Inhibition of cortisol production in isolated guinea-pig adrenal cells

Cortisol, added to 1 ml incubation medium containing 3-4 X 10(5) isolated guinea-pig adrenal cells, provoked a decrease in basal and ACTH (250 pg)-stimulated cortisol production, in correlation with the amounts used (50 ng-2,000 ng). A decrease in aldosterone production could be seen when cortisol concentrations reached or exceeded 1,000 ng/ml. There were no variations in either androgens (delta 4-androstenedione, dehydropiandrosterone) or 17-hydroxyprogesterone. Only 11-deoxycortisol was slightly increased. Using increasing concentrations of ACTH (50-250 pg), both in the absence and in the presence of 1,000 ng cortisol, it was noted that the inhibition induced by cortisol was of a competitive type and could be overcome by ACTH. This decrease in cortisol was concomitant with an increase in 11-deoxycortisol. Neither corticosterone nor dexamethasone reduced cortisol production. In addition, it was shown that the conversion of tritiated 11-deoxycortisol to radioactive cortisol increased significantly under the influence of 250 pg ACTH (mean relative variation of 21.7% +/- 7.7 (SEM), n = 6, P less than 0.05); but decreased significantly under the combined effect of 1,000 ng exogenous cortisol and the same dose of ACTH: (mean relative variation of 4.3% +/- 1 (SEM), n = 8, P less than 0.005). There is therefore reason to believe that the concentrations of cortisol at the adrenal level modulate the stimulation induced by ACTH and that this self-adjustment forms part of the control mechanisms involved in corticosteroidogenesis.     

Cortisol production by dispersed guinea-pig adrenal cells; a specific, sensitive and reproducible response to ACTH....and its fragments

Naturally occurring steroids and peptide hormones, tested at supraphysiological concentrations, were without effect on basal and human (h) 1-39 ACTH (NIBSC code 74/555, 25 ng/l (5.5 X 10(-12) mol/l] stimulated cortisol production. Further, low concentrations of angiotensin II, N-pro-opiocortin (N terminal fragment 1-76) and gamma-MSH all of which have been reported to synergise with ACTH with regard to cortisol production, were without significant effect alone or in combination with ACTH over the range 2.2 X 10(-13) to 5.5 X 10(-12) mol/l. The activity of h 1-39 was compared with that of the ACTH related peptides 1-24, 1-18, 1-17, 1-16, 1-13-NH2 (alpha MSH), 1-10 and 4-10. The dose responses were parallel and the same maximal cortisol output was observed with all the peptides except the 1-10 fragment. Half maximal stimulation occurred at 3.1 X 10(-12) (1-24), 4.4 X 10(-12) (h 1-39), 1.5 X 10(-11) (1-39), 3.3 X 10(-10) (1-18), 5 X 10(-9) (1-13-NH2), 8 X 10(-9) (1-17), 2 X 10(-7) (1-16) and 1 X 10(-5) (4-10) mol/l respectively. Interference by the above ACTH-derived peptides in cortisol secretion by the cells in response to 5.5 X 10(-12) mol/l h 1-39 ACTH was minimal over the range 5.2 X 10(-12)-2.2 X 10(-6) mol/l. The sensitivity of the adrenal cells to h 1-39 ACTH was such that 2 ng/l (4.4 X 10(-13) mol/l) provoked cortisol secretion over the control (P less than 0.05, n = 17). The coefficient of variation within assay for each dose on the full standard curve (2.2 X 10(-13)-1.1 X 10(-10) mol/l) was less than 10% (n = 6). Half maximal stimulation was given by 14.5 ng/l (3.2 X 10(-12) mol/l). Between control and 1.1 X 10(-10) mol/l ACTH there was a 32 +/- 8 (mean +/- SD, n = 9) fold change in cortisol production.     

The effects of ACTH on adrenal steroidogenesis and blood corticosteroid levels in the echidna (Tachyglossus aculeatus).

1. The effects of short-term (S.T., 30 min) and long-term (L.T., 4 days) administration of ACTH on peripheral blood corticosteroid levels and on in vitro steroidogenesis were investigated. 2. Control levels of cortisol, corticosterone and aldosterone were 58 +/- 12, 130 +/- 26 and 10 +/- 6 (SEM) ng/100 ml respectively. 3. Corticosterone was 70% higher after S.T. and 150% higher after L.T., when cortisol was 800% higher. 4. Adrenal homogenates from control echidnas converted [14C]progesterone predominantly to 11-deoxycorticosterone (45%) and 11-deoxycortisol (12%). 5. After L.T. the principal product was corticosterone (25%), but S.T. had no effect. 6. In control echidnas the Km and V for 11 beta-hydroxylation of 11-deoxycorticosterone were 20 microM and 2.8 rho mol/min/mg respectively. After L.T. V increased to 10 rho mol/min/mg.     

