Urocortin and corticotropin-releasing factor receptor expression in the human colonic mucosa

Urocortin is a newly identified member of the CRF neuropeptide family. Urocortin has been found to bind with high affinity to CRF receptors. The present study investigated urocortin and CRF receptor expression in human colonic mucosa. Non-pathologic sections of adult colorectal tissues were obtained from patients with colorectal cancer at surgery. Urocortin expression was examined using immunohistochemistry and messenger (m) RNA in situ hybridization. Isolated lamina propria mononuclear cells (LPMC) and epithelial cells were also analyzed by flow cytometry for the characterization of urocortin-positive cells, and by RT-PCR for detection of urocortin, CRF, and CRF receptor mRNA. Urocortin peptide distribution at various stages of human development (n = 35, from 11 weeks of gestation to 6 years of age) was examined by immunohistochemistry using surgical and autopsy specimens. Immunoreactive urocortin and urocortin mRNA were predominantly detected in lamina propria macrophages. Urocortin peptide expression was detected from as early as three months of age, but not before birth or in neonates. Urocortin, CRF receptor type 1 and type 2 mRNA were detected in LPMC. CRF receptor type 2β mRNA, a minor isoform in human tissues, was also detected in LPMC, but at lower levels. Urocortin is locally synthesized in lamina propria macrophages and may act on lamina propria inflammatory cells as an autocrine/paracrine regulator of the mucosal immune system. The appearance of urocortin after birth indicates that the exposure to dietary intake and/or luminal bacteria after birth may contribute to the initiation of urocortin expression in human gastrointestinal tract mucosa.     

Peripheral urocortin inhibits gastric emptying and food intake in mice: differential role of CRF receptor 2

Intraperitoneal urocortin inhibits gastric emptying and food intake in mice. We investigated corticotropin-releasing factor receptor (CRF-R) subtypes involved in intraperitoneal urocortin actions using selective CRF-R antagonists. Gastric emptying was measured 2 h after a chow meal, and food intake was measured hourly after an 18-h fast in mice. Urocortin (3 microg/kg ip) inhibited gastric emptying by 88%. The CRF-R1/CRF-R2 antagonist astressin B (30 microg/kg ip) and the selective CRF-R2 antagonist antisauvagine-30 (100 microg/kg ip) completely antagonized urocortin action, whereas the selective CRF-R1 antagonist CP-154,526 (10 mg/kg ip) had no effect. Urocortin (1-10 microg/kg ip) dose dependently decreased the 2-h cumulative food intake by 30-62%. Urocortin (3 microg/kg)-induced hypophagia was completely antagonized by astressin B (30 microg/kg ip) and partially (35 and 31%) by antisauvagine-30 (100 or 200 microg/kg ip). The CRF-R1 antagonists CP-154,526 or DMP904 (10 mg/kg ip) had no effect. Capsaicin did not alter urocortin-inhibitory actions while blocking the satiety effect of intraperitoneal CCK. These data indicate that intraperitoneal urocortin-induced decrease in feeding is only partly mediated by CRF-R2, whereas urocortin action to delay gastric emptying of a meal involves primarily CRF-R2.     

Identification of Two Corticotropin-Releasing Factor Receptors from Xenopus laevis with High Ligand Selectivity: Unusual Pharmacology of the Type 1 Receptor

