Characterization of corticotropin-releasing hormone (CRH) in human skin

We have confirmed the expression of CRH and CRH receptor type 1 genes in human skin, cultured HaCaT keratinocytes, squamous cell carcinoma, and melanoma cells. The size of CRH messenger ribonucleic acid (mRNA), estimated by Northern blot hybridization, was 1.5 kilobases. CRH peptide was identified by reverse phase high pressure liquid chromatography separation in both whole skin and cultured cells. Forskolin and dexamethasone at concentrations of 10 micromol/L stimulated and inhibited, respectively, CRH peptide production in squamous cell carcinoma and melanoma cells, but had no significant effect on the CRH mRNA level. In melanoma cells, stimulation of melanogenesis down-regulated CRH receptor type 1 mRNA expression, but was without effect on CRH mRNA production. We suggest that in human skin the CRH signaling system is both operative and under regulatory control.     

Stimulation by urocortin of growth hormone (GH) secretion in GH-producing human pituitary adenoma cells

Urocortin (Ucn) possesses high homology with CRH and is considered to be a ligand to type-2 CRH receptor. We investigated the effect of Ucn on hormone release from cultured GH-producing human pituitary adenoma cells in vitro. GH-producing human pituitary adenoma cells were superfused on a Sephadex G-25 column. Both Ucn (10 nM) and CRH (10 nM) elicited an increase in GH release from the pituitary adenoma cells in one patient with acromegaly. In contrast, GH release from the pituitary adenoma cells was stimulated by Ucn but not by CRH in the other patient with acromegaly. These preliminary findings suggest that type-2 CRH receptors are expressed in some population of GH-producing human pituitary adenoma cells and that Ucn might be involved in GH secretion from tumorous tissues in patients with acromegaly.     

Nitric oxide inhibits corticotropin-releasing hormone exocytosis but not synthesis by cultured human trophoblasts

Nitric oxide (NO) plays an important role in many cell-cell signaling systems, but its mechanism of action is variable. We have previously reported that NO reduces secretion of the peptide hormone, CRH, from cultured placental cells and the perfused placenta. Because placental CRH production seems linked to human parturition, we wished to explore the mechanism of action of NO in this setting in more detail. We report here that in the placenta, NO specifically inhibited CRH exocytosis, not synthesis, and that endogenous NO affects this process. Cytotrophoblasts were prepared from term human placentas and cultured as monolayers. CRH immunoreactivity in the cell supernatants and cell extracts were measured by RIA. CRH messenger RNA was determined by Northern blot analysis. Sodium nitroprusside (SNP; 1-100 mumol/L) and S-nitroso-N-acetyl-penicillamine (SNAP; 1-100 mumol/L), NO donors, significantly reduced basal CRH concentration in the media, while increasing the concentration of CRH in the cells (P < 0.01), suggesting that exocytosis of CRH was inhibited. These effects could be attenuated by the NO scavenger hemoglobin (20 micrograms/mL). KCl (45 mmol/L), which causes exocytosis by depolarizing the cell membrane, increased CRH release by 2- to 3-fold, and this was inhibited by SNP. Basal release of CRH was augmented by the NO synthase competitive inhibitor N omega-L-arginine methyl ester (1 mmol/L; P < 0.01) and the guanylate cyclase inhibitor, LY83583 (1 mumol/L; P < 0.01). The inhibitory effect of SNP was also blocked by LY83583. CRH messenger RNA content did not change when the placental cells were incubated with SNP, N omega-L-arginine methyl ester, and LY83583 for 6 and 24 h, and this was consistent with studies showing that total CRH immunoreactivity (cells plus media) did not change in the presence of SNP. These studies indicate that exogenous NO inhibits CRH exocytosis, rather than biosynthesis, by human trophoblasts and that endogenous NO has tonic inhibitory effects on CRH release by these cells. The inhibitory effect of NO on basal and stimulated CRH release by placental trophoblasts seems to be a guanylate cyclase-mediated inhibition of exocytosis.     

