Functional cardiac orexin receptors: role of orexin-B/orexin 2 receptor in myocardial protection.

Orexins/hypocretins exert cardiovascular effects which are centrally mediated. In the present study, we tested whether orexins and their receptors may also act in an autocrine/paracrine manner in the heart exerting direct effects. Quantitative reverse transcription-PCR (RT-PCR), immunohistochemical and Western blot analyses revealed that the rat heart expresses orexins and orexin receptors (OXR). In isolated rat cardiomyocytes, only orexin-B (OR-B) caused an increase in contractile shortening, independent of diastolic or systolic calcium levels. A specific orexin receptor-2 (OX2R) agonist ([Ala11, d-Leu15]-Orexin B) exerted similar effects as OR-B, whereas a specific orexin receptor-1 (OX1R) antagonist (SB-408124) did not alter the responsiveness of OR-B. Treatment of the same model with OR-B resulted in a dose-dependent increase in myosin light chain and troponin-I (TnI) phosphorylation. Following ischaemia/reperfusion in the isolated Langendorff perfused rat heart model, OR-B, but not OR-A, exerts a cardioprotective effect; mirrored in an in vivo model as well. Unlike OR-A, OR-B was also able to induce extracellular signal-regulated kinase (ERK) 1/2 (ERK1/2) and Akt phosphorylation in rat myocardial tissue and ERK1/2 phosphorylation in human heart samples. These findings were further corroborated in an in vivo rat model. In human subjects with heart failure, there is a significant negative correlation between the expression of OX2R and the severity of the disease clinical symptoms, as assessed by the New York Heart Association (NYHA) functional classification. Collectively, we provide evidence of a distinct orexin system in the heart that exerts a cardioprotective role via an OR-B/OX2R pathway.

Novel insights into the cardio-protective effects of FGF21 in lean and obese rat hearts.

AIMS: Fibroblast growth factor 21 (FGF21) is a hepatic metabolic regulator with pleotropic actions. Its plasma concentrations are increased in obesity and diabetes; states associated with an increased incidence of cardiovascular disease. We therefore investigated the direct effect of FGF21 on cardio-protection in obese and lean hearts in response to ischemia. METHODS AND RESULTS: FGF21, FGF21-receptor 1 (FGFR1) and beta-Klotho (ßKlotho) were expressed in rodent, human hearts and primary rat cardiomyocytes. Cardiac FGF21 was expressed and secreted (real time RT-PCR/western blot and ELISA) in an autocrine-paracrine manner, in response to obesity and hypoxia, involving FGFR1-ßKlotho components. Cardiac-FGF21 expression and secretion were increased in response to global ischemia. In contrast ßKlotho was reduced in obese hearts. In isolated adult rat cardiomyocytes, FGF21 activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt), ERK1/2(extracellular signal-regulated kinase) and AMPK (AMP-activated protein kinase) pathways. In Langendorff perfused rat [adult male wild-type wistar] hearts, FGF21 administration induced significant cardio-protection and restoration of function following global ischemia. Inhibition of PI3K/Akt, AMPK, ERK1/2 and ROR-α (retinoic-acid receptor alpha) pathway led to significant decrease of FGF21 induced cardio-protection and restoration of cardiac function in response to global ischemia. More importantly, this cardio-protective response induced by FGF21 was reduced in obesity, although the cardiac expression profiles and circulating FGF21 levels were increased. CONCLUSION: In an ex vivo Langendorff system, we show that FGF21 induced cardiac protection and restoration of cardiac function involving autocrine-paracrine pathways, with reduced effect in obesity. Collectively, our findings provide novel insights into FGF21-induced cardiac effects in obesity and ischemia.

Measuring burden of diseases in a rapidly developing economy: state of Qatar.