An oxidized metabolite of linoleic acid stimulates corticosterone production by rat adrenal cells

Oxidized derivatives of linoleic acid have the potential to alter steroidogenesis. One such derivative is 12,13-epoxy-9- keto-10-(trans)-octadecenoic acid (EKODE). To evaluate the effect of EKODE on corticosterone production, dispersed rat zona fasciculata/reticularis (subcapsular) cells were incubated for 2 h with EKODE alone or together with rat ACTH (0, 0.2, or 2.0 ng/ml). In the absence of ACTH, EKODE (26 microM) increased corticosterone production from 5.3 +/- 2.3 to 14.7 +/- 5.0 ng. 10(6) cells. h(-1). The stimulatory effect of ACTH was increased threefold in the presence of EKODE (26.0 microM). Cholesterol transport/P-450scc activity was assessed by measuring basal and cAMP-stimulated pregnenolone production in the presence of cyanoketone (1.1 microM). EKODE (13.1 and 26.0 microM) significantly increased basal and cAMP-stimulated (0.1 mM) pregnenolone production. In contrast, EKODE decreased the effect of 1.0 mM cAMP. EKODE had no effect on early or late-pathway activity in isolated mitochondria. We conclude that EKODE stimulates corticosterone biosynthesis and amplifies the effect of ACTH. Increased levels of fatty acid metabolites may be involved in the increased glucocorticoid production observed in obese humans.     

Development of the pituitary-adrenal axis in chronically cannulated ovine fetuses

In the intact, unstressed ovine fetus, both plasma immunoreactive adrenocorticotrophin (ACTH) and blood cortisol concentrations increased after 121 days gestation. The mean ACTH and cortisol concentrations in intact fetuses of 90-121, 122-135 and 136-144 days gestation were for ACTH 20.4 +/- 3.9 (50) (mean +/- SEM, n), 30.2 +/- 5.6 (26) and 56.0 +/- 6.3 pg/ml (37) respectively, and for cortisol 0.07 +/- 0.01 (24), 0.17 +/- 0.03 (21) and 0.64 +/- 0.13 microgram/100 ml (15), respectively. After 121 days ACTH and cortisol concentrations were correlated positively. Cortisol infused into intact or adrenalectomized fetuses and corticosterone infused into adrenalectomized fetuses suppressed fetal plasma ACTH concentrations. In summary, ACTH and cortisol increase concomitantly after 122 days, so that it is highly probable that ACTH is the trophic stimulus for fetal adrenal maturation. The suppression of ACTH by cortisol and corticosterone suggests that these are the natural feedback regulators. It is proposed that while the mechanism for cortisol feedback may exist early in gestation, it is not until after 121 days that feedback control of ACTH becomes evident and physiologically important.     

ACTH regulation of cholesterol movement in isolated adrenal cells

Confluent bovine adrenal cell primary cultures respond to stimulation by adrenocorticotropin (ACTH) to produce steroids (initially predominantly cortisol and corticosterone) at about one-tenth of the output of similarly stimulated rat adrenal cells. The early events of steroidogenesis, following ACTH stimulation, have been investigated in primary cultures of bovine adrenal cortical cells. Steroidogenesis was elevated 4-6-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter. Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide (AMG), even though cytochrome P-450scc only increases after 12 h. Mitochondrial-free cholesterol levels increased during the same time period (16.5-25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel. More prolonged exposure to ACTH prior to addition of AMG caused the elevation in mitochondrial cholesterol to more than double, possibly due to enhanced binding capacity. Early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial-free cholesterol. The maximum rate of cholesterol transport to mitochondria in AMG-blocked cells was consistent with the maximum rate of cellular steroidogenesis. Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450scc without affecting mitochondrial-free cholesterol. Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10(-8) M), for 24 h prior to ACTH addition decreased the rates of ACTH- and cAMP-stimulated steroidogenesis but did not affect the basal rate. In both cases, the effectiveness of TCDD increased with time of exposure to the stimulant. Although cholesterol accumulated in the presence of ACTH and AMG (13-28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13-8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). Since even in the absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h) TCDD pretreatment may increase the presence of a protein(s) that cause this mitochondrial-cholesterol depletion following stimulation by ACTH or cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of insulin-like growth factor I (IGF-I) on enzymatic activity in human adrenocortical cells. Interactions with ACTH

