Samuel Gallant

The in vivo effect of indomethacin and prostaglandin E2 on acth and Dbcamp-induced steroidogenesis in hypophysectomized rats

Abstract The level of plasma corticosterone attained in hypophysectomized rats stimulated with ACTH was significantly reduced by pretreatment with indomethacin, an inhibitor of prostaglandin synthesis. This effect was not seen in animals stimulated with dibutyryl cyclic AMP. Intraperitoneal injection of prostaglandin E2 to indomethacin treated rats restored the normal response to ACTH stimulation. However, PGE2 itself did not have any significant effect on plasma corticosterone levels. These findings suggest that prostaglandins are involved, perhaps in an allosteric fashion, in the mechanism of action of ACTH.

Serum 11-deoxycorticosterone levels in adrenal-regeneration hypertension under conditions of quiescence and stress

A time course study to measure adrenal cortical function was undertaken for the period prior to the development of hypertension until the onset of hypertension in the adrenal-regeneration hypertension (ARH) model. Quiescent rat kills were used so that all adrenal cortical parameters investigated would reflect basal or resting levels for controls. Thus a more accurate determination of the differences between control and experimental animals could be made. A radioimmunoassay procedure for deoxycorticosterone was developed to measure this steroid in individual rat serum samples. Elevated serum deoxycorticosterone levels were observed in rats with regenerating adrenals when they were killed under quiescent conditions. This agreed with our recently reported in vitro finding of restoration of cholesterol side chain cleavage activity while 11beta-hydroxylase activity remained imparied 25 days after adrenal enucleation. When rats were killed after ether stress, deoxycorticosterone levels were elevated in both control rats and in rats with regenerating adrenals but the difference was not significant. In contrast, after ether stress serum corticosterone levels were lower in rats with regenerating adrenals than in controls. These studies, in conjunction with our previous in vitro findings, point to the importance of deoxycorticosterone in the pathogenesis of adrenal regeneration hypertension and help to explain the anomalous corticosteroid secretion rate data found in this experimental hypertension model.

The Inhibition of Rat Adrenal Cytochrome P-45011β Gene Expression by Androgens

The synthetic androgen methylandrostenediol (MAD) and the naturally occurring one, testosterone, both bring about hypertensive cardiovascular disease when chronically administered to rats. The pathogenesis of this form of experimental hypertension is thought to result from inhibition of steroid 11β-hydroxylase activity. In contrast to the above androgens, 19–nortestosterone, androstenedione and dehydroepiandrosterone (DHEA) have been reported to be without effect in elevating blood pressure. To examine the mechanism(s) involved, we have in this study compared the effects of a number of androgens on adrenal cytochrome P- 45011β enzyme and mRNA steady state levels. These parameters were also correlated with the ability of adrenal mitochondria isolated from these groups to hydroxylate 11–deoxycorticosterone (DOC) to corticosterone and 18–hydroxy-11–deoxycorticosterone (18–hydroxy-DOC). Rats treated for seven days with 10 mg per day of dihydrotestosterone, testosterone, 19–nortestosterone or MAD showed a prof...

Serum levels of corticosterone and 18-hydroxy-11-deoxycorticosterone in the female rat at the high and low points of the circadian rhythm

Serum corticosterone (B) and 18-hydroxy-11-deoxycorticosterone (18-hydroxy-DOC) levels were determined in female rats at the high (1800 h) and low (0600 h) points of the circadian rhythm. In order to carry out these studies, a rapid and accurate non-chromatographic radioimmunoassay method was developed for the measurement of 18-hydroxy-DOC in peripheral blood and similar methodology was used for the B assay. In quiescent rats both steroids were dramatically elevated at 1800 h as compared to 0600 h. The serum levels of B and 18-hydroxy-DOC determined at 1800 h fifteen minutes following stress did not differ significantly from the levels determined following a similar stress at 0600 h. There was a good correlation (r = 0.91) between the levels of B and 18-hydroxy-DOC and it appears that both steroids are regulated by ACTH.

Serum corticosteroids at the high and low points of the circadian rhythm in rats with regenerating adrenals

Abstract Serum levels of 11-deoxycorticosterone (DOC), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) and corticosterone (B) were determined at the high (1800 h) and low (0800 h) points of the circadian rhythm in control rats and in rats with regenerating adrenals. The levels of DOC at 0800 h in quiescent rats with regenerating adrenals were 6.5 times greater than in the control group. The levels of 18-OH-DOC and B, however, were not significantly different between these groups. A circadian rhythm for B, 18-OH-DOC and DOC was evident in control rats with a 12,20 and 3.5 fold increase, respectively, at 1800 h as compared to 0800 h. In animals with regenerating adrenals there was only a minimal change in the levels of B and 18-OH-DOC at 1800 h. There was, however, a 2 fold further increase in the levels of DOC at 1800 h as compared with the elevated levels at 0800 h. These findings show that the decrease in 11β and 18-hydroxylase activity of the regenerating adrenal is most clearly evident at the high point of the circadian rhythm. Furthermore, only by taking into account physiological variations in adrenal activity can an accurate assessment of DOC secretion in the adrenal regeneration model of hypertension be obtained.

