefore, have been carried by a transepithelial movement of lithium itself. The inhibition by serosal lithium of SCC was overcome by exposure of the mucosal surface of the bladders to amphotericin B. Thus it reflected, predominantly, an inhibition of lithium entry to the cells across the apical membrane. It is suggested that this inhibition is a consequence of cellular lithium accumulation.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toad Bufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to th serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

Inhibition of ADH-enhanced transepithelial urea and water movement by prostaglandins

The effects of PGF_2α and PGE₂ on transepithelial urea flux and osmotic water flow were evaluated in toad bladders. Mucosal to serosal urea flux and osmotic water flow were not changed from basal values by the addition of either prostaglandin to the serosal bath. However, treatment with either PGF_2α or PGE₂ inhibited both urea flux and osmotic water flow in response to ADH stimulation in a concentration-dependent manner. The hydrosmotic response to ADH was more sensitive to prostaglandin inhibition than was urea flux. The inhibitory effect of the prostaglandins on ADH-enhanced urea flux was not dependent upon inhibition of the hydrosmotic response, since both PGF_2α and PGE₂ decreased urea flux in the absence of a trans-epithelial osmotic gradient. Prostaglandin E₂ was a more potent inhibitor than PGE_2α of both ADH-enhanced urea flux and osmotic water flow. The PGF_2α antagonism of osmotic water flow was apparently competitive, while antagonism of urea flux was apparently non-competitive. The results are consistent with the hypothesis of the existence of a “spare” population of prostaglandin receptors that modulate water flow, but the absence of a “spare” prostaglandin receptor population with respect to the modulation of urea flux.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

Summary The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toadBufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to the serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

Reversible inhibition by lanthanum of the hydrosmotic response to serosal hypertonicity in toad urinary bladder

Summary In the urinary bladder of amphibia, hypertonicity of the serosal bath (SH) evokes an increase in transepithelial water permeability, the characteristics of which resemble the response to antidiuretic hormone (ADH). The ionic dependency, in particular for Ca²⁺, appears very similar forSH- and ADH-induced water fluxes. In the present experiments La³⁺ was used as a probe to study the Ca²⁺-dependency of the hydrosmotic response toSH in isolated urinary bladder of the toadBufo marinus.Addition of La³⁺ (5mm) on the serosal side of the membrane produced a significant and reversible increase in basal transepithelial water flux. The hydrosmotic response elicited by adding 250mm mannitol to the serosal Ringer's solution was inhibited by 30% in the absence of serosal Ca²⁺. Similarly, the hydrosmotic response toSH was inhibited by 37%, 30% and 40% when 5mm La³⁺ was added to the serosal medium 30 min before, concommitantly with, or 60 min after induction ofSH. The inhibition of transepithelial water flux observed in the absence of serosal Ca²⁺ or in the presence of serosal La³⁺ was reversible.The results support a critical role for Ca²⁺ in the modulation of transepithelial water permeability in the urinary bladder of amphibia. Ca²⁺ presumably exerts its effects at a post-cyclic AMP step.     

The cellular specificity of the effect of vasopressin on toad urinary bladder

Summary Phase and electron micrographs of toad bladders were obtained following dilution of bathing media in the presence and absence of vasopressin. Dilution of the mucosal medium alone resulted in no morphologic changes. Subsequent addition of vasopressin produced an increase in the cell volume of the granular cells, manifested by some or all of the following changes: increased area of granular cell profiles as observed in sections, rounding of the cell nucleus, displacement of the two components of the nuclear envelope, loss of nuclear heterochromatin, sacculation of the endoplasmic reticulum and the Golgi apparatus, and reduction in the electron density of the cell cytoplasm. No such morphologic changes were noted in the other cell types comprising the mucosal epithelium — the mitochondria-rich, the goblet, and the basal cells. On the other hand, dilution of the serosal bathing medium in the absence of vasopressin caused a marked increase in the cell volume of all these cell types. The results demonstrate that the action of vasopressin to enhance bulk water flow across toad bladder is exerted specifically on the apical surface of the granular cells. It is suggested that the hormonal effect on sodium transport may also be limited to the granular cells. The route of osmotic water flow and the possible role of the other mucosal epithelial cells is discussed.     

