Steroid Output Research Articles

Adrenal and pancreatic endocrine responses to hypoxia in the conscious calf.

1. Pancreatic and adrenal responses to intense hypoxia have been examined in conscious unrestrained calves 3-5 weeks after birth. 2. The outputs of both cortisol and corticosterone from the right adrenal gland rose steadily in response to hypoxia and this cortical secretory response was accompanied by a pronounced increase in blood flow through the gland. The changes in both steroid output and adrenal blood flow corresponded with those which occur in response to supramaximal doses of corticotrophin in calves of the same age. 3. Neither adrenaline nor noradrenaline were released in significant amounts from the adrenal medulla until the arterial PO2 had fallen below 15 mmHg. Such severe hypoxia caused secretion of catecholamines at rates comparable with those recorded during maximal stimulation of the sympathetic innervation to the gland in anaesthetized calves. The response to intense hypoxia in these conscious calves differed from that which occurs under anaesthesia in that the amount of adrenaline released was invariably greater than that of noradrenaline. 4. Severe hypoxia produced a rapid but transient increase in plasma glucagon concentration, followed by a pronounced rise in plasma glucose concentration in animals with abundant liver glycogen. No change in plasma insulin concentration was observed during hypoxia although it rose subsequently in response to hyperglycaemia. 5. Bilateral section of the splanchnic nerves virtually abolished the release of catecholamines in response to hypoxia but the adrenal cortical and pancreatic responses did not appear to be affected.

Steroids after birth-puberty

The production of gonadal steroids in the perinatal period may have an important influence on the later development of the hypothalamo-pituitary-gonadal axis. In the prepubertal period there is an increased output of gonadal steroids and of androgens from the adrenal cortex, with a further increase as the secondary sex characters develop. The precise role of individual steroids in the maturation of the reproductive system and of the neuroendocrine regulating mechanisms will probably remain unclear unless conclusions are based on longitudinal studies in which hormone production is related precisely to different parameters of sexual development and somatic growth.

Delay of Puberty and Inhibition of Reproductive Processes in the rat by a Gonadotropin-Releasing Hormone Agonist Analog

Studies were performed in immature and mature female rats to determine the effects of chronic administration of [D-Leu6,des-Gly-NH210, Pro-ethylamide9]-gonadotropin-releasing hormone (GnRH) upon maturation and maintenance of reproductive function. Treatment of immature rats inhibited normal overian growth and maturation as evidenced by arrest of uterine growth, delay of vaginal opening, and absence of normal cycling. The primary responses in the mature animal were cessation of cycling, atrophy of the ovaries to a "prepuberal" size, and uterine weight regression. In both groups termination of treatment was followed by prompt restoration of normal ovarian mass and function, including support of breeding processes. The results presented here suggest that chronic administration of the GnRH analog inhibits ovarian steroid output either by direct action of the analog on the ovary or indirectly through qualitative or quantitative changes in levels of luteinizing hormone and follicle-stimulating hormone.

Response to ACTH and dibutyryl cyclic AMP by enucleated adrenocortical tumor cells

Y-1-L cells, a subline of the Y-1 functional murine adrenocortical tumor cell line, were enucleated with cytochalasin B and the response of these enucleated cells to ACTH and dibutyryl cyclic AMP (dbcAMP) was examined. Enucleated Y-1-L cells maintained a basal steroid output for at least 24 h and responded to either ACTH (10 mU/ml) or dbcAMP (1 mM) by a change from flat to rounded cell shape, and by increased steroidogenesis. The steroidogenic response of enucleated cells during the first 3 h after enucleation was highly significant and, though it decreased rapidly thereafter, it persisted to a limited degree for up to 12 h. On the other hand, the morphologic change could be induced even at 33 h after enucleation. The results of this study show that the nucleus is not required for the expression of the acute effects of ACTH or dbcAMP and that the cytoplasmic components necessary for cell ‘rounding’ and a steroidogenic response are stable for 36 h and 12 h, respectively.

