Multiple feedback regulatory loops upon rat hypothalamic corticotropin-releasing hormone secretion. Potential clinical implications.

To examine whether the hypothalamic corticotropin-releasing hormone (CRH) neuron is regulated by CRH, by products of the proopiomelanocortin (POMC) gene, and/or by glucocorticoids, we used a rat hypothalamic organ culture system in which rat CRH secretion from single explanted hypothalami was evaluated by an RIA (iCRH) specific for rat CRH. The effects of graded concentrations of ovine CRH (oCRH), adrenocorticotropin hormone (ACTH), beta-endorphin (beta-EP), alpha-melanocyte-stimulating hormone (alpha-MSH), corticotropin-like intermediate lobe peptide (CLIP), ovine beta-lipotropin (ovine beta-LPH), and dexamethasone (DEX) upon unstimulated and serotonin- (5HT), acetylcholine- (ACh), and norepinephrine-(NE) stimulated CRH secretion were determined. oCRH and DEX inhibited unstimulated iCRH secretion with ID50 at the 10(-8) M range. ACTH had no detectable suppressive effect at 10(-8) M. oCRH, ACTH, and DEX inhibited 5HT-, ACh-, and NE-stimulated iCRH secretion in a dose-dependent fashion. beta-EP, alpha-MSH, and CLIP also inhibited 5HT-induced iCRH secretion. Of the latter peptides, the strongest inhibitor was beta-EP and the weakest was CLIP. Ovine beta-LPH had only a weak inhibitory effect on 5HT-induced iCRH secretion. Generally, the concentrations required for 50% suppression of neurotransmitter-stimulated iCRH secretion were significantly lower than those required for a similar suppression of unstimulated iCRH secretion. In conclusion, these data suggest the presence of multiple negative feedback loops involved in the regulation of the hypothalamic CRH neuron: an ultrashort CRH-mediated loop, a short, hypothalamic POMC-derived peptide loop, and a long, glucocorticoid-mediated negative feedback loop. The potency of these negative feedback loops may be determined by the state of activation of the CRH neuron.

Catecholamine effects upon rat hypothalamic corticotropin-releasing hormone secretion in vitro.

To further our understanding of the functional role of catecholaminergic systems in regulating hypothalamic corticotropin-releasing hormone (CRH) secretion, we assessed the direct effects of a multiplicity of catecholamine agonists and antagonists on hypothalamic CRH secretion. To accomplish this, we used an in vitro rat hypothalamic organ culture system in which CRH secretion from single explants was evaluated by a specific RIA (IR-rCRH). Norepinephrine (NE) stimulated IR-rCRH secretion dose dependently, with peak effects in the nanomolar range. The effect of NE was antagonized by the mixed alpha antagonist phentolamine, the alpha 1 antagonist prazosin, and the alpha 2 antagonist yohimbine, but not by the beta blocker, L-propanolol. Compatible with these data were the findings that the alpha 1 agonist phenylephrine and the alpha 2 agonist clonidine both stimulated IR-rCRH secretion in a dose-dependent fashion. On the other hand, whereas the beta agonist, isoproterenol, caused a weak, non-dose-dependent increase in IR-rCRH secretion, this effect could not be antagonized by L-propanolol. Despite pretreatment with serotonin and acetylcholine antagonists, the effect of NE upon IR-rCRH secretion was undiminished, suggesting that NE-induced CRH secretion is not mediated by either neurotransmitter. On the other hand, pretreatment with gamma-aminobutyric acid (GABA) attenuated NE-induced IR-rCRH secretion. Whereas epinephrine (E) stimulated IR-rCRH secretion, this occurred only at higher concentrations, and was antagonized by phentolamine, but not by L-propanolol. Dopamine (DA) had a weak stimulatory effect that could be antagonized by the DA1 receptor antagonist, SCH 23390, but not by phentolamine. We conclude that NE and E stimulate hypothalamic IR-rCRH secretion via alpha 1 and alpha 2 receptors. The effect of NE upon IR-rCRH secretion is not apparently mediated by serotonergic or cholinergic interneurons, but is modulated by the inhibitory neurotransmitter, GABA. These data support the idea that the central catecholaminergic systems are excitatory rather than inhibitory upon CRH secretion when acting directly at the hypothalamic level.

