Norbormide enhances late steps of steroid-hormone synthesis in rat and mouse adrenal cortex.

Norbormide (N) is a vasoconstrictor agent, which acts selectively on the peripheral arteries of the rat, through the activation of the phospholipase C (PLC) cascade and the stimulation of Ca(2+) entrance in the vascular myocytes. Several endogenous vasoconstrictor agent (e.g. angiotensin-II (ANG-II) and endothelin-1 (ET-1)), that stimulate PLC pathway, are also able to enhance aldosterone secretion by the adrenal gland. Hence, we examined the effects of norbormide ((0.5, 1.0 or 5) x 10(-5)M) on corticosteroid-hormone secretion from adrenal slices of rats and mice. Quantitative HPLC assay showed that under basal conditions rat and mouse adrenal quarters secreted progesterone (PROG), 11-deoxycorticosterone (DOC), 18-hydroxy-DOC (18OH-DOC), corticosterone (CORT), 18-hydroxy-corticosterone (18OH-CORT) and aldosterone (ALDO), as well as large amounts of pregnenolone (PREG) when its metabolism was blocked by 10(-5)M cyanoketone. Norbormide concentration-dependently raised the secretion of all post-DOC steroids assayed, decreased progesterone and DOC production, and did not affect pregnenolone release. In conclusion, norbormide is able to enhance late steps of steroid synthesis, i.e. those leading to the transformation of DOC to corticosterone and aldosterone, without affecting early steps. This is an interesting finding because the other main endogenous adrenal secretagogues are known to stimulate both early and late steps of steroid synthesis. The mechanism underlying the selective activating action of norbormide on 11beta- and 18-hydroxylation remains to be investigated.

Dose-dependence of the effects of corticosterone and the non-glucocorticoid, 18-hydroxycorticosterone, on the brush-border Na+/K+ exchanger.

Up to now, only glucocorticoids were thought to act on the renal proximal Na+/H+ exchanger. Using fluorimetric techniques we studied the kinetics of Na+/H+ exchange in brush border vesicles from ADX rats treated with increasing doses of corticosterone (B) and 18-hydroxycorticosterone (18OHB). Significant linear correlations were obtained when the Vmax of each treatment were plotted against log doses. 18OHB exhibits a slightly higher sensitivity than B and log-dose responses were steeper for 18OHB than for B treated rats. Differences between both treatments were highly significant at the 4.8 microg/100 g level, corresponding to the physiological blood level of 18OHB. Physiological doses of both steroids elicited equal Na+/H+ exchange-responses. 18OHB is not a glucocorticoid since even 88 microg/100 g did not promote hepatic glycogen deposition while the same dose of B increases glycogen deposits 3.5-fold. These results demonstrate the importance of the Na+/H+ exchanger as a mediator between corticoid action and H+ transport and that of the non-glucocorticoid 18OHB in this process.

Acute effects of two natural corticosteroids on the kinetics of the Na+/H+ exchanger in rat renal brush-border membranes.

We studied the effect of adrenalectomy and acute hormone replacement, using physiological doses of natural corticosteroids, on the kinetics of the Na+/H+ exchanger in brush border membrane vesicles. We collected the data using the acridine orange uptake technique. Adrenalectomized (ADX) rats presented a decreased maximal rate (Vmax) when compared with sham-operated animals (30,000 versus 41,000 fluorescent units/min, respectively). Administration of corticosterone (B) to ADX rats restored Vmax to values above control (up to 66,000 fluorescent units/min). Smaller doses of 18-OH-B led to similar results. K(m) (16 mM) remained the same for all the groups. Amiloride behaved as a pure competitive inhibitor, with a Ki = 0.02 mM and an I50 = 98 microM (in the presence of 50 mM sodium gluconate). The presence of sodium in the external buffer, before adding the vesicles, inhibited the exchange, with an I50 = 2 mM. We observed, a significant decrease in the Na+/H+ exchanger under non-acidotic conditions in response to adrenalectomy. Acute administration of physiological doses of natural occurring corticosteroids reversed the effect.

The hypertensinogenic activity of 18,19-dihydroxycorticosterone in adrenalectomized rats.