IN VITRO MODIFICATION OF RAT ADRENAL ZONA FASCICULATA/RETICULARIS FUNCTION BY THE ZONA GLOMERULOSA

Following earlier findings, the possibility that the zona glomerulosa of the rat adrenal cortex may modify inner zone function was investigated. In a series of in vitro experiments designed to test this hypothesis it was found that: 1. Inner zone preparations gave significantly more steroid than either glomerulosa tissue incubated alone, or (for the first 40 min of a two hour incubation), an equivalent amount of whole adrenal tissue. Whole tissue yields were greater than glomerulosa alone only at the 120 min point. At no time did the whole tissue output approach the total anticipated by addition of separate inner zone and glomerulosa yields. 2. In 20 min incubations, inner zone steriod output generally could be depressed by the addition of a glomerulosa preincubation medium (PIM), or a steroid extract of a glomerulosa PIM, or by the addition of 100 ng aldosterone. Inner zone PIM had less effect. 3. Higher concentrations of aldosterone gave increased inhibition of inner zone corticosteroid production. The results indicated that only a part of the total corticosterone output was aldosterone sensitive in this way. 4. More specific analysis of the steroids showed that yields of corticosterone from endogenous precursors were depressed by aldosterone, but deoxycorticosterone and 18-hydroxydeoxycorticosterone were not. In the same incubations the presence of aldosterone had no effect on the formation of these three products from [3H]pregnenolone. In total the results suggest that the presence of the glomerulosa may affect inner zone function by inhibiting the secretion of corticosterone formed from endogenous precursors. It is possible that aldosterone is the agent of this effect.

Effects of Pinealectomy on Adrenal Functionin Vivoandin Vitroin Female Rats

Female rats were ovariectomized and either sham-pinealectomized or pinealectomized on day 24 of age and exposed to a photoperiod of 12 h light: 12 h dark. In vivo and in vitro measures of adrenocortical function were made at 6, 7, and 8 weeks of age (18, 25, and 32 days post-pinealectomy, respectively). Pinealectomy diminished the post-castration rise in adrenal 5alpha-reductase activity in all age groups (P less than .05). The proportionate secretion of corticosterone (compared with the total steroid output) by adrenal slices was likewise enhanced (P less than .05) although the secretion and production of corticosterone in vitro was not altered. Pinealectomy substantially diminished (P less than .05) the in vivo secretion rates of reduced metabolites of corticosterone (dihydrocorticosterone and tetrahydrocorticosterone) without altering corticosterone secretion. Consequently, proportionate secretion of corticosterone in vivo was also enhanced (P less than .05). These findings are consistent with the notion that in ovariectomized rats removal of the pineal gland diminishes adrenal 5alpha-reductase activity without affecting ACTH secretion. However, in rats with ovaries intact, estrogen modified the effects of pinealectomy. Not only was the intra-adrenal metabolism of corticosterone diminished (higher proportionate output), but also resting levels of plasma corticosterone (P less than .01), corticosterone production in vitro (P less than .05), and total adrenal steroidogenesis in vitro (P less than .01) were increased. Thus, ACTH secretion may be enhanced following pineal removal in the presence of estrogen. The data suggest that the pineal gland, together with the ovaries, plays a role in the modulation of adrenal steroidogenesis.

Effect of wheat fiber on blood lipids, fecal steroid excretion and serum iron

Adding 36 g of wheat fiber for 3 weeks to the metabolically controlled diets of six subjects produced a significant increase in daily fecal weight from 70.8 g +/- 6.2 SEM to 217 g +/- 12.1; serum iron also fell by 21 micrograms/100 ml +/- 2.1 SEM (P less than 0.001) during the added fiber period (measured in five subjects) as did mean corpuscular volume and mean corpuscular hemoglobin. Fecal neutral steroid concentration (measured in four subjects) fell from 31 to 17.3 mg/g dry weight (P less than 0.05) but the change in neutral steroid output and in acid steroid concentration and output was not significant. No change was seen in the serum levels of cholesterol and triglyceride.