Cells obtained from 6 adult human adrenals or adrenal fragments were cultured in serum-free synthetic medium (McCoy's) in order to study the isolated effects of IGF-I on steroidogenesis and its interactions with ACTH. After addition of peptide, changes in the activities of steroidogenic enzymes were assessed by measuring certain steroids in the spent medium. These included pregnenolone, 17-hydroxypregnenolone (17-OH-Preg), dehydroepiandrosterone (DHA), 17-hydroxyprogesterone (17-OH-P), androstenedione (AD), 11-deoxycortisol and glucocorticoids (chiefly cortisol and its immediate precursors, 11-deoxycortisol and 17-OH-P) and cortisol itself.

The steroid responses obtained with repeated doses of IGF-I (40 ng/ml ≈ 10⁻⁹ M), added at 0, 48 and 72 h, over 4 days' culture were quite different from those obtained with repeated doses of ACTH (0.25 ng/ml ≈ 10⁻¹⁰ M). All the steroids measured increased with time of culture under the influence of ACTH and, apart from pregnenolone which peaked, tended to reach a plateau. With IGF-I, by contrast, DHA, AD, 11-deoxycortisol and glucocorticoid production increased initially, then decreased progressively, whereas pregnenolone, 17-OH-Preg and 17-OH-P production was either absent or negative.

Cumulative steroid production over 4 days reached similar levels in response to a single dose of IGF-I and/or ACTH, with two major exceptions: pregnenolone dropped significantly with IGF-I [46% ± 6 (SEM) as opposed to 93% ± 11 with ACTH, P < 0.005, N = 5], as did 17-OH-P (48% ± 11 vs 113% ± 8 with ACTH, P < 0.001, N = 6). Increased formation of down-stream metabolites (DHA, AD, 11-deoxycortisol and glucocorticoids) would suggest that IGF-I induced stimulation of the 17-, 21- and 11β-hydroxylases.

The responses to ACTH stimulation of cells which 4 days previously had been pre-treated with an initial and single dose of IGF-I and/or ACTH emphasized the impact of IGF-I on the 3-hydroxylation steps in cortisol biosynthesis. Compared with ACTH pre-treatment, the effects of which faded in the long term, pre-treatment with IGF-I resulted in a significantly increased steroidogenic response (P between < 0.05 and < 0.01). With the single exception of pregnenolone (43% ± 4.7), production of all the metabolites was amplified: 17-OH-Preg: 348% ± 88; DHA: 643% ± 127; 17-OH-P: 193% ± 36; AD: 725% ± 200; 11-deoxycortisol: 573% ± 110; cortisol: 1000%.

Our findings strongly suggest that IGF-I plays a major rôle in the regulation of steroidogenesis by promoting and maintaining enzymatic activity (17, 21- and 11β-hydroxylases) via which the function of ACTH is achieved, viz., biosynthesis of cortisol.     

The influence of transcortin on adrenocorticotropin-stimulated corticosterone production in monolayer cultured rat adrenal cells

To verify the influence of the protein binding status of steroids adjacent to adrenal cells on steroidogenesis, the effect of transcortin, a specific binding protein of cortisol or corticosterone, on adrenocorticotropin (ACTH)-stimulated corticosterone production in monolayer cultured rat adrenal cells was studied. The transcortin in concentration of 5 x 10(-7) M was loaded with 0, 2.5, 5 and 10 pg/ml ACTH-(1-24), and the cells were incubated for 2 and 4 hours. Since molar concentrations of corticosterone produced in the medium were below the transcortin concentration at all levels of stimulation, protein-unbound corticosterone in the medium may have been largely reduced by the addition of transcortin. However, the total corticosterone production was not influenced by the transcortin added to the medium. It was speculated that protein-unbound steroid within the concentration range modulated by transcortin in the area surrounding the adrenal cells may not affect adrenal steroidogenesis.     

The relationship between adrenal vascular events and steroid secretion: the role of mast cells and endothelin.

