The role of corticotropin-releasing factor receptors in stress and anxiety

Corticotropin releasing factor (CRF) is a critical integratorof the hypothalamic-pituitary-adrenal (HPA) axis in responseto stress. CRF and its related molecule urocortin (UCN) bindCRF receptor 1 (CRFR1) and CRFR2 with distinct affinities. Micedeficient for CRFR1 or CRFR2 were generated in order to determinethe physiological role of these receptors. While CRFR1-mutantmice show a depleted stress response and display anxiolytic-likebehavior, CRFR2-mutant mice are hypersensitive to stress anddisplay anxiogenic-like behavior. Both CRFR1- and CRFR2-mutantmice show normal basal feeding and weight gain, but CRFR2-mutantmice exhibit decreased food intake following a stress of fooddeprivation. While CRFR2-mutant mice display increased levelsof CRF mRNA in the central nucleus of the amygdala (cAmyg) butnot in the paraventricular nucleus of the hypothalamus (PVN),the CRFR1-mutant mice express high levels of CRF in the PVNbut normal levels in the cAmyg. CRFR2-mutant mice also displayincreased levels of Ucn mRNA and protein in the edinger westphalnucleus (EW) as well as an increased number of cells expressingUcn. The levels of these CRF-receptor ligands reflect the stateof the receptor-deficient mice. These results demonstrate apossible modulatory function of CRFR2 in response to CRFR1 stimulationof the HPA axis or anxiety.     

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.     

Increased expression of corticotropin-releasing factor receptor mRNA in the locus coeruleus of stress-induced rat model of depression

Hypersecretion of corticotropin-releasing factor (CRF) has been hypothesized to occur in depression. To investigate CRF receptor (CRFR) response to the increased production of CRF in chronically stressed rats, we measured by in situ hybridization the expression of CRFR mRNA in the locus coeruleus (LC) concomitant with measuring plasma adrenocorticotropin (ACTH). The expression of both CRFR mRNA in the LC and the plasma level of ACTH increased significantly in "depression-model rats" which exhibit reduced activity following exposure to 14 days forced walking stress (FWS), but not in "spontaneous recovery rats" whose activity was restored after the long-term stress. These results suggest that the LC neurons continue to be stimulated by CRF, and that the hypothalamic-pituitary-adrenal (HPA) axis is hyperfunctioning in the depression-model rats.     

CRF receptor type 1 mediates continual hypoxia-induced CRF peptide and CRF mRNA expression increase in hypothalamic PVN of rats

We demonstrated previously that hypoxia activated CRF and CRF mRNA in PVN, and CRF receptor 1 (CRFR1) mRNA in rat pituitary. The aim of the study is to test whether the hypoxia-activated CRF and CRF mRNA is associated with triggering CRFR1. Rats were exposed to hypobaric hypoxia at altitude of 2 and 5 km. CRF and CRF mRNA were assayed by immunostaining and in situ hybridization. CRFR1 mRNA was assayed by RT-PCR. Results showed that 5 km continual hypoxia increased CRF and CRF mRNA in PVN, CRFR1 mRNA in pituitary, and plasma corticosterone. The hypoxia-increased CRF, CRF mRNA, CRFR1 mRNA, and corticosterone were blocked by CRFR1 antagonist (CP-154,526), suggesting that CRFR1 in PVN and pituitary are responsible for the hypoxia-increased CRF and CRF mRNA in PVN.     

低氧暴露对大鼠下丘脑CRF分泌的影响

目的和方法：利用人工模拟低氧及放射免疫测定（RIA）法,观察不同低氧（5km,7km）暴露对大鼠下丘脑分泌促肾上腺皮质激素释放激素（C orticotropin-releasing factor,CRF）的作用和血浆皮质酮水平的变化。比较低氧暴露不同时间（2h,24h,5d,15d）后,大鼠下丘脑CRF分泌和血浆皮质酮的变化。结果：低氧暴露2h,24h后下丘脑的CRF分泌明显增加,血浆皮质酮水平显著升高,这种变化随着低氧程度的加深而增强,随着低氧暴露时间的延长（5d）上述变化减弱。至慢性低氧暴露15d后,下丘脑CRF分泌及血浆皮质酮水平与对照组相比已无显著差异,基本恢复到对照水平。结论：动物暴露于低氧环境后,下丘脑CRF分泌及血浆皮质酮水平变化为：低氧暴露2h,24h后CRF分泌和皮质酮分泌被激活;低氧暴露5d后CRF分泌和皮质酮分泌活动减弱,并开始恢复;至低氧暴露15d后,这种分泌活动基本恢复,进入恢复期。     