Cholesterol metabolism in hypertriglyceridaemia and the effects of treatment

The faecal output of bile acids and endogenous neutral steroids was increased in three hypertriglyceridaemic patients. One patient had familial type IIb, one had type IV and the third had type V hyperlipoproteinaemia. The hyperlipidaemia in the type IV and type V patients was associated with diabetes and a high alcohol intake. The plasma cholesterol and triglyceride concentrations and the faecal output of bile acids decreased significantly when the type IIb patient was given d-thyroxine plus propranolol, and when the type IV and type V patients were treated by withdrawal of alcohol, a low-carbohydrate diet and insulin or glibenclamide. The findings are discussed in relation to the possibility that hypertriglyceridaemia and increased bile acid synthesis in these patients have a common metabolic origin.

Effect of combined clofibrate-cholestyramine treatment on serum and tissue cholesterol pools and on cholesterol synthesis in hypercholesterolemic swine

The effect of clofibrate on whole-body cholesterol synthesis was studied in cholestyramine-treated hypercholesterolemic male Yorkshire swine and swine with normocholesterolemia or mild hypercholesterolemia. Daily whole-body synthesis rates were calculated by methods developed for the non-steady-state condition using various parameters of cholesterol balance; dietary cholesterol intake, fecal excretion of steroids, and cholesterol retention or loss in the body. After 16 days of treatment, the cholestyramine group showed reductions in serum cholesterol level and carcass cholesterol concentration, and increases in fecal steroid excretion and whole-body cholesterol synthesis, as compared with pretreatment values. The group which received both cholestyramine and clofibrate showed the same changes in all parameters listed above, but showed a greater reduction in serum cholesterol levels and greater increases in fecal excretion and whole-body cholesterol synthesis as compared with the group receiving cholestyramine alone. Unfortunately for the therapeutic implications, the increased output of steroids resulting from treatment with the combination of the two drugs was largely offset by a corresponding increase in cholesterol synthesis. The increase in synthesis might have been caused by a direct effect of clofibrate on the biosynthetic process. However, it seemed more likely that the increased synthesis was secondary to the increased output of steroids. The effect of clofibrate alone in hypercholesterolemic swine treated for 64 days revealed restraint in increase of serum cholesterol levels as compared to untreated controls, and an increase in total steroid excretion. No reduction in the whole-body cholesterol synthesis was observed in the clofibrate-treated group. In the other experiment, clofibrate even under mildly hypercholesterolemic or normocholesterolemic conditions did not result in a reduction of whole-body cholesterol synthesis, although serum cholesterol levels were reduced under both conditions.

PRIMARY ADRENAL INSUFFICIENCY AND PRECOCIOUS PUBERTY IN A BOY 12 MONTHS OF AGE

Case report: Child prematurely born after suspected placental insufficiency (low estriol output). 2 1/2 weeks of age diarrhoea and vomi ting. Increased pigmentation. Na 110 meq/1, K 8,5 meq/1. Treatment with DOCA and Hydrocortisone for suspected CAH-syndrome. Biochemical work-up: Urinary steroid analysis shows no evidence of any known adrenal enzyme deficiency. Salt losing syndrome with low cortisol and high ACTH-plasma-concentrations. No stimulation of plasma-cortisol after prolonged (3 weeks) ACTH-stitnulation. Urinary steroid output (gaschromatography): No increased excretion of any metabolite after prolonged ACTH-stimulation. Normal renin and low aldosterone-plasma-concentrations. Diagnosis: Congenital adrenal hypoplasia. At 12 months pubarche, increased penis-size, testes>2 ml. Bone age more than 4 SD advanced. Supine length crossing from P 25 over P 97: Height velocity well over P 97 for age. Plasma and urinary testosterone in the adult male range, non-suppressable with Dexamethason. A combined insulin-TRH LH-RH-stimulation reveals a complex hypothalamo-pituitary disorder. Hypothesis: Congenital adrenal hypoplasia with true precocious puberty due to congenital hypothalamic defect.