BACKGROUND: The Global Burden of Disease (GBD) study has provided a conceptual and methodological framework to quantify and compare the health of populations. AIM: The objective of the study was to assess the national burden of disease in the population of Qatar using the disability-adjusted life year (DALYs) as a measure of disability. METHODS: We adapted the methodology described by the World Health Organization for conducting burden of disease to calculate years of life lost due to premature mortality (YLL), years lived with disability (YLD) and disability adjusted life years (DALYs). The study was conducted during the period from November 2011 to October 2012. RESULTS: The study findings revealed that ischemic heart disease (11.8%) and road traffic accidents (10.3%) were the two leading causes of burden of diseases in Qatar in 2010. The burden of diseases among men (222.04) was found three times more than of women's (71.85). Of the total DALYs, 72.7% was due to non fatal health outcomes and 27.3% was due to premature death. For men, chronic diseases like ischemic heart disease (15.7%) and road traffic accidents (13.7%) accounted great burden and an important source of lost years of healthy life. For women, birth asphyxia and birth trauma (12.6%) and abortion (4.6%) were the two leading causes of disease burden. CONCLUSION: The results of the study have shown that the national health priority areas should cover cardiovascular diseases, road traffic accidents and mental health. The burden of diseases among men was three times of women's.

Identification of a novel corticotropin-releasing hormone type 1beta-like receptor variant lacking Exon 13 in human pregnant myometrium regulated by estradiol-17beta and progesterone.

Two types of CRH receptors mediate the diverse biological functions of CRH and CRH-related peptides. The type 1 CRH-R (CRH-R1) is extensively targeted by pre-mRNA splicing mechanisms that give rise to multiple mRNA splice variants. RT-PCR amplification of CRH-R1 sequences from human myometrium yielded cDNAs that encode a novel CRH-R1 splice variant with structural characteristics identical with CRH-R1ß except a 14-amino acid deletion in the seventh transmembrane domain characteristic of the CRH-R1d. Transient expression of the hybrid CRH-R1 variant (CRH-R1ß/d) in human embryonic kidney 293 cells revealed primarily intracellular expression, although some plasma membrane protein expression was also detectable. CRH bound to CRH-R1ß/d with affinity comparable with the CRH-R1ß; however, it was unable to stimulate adenylyl cyclase or other second messengers. Using a semiquantitative RT-PCR assay, CRH-R1ß/d mRNA transcript was detected in human pregnant, but not nonpregnant, myometrium as early as 31 wk of gestation. Furthermore, in human pregnant myometrial cells, the relative expression of CRH-R1ß and CRH-R1ß/d mRNA appeared to be regulated by steroids; CRH-R1ß/d mRNA expression was increased by estradiol-17ß, whereas CRH-R1ß mRNA levels were increased by progesterone. Progesterone also substantially increased CRH-R1α mRNA levels and cellular responsiveness to CRH as determined by increased agonist binding and cAMP production as well as resistance to CRH-R heterologous desensitization by phorbol esters. These results provide novel evidence for distinct patterns of CRH-R1 splicing and identify specific steroid-mediated regulation of CRH-R1 variant expression, which might be important for modulating CRH actions during human pregnancy and labour.

Identification of nesfatin-1 in human and murine adipose tissue: a novel depot-specific adipokine with increased levels in obesity.