Adaptation during surgical stress. A reevaluation of the role of glucocorticoids.

Pharmacologic doses of glucocorticoids are administered to patients with adrenal insufficiency during operative procedures to prevent hemodynamic instability, cardiovascular collapse, and death. Since these supraphysiologic doses might not be necessary and might have adverse effects, we examined the effects of different doses of glucocorticoids on hemodynamic adaptation during surgical stress in adrenalectomized primates. Sham-adrenalectomized placebo-treated animals served as controls. Adrenalectomized monkeys were maintained for 4 mo on physiologic glucocorticoid and mineralocorticoid replacement. The adrenalectomized monkeys were then stratified into three groups receiving, respectively, subphysiological (one-tenth the normal cortisol production rate), physiological, or supraphysiological (10 times the normal cortisol production rate) cortisol (hydrocortisone) treatment. 4 d later a cholecystectomy was performed. The intraoperative hemodynamic and metabolic parameters, perioperative survival rates, and postoperative wound healing were compared. The subphysiologically treated group was hemodynamically unstable before, during, and after surgery and had a significantly higher mortality rate than control. In this group, arterial blood pressure was low, and the cardiac index, systemic vascular resistance index, and left ventricular stroke work index were all reduced, suggesting decreased cardiac contractility and blood vessel tone. In contrast, the physiologically replaced group was indistinguishable from either supraphysiologically treated animals or sham-operated controls. All groups had similar metabolic profiles and normal wound healing. These findings suggest that the permissive actions of physiologic glucocorticoid replacement are both necessary and sufficient for primates to tolerate surgical stress. Supraphysiological glucocorticoid treatment has no apparent advantage during this form of stress in the primate.

Glucocorticoid receptor-mediated effects on rat fibrosarcoma growth.

Glucocorticoid receptors are present in most normal and malignant mammalian cells. To examine the hypothesis that the growth of methylcholanthrene-induced malignant sarcoma is glucocorticoid dependent, we evaluated the behavior of malignant fibrosarcoma (MCA) in adrenalectomized rats treated with either normal saline or deoxycorticosterone acetate and in intact rats treated with placebo or with the glucocorticoid receptor antagonist RU 486. Survival, tumor weight, and loss of body weight (an index of cachexia) were measured. In MCA-bearing rats, neither survival nor loss of body weight was affected by bilateral adrenalectomy or by treatment with RU 486. Tumor weight and time-integrated tumor volume, however, were significantly less in bilaterally adrenalectomized rats without deoxycorticosterone acetate replacement than in animals treated with deoxycorticosterone acetate. Similarly, tumor weight and time-integrated tumor volume were less in intact animals treated with RU 486 than in intact animals treated with placebo. The glucocorticoid receptors in the tumor cells had similar binding capacity (Ro) and equilibrium dissociation constant (Kd) as in control rat fibroblasts. These results suggest that the growth of MCA sarcoma cells is partially dependent upon glucocorticoids. This effect of glucocorticoids, however, was not of sufficient magnitude to improve survival and prevent cachexia. We conclude that glucocorticoids appear to influence MCA sarcoma growth in the rat, and that glucocorticoid receptor blockade, perhaps in combination with other antitumor agents, merits future study in the treatment of malignant tumors.

Effects and pharmacokinetic properties of the rat/human corticotropin-releasing factor in rhesus monkeys.

The biological effects and pharmacokinetic properties of the recently sequenced rat/human corticotropin-releasing factor (r/hCRF) were evaluated in the rhesus monkey and compared to those of the previously studied ovine corticotropin-releasing factor (oCRF). An iv bolus of 0, 0.1, 1, 10, and 100 micrograms/kg r/hCRF and 1 microgram/kg oCRF were given to rhesus monkeys (four to five tests per dose). Serial blood samples were drawn before and up to 180 min after the injection for determination of plasma immunoreactive (IR) ACTH, cortisol, and IR-r/hCRF or IR-oCRF concentrations. Mean arterial blood pressure and heart rate were monitored. r/hCRF stimulated ACTH and cortisol secretion in a dose-dependent fashion. Its potency was similar to that of oCRF. r/hCRF, however, had a shorter half-life and a 3-fold higher MCR than oCRF. A dose-dependent decrease in the MCR of r/hCRF was observed, which may indicate a saturation of the clearance mechanisms. Significant decreases in mean arterial blood pressure, increases in heart rate, and a profound facial flush occurred at the dose of 100 micrograms/kg r/hCRF. We conclude that r/hCRF stimulates ACTH and cortisol secretion in a nonhuman primate with a potency similar to that of oCRF. The peptide has marked hypotensive effects at high doses and is cleared 3 times more rapidly than oCRF.