Integrin-mediated signalling of gelatinase B secretion in colon cancer cells

The progression of colon cancer has been linked to both cell adhesion molecules called integrins and matrix-degrading enzymes called metalloproteinases. Herein we report that the alpha v beta 6 integrin expressed in colon cancer cells induces gelatinase B secretion through the C-terminal cytoplasmic extension unique to the beta 6 integrin subunit, and that this ligand-independent event involves activation of the protein-kinase-C pathway.     

Nitric oxide regulation of corticotropin-releasing hormone release from the human perfused placenta in vitro

We have investigated the regulatory role of nitric oxide (NO) in corticotropin-releasing hormone (CRH) release from the human perfused placental lobule in vitro. The effects of the NO donor sodium nitroprusside, the NO synthase inhibitor N omega-nitro-L-arginine, and the NO substrate L-arginine on human (h) placental CRH secretion have been studied. Single lobules of term placentae were bilaterally perfused with Krebs solution (5 mL/min; 95% O2-5% CO2; 37 C; pH 7.3). Fetal and maternal perfusates were collected at 4 C every 30 min for 3 h. CRH immunoreactivity (CRH-IR) in perfusates was measured by RIA using the 41-residue synthetic CRH as standard, 125I-labeled Tyr-hCRH as tracer, and a rabbit anti-CRH antibody Y2BO. The sensitivity of the assay was 0.13 pmol/L. Under basal conditions, human perfused placentae in vitro continuously secreted CRH-IR, which diluted in parallel to a synthetic hCRH-(1-41) standard curve. Size-exclusion chromatography of placental perfusates using a Sephadex G-50 column indicated that placental CRH-IR predominately coeluted with hCRH-(1-41) standard. Basal maternal perfusate CRH-IR levels (27 +/- 4 pmol/L) released from perfused placental lobules were nearly 10-fold greater than fetal perfusate CRH-IR levels (3.4 +/- 0.7 pmol/L; P < 0.05). Infusion of sodium nitroprusside (30-100 mumol/L) into the maternal and fetal placental circulations inhibited CRH-IR release into maternal perfusate in a concentration-dependent manner, but did not inhibit CRH-IR release into the fetal perfusate. N omega-nitro-L-arginine (100 mumol/L) increased placental CRH-IR secretion into fetal perfusate, and this effect was reversed by the infusion of L-arginine (100 mumol/L), which also reduced release below basal levels. In contrast, maternal perfusate CRH-IR levels were not affected by N omega-nitro-L-arginine or L-arginine. These results indicate that the human perfused placenta in vitro releases a substance of similar mol wt and hCRH-IR. Moreover, modulators of the NO signaling pathway differentially affect placental secretion of CRH-IR into the maternal and fetal perfusates. These data are consistent with the involvement of NO in the regulation of placental CRH release during pregnancy.     

Cytokine-induced inhibition of lipid synthesis and hormone secretion by isolated human islets

Interleukin-1, tumor necrosis factor, and interleukin-6 inhibit insulin release and may be cytotoxic to isolated pancreatic islets. These cytokines have been postulated to play an important role in the beta cell destruction characteristic of type 1 diabetes. The present study was designed to investigate the effect of the above cytokines on insulin, glucagon, somatostatin, and thyrotropin-releasing hormone secretion by isolated human islets. In addition, we have investigated if cytokine-induced modifications in hormone secretion are accompanied by modifications in the ab initio synthesis of any specific lipidic fraction. All three cytokines studied, although not modifying insulin and somatostatin release to glucose 5 mmol/L, inhibited the response of both hormones to glucose 20 mmol/L. On the other hand, the cytokines almost completely blocked islet basal glucagon release, without affecting thyrotropin-releasing hormone secretion. The added cytokines also suppressed 20 mmol/L [U-14C]glucose incorporation into both phospholipids and diacylglycerol. Our results demonstrate a beta-, alpha-, and delta-cell, sensitivity to cytokine action. Additionally, they suggest that ab initio lipid synthesis might be implicated in the mechanism of insulin release in human islets.     

