Modulation of Ca2+ influx by corticotropin-releasing factor (CRF) family of peptides via CRF receptors in rat pancreatic beta-cells

The actions of the corticotropin-releasing factor (CRF) family of peptides are mediated by the seven transmembrane-domain G-protein-coupled receptors, the CRF receptors type 1 (CRF1 receptor) and type 2 (CRF2 receptor). In a previous study, we reported that CRF, an endogenous ligand for CRF1 receptor, modulated Ca2+ influx in rat pancreatic beta-cells. In addition to CRF, other additional members of the family, urocortins, have been identified in mammals. Urocortin 1 (UCN 1), a peptide of the CRF family, binds both CRF1 receptor and CRF2 receptor with equal affinities. Urocortin 3 (UCN 3), a highly selective ligand for CRF2 receptor with little affinity for CRF1 receptor, has been shown in rat pancreatic beta-cells. The present study focused on the effects of the CRF family peptides on intracellular Ca2+ ([Ca2+]i) concentration via CRF receptors in rat pancreatic beta-cells. Microfluorimetric experiments showed that CRF (0.2 nM) and UCN 1 (0.2 nM) elevated [Ca2+]i levels. Both CRF and UCN 1 effects were attenuated by astressin, a non-selective CRF receptor antagonist. Antisauvagine-30, a selective CRF2 receptor antagonist, appeared to enhance the UCN 1 effect on the elevation of [Ca2+]i. The CRF effect on the elevation of [Ca2+]i was inhibited by the addition of UCN 3. Taken together, the activation of CRF2 receptor antagonizes the CRF1 receptor-stimulated Ca2+ influx.     

The effects of corticotropin-releasing factor and the urocortins on hypothalamic gamma-amino butyric acid release--the impacts on the hypothalamic-pituitary-adrenal axis

Corticotropin-releasing factor (CRF) and the urocortins (UCNs) are structurally and pharmacologically related neuropeptides which regulate the endocrine, autonomic, emotional and behavioral responses to stress. CRF and UCN1 activate both CRF receptors (CRFR1 and CRFR2) with CRF binding preferentially to CRFR1 and UCN1 binding equipotently to both receptors. UCN2 and UCN3 activate selectively CRFR2. Previously an in vitro study demonstrated that superfusion of both CRF and UCN1 elevated the GABA release elicited by electrical stimulation from rat amygdala, through activation of CRF1 receptors. In the present experiments, the same in vitro settings were used to study the actions of CRF and the urocortins on hypothalamic GABA release. CRF and UCN1 administered in equimolar doses increased significantly the GABA release induced by electrical stimulation from rat hypothalamus. The increasing effects of CRF and UCN1 were inhibited considerably by the selective CRFR1 antagonist antalarmin, but were not influenced by the selective CRFR2 antagonist astressin 2B. UCN2 and UCN3 were ineffective. We conclude that CRF1 receptor agonists induce the release of GABA in the hypothalamus as well as previously the amygdala. We speculate that CRF-induced GABA release may act as a double-edged sword: amygdalar GABA may disinhibit the hypothalamic CRF release, leading to activation of the hypothalamic-pituitary-adrenal axis, whereas hypothalamic GABA may inhibit the hypothalamic CRF release, terminating this activation.     

