Autocrine or paracrine inflammatory actions of corticotropin-releasing hormone in vivo.

Corticotropin-releasing hormone (CRH) functions as a regulator of the hypothalamic-pituitary-adrenal axis and coordinator of the stress response. CRH receptors exist in peripheral sites of the immune system, and CRH promotes several immune functions in vitro. The effect of systemic immunoneutralization of CRH was tested in an experimental model of chemically induced aseptic inflammation in rats. Intraperitoneal administration of rabbit antiserum to CRH caused suppression of both inflammatory exudate volume and cell concentration by approximately 50 to 60 percent. CRH was detected in the inflamed area but not in the systemic circulation. Immunoreactive CRH is therefore produced in peripheral inflammatory sites where, in contrast to its systemic indirect immunosuppressive effects, it acts as an autocrine or paracrine inflammatory cytokine.

Cerebrospinal fluid neurochemistry in children and adolescents with obsessive-compulsive disorder.

Cerebrospinal fluid hormones, monoaminergic metabolites, and dynorphin A (1-8 sequence) were examined in 43 children with severe, primary obsessive-compulsive disorder. Cerebrospinal fluid levels of 5-hydroxyindoleacetic acid were positively correlated with one of eight obsessive-compulsive disorder severity ratings and three of seven measures of improvement following 5 weeks of treatment with clomipramine hydrochloride. Arginine vasopressin concentration was significantly and negatively correlated with several ratings of obsessive-compulsive disorder symptom severity, while oxytocin concentration was positively correlated with depressive symptoms. The ratio of arginine vasopressin to oxytocin was also negatively correlated with obsessive-compulsive disorder and depressive symptoms. Comorbid affective disorder was associated with decreased arginine vasopressin concentrations, while concomitant anxiety disorder was associated with increased oxytocin. Dynorphin A (1-8 sequence), homovanillic acid, corticotropin, 3-methoxy-4-hydroxyphenylglycol, and corticotropin releasing hormone were not significantly related to obsessive-compulsive disorder symptoms. These results seem to indicate that arginine vasopressin may be related to obsessive-compulsive disorder symptom severity, while 5-hydroxyindoleacetic acid might be associated with drug response.

Effects of immune neutralization of corticotropin-releasing hormone, adrenocorticotropin, and beta-endorphin in the surgically stressed rat.

Specific in vivo neutralization was used in an attempt to explore the roles of corticotropin-releasing hormone (CRH), ACTH, and beta-endorphin during surgical stress in Sprague-Dawley rats. Rats were randomly assigned to groups (n = 20-30/group) that received iv injections of rabbit antirat/human CRH (anti-r/hCRH), antihuman ACTH (anti-hACTH), antihuman beta-endorphin (anti-h beta-endorphin), or normal rabbit serum. Three hours later all animals were subjected to a uniform stress consisting of ether anesthesia, surgical laparotomy, and phlebotomy of 7 ml via the inferior vena cava. Survival rates were recorded, and RIAs were performed for ACTH, beta-endorphin, and corticosterone. Rats treated with anti-h beta-endorphin had a survival rate of 64%, which was significantly higher than that of the control group (33%; P less than 0.025, by analysis of variance). Anti-r/hCRH or anti-hACTH treatment was not associated with a change in survival rate. Plasma immunoreactive beta-endorphin levels were markedly decreased in the group treated with anti-h beta-endorphin (P less than 0.0001). Anti-r/hCRH had no effect on plasma immunoreactive ACTH or beta-endorphin. Plasma immunoreactive ACTH and corticosterone levels were decreased in the group treated with anti-hACTH (P less than 0.0001 and P less than 0.01, respectively). We conclude that 1) beta-endorphin immune neutralization is associated with a survival advantage during severe surgical stress, suggesting that circulating beta-endorphin might have deleterious effects during stress; 2) In severe stress, acute immune neutralization of CRH is not sufficient to inhibit ACTH, beta-endorphin, and corticosterone secretion, suggesting significant involvement of other secretagogues of the pituitary-adrenal axis; and 3) moderate decreases in corticosterone cannot affect survival, presumably because glucocorticoids play only a permissive role in maintaining cardiovascular stability during surgical stress.

