Urinary Bladder Of Bufo Marinus Research Articles

Functional Analysis of Natriuretic Peptide Receptors in the Bladder of the Toad, Bufo marinus

This study aimed to localize and characterize natriuretic peptide binding sites in the urinary bladder of Bufo marinus and to then examine the effect of natriuretic peptides on the bladder vascular tone and water reabsorption in isolated perfused bladder preparations. Specific 125I-rat atrial natriuretic peptide (125I-rANP) binding sites were present on blood vessels, muscle, and epithelium. In tissue sections and/or isolated membranes, the binding was completely displaced by frog ANP, rat ANP, and porcine C-type natriuretic peptide (CNP; membranes only). However, a reduction in binding was observed after incubation with 125I-rANP and 1 μM of the natriuretic peptide receptor-C (NPR-C) ligand C-ANF, but residual binding remained suggesting the presence of two distinct binding sites. Electrophoresis of bladder membranes cross-linked to 125I-rANP identified two bands at approximately 70 and 140 kDa that correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. Furthermore, the presence of natriuretic peptide receptor-A and NPR-C mRNA in the bladder was demonstrated with reverse transcription–polymerase chain reaction. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP generation in bladder membrane preparations, which indicated the presence of guanylate cyclase-linked receptors. In perfused bladder preparations, arginine vasotocin increased perfusion pressure and water permeability. The infusion of frog ANP or porcine CNP failed to alter perfusion pressure or water reabsorption in the presence or absence of arginine vasotocin. This study identified a well-developed natriuretic peptide receptor system in the urinary bladder of B. marinus but the function of the receptors remains unclear.

The Na+-K+-ATPase beta1 subunit is associated with the HKalpha2 protein in the rat kidney

The Na+-K+-ATPase beta1 subunit is associated with the HKalpha2 protein in the rat kidney

Ammonia transport by the urinary bladder of Bufo marinus.

All experiments were performed in vitro on toad bladders. Bladder sacs from acidotic toads produced a concentration gradient across the bladder with both [NH3] and [NH4] higher in the mucosal media. By varying the pH of the serosal media, paired sacs from normal toads were incubated with similar [NH3] in the serosal media but a 75 fold difference in [NH4] of the serosal media of the pairs. The hemibladders with the higher [NH4] had a 2.4 fold greater excretory flux than the paired sac. Both serosal to mucosal and mucosal to serosal fluxes were determined in normal bladders between chambers at various ammonium concentration gradients. The plot of mucosal to serosal flux against concentration produced a curve compatible with both carrier mediated and diffusion transport; the plot of serosal to mucosal flux produced a straight line with flux increasing when the ammonium concentration was increased. The serosal to mucosal transepithelial flux was augmented by making the serosal side of the bladder 50 mV positive. Although NH3 diffusion may occur, it cannot be the primary method of ammonia transport in the toad bladder.

Stimulation of Phosphoinositides by Agents that Stimulate Proton Secretion in Toad Urinary Bladder

The urinary bladder of Bufo marinus excretes H+ and this excretion is increased by metabolic acidosis (MA), insulin (IN), prostaglandin E2 (PGE2), increases CO2, and aldosterone. The purpose of this experiment was to determine whether MA, IN, PGE2, CO2, and aldosterone stimulate inositol phosphate's (IP) formation in isolated cells of toad urinary bladder. Cells were prepared by treating bladder sacs with collagenase. Cells were obtained from 10 toads in MA and 10 normal toads, suspended in 2 ml of Ringer's solution containing LiCl (10 mM), myo-inositol (5 mM), and [3H]myo-inositol (10 microCi), and then incubated for 2 hr at 25 degrees C. Cells were homogenized and the IP fractions quantitated by column chromatography and liquid scintillation counting. The results were expressed as dpm (mu MPO4)-1 (hr)-1. The IP in MA cells was 44,202 +/- 4,646 and in normal toad cells it was 31,637 +/- 3,613 (P < 0.05). In a separate experiment, cells from 10 paired hemibladders were isolated from normal toads. The cells were treated exactly as above except there were no LiCl in the bath. LiCl was added to all baths after 2 hr and the experimental cells were challenged with IN, PGE2, increases CO2, and aldosterone for 20 min. The IP were quantitated as above. IN treatment stimulated inositol bisphosphate and inositol triphosphate (P < 0.01). PGE2 and increases CO2 also stimulated inositol triphosphate (P < 0.05). Aldosterone did not alter formation of any of the IP fractions. We conclude that MA, IN, PGE2, and increases CO2 stimulate IP formation in cells of toad urinary bladder and inositol triphosphate may be an important second messenger in mediating the response of MA, IN, PGE2, and increases CO2.