Urocortin 1 reduces food intake and ghrelin secretion via CRF(2) receptors

Although it is known that urocortin 1 (UCN) acts on both corticotropin-releasing factor receptors (CRF(1) and CRF(2)), the mechanisms underlying UCN-induced anorexia remain unclear. In contrast, ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, stimulates food intake. In the present study, we examined the effects of CRF(1) and CRF(2) receptor antagonists (CRF(1)a and CRF(2)a) on ghrelin secretion and synthesis, c-fos mRNA expression in the caudal brain stem, and food intake following intracerebroventricular administration of UCN. Eight-week-old, male Sprague-Dawley rats were used after 24-h food deprivation. Acylated and des-acylated ghrelin levels were measured by enzyme-linked immunosorbent assay. The mRNA expressions of preproghrelin and c-fos were measured by real-time RT-PCR. The present study provided the following important insights into the mechanisms underlying the anorectic effects of UCN: 1) UCN increased acylated and des-acylated ghrelin levels in the gastric body and decreased their levels in the plasma; 2) UCN decreased preproghrelin mRNA levels in the gastric body; 3) UCN-induced reduction of plasma ghrelin and food intake were restored by CRF(2)a but not CRF(1)a; 4) UCN-induced increase of c-fos mRNA levels in the caudal brain stem containing the nucleus of the solitary tract (NTS) was inhibited by CRF(2)a; and 5) UCN-induced reduction of food intake was restored by exogenous ghrelin and rikkunshito, an endogenous ghrelin secretion regulator. Thus, UCN increases neuronal activation in the caudal brain stem containing NTS via CRF(2) receptors, which may be related to UCN-induced inhibition of both ghrelin secretion and food intake.     

Peripheral administration of CRF and urocortin: effects on food intake and the HPA axis in the marsupial Sminthopsis crassicaudata

The hypothalamic peptides corticotrophin releasing factor (CRF) and urocortin (UCN) decrease food intake and increase energy expenditure when administered either centrally or peripherally to rodents. The effects of CRF and UCN on food intake in other mammals (for example marsupials), however, are not known. Peripherally administered CRF induced cortisol release in the marsupial Sminthopsis crassicaudata via the CRF1 receptor, and central CRF administration potently decreased food intake, as in rodents. When peripherally administered, both CRF and UCN decreased food intake in S. crassicaudata, but UCN was considerably more potent ( approximately 50 fold) in this regard. The anorectic effects of CRF and UCN were not blocked by the CRF1 receptor antagonist antalarmin, suggesting that the peripheral effects of CRF and UCN on food intake are mediated primarily by the CRF2 receptor.     

Acute stress increases colonic paracellular permeability in mice through a mast cell-independent mechanism: Involvement of pancreatic trypsin

AimsIncreased colonic paracellular permeability (CPP) is a key feature of gastro-intestinal disorders as irritable bowel syndrome and inflammatory bowel diseases. Stress stimulates exocrine pancreatic secretion through cholinergic pathways, and trypsin is known to increase CPP. Consequently we have investigated in this work whether trypsin released into the gut lumen following an acute stress may participate to the short-term increase in CPP.Main methodsMice were treated with atropine or a non-selective CRF (corticotropin-releasing factor) receptor antagonist (α-helical CRF (9–41)), before being submitted to a 2-h stress session. Then, CPP and protease activity in colonic contents (total proteolytic, trypsin activity, and mouse mast cell protease (MMCP)-1 levels) were determined. The effects of colonic contents from sham-stressed or stressed animals on CPP were evaluated in mice colonic tissues mounted in Ussing chambers, in presence or not of soybean trypsin inhibitor (SBTI) or FSLLRY, a protease-activated receptor-2 (PAR2) antagonist.Key findingsAcute stress significantly increased CPP, proteolytic and trypsin activities, and MMCP-1 levels. Atropine inhibited stress-induced impairment of CPP and strongly diminished total proteolytic and trypsin activities in stressed animals, but not MMCP-1 levels. Colonic contents from stressed animals increased CPP in mice tissues, this effect being inhibited by SBTI and PAR2 antagonist.SignificanceAcute stress activates cholinergic pathways, to trigger exocrine pancreatic secretion. Trypsin, released in these conditions, may be responsible for colonic barrier alterations through the activation of PAR2.     

Urocortin II inhibits the apoptosis of mesenteric arterial smooth muscle cells via L-type calcium channels in spontaneously hypertensive rats.