Effects of short and long duration hypothyroidism and hyperthyroidism on the plasma adrenocorticotropin and corticosterone responses to ovine corticotropin-releasing hormone in rats.

We report here a study of the plasma ACTH and corticosterone responses to synthetic ovine CRH (oCRH) in hypothyroid and hyperthyroid rats studied 7, 15, and 60 days after either thyroidectomy or the administration of pharmacological doses of T4. The purpose of this study was to further clarify the time-dependent effects of alterations in thyroid status on the functional integrity of the hypothalamic-pituitary-adrenal axis and to aid in the interpretation of the oCRH stimulation test in hypo- and hyperthyroid states. Our data demonstrate that hypothyroid rats have a significant reduction in the cerebrospinal fluid (CSF) levels of corticosterone and a significant decrease in adrenal weight in association with significant increases in the plasma ACTH response to oCRH. On the other hand, the corticosterone response to the ACTH released during the oCRH stimulation test was significantly reduced in hypothyroidism. With increasing duration of thyroidectomy-induced hypothyroidism, there was a progressive fall in CSF corticosterone levels, a progressive increase in the plasma ACTH response to oCRH, and a gradual normalization of the corticosterone responses to the ACTH released during oCRH stimulation. Our findings in hyperthyroid rats were generally the converse of those seen in hypothyroidism. Hence, there was a significant increase in the CSF levels of corticosterone and a significant increase in adrenal weight in association with an initial slight decrease in the ACTH response to oCRH. On the other hand, the corticosterone response to the ACTH released during oCRH stimulation was significantly increased. There was a gradual increase in the magnitude of the rise in CSF corticosterone levels with time, as well as a gradual normalization of adrenocortical responses during oCRH stimulation. The ACTH plasma clearance rates were similar in hypo-, hyper-, and euthyroid rats. Our data do not permit definitive identification of the precise locus in the hypothalamic-pituitary-adrenal axis that is principally affected by experimentally induced alterations in thyroid status. However, these data are most compatible with a subtle hypothyroid-induced centrally mediated adrenal insufficiency and a subtle hyperthyroid-induced centrally mediated hypercortisolism. These data also suggest that alterations in hypothalamic-pituitary-adrenal function in states of disturbed thyroid function become somewhat more pronounced as the duration of thyroid dysfunction increases. The fact that pituitary-adrenal responses to oCRH are consistently altered in states of thyroid dysfunction may be relevant to the clinical interpretation of oCRH stimulation tests.(ABSTRACT TRUNCATED AT 400 WORDS)

Androstanediol glucuronide isomers in normal men and women and in men infused with labeled dihydrotestosterone.

3 alpha-Androstanediol glucuronide (Adiol G) is a major metabolite of dihydrotestosterone (DHT). Adiol G actually represents 2 different compounds, since the glucuronide can be conjugated at the 3-carbon position (Adiol 3-G) or at the 17-carbon position (Adiol 17-G). To determine which glucuronide represents the predominant physiological DHT metabolite and which isomer is the major circulating form, we developed a RIA to directly measure Adiol 3-G in serum extracts. In 10 normal men, mean serum Adiol 3-G and total Adiol G levels were 4.44 +/- 0.49 (+/- SE) nmol/L (208 +/- 23 ng/dL) and 27.9 +/- 2.8 nmol/L (1310 +/- 129 ng/dL), respectively (13.9 +/- 3.0% of Adiol G was Adiol 3-G). In 10 normal women sampled during the early follicular phase, mean serum Adiol 3-G and total Adiol G levels were 2.64 +/- 0.64 nmol/L (124 +/- 30 ng/dL) and 14.9 +/- 1.5 nmol/L (697 +/- 69 ng/dL), respectively (17.4 +/- 3.6% of Adiol G was Adiol 3-G). In 4 normal men infused for 8 h with tritiated DHT, 17.4 +/- 3.4% of the resulting tritiated Adiol G was Adiol 3-G. These results indicate that Adiol 17-G is the predominant circulating form of Adiol G in normal men and women and that it is also the major Adiol G isomer derived from DHT.

Sympathetic reactivity during a yohimbine challenge test in essential hypertension.