Adrenalectomized, spontaneously hypertensive rats (SHR) were used to test the hypertensinogenic property of recently synthesized 18,19-dihydroxycorticosterone (18,19-Di-OH-B). The steroid was given via Alzet miniosmotic pump, with and without aldosterone. Neither 18,19-Di-OH-B (5 micrograms) nor aldosterone (5 micrograms) increased blood pressure in SHR when given alone; when administered together they resulted in a significant rise in blood pressure. Results indicate a high probability that certain mineralocorticoids, which are inactive by themselves, might play a role in the etiology of hypertension when acting together under physiological conditions.

Role of corticosteroids in distal acidification of amiloride-treated rats.

The role of amiloride-dependent sodium channels in the action of adrenal cortical steroids on urine-blood PCO2 (U-B PCO2) differences was studied in bicarbonate-infused and amiloride-treated adrenalectomized rats. U-B PCO2 was significantly reduced by amiloride in bicarbonate-infused control rats. Adrenalectomy further reduced U-B PCO2 in amiloride-treated, bicarbonate-infused rats (from 27.9 +/- 1.82 mmHg in sham-operated rats to 21.3 +/- 1.58 mmHg in adrenalectomized (ADX) rats) (1 mmHg = 133.322 Pa). Acute administration of corticosterone and 18-hydroxycorticosterone (18-OH-B), but not of aldosterone, caused recovery of U-B PCO2 to the level of sham-operated animals treated with amiloride. Aldosterone did not affect U-B PCO2 in the presence of amiloride (21.9 mmHg ADX group vs. 20.98 mmHg aldosterone group). Results are compatible with aldosterone affecting distal H ion secretion mostly by a sodium and potential difference dependent mechanism, while corticosterone and 18-OH-B should act by other mechanisms (e.g., increased luminal buffer level).

Dantrolene inhibits adrenal steroidogenesis by a mechanism independent of effects on stored calcium release.

The muscle relaxant dantrolene has been widely used in signal transduction studies as an inhibitor of intracellular calcium release. However, in vivo studies have shown that the drug may inhibit steroidogenesis by a mechanism which is distinct from its effects on calcium mobilization. Using freshly isolated cells and mitochondria from the outermost regions of bovine adrenal cortex we have shown that dantrolene (0.2 mM) significantly inhibits steroid synthesis stimulated by either angiotensin II (AII) or by addition of various precursors. Our results suggest that dantrolene inhibits the rate-limiting steps of adrenocortical steroidogenesis, i.e. the intramitochondrial conversion of cholesterol to pregnenolone (for both aldosterone and cortisol) and the conversion of corticosterone to aldosterone (for aldosterone), by a mechanism independent from its known effects on calcium release. A possible alternative mechanism may involve direct inhibition of cytochrome P450-dependent hydroxylation reactions.

Amplification of mineralocorticoid activity of aldosterone by 18-hydroxy-corticosterone and 18-hydroxy-19-nor-corticosterone in adrenalectomized rats.

A combination of aldosterone (1 micrograms) with either 18-OH-corticosterone (1 micrograms) or 18-OH-19-norcorticosterone (1 micrograms) injected to adrenalectomized rats indicated an amplification of mineralocorticoid activity as expressed by Na/K ratio in urine. Without aldosterone their mineralocorticoid potency was negligible.

Early and late effects of steroid hormones on the central nervous system.

Steroids have fast and probably partly GABA-mediated central anaesthetic effects for which a strict structure-function correlation is required. They also affect short- and long-term activity in the CNS in other ways. One of these is long-term potentiation (the persistent facilitation of synaptic transmission), which occurs particularly in the hippocampus after repetitive stimulation of a fibre pathway. Two clearly distinguished components of the evoked response can be studied in the hippocampus: the excitatory postsynaptic potential (EPSP) which denotes the graded depolarization of the somadendritic region of the neuron and the population spike (PS), a manifestation of the all-or-none discharge of the cell action potential. Corticosterone had a significant depressant effect on the EPSP component of the evoked response immediately and 15 min after injection. Thereafter EPSP amplitudes were within normal values. Corticosterone significantly decreased the PS immediately after the train, the component remaining low 30 min after the train. 5 alpha-Dihydrocorticosterone (a ring A-reduced metabolite of corticosterone) significantly reduced the PS component of the response at all times after injection. 18-Hydroxydeoxycorticosterone and deoxycorticosterone significantly decreased both EPSP and PS components of the evoked response from the time of infusion. Contrary to expectation, tetrahydrodeoxycorticosterone was ineffective in decreasing, and if anything, enhanced the development of long-term potentiation. 18-Hydroxydeoxycorticosterone 21-acetate behaved like vehicle, except for the first 30 min after injection, when the EPSP was decreased. Different steroids can selectively affect different parts of a neuron and appear to show a different structure-function correlation for long-term potentiation from that required for anaesthesia.

