Incubation Of Adrenal Slices Research Articles

The β-Hydroxybutyrate-GPR109A Receptor Regulates Fasting-induced Plasticity in the Mouse Adrenal Medulla.

During fasting, increased sympathoadrenal activity leads to epinephrine release and multiple forms of plasticity within the adrenal medulla including an increase in the strength of the preganglionic → chromaffin cell synapse and elevated levels of agouti-related peptide (AgRP), a peptidergic cotransmitter in chromaffin cells. Although these changes contribute to the sympathetic response, how fasting evokes this plasticity is not known. Here we report these effects involve activation of GPR109A (HCAR2). The endogenous agonist of this G protein-coupled receptor is β-hydroxybutyrate, a ketone body whose levels rise during fasting. In wild-type animals, 24-hour fasting increased AgRP-ir in adrenal chromaffin cells but this effect was absent in GPR109A knockout mice. GPR109A agonists increased AgRP-ir in isolated chromaffin cells through a GPR109A- and pertussis toxin-sensitive pathway. Incubation of adrenal slices in nicotinic acid, a GPR109A agonist, mimicked the fasting-induced increase in the strength of the preganglionic → chromaffin cell synapse. Finally, reverse transcription polymerase chain reaction experiments confirmed the mouse adrenal medulla contains GPR109A messenger RNA. These results are consistent with the activation of a GPR109A signaling pathway located within the adrenal gland. Because fasting evokes epinephrine release, which stimulates lipolysis and the production of β-hydroxybutyrate, our results indicate that chromaffin cells are components of an autonomic-adipose-hepatic feedback circuit. Coupling a change in adrenal physiology to a metabolite whose levels rise during fasting is presumably an efficient way to coordinate the homeostatic response to food deprivation.

Ketoconazole blocks cortisol secretion in man by inhibition of adrenal 11β-hydroxylase

We investigated basal and ACTH stimulated levels of cortisol, corticosterone, 17 alpha-hydroxyprogesterone, 11-deoxycortisol and 11-deoxycorticosterone as well as plasma levels of ACTH before and during the oral administration of ketoconazole in five patients with Cushing's syndrome (3 with bilateral adrenal hyperplasia, 1 with adrenal adenoma and 1 with adrenal carcinoma) and in three controls. The influence of ketoconazole on the transformation of 3H-17 alpha-hydroxyprogesterone to 3H-11-deoxycortisol and 3H-cortisol and of 3H-11-deoxycortisol to 3H-cortisol as well as of 3H-11-deoxycorticosterone to 3H-corticosterone was also examined in slices or homogenates of normal and hyperplastic adrenal tissue from four patients. Ketoconazole induced a rise of 11-deoxycortisol and 11-deoxycorticosterone, but not of cortisol and inconsistently of corticosterone which were increased by ACTH. Thus the ratio 11-deoxycortisol/cortisol rose more after ketoconazole than after ACTH and the ratio 11-deoxycorticosterone/corticosterone rose after ketoconazole but fell after ACTH. Plasma ACTH levels were stimulated 2-50 fold by ketoconazole. Incubation studies of adrenal tissue slices with 3H-17 alpha-hydroxyprogesterone showed that ketoconazole inhibited the transformation of 3H-17 alpha-hydroxyprogesterone to 3H-cortisol but not to 3H-11-deoxycortisol so that the ratio 3H-11-deoxycortisol/3H-cortisol increased 15-80 fold. After incubation of adrenal slices with 3H-11-deoxycortisol or 3H-11-deoxycorticosterone and ketoconazole, a 2-260 fold increase of the ratios 3H-11-deoxycortisol/3H-cortisol and 3H-11-deoxycorticosterone/3H-corticosterone were also found.

