Corticosteroid Output Research Articles

How should the psychological well‐being of zoo elephants be objectively investigated?

Animal welfare (sometimes termed "well-being") is about feelings - states such as "suffering" or "contentment" that we can infer but cannot measure directly. Welfare indices have been developed from two main sources: studies of suffering humans, and of research animals deliberately subjected to challenges known to affect emotional state. We briefly review the resulting indices here, and discuss how well they are understood for elephants, since objective welfare assessment should play a central role in evidence-based elephant management. We cover behavioral and cognitive responses (approach/avoidance; intention, redirected and displacement activities; vigilance/startle; warning signals; cognitive biases, apathy and depression-like changes; stereotypic behavior); physiological responses (sympathetic responses; corticosteroid output - often assayed non-invasively via urine, feces or even hair; other aspects of HPA function, e.g. adrenal hypertrophy); and the potential negative effects of prolonged stress on reproduction (e.g. reduced gametogenesis; low libido; elevated still-birth rates; poor maternal care) and health (e.g. poor wound-healing; enhanced disease rates; shortened lifespans). The best validated, most used welfare indices for elephants are corticosteroid outputs and stereotypic behavior. Indices suggested as valid, partially validated, and/or validated but not yet applied within zoos include: measures of preference/avoidance; displacement movements; vocal/postural signals of affective (emotional) state; startle/vigilance; apathy; salivary and urinary epinephrine; female acyclity; infant mortality rates; skin/foot infections; cardio-vascular disease; and premature adult death. Potentially useful indices that have not yet attracted any validation work in elephants include: operant responding and place preference tests; intention and vacuum movements; fear/stress pheromone release; cognitive biases; heart rate, pupil dilation and blood pressure; corticosteroid assay from hair, especially tail-hairs (to access endocrine events up to a year ago); adrenal hypertrophy; male infertility; prolactinemia; and immunological changes.

Rapid and fine adjustment of corticosteroidogenesis operating in the adrenal gland of the Mongolian gerbil (Meriones unguiculatus) superfused in-vitro.

Corticosteroid release from adrenals of male adult Mongolian gerbils (Meriones unguiculatus) has been studied during continuous and discontinuous in-vitro superfusion. Corticosteroid output from glands of untreated animals with plasma corticosteroid levels below 300 ng/ml was 3.3 +/- 0.3 ng/adrenal pair/min and decreased only slightly with the length of superfusion (60 min: 3.1 +2- 0.3 ng/adrenal pair/min). In-vitro secretion was significantly higher from adrenals of animals which had corticosteroid plasma levels of over 300 ng/ml, underwent clonic-tonic seizures, or were injected with 6 IU/animal (1-24) ACTH. On the other hand, injection of 2 X 50 micrograms dexamethasone markedly decreased corticosteroid plasma levels but had no significant effect on in-vitro secretion of corticosteroids. In contrast to the slow and small, but long-lasting stimulation of corticosteroid secretion elicited by (1-24) ACTH, secretion could be changed within much shorter time periods, either by the addition of plasma proteins to superfusion medium or by stops of superfusion flow. While a significant stimulation of corticosteroid output occurred after the addition of 1% or 10% BSA or rat plasma, stops of superfusion flow for 1.5 or 10 min resulted in a strong inhibition of steroidogenesis, as is evident from corticosteroid amounts found in the first 1-min samples after re-start of superfusion. Within 4-5 min after re-start of superfusion, secretion returned to basal values. Corticosteroid amounts secreted from adrenals superfused in-vitro were significantly higher than those secreted from adrenals incubated in-vitro. In addition, prolonged incubation suppressed corticosteroidogenesis (30 min: 100%, 60 min: 64%, 90 min: 56%, 120 min: 59%). The results demonstrate that superfusion of sliced adrenal tissue gives insights into aspects of adrenal function, including the rapid changes in synthesis and secretion after flow stops which cannot be investigated by incubation of either tissue slices or isolated cells. The possibility that the observed decline in corticosteroidogenesis during flow stops may be due to a feedback inhibition resulting from corticosteroids accumulating within slices is discussed.

