Experimental cardiac fibrosis: differential time course of responses to mineralocorticoid-salt administration.

The rapid (1-4 h) responses of epithelial target tissues to mineralocorticoids contrast with the days/weeks apparently required for responses in the cardiovascular system. The present study explores the time course and pattern of early events leading to cardiac fibrosis in the mineralocorticoid-salt rat model. Uninephrectomized rats were given deoxycorticosterone (20 mg, sc, weekly) plus 0.9% NaCl/0.3% KCl to drink and were killed at 2, 4, 8, 16, and 32 d. Type III collagen increased progressively from d 2, and blood pressure from d 4, with 4 and 8 d rats showing marked perivascular inflammatory cell infiltration. Apoptosis was also noted in perivascular areas at 4 and 8 d and in scar areas at 8, 16, and 32 d. Elevation of mineralocorticoid hormone levels inappropriate for salt status thus provokes a series of changes in cardiac vessels and myocytes leading to increased collagen deposition. When mineralocorticoid levels are elevated acutely by bolus injection, changes are discernible after 2 d, in contrast with previous infusion studies in which 3-4 wk were required for measurable changes.

The serum- and glucocorticoid-induced kinase is a physiological mediator of aldosterone action.

Aldosterone plays a major role in regulating sodium and potassium flux in epithelial tissues such as kidney and colon. Recent evidence suggests that serum- and glucocorticoid-regulated kinase (SGK) is induced by aldosterone and acts as a key mediator of aldosterone action in epithelial tissues. Induction of SGK messenger RNA (mRNA) has previously been shown within 30 min of addition of supraphysiological doses of aldosterone to Xenopus A6 cells and within 4 h in rat kidney in vivo. In this study we determined the time course of SGK induction, at doses of aldosterone in the physiological range, in rat kidney and colon, using Northern and Western blot analyses and in situ hybridization and determined concurrent changes in urinary sodium and potassium excretion by Kagawa bioassay. On Northern blot analysis, SGK mRNA levels were significantly elevated in both kidney and colon 60 min after the injection of aldosterone. SGK protein in late distal colon was significantly elevated 2 and 4 h after aldosterone treatment. In situ hybridization showed SGK mRNA to be induced in renal collecting ducts and distal tubular elements in both cortex and medulla by doses of aldosterone of 0.1 microg/100 g BW or more within 30 min of steroid treatment. Significant changes in urinary composition were similarly seen with an aldosterone dose of 0.1 microg/100 g BW from 90 min after aldosterone injection. The early onset of SGK induction in kidney and colon and the correlation with urinary changes in terms of both time course and dose response suggest that SGK plays an important role in mediating the effects of aldosterone on sodium homeostasis in vivo.

Dynamin II regulates hormone secretion in neuroendocrine cells.

The dynamin family of GTP-binding proteins has been implicated as playing an important role in endocytosis. In Drosophila shibire, mutations of the single dynamin gene cause blockade of endocytosis and neurotransmitter release, manifest as temperature-sensitive neuromuscular paralysis. Mammals express three dynamin genes: the neural specific dynamin I, ubiquitous dynamin II, and predominantly testicular dynamin III. Mutations of dynamin I result in a blockade of synaptic vesicle recycling and receptor-mediated endocytosis. Here, we show that dynamin II plays a key role in controlling constitutive and regulated hormone secretion from mouse pituitary corticotrope (AtT20) cells. Dynamin II is preferentially localized to the Golgi apparatus where it interacts with G-protein betagamma subunit and regulates secretory vesicle release. The presence of dynamin II at the Golgi apparatus and its interaction with the betagamma subunit are mediated by the pleckstrin homology domain of the GTPase. Overexpression of the pleckstrin homology domain, or a dynamin II mutant lacking the C-terminal SH3-binding domain, induces translocation of endogenous dynamin II from the Golgi apparatus to the plasma membrane and transformation of dynamin II from activity in the secretory pathway to receptor-mediated endocytosis. Thus, dynamin II regulates secretory vesicle formation from the Golgi apparatus and hormone release from mammalian neuroendocrine cells.