Effects of adrenalectomy and acute replacement by corticosteroids on distal acidification.

The role of adrenocortical steroids in distal nephron acidification was studied in rats by measuring urine minus blood PCO2 differences (U-B PCO2) in control, sham-operated, and adrenalectomized (ADX) animals. Operations were performed 48 h before experiments. During the experiments, all rats received an infusion of 0.35-0.60 M NaHCO3, leading to urine bicarbonate concentrations in the order of 100-200 mM. Adrenalectomized rats had significantly decreased U-B PCO2 (11.9 +/- 1.99 mmHg; 1 mmHg = 133.3 Pa) with respect to sham-operated rats (39.9 +/- 1.26 mmHg). In another series, ADX rats received supplements of the adrenal steroids corticosterone, aldosterone, and 18-hydroxycorticosterone 100 min before the experiment. U-B PCO2 increased after hormone administration: corticosterone, 30.0 +/- 2.13 mmHg; aldosterone, 26.6 +/- 1.74 mmHg; 18-hydroxycorticosterone, 29.0 +/- 1.60 mmHg; but none restored these values to normal. Combinations of two hormones were also used; only aldosterone + corticosterone restored U-B PCO2 to normal: 39.0 +/- 1.66 mmHg. Renal phosphate excretion (but not urine phosphate levels) decreased significantly in ADX as compared with sham-operated rats. Extracellular volume was not significantly affected in ADX rats, which received ad libitum 0.9% NaCl for drinking. It is concluded that distal tubular acidification, as evaluated by U-B PCO2, is dependent on cortical steroids.

Lack of increase in sodium concentration of cerebrospinal fluid in DOCA-salt hypertensive rats.

The purpose of the present study is to determine whether DOCA-salt treatment could cause an elevation of Na concentration of cerebrospinal fluid (CSF), which may increase blood pressure (BP) as a result of enhanced activity of sympathetic nervous system (SNS) and vasopressin (AVP). Blood pressure was gradually increased by DOCA-salt treatment. Serum NA was also elevated with time by DOCA-salt, and significantly higher in DOCA rats on the 4th treatment week, as compared with controls. By contrast, DOCA-salt did not alter CSF Na at any time of treatment. No relationship was detected between BP and CSF Na at any stage of DOCA-salt hypertension. The precent fall in BP by AVP antagonist or hexamethonium was always greater in DOCA-treated rats than that in the controls. These hypotensive effects were gradually, but consistently enhanced with the development of hypertension in DOCA rats. It is concluded that the enhanced activity of both SNS and AVP system responsible for DOCA-salt hypertension may be attributed to the mechanism(s) unrelated to the increased CSF Na concentration.

[Receptor binding of corticosterone in brain structures participating in regulation of the hypothalamo-hypophyseo-adrenal system]. / Retseptornoe sviazyvanie kortikosterona v strukturakh mozga, prinimaiushchikh uchastie v reguliatsii gipotalamo-gipofizarno-nadpochechnikovoi sistemy.

Binding of corticosterone by the cytosol receptors after hydrocortisone administration was studied in different tissues of rats. A decrease in the specific binding of corticosterone occurred within 24 hrs in all the structures under study. In 3 days the decrease only persisted in the hypophysis and hypothalamus. The data suggest a connection between the long lasting decrease in the cytosol reception of corticosterone in the hypothalamus and hypophysis and the blockade of the hypothalamo-hypophyseal-adrenal system.

Effects of 18-hydroxycorticosterone and of aldosterone on acid-base parameters in the arterial blood of adrenalectomized rats.