Expression of prepro-ghrelin and related receptor genes in the rat adrenal gland and evidences that ghrelin exerts a potent stimulating effect on corticosterone secretion by cultured rat adrenocortical cells

The orexigenic peptide ghrelin (GHREL) and obestatin (OBS) originate from the same peptide precursor, preproghrelin (ppGHREL). Apart from orexigenic effect, GHREL also regulates neuroendocrine function. We investigated GHREL and OBS effects on corticosterone secretion by freshly isolated and cultured rat adrenocortical cells. Classic RT-PCR revealed the presence of ppGHREL, GHS-R1a, GPR39v1 and GPR39v2 and GOAT4 (ghrelin O-acyl transferase) mRNAs in rat adrenals and cultured for 4 days rat adrenocortical cells. Expression of ppGHREL, GHS-R1a, and GOAT genes was notably higher in the cortex than in medulla. High expression level of GOAT gene was found in the zona glomerulosa, while expression level of both GPR39v1 and GPR39v2 genes was similar in adrenal cortical zones and in medulla. In freshly isolated cells neither GHREL nor OBS had an effect on corticosteroid output. Prolonged exposure of cultured cells to GHREL resulted in a potent, comparable to ACTH, stimulating effect of GHREL on corticosterone secretion. Prolonged exposure to OBS was ineffective. Neither GHREL nor OBS had any effect on proliferation of studied cells, while ACTH notably lowered it. GHREL down regulated GHS-R1a gene expression while both ACTH and GHREL stimulated expression level of GPR39v1 gene. Expression of CYP11A1 gene was notably stimulated and that of StAR gene remained unaffected by ACTH or GHREL. Thus, our study is the first to demonstrate direct stimulating effect of GHREL on corticosterone output by cultured rat adrenocortical cells. This stimulating action differs from that evoked by ACTH and is not dependent on the presence of functional ACTH receptor.

In vitro effects of crude extracts of Parkia biglobosa (Mimosaceae), Stereospermum kunthianum (Bignoniaceae) and Biophytum petersianum (Oxalidaceae) on corticosteroid secretion in rat

Previous studies conducted in guinea pig, rat and rabbit have revealed that crude extracts from Parkia biglobosa, Stereospermum kunthianum and Biophytum petersianum exert hypotensive and/or hypoglycemic activities. Since corticosteroids are involved in the control of arterial blood pressure and glycemia, we have investigated the possible effects of these plant extracts on rat adrenal tissue in vitro. Short-term administration of crude semi-ethanolic extracts of P. biglobosa and S. kunthianum to perifused rat adrenal tissue did not induce any significant changes in corticosteroid output. Conversely, the B. petersianum extract caused a dose-dependent increase in corticosterone and aldosterone secretion. Repeated infusions or prolonged administration of B. petersianum extract did not produce any apparent attenuation of the steroid response. Altogether, these data indicate that a semi-ethanolic extract of B. petersianum dose-dependently stimulates corticosterone and aldosterone secretion in rat without any desensitization phenomenon.

Immunohistochemical localization, biochemical characterization, and biological activity of neurotensin in the frog adrenal gland.

The primary structure of neurotensin has been recently determined for the frog Rana ridibunda (Endocrinology 139: 4140-4146, 1998). In the present study, we have investigated the distribution and biochemical characterization of neurotensin-like immunoreactivity in the frog adrenal gland, using an antiserum directed against the conserved C-terminal region of the peptide. Neurotensin-like immunoreactivity was detected in two populations of nerve fibers: numerous varicose fibers coursing between adrenal cells, and a few processes located in the walls of blood vessels irrigating the gland. Reversed-phase HPLC analysis of frog adrenal gland extracts revealed the existence of a major peak of neurotensin-like immunoreactivity that exhibited the same retention time as synthetic frog neurotensin. The possible involvement of neurotensin in the regulation of steroid secretion was studied in vitro using perifused frog adrenal slices. For concentrations ranging from 10(-10) to 10(-5) M, synthetic frog neurotensin increased corticosterone and aldosterone production in a dose-dependent manner (EC50 = 1.2 x 10(-9) M and 5.8 x 10(-10) M, respectively). Repeated administration of neurotensin induced a reproducible stimulation of steroid output without any tachyphylaxis. Prolonged administration (3 h) of frog neurotensin caused a transient increase in corticosterone and aldosterone secretion followed by a decline of corticosteroid secretion. Neurotensin also produced a significant stimulation of corticosteroid secretion from dispersed frog adrenal cells. This study demonstrates that neurotensin is located in nerve processes innervating the adrenal gland of amphibians. The results also show that synthetic frog neurotensin exerts a direct stimulatory effect on corticosteroid output. Taken together, these data support the view that neurotensin, released by nerve fibers, may act as a local regulator of corticosteroid secretion.