Resistance of W/Fu rats to adrenal regeneration hypertension.

Summary The procedure for producing adrenal regeneration hypertension did not cause an increase in the systolic blood pressure of W/Fu animals. The regenerating adrenal gland in W/Fu animals was not restored to normal; reduced numbers of mitochondrial cristae were seen and the mitochondria were smaller in size; regeneration was complete in Sprague-Dawley rats of the Holtzman strain and there was a severe form of hypertensive, cardiovascular disease. The authors are grateful to Mrs. Yam Pun, Mrs. Neonile Fylypiw, Mr. Luther Joseph, Mrs. Geneva Joseph, Mrs. Elisabeth Lawson, and Mr. Robert Linsmair for skilled technical assistance.

Sex differences in cytochromes oxidase and P-45011β in the rat adrenal cortex

Abstract A comparative study of cytochrome c oxidase (COX) activity and expression as well as cytochrome P -450 11β expression has been carried out on the adrenal cortex of male and female rats. COX has also been examined in rat liver. In addition, the effect of testosterone replacement in orchiectomized male rats on adrenal COX has also been investigated. Adult male rats had higher COX activity in adrenal (255%) and liver (144%) mitochondria compared to adult female rats. Male rat adrenals and liver also had increased levels of COX II, a mitochondria-encoded COX subunit, and of COX IV, a nucleus-encoded COX subunit, as measured by Western analysis. In contrast, cytochrome P -450 11β levels were lower (48%) in adrenal mitochondria from male rats than those of female rats. There was no significant sex difference in the level COX II and COX IV mRNAs in adrenal or liver, whereas the cytochrome P -450 11β mRNA was 4-fold higher in female adrenals than in males. In male rats, orchiectomy caused a 23% decrease and testosterone replacement a 66% increase in adrenal COX activity. There were no corresponding changes in the levels of mRNAs encoding for COX subunits, suggesting post-transcriptional effects of testosterone on COX. These results are consistent with a regulatory role of testosterone on the expression of components of the respiratory and steroidogenic electron transport chains.

EFFECT OF ACTH ON CYTOCHROME P450 SYSTEMS OF THE ADRENAL CORTEX

The ability to separate the 1 1 P-hydroxylase and cholesterol side chain cleavage forms of cytochrome P450 from adrenal cortical mitochondria,l led to the development of techniques for the measurement of cholesterol association with the cytochrome P450,,.,..2-4 Furthermore, it was shown that when rats were stressed or treated with ACTH there was an increased association of cholesterol with cytochrome P450,,,,, and this was correlated with an increased rate of cholesterol side chain cleavage to yield pregnenolone from endogenous cholesterol.:<-“ The increased formation of the cholesterol-cytochrome P450,,.,. complex in adrenals from rats treated with ACTH was prevented if the rats were pretreated with an inhibitor of protein synthesis, cycloheximide.3, The above observations led to the hypothesis that the “labile factor” of Garren et al.7 has a role to play in increasing the association of cholesterol with cytochrome P450,,.,. and that the latter is an important part of the action of ACTH on corticosteroidogenesis in the adrenal. The experiments described in this paper were designed to extend these observations to include a recent finding8 that cholesterol binding to cytochrome P450,,.,. in intact rat adrenal mitochondria was temperature-dependent. In addition, preliminary studies are described of the control of corticosteroidogenesis in a cortisol secretor, the guinea pig.

Cyclic nucleotide phosphodiesterase activity in rat adrenal gland zones.

Abstract The distribution of cyclic 3′, 5′ -nucleotide phosphodiesterase activity in the rat adrenal gland has been studied. Phosphodiesterase activity was 10-fold higher in the zona glomerulosa than in the zona fasciculata-reticularis. Kinetic studies carried out at low substrate concentrations suggest the possible presence of multiple forms of phosphodiesterase activity in both zones of the adrenal; however, these forms appear to have similar apparent Kms for cAMP. Thus, the well known differences in the steroidogenic response of the two zones to ACTH stimulation may be partially explained by large differences in total activities of the various forms of phosphodiesterase.

Cytochrome c oxidase in rat adrenal and liver: effects of anti-androgen treatment and studies in testicular feminized rats.

The role of androgen receptors in androgen-induced changes in rat adrenocortical and liver cytochrome c oxidase (COX) has been investigated. The anti-androgen flutamide, blunted the increase in COX activity and COX subunits II/III and IV, that is seen with androgen treatment. Testicular feminized (Tfm) rats had levels of COX activity and COX subunits II/II and IV in adrenal cortex and liver that were intermediate between the high levels found in normal male rats and the lower levels of normal female rats. These data suggest that androgen effects on adrenal and liver COX are mediated through interactions with androgen receptors known to be present in these issues. However, the observed changes in COX activity and COX subunits were not accompanied by altered levels of mRNAs encoding for COX II or COX IV.