Acute effects of ACTH on dissociated adrenocortical cells: Quantitative changes in mitochondria and lipid droplets

To study the role of certain organelles in steroidogenesis, dissociated rat adrenocortical cells were incubated for two hours with ACTH at a concentration that induces a high level of steroid production. Sections of ACTH treated and untreated cells were photographed in the electron microscope, and morphometric analysis was undertaken to assess possible ACTH-induced changes in total cell volume, volume density and numerical denisty of lipid droplets and mitochondria. There was no change in total cell volume. Lipid droplet volume density and numerical density decreased. Mitochondrial volume density did not change, but numerical density increased. The decrease in lipid droplet volume density indicates a rapid depletion of cholesterol for steroid production. This depletion is almost entirely due to the disappearance of lipid droplets, rather than to an overall diminution in their size, as shown by the decrease in lipid droplet numerical density. The mitochondrial data suggest that the adrenocortical cell has an adedquate mitochondrial apparatus to respond to acute ACTH stimulation with increased steroid output without an increase inmitochondrial volume.

Relation of Intracellular K+ and Steroidogenesis in Isolated Adrenal Zona Glomerulosa and Fasciculata Cells

1. Intracellular K+ content, water spaces and corticosterone output were measured in isolated zona glomerulosa and zona fasciculata-reticularis cell suspensions of rat adrenal cortex, after incubation in vitro under conditions designed to alter steroidogenesis. 2. Intracellular K+ of unpurified zona glomerulosa cells was not altered after stimulation of corticosterone output with serotonin. Similarly, with zona glomerulosa cells purified by unit gravity sedimentation, no change in intracellular K+ was detected after stimulation of steroidogenesis with serotonin or angiotensin II. 3. In high-potassium medium (final concentration 8.4 mmol/1), parallel increases in intracellular K+ and corticosterone output were observed with both purified zona glomerulosa cells. However, a similar increase in intracellular K+ also occurred in high-potassium medium with zona fasciculata cells, whose steroid output is unresponsive to external potassium concentration ([K+]). 4. Ouabain at 10(-5) mol/1 depressed the intracellular [K+] of glomerulosa cells but did not alter basal or stimulated corticosterone output. Similar results were obtained with fasciculata cells. 5. Ouabain at 5 times 10(-4) mol/1 further depressed intracellular [K-+] of glomerulosa cells and inhibited basal and stimulated corticosterone output. However, this concentration of ouabain also inhibited steroidogenesis in fasciculata cells. 6. These results demonstrate a variety of situations where changes in intracellular [K+] are dissociated from those in corticosterone output and indicate that intracellular [K+] cannot be the sole mechanism regulating steroidogenesis under these conditions.

An investigation of the involvement of adenosine 3′:5′-cyclic monophosphate in steroidogenesis by using isolated adrenal cell column perfusion

The involvement of cyclic AMP in corticosteroidogenesis was investigated by using isolated adrenal cell column perfusion. Steroids were produced in response to 0.5, 1.0 and 5.0 mg of cyclic AMP/ml. Analysis of the shape of the response curves indicated an inverse relationship between rate of onset of steroid production and dose. A further increase in steroid production during the washout period after the 5 mg/ml dose was considered to indicate an intracellular inhibitory effect of cyclic AMP. Release of cyclic AMP into the perfusate only occurred in response to supramaximal steroidogenic doses of ACTH (adrenocorticotrophin). A connexion between dose and response was demonstrated over a narrow concentration range. Variation in the time-lag before cyclic AMP production and in the duration of the response was marked; further, no reproducible ratio of steroid output to cyclic AMP output was shown at any level of stimulation. These results are discussed together with those of other recent investigations. It is considered that these findings do not support an obligatory role for cyclic AMP as mediator of ACTH action in the adrenal.

The sensitivity of adrenal responses to synthetic adrenocorticotrophin in the conscious unrestrained calf.