Systemic administration of yohimbine augments sympathetic outflow and blocks presynaptic alpha 2-adrenergic receptors, releasing the sympathetic neurotransmitter norepinephrine (NE) into the bloodstream. The present study examined sympathoadrenal and hemodynamic responses to yohimbine in 19 patients with essential hypertension and 19 normotensive control subjects. Baseline mean values for arterial NE, epinephrine, dihydroxyphenylglycol (the main intraneuronal metabolite of NE), spillover of NE into arterial plasma, and corticotropin did not differ between the hypertensive and normotensive groups. Yohimbine (0.125 mg/kg i.v. bolus followed by 0.001 mg/kg/min infusion for a total of 15 minutes) increased mean arterial pressure in all but one subject (by 13 +/- 2% [SEM] in the normotensive and 17 +/- 2% in the hypertensive group) and increased arterial NE levels in all subjects (by 253 +/- 50 pg/ml in the normotensive and 312 +/- 51 pg/ml in the hypertensive group). Among hypertensive patients, pressor, cardiac, output, and arterial NE responses were distributed bimodally. Patients with large hemodynamic and NE responses to yohimbine typically reported a history of anxiety, depression, or other psychopathology and of marked pressor or tachycardic episodes during emotional stress. In the hypertensive and normotensive groups, baseline arterial NE concentrations predicted the magnitude of pressor responses to yohimbine (r = 0.59, r = 0.54,p less than 0.01), whereas baseline mean arterial pressure was unrelated to the pressor response. A yohimbine challenge test can identify patients with pressor hyperresponsiveness and can distinguish patients with pressor hyperresponsiveness due to excessive sympathoadrenal reactivity from patients with enhanced postsynaptic responsiveness to endogenous NE.

Physiological and molecular aspects of mineralocorticoid receptor action in pseudohypoaldosteronism: a responsiveness test and therapy.

Pseudohypoaldosteronism (PHA), characterized by congenital resistance to aldosterone and excessive salt loss, has been traditionally treated with salt replacement. Although the mineralocorticoid receptor (MR) has been suggested as a potential locus of the defect in this disease, no such abnormality has been identified as yet. We studied a 17-yr-old male patient with congenital multifocal target organ resistance to aldosterone. Both carbenoxolone, an 11 beta-hydroxysteroid dehydrogenase inhibitor, and a high dose of fludrocortisone normalized the patient's serum electrolyte concentrations and decreased his urinary excretion of sodium, suggesting that this patient's resistance was partial and could be overcome by high concentrations of endogenous or exogenous mineralocorticoids. We hypothesized that the beneficial effect of these treatments was mostly mediated by the MR, because the administration of dexamethasone, while this patient was receiving a therapeutic dose of carbenoxolone, caused its reversal. These findings convinced us that there was functional, albeit possibly defective, MR in this patient and led us to perform molecular studies. Both alleles of the MR gene were expressed in the patient and his clinically and biochemically normal father. A conservative heterozygous mutation (A760-->G760, Ileu180-->Val180) and a nonconservative homozygous mutation (C944-->T944, Ala241-->Val241) were identified in the complementary DNA of both the patient and his father. The first untranslated exon and 0.9 kilobase of the 5'-regulatory region were also identical in the two men. It appears that the mutations causing amino acid substitutions represent polymorphisms, as we found high frequencies of both in the general population. We conclude that carbenoxolone and fludrocortisone may help define the presence of functional MR in patients with PHA and that the former could be used in the long term therapy of this disease. We hypothesize that the defect causing PHA in this patient might be in a post-MR step of aldosterone action.

The neuroendocrine effects of interleukin-2 treatment.