The actions of ACTH on the adrenal cortex are known to be 2-fold. In addition to increased steroidogenesis, ACTH also causes marked vasodilation, reflected by an increased rate of blood flow through the gland. Our studies, using the in situ isolated perfused rat adrenal preparation, have shown that zona fasciculata function and corticosterone secretion are closely related to vascular events, with an increase in perfusion medium flow rate causing an increase in corticosterone secretion, in the absence of any known stimulant. These observations give rise to two important questions: how does ACTH stimulate blood flow; and how does increased blood (or perfusion medium) flow stimulate steroidogenesis? Addressing the first question, we have recently identified mast cells in the adrenal capsule, and shown that Compound 48/80, a mast cell degranulator, mimics the actions of ACTH on adrenal blood flow and corticosterone secretion. We have also demonstrated an inhibition of the adrenal vascular response to ACTH in the presence of disodium cromoglycate, which prevents mast cell degranulation. We conclude, therefore, that ACTH stimulates adrenal blood flow by its actions on mast cells in the adrenal capsule. Addressing the second question, we looked at the role of endothelin in the rat adrenal cortex. Endothelin 1, 2 and 3 caused significant stimulation of steroid secretion by collagenase dispersed cells from both the zona glomerulosa and the zona fasciculata. A sensitive response was seen, with significant stimulation at an endothelin concentration of 10(-13) mol/l or lower. Endothelin secretion by the in situ isolated perfused rat adrenal gland was measured using the Amersham assay kit. Administration of ACTH (300 fmol) caused an increase in the rate of immunoreactive endothelin secretion, from an average of 28.7 +/- 2.6 to 52.6 +/- 6 fmol/10 min (P less than 0.01, n = 5). An increase in immunoreactive endothelin secretion was also seen in response to histamine, an adrenal vasodilator, which stimulates corticosterone secretion in the intact gland, but has no effect on collagenase-dispersed cells. From these data we conclude that endothelin may mediate the effects of vasodilation on corticosterone secretion, and this mechanism may explain some of the differences in response characteristics between the intact gland and dispersed cells.     

Hypothalamic-pituitary disconnection in fetal sheep blocks the peripartum increases in adrenal responsiveness and adrenal ACTH receptor expression

Although it has been recognized for over a decade that hypothalamic-pituitary disconnection (HPD) in fetal sheep prevents the late gestation rise in plasma cortisol concentrations, the underlying mechanisms remain unclear. We hypothesized that reductions in adrenal responsiveness and ACTH receptor (ACTH-R) expression may be mediating factors. HPD or sham surgery was performed at 120 days of gestation, and catheters were placed for blood sampling. At approximately 138 days of gestation, fetuses were killed, and adrenals were removed for cell culture and analyses of ACTH-R mRNA and protein. After 48 h, adrenocortical cells were stimulated with ACTH for 2 h, and the medium was collected for cortisol measurement. The same cells were incubated overnight with medium or medium containing ACTH or forskolin (FSK), followed by ACTH stimulation (as above) and cortisol and cellular ACTH-R mRNA analyses. HPD prevented the late gestation increase in plasma cortisol and bioactive ACTH and reduced adrenal ACTH-R mRNA and protein levels by over 35%. HPD cells secreted significantly less cortisol than sham cells (3.2 +/- 1.2 vs. 47.3 +/- 11.1 ng.ml(-1).2 h(-1)) after the initial ACTH stimulation. Overnight incubation of HPD cells with ACTH or FSK restored cortisol responses to acute stimulation to levels seen in sham cells initially. ACTH-R mRNA levels in cells isolated from HPD fetuses were decreased by over 60%, whereas overnight incubation with ACTH or FSK increased levels by approximately twofold. Our findings indicate that the absence of the cortisol surge in HPD fetuses is a consequence, at least in part, of decreased ACTH-R expression and adrenal responsiveness.     

The ratio of plasma bioactive to immunoreactive ACTH-like activity increases with gestational age in the fetal lamb.