Unimpaired postreceptor regulation of luteinizing hormone secretion by gonadotropin-releasing hormone and estrogen in aged rat anterior pituitary cells

Delayed, attenuated, or absence of the proestrous LH surge occurs in aging rats. To assess how aging affects the positive feedback action of 17 beta-estradiol (E2) on the pituitary, we determined the responsiveness of rat pituitary cells to GnRH and the secretagogues affecting intracellular signal transduction mechanisms in the presence or absence of E2. We also correlated the LH response to pituitary LH content. Anterior pituitaries excised from ovariectomized Sprague-Dawley rats, either young (3-4 months) or old (19-20 months), were enzymatically dispersed and then pretreated with or without E2 (0.6 nM) for 48 h, followed by incubation for 3 h with or without various secretagogues. The secretagogues included GnRH (1 and 10 nM), veratridine (increases Ca2+ influx; 5 and 10 microM), and phorbol 12-myristate 13-acetate (a protein kinase-C activator; 10 and 100 nM). LH in media and cells were measured by RIA and expressed on the basis of cellular DNA. GnRH, veratridine, and phorbol 12-myristate 13-acetate at all doses stimulated (P < 0.01) LH release in cells from both young and old rats. E2 stimulated (P < 0.05 to P < 0.01) all secretagogue-induced LH release in cells from both young and old rats, but only basal LH release (P < 0.05) in cells from young rats. The magnitude of both basal and secretagogue-induced LH release in either the presence or absence of E2 was smaller (P < 0.01) in cells from old than in those from young rats. The initial cellular LH was lower (P < 0.01) in cells from old than in those from young rats. The LH-releasing ability (expressed as a percentage of total cellular LH) of cells from old rats was identical (P > 0.05) to that of cells from young rats under all conditions studied. These results suggest that the reduced magnitude of LH release by cells from old rats may be attributed to reduced cellular LH, rather than to impaired estrogen feedback or impaired signal transduction mechanisms. It remains to be determined whether LH biosynthesis per cell and/or the number of gonadotropes decrease with age.     

Peptide gene activation, secretion, and steroid feedback during stimulation of rat neuroendocrine corticotropin-releasing hormone neurons

We have used colloid-induced hypovolemia to investigate mechanisms operating in CRH neuroendocrine neurons of the hypothalamic paraventricular nucleus during a sustained stress. Specifically, three questions have been addressed using in situ hybridization and RIA. 1) Do neuropeptide secretion and gene activation share the same stimulus threshold? 2) Does corticosterone modulate mechanisms regulating CRH gene expression during sustained stress? 3) How are neuropeptides commonly colocalized with CRH affected? Our results show that the secretion of ACTH and activation of the CRH gene have distinct and separate stimulus thresholds. The threshold is higher for CRH gene activation than for ACTH secretion, suggesting some degree of mechanistic separation. In addition, corticosterone secreted during the first 3 h of sustained hypovolemia does not inhibit CRH gene expression. However, feedback inhibition may occur in the delayed time domain. Finally, neuropeptides colocalized with CRH are differentially regulated by sustained hypovolemia. Proenkephalin messenger RNA levels show a slower temporal response than those of CRH, while the vasopressin gene is not activated at any time in parvicellular neuroendocrine neurons. Our results emphasize that CRH neuroendocrine neurons respond to a stress event in a stimulus-specific manner in terms of both the profiles of secretion and gene expression, and the structure of glucocorticoid feedback.     