Observations of neuropsychiatric changes in patients receiving interleukin-2 (IL-2) led us to examine the effects of IL-2 administration on the stress-related hormones, beta-endorphin, ACTH, cortisol, and CRH. We evaluated 30 cancer patients who received immunotherapy with IL-2 or IL-2 plus lymphokine-activated killer (LAK) cells. Blood samples were taken immediately before and 4 and 8 h after infusion of IL-2 or IL-2 plus LAK cells. IL-2 stimulated increased hormone levels 4 h after infusion compared with those before therapy and with basal levels in normal volunteers at the following magnitudes: beta-endorphin, 10-fold; ACTH, 20-fold; and cortisol, 2-fold. The effect of IL-2 was not altered in patients also receiving LAK cells. An effect of treatment course was noted, with higher stimulated values seen 4 h after IL-2 in the second treatment course compared with those after the first course [change (delta) in beta-endorphin, 101 vs. 11 fmol/mL; delta ACTH, 138 vs. 8 pmol/L; delta cortisol, 414 vs. 218 nmol/L]. We conclude that IL-2 treatment induces the release of neuroendocrine hormones and that a significant increase in hormonal stimulation occurs upon reexposure to IL-2.

Plasma and cerebrospinal fluid measures of arginine vasopressin secretion in patients with bulimia nervosa and in healthy subjects.

Bulimia nervosa is a psychiatric syndrome associated with intense hunger, deficient satiety mechanisms, an obsessional preoccupation with the adverse consequences of eating, ritualistic binge eating, and subsequent purging to forestall the effects of the binge. The morbidity of this illness reflects both the psychological suffering associated with a life organized around pathological eating behaviors, as well as medical complications such as fluid and electrolyte imbalances that occur largely as a result of purging and laxative abuse. We report here a study of the osmoregulation of plasma arginine vasopressin secretion and of vasopressin levels in the cerebrospinal fluid. This study was undertaken because vasopressin not only functions as the antidiuretic hormone, and thus as a principal modulator of fluid and electrolyte balance, but also because, in animals, centrally directed vasopressin delays the extinction of behaviors acquired during aversive conditioning. Thirteen normal-weight female patients with bulimia nervosa were studied after at least 1 month of nutritional stabilization and supervised abstinence from binge eating and purging. Plasma vasopressin, plasma sodium, and subjective thirst were measured serially before and during a 2-h infusion of 3% hypertonic saline (0.1 ml/kg min). In addition, cerebrospinal fluid was obtained by lumbar puncture upon admission and at 1 week before hypertonic saline infusion in 11 of these patients and in an additional 11 female patients who did not participate in the hypertonic infusion study. Fifteen healthy normal weight individuals (4 female, 11 male) served as controls for the hypertonic saline infusion and a separate group of 11 healthy normal weight female controls underwent puncture. Compared to controls, bulimic subjects showed a significant reduction in the plasma vasopressin response to hypertonic saline; in 12/13, plasma vasopressin correlated closely with plasma sodium, whereas in one patient vasopressin fluctuated erratically, with no relation to plasma sodium. Cerebrospinal fluid vasopressin levels were significantly higher in patients, and correlated positively with basal thirst level, which was enhanced in bulimics. Compared to controls, patients showed significant polyuria. We conclude that patients with bulimia nervosa have abnormal levels of vasopressin in their plasma and cerebrospinal fluid during abstinence from binge eating and purging. The disturbance in osmoregulation may aggravate the maintenance of adequate fluid volume in these patients, while the increase in centrally directed vasopressin may have relevance to their obsessional preoccupation with the aversive consequences of eating and weight gain.

Evidence for impaired activation of the hypothalamic-pituitary-adrenal axis in patients with chronic fatigue syndrome.