Regulation of Tyrosine Hydroxylase Activity By Prostaglandin E1 in Guinea Pig Adrenal Gland

The action of prostaglandin E1 on tyrosine hydroxylase activity in adrenal slices of guinea pig was studied. The activity of tyrosine hydroxylase was decreased by the incubation of adrenal slices with prostaglandin E1 at concentrations beyond 2 µg per ml for 2 hours. The activity of tyrosine hydroxylase was stimulated by dibutyryl adenosine 3',5'-monophosphate in slices of guinea pig adrenal glands. Incubation of adrenal slices with the combination of PGE1 and DBc-AMP led to a tyrosine hydroxylase activity higher than that with PGE1 alone, but not as great as DBc-AMP alone. It was suggested that PGE1 inhibited the enzyme activity independently of the cyclic AMP level. Other prostaglandins such as PGA1 and PGB1 were deficient to the extent that the tyrosine hydroxylase activity was decreased. PGE1 inhibited the enzyme activity much to the same extent seen with protein synthesis inhibitors such as cyclo- heximide and actinomycin D. However, PGE1 did not influence the incorporation of L-leucine-14C into acid insoluble protein. The studies reported here showed that PGE1 inhibited the synthesis of tyrosine hydroxylase.

Adrenocortical function in foetal, neonatal and young pigs.

SUMMARY Absolute and relative adrenal weight, blood plasma concentrations of 17-hydroxycorticosteroids and their production in vitro on incubation of adrenal slices with corticotrophin (ACTH), were measured in foetal pigs, piglets and older pigs. These criteria all indicated that adrenocortical activity was highest on the day of birth. It developed intensely in about the last 10 days of intra-uterine life. After birth it decreased progressively with age, approaching the adult level after weaning in the second month of life. The adrenal cortex of pigs of all developmental stages investigated in this study was capable of responding markedly to stimulation with ACTH. No significant differences in adrenocortical activity were found between sexes up to 90 days of age.

Effect of 1-( o-chlorophenyl)-1-( p-chlorophenyl)-2,2-dichloroethane on adrenocorticotropic hormone-induced steroidogenesis in various preparations in vitro of dog adrenal cortex

Studies in the intact dog revealed that the intravenous administration of 60 mg/kg of 1-( o-chlorophenyl)-1-( p-chlorophenyl)-2,2-dichloroethane ( o, p′-DDD) inhibited ACTH-induced steroid production by greater than 90 per cent within 2 hr. Similar results were noted in perfused adrenals and adrenal slices obtained from o, p′-DDD-treated dogs in vivo. In these preparations, baseline steroid production was unaffected by o, p′-DDD treatment. Incubation of adrenal slices from control dogs in either Krebs-Ringer bicarbonate-glucose buffer (KRBG) of plasma containing o, p′-DDD for as long as 16 hr did not block the adrenal responsiveness to ACTH. Ligation of the blood supply to the liver or gastrointestinal tract had no effect on the ACTH-inhibitory actions of the drug when administered in vivo. A 2-hr retrograde perfusion of adrenals with KRBG or plasma containing o, p′-DDD almost completely abolished the response to ACTH. In adrenals perfused with plasma containing both a maximally stimulating concentration of ACTH and o, p′-DDD, steroid production fell at approximately the same rate as was observed in the intact dog. Thus, o, p′-DDD was found to be effective in an adrenal preparation in vitro. The drug is not effective in the adrenal slice, probably because it does not penetrate to the interior of the slice. These studies suggest that either o, p′-DDD per se is the active principle in inhibiting ACTH-induced steroidogenesis or the active principle is formed within the adrenal gland.

Cushing's syndrome with a paradoxical response to dexamethasone.

Abstract A paradoxical response to dexamethasone was observed in a patient with Cushing's syndrome due to bilateral adrenal hyperplasia. Postoperatively the patient died and was found to have a basophil adenoma of the pituitary. In two separate tests the administration of dexamethasone caused increases in urinary 17-hydroxysteroids and 17-ketosteroids. After the administration of dexamethasone, plasma ACTH activity appeared slightly increased and urinary 17-hydroxysteroid metabolites were similar to those found after the administration of ACTH. Metyrapone caused an initial rise in urinary 17-hydroxysteroids due to an increase in tetrahydro-11-deoxycortisol, as would be expected, but a much larger increase in adrenal corticoid excretion occurred on the second and third days after metyrapone treatment and was associated with the reappearance of urinary cortisol metabolites. Incubation of adrenal slices in the presence of dexamethasone did not stimulate the conversion of labeled acetate to cortisol. These findings implicate the pituitary adenoma in the paradoxical response, and suggest that it was mediated by ACTH. The early response to metyrapone indicates that there was an intact positive feedback mechanism, the late rise in cortisol metabolites suggests that ACTH secretion was also paradoxically stimulated by endogenous adrenal corticoids.

