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1.
Mol Psychiatry ; 7(5): 474-83, 433, 2002.
Article in English | MEDLINE | ID: mdl-12082565

ABSTRACT

When exposed to prolonged stress, rats develop gastric ulceration, enhanced colon motility with depletion of its mucin content and signs of physiological and behavioral arousal. In this model, we tested whether antidepressants (fluoxetine and bupropion), anxiolytics (diazepam and buspirone) or the novel nonpeptide corticotropin-releasing hormone (CRH) type-1 receptor (CRH-R1) antagonist, antalarmin, modify these responses. Fluoxetine, bupropion, diazepam and antalarmin all suppressed stress-induced gastric ulceration in male Sprague-Dawley rats exposed to four hours of plain immobilization. Antalarmin produced the most pronounced anti-ulcer effect and additionally suppressed the stress-induced colonic hypermotility, mucin depletion, autonomic hyperarousal and struggling behavior. Intraperitoneal CRH administration reproduced the intestinal but not the gastric responses to stress while vagotomy antagonized the stress-induced gastric ulceration but not the intestinal responses. We conclude that brain CRH-R1 and vagal pathways are essential for gastric ulceration to occur in response to stress and that peripheral CRH-R1 mediates colonic hypermotility and mucin depletion in this model. Nonpeptide CRH-R1 antagonists may therefore be prophylactic against stress ulcer in the critically ill and therapeutic for other pathogenetically related gastrointestinal disorders such as peptic ulcer disease and irritable bowel syndrome.


Subject(s)
Gastric Mucosa/physiopathology , Pyrimidines/therapeutic use , Pyrroles/therapeutic use , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Stomach Ulcer/drug therapy , Analysis of Variance , Animals , Bupropion/therapeutic use , Buspirone/therapeutic use , Colon/drug effects , Colon/physiology , Corticotropin-Releasing Hormone/pharmacology , Diazepam/therapeutic use , Dose-Response Relationship, Drug , Fluoxetine/therapeutic use , Gastric Mucosa/drug effects , Male , Mucins/metabolism , Rats , Rats, Sprague-Dawley , Stomach Ulcer/prevention & control
2.
Bioorg Med Chem Lett ; 11(3): 331-3, 2001 Feb 12.
Article in English | MEDLINE | ID: mdl-11212103

ABSTRACT

A high-affinity radioligand for CRHR1 has been prepared that can serve as a template for the development of SPECT imaging agents. The 5-chloro-N-cyclopropylmethyl-N-(2,6-dichloro-4-iodophenyl)-2-methyl-N-propylpyrimidine-4,6-diamine (6b, Ki = 14 nM), and the corresponding 4-bromophenyl analogue (6a, Ki = 21 nM), were synthesized in four steps from compound 3.


Subject(s)
Cyclopropanes , Pyrimidines , Radiopharmaceuticals , Receptors, Corticotropin-Releasing Hormone/metabolism , Tomography, Emission-Computed, Single-Photon/methods , Animals , Cyclopropanes/chemical synthesis , Cyclopropanes/metabolism , Humans , Iodine Radioisotopes , Ligands , Protein Binding , Pyrimidines/chemical synthesis , Pyrimidines/metabolism , Structure-Activity Relationship
3.
Proc Natl Acad Sci U S A ; 97(11): 6079-84, 2000 May 23.
Article in English | MEDLINE | ID: mdl-10823952

ABSTRACT

We evaluated the effects of the lipophilic nonpeptide corticotropin-releasing hormone (CRH) type 1 receptor antagonist antalarmin on the behavioral, neuroendocrine, and autonomic components of the stress response in adult male rhesus macaques. After oral administration, significant antalarmin concentrations were detected in the systemic circulation and the cerebrospinal fluid by a mass spectrometry-gas chromatography assay developed specifically for this purpose. Pharmacokinetic and dose-response studies suggested that an oral dose of 20 mg/kg was optimal for behavioral and endocrine effects. We then administered this dose in a double-blind, placebo-controlled fashion to monkeys exposed to an intense social stressor: namely, placement of two unfamiliar males in adjacent cages separated only by a transparent Plexiglas screen. Antalarmin significantly inhibited a repertoire of behaviors associated with anxiety and fear such as body tremors, grimacing, teeth gnashing, urination, and defecation. In contrast, antalarmin increased exploratory and sexual behaviors that are normally suppressed during stress. Moreover, antalarmin significantly diminished the increases in cerebrospinal fluid CRH as well as the pituitary-adrenal, sympathetic, and adrenal medullary responses to stress. We conclude that CRH plays a broad role in the physiological responses to psychological stress in primates and that a CRH type 1 receptor antagonist may be of therapeutic value in human psychiatric, reproductive, and cardiovascular disorders associated with CRH system hyperactivity.