Chronic fatigue syndrome is characterized by persistent or relapsing debilitating fatigue for at least 6 months in the absence of a medical diagnosis that would explain the clinical presentation. Because primary glucocorticoid deficiency states and affective disorders putatively associated with a deficiency of the arousal-producing neuropeptide CRH can be associated with similar symptoms, we report here a study of the functional integrity of the various components of the hypothalamic-pituitary-adrenal axis in patients meeting research case criteria for chronic fatigue syndrome. Thirty patients and 72 normal volunteers were studied. Basal activity of the hypothalamic-pituitary-adrenal axis was estimated by determinations of 24-h urinary free cortisol-excretion, evening basal plasma total and free cortisol concentrations, and the cortisol binding globulin-binding capacity. The adrenal cortex was evaluated indirectly by cortisol responses during ovine CRH (oCRH) stimulation testing and directly by cortisol responses to graded submaximal doses of ACTH. Plasma ACTH and cortisol responses to oCRH were employed as a direct measure of the functional integrity of the pituitary corticotroph cell. Central CRH secretion was assessed by measuring its level in cerebrospinal fluid. Compared to normal subjects, patients demonstrated significantly reduced basal evening glucocorticoid levels (89.0 +/- 8.7 vs. 148.4 +/- 20.3 nmol/L; P less than 0.01) and low 24-h urinary free cortisol excretion (122.7 +/- 8.9 vs. 203.1 +/- 10.7 nmol/24 h; P less than 0.0002), but elevated basal evening ACTH concentrations. There was increased adrenocortical sensitivity to ACTH, but a reduced maximal response [F(3.26, 65.16) = 5.50; P = 0.0015). Patients showed attenuated net integrated ACTH responses to oCRH (128.0 +/- 26.4 vs. 225.4 +/- 34.5 pmol/L.min, P less than 0.04). Cerebrospinal fluid CRH levels in patients were no different from control values (8.4 +/- 0.6 vs. 7.7 +/- 0.5 pmol/L; P = NS). Although we cannot definitively account for the etiology of the mild glucocorticoid deficiency seen in chronic fatigue syndrome patients, the enhanced adrenocortical sensitivity to exogenous ACTH and blunted ACTH responses to oCRH are incompatible with a primary adrenal insufficiency. A pituitary source is also unlikely, since basal evening plasma ACTH concentrations were elevated. Hence, the data are most compatible with a mild central adrenal insufficiency secondary to either a deficiency of CRH or some other central stimulus to the pituitary-adrenal axis. Whether a mild glucocorticoid deficiency or a putative deficiency of an arousal-producing neuropeptide such as CRH is related to the clinical symptomatology of the chronic fatigue syndrome remains to be determined.

No apparent mineralocorticoid receptor defect in a series of sporadic cases of pseudohypoaldosteronism.

Pseudohypoaldosteronism (PHA) is characterized by congenital resistance of the kidney and/or other mineralocorticoid target tissues to aldosterone, resulting in excessive salt wasting. Although the mineralocorticoid receptor (MR) was suggested as a potential locus of the defect in this disease, no such abnormality was found in 3 recently reported cases, one of whom belongs to this series of 5 patients. Molecular studies of the MR complementary DNA and gene in this series of sporadic cases of pseudohypoaldosteronism are reported. Four of these patients had multiple mineralocorticoid target tissue resistance, whereas 1 had transient isolated resistance in the kidney. A nonconservative homozygous mutation (C944-->T944, Ala241-->Val241) was identified in the complementary DNA of 4 of the patients but was also found in 62 of 100 normal alleles. One of these 4 patients had an additional conservative heterozygous mutation (A760-->G760, Ileu180-->Val180), which was also present in 11 of 100 normal alleles. None of the patients had any abnormalities in the first untranslated exon and 0.9 kilobases of the 5'-regulatory region of the MR gene, which were fully sequenced and compared with the normal sequence. It is concluded that the mutations identified in 4 of these 5 patients with PHA are polymorphisms, which on their own have no apparent pathophysiological significance. It is hypothesized that the defect causing PHA might be in a post-MR step of aldosterone action or in an unsuspected nonclassic receptor for this hormone.

Diurnal variation of cerebrospinal fluid immunoreactive corticotropin-releasing hormone levels in healthy volunteers.

CRH is not only secreted into hypophyseal protal blood where it is believed to regulate the circadian rhythm of pituitary-adrenal activity, but is also measurable in cerebrospinal fluid (CSF). Altered CSF immunoreactive CRH (IR-CRH) levels have been found in patients with a number of neuropsychiatric disorders and have been implicated in some of the symptoms of these disorders. To further study the potential functional relevance of CRH in human CSF, we examined whether a nonuniform temporal pattern of IR-CRH levels existed in CSF using hourly sampling over a 30-h period in six healthy volunteers. CSF was withdrawn continuously at 6 mL/h through a catheter placed in the lumbar subarachnoid space and connected to a miniroller pump and fraction collector. A significant diurnal variation in CSF IR-CRH levels was observed (P < 0.001), with highest levels between 1830-2330 h and lowest levels around 0730 h. This pattern was nearly opposite that of plasma cortisol levels, which showed the expected peak around 0800 h and nadir around 2000-2200 h. In addition, CSF IR-CRH levels in three of the six volunteers showed significant negative correlations with simultaneous plasma cortisol levels. These data suggest that CSF IR-CRH concentrations are negatively modulated by peripheral cortisol secretion, which may be one factor involved in the entrainment of this rhythm. Although the functional significance of this diurnal variation in CSF IR-CRH levels is unknown, the presence of a distinct temporal organization of CRH release into the CSF in humans is compatible with the idea that CSF may play a functional role in or otherwise reflect nonsynaptic information processing in the central nervous system. Diurnal factors should be taken into account in future studies of CRH concentrations in human CSF.