The Effects of Corticoptropin-Releasing Factor and the Urocortins on Striatal Dopamine Release Induced by Electrical Stimulation—An in vitro Superfusion Study

The members of the CRF peptide family, corticotropin-releasing factor (CRF), urocortin I (Ucn I), urocortin II (Ucn II) and urocortin III (Ucn III) coordinate endocrine and behavioral responses to stress. CRF has also been demonstrated to stimulate dopamine (DA) synthesis.In our study, a superfusion system was used to investigate the effects of this peptide family on striatal DA release following electrical stimulation. The involvement of the CRF receptors was studied by pretreatment of rat striatal slices with selective CRF antagonists. CRF and Ucn I increased the release of [³H]DA while Ucn II and Ucn III were ineffective. The CRFR1 antagonist antalarmin inhibited the [³H]DA release induced by electrical stimulation and enhanced by CRF and Ucn I. The CRFR2 antagonist astressin-2B was ineffective.These results suggest that CRF and Ucn I mediate DA release through the activation of CRFR1. Ucn II and Ucn III are not involved in this process.Special Issue Dedicated to Miklós Palkovits.     

低氧暴露条件下高原鼠兔和大鼠HPA轴活动的比较

采用人工模拟低气压低氧的方法比较研究了不同程度（模拟海拔5 km和7 km)和不同时间（24d和5d）低氧暴露,对大鼠和高原鼠兔（Ochotona curzoniae）下丘脑-垂体-肾上腺皮质 (hypothalamo-pituitary-adrenalcortex,HPA)轴活动的影响。结果如下：7 km低氧暴露24 h,大鼠下丘脑的促肾上腺皮质激素释放激素（corticotropin-releasing actor,CRF）和肾上腺皮质激素皮质酮分泌显著增加,大鼠HPA低氧暴露对大鼠HPA 轴活动无显著差异。低氧暴露5天后,大鼠7 km、5 km组的HPA轴活动与对照相比无明显差异。低氧暴露对高原鼠兔的HPA轴无明显影响。上述结果表明：低氧暴露的时间和程度与大鼠HPA的活动密切相关;从HPA的活动来看,高原鼠兔表现出较强的低氧耐受性。     

Molecular recognition of corticotropin-releasing factor by its G-protein-coupled receptor CRFR1

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.     

Physiological and Neurochemical Aspects of Corticotropin-Releasing Factor Actions in the Brain: The Role of the Locus Coeruleus

Corticotropin-releasing factor (CRF) is both a major regulator of the hypothalamo-pituitary-adrenal (HPA) axis and the activity of the autonomic nervous system. Besides, it exerts numerous effects on other physiological functions such as appetite control, motor and cognitive behavior and immune function. The basis for these effects is constituted by its distribution in hypothalamic and extrahypothalamic brain areas, the latter being represented by limbic structures such as the central nucleus of the amygdala or by brain stem neurons such as the locus coeruleus (LC) or nucleus of the solitary tract (NTS). The effects of CRF are mediated through recently described CRF-receptor subtypes, whose molecular biology, biochemistry and pharmacological regulation are discussed in detail. In the second part of this review, we will focus on the physiology of CRF-systems in the brain, with a particular emphasis on cardiovascular regulation, respiration, appetite control and stress-related behavior. Finally, the role of the locus coeruleus in the control of CRF-mediated behavioral activities is discussed. The interaction of noradrenergic and CRF-neurons clearly implies that CRF appears to directly activate LC neurons in a stressful situation, thus ultimately coordinating the bodily response to a stressful stimulus.     

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.     