How to write a paper for publication.

Engaging in the scientific publication process can be for both altruistic and egotistical reasons; publication advances the state of scientific knowledge while advancing your institution and your career. Writing for publication means setting aside a location and time dedicated entirely to the process of planning and writing. It is easiest to begin with the Methods section, then the Results, followed by the Discussion, which is the most challenging part of a paper. A realistic assessment of the value of the article will determine the level of journal into which it is likely to gain acceptance. If your article is rejected by a journal, be consoled by the fact that 50% of articles that are initially rejected are eventually published. Following the steps outlined here can reduce the daunting task of writing to one of manageable proportions and can help overcome the mental block and procrastination that all of us have experienced when we set out to write a scientific paper.

The glucocorticoid receptor is essential for maintaining basal and dexamethasone-induced repression of the murine corticosteroid-binding globulin gene.

We have investigated hepatic expression and glucocorticoid regulation of the corticosteroid-binding globulin (CBG) gene in mice lacking a functional glucocorticoid receptor (GR). GR-/- mice show impaired negative feedback in the hypothalamic-pituitary-adrenal axis, resulting in elevated circulating levels of ACTH and corticosterone. This is seen in the neonatal period and continues into adulthood where ACTH and corticosterone levels are increased up to 4-5 fold. Despite high elevation of corticosterone we find no change in mean arterial blood pressure in GR-/- mice and no change in the renal activity of the glucocorticoid-metabolising enzymes 11beta-hydroxysteroid dehydrogenase type-1 (HSD1) and type-2 (HSD2). We do find markedly increased hepatic expression of CBG with a 50% increase in plasma CBG levels. Increased expression of CBG was detected in adult GR-/- mice and also at birth with a greater than 10-fold increase in CBG hepatic mRNA in day-18.5 embryonic GR-/- mice. Adult GR-/- mice were also resistant to dexamethasone-induced repression of CBG expression in the liver. These results indicate that in mice, GR is essential for maintaining the basal level of CBG gene expression in the liver, and is also required for dexamethasone-induced repression of the CBG gene in the adult.

Effects of estrogen and estrous cycle on glucocorticoid and catecholamine responses to stress in sheep.

There have been relatively few studies of the effects of estrogen on hormonal responses to stress. We therefore studied changes in ACTH, cortisol, norepinephrine (NE), and epinephrine (Epi) after stress induced by a barking dog (audiovisual stressor) and insulin-induced hypoglycemia (metabolic stressor) in ovariectomized sheep treated with estradiol or placebo and in intact sheep in the follicular and luteal phases of the estrous cycle. Both stressors produced acute increases in ACTH, cortisol, Epi, and NE. A high physiological dose of estradiol significantly reduced the ACTH and cortisol responses to both stressors but did not affect Epi and NE responses. Plasma ACTH and cortisol responses to both stressors and Epi and NE responses to insulin were lower in the follicular than in the luteal phase, but catecholamine responses to the audiovisual stressor did not change during the estrous cycle. We conclude that in sheep, estrogen attenuates glucocorticoid responses to stress and that hormonal changes during the estrous cycle affect glucocorticoid responses to both metabolic and audiovisual stressors and catecholamine responses to a metabolic stressor.

cAMP modulates glucocorticoid-induced protein accumulation and glucocorticoid receptor in cardiomyocytes.