Effects of alprazolam on pituitary-adrenal and catecholaminergic responses to metabolic stress in humans.

Concurrent effects of benzodiazepines on stress-induced activation of the three classical "stress" systems: pituitary-adrenal, adrenomedullary, and sympathoneural systems have not been extensively investigated in humans. In the present study, the effects of alprazolam (1.5 mg) on plasma levels of adrenocorticotropin hormone (ACTH), epinephrine, norepinephrine, dihydroxyphenylglycol (DHPG, the intraneuronal metabolite of norepinephrine), and mood states were examined in 10 healthy volunteers undergoing glucoprivic stress. Glucoprivic stress was induced by intravenous administration of the glucose analog, 2-deoxyglucose (2DG), at a dose (50 mg/kg) that impairs cellular glucose metabolism and produces a state comparable to hypoglycemia. Alprazolam and 2DG were administered in a double-blind, placebo-controlled manner. 2DG produced robust elevations in plasma ACTH and epinephrine levels, modest elevations in plasma norepinephrine levels, and decreases in plasma DHPG levels. Alprazolam significantly attenuated the 2DG-induced increases in plasma ACTH and epinephrine, but did not significantly effect plasma norepinephrine and DHPG. These data suggest that benzodiazepines attenuate metabolic stress-induced activation of the pituitary-adrenal and adrenomedullary systems but do not effect 2DG-related effects on peripheral sympathoneural function. The possible mechanisms involved are discussed.

Association of fluoxetine treatment with reductions in CSF concentrations of corticotropin-releasing hormone and arginine vasopressin in patients with major depression.

The authors measured CSF concentrations of corticotropin-releasing hormone (CRH) and arginine vasopressin in nine depressed patients before and after fluoxetine treatment. They found significant decreases in CSF CRH, CSF arginine vasopressin, and Hamilton depression ratings. Thus, the therapeutic effect of this serotonin-uptake inhibitor may be related to diminution of these arousal-promoting neuropeptides.

CSF oxytocin in anorexia nervosa and bulimia nervosa: clinical and pathophysiologic considerations.

Oxytocin is a hypothalamic neuropeptide with both centrally and peripherally directed pathways. Data from experimental animals indicate that oxytocin impairs consolidation of aversively conditioned behaviors and is released after feeding or experimental gastric distension. The authors report that the mean CSF oxytocin level of five underweight women with restricting anorexia, but not 12 underweight bulimic anorexic women or 35 normal-weight women with bulimia nervosa, was significantly lower than the level of 11 control subjects. Restricting anorexic patients' low CSF oxytocin levels may reflect their persistently low food intake, and this behavior may exacerbate their tendency for perseverative preoccupation with adverse consequences of food intake.

The effect of clozapine on plasma norepinephrine: relationship to clinical efficacy.