Nesfatin-1 is a recently identified anorexigenic peptide derived from its precursor protein, nonesterified fatty acid/nucleobindin 2 (NUCB2). Although the hypothalamus is pivotal for the maintenance of energy homeostasis, adipose tissue plays an important role in the integration of metabolic activity and energy balance by communicating with peripheral organs and the brain via adipokines. Currently no data exist on nesfatin-1 expression, regulation, and secretion in adipose tissue. We therefore investigated NUCB2/nesfatin-1 gene and protein expression in human and murine adipose tissue depots. Additionally, the effects of insulin, dexamethasone, and inflammatory cytokines and the impact of food deprivation and obesity on nesfatin-1 expression were studied by quantitative RT-PCR and Western blotting. We present data showing NUCB2 mRNA (P < 0.001), nesfatin-1 intracellular protein (P < 0.001), and secretion (P < 0.01) were significantly higher in sc adipose tissue compared with other depots. Also, nesfatin-1 protein expression was significantly increased in high-fat-fed mice (P < 0.01) and reduced under food deprivation (P < 0.01) compared with controls. Stimulation of sc adipose tissue explants with inflammatory cytokines (TNFalpha and IL-6), insulin, and dexamethasone resulted in a marked increase in intracellular nesfatin-1 levels. Furthermore, we present evidence that the secretion of nesfatin-1 into the culture media was dramatically increased during the differentiation of 3T3-L1 preadipocytes into adipocytes (P < 0.001) and after treatments with TNF-alpha, IL-6, insulin, and dexamethasone (P < 0.01). In addition, circulating nesfatin-1 levels were higher in high-fat-fed mice (P < 0.05) and showed positive correlation with body mass index in human. We report that nesfatin-1 is a novel depot specific adipokine preferentially produced by sc tissue, with obesity- and food deprivation-regulated expression.

Association of the 894G>T polymorphism in the endothelial nitric oxide synthase gene with risk of acute myocardial infarction.

BACKGROUND: This study was designed to investigate the association of the 894G>T polymorphism in the eNOS gene with risk of acute myocardial infarction (AMI), extent of coronary artery disease (CAD) on coronary angiography, and in-hospital mortality after AMI. METHODS: We studied 1602 consecutive patients who were enrolled in the GEMIG study. The control group was comprised by 727 individuals, who were randomly selected from the general adult population. RESULTS: The prevalence of the Asp298 variant of eNOS was not found to be significantly and independently associated with risk of AMI (RR = 1.08, 95%CI = 0.77-1.51, P = 0.663), extent of CAD on angiography (OR = 1.18, 95%CI = 0.63-2.23, P = 0.605) and in-hospital mortality (RR = 1.08, 95%CI = 0.29-4.04, P = 0.908). CONCLUSION: In contrast to previous reports, homozygosity for the Asp298 variant of the 894G>T polymorphism in the eNOS gene was not found to be associated with risk of AMI, extent of CAD and in-hospital mortality after AMI.

Structural domains determining signalling characteristics of the CRH-receptor type 1 variant R1beta and response to PKC phosphorylation.

Mammalian adaptive mechanisms to stressful stimuli involve release of corticotropin-releasing hormone (CRH) and downstream activation of specific G-protein-coupled 7 transmembrane domain receptors. These CRH receptors (CRH-R) are expressed as multiple mRNA spliced variants. In contrast to other mammals, the human type 1 CRH-R gene contains an additional exon (exon 6) that needs to be spliced out in order to generate the fully active CRH-R1alpha. Transcription of all 14 exons results in a CRH-R1 variant (CRH-R1beta) with an extended 1st intracellular loop (IC1); this sequence modification impairs signalling activity and alters receptor responsiveness to PKC-induced phosphorylation that leads to signalling desensitization and receptor endocytosis. To elucidate structure-function relationships and delineate sequences involved in CRH-R1beta properties, site directed mutagenesis was used to introduce a number of specific mutations into IC1 of CRH-R1beta as well as replace specific phospho-acceptor residues within the aminoacid sequence of CRH-R1alpha and CRH-R1beta. Mutant receptors were transiently expressed in human embryonic kidney (HEK293) cells and tested for their abilities to increase intracellular cAMP and their response to PKC-induced phosphorylation. Results identified a penta-aminoacid cassette within the 29-aminoacid insert of CRH-R1beta, which contains multiple positive charged aminoacids (F170-R174), as an important structural determinant for the impaired cAMP response. Furthermore, serine at position 408 in the carboxy-terminus appears to be important for mediating CRH-R1alpha resistance, but not CRH-R1beta susceptibility, to PKC-induced desensitization and internalization. These findings provide new insights about the structural determinants of CRH-R1 coupling to Gs proteins and response to protein kinase phosphorylation.

