Effects of adrenalectomy and spironolactone on urinary metabolites of aldosterone in rats

The quantities and temporal sequences of appearance of aldosterone metabolites in the urine of adrenalectomized rats, and adrenalectomized rats treated with spironolactone, were compared following subcutaneous administration of a physiological dosage (0.05 microgram) of [1,2,-3H]aldosterone. Large amounts of radiometabolites were rapidly excreted during 0-1 and 1-3 h and only small quantities by 3-4 h in urine of both groups of rats. The majority of the urinary radiometabolites (70-85%) were identified by Sephadex DEAP-LH-20 chromatography as neutral metabolites of aldosterone (NMA), together with lesser quantities of acidic, sulfate, and glucuronide conjugates. Further characterization by high pressure liquid chromatography (HPLC) showed that 90% of the NMA excreted by adrenalectomized rats were polar metabolites which could be separated into at least 15 peaks eluting in regions of increasing polarity (designated A, B, C, and D). Only small quantities of unaltered [3H]aldosterone and no ring-A-reduced metabolites were excreted by the adrenalectomized rats. Spironolactone treatment caused large changes in the excretion of acidic and sulfate derivatives of aldosterone, as well as discrete alterations in the HPLC patterns of the polar NMA (particularly those metabolites in regions A and B). Such discrete changes in these metabolic pathways which occur at the same time as the hormonal actions of aldosterone in the kidney may provide further insight into understanding the biological role of aldosterone metabolism.     

Regulation of ODC activity in the thymus and liver of rats by adrenal hormones

The activity of L-ornithine decarboxylase (EC 4.1.1.17, ODC) has become a useful indicator of hormone responsiveness. Various regimens of dexamethasone, aldosterone and epinephrine, alone or in combination, were administered to adrenalectomized rats either in acute or chronic doses. In addition, adrenalectomized rats, which were chronically treated with aldosterone and epinephrine, were given a single injection of 50 micrograms dexamethasone and sacrificed at various time intervals after hormone treatment. Hepatic and thymic ODC activity was measured. The expected dexamethasone effect, an increase in hepatic and a decrease in thymic ODC, was observed. This study also revealed that aldosterone induced similar responses in these tissues. Epinephrine had the opposite effect since chronic administration of dexamethasone or aldosterone with epinephrine resulted in control levels of ODC. Furthermore, when aldosterone and epinephrine were chronically administered to adrenalectomized rats, to study the acute effects of dexamethasone on rat thymus and liver, the time course of the response in each tissue was found to be distinct. The influence of the adrenal gland on rat thymus and liver is not restricted only to glucocorticoids, but may also involve other hormones which it secretes.     

Glucocorticoid independent mediation of interleukin-1 induced changes in serum zinc and liver metallothionein levels

Interleukin-1 (IL-1) causes changes in zinc metabolism which have been attributed to mediation, at least in part, by glucocorticoids. However, IL-1 was found to actually lower serum corticosterone levels in rats. In addition, adrenalectomy only partially inhibited the ability of IL-1 to depress serum zinc levels and increase the amount of zinc associated with hepatic metallothionein. Furthermore, IL-1 increased total liver metallothionein protein to similar levels in both adrenalectomized and normal rats. Administering the synthetic glucocorticoid dexamethasone with IL-1 to adrenalectomized rats produced additive, but not synergistic effects on serum zinc and metallothionein concentrations. Studies with actinomycin D suggested that IL-1 induction of metallothionein might involve glucagon.     

Corticosteroid regulation of Na+ and K+ transport in the rat distal colon during postnatal development