Ca(2+)-blockable, poorly selective cation channels in the apical membrane of amphibian epithelia. Tetracaine blocks the UO2(2+)-insensitive pathway.

We examined the effect of the local anesthetic tetracaine on the Ca(2+)-blockable, poorly selective cation channels in the isolated skin of Rana temporaria and the urinary bladder of Bufo marinus using noise analysis and microelectrode impalements. Experiments with frog skin demonstrated that mucosal concentrations of the compound up to 100 microM did not affect the Na+ current through type S channels (slowly fluctuating, UO2(2+)-blockable channels) and the associated noise. On the other hand, 20 microM mucosal tetracaine already suffices to inhibit approximately 50% of the current carried by Cs+ and Na+ through channel type F (fast fluctuating, UO2(2+)-insensitive channel) and So of the associated Lorentzian component. With 100 microM of the inhibitor the current and So values were reduced by at least 70-80%. The time course of the response to serosal tetracaine was markedly slower and the effects on the current and So were smaller. Possible effects on the basolateral K+ conductance were excluded on the basis of the lack of response of transepithelial K+ movements to 100 microM tetracaine. UO2(2+) and tetracaine together blocked the poorly selective cation pathways almost completely. Moreover, both agents retain their inhibitory effect in the presence of the other. In toad urinary bladder, the Ca(2+)-blockable channel is also tetracaine blockable. The concentration required for half-maximal inhibition is approximately 100 microM in SO4(2-) and approximately 20 microM in Cl-. The data with tetracaine complement those obtained with UO2(2+) and support the idea that the Ca(2+)-blockable current proceeds through two distinct classes of cation channels. Using tetracaine and UO2(2+) as channel-specific compounds, we demonstrated with microelectrode measurements that both channel types are located in the granulosum cells.

Prostaglandins as Mediators of Acidification in the Urinary Bladder of Bufo marinus

Experiments were performed to determine whether prostaglandins (PG) play a role in H+ and NH4+ excretion in the urinary bladder of Bufo marinus. Ten paired hemibladders from normal toads were mounted in chambers. One was control and the other hemibladder received PGE2 in the serosal medium (10(-5) M). H+ excretion was measured by change in pH in the mucosal fluid and reported in units of nmol (100 mg tissue)-1 (min)-1. NH4+ excretion was measured colorimetrically and reported in the same units. The control group H+ excretion was 8.4 +/- 1.67, while the experimental group was 16.3 +/- 2.64 (P less than 0.01). The NH4+ excretion in the experimental and control group was not significantly different. Bladders from toads in a 48-hr NH4+Cl acidosis (metabolic) did not demonstrate this response to PGE2 (P greater than 0.30). Toads were put in metabolic acidosis by gavaging with 10 ml of 120 mM NH4+Cl 3 x day for 2 days. In another experiment, we measured levels of PG in bladders from control (N) and animals placed in metabolic acidosis (MA). Bladders were removed from the respective toad, homogenized, extracted, and PG separated using high-pressure liquid chromatography and quantified against PG standards. The results are reported in ng (mg tissue)-1. PGE2 fraction in N was 1.09 +/- 0.14 and in MA was 3.21 +/- 0.63 (P less than 0.01). PGF1 alpha, F2 alpha and I2 were not significantly different in N and MA toads. Bladders were also removed from N and MA toads, and incubated in Ringer's solution containing [3H]arachidonic acid (0.2 microCi/ml) at 25 degrees C for 2 hr. Bladders were then extracted for PG and the extracts separated by thin layer chromatography. PG were identified using standards and autoradiography, scraped from plates, and counted in a scintillation detector. The results are reported in cpm/mg tissue x hr +/- SEM. In MA toads, PG6-keto-F1 alpha = 1964 +/- 342, PGF2 alpha = 1016 +/- 228, and PGE2 = 904 +/- 188; in N animals PG6-keto-F1 alpha = 625 +/- 280, PGF2 alpha = 364 +/- 85, and PGE2 = 404 +/- 104; (P less than 0.01, less than 0.025, less than 0.05, respectively). We conclude that PGE2 may be an important mediator of H+ excretion in toad urinary bladder and that endogenous PGE2 levels are increased in response to MA.