Subject(s)
Anti-Anxiety Agents/therapeutic use , Anxiety/drug therapy , Corticotropin-Releasing Hormone/physiology , Pyrimidines/therapeutic use , Pyrroles/therapeutic use , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Stress, Psychological/drug therapy , Administration, Oral , Adrenocorticotropic Hormone/blood , Animals , Anti-Anxiety Agents/administration & dosage , Anti-Anxiety Agents/pharmacology , Anxiety/etiology , Arginine Vasopressin/cerebrospinal fluid , Corticotropin-Releasing Hormone/cerebrospinal fluid , Double-Blind Method , Drug Evaluation, Preclinical , Epinephrine/blood , Exploratory Behavior/drug effects , Fear/drug effects , Hydrocortisone/blood , Hypothalamo-Hypophyseal System/drug effects , Hypothalamo-Hypophyseal System/physiopathology , Macaca mulatta , Male , Norepinephrine/blood , Pituitary-Adrenal System/drug effects , Pituitary-Adrenal System/physiopathology , Pyrimidines/administration & dosage , Pyrimidines/pharmacology , Pyrroles/administration & dosage , Pyrroles/pharmacology , Receptors, Corticotropin-Releasing Hormone/physiology , Sexual Behavior, Animal/drug effects , Social Dominance , Stress, Psychological/metabolism , Stress, Psychological/physiopathology , Stress, Psychological/psychology
4.
Bioorg Med Chem Lett ; 10(8): 707-10, 2000 Apr 17.
Article in English | MEDLINE | ID: mdl-10782669

ABSTRACT

A series of fluoro-substituted 4-(dialkylamino)pyrrolo[2,3-d]pyrimidines was synthesized and their binding affinity for corticotropin-releasing hormone type 1 receptor (CRHR1) was investigated. Compounds 11a and 11b possessed very high CRHR1 affinity (Ki=3.5, 0.91 nM, respectively). They are promising candidates for the development of 18F-containing nonpeptide PET radioligands for CRHR1.


Subject(s)
Contrast Media/chemical synthesis , Contrast Media/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Receptors, Corticotropin-Releasing Hormone/metabolism , Animals , Cerebellum/metabolism , Contrast Media/metabolism , Fluorine/chemistry , Pyrimidines/metabolism , Pyrroles/chemistry , Radioligand Assay , Rats , Tomography, Emission-Computed
5.
Ann N Y Acad Sci ; 876: 1-11; discussion 11-3, 1999 Jun 22.
Article in English | MEDLINE | ID: mdl-10415589

ABSTRACT

Corticotropin-releasing hormone (CRH) influences the immune system indirectly, through activation of the hypothalamic-pituitary-adrenal axis and sympathetic system, and directly, through local modulatory actions of peripheral (immune) CRH. We recently demonstrated that catecholamines and histamine potently inhibited interleukin (IL)-12 and stimulated IL-10, whereas glucocorticoids suppressed IL-12, but did not affect IL-10 production ex vivo. Thus, both glucocorticoids and catecholamines, the end products of the stress system, and histamine, a product of activated mast cells, may selectively suppress cellular immunity and favor humoral immune responses. We localized immunoreactive CRH in experimental carrageenin-induced aseptic inflammation and, in humans, in inflamed tissues from patients with several autoimmune disease. In addition, we demonstrated that CRH activated mast cells via a CRH receptor type 1-dependent mechanism, leading to release of histamine and hence vasodilatation and increased vascular permeability. Thus, activation of the stress system, through direct and indirect effects of CRH, may influence the susceptibility of an individual to certain autoimmune, allergic, infectious or neoplastic diseases. Antalarmin, a novel nonpeptide CRH antagonist, prevented several proinflammatory effects of CRH, thus revealing its therapeutic potential in some forms of inflammation.