To study the role of corticosteroids in the regulation of colonic electrogenic amiloride-sensitive Na+ absorption (ISCNa) and barium-sensitive K+ secretion (ISCK) during development, we investigated suckling (10-day old), weanling (25-day old) and adult (90-day old) adrenalectomized rats after they had received aldosterone, dexamethasone or corticosterone. Adrenalectomy reduced markedly ISCNa in suckling rats and completely inhibited ISCNa in weanling animals; the ISCNa was absent in intact adult rats. The doses of aldosterone, corticosterone and dexamethasone estimated to be equivalent to the endogenous production rate of aldosterone and corticosterone restored ISCNa after 1 day in both suckling and weanling rats. Compared with aldosterone, glucocorticoids produced a greater increase in ISCNa. Concurrent spironolactone treatment (a mineralocorticoid antagonist) completely prevented the effect of aldosterone but had no effect in dexamethasone-treated rats. The glucocorticoid antagonist RU 38 486 inhibited the dexamethasone-induction of ISCNa but had no effect on aldosterone. The response to corticosteroids, measured as the increase of ISCNa, declined from suckling to adult rats. In contrast to ISCNa, the same time of treatment and the same doses of corticosteroids did not influence ISCK. ISCK was stimulated only after chronic treatment (4 days). These findings suggest that, in the distal colon of young rats, (1) both corticosteroids may regulate amiloride-sensitive Na+ absorption and barium-sensitive K+ secretion, (2) different receptors mediate the colonic effects of glucocorticoids and mineralocorticoids, (3) immature rats are more sensitive to corticosteroids than adult animals, and (4) the acute effect of corticosteroids is an increase in Na+ absorption which is followed by delayed stimulation of K+ secretion.     

Natriuretic and diuretic action of centrally administered rat atrial natriuretic peptide (99-126): possible involvement of aldosterone and the sympathoadrenal system

Intracerebroventricular (ICV) administration of rat atrial natriuretic peptide (99-126) (rANP) to conscious male hydrated rats resulted in a dose-related increase in urinary volume and sodium excretion over a 6-h period of urine collection. A diminished mineralocorticoid effect on the kidneys may explain the natriuretic phenomenon. This hypothesis was tested by ICV rANP injection (1.25 microgram/5 microL) in conscious hydrated rats pretreated beforehand with d-aldosterone (20 micrograms/kg, ip). Although the absolute amount of sodium excreted was reduced, aldosterone did not affect rANP-induced sodium output at 1 and 3 h. Rats that were sham-operated or bilaterally adrenalectomized after 4 days were pretreated with aldosterone and given an oral water load followed by ICV rANP or saline. The possible participation of the peripheral sympathetic nervous system in the central action of rANP was evaluated in rats pretreated with 6-hydroxydopamine. In sympathectomized and adrenalectomized rats natriuresis and diuresis were still evident after rANP. Our results indicate that the natriuretic effect of ICV rANP is independent of mineralocorticoids. Likewise, diuresis and natriuresis can occur in the absence of the adrenal glands and are independent from the neural tone that the adrenergic system exerts on sodium reabsorption.     

Sex-dependence of the metabolism of aldosterone in adrenalectomized and intact rats

The metabolism of aldosterone at physiological levels was shown to be significantly different in male and female rats: more aldosterone was metabolized in the male rats leading to significantly higher quantities of non-extractable polar derivatives of aldosterone in all subcellular fractions of kidney, particularly in the cytosol fractions. These results may be correlated with our findings in which it was shown that the physiological responses to aldosterone in both adrenalectomized and intact rats were significantly greater in the males than the females. These sex differences support the concept that the metabolism of aidosterone may be essential for the components of the physiological response to aldosterone to occur. Furthermore, the sex-dependence of the metabolism of aldosterone appears to be independent of the presence of the previously identified protein receptor-hormone complexes. At all dosages within the physiological range, no significant differences were observed between the extent of the ³H-aldosterone labeling of these binding proteins in the kidney cytosol fractions of male and female adrenalectomized rats, even at dosages where no physiological response was demonstrable in the females.     

Acute glucocorticoid treatment increases urinary biotin excretion and serum biotin

Previous studies have demonstrated that glucocorticoids alter biotin metabolism. To extend these studies, the effect of dexamethasone on biotin pools was analyzed in rats consuming a purified diet containing a more physiological level of dietary biotin intake (0.06 mg/kg). Acute (5 h) dexamethasone administration (0.5 mg/kg) elicited elevated urinary glucose output as well as elevated urinary biotin excretion and serum biotin. Renal and hepatic free biotin was also significantly elevated by acute dexamethasone administration. Chow-fed rats treated with an acute administration of dexamethasone demonstrated significantly elevated urinary glucose excretion, urinary biotin excretion, and serum biotin, but no change in tissue associated biotin was detected. Chronic administration of dexamethasone (0.5 mg/kg ip) over 4 days significantly elevated urinary glucose excretion 42% but had no effect on urinary biotin excretion, serum biotin, or hepatic- or renal-associated free biotin. These results demonstrate the existence of potentially novel regulatory pathways for total biotin pools and the possibility that experimental models with high initial biotin status may mask potentially important regulatory mechanisms.     