Urocortin (UCN) II, a newly isolated corticotropinreleasing- factor (CRF) related peptide, has been found to have potent cardiovascular protective effects. To investigate the mechanisms of its vascular protective effects, we exposed mesenteric arterial smooth muscle cells (MASMC) from spontaneously hypertensive rats (SHR) to UCN II to observe the change in cell apoptosis using TUNEL assay and measured intracellular calcium concentration ([Ca2+]i) using confocal laser scanning microscope. In addition, effects of UCN II on L-type calcium currents (ICa,L) were also measured using whole-cell patch clamp. Our results showed that UCN II concentration-dependently, but time-independently inhibited cell apoptosis. Astressin 2B, a special CRF 2 receptor antagonist, had no influence on this inhibition. Hypoxia or Bay K8644, the L-type calcium channel activator, induced the apoptosis of MASMC from SHR. Pretreatment of the cells with UCN II diminished the effects of hypoxia or Bay K8644. UCN II was also observed to reduce [Ca2+]i increase induced by KCl or Bay K8644. UCN II concentration-dependently inhibited ICa,L, which was not affected by astressin 2B. It did not affect the activation of ICa,L, but markedly shifted the inactivation curve to the left. In conclusion, UCN II inhibits the apoptosis of MASMC from SHR via inhibiting L-type calcium channels.     

Effects of gamma-aminobutyric acid on secretagogue-induced exocrine secretion of isolated, perfused rat pancreas

Because GABA and its related enzymes have been determined in beta-cells of pancreas islets, effects of GABA on pancreatic exocrine secretion were investigated in the isolated, perfused rat pancreas. GABA, given intra-arterially at concentrations of 3, 10, 30, and 100 microM, did not exert any influence on spontaneous or secretin (12 pM)-induced pancreatic exocrine secretion. However, GABA further elevated CCK (10 pM)-, gastrin-releasing peptide (100 pM)-, or electrical field stimulation-induced pancreatic secretions of fluid and amylase dose dependently. The GABA (30 microM)-enhanced CCK-induced pancreatic secretions were completely blocked by bicuculline (10 microM), a GABA(A) receptor antagonist, but were not affected by saclofen (10 microM), a GABA(B) receptor antagonist. The enhancing effects of GABA (30 microM) on CCK-induced pancreatic secretions were not changed by tetrodotoxin (1 microM) but were partially reduced by cyclo-(7-aminoheptanonyl-Phe-D-Trp-Lys-Thr[BZL]) (10 nM), a somatostatin antagonist. In conclusion, GABA enhances pancreatic exocrine secretion induced by secretagogues, which predominantly induce enzyme secretion, via GABA(A) receptors in the rat pancreas. The enhancing effect of GABA is partially mediated by inhibition of islet somatostatin release.     

Roles of 5-HT receptors in the release and action of secretin on pancreatic secretion in rats

5-Hydroxytryptamine (serotonin, 5-HT) is a hormone and neurotransmitter regulating gastrointestinal functions. 5-HT receptors are widely distributed in gastrointestinal mucosa and the enteric nervous system. Duodenal acidification stimulates not only the release of both 5-HT and secretin but also pancreatic exocrine secretion. We investigated the effect of 5-HT receptor antagonists on the release of secretin and pancreatic secretion of water and bicarbonate induced by duodenal acidification in anesthetized rats. Both the 5-HT(2) receptor antagonist ketanserin and the 5-HT(3) receptor antagonist ondansetron at 1-100 microg/kg dose-dependently inhibited acid-induced increases in plasma secretin concentration and pancreatic exocrine secretion. Neither the 5-HT(1) receptor antagonists pindolol and 5-HTP-DP nor the 5-HT(4) receptor antagonist SDZ-205,557 affected acid-evoked release of secretin or pancreatic secretion. None of the 5-HT receptor antagonists affected basal pancreatic secretion or plasma secretin concentration. Ketanserin or ondansetron at 10 microg/kg or a combination of both suppressed the pancreatic secretion in response to intravenous secretin at 2.5 and 5 pmol x kg(-1) x h(-1) by 55-75%, but not at 10 pmol x kg(-1) x h(-1). Atropine (50 microg/kg) significantly attenuated the inhibitory effect of ketanserin on pancreatic secretion but not on the release of secretin. These observations suggest that 5-HT(2) and 5-HT(3) receptors mediate duodenal acidification-induced release of secretin and pancreatic secretion of fluid and bicarbonate. Also, regulation of pancreatic exocrine secretion through 5-HT(2) receptors may involve a cholinergic pathway in the rat.     