Subject(s)
Corticotropin-Releasing Hormone/physiology , Glucocorticoids/physiology , Immune System/physiopathology , Inflammation/etiology , Stress, Physiological/complications , Stress, Physiological/physiopathology , Animals , Humans , Time Factors
6.
Life Sci ; 65(4): PL53-8, 1999.
Article in English | MEDLINE | ID: mdl-10421433

ABSTRACT

Antalarmin is a pyrrolopyrimidine compound that antagonizes corticotropin-releasing hormone (CRH) type 1 receptors (CRHR1). In order to assess the effects of antalarmin treatment on hypothalamic-pituitary-adrenal (HPA) function we measured the plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone in animals treated with either antalarmin or vehicle for 1 week or for 8 weeks. We found that antalarmin treatment for 1 week did not affect basal concentrations of ACTH or corticosterone. In contrast, treatment for 8 weeks significantly lowered basal ACTH and corticosterone concentrations and also significantly decreased the basal corticosterone to ACTH ratio, indicating decreased basal adrenocortical responsiveness to ACTH. However, immobilization stress resulted in ACTH and corticosterone concentrations that were the same in animals treated with vehicle or antalarmin for either 1 or 8 weeks. We conclude that even though 8-week antagonism of CRHR1 by the non-peptide antalarmin blunts basal concentrations of ACTH and corticosterone, and affects the adrenal responsiveness to ACTH, it does not blunt the HPA response to acute stress, and it does not appear to cause stress-induced adrenal insufficiency.


Subject(s)
Hypothalamo-Hypophyseal System/drug effects , Pituitary-Adrenal System/drug effects , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Stress, Physiological/metabolism , Adrenocorticotropic Hormone/blood , Animals , Corticosterone/blood , Hypothalamo-Hypophyseal System/metabolism , Pituitary-Adrenal System/metabolism , Restraint, Physical
7.
Neuroimmunomodulation ; 6(3): 182-6, 1999.
Article in English | MEDLINE | ID: mdl-10213916

ABSTRACT

Urocortin, a newly isolated 40-amino-acid mammalian peptide homologous to corticotropin-releasing hormone (CRH), activates both CRH type 1 and 2 receptors, but may be an endogenous ligand for CRH receptor type 2. Urocortin given systemically inhibited heat-induced paw edema in the rat, and was therefore ascribed anti-inflammatory properties. We examined the effects of urocortin in the carrageenin-induced subcutaneous inflammation model. Rats were treated with urocortin 200 (n = 6) or 20 nmol/kg (n = 6); inflammatory exudates were reduced by approximately 30% compared to controls (n = 7) at both doses. However, since subcutaneous urocortin has been shown to reduce arterial blood pressure, we tested the hypothesis that its antiedema and antiextravasatory effects were secondary to arterial hypotension. Therefore, we examined the parallel effects of urocortin- and hydralazine-induced hypotension on acute inflammation induced by carrageenin in the rat. Rats were treated with subcutaneous carrageenin and control injections (n = 8), carrageenin and urocortin (20 nmol/kg, n = 9), or carrageenin and intraperitoneal hydralazine (10 mg/kg, n = 8). Mean arterial blood pressure was measured hourly for 7 h in 12 animals, and after 2 h, the nadir of treatment, in a further 13 animals. Rats were then sacrificed, and the inflammatory exudate volume and leukocyte count were measured. Mean exudate volumes were reduced from 4.8 +/- 0.5 ml (controls) to 2.4 +/- 0.3 ml (p = 0.004) and 2.9 +/- 0.6 ml (p = 0.007) in urocortin- and hydralazine-treated animals, respectively. Urocortin and hydralazine both produced a significant fall in blood pressure compared to controls, with mean arterial pressure 2 h after carrageenin injection falling to 51.0 +/- 4.1 (p < 0.001) and 34.6 +/- 4.6 (p < 0.001) vs. 92.9 +/- 3.7 mm Hg in controls, respectively. A significant positive correlation was noted between blood pressure and inflammatory exudate volume (r = 0. 52, p = 0.007). As both hydralazine and urocortin lowered blood pressure and inflammatory exudate volume, we suggest that the anti-inflammatory effects of urocortin and related neuropeptides may be nonspecific, acting through hypotension rather than through direct anti-inflammatory mechanisms. The use of inflammatory models which rely on extravasation may be inappropriate for the study of substances that produce hypotension.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Corticotropin-Releasing Hormone/physiology , Hypotension/complications , Inflammation/physiopathology , Receptors, Corticotropin-Releasing Hormone/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/toxicity , Blood Pressure/drug effects , Carrageenan/toxicity , Corticotropin-Releasing Hormone/pharmacology , Corticotropin-Releasing Hormone/toxicity , Edema/prevention & control , Exudates and Transudates/chemistry , Exudates and Transudates/cytology , Foot , Hydralazine/toxicity , Hypotension/chemically induced , Inflammation/complications , Inflammation/drug therapy , Leukocyte Count/drug effects , Male , Rats , Rats, Sprague-Dawley , Receptors, Corticotropin-Releasing Hormone/physiology , Urocortins
8.
Endocrinology ; 139(7): 3357-60, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9645712