The Effect of the Endozepine Triakontatetraneuropeptide on Corticosteroid Secretion by the Frog Adrenal Gland Is Mediated by Activation of Adenylyl Cyclase and Calcium Influx through T-Type Calcium Channels

We have recently found that, in the frog adrenal gland, endozepines are present in chromaffin cells and we have shown that the triakontatetraneuropeptide TTN is a potent stimulator of corticosteroid secretion in vitro. In the present study, we have investigated the transduction mechanisms mediating the corticotropic effect of TTN on adrenocortical cells. Incubation of adrenal explants with graded concentrations of TTN induced a dose-dependent increase in cAMP formation, but did not affect polyphosphoinositide metabolism. Pretreatment of adrenal cells with the protein kinase A inhibitor H-89 markedly reduced the stimulatory effect of TTN on corticosterone and aldosterone secretion by perifused cells, whereas the phospholipase C inhibitor U-73122 did not affect the TTN-evoked stimulation of corticosteroid output. Incubation of adrenal cells with cholera toxin abolished the stimulatory effect of TTN on steroid secretion. Administration of a brief pulse of TTN (10−6m) in the vicinity of cultured adrenocortical cells induced a robust increase in the concentration of intracellular calcium ([Ca2+]i). Repeated pulses of TTN resulted in a gradual attenuation of the responses, indicating the existence of a desensitization phenomenon. Incubation of the cells with the T-type calcium channel blocker mibefradil significantly reduced the TTN-evoked[ Ca2+]i increase, whereas the L-type calcium channel blocker nifedipine and the N-type calcium channel blockerω -conotoxin GVIA had no effect. Incubation of adrenal cells with H-89 markedly reduced the stimulatory effect of TTN on[ Ca2+]i. The involvement of calcium in steroid secretion induced by TTN has also been investigated. Administration of mibefradil significantly reduced the TTN-evoked stimulation of steroid production, whereas nifedipine was devoid of effect. Taken together, these data indicate that in frog adrenocortical cells, the endozepine TTN stimulates cAMP formation and calcium entry through T-type calcium channels. The effects of TTN on the adenylyl cyclase/protein kinase A pathway and calcium influx both contribute to the stimulatory action of the peptide on corticosteroid secretion.

The effect of the endozepine triakontatetraneuropeptide on corticosteroid secretion by the frog adrenal gland is mediated by activation of adenylyl cyclase and calcium influx through T-type calcium channels.

We have recently found that, in the frog adrenal gland, endozepines are present in chromaffin cells and we have shown that the triakontatetraneuropeptide TTN is a potent stimulator of corticosteroid secretion in vitro. In the present study, we have investigated the transduction mechanisms mediating the corticotropic effect of TTN on adrenocortical cells. Incubation of adrenal explants with graded concentrations of TTN induced a dose-dependent increase in cAMP formation, but did not affect polyphosphoinositide metabolism. Pretreatment of adrenal cells with the protein kinase A inhibitor H-89 markedly reduced the stimulatory effect of TTN on corticosterone and aldosterone secretion by perifused cells, whereas the phospholipase C inhibitor U-73122 did not affect the TTN-evoked stimulation of corticosteroid output. Incubation of adrenal cells with cholera toxin abolished the stimulatory effect of TTN on steroid secretion. Administration of a brief pulse of TTN (10(-6) M) in the vicinity of cultured adrenocortical cells induced a robust increase in the concentration of intracellular calcium ([Ca2+]i). Repeated pulses of TTN resulted in a gradual attenuation of the responses, indicating the existence of a desensitization phenomenon. Incubation of the cells with the T-type calcium channel blocker mibefradil significantly reduced the TTN-evoked [Ca2+]i increase, whereas the L-type calcium channel blocker nifedipine and the N-type calcium channel blocker omega-conotoxin GVIA had no effect. Incubation of adrenal cells with H-89 markedly reduced the stimulatory effect of TTN on [Ca2+]i. The involvement of calcium in steroid secretion induced by TTN has also been investigated. Administration of mibefradil significantly reduced the TTN-evoked stimulation of steroid production, whereas nifedipine was devoid of effect. Taken together, these data indicate that in frog adrenocortical cells, the endozepine TTN stimulates cAMP formation and calcium entry through T-type calcium channels. The effects of TTN on the adenylyl cyclase/protein kinase A pathway and calcium influx both contribute to the stimulatory action of the peptide on corticosteroid secretion.