The effects of infusions of ring-A-reduced derivatives of aldosterone on the antinatriuretic and kaliuretic actions of aldosterone

Infusion of Ring-A-reduced metabolites of aldosterone in adrenalectomized male rats for 4 days revealed that 5 alpha-Ring-A-reduced derivatives, 5 alpha-dihydroaldosterone (5 alpha-DHAldo; 2.5-5.0 micrograms/day), 3 alpha,5 alpha-tetrahydroaldosterone (3 alpha,5 alpha-THAldo; 5-25 micrograms/day), and 3 beta,5 alpha-THAldo (50-175 micrograms/day) possessed intrinsic Na+-retaining activity. The same infusions of 5 alpha-DHAldo, 3 alpha,5 alpha-THAldo, and 3 beta,5 alpha-THAldo, also lowered the urinary excretion of potassium. The 5 beta-Ring-A-reduced derivative 3 alpha,5 beta-THAldo did not demonstrate either of these biological properties. In another set of experiments, on the fourth day of infusion, aldosterone (0.1 microgram/rat) was administered acutely subcutaneously; none of the Ring-A-reduced derivatives altered the Na+-retaining activity of aldosterone. However, in a dose-dependent manner, both 3 alpha,5 alpha-THAldo and 3 beta,5 alpha-THAldo blunted the urinary K+-secretory effect of aldosterone; low dosages of 5 alpha-DHAldo and larger dosages of 3 alpha,5 beta-THAldo did not. Thus, the 5 alpha-reduced derivatives of aldosterone not only lowered urinary Na+ and K+ excretion in their own right, but two of them blunted the kaliuretic response of the parent mineralocorticoid, aldosterone. Further experiments will be required to determine whether these aldosterone metabolites are further metabolized or interconverted during the expression of the regulatory properties described here and whether these properties are physiologically relevant.     

High phosphorus diet changes phosphorus metabolism regardless of PTH action in rats

In this study, we ascertained whether the parathyroid hormone (PTH) dominantly regulated the effects of high phosphorus (P) intakes on urinary excretion of P and bone metabolism in rats. To maintain serum PTH level equally, parathyroidectomy (PTX) and sham-operated rats were constantly exposed to rPTH(1-34) and fed both control (0.3% P) and high P (1.2% P) diet for 7 days, respectively. Urinary excretions of P and C-terminal telopeptides of type I collagen were significantly increased in both PTX and sham rats by the high P diet. These results suggest that high P diet increased urinary P excretion while promoting bone resorption regardless of PTH-dependent regulation.     

Responses of plasma immunoreactive atrial natriuretic factor, aldosterone and urinary dopamine to salt-loading in a patient with severe idiopathic edema

A patient with severe idiopathic edema and long history of diuretic abuse had, in response to salt loading, an inability to increase urinary sodium excretion associated with a paradoxical response (decrease) of urinary dopamine excretion, a non suppressible aldosterone and non stimulable immunoreactive atrial natriuretic factor in plasma. These patterns distinguished this patient from those with a milder form of idiopathic edema who did not abuse diuretics and had, in comparison with controls, marginally decreased urinary sodium and dopamine responses but normal aldosterone suppressibility and ANF stimulability. Since the natriuretic action of ANF appears to be mediated by dopaminergic mechanisms, this severe natriuretic handicap may be due to a chronic diuretic abuse-induced combined ANF and dopamine deficiency.     

19-Norandrost-4-ene-3, 17-dione amplifies the action of aldosterone only in sodium-loaded conditions: Evidence for a new class of amplifiers of aldosterone

The amplification effect of 19-norandrost-4-ene-3, 17-dione (19-nor-A-dione) on aldosterone in normal and sodium-loaded conditions was evaluated using adrenalectomized rats fed a normal or high sodium diet. The administration of 19-nor-A-dione in normal or sodium-loaded conditions did not cause any significant change in urinary $\text{Na}\text{K}$ ratio. The simultaneous administration of subthreshold doses of aldosterone and 19-nor-A-dione in normal conditions also did not cause any significant change in urinary $\text{Na}\text{K}$ ratio. However, the simultaneous administration of subthreshold doses of aldosterone and 19-nor-A-dione in sodium-loaded conditions caused a significant decrease in urinary $\text{Na}\text{K}$ ratio. The decrease in urinary $\text{Na}\text{K}$ ratio was caused by a decrease in urinary Na excretion. These results demonstrate that 19-nor-A-dione, which did not amplify the action of aldosterone in normal conditions, amplified the action of subthreshold doses of aldosterone in sodium-loaded conditions. 19-Nor-A-dione is considered to be an amplifier of aldosterone which works only in sodium-loaded conditions.     