ABSTRACT

In sheep, corticotropin-releasing hormone (CRH) can stimulate the fetal release of ACTH to produce a cortisol surge which leads to the onset of parturition. We tested the hypothesis that fetal CRH is a primary factor in the onset of parturition in sheep by using a Type I CRH receptor antagonist, antalarmin, to block the endogenous action of CRH. Pregnant ewes were cannulated at 130-135 days of gestation. Five catheters were placed into the amniotic sac, fetal femoral artery, fetal tarsal vein, maternal jugular vein and carotid artery. After 5 days' recovery, blood samples from maternal and fetal vessels were collected at the following times: a day before the start of infusion, at [-1, 0, 1, 2, 4, 8 and 24]h, on the first day of infusion, and thereafter daily throughout a 10-day infusion. Animals (n=6 per group) received infusions into a fetal vein of either a vehicle comprising 1:1 mixture of ethanol and polyethoxylated castor oil (Cremophor EL) or antalarmin (50 g/L) in the vehicle at a rate of 0.3 mL/h. The plasma samples were assayed for ACTH and cortisol using commercial RIA kits. Fetuses infused with vehicle delivered at a mean gestational age of 141.8 +/- 0.9 days compared with antalarmin-infused sheep at 148.8 +/- 1.6 days (P = 0.0036, unpaired Student's t-test). Fetal ACTH and cortisol did not change in the antalarmin-infused sheep after 3 days' infusion compared to significant increases in vehicle-infused sheep (P=0.004 and P = 0.016 respectively, ANOVA). These data show that CRH receptor antagonism in the fetus can delay the onset of parturition. It supports the hypothesis that hypothalamic CRH drives fetal production of ACTH and is essential for the onset of parturition triggered by a surge in fetal cortisol.


Subject(s)
Labor, Obstetric/drug effects , Pregnancy, Animal/physiology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Sheep/physiology , Adrenocorticotropic Hormone/blood , Animals , Female , Fetal Blood/drug effects , Fetal Blood/metabolism , Fetus/drug effects , Hydrocortisone/blood , Injections, Intravenous , Pharmaceutical Vehicles/pharmacology , Pregnancy
9.
Ann N Y Acad Sci ; 840: 21-32, 1998 May 01.
Article in English | MEDLINE | ID: mdl-9629233

ABSTRACT

Corticotropin-releasing hormone (CRH) is a major regulator of the hypothalamic-pituitary-adrenal axis (HPA) and principal coordinator of the stress response. As in stress, intracerebroventricular administration of CRH suppresses the immune system indirectly, via glucocorticoid and/or sympathetic system-mediated mechanisms. Also, during inflammatory stress, the cytokines TNF alpha, IL-1, and IL-6 stimulate hypothalamic CRH and/or vasopressin secretion as a way of preventing the inflammatory reaction from overreacting. Recently, CRH receptors were described in peripheral sites of the immune system, and CRH was found to promote several immune functions in vitro. We demonstrated a direct role of CRH in the inflammatory immune process in vivo, by first studying the effect of systemic CRH immunoneutralization in an experimental model of carrageenin-induced aseptic inflammation in Spague-Dawley rats. We extended these observations to other forms of experimental inflammation, including streptococcal cell wall polysaccharide- and adjuvant-induced arthritides and peptide R16 (epitope of the interphotoreceptor retinoid-binding protein)-induced uveitis in Lewis rats. We also studied human disease states, including rheumatoid arthritis, Hashimoto thyroiditis, and ulcerative colitis. Inflamed tissues contained large amounts of IR CRH, reaching levels similar to those observed in the hypophyseal portal system. We also demonstrated the presence of CRH mRNA and CRH receptors in inflammatory cells and identified the mast cells as a major immune target for CRH. In addition to production by immune cells, the peripheral nervous system, including the postganglionic sympathetic neurons and the sensory fibers type C, appears to contribute to IR CRH production in inflammatory sites. The production of CRH from the postganglionic sympathetic neurons may be responsible for the stress-induced activation of allergic/autoimmune phenomena, such as asthma and eczema, via mast cell degranulation. Antalarmin, a novel nonpeptide CRH receptor antagonist, displaced 125I-labeled ovine CRH binding in rat pituitary, frontal cortex, and cerebellum, but not heart, consistent with antagonism at the CRHR1 receptor. In vivo antalarmin significantly inhibited CRH-stimulated ACTH release and carrageenin-induced subcutaneous inflammation in rats. Thus, antalarmin and other related compounds that antagonize CRH at the level of its own receptor have therapeutic potential in some forms of inflammation directly mediated by type 1 CRH receptors and promise to enhance our understanding of the many roles of CRH in immune/inflammatory reactions.