Effect of the triakontatetraneuropeptide (TTN) on corticosteroid secretion by the frog adrenal gland

Diazepam-binding inhibitor (DBI) was initially isolated from the rat brain as a result of its ability to compete with benzodiazepines for their receptors. Immunohistochemical studies have recently shown the presence of peripheral-type benzodiazepine receptor (PBR)- and DBI-like immunoreactivity in the frog adrenal gland. The aim of the present study was to investigate the effect of two biologically active DBI-derived peptides, the triakontatetraneuropeptide [TTN; DBI(17-50)] and the octadecaneuropeptide [ODN; DBI(33-50)], on corticosteroid secretion by frog adrenocortical cells. Exposure of frog adrenal explants to graded concentrations of TTN (3.16 x 10(-8) to 3.16 x 10(-6) M) induced a dose-related increase in corticosterone and aldosterone secretion. In contrast, ODN did not modify corticosteroid output. When repeated pulses of TTN (10(-6) M) were administered at 2-h intervals, the response of the adrenal explants to the second dose of TTN was markedly reduced, suggesting the existence of a desensitization phenomenon. Exposure of dispersed adrenal cells to TTN also induced a marked stimulation of corticosteroid secretion, indicating that TTN acts directly on adrenocortical cells. The central-type benzodiazepine receptor (CBR) agonist, clonazepam, did not stimulate corticosteroid secretion and the CBR antagonist, flumazenil, did not block the stimulatory action of TTN. Similarly, the PBR agonist, Ro5-4864, did not mimic the stimulatory effect of TTN and the PBR antagonist, flunitrazepam, did not affect the stimulatory action of TTN. The present study provides the first evidence for a stimulatory effect of TTN on intact adrenocortical cells. The receptor mediating the corticotropic action of TTN is not related to central- or peripheral-type benzodiazepine receptors. Our data suggest that TTN, released by chromaffin cells, may act as a paracrine factor regulating the activity of adrenocortical cells.

Adrenal vasoactive intestinal peptide participates in neonatal corticosteroid production in the rat.

Neonatal rats (3-14 days old) exhibit a period of adrenal hyporesponsiveness characterized by blunted corticosterone (B) responses to stress and reduced adrenal sensitivity to adrenocorticotropic hormone (ACTH). Several adrenomedullary peptidergic systems like vasoactive intestinal peptide (VIP) are postulated to influence cortical function. VIP is known to stimulate corticosterone secretion in vitro and to be released from the adrenal medulla following splanchnic nerve stimulation. Here, we tested whether 1) accelerated sympathetic innervation of the adrenal gland by daily L-thyroxine (T4) treatment modified the ontogeny of adrenal VIP and 2) an increase in VIP synthesis could prematurely increase adrenal sensitivity and corticosteroid output during neonatal life. Immunohistochemical VIP staining revealed a different ontogenetic pattern between adrenal regions from days 2-18 and different sensitivities to T4 treatment. Capsular staining was most abundant at all ages and increased with T4 treatment, whereas medullary staining was seen by day 18 and was not affected by T4. Throughout development, VIP receptors were detected mostly in the capsular region, but not in the adrenal cortex. Although receptor levels were not modified by T4 injections, T4 significantly enhanced VIP mRNA levels in the whole adrenal at all ages. In vivo administration of VIP (0.1-2.0 mg/kg body wt ip) to 9- to 12-day-old neonates increased pituitary ACTH, adrenal B, and aldosterone secretion significantly. Corticotropin-releasing factor immunoneutralization before VIP injection diminished VIP-induced ACTH release but still produced small but significant B and aldosterone secretion. Our results show that 1) VIP innervation of the adrenal capsule is present soon after birth and is increased by sympathetic activity whereas VIP appears only much later in the medulla and does not coincide with the onset of splanchnic innervation and 2) exogenous VIP stimulates ACTH, B, and aldosterone release during development and the effect of VIP on steroidogenic secretion is occurring through ACTH secretion, but also, at least in part, directly at the level of the adrenal gland.