1. Changes in cortisol and corticosterone output and blood flow from the adrenal gland have been determined in the conscious unrestrained calf during I.V. infusions of synthetic adrenocorticotrophin (Synacthen) at 0-5 ng-kg(-1) min- minus 1 (low dose), 5 ng-kg- minus 1 (medium dose), 50 ng-kg- minus 1 min- minus 1 (high dose) and 500 ng-kg- minus 1 min- minus 1 (medium dose), 50 ng-kg- minus 1 min- minus 1 (high dose) and 500 ng-kg- minus 1 min- minus 1(very high dose). 2. Infusions at the low dose produced a rise in adrenal output of both cortisol and corticosterone to maximum values of approximately 100 and 30 ng-kg- minus 1 min- minus 1 respectively. Mean output of both steroids was significantly increased within 5 min, reached a maximum within 10 min and had fallen to resting levels 10 min after the infusion was discontinued. 3. The effects of infusions at both the high and very high doses were closely similar; maximal cortisol outputs were within the range 600-800 ng-kg- minus 1 min- minus 1 and corticosterone 350-500 ng-kg- minus 1 min- minus 1 in both groups. 4. When the infusions were terminated, pronounced differences were observed in the rates at which steroid outputs declined. Basal levels were restored within 10 min following the low dose and within 60 min in medium dose animals, but both cortisol and corticosterone output were still elevated 2 hr after infusion in high dose animals. In calves infused at the very high dose, cortisol output did not fall significantly during 2 hr period. The ratio of cortisol: corticosterone released from the adrenal gland immediately before infusion (3-2 +/- 0-3) approximated to the proportions in which the two steroids were found in the arterial plasma, but fell progressively to a minimum (1-3 +/- 0-1) with increasing doses of Synacthen. Conversely, the ratio of the steroids in the arterial plasma was increased during infusions at the low dose, but not at the higher doses. 6. No significant change in adrenal blood flow occurred during Synacthen infusion in low dose animals despite the increase in steroid output. In medium dose animals blood flow through the gland rose during infusion by approximately 75 per cent while in both high and very high dose animals the flow increased by up to 300 percent. 7. In the three groups in which adrenal hyperaemia occurred, blood flow had fallen to within the resting range 45 min after infusion: in each case this fall was much more rapid than the fall in steroid output. No significant increase in aortic blood pressure or heart rate accompanied infusion of Synacthen, indicating that adrenal hyperaemia was dependent upon vasodilatation with the gland. 8. Administration of cycloheximide (10 mg/kg) by I.V. injection either before or during an infusion of Synacthen, inhibited steroidogenesis without affecting the vasodilator response.

Endocrine responses to insulin hypoglycaemia in the young calf.

1. Variations in the output of glucocorticoids and catecholamines from the right adrenal gland, in response to insulin hypoglycaemia, have been investigated in calves 2-5 weeks after birth. These have been correlated with changes in the concentration of glucocorticoids and glucagon in arterial plasma. 2. Moderate hypoglycaemia for a limited period (0-1 u. insulin/kg), elicited a prompt increase in steroid output from the adrenal gland followed by a significant rise in plasma glucagon concentration. By comparison, changes in both catecholamine output and peripheral plasma glucocorticoid concentrations were found to be trivial in this group of animals. 3. Administration of a larger dose of insulin (0-5 u./kg) produced a more substantial fall in plasma glucose concentration followed by spontaneous recovery within 2-3 hr. This stimulus elicited the release of greater amounts of both cortisol and corticosterone, followed by a significant increase both in the output of adrenaline and in plasma glucagon concentration. Increase in steroid output was accompanied by an increase in adrenal blood flow and was associated with elevated concentrations of both steroids in arterial plasma. 4. The adrenal cortical response and associated changes in plasma steroid concentration were found to be transient even in response to persistent and intense hypoglycaemia (4 u. insulin/kg). The increase in plasma glucagon concentration in this group of animals was not significantly greater than that produced by smaller doses of insulin. However, substantial amounts of adrenaline (78 plus or minus 14 ng. kg-minus 1 min-minus 1; maximum; n equals 9) together with a little noradrenaline (10 plus or minus 3 ng.kg-minus 1 min-minus 1; maximum; n equals 9) were released from the right adrenal gland under these conditions. 5. Changes in adrenal blood flow could be related to adrenal glucocorticoid output in calves given 0-1 or 0-5 u. insulin/kg. In animals given the largest dose of insulin adrenal blood flow was found to increase coincidentally with rising steroid output but this hyperaemia then persisted after steroid output had subsided to values within the normal range. 6. Calves given the largest dose of insulin (4-0 u./kg) invariably collapsed and convulsed after 2-3 hr, but these symptoms could not be related to any particular endocrine response. No clinical signs of hypoglycaemia were observed in the other animals. 7. The results are discussed in relation to previous studies of adrenal function in this and other species.