Steroid metabolism determines mineralocorticoid specificity in the toad bladder

Edwards et al. (C. R. W. Edwards, P. M. Stewart, D. Burt, L. Brett, M. A. McIntyre, W. S. Sutanto, E. R. de Kloet, and C. Monder, Lancet 2: 986-989, 1988) proposed that 11 beta-hydroxysteroid-dehydrogenase (11 beta-OHSD) plays a key role in the kidney by converting glucocorticoids (cortisol or corticosterone), which display a high affinity for type 1 mineralocorticoid receptors, into their inactive metabolites (cortisone or 11-dehydroxy-corticosterone), thus preventing their illicit occupation of the receptor in the target cell for aldosterone. We have tested this hypothesis in the urinary bladder of Bufo marinus by measuring the sodium transport responses to aldosterone and corticosterone. Aldosterone (10 nM) on the serosal side elicited a quarter of the maximal increase in sodium transport. At the same concentration, corticosterone (10 nM, serosal side) was ineffective. Adding corticosterone (10 nM) on the mucosal side elicited a response equivalent to that of aldosterone, suggesting that corticosterone was inactivated in the serosal or underlying tissue of the toad bladder. Carbenoxolone (10 microM, serosal side), a potent inhibitor of 11 beta-OHSD, did not modify the base-line sodium transport. However, in the presence of carbenoxolone (10 microM, serosal side, 2 h pretreatment) corticosterone (10 nM, serosal side) became as potent as aldosterone in eliciting the mineralocorticoid response. Our data are consistent with the idea that corticosterone is converted into an inactive metabolite in the mucosal and/or submucosal tissue of the toad bladder. These studies are consistent with our concept that 11 beta-OHSD is crucial in protecting the nonspecific mineralocorticoid receptor from glucocorticoid.

Phospholipid Changes during Adaptation to Acidosis in Urinary Bladder of Bufo marinus

The purpose of this study was to determine whether phospholipids (PL) play a role in the adaptation to metabolic acidosis by toad urinary bladder epithelium. Toads were placed in an NH4Cl acidosis for 48 hr. Quarter bladders were removed and incubated with [32P]orthophosphate or [3H]arachidonic acid for 1 hr at 25 degrees C. PL were detected by thin layer chromatography, autoradiography, and quantitated by liquid scintillation counting or fractional amounts were determined from phosphate content and expressed as counts per minute per micromolar of total phosphate or as percentage of fraction of total PL. Incorporation of [3H]arachidonic acid into urinary bladder PL was measured in acidotic and normal toads. There was a higher rate of arachidonic acid incorporation into several PL in acidotic animals. Phosphatidic acid and phosphatidylserine fraction in acidosis was 37,705 +/- 6,821 and in normal bladders was 9,254 +/- 2,652 (P less than 0.005); phosphatidylcholine fraction in acidotic toads was 80,462 +/- 16,862 and in normal bladders was 26,892 +/- 5,198 (P less than 0.025); and the phosphatidylethanolamine (PE) fraction in acidotic was 48,665 +/- 10,998 and in normal animals was 17,441 +/- 3,905 (P less than 0.025). 32P labeling revealed a higher rate of incorporation in bladders from acidotic toads compared with normal toads. In the acidotic bladders, the phosphatidic acid and phosphatidylserine fraction was 19,754 +/- 3,597 and in normal bladders was 12,980 +/- 1,394 (P less than 0.05) and for PE acidotic bladders was 9,129 +/- 1,304 and in normal bladders was 3,285 +/- 416 (P less than 0.001). Fractional PL (reported as percentage of fraction of total PL based on total lipid phosphorus) analysis in normal toads revealed phosphatidylinositol = 8.1 +/- 0.6% and PE = 27 +/- 1.2%, whereas for acidotic toads phosphatidylinositol = 11 +/- 0.6% and PE = 32 +/- 1.0% (P less than 0.01 for both). Aldosterone, a known stimulator of acidification, had no effect on 32P incorporation into PL fractions of the bladder. The increase in PL turnover following induction of acidosis is consistent with increased membrane synthesis or turnover during metabolic acidosis and this may reflect an increased transport of vesicular H+-ATPase into the apical membrane or the result of a proliferation of acid-secreting mitochondria-rich cells or both.