Ovine corticotropin-releasing hormone stimulation test in normal children.

We administered ovine corticotropin-releasing hormone (CRH) as a bolus iv injection (1 microgram/kg) to 21 normal boys and girls, aged 6-15 yr. CRH stimulated release of immunoreactive ACTH and cortisol in all children. The peak plasma ACTH and cortisol levels after CRH were 15.7 +/- 9.4 (SD) pg/ml and 14.3 +/- 3.6 micrograms/dl, respectively, in the girls, and 20.7 +/- 9.7 pg/ml and 16.6 +/- 3.3 micrograms/dl, respectively, in the boys. Plasma ACTH and cortisol responsiveness to CRH did not differ between girls or boys, or between children and adults. Cortisol-binding globulin concentrations in plasma did not change with age. We conclude that CRH provides a safe means of stimulating the pituitary-adrenal axis in children.

Effects of carbamazepine on pituitary-adrenal function in healthy volunteers.

Carbamazepine (CBZ) is a widely used therapeutic agent in seizure, pain, and mood disorders. Although CBZ has been shown to inhibit hypothalamic CRH secretion in vitro, limited data suggest that systemic CBZ induces pituitary-adrenal activation. Few data are available to reconcile these effects or clarify their mechanism(s), particularly in healthy human subjects. We report here a study of basal ACTH and cortisol secretion and their responses to ovine CRH administration in nine healthy volunteers, studied both during repeated (2-3 weeks) administration of CBZ and while medication free. CBZ significantly increased mean 24-h urinary free cortisol (mean +/- SE, 197 +/- 17 vs. 137 +/- 24 nmol/day; P less than 0.02) and evening basal total plasma cortisol (113 +/- 17 vs. 83 +/- 14 nmol/L; P less than 0.05) as well as cortisol-binding globulin-binding capacity (497 +/- 36 vs. 433 +/- 28 nmol/L; P less than 0.01). Despite the CBZ-induced hypercortisolism, plasma ACTH responses to CRH during CBZ treatment remained robust, rather than being suppressed by basal hypercortisolism. In fact, during CBZ treatment, we noted a positive correlation between the increase in basal plasma cortisol and the increase in the plasma ACTH response to CRH (r = 0.65; P less than 0.05). We also observed a reduction in cortisol-binding globulin-binding capacity after CRH administration (315 +/- 25 vs. 433 +/- 28 nmol/L; P less than 0.001), which was accentuated by CBZ treatment (342 +/- 19 vs. 497 +/- 36 nmol/L; P less than 0.001; magnitude of fall, -155 +/- 22 nmol/L on CBZ vs. -118 +/- 11 nmol/L off CBZ; P less than 0.05). We conclude that CBZ increases plasma cortisol secretion in healthy volunteers independent of its effect on plasma cortisol-binding capacity. This pituitary-adrenal activation seems to reflect a pituitary, rather than a hypothalamic, effect of CBZ. Hence, despite CBZ-induced hypercortisolism, the ACTH response to CRH remained robust in direct proportion to the CBZ-induced rise in basal plasma cortisol. Thus, we propose that the increased cortisol secretion observed during CBZ treatment reflects a relative inefficacy of glucocorticoid negative feedback at the pituitary. This pituitary-driven increase in cortisol secretion combined with the expected reduction in centrally directed CRH secretion could contribute to the anticonvulsant properties of CBZ.

Interaction of epidermal growth factor with the hypothalamic-pituitary-adrenal axis: potential physiologic relevance.