Differential responses of corticotropin-releasing hormone receptor type 1 variants to protein kinase C phosphorylation.

Corticotropin-releasing hormone (CRH) regulates diverse biological functions in mammals, through activation of two types of specific G protein-coupled receptors that are expressed as multiple mRNA spliced variants. In most cells, the type 1alpha CRH receptor (CRH-R1alpha) preferentially activates the G(s)-adenylyl cyclase signaling cascade. CRH-R1alpha-mediated signaling activity is impaired by insertion of 29 amino acids in the first intracellular loop, a sequence modification that is characteristic of the human-specific CRH-R1beta variant. In various tissues, CRH signaling events are regulated by protein kinase C (PKC). The CRH receptors contain multiple putative PKC phosphorylation sites that represent potential targets. To investigate this, we expressed recombinant CRH-R1alpha or CRH-R1beta in human embryonic kidney 293 cells and analyzed signaling events after PKC activation. Agonist (oxytocin) or phorbol 12-myristate 13-acetate-induced activation of PKC led to phosphorylation of both CRH-R1 variants. However, CRH-R1alpha and CRH-R1beta exhibited different functional responses to PKC-induced phosphorylation, with only the CRH-R1beta susceptible to cAMP signaling desensitization. This was associated with a significant decrease of accessible CRH-R1beta receptors expressed on the cell surface. Both CRH-R1 variants were susceptible to homologous desensitization and internalization following treatment with CRH; however, PKC activation increased internalization of CRH-R1beta but not CRH-R1alpha in a beta-arrestin-independent manner. Our findings indicate that CRH-R1alpha and -R1beta exhibit differential responses to PKC-induced phosphorylation, and this might represent an important mechanism for functional regulation of CRH signaling in target cells.

The molecular mechanisms underlying the regulation of the biological activity of corticotropin-releasing hormone receptors: implications for physiology and pathophysiology.

The CRH receptor (CRH-R) is a member of the secretin family of G protein-coupled receptors. Wide expression of CRH-Rs in the central nervous system and periphery ensures that their cognate agonists, the family of CRH-like peptides, are capable of exerting a wide spectrum of actions that underpin their critical role in integrating the stress response and coordinating the activity of fundamental physiological functions, such as the regulation of the cardiovascular system, energy balance, and homeostasis. Two types of mammal CRH-R exist, CRH-R1 and CRH-R2, each with unique splicing patterns and remarkably distinct pharmacological properties, but similar signaling properties, probably reflecting their distinct and sometimes contrasting biological functions. The regulation of CRH-R expression and activity is not fully elucidated, and we only now begin to fully understand the impact on mammalian pathophysiology. The focus of this review is the current and evolving understanding of the molecular mechanisms controlling CRH-R biological activity and functional flexibility. This shows notable tissue-specific characteristics, highlighted by their ability to couple to distinct G proteins and activate tissue-specific signaling cascades. The type of activating agonist, receptor, and target cell appears to play a major role in determining the overall signaling and biological responses in health and disease.

Progesterone signaling in human myometrium through two novel membrane G protein-coupled receptors: potential role in functional progesterone withdrawal at term.

Progestin withdrawal is a crucial event for the onset of labor in many mammalian species. However, in humans the mechanism of a functional progestin withdrawal is unclear, because progestin concentrations do not drop in maternal plasma preceding labor. We report the presence of two novel functional membrane progestin receptors (mPRs), mPRalpha and mPRbeta, in human myometrium that are differentially modulated during labor and by steroids in vitro. The mPRs are coupled to inhibitory G proteins, resulting in a decline in cAMP levels and increased phosphorylation of myosin light chain, both of which facilitate myometrial contraction. Activation of mPRs leads to transactivation of PR-B, the first evidence for cross-talk between membrane and nuclear PRs. Progesterone activation of the mPRs leads also to a decrease of the steroid receptor coactivator 2. Our data indicate the presence of a novel signaling pathway mediated by mPRs that may result in a functional progestin withdrawal, shifting the balance from a quiescent state to one of contraction.