Abstract: Two cDNA clones encoding distinct members of the corticotropin-releasing factor (CRF) receptor family have been isolated from Xenopus laevis with PCR-based approaches. The first full-length cDNA amplified from Xenopus brain encoded a 415-amino acid protein with ∼80% identity to mammalian CRF receptor type 1 (CRF-R1). The second full-length cDNA isolated from Xenopus brain and heart encoded a 413-amino acid protein with ∼81% identity to the α-variant of mammalian CRF receptor, type 2 (CRF-R2). No evidence could be obtained that the β-variant of CRF-R2 existed in Xenopus laevis . Binding studies using human embryonic kidney 293 (HEK 293) cells stably transfected with xenopus CRF-R2 showed that the CRF analogues urotensin I, urocortin, and sauvagine were bound with higher affinities than human/rat CRF, xenopus CRF, and ovine CRF. In contrast to human CRF-R1, xenopus CRF-R1 (xCRF-R1) was very selective for different CRF ligands. Urotensin I, urocortin, human/rat CRF, and xenopus CRF were bound with significantly (10–22-fold) higher affinities than ovine CRF ( K _D = 31.7 n M ) and sauvagine ( K _D = 51.4 n M ). In agreement with these binding data, EC₅₀ values of 39.7 and 1.1 n M were found for sauvagine and for human/rat CRF or xenopus CRF, respectively, when the cyclic AMP production in HEK 293 cells stably transfected with xCRF-R1 was determined.     

The relaxant effect of urocortin in rat pulmonary arteries

Urocortin is a potent vasodilator, which plays physiological or pathophysiological roles in systemic circulation. However, little is known about its action on pulmonary circulation. The present study was aimed to characterize some cellular mechanisms underlying the relaxant effect of urocortin in isolated rat pulmonary arteries. Changes in isometric tension were measured on small vessel myographs. Urocortin inhibited U46619-induced contraction with reduction of the maximal response. Urocortin-induced relaxation was independent of the presence of endothelium. Inhibitors of nitric oxide (NO)-dependent dilator, NG-nitro-L-arginine methyl ester or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one, did not affect the relaxation. Astressin (100-500 nM), a corticotropin-releasing factor (CRF) receptor antagonist and KT5720, a protein kinase A (PKA) inhibitor reduced urocortin-induced relaxation. Urocortin produced less relaxant effect in 30 mM K+- than U46619-contracted arterial rings. Urocortin did not reduce CaCl2-induced contraction in 60 mM K+-containing solution. Ba2+ (100-500 microM) but not other K+ channel blockers reduced the relaxant responses to urocortin. Urocortin also relaxed the rings preconstricted by phorbol 12,13-diacetae in normal Krebs solution while this relaxation was less in a Ca2+-free solution. Our results show that urocortin relaxed rat pulmonary arteries via CRF receptor-mediated and PKA-dependent but endothelium/NO or voltage-gated Ca2+ channel-independent mechanisms. Stimulation of Ba2+-sensitive K+ channel may contribute to urocortin-induced relaxation. Finally, urocortin relaxed pulmonary arteries partly via inhibition of a PKC-dependent contractile mechanism.     

Modulation of T-type Ca2+ channels by corticotropin-releasing factor through protein kinase C pathway in MN9D dopaminergic cells

Corticotrophin-releasing factor (CRF) is the main regulator of the body's stress axis and its signal is translated through G-protein-coupled CRF receptors (CRF-R1, CRF-R2). Even though CRF receptors are present in the midbrain dopamine neurons, the cellular mechanism of CRF action is not clear yet. Since voltage-dependent Ca(2+) channels are highly expressed and important in dopamine neuronal functions, we tested the effect of CRF on voltage-dependent Ca(2+) channels in MN9D cells, a model of dopamine neurons. The application of CRF-related peptide, urocortin 1, reversibly inhibited T-type Ca(2+) currents, which was a major Ca(2+) channel in the cells. The effect of urocortin was abolished by specific CRF-R1 antagonist and was mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate. PKC inhibitors abolished the effect of urocortin. These results suggest that urocortin modulates T-type Ca(2+) channel by interacting with CRF-R1 via the activation of PKC signal pathway in MN9D cells.     

Localization and physiological roles of urocortin

Urocortin, a 40 amino acid peptide, is a corticotropin-releasing factor (CRF) related peptide, and can bind to all three types of CRF receptors (CRF type 1, type 2a and type 2b receptors) with higher affinities for these receptors than CRF. Immunoreactivity of urocortin is widely distributed in central nervous, digestive, cardiovascular, reproductive, immune and endocrine systems. Urocortin plays important roles in appetite-suppression, immunomodulation, steroidogenesis in the ovary, maintenance of the placental function, labor, and cardioprotection via CRF receptors. Although urocortin has potent adrenocorticotropin (ACTH) releasing activity in vitro, endogenous urocortin does not act on pituitary ACTH secretion in vivo.     