The preparation and use of purified and unpurified dispersed adrenal cells and a study of the relationship of their camp and steroid output

Zona glomerulosa cells purified from zona fasciculata contamination by unit gravity sedimentation have previously been characterized. Similarly, zona fasciculata cells have now been separated from zona glomerulosa and zona reticularis cells. Purified zona fasciculata cells respond to valine angiotensin II or ACTH. The maximal steroid response is equivalent for both stimuli (about one hundred fold) and also is about the same using purified zona fasciculata cells or unpurified cells from decapsulated tissue. Preparations consisting mainly of zona reticularis cells respond to a lesser extent than pure zona fasciculata cells to both stimuli. The cAMP output for purified glomerulosa cells is increased by high amounts of ACTH, angiotensin II, serotonin and K + (8.4 mM). Submaximal doses of these stimuli, which markedly increase corticosteroid output, do not detectably raise cAMP output. This could be due to kinetic or compartmental factors. However when a maximal steroidogenic dose of cAMP or serotonin (‘pure’ internal cAMP generator) was used to stimulate unpurified or purified glomerulosa cells, steroid output could still be altered by varying K + concentration. This suggests that alteration in cAMP output production is not the only mechanism for controlling steroidogenesis; alteration in cAMP activity by K + or a K + control mechanism independent of cAMP production are possible extra or alternative mechanisms. A formal treatment of the discussion of the role of cAMP in mediating steroidogenesis is presented.

Effects of premature weaning on adrenal function in intact and gonadectomized rats.

SUMMARY Premature weaning of intact or gonadectomized male and female rat pups did not alter adrenal function at day 49 provided the level of nutrition was sufficient to prevent loss of body weight. However, when nutrition was limited during the early weaning period (day 16–21) both intact male and female rats exhibited decreased production of corticosterone in vitro. Mechanisms mediating this decreased capacity may differ according to sex. In the male rat, production of total steroids in vitro was decreased as were resting plasma levels of corticosterone, suggesting that the set-point for regulation of corticosterone is altered by diminished adrenocorticotrophic hormone secretion. On the other hand, early weaning of the female did not affect plasma levels of corticosterone or total steroid production in vitro; however, the proportionate production of corticosterone compared with total steroid output in vitro was lowered. This effect could be mediated by depressed plasma levels of luteinizing hormone which obtained in prematurely weaned females whereby a paucity of oestrogen could alter the balance between corticosterone and total steroid production.

Effects of clofibrate on sterol metabolism in the rat

Sterol balance measurements using isotopic and Chromatographie techniques were carried out in rats on a stock diet and a stock diet plus 0.3% clofibrate ( α- p-chlorophenoxyisobutyryl ethyl ester (CPIB)). At the end of the feeding period the activity of hepatic microsomal β-hydroxy-β-methylglutaryl-CoA reductase (EC 1.1.1.34), the rate limiting enzyme of cholesterol synthesis was determined in each animal. Administration of the drug caused a significant decrease in fecal bile acid output (10.5–7.6 mg/day), no change in fecal neutral steroid output (11–10 mg/day) and a decrease in cholesterol synthesis (16.6–12.6 mg/day). Biliary cholesterol concentration was reduced from 0.23 mg/ml in the controls to 0.11 mg/ml in the clofibrate treated group. A decrease in both liver cholesterol and plasma cholesterol was also observed in response to the drug. Measurement of the activity of the rate limiting enzyme of cholesterol synthesis showed a 2-fold decrease of activity when cholesterol synthesis was reduced 25%.