Subject(s)
Corticotropin-Releasing Hormone/physiology , Inflammation/physiopathology , Animals , Corticotropin-Releasing Hormone/antagonists & inhibitors , Humans , Immune System/physiology , Immune System/physiopathology , Stress, Physiological/physiopathology
10.
Endocrinology ; 139(4): 1546-55, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9528933

ABSTRACT

CRH, the principal regulator of the hypothalamic-pituitary-adrenal axis and modulator of autonomic nervous system activity, also participates in the regulation of appetite and energy expenditure. Antalarmin, a pyrrolopyrimidine compound, antagonizes CRH type 1 receptor-mediated effects of CRH, including pituitary ACTH release, stress behaviors, and acute inflammation. We administered antalarmin chronically to evaluate its effects on hypothalamic-pituitary-adrenal axis function and metabolic status. Adult male rats were treated twice daily with 20 mg/kg of i.p. antalarmin or placebo over 11 days. The animals were weighed; plasma ACTH, corticosterone, leptin, and blood glucose levels were determined; and morphometric analyses were performed to determine adrenal size and structure, including sizing, histochemistry, immunohistochemistry, and electron microscopy. Leptin messenger RNA expression in peripheral fat was analyzed by Northern blot. Antalarmin decreased plasma ACTH (mean +/- SD, 2.62 +/- 0.063 pg/ml) and corticosterone concentrations (10.21 +/- 1.80 microg/dl) compared with those in vehicle-treated rats [respectively, 5.3 +/- 2.0 (P < 0.05) and 57.02 +/- 8.86 (P < 0.01)]. Antalarmin had no significant effect on body weight, plasma leptin, or blood glucose concentrations or fat cell leptin messenger RNA levels. The width of the adrenal cortex of animals treated with antalarmin was reduced by 31% compared with that in controls without atrophy of the gland. On the ultrastructural level, adrenocortical cells were in a hypofunctional state characterized by reduced vascularization, increased content of lipid droplets, and tubulovesicular mitochondria with fewer inner membranes. The apoptotic rate was increased in the outer zona fasciculata of animals treated with the antagonist (26.6 +/- 3.58%) compared with that in placebo-treated controls (6.8 +/- 0.91%). We conclude that chronic administration of antalarmin does not affect body weight, carbohydrate metabolism, or leptin expression, whereas it reduces adrenocortical function mildly, without anatomical, clinical, or biochemical evidence of causing adrenal atrophy. These results are promising for future uses of such an antagonist in the clinic.


Subject(s)
Adrenal Glands/drug effects , Body Weight/drug effects , Metabolism/drug effects , Pituitary Gland/drug effects , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Adipose Tissue/chemistry , Adrenal Glands/physiology , Adrenal Glands/ultrastructure , Adrenocorticotropic Hormone/blood , Animals , Apoptosis , Blotting, Northern , Corticosterone/blood , Leptin , Male , Microscopy, Electron , Pituitary Gland/physiology , Pituitary Gland/ultrastructure , Proteins/genetics , Proteins/metabolism , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley
12.
Mol Psychiatry ; 2(5): 368-72, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9322225