Adrenalectomized rats were injected twice with either 2 micrograms aldosterone or 6 micrograms 18-hydroxycorticosterone (18-OH-B) and were then kept either under normal, or high-CO2 respiratory conditions. Arterial blood samples were withdrawn sequentially from T1 (i.e., 70 minutes after the first injection) on, and were then submitted to determinations of pH and PCO2. Bicarbonate levels were calculated from these data. 18-OH-B: 1) increased pH under both conditions; 2) had a tendency to decrease PCO2 in both conditions; the decrease was significant at 130 minutes after injection, under normal conditions; 3) increased CO3H levels at T1 under high-CO2 atmospheres. Aldosterone did not produce changes in pH values, even if injected in doses equimolar to those of 18-OH-B, but showed a tendency--at these higher doses--to decrease PCO2 values.

Evaluation of the mineralocorticoid activity of 18-hydroxycorticosterone.

1. The mineralocorticoid activity of 18-hydroxycorticosterone (18-OH-B) has been compared with that of aldosterone by using human bioassay in vivo with measurement of rectal potential difference and urinary log 10 Na+/K+ ratio. 2. A log-linear relationship was found between maximum change in rectal potential difference and increasing doses of aldosterone. 3. No mineralocorticoid activity could be demonstrated after an intravenous bolus and infusion of 18-OH-B. 4. The half-life of clearance of 18-OH-B was measured in three subjects and found to be 28, 48 and 24 min.

Peripheral serum corticosteroid concentrations in relation to the rat adrenal cortical circadian rhythm in androgen-induced hypertension.

Adrenal function was assessed in control rats and in rats treated for 2 and 4 weeks with 17 alpha-methylandrostenediol (MAD; 17 alpha-methyl-5-androstene-3 beta-diol), a synthetic androgen known to produce hypertensive cardiovascular disease. In both groups and at both time periods, a circadian rhythm of blood corticosteroid concentrations was observed. The high point for serum corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18-hydroxy-DOC), and 11-deoxycorticosterone (DOC) concentrations occurred at the beginning of the dark period (1800 hours), and the low point occurred at the onset of the light period (0600 hours). Serum concentrations of DOC were always found to be higher in MAD-treated rats as compared with controls. The serum concentrations of B and 18-hydroxy-DOC were lower than control values at 1800 hours but higher than control concentrations at 0600 hours. The in vitro 11 beta- and 18-hydroxylation of DOC was markedly reduced with MAD treatment. In contrast, cholesterol side-chain cleavage activity was higher in animals treated with MAD. These in vitro findings correlated with spectral studies that showed a decreased binding of DOC to cytochrome P450(11) beta and increased binding of cholesterol to cytochrome P450scc. These studies suggest that MAD treatment selectively decreases 11 beta- and 18-hydroxylation in adrenal mitochondria, and this results in an increased serum concentration of DOC, a hypertensinogenic steroid. This effect of MAD on peripheral serum DOC concentration is most readily observed in quiescent animals at the high point of the circadian rhythm.

Acute effects of three natural corticosteroids on the acid-base and electrolyte composition of urine in adrenalectomized rats.

In acute experiments, aldosterone (aldo), 18-hydroxycorticosterone (18 OH B) or corticosterone (B) were administered to adrenalectomized rats and parameters related to acid-base balance measured in urine samples collected for 3.5 hours after injection. Aldo reduced sodium excretion but did not affect pH nor the outputs of K, NH4+, CO3H-, phosphates nor titratable acidity. 18 OH B increased the excretion of titratable acidity and reduced drastically that of CO3H-. The lowest effective dose (3 micrograms) promoted Na excretion while the highest dose employed (6 micrograms) reduced pH and Na excretion. B increased the excretions of phosphates and ammonium, the former drastically. Potassium output either increased or did not change, and pH augmented marginally. It is postulated that a) 18 OH B is a naturally occurring steroid eliciting urine-acidification not necessarily accompanied by sodium retention; and b) at least B and 18 OH B in the rat, possess hormonal roles according to which the latter promotes the presence of protons, and the former, that of acute proton-acceptors in the lumen of tubuli.