Electrolyte excretion in alcohol preferring and alcohol avoiding rats

Water and electrolyte metabolism was studied in alcohol preferring (AA) and alcohol avoiding (ANA) rats. During water diuresis AA rats had higher Mg, cAMP, creatinine and inorganic phosphate excretion, but lower urine and urinary protein output. During ethanol diuresis AA rats had lower Na, K, Ca, protein and urine output, but higher cAMP and inorganic phosphate excretion. Ethanol increased K, Ca and urine output in ANA rats only. A slight increase of blood pH was observed only in AA rats. Before ethanol ANA rats had higher plasma Ca concentration. Plasma aldosterone level was higher in AA rats. High salt excretion of ANA rats may lead them to prefer salt containing energy sources and therefore to avoid ethanol. On the other hand, renal salt conservation in AA rats may lead them to prefer ethanol.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Role of Some Hormones in Protein Sparing Action of Dietary Fat

Feeding rats with a fat meal caused marked reduction in the level of plasma urea and urinary output of urea and total nitrogen. Experiments were carried out to examine the possible intervention of some hormones in these phenomena. Protein sparing action of fat was exerted even in the alloxan-diabetic, adrenalectomized, hypophysectomized and thyroidectomized rats. Feeding rats with a fat meal caused no appreciable change in the level of cyclic AMP in liver and gastrocnemius muscle. The overall results obtained here are through! to suggest that the action of fat may be exerted independently of any hormones examined; insulin, glucocorticoids, cyclic AMP and other hormones excreted from adrenal, hypophysis and thyroid gland.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Influence of spironolactone on urinary prostaglandin E2 and kinin excretion in rats

Spironolactone was administered to spontaneously hypertensive rats (SHRs) in order to examine the urinary excretions of prostaglandin E2 (PGE2) and kinin. Thirteen SHRs were divided into 2 groups: 0.1 ml of sesame oil was administered to one group (the spironolactone-lactone-untreated group, n = 6) and 20 mg of spironolactone in 0.1 ml of sesame oil was administered to the other group (the spironolactone-treated group, n = 7) by the subcutaneous route for 10 days in succession. Determinations were then made of the body weight, blood pressure, urine volume, and excretion levels of Na, K, kinin and PGE2 in the 24-hour urine. After the animals had been killed by decapitation, blood samples were drawn for determination of the plasma renin activity (PRA). The results obtained indicated decreased blood pressure and increased urinary Na excretion in the spironolactone-treated group. On the other hand, the PGE2 excretion level in the 24-hour urine decreased markedly immediately after administration of spironolactone (p less than 0.05) and was maintained at lower levels up to the end of the experiment. However, the 24-hour urinary kinin levels showed similar changes in both the spironolactone-treated group and the untreated group with no significant difference between them. These findings suggest that spironolactone has a suppressive effect on urinary PGE2 excretion, the activity of which is not mediated by kinin production in the kidneys but is the result of a direct action of spironolactone itself.     

Effects of adrenalectomy and aldosterone on the action of centrally administered rat atrial natriuretic peptide-(99-126)

Intracerebroventricular (i.v.t.) administration of rat atrial natriuretic peptide-(99-126) (rANP) increases urinary volume and sodium excretion, but the mechanism is undefined. A diminished mineralocorticoid effect on the kidneys may explain the natriuretic phenomenon. This hypothesis was tested by i.v.t. rANP injection (1.25 micrograms/5 microliters) in conscious, hydrated rats pretreated beforehand with d-aldosterone (20 micrograms/kg, i.p.). Although the absolute amount of sodium excreted was reduced, aldosterone did not affect rANP-induced sodium output at 1 and 3 h. Rats which were sham-operated or bilaterally adrenalectomized (ADX) after four days were pretreated with aldosterone and given an oral water load followed by i.v.t. rANP or saline. In ADX rats natriuresis and diuresis after rANP were still evident. Our results indicate that the natriuretic effect of i.v.t. rANP is unrelated to plasma levels of mineralocorticoids. Likewise, diuresis and natriuresis can occur in the absence of the adrenal glands.     