The fetal ovine pituitary-adrenal axis plays an important role in the timing of parturition, in fetal lung maturation, and in fetal and neonatal responses to stress. While the ovine pituitary during the last third of gestation (term = 145 days) is capable of secreting immunoreactive ACTH (iACTH) in response to various stimuli, plasma cortisol levels frequently do not reflect the rise in plasma ACTH. Therefore, we examined the relationship between plasma iACTH and steroidogenic ACTH-like activity (bACTH) in a group of immature fetal lambs (Group I: gestational age = 97 +/- 2 days, mean +/- SEM, n = 16) and a group of near-term fetuses (Group II: gestational age = 136 +/- 1 days, n = 13) following acute exteriorization. Plasma iACTH was determined by RIA. Plasma bACTH was determined by the ability of glass-extracted material to stimulate corticosterone (B) production in an acutely dispersed rat adrenal bioassay. Plasma iACTH and bACTH levels varied among animals within age groups, with iACTH tending to be higher in immature fetal lambs (Group I) than near-term lambs (Group II) and bACTH being higher (P < 0.05) near term than earlier (Group I: iACTH = 807 +/- 273 pg/ml, bACTH = 173 +/- 44 pg/ml; Group II: iACTH = 405 +/- 85 pg/ml, bACTH = 371 +/- 96 pg/ml). The proportion of iACTH that had biologic activity (e.g. B/I ratio) was significantly greater in the older than in the younger fetuses (Group II: B/I = 0.862 +/- 0.109; Group I: B/I = 0.462 +/- 0.105 P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Adrenal adenylate cyclase and steroidogenic activities of 63 day old ovine fetuses: in vitro effects of ACTH1-24 and forskolin

This study examines the activity of the adenylate cyclase system and that of some enzymes of the steroidogenic pathway of adrenal cells from 62-63 day old ovine fetuses. Synthetic corticotropin (ACTH1-24), cholera toxin and forskolin stimulated both cAMP and corticoid productions by freshly isolated adrenal cells. The cAMP response to ACTH1-24 was lower than that to forskolin. However, forskolin-induced steroidogenesis was significantly lower than the ACTH1-24-induced steroid output. Freshly isolated cells metabolized quickly [14C]-labeled pregnenolone mainly through the 17-deoxy pathway. The amounts of cortisol and of corticosterone formed, in the presence of exogenous pregnenolone, were roughly 15-fold higher than under maximal stimulation by ACTH1-24. When the cells were cultured for 6 days in the absence or presence of ACTH1-24 (10(-8) M) or forskolin (10(-5) M), a small development of the cAMP response to these factors was observed in the course of the experiment. However, the mechanism of this development appeared different, according to the conditions of culture. The amounts of corticosterone secreted on day 6 by ACTH1-24- or forskolin-treated cells were 2- to 4-fold higher than on day 1, whereas cortisol outputs were much lower on day 6 than on day 1. The response to ACTH1-24 of cells maintained in ACTH-free media decreased dramatically during the culture in terms of both cortisol and of corticosterone. On day 6 of the experiment, the metabolism of [14C]pregnenolone was lower than on day 1 under all 3 conditions of culture. Only the 3 beta-hydroxysteroid dehydrogenase/isomerase activity could be maintained by continuous treatment with forskolin. However, both ACTH1-24 and forskolin enhanced the production of pregnenolone from an endogenous substrate. In conclusion, these results present evidence that: 1) the adenylate cyclase system is not a bottleneck in the steroidogenic response to ACTH1-24 of freshly isolated adrenal cells from 62-63 day old ovine fetuses; 2) the main rate-limiting step for steroidogenesis by these cells is the availability of pregnenolone; 3) neither ACTH1-24 nor forskolin is able to maintain the activity of most enzymes involved in the metabolization of pregnenolone by cultured cells while increasing pregnenolone availability; 4) some inhibiting factors are involved in the loss of adrenal cells responsiveness to ACTH between days 50 and 100 of gestation, and they probably act mainly on the adenylate cyclase system.     

Steroidogenic acute regulatory protein expression is decreased in the adrenal gland of the growth-restricted sheep fetus during late gestation