Asymmetry of canine tracheal epithelium: osmotically induced changes

The symmetry of osmotic conductivity of the canine tracheal epithelial cells was examined in vitro. When an osmotic load of 100 mosM sucrose was added to the serosal bathing solution, no change in the transepithelial potential difference was observed in 15 tissue preparations. In contrast, when the same osmotic load was added to the mucosal bathing solution, there was a rapid decrease in the transepithelial potential difference of 3.9 +/- 0.5 mV (n = 23); ouabain (10(-4) M) eliminated this change. Tissues that had been exposed to the osmotic load added to either the mucosal or serosal side were compared with the control using light and electron microscopy. When the osmotic load was added to the mucosal fluid, there was no change in the nuclear-to-cytoplasmic area ratio of the cell types examined. However, when the same osmotic load was added to the serosal fluid, a marked increase in the nuclear-to-cytoplasmic area ratio of the ciliated cells was observed. This finding indicated cell shrinkage. Dilution potentials measured by substituting NaCl with mannitol also showed asymmetry. The morphological features are probably caused by differences in the osmotic conductivity (Lp) of the basolateral and apical cell membranes, with the Lp of the apical membrane being less than that of the basolateral membrane. The basis for osmotically induced potentials remained undetermined.     

Ion selectivity of the apical membrane Na channel in the toad urinary bladder

The ion selectivity of the apical membrane Na channel in the toad urinary bladder was investigated. The electrical potential difference and resistance across the basal-lateral membrane were reduced using high concentrations of KCl in the serosal bathing medium, and gradients for various ions were imposed across the apical membrane by altering the composition of the mucosal bathing medium. Ion fluxes through the channel were measured as the transepithelial current inhibited by amiloride, a specific blocker of the channel's Na conductance. The selectivity sequence for alkali metal cations was H greater than Li greater than Na much greater than K. K permeability was barely detectable; the selectivity for Na over K was about 1000:1. Ammonium, hydroxyl ammonium and hydrazinium ions were, like K, virtually impermeant. The results suggest that the size of the unhydrated ion is an important factor in determining permeability in this channel.     

Intracellular solute gradients during osmotic water flow: An electron-microprobe analysis

Summary In an attempt to quantify possible intracellular water activity gradients during ADH-induced osmotic water flow, we employed energy dispersive X-ray microanalysis to thin, freezedried cryosections obtained from fresh, shock-frozen tissue of the toad urinary bladder. The sum of all detectable small ions (Na + K + Cl) in the cellular water space was taken as an index of the intracellular osmolarity. Presuming that all ions are osmotically active, they comprise about 90% of the cellular solutes. When the cells were exposed to dilute serosal medium, the reduction in the sum of the ions agreed well with the expected reduction in osmolarity. After inducing water flow by addition of ADH and dilution of the mucosal medium, all epithelial cells showed a fall in osmolarity. The change was more pronounced in granular cells than in basal or mitochondria-rich cells, consistent with the notion that granular cells represent the main transport pathway. Most significantly, intracellular osmolarity gradients, largely caused by an uneven distribution of K and Na, were detectable in granular cells. The gradients were not observed after ADH or mucosal dilution alone, or when the direction of transepithelial water flow was reversed. We conclude from these results that there is a significant cytoplasmic resistance to water flow which may lead to intracellular gradients of water activity. Concentration gradients of diffusible cations can be explained by a flow-induced Donnan-type distribution of fixed negative charges. With regard to transepithelial Na transport, the data suggest that ADH stimulates transport by increasing the Na permeability of the apical membranes of granular cells specifically.     

On the role of calcium in the mechanism of ADH action

With an increased influx of Ca2+ in the cytoplasm, the response of cells to ADH in the urinary bladder of the frog was lowered by addition of ionophore A23187 from the side of the basolateral cell membrane, but inhibited when it was added from the apical cell membrane. The removal of calcium by EGTA from the serosal surface was accompanied by a sharp increase of osmotic permeability not only to water, but also to inulin; while when calcium was removed from the mucosal surface of the urinary bladder, osmotic permeability was not changed. After being added to the Ringer solution from the outer surface of the apical cell membrane, the inhibitors of Ca2+ channels (verapamil, Ni2+, Mn2+, Co2+) decreased the effect of ADH. These data indicate that Ca2+ applied onto the outer surface of apical plasma membrane plays an important role in the action of ADH.     