Insulin-mediated H+ and NH+4 excretion in the urinary bladder of Bufo marinus.

This study was done to determine if insulin mediates H+ and NH+4 excretion in the urinary bladder of Bufo marinus. Acidosis was induced by gavaging with 10 ml of 120 mM NH4Cl 3X daily for 2 days. Hemibladders were mounted between Lucite chambers. Insulin (porcine) was added to the serosal solution of the experimental bladder (10(2) mU/ml). After a 15-min equilibration the flux was measured for 2 hr. H+ excretion was measured from change in pH of the mucosal fluid and the NH+4 measured colorimetrically. The excretion was normalized for weight of bladder and reported in units of nanomoles (100 mg bladder)-1(min)-1. Plasma insulin was determined by radioimmunoassay and glucose by the glucose oxidase method. In 14 control bladders H+ excretion was 8.75 +/- 1.28 and experimental was 16.35 +/- 2.50 (P less than 0.025), while NH+4 excretion in control bladder was 3.29 +/- 0.95 and experimental was 6.58 +/- 1.89 (P less than 0.01). This response was absent when the insulin was heat inactivated (P greater than 0.2 and P greater than 0.3 respectively). Plasma insulin-like levels in 10 normal toads was 0.57 +/- 0.16 ngm/ml and in acidotic toads 1.25 +/- 0.16 ng/ml (P less than 0.025). Plasma glucose levels in 10 normal toads were 22.0 +/- 3.5 mg/dl and in 12 acidotic toads 17.8 +/- 0.75 mg/dl (P less than 0.025). We conclude that plasma insulin is increased in acidosis and that insulin stimulates excretion of H+ and NH+4 in the toad urinary bladder.

Characteristics of proton excretion in normal and acidotic toad urinary bladder

This study, performed on the urinary bladder of Bufo marinus, was to investigate the characteristics of H + excretion in the normal and metabolic acidotic toad. Experiments were run in modified Ussing chambers in the presence and absence of exogenous CO 2. Amiloride in the mucosal medium (1.5 · 10 −4 M) inhibited H + excretion in the acidotic bladder but this inhibition was reversed in the presence of 5% CO 2. Na +-free muscosal medium revealed a component of H + excretion in the normal toad that is Na +-dependent and not reversed by 5% CO 2. Acetazolamide (10 −3 M) had no effect on H + excretion in normal toad but inhibits excretion in the acidotic toad both in the presence and absence of exogenous CO 2. In both the presence and absence of exogenous CO 2 and in the presence of varying pH gradients, the bladders from acidotic toads excreted H + at a greater rat and were able to generate greater pH gradients than in normal bladders. Voltage clamp experiments revealed that H + excretion in the acidotic toad was inhibited by a mucosal potential difference of −20 to −100 mV but this inhibition was completely reversed by 5% CO 2. In the normal toad bladder H + excretion was stimulated by a mucosal potential difference of −20 to −100 mV in both the presence and absence of exogenous CO 2. Our evidence suggests the possibility of two H + excretory mechanisms in toad urinary bladder each with its' own set of characteristics dependent on the acid-base state of the animal. Proposed models are presented for these mechanisms.