Microinjection of exogenous somatostatin in the dorsal vagal complex inhibits pancreatic secretion via somatostatin receptor-2 in rats

Previous studies have suggested that somatostatin inhibits pancreatic secretion at a central vagal site, and the dorsal vagal complex (DVC) is involved in central feedback inhibition of the exocrine pancreas. The aim of this study was to investigate the effect of exogenous somatostatin in the DVC on pancreatic secretion and the somatostatin receptor subtype(s) responsible for the effect. The effects of somatostatin microinjected into the DVC on pancreatic secretion stimulated by cholecystokinin octapeptide (CCK-8) or 2-deoxy-d-glucose (2-DG) were examined in anesthetized rats. To investigate the somatostatin inhibitory action site, a somatostatin receptor antagonist [SRA; cyclo(7-aminoheptanoyl-Phe-d-Trp-Lys-Thr)] was microinjected into the DVC before intravenous infusion of somatostatin and CCK-8/2-DG. The effects of injection of a somatostatin receptor-2 agonist (seglitide) and combined injection of somatostatin and a somatostatin receptor-2 antagonist (CYN 154806) in the DVC on the pancreatic secretion were also investigated. Somatostatin injected into the DVC significantly inhibited pancreatic secretion evoked by CCK-8 or 2-DG in a dose-dependent manner. SRA injected into the DVC completely reversed the inhibitory effect of intravenous administration of somatostatin. Seglitide injected into the DVC also inhibited CCK-8/2-DG-induced pancreatic protein secretion. However, combined injection of somatostatin and CYN 154806 did not affect the CCK-8/2-DG-induced pancreatic secretion. Somatostatin in the DVC inhibits pancreatic secretion via somatostatin receptor-2, and the DVC is the action site of somatostatin for its inhibitory effect.     

Expression, binding, and signaling properties of CRF2(a) receptors endogenously expressed in human retinoblastoma Y79 cells: passage-dependent regulation of functional receptors

Endogenous expression of the corticotropin-releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF(1)-preferring or non-selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of approximately 100 pmoles/10(5) cells, whereas the exclusive CRF2 receptor-selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of approximately 25-30 pmoles/10(5) cells. UCN2 and 3-mediated cAMP stimulation was potently blocked by the approximately 300-fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 +/- 1.6 nmol/L), whereas the CRF(1)-selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 microL. When the CRF(1)-preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 +/- 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 +/- 0.2 microL). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2a receptors and that the CRF2(a) receptor protein is up-regulated during prolonged culture.     

Enhanced intracellular calcium induced by urocortin is involved in degranulation of rat lung mast cells.