Glucocorticoids have complex effects on cardiac muscle growth in vivo, and one possible reason may the regulatory cross talk between glucocorticoids and second messengers. In this study we investigated the effect of adenosine 3',5'-cyclic monophosphate (cAMP), shown to affect cardiomyocyte growth and glucocorticoid action in several systems, on glucocorticoid-induced protein accumulation and glucocorticoid receptor (GR) in neonatal rat cardiomyocytes. Dexamethasone (DEX) decreased the protein-to-DNA ratio, and 8-bromoadenosine 3',5'-cyclic monophosphate (BrcAMP) or forskolin increased this ratio. The inhibitory effect of DEX was potentiated by an elevated cAMP, despite the stimulatory effect of cAMP alone. Nuclear GR binding was increased by BrcAMP, with no change in GR mRNA or protein levels, via increased affinity of nuclear GR. H-89 blocked the effects of BrcAMP. In conclusion, glucocorticoids have an inhibitory effect on protein accumulation in cardiomyocytes via GR, an effect potentiated by elevated cAMP via increased nuclear GR binding. These results suggest that glucocorticoid effects on cardiomyocytes may be modulated by cAMP-mediated mechanisms, which may produce the complex effects of glucocorticoids on cardiomyocyte growth in vivo.

Glucocorticoid receptor expression is down-regulated by Lp(a) lipoprotein in vascular smooth muscle cells.

Glucocorticoids have been reported to protect against atherosclerosis and have been used clinically as protective therapy for restenosis after balloon angioplasty. Recently, Lp(a) lipoprotein [Lp(a)] levels have been suggested to be an independent risk factor for atherosclerosis, although its mechanisms of action are still uncertain. To clarify this atherogenic mechanism of Lp(a), we investigated the effects of Lp(a) on glucocorticoid receptor (GR) expression in human vascular smooth muscle cells (SMC). Levels of nuclear GR in SMC began to decrease after 12-h incubation with Lp(a), to 55 +/- 8% of the control value at 48 h; binding affinity did not change. Lp(a) had no effect on estrogen receptor binding in SMC. Moreover, low, very low, and high density lipoproteins had no effect on GR binding in SMC. The effects of Lp(a) on nuclear GR in rat SMC were very similar to those in human SMC; in contrast, Lp(a) did not alter GR or estrogen receptor levels in rat endothelial cells. GR messenger RNA levels in SMC decreased after 1-h treatment with Lp(a) to 23% of the control value after 12 h. Further, the antiproliferative effect of glucocorticoids on SMC was blunted by exposure to Lp(a). We conclude that Lp(a) down-regulates GR gene expression, resulting in a decreased number of GR in SMC. These findings suggest the possibility of a novel atherogenic mechanism of Lp(a) via inhibition of a protective action of glucocorticoids on SMC.

Aldosterone and cardiac fibrosis: in vitro studies.

OBJECTIVE: Increased cardiac collagen is seen in uninephrectomised rats made hypertensive by aldosterone infusion and high salt, with the possibility of a direct effect of aldosterone at the level of the cardiac fibroblast. The aim of this study was to examine the effects of aldosterone, glucocorticoids, and angiotensin II on collagen production in a series of rat cardiac fibroblast cultures. METHODS: Cardiac fibroblast cultures were established from neonatal and adult rats of Sprague-Dawley, WKY, and SHR strains, with collagen synthesis measured by incorporation of [3H]proline into collagen. Cells were grown alone or co-cultured with cardiomyocytes, and exposed to aldosterone, glucocorticoids, or angiotensin II for 24-28 h before determination of [3H]proline incorporation. RESULTS: Aldosterone did not affect production of collagen; in contrast, angiotensin II increased, and the glucocorticoid agonist RU28362 suppressed, collagen production in cultured cardiac fibroblasts in the various rat strains. CONCLUSIONS: Aldosterone does not exert a direct effect on collagen synthesis in rat cardiac fibroblasts grown in culture. The increased cardiac collagen observed in vivo in aldosterone treated, salt loaded rats may thus represent secondary effects of aldosterone in this model.

Medullary pathways for adrenocorticotropic hormone and vasopressin secretion in rabbits.