Involvement of CRH receptors in urocortin-induced hyperthermia

The actions of individual corticotropin-releasing hormone (CRH) receptor (CRHR1 and CRHR2) were studied on the hyperthermia caused by urocortin 1, urocortin 2 and urocortin 3 in rats. Urocortin 1, urocortin 2 or urocortin 3 was injected into the lateral brain ventricle in conscious rats and the colon temperature was measured at different times following injection, up to 6h. In order to study the possible role of CRH receptors, the animals were treated with a urocortins together with the urocortin receptor inhibitors CRF 9-41, antalarmin and astressin 2B to influence the action of urocortins in initiating hyperthermia. Urocortin 1 at a dose of 2microg caused an increase in colon temperature, maximal action being observed in body temperature at 3h. CRH 9-41 and antalarmin, CRHR1 receptor antagonists, prevented the urocortin-induced increase in colon temperature while astressin 2B (CRHR2 receptor antagonist) was ineffective. Urocortin 2 at a dose of 2microg showed a byphasic action in increase in colon temperature having the first peak between 30 min and 1h and the second peak at 4h following treatment. CRF (9-41) and antalarmin was ineffective while astressin 2B fully blocked the action of urocortin 2. Urocortin 3 in a dose of lmicrog increased colon temperature; the maximal effect was observed at 2h. CRF (9-41) and antalarmin was ineffective while astressin 2B fully blocked the action of urocortin 3. The results demonstrated that urocortin 1, 2 or 3 when injected into the lateral brain ventricle caused increases in body temperature is mediated by urocortin receptors. The action of urocortin 1 is mediated by CRHR1 receptor, while in the action of urocortin 2 and urocortin 3 CRHR2 receptor is involved.     

Urocortin 3 elevates cytosolic calcium in nucleus ambiguus neurons

J. Neurochem. (2012) 122, 1129-1136. ABSTRACT: Urocortin 3 (also known as stresscopin) is an endogenous ligand for the corticotropin-releasing factor receptor 2 (CRF(2) ). Despite predominant G(s) coupling of CRF(2) , promiscuous coupling with other G proteins has been also associated with the activation of this receptor. As urocortin 3 has been involved in central cardiovascular regulation at hypothalamic and medullary sites, we examined its cellular effects on cardiac vagal neurons of nucleus ambiguus, a key area for the autonomic control of heart rate. Urocortin 3 (1?nM-1000?nM) induced a concentration-dependent increase in cytosolic Ca(2+) concentration that was blocked by the CRF(2) antagonist K41498. In the case of two consecutive treatments with urocortin 3, the second urocortin 3-induced Ca(2+) response was reduced, indicating receptor desensitization. The effect of urocortin 3 was abolished by pre-treatment with pertussis toxin and by inhibition of phospolipase C with U-73122. Urocortin 3 activated Ca(2+) influx via voltage-gated P/Q-type channels as well as Ca(2+) release from endoplasmic reticulum. Urocortin 3 promoted Ca(2+) release via inositol 1,4,5 trisphosphate receptors, but not ryanodine receptors. Our results indicate a novel Ca(2+) -mobilizing effect of urocortin 3 in vagal pre-ganglionic neurons of nucleus ambiguus, providing a cellular mechanism for a previously reported role for this peptide in parasympathetic cardiac regulation.     