Suppression of medical conditions by Cushing's syndrome

EDITOR,—Alan Steuer and colleagues describe the unusual occurrence of a medical condition (sarcoidosis) that was suppressible by steroids and presented shortly after surgical treatment of Cushing's syndrome.1 The corollary of this—namely, that a pre-existing medical condition may be suppressed by the onset of Cushing's syndrome, reflecting the increased output of corticosteroids—is also true and may help diagnosis. The case of a 66 year old man who presented with diarrhoea and …

Immunohistochemical distribution, biochemical characterization, and biological action of tachykinins in the frog adrenal gland.

The distribution of tachykinin-like immunoreactivity (LI) was studied in the adrenal gland of the frog Rana ridibunda using the immunofluorescence technique. A dense network of varicose fibers immunoreactive to both substance-P (SP) and neurokinin-A (NKA) was found in the adrenal tissue. In contrast, no positive fibers could be detected using antineurokinin-B (NKB) antibodies. At the electron microscope level, the immunogold technique revealed that tachykinin-LI was sequestered in dense core vesicles of 50-70 nm. Bilateral transection of either splanchnic or vagus nerves or total lesion of celiac sympathetic ganglion did not suppress tachykinin-LI. A combination of HPLC analysis and RIA detection was used to characterize tachykinin-LI in frog adrenal extracts. Two major peaks were resolved, which coeluted, respectively, with synthetic ranakinin, a novel tachykinin previously isolated from the frog brain, and [Leu3,Ile7]NKA previously isolated from the frog gut. No NKB could be detected in the extracts. The effects of various synthetic tachykinins on corticosteroid secretion were studied using perifused frog adrenal slices. For concentrations ranging from 10(-8)-10(-4) M, SP induced a dose-dependent stimulation of corticosterone and aldosterone release. A desensitization phenomenon was observed when iterative or prolonged infusions of SP were administered to the tissue. All mammalian or amphibian tachykinin-related peptides tested in our model also enhanced corticosteroid production. The effectiveness of the tachykinins tested was: [Pro7] NKB > NKA > ranakinin > [Pro9]SP > SP > kassinin > physalaemin > NKB > [Leu3,Ile7]NKA. SP also enhanced prostaglandin E2 and prostacyclin release in the effluent perifusate and the response preceded by 10-15 min the increase in corticosteroid output. Indomethacin (5 x 10(-6) M), a specific blocker of cyclooxygenase activity, totally suppressed SP-evoked steroid secretion. These data indicate that tachykinin-induced stimulation of steroidogenesis was mediated through activation of the arachidonic acid cascade. Taken together, our results show that the frog adrenal gland is innervated by a dense network of peptidergic fibers containing both ranakinin and [Leu3,Ile7]NKA, which, in vitro, stimulates corticosteroid secretion by adrenocortical cells through a prostaglandin-dependent mechanism. The present results support the view that tachykinins released by nerve fibers exert a neuroendocrine control on corticosteroid release in amphibians.

Feast and Famine: Critical Role of Glucocorticoids with Insulin in Daily Energy Flow