Mechanism of inhibition by lithium of sodium transport in the toad bladder

Lithium transport across the urinary bladder of Bufo marinus has been studied by means of the short-circuit current technique, as well as unidirectional ion flux measurements. Exposure to lithium of the epithelial (mucosal) surface of this preparation led to a slow, progressive decrease of ion transport, with increasing discrepancy between short-circuit current and lithium influx; in fact there was still an appreciable lithium influx across bladder exposed to amiloride even though short-circuit current was suppressed. Ohmic conductance and sodium efflux barely increased under these circumstances. Upon replacement of lithium by sodium on the epithelial side, the preparations recovered slowly indeed, and residual lithium could be detected in bladder tissue for more than 2 hr while the rate of sodium extrusion at the basal-lateral cell border was slowed down. Recovery from exposure to lithium was accelerated by vasopressin and amphotericin, both of which facilitate sodium entry at the apical border of the epithelium. Thus the lasting deleterious influence of lithium on sodium transport might result from the fact that this ion, once trapped in the cytoplasm, closes the sodium channels.

The role of the adrenal gland in control of H+ and NH4+ excretion by toad urinary bladder.

The urinary bladder of Bufo marinus has been shown to excrete H+ and NH4+ and this excretion is increased by metabolic acidosis. The involvement of the adrenal gland and its steroid secretions in the adaptation for increased acid and ammonia excretion by the bladder was tested during the course of this study. Groups of toads were adrenalectomized and maintained in chronic NH4Cl-induced acidosis. Three other groups of toads were adrenalectomized and put in acidosis but repleted with 2.5 mg/day of either cortisol (CT), dexamethasone (Dexa), or deoxycorticosterone acetate (DOCA). All control groups were sham-operated. The bladders were excised after 3 days and mounted between 2-ml Lucite chambers. Net H+ and NH4+ fluxes into the mucosal media were measured and reported in units of nanomoles per 100 mg bladder per minute. In control acidotic toads H+ excretion was 20.1 +/- 2.0 and the adrenalectomized nonreplete group H+ excretion was 14.2 +/- 1.87 (P less than 0.04). For the same groups NH4+ excretion was 2.90 +/- 0.26 for the controls and 1.38 +/- 0.19 for the adrenalectomized (P less than 0.001). The H+ excretion in CT-, Dexa-, and DOCA-repleted toads was not significantly different from the control group. NH4+ excretion, however, showed a 55% decrease (P less than 0.001) in the CT group, and a 45% decrease (P less than 0.05) in the Dexa group. The NH4+ excretion in the DOCA repleted group was significantly different from the control group. Therefore, we conclude that the adrenal gland plays a role in the adaptive increase of H+ and NH4+ excretion by the urinary bladder in acidosis through the secretion of steroid hormones. The increase in NH4+ excretion appears to be a mineralocorticoid-stimulated process. We were not able to determine in this study if the steroid hormones had an exacting regulatory role or one of a permissive role over H+ and NH4+ excretion in the toad urinary bladder.

Modulation of vasopressin action by reducing agents in Bufo marinus.

The effects of the reducing agent dithiothreitol (DTT) on vasopressin (AVP)-stimulated osmotic water flow and adenylate cyclase activity were studied in the urinary bladder of Bufo marinus. DTT produced concentration-dependent inhibition of the hydroosmotic water permeability response to 10 mU/ml AVP and 10 mM theophylline but did not inhibit the response to 10 mM adenosine 3',5'-cyclic monophosphate (cAMP). The inhibitory effects of DTT on AVP responsiveness were partially reversed by washing in DTT-free Ringer solution or by addition of oxidizing agents such as dehydroascorbic acid (DHA) or H2O2. The inhibitory effects of DTT were completely reversed by washing in DTT-free Ringer plus addition of DHA. In addition, the inhibitory effects of DTT on AVP-induced osmotic water flow were partially reversed by the GTP analogue 5'-guanylyl imidodiphosphate [Gpp(NH)p]. DTT also inhibited the adenylate cyclase response to AVP but did not alter the response to AVP plus Gpp(NH)p or the response to NaF. These observations suggest that the inhibitory effect of thiol compounds on AVP responsiveness may be modulated through alterations of a redox system distal to the hormone receptor but proximal to the catalytic subunit of adenylate cyclase. Inasmuch as Gpp(NH)p partially reversed the inhibitory effects of DTT on AVP-stimulated osmotic water permeability and prevented the inhibitory effect of DTT on AVP-stimulated adenylate cyclase, an effect on either GTPase or binding of GTP to the regulatory protein of adenylate cyclase is suggested by these observations.