CRF type 1 receptors of the medial amygdala modulate inhibitory avoidance responses in the elevated T-maze

Corticotropin-releasing factor (CRF) plays a critical role in the mediation of physiological and behavioral responses to stressors. In the present study, we investigated the role played by the CRF system within the medial amygdala (MeA) in the modulation of anxiety and fear-related responses. Male Wistar rats were bilaterally administered into the MeA with CRF (125 and 250 ng/0.2 μl, experiment 1) or with the CRFR1 antagonist antalarmin (25 ng/0.2 μl, experiment 2) and 10 min later tested in the elevated T-maze (ETM) for inhibitory avoidance and escape measurements. In clinical terms, these responses have been respectively related to generalized anxiety and panic disorder. To further verify if the anxiogenic effects of CRF were mediated by CRFR1 activation, we also investigated the effects of the combined treatment with CRF (250 ng/0.2 μl) and antalarmin (25 ng/0.2 μl) (experiment 3). All animals were tested in an open field, immediately after the ETM, for locomotor activity assessment. Results showed that CRF, in the two doses administered, facilitated ETM avoidance, an anxiogenic response. Antalarmin significantly decreased avoidance latencies, an anxiolytic effect, and was able to counteract the anxiogenic effects of CRF. None of the compounds administered altered escape responses or locomotor activity measurements. These results suggest that CRF in the MeA exerts anxiogenic effects by activating type 1 receptors, which might be of relevance to the physiopathology of generalized anxiety disorder.     

The stress response neuropeptide CRF increases amyloid‐β production by regulating γ‐secretase activity

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid‐β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ‐secretase internalization. Co‐immunoprecipitation studies establish that γ‐secretase associates with CRFR1; this is mediated by β‐arrestin binding motifs. Additionally, CRFR1 and γ‐secretase co‐localize in lipid raft fractions, with increased γ‐secretase accumulation upon CRF treatment. CRF treatment also increases γ‐secretase activity in vitro, revealing a second, receptor‐independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ‐secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ‐secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ‐secretase.     

Mapping the CRF-urocortins system in human osteoarthritic and rheumatoid synovial fibroblasts: effect of vasoactive intestinal peptide

In addition to the brain and pituitary gland, the corticotrophin‐releasing factor (CRF) system is expressed in peripheral tissues. In this study we characterize the expression of CRF, urocortins (UCN1, UCN2, and UCN3), and their receptors (CRFR1 and CRFR2) in osteoarthritis (OA) and rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS). Moreover, we analyze the vasoactive intestinal peptide (VIP) effect on the CRF system, as well as its physiological consequences on mediators of inflammatory/destructive processes. CRF and UCNs exhibit differential pattern in OA and RA‐FLS. By real‐time PCR we detected more expression of CRF and UCN1 in RA, and UCN2 and UCN3 in OA, while the CRFR2 expression was similar. In RA‐FLS VIP treatment resulted in a significant decrease of the proinflammatory peptides, CRF and UCN1, and a significant increase of the potential anti‐inflammatory agents, UCN3 and CRFR2. Using Western blot assays, we showed that the ratio between phospho‐CREB (p‐CREB) and c‐AMP response element‐binding (CREB) is higher in OA and significantly lower in RA‐FLS after VIP treatment, with consequences upon cAMP response element in CRF and UCN1 genes. Real‐time PCR and EIA proved that VIP significantly inhibits cycloxygenase‐2 (COX‐2) and prostaglandin E2 (PGE2) in RA‐FLS. In all cases, we consider significant data when P < 0.05. These data indicate a role of endogenous CRF, UCNs, and CRFR2 in the OA and RA joint microenvironment. We confirm the anti‐inflammatory function of VIP, through the modulation of the expression of CRF system that impacts in a reduction of mediators with inflammatory/destructive functions, supporting its therapeutic potential in rheumatic diseases. J. Cell. Physiol. 226: 3261–3269, 2011. © 2011 Wiley Periodicals, Inc.     

CRF-dependent and CRF-independent mechanisms involved in hypophysial-adrenal system activation by bacterial endotoxin.

The immune system and the hypothalamic-pituitary-adrenal (HPA) axis play important role in the overall inflammatory response. The mechanism through which lipopolysaccharide (LPS, endotoxin) stimulates the HPA axis is not well understood. In order to clarify the role of hypophysiotropic peptides of paraventricular origin in the effect of LPS on ACTH and corticosterone secretion, the effect of LPS was studied on rats with lesions of hypothalamic paraventricular nucleus (PVN). It was shown that 90 min after 2 mg/kg LPS i.p. the ACTH, but not the corticosterone response was effectively blunted in PVN-lesioned rats, as compared to sham operated animals. However, in PVN-lesioned rats 240 min after treatment with LPS a significantly higher plasma ACTH and corticosterone level was monitored. It is, therefore, suggested that in response to LPS activation of HPA both CRF(s)-dependent and CRF(s)-independent mechanisms are involved, even a direct effect of the adrenal cortex should be taken into account.     