Clozapine is an atypical neuroleptic medication with superior efficacy to conventional antipsychotic agents for patients with chronic, symptomatic schizophrenia. Neurochemical characteristics that distinguish clozapine from other neuroleptics and contribute to its differential efficacy are not known. We assessed the effects of clozapine on plasma levels of norepinephrine (NE) in a double-blind, parallel groups comparison of clozapine (n = 11) and haloperidol (n = 15) in chronic schizophrenic outpatients who had been previously treated with fluphenazine. Simultaneous measurements were obtained for plasma levels of the catecholamine precursor dopa, the dopamine metabolite dihydroxyphenylacetic acid (DOPAC), the NE metabolite 3,4-dihydroxyphenylglycol (DHPG), adrenocorticotropin (ACTH), cortisol, and hemodynamic parameters. Clozapine produced marked increases (471%) in plasma NE levels, whereas haloperidol had no significant effects on plasma NE levels. Clozapine also increased dopa and tended to increase DOPAC levels, without effects on DHPG, ACTH, or cortisol levels and without consistent changes in blood pressure. Across patients, the magnitude of clozapine-induced increments in plasma NE levels was positively related to improvement in positive symptoms and global symptomatology and was unrelated to the occurrence of extrapyramidal symptoms. The results suggest that clozapine differs importantly from other neuroleptics in increasing plasma NE levels, with the peripheral noradrenergic stimulation related to its superior efficacy profile. The unchanged DHPG levels and absence of hypertension suggest a more complex mechanism of action of clozapine than heightened NE release alone.

Lipopolysaccharide-induced oestrogen receptor regulation in the paraventricular hypothalamic nucleus of lewis and Fischer rats.

Oestrogen receptor (ER) regulation of gene transcription in neurosecretory and pituitary cells has been proposed as an important mechanism for increased hypothalamic-pituitary-adrenal (HPA) axis responses in females of several mammalian species, including humans. Inbred female Fischer (F344/N) and Lewis (LEW/N) rats have similar oestrogen levels, although Fischer rats exhibit hyper- and Lewis rats hypo-HPA axis responses. The blunted HPA axis response of Lewis rats has been associated with their blunted hypothalamic corticotropin releasing hormone (CRH) expression. To determine if the female CRH expression deficiency in Lewis rats is associated with defective ER expression and regulation, hypothalamic paraventricular nucleus (PVN) transcript levels of CRH and ER were determined under basal conditions and after immune challenge. Microdissected PVN were obtained from control and lipopolysaccharide (LPS) treated Lewis and Fischer rats and CRH, ERalpha and beta mRNA levels were determined by semiquantitative reverse-transcriptase-polymerase chain reaction. In addition, ERalpha and beta protein levels were determined by semiquantitative Western blots. ERalpha and beta mRNA and protein levels in the PVN of control Fischer rats were significantly higher than in control Lewis rats. ERalpha and beta mRNA and protein levels in Fischer rats were reduced by LPS administration at the time of maximal CRH mRNA levels but did not change in Lewis rats, an effect independent of oestrogen levels. These data indicate that defective neuroendocrine HPA axis responses are associated with defective ER expression and regulation in Lewis PVN despite oestrogen concentrations.

Role of CRH in glucopenia-induced adrenomedullary activation in rats.

Acute glucoprivation profoundly stimulated hypothalamic-pituitary-adrenocortical (HPA) and adrenomedullary outflows. Whether these responses reflect a single central mechanism regulated by corticotropin-releasing hormone (CRH) has been unclear. This study examined the role of endogenous CRH in HPA and adrenomedullary responses to hypoglycemia in Sprague-Dawley rats, by using anti-CRH immune serum or a CRH antagonist (alpha-helical h/r CRH9-41, and in Lewis rats, a strain characterized by deficient hypothalamic CRH responses during stress. In conscious Sprague-Dawley rats with indwelling arterial and venous cannulas, insulin (0.3 U/kg was injected iv, and responses of serum glucose concentrations and plasma levels of corticotropin (ACTH) and catechols (including epinephrine, EPI; norepinephrine, NE; dihydroxyphenylalanine, DOPA; dihydroxyphenylglycol, DHPG; and dihydroxyphenylacetic acid, DOPAC) were assessed, with or without pretreatment with anti-CRH immune serum (0.5 or 1.0 ml iv or 10 microl icv) or alpha-helical h/r CRH9-41 (130 nmol iv or 13 nmol icv). Responses to insulin (1.0 U/kg iv) were also measured in conscious juvenile Lewis and Fischer 344/N rats. Insulin-induced hypoglycemia markedly increased plasma levels of EPI and ACTH in all groups. Pretreatment iv with 1/0 ml of anti-CRH immune serum blocked the ACTH response to insulin but failed to attenuate the EPI response. alpha-helical h/r CRH9-41, whether given iv or icv, failed to alter ACTH or EPI responses to insulin, although the antagonist did block EPI responses to icv CRH. Hypoglycemia elicited similar increments in ACTH levels in Lewis rats and Fischer 344/N control rats; and although Lewis rats had lower baseline EPI and smaller responses of NE, DHPG, DOPA, and DOPAC levels, the groups did not differ in proportionate increments in EPI levels. The results indicate that the ACTH response to hypoglycemia depends on availability of CRH outside the blood-brain barrier--presumably in the pituitary gland. The findings with icv alpha-helical h/r CRH9-41 can be explained by failure of the antagonist to reach effective concentrations at central sites of action of endogenous CRH, or by mechanisms other than CRH release determining the adrenomedullary response to hypoglycemia. Lewis rats seem to have less adrenomedullary secretion at baseline and smaller responses of NE synthesis and release during hypoglycemia than do Fischer 344/N rats. Neurochemical evidence for differential adrenomedullary and sympathoneural responses during hypoglycemia in all three rat strains is inconsistent with Cannon's view of a functionally unitary sympathoadrenal system. Lewis rats have deficient CRH responses to some stressors but not to others, or else pituitary-adrenomedullary responses in this setting depend on mechanisms other than CRH release in the brain. Both explanations are inconsistent with the doctrine of non-specificity, the main tenet of Selye's stress theory.