Regulation of corticotropin-releasing factor receptor type 2beta mRNA via cyclic AMP pathway in A7r5 aortic smooth muscle cells

Corticotropin-releasing factor receptor type 2beta (CRF R2beta) is a member of the Class B heptahelical G protein-coupled receptors. This receptor is positively coupled to adenylate cyclase and is bound preferentially by the CRF-related peptides, urocortin (Ucn), Ucn II and Ucn III. In the rodent, CRF R2beta messenger RNA (mRNA) is expressed in the cardiovascular system, where its levels can be modulated by Ucn. In the present study, we investigated regulation of CRF R2beta levels by Ucn in A7r5 aortic smooth muscle cells. Ribonuclease protection assays show that A7r5 cells expressed the CRF R2beta subtype, which had two isoforms differing in one codon at the junction of exons 3 and 4. Ucn induced accumulation of intracellular cAMP via CRF R2beta in this cell line. In addition to the treatment with Ucn, cAMP agonists or analogues themselves caused a significant decrease in CRF R2beta mRNA levels. Blockade of Ucn- or cAMP-induced decreases in CRF R2beta mRNA levels by H7, a broad protein kinase inhibitor, suggested that a protein kinase pathway might be involved in this regulation. H89, a protein kinase A inhibitor, partially blocked Ucn- or cAMP-induced decreases in CRF R2beta mRNA levels. Thus, Ucn induces intracellular cAMP to downregulate CRF R2beta mRNA expression in A7r5 cells.     

Urocortin has cell-proliferative effects on cardiac non-myocytes

Urocortin (Ucn) is a member of the corticotropin-releasing hormone (CRH)-related peptides that has been reported to have cardiac inotropic and hypertrophic effects. In addition, Ucn mRNA was expressed in cardiac myocytes (MCs) and Ucn was suggested to have cardioprotective effects. Recently, it was reported that Ucn mRNA was expressed in cardiac non-myocytes (NMCs). Based on these facts, Ucn is assumed to affect not only MCs but also NMCs in an autocrine fashion. The present study was designed to elucidate a pathophysiological role of Ucn on NMCs. NMCs were prepared by the discontinuous Percoll gradient and adhesion method. Ucn increased [(3)H]-thymidine uptake into NMCs. Ucn also enhanced endothelin-1-induced increase of [(3)H]-thymidine uptake into NMCs. Effects of Ucn on [(3)H]-thymidine uptake into NMCs were significantly abolished by the protein kinase A inhibitor, H89 (10(-5) M), but not by a competitive antagonist of CRH receptors, astressin (10(-5) M). Ucn also increased intracellular cAMP accumulation more potently than CRH on a molar basis. Finally, both MCs and NMCs also secreted Ucn. Together with the recent findings, at least in NMCs, these data suggest that Ucn could exert its own actions via the cAMP signaling pathway, but not through known CRH type 2 receptors, in an autocrine fashion. In conclusion, the present study indicated that Ucn was secreted not only from MCs but also from NMCs and that the primary source of Ucn acting on heart was the heart itself. On the other hand, Ucn could proliferate NMCs as well as MCs, suggesting that Ucn could be involved in cardiac hypertrophy and fibrosis, i.e., cardiac remodeling, in spite of its putative cardioprotective actions.     

Effects of urocortin II on neonatal rat cardiac myocytes and non-myocytes

Urocortin (Ucn) II and III, homologous peptides of Ucn that are specific ligands for corticotropin-releasing hormone (CRH) type 2 receptor (CRH-R2), have recently been identified. The present study was designed to elucidate the effects of Ucn II, which is predominantly expressed in rodent heart, on neonatal rat cardiac myocytes (MCs) and cardiac non-myocytes (NMCs). Ucn II increased the incorporation of [³H]-leucine into MCs, as well as the accumulation of cAMP and the secretion of atrial natriuretic peptide. However, no significant changes were demonstrated in NMCs or an MC/NMC co-culture system. The effects of Ucn II were attenuated by astressin2-B, a specific antagonist of CRH-R2, and/or H89, an inhibitor of protein kinase A (PKA). These results indicate that Ucn II may be another endogenous cardiovascular substance that acts via CRH-R2 and the cAMP-dependent PKA pathway.     

Neurophysiological effects of intracerebroventricular administration of urocortin.