We determined, in urethan-anesthetized rabbits, whether pharmacological alteration of neuronal function in the ventrolateral medulla oblongata, including the A1 area, and in the nucleus tractus solitarii (NTS), alters plasma adrenocorticotropic hormone (ACTH) and vasopressin and whether inhibition of neuronal function in the ventrolateral medulla impairs the secretion of ACTH normally observed in response to hemorrhage or constriction of the inferior vena cava. We also tested whether the increase in plasma ACTH and vasopressin after pharmacological inhibition of neuronal function in the NTS is dependent on a pathway that synapses in the A1 area of the ventrolateral medulla. Activation of the A1 area with bicuculline increased both ACTH and vasopressin. Inhibition of the NTS with muscimol increased levels of both hormones, as did hemorrhage and constriction of the inferior vena cava. Inhibition of neuronal function within the A1 area with muscimol eliminated the secretion of vasopressin but did not significantly alter the secretion of ACTH, obtained by injecting muscimol into the NTS. Injection of muscimol into the A1 area eliminated the secretion of both ACTH and vasopressin in response to constriction of the inferior vena cava and, in the case of vasopressin, in response to hemorrhage. Although hemorrhage-initiated secretion of ACTH was significantly reduced by injection of muscimol into the A1 area, it was not completely eliminated by these injections or by injections of muscimol into a more rostrocaudally extensive region of the medulla oblongata. We conclude that the net output from the NTS tonically inhibits secretion of both ACTH and vasopressin, reflecting tonic baroreceptor tone. For vasopressin, the pathway from the NTS to the hypothalamus is dependent on a synapse in the A1 area. For ACTH, there are pathways to the hypothalamus that do not synapse in the A1 area, but neurons in this region do have an excitatory effect on secretion of ACTH.

Adrenalectomy and dexamethasone administration: effect on atrial natriuretic peptide synthesis and circulating forms.

Previous in vivo and in vitro studies have reported a variety of glucocorticoid effects on the synthesis and secretion of immunoreactive atrial natriuretic peptide (ir-ANP) into plasma. To further define glucocorticoid modulation of ir-ANP, we have measured ir-ANP levels in plasma and the four cardiac chambers, and tissue ANP mRNA levels, in intact rats and adrenalectomized rats with or without dexamethasone treatment for 1, 2, 4, 8 and 16 days. Plasma levels fell by 50% between 8 and 16 days post-adrenalectomy; in contrast, dexamethasone treatment caused a 3-fold rise in plasma ANP 1-2 days post-adrenalectomy, with levels gradually returning to control by day 16. Circulating forms of ANP were unchanged by adrenalectomy or dexamethasone treatment, as were atrial ANP concentrations and ANP mRNA levels. Left ventricular ANP concentrations rose with dexamethasone treatment, and ventricular ANP mRNA levels changed in parallel with those of circulating ANP. The in vivo effect of glucocorticoids (at moderate rather than very high doses) on ANP synthesis and secretion thus appears to be predominantly but not exclusively upon the left ventricle rather than the atria.

Studies of the regulation of the hypothalamic-pituitary-adrenal axis in sheep with hypothalamic-pituitary disconnection. II. Evidence for in vivo ultradian hypersecretion of proopiomelanocortin peptides by the isolated anterior and intermediate pituitary.

Studies were performed to determine whether the isolated ovine anterior and intermediate pituitary might rhythmically secrete three POMC peptides, ACTH, ir-beta-endorphin (ir-beta-EP), and ir-alpha-melanocyte stimulating hormone (ir-alpha-MSH) in vivo. When blood was taken at 10-min intervals from four ewes with hypothalamo-pituitary-disconnection (HPD), a distinct POMC-peptide and cortisol ultradian rhythm was noted. A comparison of the four HPD ewes with five nonstressed hypothalamopituitary-intact (HPI) ewes revealed that the mean plasma levels of the three POMC-peptides and cortisol were increased, the mean ACTH and ir-alpha-MSH pulse amplitudes were increased, and the mean ir-beta-EP and ir-alpha-MSH interpulse intervals were decreased. When four HPI ewes were subjected to a mild stress, plasma POMC-peptide and cortisol levels increased significantly when compared with the five unstressed HPI animals. In addition, the ACTH and cortisol pulse amplitudes increased and the ir-beta-EP and ir-alpha-MSH interpulse intervals decreased. Although plasma ACTH levels in the stressed HPI and HPD ewes were comparable, mean plasma cortisol levels were 2-fold greater in the stressed HPI animals. To determine whether the ACTH hypersecretion in the HPD ewe might reflect a net reduction in hypothalamic inhibitory influence over ACTH secretion, we examined the effects of dopamine (DA), somatostatin (SS-14), and rat atrial natriuretic peptide [rANF(1-28)] on the secretion of ACTH from cultured ovine anterior pituitary cells. DA and SS-14 did not exert a discernible effect on basal, CRF-, or arginine vasopressin (AVP)-stimulated ACTH secretion. Although basal ACTH secretion was unaffected by rANF(1-28) (10(-12)-10(-8) M), a significant inhibition of CRF- and AVP-stimulated ACTH release was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered thyroidal status and the in vivo synthesis of atrial natriuretic peptide in the rat heart.