Preeclampsia is associated with impaired regulation of the placental nitric oxide-cyclic guanosine monophosphate pathway by corticotropin-releasing hormone (CRH) and CRH-related peptides.

During pregnancy, CRH and CRH-related peptides appear to regulate the fetoplacental circulation via activation of the nitric oxide (NO)/cGMP pathway. Pregnancies with abnormal placental function such as preeclampsia (PE) are characterized by increased maternal plasma CRH concentrations and reduced placental CRH-receptor 1alpha (CRH-R1alpha) expression. In this study, we investigated the actions of CRH/CRH-related peptides on the NO/cGMP system in normal and PE placentas (n = 8 for each group). Fluorescent in situ hybridization, RT-PCR, and immunofluorescence experiments in human term placenta detected mRNAs expression for both R1 and R2 types of CRH-R, as well as urocortin (UCN) II and UCN III and showed CRH-R protein expression mainly in syncytiotrophoblast, whereas the endothelial NO synthase (eNOS) expression was confined within the cytoplasm of the chorionic villi. In placental explants, CRH and UCN induced mRNA and protein expression of eNOS, but not inducible NOS, and also caused an acute increase in cGMP levels (maximal stimulation, 80-90% above basal; P < 0.05). UCN II also induced a modest induction of cGMP (42% above basal; P < 0.05). These responses were attenuated by the NOS and soluble guanylyl cyclase inhibitors, l-N(G)-nitro-l-arginine methyl ester and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. In PE placental explants there was a significant reduction in CRH/CRH-related peptide-induced cGMP response; however, changes in the mRNA content of eNOS, inducible NOS, and soluble guanylyl cyclase (assessed by quantitative RT-PCR) between normal and PE placentas were not altered. In conclusion, we demonstrated that CRH and CRH-related peptides can positively regulate the placental NO/cGMP system. This pathway appears to be impaired in PE and may contribute toward dysregulation of the balance controlling vascular resistance.

Food deprivation differentially modulates orexin receptor expression and signaling in rat hypothalamus and adrenal cortex.

Although starvation-induced biochemical and metabolic changes are perceived by the hypothalamus, the adrenal gland plays a key role in the integration of metabolic activity and energy balance, implicating feeding as a major synchronizer of rhythms in the hypothalamic-pituitary-adrenal (HPA) axis. Given that orexins are involved in regulating food intake and activating the HPA axis, we hypothesized that food deprivation, an acute challenge to the systems that regulate energy balance, should elicit changes in orexin receptor signaling at the hypothalamic and adrenal levels. Food deprivation induced orexin type 1 (OX1R) and 2 (OX2R) receptors at mRNA and protein levels in the hypothalamus, in addition to a fivefold increase in prepro-orexin mRNA. Cleaved peptides OR-A and OR-B are also elevated at the protein level. Interestingly, adrenal OX1R and OX2R levels were significantly reduced in food-deprived animals, whereas there was no expression of prepro-orexin in the adrenal gland in either state. Food deprivation exerted a differential effect on OXR-G protein coupling. In the hypothalamus of food deprived rats compared with controls, a significant increase in coupling of orexin receptors to Gq, Gs, and Go was demonstrated, whereas coupling to Gi was relatively less. However, in the adrenal cortex of the food-deprived animal, there was decreased coupling of orexin receptors to Gs, Go, and Gq and increased coupling to Gi. Subsequent second-messenger studies (cAMP/IP3) have supported these findings. Our data indicate that food deprivation has differential effects on orexin receptor expression and their signaling characteristics at the hypothalamic and adrenocortical levels. These findings suggest orexins as potential metabolic regulators within the HPA axis both centrally and peripherally.