Modulation of binding sites for corticotropin-releasing hormone by chronic psychosocial stress

This study was conducted to determine whether long lasting psychosocial stress would affect corticotropin-releasing hormone (CRH) binding sites in the brain, the pituitary, and the adrenal gland. As a model for sustained emotional stress we used chronic psychosocial conflict in male tree shrews. In subordinate tree shrews, repeated confrontation with a dominant conspecific results in constant hyperactivity of the HPA-axis and an elevated neurosympathetic tone. After 24 days of psychosocial conflict, CRH binding sites were quantified by in vitro-autoradiography with 125I-ovine CRH in 23 discrete brain regions, the pituitaries, and the adrenal glands of subordinate and control animals. Chronic stress significantly reduced the number of binding sites (Bmax) in the anterior lobe of the pituitary, the dentate gyrus, the CA1-CA3 areas of the hippocampus, and in both the stratum griseum superficiale and the stratum opticum of the superior colliculus. In cortical area 17, the reduction of Bmax was counterbalanced by an increase in the affinity (Kd) of the radioligand for the binding sites. A significant stress-induced enhancement of Bmax was observed in the frontal cortex, cingulate cortex, claustrocortex, the central and lateral nucleus of the amygdala, and in the choroid plexus. This increase was accompanied by a significant decrease of Kd-values in the frontal and cingulate cortex, the lateral nucleus of the amygdala, and the choroid plexus. These findings represent the first in vivo demonstration of a modulation of extrahypothalamic CRH receptors by a naturally occurring form of stress. The different response patterns of the central CRH binding sites reflect distinct neuroendocrine processes which are presumed to coordinate behavioral, autonomic, endocrine, and immune responses to long-lasting psychosocial conflict.     

Maternal corticotropin-releasing hormone levels in the early third trimester predict length of gestation in human pregnancy

OBJECTIVE: Corticotropin releasing hormone, a hypothalamic neuropeptide, plays a major role in regulating pituitary-adrenal function and the physiologic response to stress. During pregnancy corticotropin-releasing hormone is synthesized in large amounts by the placenta and released into the maternal and fetal circulations. Various endocrine, autocrine, and paracrine roles have been suggested for placental corticotropin-releasing hormone. The aim of this study was to prospectively assess the relationship between maternal plasma concentrations of corticotropin-releasing hormone in the early third trimester of pregnancy and the length of gestation in two groups of deliveries, with and without spontaneous labor. STUDY DESIGN: In a sample of 63 women with singleton intrauterine pregnancies, maternal plasma samples were collected between 28 and 30 weeks' gestation and corticotropin-releasing hormone concentrations were determined by radioimmunoassay. Each pregnancy was dated on the basis of last menstrual period and early ultrasonography. Parity, antepartum risk conditions, presence or absence of spontaneous labor, and birth outcomes were abstracted from the medical record. RESULTS: Maternal corticotropin-releasing hormone levels between 28 and 30 weeks' gestation significantly and negatively predicted gestational length (P < .01) after adjustment for antepartum risk. Moreover, subjects who were delivered preterm had significantly higher corticotropin-releasing hormone levels in the early third trimester (P < .01) than did those who were delivered at term. In deliveries preceded by spontaneous onset of labor, maternal third-trimester corticotropin-releasing hormone levels significantly and independently predicted earlier onset of labor (P < .01) and preterm labor (P < .05), whereas in deliveries effected by induction of labor or cesarean delivery, maternal corticotropin-releasing hormone levels were a marker of antepartum risk but not a statistically independent predictor of gestational length. CONCLUSION: These findings support the premise that placental corticotropin-releasing hormone is potentially implicated in the timing of human delivery in at least two ways. First, placental corticotropin-releasing hormone may play a role in the physiology of parturition. Premature or accelerated activation of the placental corticotropin-releasing hormone system, as reflected by precocious elevation of maternal corticotropin-releasing hormone levels, may therefore be associated with earlier onset of spontaneous labor and resultant delivery. Second, placental corticotropin-releasing hormone may be a marker of antepartum risk for preterm delivery and therefore an indirect predictor of earlier delivery. The implications of these findings are discussed in the context of the neuroendocrinology of placental corticotropin-releasing hormone and human parturition. Furthermore, the role of corticotropin-releasing hormone as a possible effector of prenatal stress in producing alterations in the timing of normal delivery is detailed.     