The effect of altered thyroidal status on levels of immunoreactive (ir)- atrial natriuretic peptide (ANP) in serum and the four cardiac chambers, and of tissue ANP mRNA, was determined in groups of rats given vehicle, thyroxine (T4), propylthiouracil (PTU) or T4 plus PTU for 3 weeks. Serum levels of ir-ANP were approximately 3-fold higher in T4-treated animals compared with control; levels in PTU or PTU/T4 groups were not different from control. Right ventricular ANP mRNA was below detection; in other chamber, levels rose with T4, alone or plus PTU, and fell after PTU compared with control. Atrial ir-ANP levels were unchanged by T4, but increased (left atrium, LA) or decreased (right atrium, RA) after PTU alone. After PTU/T4, some indices (e.g. tissue weight) remained at control levels, others (e.g. ANP mRNA levels) were equivalent to levels in the T4-alone group, and others (e.g. LA ir-ANP) were equivalent to those seen with PTU alone. We conclude that the role of thyroid hormones on ANP synthesis may be similar between chambers but their effects on release appear to differ widely. The extent to which this represents secondary rather than direct effects, or possible T3-versus T4-specific events, awaits elucidation.

Induction of ventricular morphogenesis and atrial natriuretic factor synthesis by thyroid hormone.

Thyroid hormones have been shown to increase atrial natriuretic peptide (ANP) synthesis in atria both in vitro and in vivo. In this study the effects of triiodothyronine (T3) administration on primary cultures of 6-day-old atrial and ventricular myocytes were studied. Levels of immunoreactive (ir-) ANP were determined by radioimmunoassay over 9 days of culture in cells cultured in the presence and absence of 10(-8) M T3. The proportion of immunostained nuclei was also determined and changes in cell characteristics noted over this time. Over 9 days T3 had no effect on the proportion of atrial cells immunostained for ANP, while cell and medium content of ir-ANP in treated wells doubled that of the untreated. In treated ventricular myocytes, cell and medium ir-ANP similarly increased; in addition, the proportion of immunostained cells increased by 1-2 orders of magnitude. The formation of dendritic processes was enhanced in these cultures, reflected in increased rates of spontaneous contractility. Thus it would appear that the 6-day-old myocardium exhibits profound differences in response to T3 administration, in that ventricular cells dedifferentiate and undergo morphological changes which are not seen in atriocytes.

Evidence for an ultradian secretion of adrenocorticotropin, beta-endorphin and alpha-melanocyte-stimulating hormone by the ovine anterior and intermediate pituitary.