Measurement of the dynamics of stimulation and inhibition of steroidogenesis in isolated rat adrenal cells by using column perfusion.

Isolated adrenal cells were perfused in a small column by using Bio-Gel polyacrylamide beads as an inert supporting matrix, and the time-course of the response to various stimuli was observed by measuring fluorogenic 11-hydroxycorticosteroids in the effluent. A small but significant response was observed 1 min after stimulation with physiological concentrations of ACTH (adrenocorticotrophin), but the response did not start to build up rapidly for 3-4min and eventually reached a plateau after 9-10min. A similar pattern of events was observed for the decay of the steroid output on removal of ACTH. ACTH analogues, including one with a long duration of action in vivo, were found to produce responses with similar kinetics. However, cyclic AMP caused a more rapid increase in steroidogenesis and its effects were more short-lived after withdrawal. If, as present evidence suggests, cyclic AMP is produced rapidly after ACTH stimulation the delayed build-up of the steroidogenic response to ACTH would indicate that cyclic AMP may not be the intracellular mediator. When inhibitors were applied during ACTH stimulation, aminoglutethimide, which blocks mitochondrial conversion of cholesterol into pregnenolone (3beta-hydroxypregn-5-en-20-one), caused a rapid fall in steroid output (1 min), whereas cycloheximide took longer to achieve its full effect. Nevertheless, the response had fallen by 50% in 2 min, indicating a much shorter half-life than that previously reported for the labile protein implicated in steroidogenesis. In addition the rapid response to cyclic AMP makes it unlikely that steroid production is induced as a result of initiation of protein synthesis. This suggests that the labile protein plays an obligatory but permissive role in the development of the response. Column perfusion has proved to be a simple technique which can readily yield accurate data on responses of cells to stimulants and inhibitors.

The effects of potassium, 5-hydrocytryptamine, adrenocorticotrophin and angiotensin II on the concentration of adenosine 3':5'-cyclic monophosphate in suspensions of dispersed rat adrenal zona glomerulosa and zona fasciculata cells.

Dispersed rat adrenal cells prepared from both the capsule and the decapsulated gland were used to investigate the effects on cyclic AMP accumulation of known stimuli of steroidogenesis [ACTH (adrenocorticotrophin), angiotensin II, K(+) ions and 5-hydroxytryptamine]. Since glomerulosa-cell preparations from capsular strippings are normally contaminated with a proportion of fasciculata cells, cells purified by fractionation on a bovine serum albumin gradient were also used. The results showed that: (1) ACTH and angiotensin II stimulated cyclic AMP accumulation in both fractionated and unfractionated zona fasciculata cells; (2) 5-hydroxytryptamine and an increased extracellular K(+) concentration (from 3.6 to 8.4mm) had no effect on cyclic AMP concentrations in fasciculata cell preparations; (3) the addition of ACTH, angiotensin II, 5-hydroxytryptamine or K(+) to the incubation medium resulted in increased cyclic AMP concentrations in unpurified zona glomerulosa cell preparations; (4) fractionation and hence the virtual elimination of fasciculata contamination, did not affect the response to 5-hydroxytryptamine and increased K(+) concentration. However, the responses to ACTH and angiotensin II were markedly lowered but not abolished. These results strongly suggest a link between cyclic AMP production and steroidogenesis in the zone of the adrenal gland that specifically secretes aldosterone. All four agents used stimulated both steroid output and cyclic AMP accumulation. However, at certain doses of 5-hydroxytryptamine, K(+) and angiotensin II the significant increases in corticosterone output were not accompanied by measurable increases in cyclic AMP accumulation.