Epidermal growth factor (EGF), a polypeptide mitogen that participates in wound healing, has ACTH-like activity in ewes. We examined its effects on the primate hypothalamic-pituitary-adrenal (HPA) axis by administering mouse EGF (mEGF) iv (0-100 micrograms/kg) to rhesus monkeys. mEGF caused dose-dependent elevations of plasma ACTH and cortisol in these animals. To define whether the locus of stimulation was the hypothalamus and/or the pituitary gland we examined the capacity of mEGF to directly stimulate hypothalamic CRH or pituitary ACTH secretion in a rat hypothalamic organ culture system and a rat pituitary cell system, respectively. mEGF stimulated hypothalamic CRH release in a dose-dependent manner, but failed to cause pituitary ACTH release. We conclude that EGF is a stimulator of the HPA axis in primates. Thus, EGF, and/or one of its naturally occurring analogs, may participate in the physiological activation of the HPA axis at times during which the concentrations of these factors are raised in the systemic circulation and/or locally in the hypothalamus. Such states may include trauma, surgery, and possibly emotional stress.

Dissociation between cortisol and adrenal androgen secretion in patients receiving alternate day prednisone therapy.

To evaluate the hypothesis that chronic, low dose, alternate day prednisone treatment may suppress adrenal androgen secretion without causing long term suppression of the hypothalamic-pituitary-adrenal axis we studied seven patients with systemic lupus erythematosus who had been taking low dose (5-20 mg), alternate day prednisone therapy for at least 1 yr. Basal and ovine CRH (oCRH)-stimulated plasma ACTH, cortisol, and adrenal androgen levels were measured 12 h (day on) and 36 h (day off) after the most recent dose of prednisone, and the results were compared to those in seven age- and sex-matched normal subjects. The patients' basal ACTH and cortisol levels did not differ significantly from those in the normal subjects on either the day on or the day off prednisone treatment. By contrast, their basal adrenal androgen levels were significantly decreased compared to those in normal subjects on both the day on and the day off prednisone (P less than 0.05). The patients' oCRH-stimulated ACTH and cortisol levels on the day off prednisone did not differ from normal levels, but were significantly blunted during the day on prednisone (P less than 0.05). In contrast, the patient's oCRH-stimulated adrenal androgen levels were significantly decreased during both the day off and the day on prednisone (P less than 0.05). These findings are consistent with the hypothesis that chronic alternate day prednisone therapy, at doses close to or below replacement, suppresses adrenal androgen levels without long term suppression of the hypothalamic-pituitary-adrenal axis. Based upon these findings, we postulate that an alternate day regimen of prednisone might maintain the benefits while reducing the risks of glucocorticoid therapy of adrenal hyperandrogenism.

Human corticotropin-releasing factor in man: pharmacokinetic properties and dose-response of plasma adrenocorticotropin and cortisol secretion.

The responses of plasma immunoreactive ACTH (IR-ACTH), cortisol, GH, PRL, and LH to a single iv injection of 0.01-5 micrograms/kg human corticotropin-releasing factor (hCRF) were investigated in healthy volunteers. The lowest effective dose of hCRF was 0.5 micrograms/kg. hCRF caused brief pulse-like elevations of plasma IR-ACTH and cortisol without any effect on the plasma concentrations of GH, PRL, and LH. By contrast, ovine CRF (oCRF), given for comparison, produced long-lasting stimulation of the human pituitary-adrenal axis. The difference in duration of effect between hCRF and oCRF may be attributed to an approximately 3-4 times higher MCR of hCRF [7.9 +/- 1.2 (+/- SE) ml/kg min; n = 14] than oCRF (1.9 +/- 0.1 ml/kg min; n = 9) in man. No serious side effects occurred at any of the doses of hCRF tested. The highest dose (5 micrograms/kg) caused a slight increase of heart rate that was not associated with significant changes in arterial blood pressure. All subjects receiving 5 micrograms/kg and one third of the subjects receiving 1 micrograms/kg hCRF experienced a transient facial flush. We conclude that hCRF causes brief plasma ACTH and cortisol secretory episodes in man, similar to the physiological plasma ACTH and cortisol secretory episodes. This is in contrast to oCRF, which causes prolonged ACTH and cortisol secretion. These differences between the two peptides may be explained by the higher MCR of hCRF than oCRF.