The recently isolated Corticotropin Releasing Factor (CRF) related peptide, urocortin, has been reported to elicit a different behavioral profile than that of CRF. CRF is a potent anxiogenic agent and stimulant of motor activity whereas under similar conditions urocortin is a potent anorectic and mild locomotor stimulant. The neurophysiological effects of this newly synthesized peptide have not yet been examined. The present study evaluated the effects of intracerebroventricular administration of 3 doses of urocortin on the electroencephalogram (EEG) and on Event-Related Potentials (ERPs) in rats. Twenty male Wistar rats were implanted with electrodes in the amygdala and dorsal hippocampus, a cannula into the lateral ventricle, and skull surface electrodes over the frontal and parietal cortices. Following recovery from surgery, urocortin (0.01-1.0 microg) was infused into the lateral ventricle 5 min prior to the recording of EEG (10 min) and ERPs (10 min). Urocortin at any of the doses, did not produce any electrographic or behavioral signs of seizure activity. The predominant effect of urocortin infusion on EEG spectral activity was an increase in mean power in the 4-16 Hz range in the frontal cortex and a decrease in EEG stability in the frontal cortex and amygdala. Urocortin administration also decreased the latency of the P3 component of the ERP in the amygdala and hippocampus. These neurophysiological effects, that only partially overlap with those of CRF, are consistent with the behavioral profile described following urocortin administration in rats. Overall, these data further support the assertion that urocortin functions as a mild CNS stimulant enhancing arousal, as measured by EEG, and modulating the speed of stimulus evaluation as measured by ERPs.     

Down-regulation of corticotropin-releasing hormone receptor type 2beta mRNA expression in the rat cardiovascular system following food deprivation

The present study was conducted to assess the effect of nutritional stress induced by food deprivation on expression of messenger ribonucleic acid (mRNA) for corticotropin-releasing hormone receptor type 2beta (CRH-R2beta) in the rat cardiovascular system in the presence or absence of changes in circulating corticosterone. Food deprivation for 96 h caused a robust increase in plasma corticosterone levels and a significant decrease in CRH-R2beta mRNA expression in the rat heart. Starvation for 48 and 96 h decreased CRH-R2beta mRNA expression in the atria, ventricle as well as aorta of sham-adrenalectomized (sham) rats. Surprisingly, clamping plasma glucocorticoids at low levels by adrenalectomy with corticosterone pellet replacement (ADX+B) did not completely prevent starvation-induced decreases of CRH-R2beta mRNA expression in the rat cardiovascular system. Urocortin (Ucn) mRNA expression was increased significantly by food deprivation in the heart of sham as well as ADX+B rats. We speculate that food deprivation may increase urocortin, which in turn down-regulates CRH-R2beta mRNA expression in cardiovascular system. These data indicate that food deprivation despite the presence or absence of changes in circulating corticosterone may have an inhibitory effect on CRH-R2beta mRNA expression in the rat cardiovascular system.     

Regulation of Corticotropin-Releasing Factor Receptor Function in Human Y-79 Retinoblastoma Cells: Rapid and Reversible Homologous Desensitization but Prolonged Recovery

Abstract: Homologous receptor desensitization is an important regulatory response to continuous activation by agonist that involves the uncoupling of a receptor from its G protein. When human retinoblastoma Y-79 cells expressing corticotropin-releasing factor (CRF) receptors were preincubated with CRF for 10 min-4 h, a time-dependent reduction in both the peak and sensitivity of CRF-stimulated intracellular cyclic AMP (cAMP) accumulation developed with a t _1/2 of 38 min and an EC₅₀ of 6–7 n M CRF. CRF receptor desensitization was slowly reversible after a 4-h CRF preincubation with a t _1/2 of 13 h and a full restoration of cAMP responsiveness to CRF at 24 h following the removal of 10 n M CRF. Because the ability of vasoactive intestinal peptide, forskolin, or (−)-isoproterenol to stimulate cAMP accumulation was not diminished in Y-79 cells desensitized with 10 n M CRF, the observed desensitization was considered to be a specific homologous action of CRF. CRF receptor desensitization was markedly attenuated by CRF receptor antagonists, which alone did not produce any appreciable reduction in CRF-stimulated cAMP accumulation. Although recent reports have demonstrated a rapid decline in steady-state levels of CRF receptor type 1 (CRF-R1) mRNA in anterior pituitary cells during several hours of exposure to CRF, there was no observed reduction in CRF-R1 mRNA levels when Y-79 cells were preincubated with 10 n M CRF for 10 min-24 h despite a rapid time- and concentration-dependent loss of CRF receptors from the retinoblastoma cell surface.     