Summary: The hypothesis proposed in this review is that normal diurnal rhythms in the hypothalamic-pituitary-adrenal (HPA) axis are highly regulated by activity in medial hypothalamic nuclei to effect an interaction between corticosteroids and insulin such that optimal metabolism results in response to changes in the fed or fasted state of the animal. There are marked diurnal rhythms in function of the HPA axis under both basal and stress conditions. The HPA axis controls corticosteroid output from the adrenal and, in turn, forward elements of this axis are inhibited by feedback from circulating plasma corticosteroid levels. Basal activity in the HPA axis of mammals fed ad lib peaks about 2 h before the peak of the diurnal feeding rhythm, and is controlled by input from the suprachiasmatic nuclei. The rhythm in stress responsiveness is lowest at the time of the basal peak and highest at the time of the basal trough in the HPA axis activity. There are also diurnal rhythms in corticosteroid feedback sensitivity of basal and stress-induced ACTH secretion which peak at the time of the basal trough. These rhythms are all overridden when feeding, and thus insulin secretion, is disrupted. Corticosteroids interact with insulin on food intake and body composition, and curticosteroids also increase insulin secretion. Corticosteroids stimulate feeding at low doses but inhibit it at high doses; however, it is the high levels of insulin, induced by high levels of corticosteroids, that may inhibit feeding. The effects of corticosteroids on liver, fat, and muscle cell metabolism, with emphasis on their interactions with insulin, are briefly reviewed. Corticosteroids both synergize with and antagonize the effects of insulin. The effects of stress hormones, and their interactions with insulin on lipid and protein metabolism, followed by some of the metabolic effects of injury stress, with or without nutritional support, are evaluated. In the presence of elevated insulin stimulated by glucocorticoids and nutrition, stress causes less severe catabolic effects. In the central nervous system, regulation of function in the HPA axis is clearly affected by the activity of medial hypothalamic nuclei that also alter feeding, metabolism, and obesity in rats. Lesions of the arcuate (ARC) and ventromedial (VMN) paraventricular (PVN) nuclei result in obesity and hyperactivity in the HPA axis. Moreover, adrenalectomy inhibits or prevents development of the lesion-induced obesity. There are interactions among these nuclei; one mode of communication is via inputs of neuropeptide Y (NPY) cells in the ARC to the VMN, dorsomedial nuclei, and PVN. We suggest that these NPY-ergic pathways, the activity of which is increased by fasting, and reduced by feeding, insulin, and lack of glucocorticoids, may mediate the fasting-induced override of diurnal rhythms in the HPA axis. Supporting data are drawn from studies of diabetic rats and rodents with genetic obesities. We conclude that (a) There are marked interactions between glucocorticoids and insulin on most aspects of metabolism; (b) The NPY-ergic pathway from arcuate to paraventricular nuclei probably mediates fasting-induced alterations in hunger and the HPA axis activity; (c) modulation of NPY-ergic activity in the arcuate is mediated by glucocorticoids through stimulation of insulin secretion; insulin, in turn, inhibits NPY-ergic activity in the arcuate; (d) the interaction between corticosteroids and insulin serves as a peripheral hormonal feedback loop that regulates this NPY-ergic feeding-fasting system.

The differential regulation of aldosterone output in hamster adrenal by angiotensin II and adrenocorticotropin

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 ± 1.9 ng and 8.0 ± 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K + concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K +. Angiotensin II (A II) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10 −8 M A II, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10 −8 M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of A II and ACTH on adrenal cytochrome P-450 11 β involved in the last steps of aldosterone formation were evaluated by c combined in vivo and in vitro experiments. The P-450 11 β mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and A II differentially regulate P-450 11 β . It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.

Chronic Effects of Recombinant Porcine Growth Hormone on Adrenal Weight and Activity in Pigs

This study was conducted to examine serum cortisol concentrations and adrenal cortisol output in pigs treated chronically with recombinant pGH (rpGH) at a maximally effective anabolic dose. Recombinant pGH (140 micrograms/kg body weight) was administered daily to eight barrows for 77 d. At slaughter, adrenal glands were removed, weighed and sliced; slices of fresh adrenal tissue were incubated for 1 h in the presence or absence of ACTH. Recombinant pGH increased adrenal weight by 39%. This change was accompanied by an inversely proportional reduction of in vitro cortisol output per gram of tissue, with the net result that total cortisol output per adrenal per kilogram of BW was unaltered, as was cortisol output in the presence of ACTH. Serum cortisol concentrations were measured in 10 barrows fitted with femoral artery catheters and treated daily with 0 or 140 micrograms rpGH/kg BW for 8 d. Basal and ACTH-stimulated cortisol concentrations were not altered by rpGH treatment. These results do not support our earlier speculation that a pGH-dependent increase in adrenal weight is associated with a chronic increase in adrenal activity, but rather demonstrate that corticosteroid output is tightly regulated and remains constant despite a marked increase in the size of the adrenal glands.