In vitro and in vivo effects of the triazolobenzodiazepine alprazolam on hypothalamic-pituitary-adrenal function: pharmacological and clinical implications.

We report here a study of the effects of alprazolam on in vivo pituitary-adrenal function in jacketed nonrestrained nonhuman primates and on in vitro CRH release from rat hypothalami and ACTH release from rat dispersed anterior pituicytes. We undertook this study because alprazolam is the only benzodiazepine effective in treating both major depressive and anxiety disorders, and recent data suggest that the hypercortisolism of major depression reflects hypersecretion of CRH. Moreover, the intracerebroventricular administration of CRH can reproduce many of the components of the symptom complex of major depression, including not only hypercortisolism, but also hypothalamic hypogonadism, decreased libido, anorexia, and intense anxiety. As a comparison, we also assessed the effects of diazepam on in vitro CRH release, because in contrast to alprazolam, diazepam is effective in anxiety states but not in depression. Alprazolam (0.01-0.3 mg/kg, iv) produced a dose-dependent inhibition of both plasma ACTH and cortisol secretion in non-restrained adult male rhesus monkeys. Our in vitro studies showed that alprazolam significantly inhibited serotonin (5HT)-induced CRH release in a dose-dependent fashion (10(-10)-10(-5) M). Diazepam also inhibited 5HT-induced CRH release, but was 40 times less potent than alprazolam. Alprazolam was ineffective in blocking basal or CRH-induced ACTH release from rat dispersed anterior pituicytes, suggesting that its in vivo effects are through inhibition of CRH secretion. As expected, the inactive benzodiazepine ligand Ro 15-1788 inhibited the effects of alprazolam on 5HT-induced CRH release, but this occurred only at doses below 10(-7) M. Interestingly, when incubated alone in higher doses with our rat hypothalamic organ culture, Ro 15-1788, like alprazolam, produced a dose-dependent inhibition of 5HT-induced CRH release (10(-7)-10(-5) M), suggesting an agonistic action of Ro 15-1788 at the benzodiazepine receptor at higher concentrations. We conclude that alprazolam is capable of suppressing the primate pituitary-adrenal axis, and that this suppression most likely reflects suppression of the CRH neuron rather than of the pituitary corticotroph cell. We speculate that the enhanced capacity of alprazolam to suppress the CRH neuron relative to other benzodiazepines may contribute to its unique efficacy among this class of drugs in the treatment of major depression. The capacity of Ro 15-1788 to reverse alprazolam-induced suppression of the CRH neuron indicates that the effects of alprazolam are mediated at least in part via its interaction with the benzodiazepine component of the gamma-aminobutyric acidA macromolecular complex.

The ovine corticotropin-releasing hormone-stimulation test in type I diabetic patients and controls: suggestion of mild chronic hypercortisolism.

We examined hypothalamic-pituitary-adrenal (HPA) axis function in insulin-dependent diabetic outpatients (N = 22) and age-, sex-, and weight-matched normal controls (N = 22). The evaluation included measurements of 9:00 AM fasting plasma cortisol and cortisol-binding globulin (CBG) levels, 24-hour urinary free cortisol (UFC) excretion, and plasma corticotropin and cortisol responses to intravenously administered ovine corticotropin-releasing hormone ([CRH] 1 microgram/kg given as a bolus at 8:00 PM). Diabetic patients had significantly elevated 9:00 AM plasma cortisol levels (mean +/- SE, 300.7 +/- 99.3 v 237.3 +/- 99.3 nmol/L, P < .04), higher 24-hour UFC excretion (313.2 +/- 112.6 v 244.2 +/- 69.3 nmol/24 h, P < .02), and greater cortisol responses to CRH infusion (time-integrated values: 49,408.2 +/- 11,289.8 v 40,217.9 +/- 7,228.6 nmol/L.120 min, P < .004; peak cortisol values: 529.7 +/- 107.6 v 438.7 +/- 77.3 nmol/L, P < .002) than controls. UFC excretion values were positively correlated with both 5-year averaged hemoglobin A1c level (P = .03) and total number of insulin units administered per day (P = .03). These results suggest that insulin-dependent diabetic outpatients have mild chronic hypercortisolism, which might influence the control of the disease and play a role in the development of its chronic complications.