Na(+) and Cl(-) transport by the urinary bladder of the freshwater rainbow trout (Oncorhynchus mykiss)

Freshwater (FW) rainbow trout (Oncorhynchus mykiss) urinary bladders mounted in vitro under symmetrical saline conditions displayed electroneutral active absorption of Na(+) and Cl(-) from the mucosal side; the transepithelial potential (V(t)) was 0.1 mV, and the short-circuit current was less than 1 microA cm(-2). Removal of Na(+) from mucosal saline decreased Cl(-) absorption by 56% and removal of Cl(-) decreased Na(+) absorption by 69%. However, active net absorption of both Na(+) and Cl(-) was not abolished when Cl(-) or Na(+) was replaced with an impermeant ion (gluconate or choline, respectively). Under physiological conditions with artificial urine (?Na(+) = 2.12 mM, ?Cl(-) = 3.51 mM) bathing the mucosal surface and saline bathing the serosal surface, transepithelial potential (V(t)) increased to a serosal positive approximately +7.6 mV. Unidirectional influx rates of both Na(+) and Cl(-) were 10-20-fold lower but active absorption of both ions still occurred according to the Ussing flux ratio criterion. Replacement of Na(+) with choline, or Cl(-) with gluconate, in the mucosal artificial urine yielded no change in unidirectional influx of Cl(-) or Na(+), respectively. However, kinetic analyses indicated a decrease in maximum Na(+) transport rate (J(max)) of 66% with no change in affinity (K(m)) in the low Cl(-) mucosal solution relative to the control solution. Similarly, there was a 79% decrease in J(max) values for Cl(-), again with no change in K(m), in the low-Na(+) mucosal bathing. The mucosal addition of DIDS, amiloride or bumetanide (10(-4) M) had no effect on either Na(+) or Cl(-) transport, under either symmetrical saline or artificial urine/saline conditions. Addition of the three drugs simultaneously (10(-4) M), or chlorothiazide (10(-3) M), under symmetrical saline conditions also had no effect on Na(+) or Cl(-) transport rates. Cyanide (10(-3) M) addition to mucosal artificial urine caused a slowly developing decrease of Na(+) influx to 59% and Cl(-) influx to 50% in the period after drug addition. Na(+) and Cl(-) reabsorption appears to be a partially coupled process in the urinary bladder of O. mykiss; transport mechanisms are both dependent upon and independent of the other ion.     

Pathways for movement of ions and water across toad urinary bladder

Hypertonicity of the mucosal bathing medium increases the electrical conductance of toad urinary bladder by osmotic distension of the epithelial "tight" or limiting junctions. However, toad urine is not normally hypertonic to plasma. In this study, the transmural osmotic gradient was varied strictly within the physiologic range; initially hypotonic mucosal bathing media were made isotonic by addition of a variety of solutes. Mucosal NaCl increased tissue conductance substantially. This phenomenon could not have reflected soley an altered conductance of the transcellular active transport pathway since mucosal KCl also increased tissue conductance, whether or not Na+ was present in the bathing media. The effect of mucosal NaCl could not have been mediated solely by a parallel transepithelial pathway formed by damaged tissue since mucosal addition of certain nonelectrolytes also increased tissue conductance. Finally, the osmotically-induced increase in conductance could not have occurred soley in transcellular transepithelial channels in parallel with the active pathway for Na+, since the permeability to 22Na from serosa to mucosa (s to m) was also increased by mucosal addition of NaCl; a number of lines of evidence suggest that s-to-m movement of Na+ proceeds largely through paracellular transepithelial pathways. The results thus establish that the permeability of the limiting junctions is physiologically dependent on the magnitude of the transmural osmotic gradient. A major role is proposed for this mechanism, serving to conserve the body stores of NaCl from excessive urinary excretion.     

Metabolic evidence that serosal sodium does not recycle through the active transepithelial transport pathway of toad bladder

Summary The possibility that sodium from the serosal bathing medium back-diffuses into the active sodium transport pool within the mucosal epithelial cell of the isolated toad bladder was examined by determining the effect on the metabolism of the tissue of removing sodium from the serosal medium. It was expected that if recycling of serosal sodium did occur through the active transepithelial transport pathway of the isolated toad bladder, removal of sodium from the serosal medium would reduce the rate of CO₂ production by the tissue and enhance the stoichiometric ratio of sodium ions transported across the bladder per molecule of sodium transport dependent CO₂ produced simultaneously by the bladder (J _Na/J _{CO 2}). The data revealed no significant change in this ratio (17.19 with serosal sodium and 16.13 after replacing serosal sodium with choline). Further, when transepithelial sodium transport was inhibited (a) by adding amiloride to the mucosal medium, or (b) by removing sodium from the mucosal medium, subsequent removal of sodium from the serosal medium, or (c) addition of ouabain failed to depress the basal rate of CO₂ production by the bladder [(a) rate of basal, nontransport related, CO₂ production (J _CO2 ^b ) equals 1.54±0.52 with serosal sodium and 1.54±0.37 without serosal sodium; (b)J _CO2 ^b equals 2.18±0.21 with serosal sodium and 2.09±0.21 without serosal sodium; (c) 1.14±0.26 without ouabain and 1.13±0.25 with ouabain; unite ofJ _CO2 ^b are nmoles mg d.w.^–1 min^–1]. The results support the hypothesis that little, if any, recycling of serosal sodium occurs in the toad bladder.     