Reversal of copper-induced inhibition of vasopressin responsiveness by reducing agents

Copper inhibits the hydro-osmotic response to vasopressin in the urinary bladder of Bufo marinus at a site proximal to cyclic AMP production. This effect is not reversed by washing in Cu 2+-free Ringer's solution but is overcome by serosal addition of reducing agents, suggesting that vasopressin responsiveness in this tissue is modulated by either the redox potential or the sulfhydryl content of the serosal membrane.

Hormone effects on transport in cultured epithelia with high electrical resistance.

Three continuous lines of amphibian epithelial cells form epithelia with a high transepithelial resistance (greater than 4,000 omega . cm2) in culture. The cell lines are TB-M and TB-6c, derived from the urinary bladder of Bufo marinus, and A6, derived from the kidney of Xenopus laevis. Short-circuit current is equivalent to net mucosa-to-serosa sodium transport in two cell lines and slightly exceeds sodium transport in epithelia formed by TB-6c cells. None of the cell lines has an adenylate cyclase response or a transport or permeability response to vasopressin. Water permeability is low in all three cell lines and is not affected by adenosine 3',5'-cyclic monophosphate (cAMP). In the three lines of cells, cAMP and aldosterone each increases short-circuit current with a time course similar to that seen in naturally occurring epithelia. In contrast to the toad urinary bladder and epithelia of line TB-M in which the aldosterone stimulation of short-circuit current is associated with a fall in transepithelial resistance, there is no change in resistance across epithelia of lines TB-6c and A6. There is also a striking difference in the sensitivity of the three lines to inhibition of short-circuit current by amiloride.

Ammoniuretic activity of dog plasma when tested on urinary bladder of Bufo marinus.

AbstractHemibladders from the toad Bufo marinus were used in all experiments. A protein free preparation of plasma was made from dogs in metabolic acidosis, respiratory acidosis, and metabolic alkalosis. These plasma preparations were tested for ammoniuretic activity by placing them on the serosal side of the urinary hemibladders of Bufo marinus mounted in vitro. Both acidotic plasma preparations caused increased ammonia excretion into the mucosal media as compared to control hemibladders. Metabolic alkalotic plasma preparations did not produce significant ammoniuretic activity. Using paired hemibladders, the effect of metabolic acidotic plasma preparations showed increased ammoniuretic activity when compared to metabolic alkalotic plasma preparations (P < 0.05).

Isolation and separation of toad bladder epithelial cells

The epithelium of the urinary bladder of Bufo marinus is composed of 5 cell types, i.e., granular (Gr), mitochondria-rich (MR) and goblet (G) cells which face the urinary lumen, microfilament-rich (MFR) and undifferentiated cells (Un) located basally. The epithelium was dissociated by collagenase and EGTA treatment. Fractionation of dispersed cells by isopycnic centrifugation on dense serum albumin solutions yielded 4 fractions: (i) a very light fraction (p approximately equal to 1.025) enriched in MR and MFR cells; (ii) a light fraction (p approximately equal to 1.045) enriched in vacuolated Gr cells; (iii) a heavy fraction (p approximately equal to 1.065) composed essentially of aggregated Gr cells, and (iv) a pellet (p approximately equal to 1.085) enriched in G and undifferentiated cells. Recoveries were based on cell counts and DNA measurements. DNA content per cell was 13.2 pg +/- 0.9 (n = 37). From 1 g fresh tissue, 62 +/- 5 x 10(6) (n = 10) cells were recovered before isopycnic centrifugation of which about 70% excluded Trypan blue. After centrifugation, 90 to 95% of the cells excluded the vital dye and approximately 3(9) x 10(6) cells were recovered from the gradient. Cell metabolism in each fraction was estimated by oxygen consumption measurements in absence or presence of ouabain, acetazolamide, and dinitrophenol. The consumption measurements in absence or presence of ouabain, acetazolamide, and dinitrophenol. The consumption was threefold higher in the very light and light fractions when compared to the heavy and pellet fractions. Ouabain sensitive oxygen consumption (QO2) represented 12 to 35% of the total O2 consumption depending on the cell fraction, and acetazolamide sensitive QO2 varied from -0.8% in the heavy fractions to 20% in the lighter fractions. DNP increased QO2 in all fractions by 20 to 50%. Finally, the cells were able to reaggregate and form junctional complexes upon addition of calcium to the medium.

