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

Summary The urinary bladder ofBufo 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 g/ml was found to increase H⁺ excretion by 50% 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 g/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.     

The effect of catecholamines on H+ and NH+4 excretion in toad urinary bladder

The catecholamines epinephrine and norepinephrine, when placed on the toad urinary bladder in vitro, at a final concentration of 50 microM, caused a significant increase in H+ and NH+4 excretion by the bladder. Isoprenaline in a final concentration of 50 microM also increased H+ and NH+4 excretion in the bladder. Propranolol at a concentration of 50 microM blocked the stimulation of H+ excretion by isoprenaline but propranolol at 100 microM was required to block the stimulation of NH+4 by isoprenaline. The dose-response analysis indicates that the concentration of epinephrine used (50 microM) is at or near the maximal effective dose. These findings indicate that catecholamines stimulate H+ and NH+4 excretion in the toad urinary bladder and evidence suggests this may be mediated via the beta receptor mechanism.     

Action of steroids on H+ and NH 4 + excretion in the toad urinary bladder

Summary This study was done to determine if steroid compounds will stimulate the urinary bladder of the toad to increase its capacity to acidify the urine and excrete NH ₄ ⁺ , Aldosterone, 17-estradiol, dexamethasone, pregnenolone, and cholesterol were tested on the bladder. All compounds tested were found to stimulate the rate of acidification by the bladder, above that of a paired control hemibladder. In contrast, only the steroids aldosterone and 17-estradiol were found to stimulate NH ₄ ⁺ excretion in the bladder. Cycloheximide was found to block the action of aldosterone on the NH ₄ ⁺ excretion, but did not have a significant effect on the stimulation of acidification by aldosterone. We conclude that steroids stimulate H⁺ and NH ₄ ⁺ excretion in the toad urinary bladder. In addition, the NH ₄ ⁺ excretory system seems to be more specific to this effect than is the H⁺ excretory system.This work was presented in part at the 62nd annual meeting, Federation of American Societies for Experimental Biology, Atlantic City, N.J., April 1978.     

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.     

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.     

Effects of tetracyclines on aldosterone- and insulin-mediated Na+ transport in the toad urinary bladder.

The effect of oxytetracycline and demethylchlortetracycline on aldosterone- and insulin-mediated Na+ transport (short-circuit current) were examined in toad urinary bladders mounted in modified Ussing chambers. Oxytetracycline had little or no effect on either basal or aldosterone-mediated Na+ transport. In contrast, demethylchlortetracycline markedly inhibited both basal and aldosterone-mediated Na+ transport. Furthermore, demethylchlortetracycline inhibited the aldosterone response significantly out of proportion to its effects on basal Na+ transport. Neither of the drugs had an effect on insulin-mediated Na+ transport. Consequently, the natriuresis observed in certain patients treated with demethylchlortetracyline may be related to drug-induced renal resistance to the effects of aldosterone.     

Characteristics of proton excretion in normal and acidotic toad urinary bladder

Leupeptin, an inhibitor of lysosomal cathepsin activity, was injected intravenously into male rats. Tissues obtained from leupeptin-treated animals showed a depressed cathepsin activity when compared with tissues from saline-treated control animals. Leupeptin treatment did not change the hepatic activities and subcellular distribution of marker enzymes for mitochondria, microsomes and plasma membranes. Hepatic lysosomal cathepsin activity was specifically inhibited, but the subcellular distribution of all lysosomal marker enzymes tested was changed, indicating the occurrence of enlarged lysosomes in the leupeptin-treated animals. No significant differences were observed in the serum concentrations of protein, cholesterol, cholesteryl esters, phospholipids and apolipoproteins A-I, A-IV and E between leupeptin-treated rats and control animals. When radioiodinated asialofetuin was injected intravenously, the radiolabel was retained for an extended period of time in the liver of leupeptin-treated animals, indicating diminished catabolism of this protein in the liver. When rat high-density lipoprotein, labelled specifically in the apolipoprotein A-I or E moiety was injected intravenously, only the kidneys and the liver showed a leupeptin-induced accumulation of radioactivity. These studies provide evidence for an important contribution of the kidneys and the liver to the in vivo catabolism of high-density lipoprotein apolipoproteins, using a method completely different from sugar-containing labelling compounds.     