Osmotic water permeability of Necturus gallbladder epithelium

An electrophysiological technique that is sensitive to small changes in cell water content and has good temporal resolution was used to determine the hydraulic permeability (Lp) of Necturus gallbladder epithelium. The epithelial cells were loaded with the impermeant cation tetramethylammonium (TMA+) by transient exposure to the pore-forming ionophore nystatin in the presence of bathing solution TMA+. Upon removal of the nystatin a small amount of TMA+ is trapped within the cell. Changes in cell water content result in changes in intracellular TMA+ activity which are measured with intracellular ion-sensitive microelectrodes. We describe a method that allows us to determine the time course for the increase or decrease in the concentration of osmotic solute at the membrane surface, which allows for continuous monitoring of the difference in osmolality across the apical membrane. We also describe a new method for the determination of transepithelial hydraulic permeability (Ltp). Apical and basolateral membrane Lp's were assessed from the initial rates of change in cell water volume in response to anisosmotic mucosal or serosal bathing solutions, respectively. The corresponding values for apical and basolateral membrane Lp's were 0.66 x 10(-3) and 0.38 x 10(-3) cm/s.osmol/kg, respectively. This method underestimates the true Lp values because the nominal osmotic differences (delta II) cannot be imposed instantaneously, and because it is not possible to measure the true initial rate of volume change. A model was developed that allows for the simultaneous determination of both apical and basal membrane Lp's from a unilateral exposure to an anisosmotic bathing solution (mucosal). The estimates of apical and basal Lp with this method were 1.16 x 10(-3) and 0.84 x 10(-3) cm/s.osmol/kg, respectively. The values of Lp for the apical and basal cell membranes are sufficiently large that only a small (less than 3 mosmol/kg) transepithelial difference in osmolality is required to drive the observed rate of spontaneous fluid absorption by the gallbladder. Furthermore, comparison of membrane and transepithelial Lp's suggests that a large fraction of the transepithelial water flow is across the cells rather than across the tight junctions.     

Inhibition of the permeability response to vasopressin and oxytocin in the toad bladder: Effects of bradykinin,kallidin, eledoisin,and physalaemin

Summary It has been shown by means of Bentley'sin vitro preparation of the isolated urinary bladder of the toad,Bufo marinus paracnemis Lutz, that bradykinin reversibly inhibited the increase brought about by vasopressin on the permeability to water of the toad bladder. The increased hydro-osmotic response of the bladder to oxytocin was also inhibited by the kinin. The effect on water permeability was observed when bradykinin was added either to the serosal Ringer's solution or to the mucosal solution. The addition of bradykinin alone did not alter the basal osmotic water transfer across the bladder. In this context, bradykinin acted as a competitive antagonist of vasopressin (and oxytocin). Although lacking intrinsic activity, bradykinin exhibited affinity for receptor sites that are also common to the neurohypophysial hormones, causing a parallel shift of the log-dose/response curve for vasopressin without changing the maximal responses. The effects of other kinins (namely kallidin, eledoisin and physalaemin) on the toad bladder were also tested. Each of these drugs alone did not change the basal water flux across the bladder wall. Like bradykinin, these peptides inhibited the increase in water permeability evoked by vasopressin and oxytocin in the bladder. In view of the importance of neurohypophysial hormones and their target tissues to the osmotic homeostasis of amphibians, and the observation of antagonism between the kinins and the pituitary hormones coupled to the abundance of kinins in the amphibian organism, particularly in the skin and urinary bladder, teleological reasoning predicts a physiological role for the kinins, possibly functioning to dampen excesses and oscillations in membrane permeability that could occur in face of a constant and variable secretion of neurohypophysial hormone, thus adding to the homeostatic response of the amphibian organism.