These studies were undertaken to characterize the secretion of the pro-opiomelanocortin (POMC)-peptides adrenocorticotropin (ACTH), immunoreactive (ir)-beta-endorphin (beta-EP), and ir-alpha-melanocyte-stimulating hormone (ir-alpha-MSH) in the conscious sheep. When blood samples were taken at 10-min intervals, a distinct POMC-peptide and cortisol ultradian rhythm was observed, and 4 of the 7 possible POMC-peptide pulse patterns were defined. Three of these patterns included pulsatile ACTH secretion, and 70% were followed by a rise in plasma cortisol. The fourth pulse pattern consisted of concordant ir-beta-EP and ir-alpha-MSH secretion and was not temporally correlated with cortisol secretion. When an intensified rate of venous sampling was used (2-min intervals over 1 h), the POMC-peptide and cortisol pulse frequencies were increased. Although the mean POMC-peptide and cortisol pulse amplitudes remained unaltered, the mean cortisol pulse amplitude was decreased. Five of the 7 possible POMC-peptide pulse patterns were noted, and 3 of these were identical to those observed during the 10-min sampling procedure. Chronic bromocriptine treatment decreased the plasma levels and pulse amplitudes of ir-beta-EP and ir-alpha-MSH, increased the ir-beta-EP pulse interval, and abolished concordant ir-beta-EP and ir-alpha-MSH secretion. Neither the plasma levels nor the ultradian rhythms of ACTH and cortisol were affected by the treatment. Chronic dexamethasone administration abolished the ACTH ultradian rhythm, reduced the plasma levels of ACTH, ir-beta-EP, and ir-alpha-MSH, while plasma cortisol levels became undetectable. Although the ir-alpha-MSH interpulse interval was increased, the ir-beta-EP and ir-alpha-MSH ultradian rhythms were not abolished. We conclude that: (1) an ultradian rhythm characterizes the secretion of ACTH, ir-beta-EP, and ir-alpha-MSH from the ovine pituitary; (2) this rhythm is due to the pulsatile secretion of peptides from the adenohypophysis and intermediate lobe; (3) the adenohypophysis and intermediate lobes appear to secrete POMC-peptides in synchrony and asynchrony, and (4) intensified rates of venous sampling unmask the appearance of high-frequency POMC-peptide and cortisol pulses in vivo in the sheep.

Studies of the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophysial-portal circulation of the conscious sheep. I. Effect of an audiovisual stimulus and insulin-induced hypoglycemia.

Studies were undertaken to characterize the secretion of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) into the hypophysial-portal circulation of the conscious sheep. In addition, we examined the temporal relationship between the secretion of these two hypothalamic peptides and the secretion of three pro-opiomelanocortin peptides--adrenocorticotropic hormone (ACTH), ir-beta-endorphin, and ir-alpha-melanocyte-stimulating hormone--and cortisol and determined the effects of an audiovisual emotional stimulus and insulin-induced hypoglycemia on the entire hypothalamic-pituitary-adrenal axis. In the basal state, the secretion of CRF, AVP, the three pro-opiomelanocortin peptides, and cortisol was pulsatile in nature, and three CRF and AVP pulse patterns were observed: a concordant increase in CRF and AVP, an isolated rise in CRF, and an isolated increase in AVP. In 4 of the 5 animals, a 3-min audiovisual stress (barking dog) rapidly increased the plasma levels of all the measured substances, although the magnitude and duration of the effect differed markedly between the animals. Insulin-induced hypoglycemia markedly increased AVP and, to a lesser extent, CRF concentrations in portal plasma and thereby altered the CRF:AVP molar ratio. Although pituitary-adrenal activation was closely correlated with the increased hypothalamic activity, a strict 1:1 concordance between CRF/AVP secretion and ACTH secretion was not seen. The anesthetic ketamine selectively increased portal AVP concentrations to levels which exceeded those attained during hypoglycemia and rapidly activated the pituitary-adrenal axis. We conclude the following: (1) CRF and AVP are secreted by the hypothalamus in a pulsatile fashion; (2) ACTH secretion can be stimulated by increases in either CRF or AVP; (3) the absence of a strict 1:1 concordance between hypothalamic CRF/AVP release and pituitary ACTH secretion during stress may be partly due to the release of additional hypothalamic ACTH secretagogues; (4) the ability of both audiovisual stimuli and insulin-induced hypoglycemia to augment CRF and AVP secretion indicates that the paraventricular hypothalamus may be activated by a variety of neural inputs, and (5) the marked alteration of the CRF:AVP molar ratio during stress suggests that AVP may be an important ACTH secretagogue in vivo in the sheep.