Regulation of corticotropin-releasing hormone receptor type 1alpha signaling: structural determinants for G protein-coupled receptor kinase-mediated phosphorylation and agonist-mediated desensitization.

Attenuation of CRH receptor type 1 (CRH-R1) signaling activity might involve desensitization and uncoupling of CRH-R1 from intracellular effectors. We investigated the desensitization of native CRH-R in human myometrial cells from pregnant women and recombinant CRH-R1alpha stably overexpressed in human embryonic kidney (HEK) 293 cells. In both cell types, CRH-R1-mediated adenylyl cyclase activation was susceptible to homologous desensitization induced by pretreatment with high concentrations of CRH. Time course studies showed half-maximal desensitization occurring after approximately 40 min of pretreatment and full recovery of CRH-R1alpha functional response within 2 h of removal of CRH pretreatment. In HEK 293 cells, desensitization of CRH-R1alpha was associated with receptor phosphorylation and subsequent endocytosis. To analyze the mechanism leading to CRH-R1alpha desensitization, we overexpressed a truncated beta-arrestin (319-418) and performed coimmunoprecipitation and G protein-coupled receptor kinase (GRK) translocation studies. We found that GRK3 and GRK6 are the main isoforms that interact with CRH-R1alpha, and that recruitment of GRK3 requires Gbetagamma-subunits as well as beta-arrestin. Site-directed mutagenesis of Ser and Thr residues in the CRH-R1alpha C terminus, identified Thr399 as important for GRK-induced receptor phosphorylation and desensitization.We conclude that homologous desensitization of CRH-R1alpha involves the coordinated action of multiple GRK isoforms, Gbeta gamma dimers and beta-arrestin. Based on our identification of key amino acid(s) for GRK-dependent phosphorylation, we demonstrate the importance of the CRH-R1alpha carboxyl tail for regulation of receptor activity.

Promoter analysis of human corticotropin-releasing factor (CRF) type 1 receptor and regulation by CRF and urocortin.

We report the full genomic organization of the human gene for the corticotropin-releasing factor (CRF) receptor type 1 (CRFR1), with complete mapping of exons 1-14. The 5' flanking region (2.4 kb) of the gene encoding for human CRFR1 was isolated, sequenced, and characterized. Two major transcriptional start sites were determined at -265 and -238, relative to the ATG start site (+1). Transient expression of constructs containing sequentially deleted 5'-flanking sequences of CRFR1 fused to luciferase, revealed the minimal promoter sequence 370 bp in size, as shown by assays in neuroblastoma (SH-5YSY), teratocarcinoma (NT2), and adenocarcinoma (MCF 7) cell lines. CRF and UCN markedly increased promoter activity during transient CRFR1 expression studies. Similarly, CRF and UCN up-regulate the endogenous CRFR1 at the mRNA level in NT2 and MCF 7 cells. To dissect further the mechanisms involved, we have used primary myometrial cells transfected with the CRFR1 promoter. CRF and UCN increased the promoter activity, an effect blocked by protein kinase (PK)A and PKC inhibitors. Both CRF and UCN cause a positive feedback effect in primary cultures of human pregnant myometrial cells, by increasing mRNA expression of CRFR1. This effect appears to be dependent on activation of both PKA and PKC by CRF, whereas UCN's effect was mediated solely via PKC activation. Collectively, our data suggest that the CRFR1 gene is under the influence of both CRF and UCN, acting via distinct signaling pathways to create a positive feedback loop and regulate further the transcription of the receptor.

Growth hormone replacement decreases plasma levels of matrix metalloproteinases (2 and 9) and vascular endothelial growth factor in growth hormone-deficient individuals.