Surfactant homeostasis in corticotropin-releasing hormone deficiency in adult mice

Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant disease caused by deficiency in the antidiuretic hormone arginine vasopressin (AVP) encoded by the AVP-neurophysin II (AVP-NPII) gene on chromosome 20p13. In this study, we analyzed two families with FNDI using direct automated fluorescent, solid phase, single-stranded DNA sequencing of PCR-amplified AVP-NPII DNA. In one of the families, affected individuals presented a novel nonsense mutation in exon 3 of the gene, consisting in a G to T transition at nucleotide 2101, which produces a stop signal in codon 82 (Glu) of NPII. The premature termination eliminates part of the C-terminal domain of NPII, including a cysteine residue in position 85, which could be involved in the correct folding of the prohormone. In the second family, a G279A substitution at position -1 of the signal peptide was observed in all affected individuals. This missense mutation, which replaces Ala with Thr, is frequent among FNDI patients and is thought to reduce the efficiency of cleavage by signal peptidases.     

Potentiation of photodynamic therapy by mitomycin C in cultured human colon adenocarcinoma cells

Severe ketotic diabetes induced in rats by streptozotocin resulted in a reduction in activity of the hepatic branched-chain alpha-ketoacid dehydrogenase complex, regardless of whether activity was expressed on the basis of liver wet weight, total liver, liver protein, or liver DNA. A decrease in enzyme specific activity (units of enzyme activity per mg of enzyme protein) was found responsible for the reduction in measurable enzyme activity of the complex. Insulin treatment reversed the decrease in enzyme specific activity. Treatment of tissue extracts with phosphoprotein phosphatase had no effect, indicating that activity of the complex was decreased by some mechanism other than reversible phosphorylation. Specific protein components of the complex were also not found reduced by the diabetic state. Induction of severe ketotic diabetes in rats previously fed a low-protein diet resulted in activation of the enzyme as a consequence of dephosphorylation. Nevertheless, the specific activity of the dephosphorylated enzyme of diabetic, low-protein-fed rats was decreased relative to that of control, low-protein-fed animals. Reconstitution studies with tissue extracts fortified with the purified E1 component indicate that severe diabetes induces a defect in this component of the hepatic branched-chain alpha-ketoacid dehydrogenase complex.     

Effects of thyrotropin, follicle stimulating hormone and luteinizing hormone on sIL-2R in vitro secretion from human peripheral blood mononuclear cells

The effect of thyroid stimulating hormone (TSH) or thyrotropin (0.06, 0.6, 6, and 60 microIU/ml), follicle stimulating hormone (FSH) and luteinizing hormone (0.1, 1, 10, and 100 mIU/ml) on soluble interleukin-2 receptor (sIL-2R) release in vitro from resting or phytohaemagglutinin (PHA) activated human peripheral blood mononuclear cells (PBMC) was evaluated. sIL-2R concentrations were measured in supernatants of cultured cells by quantitative sandwich enzyme immunoassay method (ELISA). TSH in a dilution of 0.6 microIU/ml and FSH in a concentration of 1 mIU/ml inhibited the secretion of sIL-2R only (p < 0.01) into supernatants from PHA activated PBMC cultures.     

Determinants of trimetrexate lethality in human colon cancer cells

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.     

The growth hormone secretagogue, L-692,429, induces phosphatidylinositol hydrolysis and hormone secretion by human pituitary tumors

L-692,429 is a non-peptidyl GH secretagogue. We examined the effects of L-692,429 on cultured human pituitary tumors removed from patients with acromegaly. Dose-dependent stimulation of GH secretion was observed, with 1 mumol/L leading to 2 or 3-fold increases. Prolactin (PRL) secretion by a mixed somatotrophic-lactotrophic tumor was also stimulated. The effects of L-692,429 were abolished by phloretin and W7 but not Rp-cAMPS. Rate of phosphatidylinositol turnover was markedly increased up to 3-fold by L-692,429. These results show that L-692,429 increases hormone secretion by human pituitary cells via a protein kinase C and Ca2+ dependent mechanism.     