Functional development of the adrenal cortex is critical for fetal maturation and postnatal survival. In the present study, we have determined the developmental profile of expression of the mRNA and protein of an essential cholesterol-transporting protein, steroidogenic acute regulatory protein (StAR), in the adrenal of the sheep fetus. We have also investigated the effect of placental restriction (PR) on the expression of StAR mRNA and protein in the growth-restricted fetus. Adrenal glands were collected from fetal sheep at 82-91 days (n = 10), 125-133 days (n = 10), and 140-144 days (n = 9) and from PR fetuses at 141-145 days gestation (n = 9) (term = 147 +/- 3 days gestation). The adrenal StAR mRNA:18S rRNA increased (P < 0.05) between 125 days (7.44 +/- 1.61) and 141-144 days gestation (13.76 +/- 1.88). There was also a 13-fold increase (P < 0.05) in the amount of adrenal StAR protein between 133 and 144 days gestation in these fetuses. However, the amount of StAR protein (6.9 +/- 1.7 arbitrary densitometric units [AU]/microg adrenal protein) in the adrenal of the growth-restricted fetal sheep was significantly reduced, when compared with the expression of StAR protein (17.1 +/- 1.9 AU/microg adrenal protein) in adrenals from the age-matched control group. In summary, there is a developmental increase in the expression of StAR mRNA and protein in the fetal sheep adrenal during the prepartum period when adrenal growth and steroidogenesis is dependent on ACTH stimulation. We have found that, while the level of expression of StAR protein is decreased in the adrenal gland of the growth-restricted fetus during late gestation, this does not impair adrenal steroidogenesis. Our data also suggest that the stimulation of adrenal growth and steroidogenesis in the growth-restricted fetus may not be ACTH dependent.     

Transmural gradients in Na/K pump activity and [Na+]I in canine ventricle

There are well-documented differences in ion channel activity and action potential shape between epicardial (EPI), midmyocardial (MID), and endocardial (ENDO) ventricular myocytes. The purpose of this study was to determine if differences exist in Na/K pump activity. The whole cell patch-clamp was used to measure Na/K pump current (I(P)) and inward background Na(+)-current (I(inb)) in cells isolated from canine left ventricle. All currents were normalized to membrane capacitance. I(P) was measured as the current blocked by a saturating concentration of dihydro-ouabain. [Na(+)](i) was measured using SBFI-AM. I(P)(ENDO) (0.34 +/- 0.04 pA/pF, n = 17) was smaller than I(P)(EPI) (0.68 +/- 0.09 pA/pF, n = 38); the ratio was 0.50 with I(P)(MID) being intermediate (0.53 +/- 0.13 pA/pF, n = 19). The dependence of I(P) on [Na(+)](i) or voltage was essentially identical in EPI and ENDO (half-maximal activation at 9-10 mM [Na(+)](i) or approximately -90 mV). Increasing [K(+)](o) from 5.4 to 15 mM caused both I(P)(ENDO) and I(P)(EPI) to increase, but the ratio remained approximately 0.5. I(inb) in EPI and ENDO were nearly identical ( approximately 0.6 pA/pF). Physiological [Na(+)](i) was lower in EPI (7 +/- 2 mM, n = 31) than ENDO (12 +/- 3 mM, n = 29), with MID being intermediate (9 +/- 3 mM, n = 22). When cells were paced at 2 Hz, [Na(+)](i) increased but the differences persisted (ENDO 14 +/- 3 mM, n = 10; EPI 9 +/- 2 mM, n = 10; and MID intermediate, 11 +/- 2 mM, n = 9). Based on these results, the larger I(P) in EPI appears to reflect a higher maximum turnover rate, which implies either a larger number of active pumps or a higher turnover rate per pump protein. The transmural gradient in [Na(+)](i) means physiological I(P) is approximately uniform across the ventricular wall, whereas transporters that utilize the transmembrane electrochemical gradient for Na(+), such as Na/Ca exchange, have a larger driving force in EPI than ENDO.     

The role of ether-a-go-go-related gene K(+) channels in glucocorticoid inhibition of adrenocorticotropin release by rat pituitary cells

The present study investigated the role of K(+) channels in the inhibitory effect of glucocorticoid on adrenocorticotropin (ACTH) release induced by corticotropin-releasing hormone (CRH) using cultured rat anterior pituitary cells. Apamin and charybdotoxin (CTX) did not have a significant effect on ACTH release induced by CRH (1 nM). Tetraethylammonium (TEA), a broad spectrum K(+) channel blocker, increased the ACTH response to CRH only at the highest concentration (10 mM). The exposure to 100 nM corticosterone for 60 min inhibited the CRH-induced ACTH release. Neither TEA, apamin, nor CTX affected the inhibitory effect of corticosterone. In contrast, astemizole (Ast) and E-4031, ether-a-go-go-related gene (erg) K(+) channel blockers, abolished the inhibitory effect of corticosterone on CRH-induced ACTH release (1.25+/-0.10 vs. 1.45+/-0.11 ng/well at 10 microM Ast, p>0.05, 1.71+/-0.16 vs. 1.91+/-0.32 ng/well at 10 microM E-4031, p>0.05, vehicle vs. corticosterone). RT-PCR demonstrated all three subtypes of rat-erg mRNAs in the pituitary and corticosterone increased only erg1 mRNA up to 2.47+/-0.54 fold. In conclusion, erg K(+) channels were up-regulated by glucocorticoid, and have indispensable roles in delayed glucocorticoid inhibition of CRH-induced ACTH release by rat pituitary cells.     