ABSTRACT

Neuroendocrine-immune interactions are profoundly regulated by corticotropin-releasing hormone (CRH) indirectly, through activation of a global stress response, and directly, through pro-inflammatory actions on peripheral immune functions. The indirect effects of stress on immune/inflammatory responses occur via the stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic/adrenomedullary system. We have demonstrated that glucocorticoids and catecholamines favor T helper 2 (TH2) over T helper 1 (TH1) immune cells and mediators, by controlling the production of specific key regulatory cytokines. This could explain the influences of chronic stress on the development, course, and pathology of certain allergic, autoimmune/inflammatory, infectious, and neoplastic diseases. We have also shown that 'immune CRH' is secreted peripherally and plays a direct immunomodulatory role as an autocrine or paracrine mediator of inflammation. Upon release from immune cells and peripheral sensory afferent and/or postganglionic sympathetic nerves, CRH acts locally to elicit pro-inflammatory responses. This would explain the triggering or exacerbation of certain allergic or vasokinetic states by acute stress.


Subject(s)
Adrenal Glands/drug effects , Corticotropin-Releasing Hormone/pharmacology , Hypothalamo-Hypophyseal System/drug effects , Neurons/drug effects , Animals , Stress, Physiological/immunology
14.
Endocrinology ; 137(12): 5747-50, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8940412

ABSTRACT

Corticotropin-releasing hormone (CRH) secreted from the hypothalamus is the major regulator of pituitary ACTH release and consequent glucocorticoid secretion. CRH secreted in the periphery also acts as a proinflammatory modulator. CRH receptors (CRH-R1, R2alpha, R2beta) exhibit a specific tissue distribution. Antalarmin, a novel pyrrolopyrimidine compound, displaced 12SI-oCRH binding in rat pituitary, frontal cortex and cerebellum, but not heart, consistent with antagonism at the CRHR1 receptor. In vivo antalarmnin (20 mg/kg body wt.) significantly inhibited CRH-stimulated ACTH release and carageenin-induced subcutaneous inflammation in rats. Antalarmin, or its analogs, hold therapeutic promise in disorders with putative CRH hypersecretion, such as melancholic depression and inflammatory disorders.


Subject(s)
Adrenocorticotropic Hormone/antagonists & inhibitors , Pituitary Gland/metabolism , Pyrimidines/pharmacology , Pyrroles/pharmacology , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Adrenocorticotropic Hormone/metabolism , Animals , Binding, Competitive , Carrageenan/administration & dosage , Carrageenan/pharmacology , Cerebellum/metabolism , Corticotropin-Releasing Hormone/metabolism , Frontal Lobe/metabolism , Inflammation/chemically induced , Inflammation/prevention & control , Injections, Subcutaneous , Male , Myocardium/metabolism , Rats , Rats, Sprague-Dawley , Sheep
15.
J Clin Invest ; 92(2): 961-8, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8394389

ABSTRACT

Corticotropin-releasing hormone (CRH), the principal neuropeptide regulator of pituitary ACTH secretion, is also produced at peripheral inflammatory sites, where it acts as a proinflammatory cytokine, and by the Leydig cell of the testis, where it exerts autocrine inhibition of testosterone biosynthesis. Because key ovarian functions, such as ovulation and luteolysis, represent aseptic inflammatory responses, and because the theca cell is the functional equivalent of the Leydig cell, we explored the CRH presence in the ovary, first, by specific CRH immunohistochemistry of adult cycling female Sprague-Dawley rat ovaries. We detected cytoplasmic immunoreactive CRH (IrCRH) in theca and stromal cells and in cells within the corpora lutea, at all phases of the estrous cycle. Using a specific radioimmunoassay, we measured IrCRH in extracts of rat ovaries (0.042-0.126 pmol/g wet tissue). The mobility of the ovarian IrCRH molecule was similar to that of rat/human CRH by reverse phase HPLC. To investigate the CRH action in the ovary, we identified, characterized, and localized CRH receptors in the rat ovary. Binding was linear with increasing tissue concentration, saturable, and of high affinity. Scatchard analysis of 125I-Tyr-ovine CRH competitive displacement curves indicated a high affinity binding site with a Kd of approximately 6 nM and a Bmax value of approximately 61 fM/mg protein. Autoradiographic studies revealed CRH receptors primarily in ovarian theca and stroma. We conclude that IrCRH and CRH receptors are present in rat ovaries, suggesting that this neuropeptide may play a regulatory role in this gonad, perhaps through its proinflammatory properties and/or by participating in the auto/paracrine regulation of steroid biosynthesis. Functional studies are necessary to define the role(s) of CRH in the ovary.