Binding and transcytosis of botulinum neurotoxin by polarized human colon carcinoma cells

T-84 and Caco-2 human colon carcinoma cells and Madin-Darby canine kidney (MDCK) cells were used to study binding and transcytosis of iodinated Clostridium botulinum neurotoxin serotypes A, B, and C, as well as tetanus toxin. Specific binding and transcytosis were demonstrated for serotypes A and B in intestinal cells. Using serotype A as an example, the rate of transcytosis by T-84 cells was determined in both apical to basolateral (11.34 fmol/h/cm2) as well as basolateral to apical (8.98 fmol/h/cm2) directions, and by Caco-2 cells in the apical to basolateral (8.42 fmol/h/cm2) direction. Serotype A retained intact di-chain structure during transit through T-84 or Caco-2 cells, and when released on the basolateral side was toxic in vivo to mice and in vitro on mouse phrenic nerve-hemidiaphragm preparations. Serotype C and tetanus toxin did not bind effectively to T-84 cells, nor were they efficiently transcytosed (8-10% of serotype A). MDCK cells did not bind or efficiently transcytose (0.32 fmol/h/cm2) botulinum toxin. Further characterization demonstrated that the rate of transcytosis for serotype A in T-84 cells was increased 66% when vesicle sorting was disrupted by 5 microM brefeldin A, decreased 42% when microtubules were disrupted by 10 microM nocodazole, and decreased 74% at 18 degreesC. Drugs that antagonize toxin action at the nerve terminal, such as bafilomycin A1 (which prevents acidification of endosomes) and methylamine HCl (which neutralizes acidification of endosomes), produced only a modest inhibitory effect on the rate of transcytosis (17-22%). These results may provide an explanation for the mechanism by which botulinum toxin escapes the human gastrointestinal tract, and they may also explain why specific serotypes cause human disease and others do not.     

Activation of protein kinase C by oxytocin inhibits the biological activity of the human myometrial corticotropin-releasing hormone receptor at term

The role of placental CRH in human pregnancy is currently unknown. The myometrium expresses CRH receptors that during pregnancy become coupled to adenylate cyclase. Oxytocin (OT) is one of the main regulators of uterine activity, acting via activation of the inositol triphosphate pathway. In view of the possible cross-talk between the CRH and OT signal transduction pathways we have sought to examine in more detail the second messenger mechanisms involved. CRH receptor binding affinity for CRH and activation of adenylate cyclase were reduced in the presence of OT in pregnant (at term, but not preterm) human myometrium. OT action was mediated via pertussis toxin-sensitive G proteins, which directly inhibit adenylate cyclase and, via activation of protein kinase C, phosphorylate the CRH receptor, leading to desensitization. Activation of protein kinase C by OT could be partially inhibited in human pregnant myometrial cells by OT antagonists (F327 and CAP476; 1 microM) or phospholipase C inhibitors (U73122; 10 microM). These results suggest that in term myometrium, CRH receptor function is modulated by OT, leading to reduced biological activity, lower cAMP levels, and a subsequent shift in favor of contractility rather than relaxation.     

Anxiogenic effect of corticotropin-releasing hormone in the dorsal periaqueductal grey

To investigate whether the dorsal periaqueductal grey (DPAG) might be involved in the anxiogenic effect of intracerebroventricular (i.c.v.) administered corticotropin-releasing hormone (CRH), rats (n = 6-13) received microinjections into the DPAG of CRH (1 or 2 microg) or saline and were tested on the elevated plus maze. The drug caused a dose-dependent decrease in plus maze exploration (percentage of entries into open arms: saline 31.9 +/- 8.6, CRH 1 microg 19.2 +/- 4.0, CRH 2 microg 6.9 +/- 3.7; p < 0.01, ANOVA). In a second experiment the anxiogenic effect of intra-DPAG CRH (2 microg) was prevented by previous microinjection of alpha-helical-CRH(9-41) (0.5 microg), a CRH antagonist (percentage of entries into open arms: saline + CRH 20.3 +/- 3.7, alpha-helical-CRH(9-41) + CRH 45.7 +/- 1.6). These results suggest that the DPAG may be a site of the anxiogenic effect of i.c.v. injected CRH.