Enhanced circadian ACTH release in obese premenopausal women: reversal by short-term acipimox treatment

Several studies suggest that the hypothalamo-pituitary-adrenal (HPA) axis is exceedingly active in obese individuals. Experimental studies show that circulating free fatty acids (FFAs) promote the secretory activity of the HPA axis and that human obesity is associated with high circulating FFAs. We hypothesized that HPA axis activity is enhanced and that lowering of circulating FFAs by acipimox would reduce spontaneous secretion of the HPA hormonal ensemble in obese humans. To evaluate these hypotheses, diurnal ACTH and cortisol secretion was studied in 11 obese and 9 lean premenopausal women (body mass index: obese 33.5 +/- 0.9 vs. lean 21.2 +/- 0.6 kg/m(2), P < 0.001) in the early follicular stage of their menstrual cycle. Obese women were randomly assigned to treatment with either acipimox (inhibitor of lipolysis, 250 mg orally four times daily) or placebo in a double-blind crossover design, starting one day before admission until the end of the blood-sampling period. Blood samples were taken during 24 h with a sampling interval of 10 min for assessment of plasma ACTH and cortisol concentrations. ACTH and cortisol secretion rates were estimated by multiparameter deconvolution analysis. Daily ACTH secretion was substantially higher in obese than in lean women (7,950 +/- 1,212 vs. 2,808 +/- 329 ng/24 h, P = 0.002), whereas cortisol was not altered (obese 36,362 +/- 5,639 vs. lean 37,187 +/- 4,239 nmol/24 h, P = 0.912). Acipimox significantly reduced ACTH secretion in the obese subjects (acipimox 5,850 +/- 769 ng/24 h, P = 0.039 vs. placebo), whereas cortisol release did not change (acipimox 33,542 +/- 3,436 nmol/24 h, P = 0.484 vs. placebo). In conclusion, spontaneous ACTH secretion is enhanced in obese premenopausal women, whereas cortisol production is normal. Reduction of circulating FFA concentrations by acipimox blunts ACTH release in obese women, which suggests that FFAs are involved in the pathophysiology of this neuroendocrine anomaly.     

Activation of adrenal function in fetal sheep by the infusion of adrenocorticotropin to the fetus in utero

We determined whether ACTH1-24, infused into fetal lambs at a rate that is known to cause premature labor, elicits changes in the responsiveness of the fetal adrenal glands, and alters the pattern of corticosteroid output. Plasma cortisol (F), corticosterone (B) and progesterone (P4) were measured during 72 h of infusion of saline or ACTH (10 micrograms/h) beginning on Day 127 of pregnancy. Adrenals were then dispersed into isolated cells, and the output of F, B and P4 after exogenous ACTH determined in vitro. Plasma concentrations of F and B were higher in ACTH-treated fetuses. The increment in F (5-to 7-fold) was greater than that in B (2-fold) such that the F:B ratio in plasma of ACTH-treated fetuses on Days 2 and 3 of infusion was 2.5 times higher than in controls. After 72 h of infusion, the adrenal weights in ACTH-treated fetuses (741 +/- 38 mg, +/- SEM; n = 4) were greater than in the control animals (349 +/- 11 mg). There was a significant effect of ACTH pretreatment in vivo on F output by isolated adrenal cells in vitro. Mean increments in F output after addition of ACTH1-24 (5000 pg/ml) in vitro rose from 368 +/- 235 pg/50,000 cells in controls, to 64,639 +/- 19,875 pg/50,000 cells after ACTH in vivo. There was no significant effect of ACTH in vivo on B output in vitro; the ratio of F:B output, either in the absence or presence of ACTH in vitro, was significantly higher in cells from ACTH-pretreated fetuses. There was a significant effect of in vivo ACTH on in vitro P4 output. After ACTH treatment in vivo there was an increase in the vitro output ratio of F:P4, but no change in the output ratio of B:P4. We conclude that ACTH treatment of the fetal lamb in vivo results in activation of fetal adrenal function, increased fetal adrenal responsiveness to ACTH, and directed corticosteroid biosynthesis towards cortisol. Our results are consistent with an increase in fetal adrenal 17 alpha-hydroxylase activity after ACTH treatment.     