BACKGROUND: Matrix metalloproteinases (MMP) are implicated in cardiovascular disease. Growth hormone (GH) deficiency is associated with increased cardiovascular mortality. We assessed whether GH replacement, in GH-deficient adults, has any effect on plasma levels of MMP-2 and MMP-9 and on vascular endothelial growth factor (VEGF), known to activate MMPs. METHODS AND RESULTS: The study comprised 66 GH-deficient adults, 37.8+/-14.7 years of age (37 female). Plasma MMP-2 and MMP-9, VEGF, and insulin-like growth factor-1 (IGF-1) were measured at baseline (V1), at 12 months (V2), and at 24 months of GH treatment (V3). IGF-1 levels rose under GH replacement (mean+/-SD): V1, 151.6+/-91.9 microg/mL; V2, 270.2+/-114.8 microg/mL; and V3, 266.2+/-109.8 (V1 versus V2; P<0.001: V2 versus V3; P=0.76). MMP-9 exhibited the most pronounced and sustained decline from 1248.0+/-651.1 ng/mL at V1, 949.2+/-457.7 ng/mL at V2, and 760.8+/-386.1 ng/mL at V3 (P<0.001 at all time points). A similar pattern was detected for VEGF levels: 358.5+/-209.0 pg/mL at V1, 310.6+/-225.7 pg/mL at V2 (P<0.001), and 283.7+/-202.7 pg/mL at V3 (V2 versus V3; P=0.005). MMP-2 demonstrated a significant decline initially from V1 to V2 (1134.4+/-217.8 ng/mL versus 1074.5+/-203.0 ng/mL, respectively; P=0.031), reaching a plateau at V3 (1072.3+/-220.2 ng/mL) (V2 versus V3; P=0.93). A negative relation existed between MMP-9 versus IGF-1 and MMP-2 versus IGF-1 (P<0.001 and P=0.007, respectively) as well as between VEGF and IGF-1 (P<0.001). CONCLUSIONS: These changes in MMPs and VEGF may contribute to the anticipated reduction in vascular mortality in hypopituitary adults receiving GH replacement.

Protein kinase A-induced negative regulation of the corticotropin-releasing hormone R1alpha receptor-extracellularly regulated kinase signal transduction pathway: the critical role of Ser301 for signaling switch and selectivity.

Activation of CRH receptors type 1 (CRH-R1) by CRH or urocortin (UCN) leads to stimulation of multiple G proteins with consequent effects on diverse signaling cascades in a tissue-specific manner. In human myometrium and human embryonic kidney (HEK)293 cells, binding of UCN to CRH-R1alpha receptors activates both the Gs and Gq, leading to activation of the adenylyl cyclase/protein kinase A (PKA) and the phospholipase C/protein kinase C and ERK1/2 signaling pathways, respectively. The overall result of these signals is often unpredictable, as these two signaling pathways can interact in many cellular systems, with either potentiation or inhibition of ERK1/2 activity. In the present studies we investigated potential signaling interactions after stimulation of CRH-R1alpha receptors in human cultured pregnant myometrial cells or HEK293 cells overexpressing recombinant CRH-R1alpha receptors. We found that the adenylyl cyclase/PKA pathway has the capacity to markedly decrease UCN-induced ERK1/2 activation, and that these effects were due in part to the ability of PKA to phosphorylate the CRH-R1alpha at position Ser(301) in the third intracellular loop. Mutant CRH-R1alpha receptors with substitutions at position Ser(301), which is the only potential PKA phosphorylation site, were resistant to PKA-dependent phosphorylation and showed altered signaling characteristics, which were dependent upon the amino acid substitution at this position. We conclude that Ser(301), which is located in the third intracellular loop of CRH-R1alpha, is critical for efficient coupling of the receptor to G proteins and to second messenger generation. Phosphorylation by PKA prevents maximal coupling of the CRH-R1alpha to Gq-protein, and thereby reduces activation of ERK 1/2.

Urocortin II is expressed in human pregnant myometrial cells and regulates myosin light chain phosphorylation: potential role of the type-2 corticotropin-releasing hormone receptor in the control of myometrial contractility.