Effects of anions on cellular volume and transepithelial Na+ transport across toad urinary bladder

Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl^– on transepithelial Na⁺ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V _T) decreased rapidly, transepithelial resistance (R _T) increased, and calculated short-circuit current (I _sc) decreased. CalculatedE _Na was unaffected, indicating that the inhibition of Na⁺ transport was a consequence of a decreased apical membrane Na⁺ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV _T decreased,R _T increased andI _sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE _Na fell substantially (from 130 to 68 mV on average). Ba²⁺ (3mm) reduced calculatedE _Na in Cl^– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl^– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE _Na did not fall. Replacement of serosal isosmotic Cl^– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na⁺ transport. Thus the inhibition of Na⁺ transport can be correlated with changes in cell volume rather than with the change in Cl^– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K⁺ gluconate mucosa, Na⁺ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na⁺/K⁺ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl^– results in a loss of highly specific Ba²⁺-sensitive K⁺ conductance channels from the basolateral plasma membrane. It is possible that the number of Na⁺ pump sites in this membrane is also decreased.     

The membrane action of antidiuretic hormone (ADH) on toad urinary bladder.

Radioactive tracer and electrical techniques were used to study the transport of nonelectrolytes and sodium, respectively, across toad urinary bladders in the presence and absence of ADH. The permeability of lipophilic molecules was roughly proportional to bulk phase oil/water partition coefficients both in the presence and absence of hormone; i.e., ADH elicited a general nonselective increase in the permeation of all nine solutes tested. The branched nonelectrolyte, isobutyramide, was less permeable than its straight-chain isomer, n-butyramide, in control tissues. ADH reduced the discrimination between these structural isomers. Hydrophilic solutes permeated more rapidly than expected. In the presence of hormone, there was no change in the permeation of large hydrophilic solutes considered to move via an extracellular pathway, but there was a marked increase in the permeability of water and other small hydrophilic solutes. Collectively, these results suggest that ADH acts to increase the motional freedom or fluidity of lipids in the cell membrane which is considered to be the preferred pathway for the permeation of lipophilic and small hydrophilic molecules. At concentrations of cAMP and ADH which elicit equivalent increments in the shortcircuit current, the effects of these agents on nonelectrolyte transport and membrane electrical conductance are divergent. Such observations suggest that some membrane effects of ADH may not be directly dependent upon cAMP. ADH in the mucosal solution increased the permeability of the toad bladder when the surface charge on the outer surface of the apical membrane was screened with the polyvalent cation, La-3+. These experiments emphasize that interaction of ADH with membranes of toad urinary bladder may account for at least some effects of this hormone.     

The role of cyclic AMP in parathyroid hormone action in the toad bladder

Summary Parathyroid hormone (PTH) inhibited active transport of inorganic phosphate and stimulated an increase in cyclic AMP concentration in the urinary bladder of the toad,Bufo marinus. Active transport of phosphate in the toad bladder was also inhibited by an analog of cyclic AMP (dibutyryl cyclic AMP) and by other drugs (pitressin and theophylline) which increase toad bladder intracellular cyclic AMP concentration. These data support the concept that cyclic AMP may be the mediator of PTH-induced phosphate transport inhibition in the toad bladder.     

Urea uptake and translocation in toad urinary bladder: the effect of antidiuretic hormone.

The uptake of C14-urea into everted and noneverted bladder sacs was compared, over short time periods (up to 2 min), with the transepithelial urea fluxes. This method allowed the study of the time course of urea uptake and distribution, while previously this problem was only studied in steady-state conditions. When mucosal uptake was studied no accumulation of C14-urea inside the tissue was observed, indicating that the mucosal border could be the limiting step. Comparative studies of urea and inulin uptake from the serosal side showed that urea equilibrated with the water epithelial cells in less than 30 sec. This accumulation suggested again that the mucosal border is an effective barrier for urea translocation. The kinetics of the increase in urea permeability induced by antidiuretic hormone was also studied and it was similar (T1/2:4.3 min) to the kinetics of the increase in water permeability induced by the hormone (T1/2:5.6 min). A strong parallelism was also observed between the time course of the increases in water and urea permeabilities induced by medium hypertonicity (T1/2 25 and 26 min, respectively). The values obtained for the permeability coefficient ktrans), either at rest or under ADH were similar to those previously reported employing steady-state techniques (28+/-8 and 432+/-25 cm-sec-1-10(-7), respectively).