Subject(s)
Corticotropin-Releasing Hormone/metabolism , Ovary/metabolism , Receptors, Neurotransmitter/metabolism , Animals , Autoradiography , Binding, Competitive , Chromatography, High Pressure Liquid , Corpus Luteum/cytology , Corpus Luteum/metabolism , Corticotropin-Releasing Hormone/analysis , Female , Granulosa Cells/cytology , Granulosa Cells/metabolism , Humans , Immunohistochemistry , Iodine Radioisotopes , Ovarian Follicle/cytology , Ovarian Follicle/metabolism , Ovary/cytology , Radioimmunoassay , Rats , Rats, Sprague-Dawley , Receptors, Corticotropin-Releasing Hormone , Receptors, Neurotransmitter/analysis , Theca Cells/cytology , Theca Cells/metabolism
16.
J Vasc Surg ; 18(2): 266-70, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8350436

ABSTRACT

PURPOSE: The tissue inhibitors of metalloproteinases (TIMPs) are major inhibitors of several enzymes that are destructive to connective tissue, and TIMP-1 has been reported to be deficient in the wall of abdominal aortic aneurysms. This deficiency could represent failure of expression resulting from either local tissue conditions or mutation in the primary structure of the gene (or one of its regulatory elements). METHODS: Southern blotting techniques were used to examine the possibility of global deletions or inserts in the gene (14 patients); Northern blot techniques were performed to examine the expression of the mRNAs in cultured fibroblasts under basal conditions (six patients); and sequence analysis of the cDNA derived from fibroblast mRNAs was done after amplification by polymerase chain reaction (six patients). RESULTS: The Southern blots revealed a normal distribution of the known alleles of the gene without unique restriction-length polymorphisms. Fibroblast expression of TIMP mRNA was normal under basal conditions. Sequence analysis of the cDNAs revealed an identical-point polymorphism in two of the six patients (a single base pair substitution of C-->T at the third position in codon 101), but the amino acid was conserved. CONCLUSION: The studies reported here do not support the hypothesis that deficiency of TIMP-1 in specimens of aorta of patients with abdominal aortic aneurysms results from a primary genetic defect.


Subject(s)
Aortic Aneurysm, Abdominal/metabolism , Glycoproteins/genetics , Metalloendopeptidases/antagonists & inhibitors , RNA, Messenger/genetics , Aortic Aneurysm, Abdominal/genetics , Base Sequence , Blotting, Northern , Blotting, Southern , DNA Mutational Analysis , Gene Expression , Glycoproteins/deficiency , Humans , Male , Molecular Sequence Data , Polymerase Chain Reaction , Tissue Inhibitor of Metalloproteinases
18.
J Clin Invest ; 90(6): 2555-64, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1281840

ABSTRACT

Corticotropin-releasing hormone (CRH), the principal regulator of the hypothalamic-pituitary-adrenal axis, is also secreted in peripheral inflammatory sites, where it acts as a local proinflammatory agent. Arthritis-susceptible LEW/N rats have profoundly deficient hypothalamic CRH responses to inflammatory stimuli and other stressors. Arthritis-resistant F344/N rats, on the other hand, have a robust increase in hypothalamic CRH in response to the same stimuli. Contrasting with these hypothalamic CRH responses, we now show that CRH expression is markedly increased in the joints and surrounding tissues of LEW/N rats with streptococcal cell wall- and adjuvant-induced arthritis, whereas it is not increased in similarly treated F344/N rats and is only transiently increased in congenitally athymic nude LEW.rnu/rnu rats. Glucocorticoid treatment suppressed, but did not eliminate, CRH immunoreactivity in the joints of LEW/N rats. CRH mRNA was present in inflamed synovia, as well as in spinal cord, and inflamed synovia also expressed specific CRH-binding sites. We compared CRH expression in inflamed joints with another well-characterized proinflammatory neuropeptide, substance P (SP), and found that SP immunoreactivity paralleled that of CRH. In summary, although LEW/N rats have deficient hypothalamic CRH responses to inflammatory stimuli compared with F344/N rats, they express relatively high levels of CRH at the site of inflammation. Analogous to SP, CRH may be delivered to the inflammatory site by peripheral nerves and/or synthesized at the inflammatory site. These data provide further support for the concept that CRH not only triggers the pituitary-adrenal antiinflammatory cascade, but also functions as an antithetically active local mediator of acute and chronic inflammatory arthritis. These data also illustrate the complex interrelationships of the nervous, endocrine, immune, and inflammatory systems.