Adrenocortical responses to ACTH in neonatal rats: effect of hypoxia from birth on corticosterone,StAR, and PBR

The adrenocortical response to hypoxia may be a critical component of the adaptation to this common neonatal stress. Little is known about adrenal function in vivo in hypoxic neonates. The purpose of this study was to evaluate adrenocortical responses to ACTH in suckling rat pups exposed to hypoxia from birth to 5-7 days of age compared with normoxic controls. We also evaluated potential cellular controllers of steroidogenic function in situ. In 7-day-old pups at 0800, hypoxia from birth resulted in increased basal (12.2 +/- 1.4 ng/ml; n = 12) and ACTH-stimulated (94.0 +/- 9.4 ng/ml; n = 14) corticosterone levels compared with normoxic controls (basal = 8.3 +/- 0.5 ng/ml; n = 11; stimulated = 51.3 +/- 3.8 ng/ml; n = 8). This augmentation occurred despite no significant difference in plasma ACTH levels in normoxic vs. hypoxic pups before (85 +/- 4 vs. 78 +/- 8 pg/ml) or after (481 +/- 73 vs. 498 +/- 52 pg/ml) porcine ACTH injection (20 microg/kg). This effect was similar in the afternoon at 6 days of age and even greater at 5 days of age at 0800. The aldosterone response to ACTH was not augmented by exposure to hypoxia from birth. Adrenocortical hypoxia-inducible factor (HIF)-1alpha mRNA was undetectable by RT-PCR. Steroidogenic acute regulatory (StAR) protein in adrenal subcapsules (zona fasciculata/reticularis) was augmented by exposure to hypoxia; this effect was greatest at 5 days of age. Peripheral-type benzodiazepine receptor (PBR) protein was also increased at 6 and 7 days of age in pups exposed to hypoxia from birth. We conclude that hypoxia from birth results in an augmentation of the corticosterone but not aldosterone response to ACTH. This effect appears to be mediated at least in part by an increase in controllers of mitochondrial cholesterol transport (StAR and PBR) and to occur independently of measurable changes in endogenous plasma ACTH. The augmentation of the corticosterone response to acute increases in ACTH in hypoxic pups is likely to be an important component of the overall physiological adaptation to hypoxia in the neonate.     

Structure-activity relationships of monomeric and dimeric synthetic ACTH fragments in perifused frog adrenal slices

The effect of synthetic monomeric and dimeric ACTH fragments on spontaneous and ACTH(1-39)-evoked steroidogenesis in frog interrenal tissue was studied in vitro. Infusion of ACTH fragment 11-24 (10(-6) M) or its dimeric conjugates, attached either by their N-terminal, Glu(11-24)2, or their C-terminal amino acid, (11-24)2Lys, had no effect on the spontaneous release of corticosteroids. The monomer ACTH(11-24) and the dimer Glu(11-24)2 were also totally devoid of effect on the steroidogenic response to ACTH(1-39) (10(-9)M). In contrast, the (11-24)2Lys conjugate (10(-6)M) significantly decreased ACTH-induced stimulation of corticosterone and aldosterone (-63 and -62%, respectively). The dimeric conjugate of the fragment ACTH(7-24), linked through the C-terminal ends, (7-24)2Lys (10(-6)M), was also completely devoid of effect on basal steroidogenesis but caused a marked decrease of ACTH-evoked corticosterone and aldosterone release (-72 and -80%, respectively). Conversely, infusion of the dimer (1-24)2Lys gave rise to a dose-related stimulation of corticosterone and aldosterone release. The time-course of the steroidogenic response to the dimer was similar to that of ACTH(1-24). The 1-24 conjugate was 70 times less potent than the monomers ACTH(1-24) and ACTH(1-39). These results suggest that amphibian adrenocortical cells contain only one class of ACTH receptor which recognizes the 11-24 domain of ACTH with an affinity which depends on the presence of a strong potentiator segment, located at the N-terminus end of ACTH(1-39). Since the ACTH-dimers are thought to induce cross-linking of the receptors, our results suggest that aggregation of ACTH receptors causes a down-regulation of the receptors.