Regulation of corticotropin-releasing factor receptor type 2beta mRNA by mitogen-activated protein kinases in aortic smooth muscle 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. CRF receptor type 2beta (CRFR2beta) messenger RNA (mRNA) is expressed primarily in the cardiovascular system, where its levels are decreased by urocortin 1 (Ucn1), a novel peptide in the CRF family. In a previous study, we reported that CRFR2beta mRNA levels were partially down-regulated via the cAMP-protein kinase A pathway. This study focused on the involvement of the intracellular mitogen-activated protein (MAP) kinase pathway in the modulation of CRFR2beta mRNA levels. Ribonuclease protection assays showed that decreases in CRFR2beta mRNA levels induced by Ucn1 and cAMP were attenuated by the p38 MAP kinase inhibitor SB202190 or SB203580. This finding suggested that the p38 MAP kinase pathway was involved in this regulation. Anisomycin, a classic p38 kinase activator, increased CRFR2beta mRNA levels in A7r5 cells. This effect of anisomycin was completely reversed by H7, a serine/threonine kinase inhibitor, while both p38 kinase and MAP kinase kinase inhibitors failed to block the increase in CRFR2beta mRNA levels caused by anisomycin. As anisomycin can activate Jun amino terminal kinases, as well as p38 MAP kinase, it is possible that other MAP kinases, such as Jun amino terminal kinases, also contribute to the increase in gene levels. Alternatively, anisomycin may increase CRFR2beta mRNA levels indirectly as a consequence of blocking protein synthesis.     

Chronic disruption of body weight but not of stress peptides or receptors in rats exposed to repeated restraint stress

Rats exposed to restraint stress for 3 h on each of 3 days lose weight and do not return to the weight of their non-stressed controls for extended periods of time. Studies described here demonstrate that the initial weight loss is associated with increased energy expenditure and reduced food intake on the days of restraint but that there is no difference between stressed and control rats once stress ends. The failure to compensate for this energy deficit accounts for the sustained reduction in weight which lasts for up to 80 days after the end of restraint. In an additional experiment, in situ hybridization was used to measure mRNA expression of corticotrophin releasing factor (CRF) and CRF receptors in hypothalamic nuclei, of urocortin (UCN) in the Edinger Westphal nucleus and of UCN III in the rostral perifornical area and medial amygdaloidal nucleus. Immediately after the second 3 h bout of restraint stress, there was a significant increase in expression of UCN in the Edinger Westphal nucleus and of CRF-R1 in the paraventricular nucleus of the hypothalamus and a less pronounced decrease in CRF-R2 expression in the ventromedial nucleus of the hypothalamus. There were no differences in expression of stress-related peptides or their receptors 40 days after the end of repeated restraint. These results suggest that the sustained reduction in body weight and increased responsiveness to subsequent stressors in rats that have been exposed to repeated restraint are not associated with prolonged changes in mRNA expression of CRF receptors or their ligands.     