Gonadal steroids and anterior lobe dynorphin in the male rat.

Immunoreactive (ir)-dynorphin levels were measured, and the species characterized by high performance liquid chromatography (HPLC), in the pituitary and hypothalamus of intact and castrate male rats. On HPLC, ir-dynorphin co-eluted with authentic dynorphin A 1-8, dynorphin A 1-17 and dynorphin 1-32 in the hypothalamus and intermediate lobe; in two different reversed phase (RP)-HPLC systems, anterior lobe ir-dynorphin co-eluted uniquely with dynorphin 32 (4K dynorphin). Anterior lobe levels of total ir-dynorphin were significantly lowered 7 days after castration, while HPLC profiles in all tissues remained unchanged. The change in anterior pituitary ir-dynorphin levels was reversed in a dose-related manner by dihydrotestosterone (15-500 micrograms/100 g b. wt/day); estradiol benzoate (3 micrograms/100 g/day) was without effect. The changes on castration and androgen administration suggest that gonadal steroids play a role in the regulation of dynorphin, as well as gonadotrophins and prolactin, within the anterior pituitary gland.

Studies of the regulation of the hypothalamic-pituitary-adrenal axis in sheep with hypothalamic-pituitary disconnection. I. Effect of an audiovisual stimulus and insulin-induced hypoglycemia.

These studies were undertaken to characterize the secretion of adrenocorticotropin (ACTH), immunoreactive (ir) beta-endorphin (ir-beta-EP) and ir alpha-melanocyte-stimulating hormone (ir-alpha-MSH) from the surgically isolated ovine pituitary in response to an audiovisual stress (barking dog, 3 min) and insulin hypoglycemia. The studies were performed in 4 ovariectomized, hypothalamo-pituitary-disconnected (HPD) and 4 sham-HPD ewes bearing indwelling jugular venous catheters. Basal concentrations of the three pro-opiomelanocortin (POMC) peptides and plasma cortisol were significantly increased in the HPD animals. When the control ewes were exposed to the audiovisual stimulus, plasma ACTH, ir-beta-EP and ir-alpha-MSH levels were increased 2.5-, 10-, and 5-fold 1 min after the stress; plasma cortisol attained maximal values at 5 min. In contrast, plasma levels of the three POMC peptides were not significantly increased in the HPD animals, although a rise in plasma cortisol occurred. The administration of regular insulin (5 units/kg i.v.) to control ewes caused plasma ACTH, ir-beta-EP, and ir-alpha-MSH levels to increase 17-, 22-, and 67-fold at 50 min; plasma cortisol values were maximal at 60 min. In contrast, the elevated basal levels of POMC peptides in the HPD animals were not significantly increased by the hypoglycemia, but a significant elevation of plasma cortisol was seen. We conclude that: (1) the increase in ACTH in intact animals after an audiovisual emotional stress and hypoglycemia, and the abolition of this increase by HPD, indicates that both stimuli, each acting through distinct neuroanatomical pathways, increase the net corticotropin-releasing activity of the hypothalamus; (2) the rise in plasma cortisol in HPD animals after stress suggests that peripheral humoral factors may release additional small amounts of ACTH from the anterior pituitary, and (3) the finding of increased basal ACTH levels after HPD suggests that POMC peptide synthesis and secretion by the anterior pituitary is tonically regulated by an inhibitory factor of hypothalamic origin.

Immunoreactive dynorphin is regulated by estrogen in the rat anterior pituitary.