Sites of action of sodium depletion on aldosterone biosynthesis in the dog.

Secretion of cortisol, corticosterone, and aldosterone was measured in vivo in normal and sodium-depleted hypophysectomized dogs. Biogenesis of steroids was then measured in vitro with outer slices of the adrenals of the same dogs. In some studies, metyrapone or puromycin was added. In vivo, sodium depletion stimulated the production of cortisol, corticosterone, and aldosterone. In vitro, tissues from sodium-depleted animals released more aldosterone, but less corticosterone than those from sodium-replete controls. The results are interpreted to indicate that (a) biosynthesis of aldosterone is regulated at at least two sites in the biosynthetic pathway. The final conversion, that of corticosterone to aldosterone, is stimulated by sodium depletion. This effect persists for at least 3 hr while slices from sodium-depleted dogs are incubated in vitro. Stimulation at this site is thus relatively stable in vitro; its activation by sodium depletion is not inhibited by puromycin in the dog. Stimulation at this site can explain, at least in part, the increased effectiveness of adrenocorticotropin (ACTH) on aldosterone biogenesis during sodium depletion.(b) the earlier site at which sodium depletion stimulates the secretion of aldosterone is "above" the position of desoxycorticosterone in the pathway; it is probably at the conversion of cholesterol to pregnenolone. Stimulation at this site is quickly lost during incubation of adrenal slices. It is thus relatively unstable in vitro; its activation by sodium depletion is inhibited by puromycin in the dog.

Adrenal function in cockerels.

Corticosterone concentration, as determined by a validated fluorometric method, in adrenal vein effluent plasma of adenohypophysectomized cockerels was partially maintained, at 37% of control. Similar results were obtained from incubation of adrenal slices of both intact and adenohypophysectomized cockerels in vitro. Steroid concentration in adrenal vein plasma decreased sharply in both groups of cockerels following Metopirone treatment. Arginine vasotocin had no adrenocorticotrophic effect upon either group. Corticosterone concentration increased after 1 hr of surgical stress in adrenal plasma of adenohypophysectomized cockerels but decreased after 4 hr of surgical stress. The adrenals of intact cockerels responded positively in both cases. Treatment with dexamethasone phosphate eliminated corticosterone from adrenal vein plasma of both groups of cockerels, but failed to inhibit corticosterone synthesis from endogenous precursors at 3 different dose levels in incubated adrenal slices of intact cockerels....

In vitro studies and adrenal steroidegenesis by the golden hamster.

No detectable amounts of steroid hormones can be recovered by direct extraction of hamster adrenal tissue or after incubation of adrenal slices in Krebs-Ringer Bicarbonate medium. Adrenal slices or cell-free homogenate preparations can. however, readily convert various steroid precursors to hydrocortisone (Cpd. F), cortisone (Cpd. E), corticosterone (Cpd. B), dehydrocorticosterone (Cpd. A) 17-hydroxy-ll-desoxycorticosterone (Subst. S) and at least three other unknown alpha ketols. It was demonstrated that hamster adrenal glands contain the 3β-ol dehydrogenease system, the C-llβ, C-17 and C-21 hydroxylase enzyme systems. Sodium acetate was not converted to adrenal corticoids either by incubation with adrenal slices or by incubation with cell-free homogenate preparations. On the other hand, sodium acetate-1-C14 was readily converted to radioactive Cpd. F in vivo, thus demonstrating that the hamster can incorporate acetate into corticoids. A cell-free homogenate preparation with added cofactors was able to c...