The human mineralocorticoid receptor gene promoter: its structure and expression.

The structure and expression of a clone containing the promoter region, all of exon 1, and part of the first intron of the human mineralocorticoid receptor (hMR) gene is presented. The clone has three sets of CAAT and TATA elements, one located at the very 5'-end of the clone, one located just 5'- to the start of transcription, and one set located in intron A, approximately 300 bp into the intron. The major start of transcription site by primer extension analysis and ribonuclease protection assays is located 26 bp downstream of a TATA-like box (TTTAA) and 90 and 143 bp downstream, respectively, of two CCAAT boxes. Putative cis-transcription factor binding sites are as follows: two potential AP1 sites, one potential AP2 site, two ATF/CREB sites, six potential GC boxes or SP1 sites, one potential perfect half-palindromic estrogen response element, and three potential PEA3 sites. Therefore, the hMR promoter region contains elements characteristic of both regulated genes and "housekeeping" genes. CAT assays of overlapping deletions of the promoter region demonstrated tissue-specific regulation in human neuroepithelioma (SK-N-MC-IXC) and non-neuronal, peripheral choriocarcinoma cell lines (JEG-3).

Pulsatile activation of the hypothalamic-pituitary-adrenal axis during major surgery.

To examine the response of the hypothalamic-pituitary-adrenal (HPA) axis to severe surgical stress, we measured the immunoreactive plasma levels of corticotropin-releasing hormone (CRH), corticotropin, cortisol, arginine-vasopressin (AVP), atrial natriuretic factor (ANF), neuropeptide Y (NPY), interleukin-1 (IL-1), IL-6, interferon gamma (INF), and tumor necrosis factor-alpha (TNF-alpha) in eight patients with Zollinger-Ellison syndrome (ZES) or mediastinal parathyroid carcinoma, all undergoing major surgery with a standardized anesthetic technique. Blood samples were drawn the morning before surgery, every 10 to 30 minutes throughout surgery (average, 308.7 +/- 15 minutes), and every morning for the next 4 postoperative days (POD). During surgery, plasma CRH concentrations were slightly but not significantly elevated compared with those before surgery and with those of the next 4 POD. However, the values were within the normal range (less than 2.2 pmol/L) and showed 8.9 +/- 0.6 pulses (one pulse every 34.7 +/- 1.6 minutes). Plasma corticotropin, on the other hand, was quite elevated, but was also released in a pulsatile fashion during the surgical procedure (one pulse every 36.7 +/- 1.6 minutes). Most of these secretory episodes of corticotropin were temporally related to those of CRH. Corticotropin returned to basal levels on the first POD and remained so for all 4 POD. Plasma cortisol concentrations increased steadily during surgery and remained elevated the first POD. Cortisol showed 6.2 +/- 1.1 pulses during the operative sampling period (one pulse every 71.8 +/- 13 minutes). Plasma AVP concentrations were also markedly elevated during surgery, but individual secretory pulses were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)