Hemodynamic effects of corticotropin releasing hormone in the anesthetized cynomolgus monkey.

Right atrial bolus administration of rat/human corticotropin releasing hormone (r/hCRH) at a dose of 90 micrograms/kg to anesthetized cynomolgus monkeys caused a dramatic and prolonged fall in both the peripheral vascular resistance (48% reduction) and mean systemic blood pressure (36% reduction). An associated tachycardia could be blocked with prior propranolol administration and thus was probably reflexic. A mean 43 and 37% increase in the flow of the superior mesenteric and common iliac arteries, respectively, was demonstrated with electromagnetic flow probes. These changes were associated with a concomitant 38 and 40% diminution in the respective vascular resistance. Similar blood flow changes were noted in the carotid artery, however, these were of a much shorter duration. None of these changes occurred in placebo-treated animals. Plasma adrenocorticotropic hormone and cortisol concentrations were elevated basally and throughout the procedure and were similar in the experimental and control groups, suggesting maximal activity of the hypothalamic-pituitary-adrenal axis. Plasma renin activity, however, gradually increased in the r/hCRH-treated animals, probably as a result of the systemic hypotension. We speculate that CRH or a CRH-like substance may function as a paracrine hormone modulating local blood vessel tone and may be important in directing blood flow during stress and injury. The vasoactive properties of exogenous r/hCRH may be of clinical use in man.

Effects of serotonergic agonists and antagonists on corticotropin-releasing hormone secretion by explanted rat hypothalami.

Experimental evidence suggests that serotonin (5HT) is excitatory to the hypothalamic-pituitary-adrenal axis and that this effect involves activation of both hypothalamic corticotropin-releasing hormone (CRH) and pituitary ACTH secretion. The present study was undertaken to examine the mechanism by which 5HT stimulates the central component of the HPA axis. To accomplish this we employed an in vitro rat hypothalamic organ culture system in which CRH secretion from single explanted hypothalami was measured by specific radioimmunoassay (IR-rCRH). All experiments were performed after an overnight (15-18 hr) preincubation. Serotonin stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped and the peak effect was observed at the concentration of 10(-9) M. The stimulatory effect of 10(-9) M 5HT was antagonized by the 5HT1 and 5HT2 receptor metergoline and by the selective 5HT2 receptor antagonists ketanserin and ritanserin. The muscarinic antagonist atropine, the nicotinic antagonist hexamethonium and the alpha-adrenergic receptor antagonist phentolamine, on the other hand, did not inhibit 5HT-induced IR-rCRH secretion. The specific 5HT2 receptor agonist 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped with peak of effect reached at the concentration of 10(-9) M. We also tested the ability of the 5HT agonist meta-chlorophenylpiperazine (m-CPP) and of the selective 5HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) to cause CRH secretion. Although both m-CPP and 8-OH-DPAT stimulated IR-rCRH secretion in a dose-dependent fashion, several differences were observed when their effect was compared to that of 5HT. These included a different shape of the dose-response curve, a lower maximal stimulatory effect and a different maximal stimulatory concentration. These findings suggest that serotonin stimulates CRH secretion by explanted rat hypothalami and that this effect appears to be mediated mainly through a 5HT2 receptor mechanism.

Interaction between GABAergic neurotransmission and rat hypothalamic corticotropin-releasing hormone secretion in vitro.