Corticotropin-releasing factor (CRF), which activates the hypothalamic-pituitary- adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. The purpose of this study was to investigate the effect of urocortin (UCN), a 40-amino-acid CRF family peptide, on degranulation and intracellular calcium of rat lung mast cells. The activation and degranulation of mast cells were observed by Toluidine blue staining and transmission electron microscope. The intracellular calcium was investigated using confocal laser scanning microscopy and flow cytometry. The results indicated that all the three different concentrations of UCN (0.1, 1 and 10 microM) significantly induced the activation and degranulation of rat lung mast cells in vitro. This effect was markedly blocked by selective CRF receptor 1 (CRF-R1) antagonist antalarmin, but not by specific CRF receptor 2 (CRF-R2) antagonist antisauvagine-30 (anti-Svg-30). The results also showed that UCN caused a rapid peak increase in [Ca(2+)](i) at point of 300s after UCN treatment, followed by a decrease to a sustained plateau phase. The peak increase in [Ca(2+)](i) induced by UCN was significantly inhibited by antalarmin, but not by anti-Svg-30. This effect of UCN on [Ca(2+)](i) in rat lung mast cells was also found by flow cytometry. Regression analysis revealed a positive correlation between mast cells degranulation extent and the maximum value of [Ca(2+)](i) (P < 0.01). Taken together, our present study suggested that UCN induced the increase of [Ca(2+)](i) and degranulation of rat lung mast cells through CRF-R1. These findings may have implications for the pathophysiology of allergic and inflammatory lung disorders such as asthma, which is closely associated with mast cell activation and degranulation.     

Stimulatory effects of islet amyloid polypeptide (amylin) on exocrine pancreas and gastrin release in conscious rats.

A rat islet amyloid polypeptide (amylin), 37-residue peptide amide was synthesized by the Fmoc-based solid phase method and the biological activity of synthetic rat amylin on exocrine pancreas was evaluated for the first time in conscious rat. Amylin (1, 10 nmol/kg/h) stimulated pancreatic exocrine secretion and plasma gastrin concentration. CR-1409, a CCK receptor antagonist, did not change amylin-stimulated pancreatic secretion. However, omeprazole (proton pump inhibitor) and atropine inhibited amylin-stimulated pancreatic secretion. This study suggests that amylin may play a role in biological action in the exocrine pancreas possibly mediated by gastric acid hypersecretion.     

Separate locations of urocortin and its receptors in mouse testis: function in male reproduction and the relevant mechanisms.

Urocortin (UCN), a newly identified corticotrophin-releasing-factor (CRF) related peptide, has been demonstrated to play important roles in female reproductive system. However, few studies were reported about its effects on male reproduction. This study aimed to investigate the expression profile of UCN and CRF receptors (CRFR) in mouse testis and functions of UCN in male reproduction. Expression of UCN and CRFR mRNA was detected by RT-PCR. Localization of UCN peptide was determined by immunohistochemistry and double-immunostaining. We found that both UCN mRNA and peptide were obviously expressed in mature spermatozoa, whereas CRFR1 and CRFR2 were expressed respectively in spermatocytes and spermatogonia. Double-immunostaining results showed that UCN expression decreased with acrosome reaction (AR) proceeding. UCN significantly inhibited AR initiated by progesterone with chlortetracycline staining and decreased spermatozoa motility concentration-dependently. Pre-incubation of spermatozoa with astressin, a CRFR antagonist, did not affect these inhibitions. In addition, flow cytometry showed that UCN concentration-dependently decreased intracellular Ca(2+) [Ca(2+)](i) in spermatozoa. In summary, UCN located in mouse spermatozoa and exerted inhibitory effects on male reproductive functions including motility and AR. UCN's inhibition on [Ca(2+)](i) via T-type calcium channels might be responsible for these effects.     

Biological effects of a proglumide derivative as cholecystokinin antagonist in conscious dogs

In conscious dogs we studied the effects of a new cholecystokinin (CCK) antagonist (coded CR 1505) on CCK8-stimulated exocrine pancreatic secretion and release of pancreatic polypeptide (PP). Graded doses of CCK8 (25-400 ng kg-1h-1) were infused i.v. Experiments were repeated against a background infusion of CR 1505 at different doses (0.1, 1 and 10 mg kg-1h-1). The lowest dose of CR 1505 had no biological effects. However, at the upper two doses the compound significantly inhibited the CCK8-stimulated PP release. Furthermore, a significant inhibition of exocrine pancreatic protein secretion was observed with 10 mg kg-1h-1 of CR 1505 (P less than 0.05). The results suggest that CR 1505 could be a useful tool in defining the physiological role of CCK in vivo.     