The family of CRH-related peptides are suggested to play important roles in the control of myometrial contractility during pregnancy and labor. In this study we investigated the expression of urocortin II (UCN II) in human myometrium and its ability to phosphorylate intracellular components that can be involved in modulating myometrial contractility. Using RT-PCR and fluorescent in situ hybridization, we demonstrated that UCN II and type-2 CRH receptor (CRH-R2) mRNAs were expressed in human nonpregnant and pregnant myometrium. Immunofluorescent studies confirmed protein expression of UCN II in human pregnant myometrial cells, whereas chemical cross-linking studies with radiolabeled UCN II confirmed the presence of CRH-R2 sites with an apparent molecular mass of 50 kDa. Treatment of primary human myometrial cells with UCN II to specifically activate CRH-R2 resulted in a dose-dependent increase of myosin light chain (MLC(20)) phosphorylation. Activation of protein kinase C (PKC) and ERK1/2 was required for the UCN II-induced activation of MLC(20), because treatment of myometrial cells with inhibitors of MAPK kinase 1 (U0126) and PKC (bisindolylmaleimide) inhibited the UCN II-induced phosphorylation of MLC(20). Furthermore, the UCN II effect on MLC(20) was dependent on RhoA translocation to the membrane and subsequent activation of RhoA-associated kinase, as shown by the use of the specific inhibitors exoenzyme C3 and Y27632. Collectively, our data suggest a distinctive role for CRH-R2- specific agonists like UCN II in the control of myometrial contractility during human pregnancy involving sequential activation of PKC, MAPK kinase 1, ERK1/2, RhoA, and RhoA-associated kinase, leading to the MLC(20) phosphorylation.

Plasma total homocysteine concentrations in adults with growth hormone (GH) deficiency: effects of GH replacement.

Growth hormone (GH) deficiency is associated with increased cardiovascular morbidity and mortality. GH treatment improves the profile of many cardiovascular risk markers in individuals with GH deficiency (GHD). The aim of the present was to assess whether GH replacement may decrease plasma total homocysteine, an independent cardiovascular risk factor, thus potentially contributing to benefits of GH replacement in adult subjects with GHD. Twenty-five patients (17 female, 8 male), mean age 39-years, with GHD were studied. GH status had been determined by an insulin tolerance test and/or arginine stimulation test. After an overnight fast, plasma insulin, IGF-1, total homocysteine (Hcy), free thyroxine (FT4), creatinine, vitamin B12, and folate were measured at baseline (V1), 3 months (V2) and then at 6 months (V3) on GH treatment. The data were analysed by hierarchical statistical models, univariate and multivariate correlation. GH treatment resulted in an increase in IGF-1 (p<0.001, p<0.001), and insulin (p=0.068, p<0.001), at each visit, respectively. Hcy levels increased from V1 to V2 (7.7+/-0.53 to 9.15+/-0.45 micromol/L; p=0.051), but this was followed by a decline at V3 (to 8.8+/-0.59), so that the overall change of Hcy levels from V1 to V3, once individuals had achieved 'adequate' GH replacement, was no longer significantly different (p=0.090). When separated by gender, at 6 months (V3) there was a small, but significant increase in Hcy in men (p=0.028), but not in women (p=0.58). There was no significant change in B12, folate, free T4 or creatinine levels. Univariate analysis revealed that only B12 and folate showed significant negative relationships with Hcy (B12: parameter= -0.013, p<0.001; folate: parameter=-1.31, p<0.001), but not between Hcy and IGF-1 (p=0.18). In a multiple variable model, both B12 and folate remained significantly negatively associated with plasma total homocysteine (p=0.018; p<0.001, respectively). In this observational study normalisation of IGF-1 levels in adult subjects with growth hormone deficiency was not associated with a fall in total homocysteine. Before firm conclusions can be drawn about the contribution of changes in plasma homocysteine concentrations to cardiovascular prognosis in adult GHD patients receiving GH replacement, further controlled studies are required.