The pituitary and hypothalamic content of dynorphin was determined by radioimmunoassay and characterized by high-performance liquid chromatography (HPLC) in adult female Sprague-Dawley rats, intact and ovariectomized with and without estrogen treatment. Animals were given estradiol benzoate, or vehicle (oil) by six daily intramuscular injections. Anterior pituitary content of immunoreactive (ir)-dynorphin in ovariectomized rats was approximately twice that of intact animals, and consisted of a single HPLC peak co-eluting with dynorphin 32. Administration of estradiol benzoate (0.06-6 micrograms/day) caused a marked decrease of ir-dynorphin in the anterior lobe of castrate female rats, with a half-maximal effect at 0.2 microgram/day; levels were restored to those seen in intact animals with 6 micrograms estradiol benzoate per day, an effect which was not influenced by concomitant administration of progesterone (1 mg/day), or bromocriptine (100 micrograms/day). In the hypothalamus and neuro-intermediate lobe multiple peaks of immunoreactive dynorphin were seen, coeluting with dynorphin A 1-8, dynorphin A 1-17 and dynorphin 32. Neither castration nor estrogen treatment altered ir-dynorphin content in these tissues. These findings suggest that the ovary exerts a specific modulating influence on AP ir-dynorphin in the rat, and that in addition this inhibition appears to be mediated by ovarian estrogen.

Posttranslational processing of corticotropin-releasing factor in the ovine tuberoinfundibular system and pituitary.

The present studies were undertaken to characterize the immunoreactive-corticotropin-releasing factor (ir-CRF) in two areas of the ovine tuberoinfundibular system, hypophysial portal blood, and pituitary. With an antiserum raised against synthetic ovine (o)CRF(1-41) and 125I-Tyro-oCRF(1-41) as the tracer, concentrations of ir-CRF (pg/mg wet weight, n = 5) were: paraventricular hypothalamus (PVN), 11.7 +/- 2.5; median eminence (ME), 2276 +/- 296; anterior pituitary (AP), less than 0.5; posterior pituitary (PP), 10.0 +/- 2.2. Analysis of the ir-CRF in these areas on G-75 Sephadex chromatography revealed two main peaks--a 'major' peak which coeluted with synthetic oCRF(1-41) and a 'minor' peak which eluted eight fractions later. These two immunoreactive species of CRF were also found in hypophysial portal blood. When ME extract was analyzed by reverse-phase high performance liquid chromatography (HPLC), the 'minor' peak of ir-CRF eluted before that of CRF(1-41). Since CRF contains Arg35-Lys36 within its sequence, we tested the hypothesis that the 'minor' peak of ir-CRF represented a fragment, or fragments, of the molecule derived by proteolytic cleavage at this site. Tyro-oCRF(34-41) was digested with trypsin and the reaction products were identified by amino acid analysis. Two of these products were CRF(36-41) and CRF(37-41), and both migrated in the 'minor' peak area on G-75 Sephadex chromatography and HPLC. In the CRF(1-41) RIA, serial dilution of both fragments yielded nonparallel displacement curves. However, with 125I-Tyro-oCRF(34-41) as the radiolabeled ligand and Tyro-oCRF(34-41) as the standard, serial dilutions of CRF(1-41), CRF(36-41), and CRF(37-41) generated parallel displacement curves, and the molar cross-reactivities were 90%, 45% and 10% respectively. When the ir-CRF in HPLC fractions of ovine ME was measured in the Tyro-oCRF(34-41) RIA, the molar abundance of the hexapeptide and pentapeptide could be obtained. Calculations based on the premise that the 'minor' peak was solely composed of either the hexapeptide or pentapeptide indicated that CRF(36-41) could account for up to 37% of the total ir-CRF, or that CRF(37-41) could account for up to 73% of the total immunoreactivity. On more discriminating HPLC systems, immunoreactive (ir-) oCRF in the sheep median eminence (ME) could be resolved into five different molecular forms. On three distinct chromatographic systems, four of these immunoreactive species shared retention characteristics identical with synthetic oCRF(37-41), oCRF(36-41), oCRF(16-41) and oCRF(1-41), with the fifth immunoreactive peak as yet unidentified.(ABSTRACT TRUNCATED AT 400 WORDS)