Corticotropin-releasing factor receptor subtypes mediating nutritional suppression of estrous behavior in Syrian hamsters

Caloric deprivation inhibits reproduction, including copulatory behaviors, in female mammals. Decreases in metabolic fuel availability are detected in the hindbrain, and this information is relayed to the forebrain circuits controlling estrous behavior by neuropeptide Y (NPY) projections. In the forebrain, the nutritional inhibition of estrous behavior appears to be mediated by corticotropin-releasing factor (CRF) or urocortin-signaling systems. Intracerebroventricular (ICV) infusion of the CRF antagonist, astressin, prevents the suppression of lordosis by food deprivation and by NPY treatment in Syrian hamsters. These experiments sought to determine which CRF receptor type(s) is involved. ICV infusion of the CRF receptor subtype CRFR2-selective agonists urocortin 2 and 3 (UCN2, UCN3) inhibited sexual receptivity in hormone-primed, ovariectomized hamsters. Furthermore, the CRFR2-selective antagonist, astressin 2B, prevented the inhibition of estrous behavior by UCN2 and by NPY, consistent with a role for CRFR2. On the other hand, astressin 2B did not prevent the inhibition of behavior induced by 48-h food deprivation or ICV administration of CRF, a mixed CRFR1 and CRFR2 agonist, suggesting that activation of CRFR1 signaling is sufficient to inhibit sexual receptivity in hamsters. Although administration of CRFR1-selective antagonists (NBI-27914 and CP-154,526) failed to reverse the inhibition of receptivity by CRF treatment, we could not confirm their biological effectiveness in hamsters. The most parsimonious interpretation of these findings is that, although NPY inhibits estrous behavior via downstream CRFR2 signaling, food deprivation may exert its inhibition via both CRFR1 and CRFR2 and that redundant neuropeptide systems may be involved.     

Effects of human corticotropin releasing factor (CRF) on gastric and pancreatic secretion in vivo and in vitro

Human CRF given IV inhibited dose-dependently pentagastrin- but not histamine-induced gastric acid secretion. When added to the incubation medium of the isolated gastric glands, CRF did not alter the formation of HCl under basal conditions or after stimulation with histamine or DBcAMP. CRF caused a small but significant increase in pancreatic HCO3 and protein secretion. It augmented CCK-induced pancreatic protein and secretin-induced HCO3 secretion in vivo but failed to affect basal or stimulated (CCK and urecholine) amylase release by the in vitro dispersed pancreatic acini. This study indicates that CRF inhibits gastric and stimulates pancreatic secretion in vivo but not in vitro and these effects are indirect involving, at least in part, alterations in the pancreatic circulation.     

Role of cholecystokinin in mediating GRP-stimulated gastric, biliary and pancreatic functions in man.

To explore the mechanisms of gastrin-releasing peptide (GRP)-induced gut functions in man, we investigated the effect on gallbladder contraction, exocrine pancreatic secretion and gastric acid secretion of a recently developed CCK receptor antagonist, loxiglumide, on GRP-stimulated effects in six healthy human subjects. Intravenous infusion of graded doses of synthetic human GRP (1-27 pmol/kg per h) caused significant and dose-dependent increases in pancreatic enzyme and gastric acid secretions and in gallbladder contraction. Intravenous administration of loxiglumide (10 mg/kg per h) abolished GRP-stimulated gallbladder contraction, augmented gastric acid secretion, but did not affect exocrine pancreatic secretion. The results suggest that endogenously released CCK is (1) responsible for GRP-stimulated gallbladder contraction, and (2) involved in regulating gastric acid secretion. The results further suggest that GRP-stimulated pancreatic secretion is not mediated by CCK, but has a direct response of GRP on the exocrine pancreas.