Glucocorticoid enhancement of glucocorticoid production by cultured ovine adrenocortical cells

The present study examines the effect of chronic treatment with glucocorticoids on the steroidogenic activity of ovine adrenocortical cells in vitro. Cells cultured in the presence of 10(-9) to 10(-5) M dexamethasone produced more glucocorticosteroids in response to ACTH1-24, forskolin or 8 BrcAMP than did control cells. Such an enhancing effect required more than 5 h of treatment and was maximal at 30 h; it was both concentration-dependent and steroid-specific. The maximal secretion of corticosteroids was observed when cells were exposed to 10(-7) M dexamethasone; with higher concentrations the response to ACTH1-24 decreased steadily; the ED50 was 2.8 +/- 0.8 nM. Cortisol and corticosterone enhanced ACTH1-24-induced steroidogenesis to the same extent as dexamethasone, but at concentrations roughly 100-fold higher than for dexamethasone. Testosterone and 17 beta-oestradiol had no enhancing effect. Dexamethasone not only enhanced the maximal steroidogenic response to ACTH1-24 but also decreased its ED50 3-fold. Treatment of cultures with the antiglucocorticoid RU 38486 resulted in a dose-dependent, time-dependent, decrease in ACTH1-24-induced corticosteroid output. Moreover, RU 38486 antagonized the enhancing effect of dexamethasone. The production of corticosteroids by dexamethasone-treated cells incubated in the presence of 22(R)-hydroxycholesterol or of exogenous pregnenolone was similar to that of control cells. The enhancing effect of dexamethasone was also observed when cultures were performed in the absence of insulin and/or in serum-free media. These data suggest that chronic exposure to glucocorticoids is necessary for the full steroidogenic activity of ovine adrenocortical cells. Moreover, they indicate that glucocorticoids exert their effect at least at two different levels in the cell: (i) on the adenylate cyclase system and (ii) at step(s) beyond cAMP but before pregnenolone formation.

Involvement of the cytoskeleton in the steroidogenic response of frog adrenal glands to angiotensin II, acetylcholine and serotonin.

In order to determine the role of the cytoskeleton in adrenal steroidogenesis, we have studied the effect of cytochalasin B (a microfilament-disrupting agent) and vinblastine (an antimicrotubular drug) on corticosteroid secretion by frog interrenal tissue in vitro. Perifusion of interrenal fragments with cytochalasin B (50 mumol/l) induced a marked inhibition of basal corticosteroid output. In addition, stimulation of corticosteroidogenesis by all corticotrophic factors was also inhibited by cytochalasin B. Using an immunohistochemical technique and specific anti-tubulin antiserum, we verified that vinblastine (10 mumol/l) was responsible for the disappearance of the microtubular network in adrenocortical cells. Administration of vinblastine (10 mumol/l) did not affect the spontaneous secretion of corticosterone and aldosterone and had no effect on the steroidogenic response of interrenal glands to angiotensin II and acetylcholine. In contrast, vinblastine was responsible for a marked decrease in serotonin-induced stimulation of corticosteroid production. On the other hand, data from high-performance liquid chromatography showed that infusion of cytochalasin B or vinblastine was not associated with the production of any new steroid which could interfere in the radioimmunoassays. Taken together, these data suggest that microfilaments are involved in a late and common step of corticosteroidogenesis while microtubules are only required for the coupling of the secretory response to certain corticotrophic factors such as ACTH and serotonin.

Biocultural perspectives on stress in prehistoric, historical, and contemporary population research

Stress, a concept addressing the consequences of disruptive events on individuals and populations, can be a useful integrative idea. The stress process has much in common with its sister concept of adaptation. However, where adaptation focuses on “adaptive” or positive consequences, stress redresses an imbalance by focusing on the costs and limits of adaptation. In this paper we first review the interdisciplinary roots of the stress concept. While most stress research derives from research in environmental physiology, Selyean concepts of stress (involving increased catecholamine and corticosteroid output) have forced an expansion toward greater concern for perceptual and psychosocial stressors. What is largely missing from all traditions, however, is concern for sociopolitical processes which are not easily adapted to and consequently are persistent and pervasive causes of stress. Studies of stress in prehistoric, historical, and contemporary populations by biological anthropologists vary, in a complementary way, as to ability to delineate aspects of the stress process. Whereas the paleopathological methods of the prehistorian provide a suite of skeletal indicators of stress response, and the demographic measures of the historian provide a detailed analysis of consequence, a wide variety of techniques for examining all levels of the stress process are potentially available to those studying contemporary populations. In order to better utilize information from different levels of analysis one needs to focus on measures of stress, such as infant mortality, which are accessible at all levels. Biological anthropologists are in a unique position to elucidate the human condition if, via concepts such as stress, attention is paid to both human adaptive and political economic processes.