Effects of corticosterone on muscle mitochondria identifying different sensitivity to glucocorticoids in Lewis and Fischer rats

Previous studies in rat have demonstrated decreased number of mitochondria and uncoupling of oxidative phosphorylation after administration of glucocorticoids but at supraphysiological doses and using synthetic glucocorticoids. To analyze the relationships between corticosterone levels (the natural glucocorticoid in rat) and muscle mitochondrial metabolism, Lewis and Fischer 344 rats were bilaterally adrenalectomized and implanted with different corticosterone pellets (0, 12, 50, 100, and 200 mg of corticosterone). Rats bearing a corticosterone pellet delivering corticosterone at concentrations in the range of chronic stress-induced levels presented a lower amount of functional muscle mitochondria with a decrease in cytochrome c oxidase and citrate synthase activities and a depletion of mitochondrial DNA. Moreover, a strain difference in tissue sensitivity to corticosterone was depicted both in end-organ sensitive to glucocorticoids (body, thymus, and adrenal weights) and in muscle mitochondrial metabolism (Lewis > Fischer). Interestingly, this strain difference was also observed in the absence of corticosterone, with a deleterious effect on muscle mitochondrial metabolism in Fischer rats, whereas no effects were observed in Lewis rats. We therefore postulate that corticosterone is necessary for muscle mitochondrial metabolism exerting its effects in Fischer rats with an inverted U curve, whereby too little (only Fischer) or too much (Fischer and Lewis) corticosterone is deleterious to muscle mitochondrial metabolism. In conclusion, we propose a general model of coordinate regulation of mitochondrial energetic metabolism by glucocorticoids.     

Effect of the exercise-induced increase in glucocorticoids on endurance in the rat

To investigate the effect of the increase in glucocorticoids during exercise on endurance, rats were either sham operated (SO) or adrenalectomized. All adrenalectomized rats were given a subcutaneously implanted corticosterone pellet at the time of adrenalectomy. Adrenalectomized rats were injected with corticosterone (ADX Cort) or corn oil (ADX) 5 min before exercise. Rats were killed at rest or after running on a treadmill (21 m/min, 15% grade) until exhaustion. SO rats ran 138 +/- 6 min compared with 114 +/- 9 min for ADX Cort and 89 +/- 8 min for ADX. All differences in run times were significant (P less than 0.05). Corticosterone levels were similar in exhausted SO and ADX Cort groups. ADX exhausted rats had corticosterone levels similar to resting values in SO and ADX rats. Inhibition of the rise in glucocorticoids during exercise had no effect on liver glycogen, liver adenosine 3',5'-cyclic monophosphate, plasma insulin, blood glucose, lactate, glycerol, or 3-hydroxybutyrate, plasma norepinephrine, or red quadriceps and soleus glycogen. Plasma free fatty acids were significantly depressed at exhaustion in ADX rats compared with SO. These data show that glucocorticoids exert effects within the time frame of a prolonged exercise bout and play a role in increasing endurance.     

Role of glucocorticoids and catecholamines on hepatic thiobarbituric acid reactants in basal and stress conditions in the rat.

Thiobarbituric acid-reactants (TBARs) are considered to be an index of lipid peroxidation. In the present experiments, the effect of stress and hormones on hepatic TBARs levels was studied in Sprague-Dawley rats. In unstressed conditions adrenalectomized rats showed higher TBARs levels than sham-adrenalectomized rats. The effect of adrenalectomy was reverted by the administration of corticosterone but not by that of aldosterone, indicating that glucocorticoids exert a negative role on the regulation of liver TBARs. The effect of these hormones appears to be a permissive one, since the administration of a long lasting ACTH preparation did not reduce liver TBARs. In contrast to that observed in unstressed rats, glucocorticoids appeared to increase liver TBARs in stressed rats. Nevertheless, other alternative explanations are possible. Finally, no evidence for a role of catecholamines in the regulation of hepatic TBARs was found.