Aldosterone receptor occupancy and sodium transport in the urinary bladder of Bufo marinus

The concentration dependence of binding of [3H]aldosterone to cytoplasmic and nuclear receptors was evaluated in urinary bladder epithelial cells of Colombian toads. One class of specific sites (sensitive to displacement by excess aldosterone) was detected in the cytosol. However, two classes of specific nuclear [3H]aldosterone binding sites were evident. In the nucleus, high-affinity (Kd = 2.7 X 10(-9) M), low-capacity (N = 15 X 10(-14) mol/mg DNA) sites (type I) were completely saturated at approximately 4 X 10(-8) M aldosterone, a concentration which gave a maximal increase in short-circuit current (SCC). Occupancy of low-affinity (Kd = 4.6 X 10(-7) M), high-capacity sites (N = 150 X 10(-14) mol/mg DNA) (type II) occurred at higher steroid concentrations and did not correlate with further increase in SCC. Binding parameters of Colombian and Dominican variants of Bufo marinus were compared. In both variants, the SCC increase elicited by aldosterone correlated with accumulation of type I complexes in the nucleus, but the relationship was markedly nonlinear. Various alternatives were considered as the basis for a curvilinear dependence of the increment in Na+ transport on abundance of nuclear type I complexes.

Cellular changes in the toad urinary bladder in response to metabolic acidosis.

The urinary bladder of Bufo marinus excretes H+ and NH+4, and the H+ excretion is increased when the animal is placed in metabolic acidosis. The mitochondria-rich (MR) cells mediate the H+ excretion by the bladder. The purpose of this study was to determine if there is a change in MR cells of the bladder during metabolic acidosis. Bladders from normal toads and from toads that had been placed in metabolic acidosis were used. The bladders were mounted between plastic chambers and H+ excretion measured. The bladder was then fixed and prepared for scanning (SEM) and transmission (TEM) electron micrograph studies. SEM's at low magnification were used to count the various cell types and the TEM's were used to confirm the different cell types. Fields were randomly selected and a total of 2500 cells counted in each group. The bladders from toads in metabolic acidosis had a consistently higher ratio of MR cells to granular cell than did the normal bladders. These results indicate that during metabolic acidosis there is an increased number of MR cells in the bladder, and this increased the bladder's capacity to excrete H+.

Effects of parathyroid hormone on H+ and NH+4 excretion in toad urinary bladder.

The urinary bladder of Bufo marinus excretes H+ and NH+4, and the H+ excretion is increased after the animal is placed in metabolic acidosis. The present study was done to determine if parathyroid hormone could stimulate the bladder to increase the excretion of H+ and/or NH+4. Parathyroid hormone added to the serosal solution in a final concentration of 10 mug/ml was found to increase H+ excretion by 50 per cent above the control hemibladders, while there was no effect on NH+4 excretion. Parathyroid hormone had no effect on H+ excretion when added to the mucosal solution. We also performed experiments utilizing theophylline and dibutyryl cyclic AMP which mimicked those of the parathyroid hormone experiments. A dose-response analysis was performed and the results indicate that 1 mug/ml of parathyroid hormone was the minimal effective dose. These results suggest that parathyroid hormone can stimulate H+ excretion in the toad urinary bladder and this effect seems to be mediated by cyclic AMP. In addition, it was found that parathyroid hormone has no effect on NH+4 excretion.