Type 1 corticotropin-releasing factor receptors in the ventromedial hypothalamus promote hypoglycemia-induced hormonal counterregulation

Type 2 corticotropin-releasing factor (CRF) receptors (CRFR2) within the ventromedial hypothalamus (VMH), a key glucose-sensing region, play a major role in regulating the hormonal counterregulatory responses (CRRs) to acute hypoglycemia. The VMH expresses both subtypes of CRF receptors, CRFR1 and CRFR2. The objective of this study was to examine the role of the CRFR1 receptor in the VMH in the regulation of the CRR to acute hypoglycemia. To compare the hormonal CRR to hypoglycemia, awake and unrestrained Sprague-Dawley rats were bilaterally microinjected to the VMH with either 1) aECF, 2) CRF (1 pmol/side), 3) CRFR1 antagonist Antalarmin (500 pmol/side), or 4) CRF + Antalarmin prior to undergoing a hyperinsulinemic hypoglycemic (2.8 mM) clamp. A second series of studies also incorporated an infusion of [(3)H]glucose to allow the calculation of glucose dynamics. In addition the effect of CRFR1 antagonism in the paraventricular nucleus (PVN) was studied. Activation of VMH CRFR1 increased, whereas inhibition of CRFR1 suppressed hypoglycemia-induced CRRs. Inhibition of VMH CRFR1 also increased peripheral glucose utilization and reduced endogenous glucose production during hypoglycemia, whereas VMH CRF reduced peripheral glucose utilization. In contrast CRFR1 inhibition in the PVN blunted corticosterone but not epinephrine or glucagon CRR to hypoglycemia. In contrast to CRFR2 activation, CRFR1 activation within the VMH amplifies CRRs to acute hypoglycemia. The balance between these two opposing CRFRs in this key glucose-sensing region may play an important role in determining the magnitude of CRRs to acute hypoglycemia.     

Residues of corticotropin releasing factor-binding protein (CRF-BP) that selectively abrogate binding to CRF but not to urocortin 1

Corticotropin releasing factor-binding protein (CRF-BP) binds CRF and urocortin 1 (Ucn 1) with high affinity, thus preventing CRF receptor (CRFR) activation. Despite recent progress on the molecular details that govern interactions between CRF family neuropeptides and their cognate receptors, little is known concerning the mechanisms that allow CRF-BP to bind CRF and Ucn 1 with picomolar affinity. We conducted a comprehensive alanine scan of 76 evolutionarily conserved residues of CRF-BP and identified several residues that differentially affected the affinity for CRF over Ucn 1. We determined that both neuropeptides derive their similarly high affinity from distinct binding surfaces on CRF-BP. Alanine substitutions of arginine 56 (R56A) and aspartic acid 62 (D62A) reduce the affinity for CRF by approximately 100-fold, while only marginally affecting the affinity for Ucn 1. The selective reduction in affinity for CRF depends on glutamic acid 25 in the CRF peptide, as substitution of Glu(25) reduces the affinity for CRF-BP by approximately 2 orders of magnitude, but only in the presence of both Arg(56) and Asp(62) in human CRF-BP. We show that CRF-BP(R56A) and CRF-BP(D62A) have lost the ability to inhibit CRFR1-mediated responses to CRF that activate luciferase induction in HEK293T cells and ACTH release from cultured rat anterior pituitary cells. In contrast, both CRF-BP mutants retain the ability to inhibit Ucn 1-induced CRFR1 activation. Collectively our findings demonstrate that CRF-BP has distinct and separable binding surfaces for CRF and Ucn 1, opening new avenues for the design of ligand-specific antagonists based on CRF-BP.     