Corticotropin-releasing hormone (CRH) has been considered a major coordinator of the overall physical and behavioral response to stress. Moreover, prolonged hypersecretion of CRH has been implicated in the pathogenesis of disorders characterized by anxiety and/or depression. Drugs acting through the gamma-aminobutyric acid/benzodiazepine (GABA/BZD) receptor system have anxiolytic and/or antidepressant properties whereas benzodiazepine inverse agonists cause anxiety and stimulate the pituitary-adrenal axis in vivo. To examine the involvement of the GABA/BZD system in the regulation of hypothalamic CRH secretion, we studied the effects of various agonists and antagonists of GABAA and GABAB receptors using a sensitive rat hypothalamic organ culture with radioimmunoassayable CRH (IR-rCRH) as endpoint. The GABAA and GABAB receptor agonist GABA inhibited serotonin (5-HT)-induced IR-rCRH secretion from 10(-9) to 10(-6) M, but failed to do so at 10(-5) M. The GABAA receptor agonist muscimol was a weak inhibitor of 5-HT-induced IR-rCRH secretion, being effective only at the concentration of 10(-6) M. In contrast, the specific GABAB receptor agonist baclofen was able to inhibit 5-HT-induced IR-rCRH secretion from 10(-7) to 10(-5) M. The rank of potency was thus, GABA much greater than baclofen greater than muscimol. Bicuculline, a GABAA receptor antagonist, partially reversed the inhibitory effects of GABA. Diazepam, a classic benzodiazepine which interacts with the benzodiazepine-site of the GABAA receptor complex, inhibited 5-HT-induced IR-rCRH secretion from 3.3 X 10(-9) to 10(-5) M, an effect that could be reversed by the BZD inactive ligand Ro15-1788.(ABSTRACT TRUNCATED AT 250 WORDS)

Cocaine stimulates rat hypothalamic corticotropin-releasing hormone secretion in vitro.

Acute or chronic cocaine administration exerts multiple behavioral and physiologic effects including stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. Pharmacologically, cocaine shares major properties with at least 2 classes of pharmaceuticals. It is a local anesthetic and also a potent psychomotor stimulant. The psychomotor stimulant properties of cocaine are thought to be related to its ability to modify the metabolism and the activity of many neurotransmitter systems, such as acetylcholine (ACh), serotonin (5-HT), norepinephrine (NE), and dopamine (DA). We and others have shown that all these neurotransmitters are potent stimulants of hypothalamic corticotropin-releasing hormone (CRH) secretion. The present study was undertaken to examine whether cocaine stimulates hypothalamic CRH secretion and whether or not such an effect is mediated by any of the above neurotransmitters. To accomplish this task, we employed a rat hypothalamic organ culture system, in which CRH secretion form single explanted hypothalami was evaluated by specific radioimmunoassay (iCRH). Cocaine stimulated iCRH secretion in a dose-dependent fashion with peak of activity at 10(-8) M. Isolated or simultaneous pharmacologic blockade of cholinergic (atropine plus hexamethonium), serotonergic (ritanserin), alpha-adrenergic (phentolamine) and/or dopaminergic (compound SCH 23390) receptor subtypes failed to inhibit cocaine-induced iCRH secretion. On the other hand, cocaine-induced iCRH secretion was inhibited by GABA, a potent inhibitor of CRH secretion, dexamethasone, verapamil, a calcium channel blocker, tetrodotoxin, a sodium channel blocker, and carbamazepine, an antiepileptic and antidepressive agent.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal ACTH and cortisol responses to ovine corticotropin releasing factor in patients with primary affective disorder.

To further explore hypothalamic pituitary adrenal regulation in patients with affective illness, we administered 1 microgram/kg of synthetic ovine corticotropin releasing factor at 2000h to 26 drug-free patients with this disorder and to 15 healthy controls. Compared to controls, depressed patients (N = 12) showed a significant elevation in baseline cortisol and significant reductions in the net ACTH and cortisol responses to corticotropin releasing factor. These findings were normal in manic (N = 6) and improved (N = 8) subjects. An additional finding was that baseline cortisol and net ACTH and cortisol responses to CRF were negatively correlated in the entire group of patients and controls as well as in the patients alone. These data indicate that the reduced ACTH and cortisol responses to CRF in depression reflect normal functioning of the pituitary corticotroph cell (i.e., that the negative feedback effect of cortisol on ACTH secretion in depression is physiologically intact, effectively serving as a brake on the ACTH response to exogenous CRF. Thus, the hypercortisolism of depression may be due to a hypothalamic defect, possibly involving hypersecretion of endogenous CRF. This possibility may be of particular interest in light of clinical observations that depression can often be precipitated by stress and by data in experimental animals that CRF may influence several processes known to be altered in the overall symptom complex of depression.

Sex differences in sensitivity of the hypothalamic-pituitary-adrenal axis.