Subject(s)
Arthritis/metabolism , Corticotropin-Releasing Hormone/metabolism , Animals , Arthritis, Experimental/metabolism , Dexamethasone/pharmacology , Gene Expression , Inflammation/metabolism , Joints/metabolism , Male , RNA, Messenger/genetics , Rats , Rats, Inbred F344 , Rats, Inbred Lew , Rats, Nude , Substance P/metabolism , Synovial Membrane/physiopathology
19.
Endocrinology ; 129(5): 2796-8, 1991 Nov.
Article in English | MEDLINE | ID: mdl-1834455

ABSTRACT

Treatment of AtT-20 cell cultures with increasing concentrations of rat/human corticotropin-releasing factor (r/hCRF) for 24 h resulted in a dose-dependent 2-3 fold increase in specific 125I-labelled recombinant human IL-1 alpha (125I-IL-1 alpha) binding that was paralleled by a 70-80% decrease in 125I-Tyro-ovine CRF binding. Saturation analysis of 125I-IL-1 alpha binding in control and CRF-treated cultures indicated that CRF produced an increase in the density (Bmax) of IL-1 receptors without altering their affinity (KD). The CRF-induced upregulation of IL-1 receptors appears to be mediated through specific membrane receptors for CRF since the CRF receptor antagonist, alpha-helical oCRF (9-41), blocked the CRF-induced upregulation of IL-1 receptors without producing any effect on 125I-IL-1 alpha binding by itself. In summary, these data demonstrate complex interactions between CRF and IL-1 at the pituitary level and identify potential novel mechanisms for cytokines to alter neuroendocrine function.


Subject(s)
Corticotropin-Releasing Hormone/pharmacology , Pituitary Neoplasms/metabolism , Receptors, Immunologic/metabolism , Up-Regulation , Animals , Corticotropin-Releasing Hormone/metabolism , Dose-Response Relationship, Drug , Interleukin-1/metabolism , Mice , Pituitary Neoplasms/pathology , Receptors, Immunologic/drug effects , Receptors, Interleukin-1 , Recombinant Proteins , Tumor Cells, Cultured
20.
Methods Achiev Exp Pathol ; 14: 23-44, 1991.
Article in English | MEDLINE | ID: mdl-1658548

ABSTRACT

CRF plays a fundamental role in integrating stress-related responses throughout the neuro-immuno-endocrine axis. Its endocrine effects include actions at the pituitary level to stimulate the synthesis and release of POMC-derived peptides. CRF acts within the CNS to integrate the autonomic, behavioral, endocrine and immune responses to stress. Furthermore, recent evidence suggests that CRF may have direct actions on immunocytes to modulate immune function in the periphery. The actions of CRF in CNS, pituitary, and spleen are mediated by specific, high-affinity membrane receptors with similar kinetic and pharmacological properties. CRF receptors in these various tissues are functionally linked to a guanine nucleotide binding protein mediating stimulation of adenylate cyclase activity. Chemical affinity cross-linking studies demonstrated that the molecular weight of the CRF receptor-binding protein is different in central versus peripheral tissues and that the differences observed in molecular weights are due to the microheterogeneity of the carbohydrate moieties on the receptors in the two types of tissues. In autoradiographic studies, CRF receptors were localized in highest densities in anterior and intermediate lobes of the pituitary, and in brain regions involved in cognitive function, in limbic areas involved in emotion and in brain areas regulating autonomic and other stress-related responses. In spleen, CRF binding sites were localized in the macrophage-rich red pulp and marginal zones surrounding the white pulp regions. Studies examining the effects of CRF administration on local cerebral glucose utilization demonstrated differential changes in glucose utilization in brain regions that have been implicated in mediating the effects of CRF in a variety of homeostatic systems and the organism's ability to respond to stress. Overall, these data provide additional evidence for a physiological role for CRF in the brain-endocrine-immune axis and further support the importance of this neuro-peptide in coordinating the response to stress.


Subject(s)
Receptors, Neurotransmitter/physiology , Stress, Physiological/physiopathology , Adrenalectomy , Animals , Brain/metabolism , Humans , Neuroimmunomodulation , Pituitary Gland/metabolism , Receptors, Corticotropin-Releasing Hormone , Spleen/metabolism
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