GRK3 regulation during CRF- and urocortin-induced CRF1 receptor desensitization

The EC(50) values for concentration-dependent stimulation of cAMP accumulation by CRF (1.3nM) and urocortin (1.0nM) were equivalent in human retinoblastoma Y79 cells. The time course and magnitude of CRF- and urocortin-induced CRF(1) receptor desensitization were similar. A significant 3-fold increase in GRK3, but not GRK2, mRNA levels accompanied the emergence of CRF(1) receptor desensitization in Y79 cells exposed to CRF. In preliminary experiments, retinoblastoma GRK3 protein expression became upregulated during a 48-h CRF exposure. Neither GRK3 nor GRK2 expression increased in Y79 cells exposed to urocortin for 10 min to 48 h. We hypothesize that GRK3 upregulation may be a cellular negative feedback process directed at maximizing CRF(1) receptor desensitization by heightening GRK3 phosphorylating capacity during prolonged exposure to high CRF. Regulation of GRK expression associated with urocortin- and CRF-induced CRF(1) receptor desensitization appears to differ, despite a similar level of signaling via the cAMP-protein kinase A pathway.     

Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness

The aim for this study was to examine whether the F4 generation of two strains of rainbow trout divergent in their plasma cortisol response to confinement stress (HR: high responder or LR: low responder) would also differ in stress-induced effects on forebrain concentrations of mRNA for corticotropin-releasing factor (CRF), arginine vasotocin (AVT), CRF receptor type 1 (CRF-R1), CRF receptor type 2 (CRF-R2) and AVT receptor (AVT-R). In addition, plasma cortisol concentrations, brainstem levels of monoamines and monoamine metabolites, and behaviour during confinement were monitored. The results confirm that HR and LR trout differ in their cortisol response to confinement and show that fish of these strains also differ in their behavioural response to confinement. The HR trout displayed significantly higher locomotor activity while in confinement than LR trout. Moreover, following 180 min of confinement HR fish showed significantly higher forebrain concentrations of CRF mRNA than LR fish. Also, when subjected to 30 min of confinement HR fish showed significantly lower CRF-R2 mRNA concentrations than LR fish, whereas there were no differences in CRF-R1, AVT or AVT-R mRNA expression between LR and HR fish either at 30 or 180 min of confinement. Differences in the expression of CRF and CRF-R2 mRNA may be related to the divergence in stress coping displayed by these rainbow trout strains.     

Losartan attenuates phospholipase C isozyme gene expression in hypertrophied hearts due to volume overload

Because the left ventricular (LV) hypertrophy due to volume overload induced by arteriovenous (AV) shunt was associated with an increase in phospholipase C (PLC) isozyme mRNA levels, PLC is considered to be involved in the development of cardiac hypertrophy. Since the renin-angiotensin system (RAS) is activated in cardiac hypertrophy, the role of RAS in the stimulation of PLC isozyme gene expression in hypertrophied heart was investigated by inducing AV shunt in Sprague-Dawley rats. The animals were treated with or without losartan (20 mg/kg, daily) for 3 days as well as 1, 2 and 4 weeks, and atria, right ventricle (RV) and LV were used for analysis. The increased muscle mass as well as the mRNA levels for PLC beta1 and beta3 in atria and RV, unlike PLC beta3 gene expression in LV, at 3 days of AVshunt were attenuated by losartan. The increased gene expression for PLC beta1 at 2 weeks in atria, at 1 and 4 weeks in RV, and at 2 and 4 weeks in LV was also depressed by losartan treatment. Likewise, the elevated mRNA levels for PLC beta3 in RV at 1 week and in LVat 4 weeks of cardiac hypertrophy were decreased by losartan. On the other hand, the increased levels of mRNA for PLC gamma1 in RV and LV at 2 and 4 weeks of inducing hypertrophy, unlike in atria at 4 weeks were not attenuated by losartan treatment. While the increased mRNA level for PLC delta1 in LV was reduced by losartan, gene expression for PLC delta1 was unaltered in atria and decreased in RV at 3 days of inducing AV shunt. These results suggest that changes in PLC isozyme gene expression were chamber specific and time-dependent upon inducing cardiac hypertrophy due to AV shunt. Furthermore, partial attenuation of the increased gene expression for some of the PLC isozymes and no effect of losartan on others indicate that both RAS dependent and independent mechanisms may be involved in hypertrophied hearts due to volume overload.     

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.