Two studies examined sex differences in responsiveness of the hypothalamic-pituitary-adrenal cortical axis, a major component of the stress response. The first measured pituitary-adrenal responses to ovine corticotropin-releasing hormone (oCRH) in 24 health men and 19 healthy women. Plasma adrenocorticotropin hormone (ACTH) response to oCRH were significantly greater among women than among men. In contrast, cortisol concentrations were similar in both groups, though elevations were more prolonged in women. Differences in corticotropin-releasing activity between men and women may help account for these findings; such differences in central components of the stress response might play a role in the known epidemiological differences in diseases of stress system dysregulation between men and women.

Effect of cholinergic agonists and antagonists on rat hypothalamic corticotropin-releasing hormone secretion in vitro.

Several lines of experimental evidence suggest that acetylcholine (ACh) is excitatory to the hypothalamic-pituitary-adrenal (HPA) axis. Since previous experiments have shown that ACh does not affect pituitary adrenocorticotropin secretion in vitro, we hypothesized that ACh stimulates the HPA axis by causing hypothalamic corticotropin-releasing hormone (CRH) secretion. We examined this hypothesis using an organ culture system that measures the ability of single rat hypothalami to secrete immunoreactive CRH (IR-rCRH) in vitro. ACh stimulated hypothalamic IR-rCRH secretion in a dose-dependent fashion, at concentrations ranging from 3.3 x 10(-10) to 10(-5) M. This effect was antagonized by the simultaneous presence of atropine and hexamethonium, a muscarinic and a nicotinic receptor antagonist, respectively (p less than 0.05). Further evidence for the cholinergic regulation of the CRH neuron was provided by the findings that both carbachol, a muscarinic receptor agonist, and nicotine, a nicotinic receptor agonist, stimulated IR-rCRH secretion in a dose-dependent fashion. These effects were antagonized by atropine and hexamethonium, respectively, suggesting that both muscarinic and nicotinic receptors are involved in the process. ACh stimulated hypothalamic IR-rCRH secretion in the presence of phentolamine, an alpha-adrenergic antagonist, and ritanserin, a serotonin2 receptor antagonist, suggesting that the cholinergic stimulation of CRH secretion is not mediated by alpha-adrenergic or serotonergic interneurons. We conclude that ACh stimulates hypothalamic CRH secretion via both muscarinic and nicotinic receptor mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Procaine and lidocaine stimulate corticotropin-releasing hormone secretion by explanted rat hypothalami through a sodium conductance-independent mechanism.

We have previously shown that procaine and lidocaine stimulate corticotropin-releasing hormone (CRH) secretion by explanted rat hypothalami. This effect was of interest in light of the fact that both lidocaine and CRH administration to experimental animals can produce kindled seizures which cross-sensitize with electrically kindled seizures, and of recent data suggesting that limbic hyperexcitability, perhaps mediated through CRH, may be involved in the pathophysiology of affective illness. Because a prominent effect of the local anesthetics is to decrease neuronal firing by blocking sodium conductance, we were surprised by the capacity of these agents to cause CRH secretion and pituitary-adrenal activation and wished to further elucidate the possible mechanism(s) of these effects. To accomplish this, we first explored the effect of the sodium channel blocker tetrodotoxin (TTX) on basal and stimulated immunoreactive CRH (iCRH) secretion by explanted rat hypothalami. In contrast to procaine and lidocaine, TTX inhibited rather than stimulated iCRH secretion. Moreover, TTX inhibited lidocaine-induced iCRH secretion but had no influence on the response of the CRH neuron to procaine. To explore other potential mechanisms of action, we examined the effect of the calcium channels blocker verapamil and of pharmacologic antagonists to serotonergic, alpha-adrenergic and cholinergic receptors. The latter was particularly of interest because of structural similarities between procaine or lidocaine and acetylcholine (ACh) and because it has been shown that these anesthetic agents interact with the ACh receptor. Verapamil and blockade of serotonergic, alpha-adrenergic and cholinergic receptors did not inhibit the effects of procaine or lidocaine on iCRH secretion, whereas both GABA and dexamethasone exerted inhibitory effects.(ABSTRACT TRUNCATED AT 250 WORDS)