The influence of plasma lipoproteins on steroidogenesis of cultured ovine fetal and neonatal adrenal cells

The present study examined the effects of serum and lipoproteins on the function of cultured adrenal cells from 115–127-day-old ovine fetuses and from newborn lambs. On day 1 of culture, corticosteroid output was similar in medium containing 2% horse serum or in serum-free medium, both for fetal and neonatal cells. However, on day 5, cells cultured in the absence of serum produced smaller amounts of these steroids than cells maintained in medium containing serum; the difference was more marked under ACTH 1–24 stimulation. Conversely, cAMP production was never lower in the absence than in the presence of serum. When stimulated by ACTH 1–24 on day 2 of culture, fetal or neonatal adrenal cells incubated in the presence of a saturating concentration of ovine LDL produced more corticosteroids than cells incubated in serum-free medium; HDL also enhanced ACTH 1–24-induced steroidogenesis, but to a lesser extent. VLDL was effective only with neonatal cells. In fetal and neonatal cells cultured for 6 days in ACTH-free medium, VLDL and LDL increased ACTH-induced steroidogenesis, but HDL did not. On the other hand, when cells were cultured in the presence of ACTH 1–24, LDL and HDL were equipotent in supporting ACTH 1–24-induced steroid output. Three major lipoprotein fractions were observed in serum of fetal and newborn lambs. The concentration of cholesterol was very low in the VLDL fraction of fetuses, but it was similar to that of newborns in LDL. Conversely, 4 times more cholesterol was present in HDL of newborns than in HDL of fetuses. These results suggest that: (i) after several days of cell culture, cholesterol availability is an important limiting factor for the steroidogenesis of cells maintained under serum-free conditions; (ii) both an “LDL pathway” and an “HDL pathway” are operating in adrenal cells from fetal as well as newborn sheep; (iii) LDL and HDL are important physiological sources of cholesterol to support steroidogenesis by fetal and neonatal adrenal cells.

Transduction of acth signal from plasma membrane to mitochondria in adrenocortical steroidogenesis. Effects of peptide, phospholipid, and calcium

The conversion of cholesterol to pregnenolone by adrenocortical mitochondria is the rate-limiting step in steroidogenesis. This process is stimulated dramatically by the action of ACTH through the sequential reactions, in which adenyl cyclase, cAMP-dependent protein kinase, cholesterol esterase and ribosomal protein synthesis are all involved. The de novo synthesized protein, the so-called labile protein with a half-life of approx 10 min, is believed to stimulate the cholesterol side chain cleavage reaction by an unknown mechanism. Available evidence indicates that the electron transfer reaction from NADPH to P-450 scc is mediated rapidly by adrenodoxin reductase and p-450 scc. In addition, these redox components are inactivated slowly with a half-life of 3.5 days after hypophysectomy. It is known that the corticoid output from adrenocortical cells starts within 5 min and reaches the maximum after 10–15 min of ACTH administration to animals. One can assume that under normal physiological conditions, both O 2 and NADPH are not limiting. Additionally, mitochondrial inner membranes are poor in cholesterol. In this context, the availability of substrate cholesterol to P-450 sccis the most likely candidate for the regulatory mechanism.

Involvement of cycloheximide-sensitive mediators in the steroidogenic action of adrenocorticotropin and angiotensin II

The involvement of short-lived proteins in the steroidogenic action of corticotropic peptides has been investigated in vitro by means of a Perifusion technique using frog adrenal glands. Graded concentrations of cycloheximide (10 −7 M to 10 −5 M) led to a dose-related inhibition of corticosterone and aldosterone production. The Perifusion model gives detailed information on the kinetics of the inhibitory effect of cycloheximide. This effect was rapidly observed (the lag period was about 15 min), maximum inhibition being obtained 25 min after the end of administration of the protein synthesis inhibitor. Whatever the concentration of cycloheximide, corticosteroid output returned to basal values 2 h after the onset of cycloheximide infusion. Stimulation of steroidogenesis by ACTH and angiotensin II was totally inhibited by cycloheximide (10 −6 M) indicating that the synthesis of a labile protein was required for the adrenal response to corticotropic peptides. In addition, the stimulatory effect of cAMP and PGE1, which are considered to be the second messengers of ACTH and angiotensin II in amphibian interrenal gland, was blocked by cycloheximide. Taken together, these data suggest that a labile protein is involved in an early step of corticosteroid biosynthesis in the frog.