Stability and change: Stress responses and the shaping of behavioral phenotypes over the life span

In mammals, maternal signals conveyed via influences on hypothalamic-pituitary-adrenal (HPA) activity may shape behavior of the young to be better adapted for prevailing environmental conditions. However, the mother's influence extends beyond classic stress response systems. In guinea pigs, several hours (h) of separation from the mother activates not only the HPA axis, but also the innate immune system, which effects immediate behavioral change, as well as modifies behavioral responsiveness in the future. Moreover, the presence of the mother potently suppresses the behavioral consequences of this innate immune activation. These findings raise the possibility that long-term adaptive behavioral change can be mediated by the mother's influence on immune-related activity of her pups. Furthermore, the impact of social partners on physiological stress responses and their behavioral outcomes are not limited to the infantile period. A particularly crucial period for social development in male guinea pigs is that surrounding the attainment of sexual maturation. At this time, social interactions with adults can dramatically affect circulating cortisol concentrations and social behavior in ways that appear to prepare the male to best cope in its likely future social environment. Despite such multiple social influences on the behavior of guinea pigs at different ages, inter-individual differences in the magnitude of the cortisol response remain surprisingly stable over most of the life span. Together, it appears that throughout the life span, physiological stress responses may be regulated by social stimuli. These influences are hypothesized to adjust behavior for predicted environmental conditions. In addition, stable individual differences might provide a means of facilitating adaptation to less predictable conditions.     

Characterization of CRF, AVT, and ACTH cDNA and pituitary-adrenal axis function in Japanese quail divergently selected for tonic immobility

Higher corticosterone (CORT) responses to acute stress have previously been reported in quail selected for short (STI) duration of tonic immobility (TI) than for long TI (LTI), although behavioral studies indicated that LTI quail were more fearful. To investigate adrenal and pituitary function in these quail lines and their possible involvement in the differences in hypothalamic-pituitary-adrenal (HPA) axis reactivity, we measured CORT responses to adrenocorticotropin (1-24 ACTH), corticotropin-releasing factor (CRF), and arginine vasotocin (AVT) after characterizing the nucleotide acid sequences of these peptides in quail. Although maximum adrenal responses, assessed by ACTH challenge, were higher in STI quail, adrenal sensitivity was comparable for the two genotypes. It is therefore unlikely that differences in HPA axis reactivity involved the adrenal level. AVT and ACTH induced comparable CORT responses in both genotypes, whereas those induced by CRF were much lower. AVT is thus more potent than CRF in quail, but the respective maximum pituitary capacity of both genotypes to secrete ACTH was similar, and it is doubtful that the AVT pathway is involved in the difference in HPA axis reactivity between genotypes. On the other hand, the higher CORT responses induced by CRF in STI quail suggest that CRF might be involved in the differences in HPA axis reactivity between LTI and STI genotypes.     

Activation of corticotropin releasing factor receptor type 2 in the heart by corticotropin releasing factor offers cytoprotection against ischemic injury via PKA and PKC dependent signaling

Corticotrophin-releasing factor receptor 2β (CRFR2β) is expressed in the myocardium. In the present study we explore whether acute treatment with the neuropeptide corticotrophin-releasing factor (CRF) could induce cytoprotection against a lethal ischemic insult in the heart (isolated murine neonatal cardiac myocytes and the isolated Langendorff perfused rat heart) by activating CRFR2. In vitro, CRF offered cytoprotection when added prior to lethal simulated ischemic stress by reducing apoptotic and necrotic cell death. Ex vivo, CRF significantly reduced infarct size from 52.1±3.1% in control hearts to 35.3±3.1% (P<0.001) when administered prior to a lethal ischemic insult. The CRF peptide did not confer cytoprotection when administered at the point of hypoxic reoxygenation or ischemic reperfusion. The acute effects of CRF treatment are mediated by CRF receptor type 2 (CRFR2) since the cardioprotection ex vivo was inhibited by the CRFR2 antagonist astressin-2B. Inhibition of the mitogen activated protein kinase-ERK1/2 by PD98059 failed to inhibit the effect of CRF. However, both protein kinase A and protein kinase C inhibitors abrogated CRF-mediated protection both ex vivo and in vitro. These data suggest that the CRF peptide reduces both apoptotic and necrotic cell death in cardiac myocytes subjected to lethal ischemic induced stress through activation of PKA and PKC dependent signaling pathways downstream of CRFR2.