Ryanodine receptor expression in trophoblasts

Trophoblasts of the placenta are the frontline cells involved in communication and exchange of materials between the mother and fetus. Within trophoblasts, calcium signalling proteins are richly expressed. Intracellular free calcium ions are a key second messenger, regulating various cellular activities. Transcellular Ca2+ transport through trophoblasts is essential in fetal skeleton formation. Ryanodine receptors (RyRs) are high conductance cation channels that mediate Ca2+ release from intracellular stores to the cytoplasm. To date, the roles of RyRs in trophoblasts have not been reported. By use of reverse transcription PCR and western blotting, the current study revealed that RyRs are expressed in model trophoblast cell lines (BeWo and JEG-3) and in human first trimester and term placental villi. Immunohistochemistry of human placental sections indicated that both syncytiotrophoblast and cytotrophoblast cell layers were positively stained by antibodies recognising RyRs; likewise, expression of RyR isoforms was also revealed in BeWo and JEG-3 cells by immunofluorescence microscopy. In addition, changes in [Ca2+]i were observed in both BeWo and JEG-3 cells upon application of various RyR agonists and antagonists, using fura-2 fluorescent videomicroscopy. Furthermore, endogenous placental peptide hormones, namely angiotensin II, arginine vasopressin and endothelin 1, were demonstrated to increase [Ca2+]i in BeWo cells, and such increases were suppressed by RyR antagonists and by blockers of the corresponding peptide hormone receptors. These findings indicate that 1) multiple RyR subtypes are expressed in human trophoblasts; 2) functional RyRs in BeWo and JEG-3 cells response to both RyR agonists and antagonists; 3) RyRs in BeWo cells mediate Ca2+ release from intracellular store in response to the indirect stimulation by endogenous peptides. These observations suggest that RyR contributes to trophoblastic cellular Ca2+ homeostasis; trophoblastic RyRs are also involved in the functional regulation of human placenta by coupling to endogenous placental peptide-induced signalling pathways.

Identifying novel molecular mechanisms of ghrelin receptor signalling underlying neural control of food intake: interaction with stress and impulsivity

The gut-hormone, ghrelin, activates the centrally expressed growth hormone secretagogue 1a (GHS-R1a) receptor, or ghrelin receptor. The ghrelin receptor is a G-protein coupled receptor (GPCR) expressed in several brain regions, including the arcuate nucleus (Arc), lateral hypothalamus (LH), ventral tegmental area (VTA), nucleus accumbens (NAcc) and amygdala. Activation of the GHS-R1a mediates a multitude of biological activities, including release of growth hormone and food intake. The ghrelin signalling system also plays a key role in the hedonic aspects of food intake and activates the dopaminergic mesolimbic circuit involved in reward signalling. Recently, ghrelin has been shown to be involved in mediating a stress response and to mediate stress-induced food reward behaviour via its interaction with the HPA-axis at the level of the anterior pituitary. Here, we focus on the role of the GHS-R1a receptor in reward behaviour, including the motivation to eat, its anxiogenic effects, and its role in impulsive behaviour. We investigate the functional selectivity and pharmacology of GHS-R1a receptor ligands as well as crosstalk of the GHS-R1a receptor with the serotonin 2C (5-HT2C) receptor, which represent another major target in the regulation of eating behaviour, stress-sensitivity and impulse control disorders. We demonstrate, to our knowledge for the first time, the direct impact of GHS-R1a signalling on impulsive responding in a 2-choice serial reaction time task (2CSRTT) and show a role for the 5-HT2C receptor in modulating amphetamine-associated impulsive action. Finally, we investigate differential gene expression patterns in the mesocorticolimbic pathway, specifically in the NAcc and PFC, between innate low- and high-impulsive rats. Together, these findings are poised to have important implications in the development of novel treatment strategies to combat eating disorders, including obesity and binge eating disorders as well as impulse control disorders, including, substance abuse and addiction, attention deficit hyperactivity disorder (ADHD) and mood disorders.

Chromosomal organization of adrenergic receptor genes.

The adrenergic receptors (ARs) (subtypes alpha 1, alpha 2, beta 1, and beta 2) are a prototypic family of guanine nucleotide binding regulatory protein-coupled receptors that mediate the physiological effects of the hormone epinephrine and the neurotransmitter norepinephrine. We have previously assigned the genes for beta 2- and alpha 2-AR to human chromosomes 5 and 10, respectively. By Southern analysis of somatic cell hybrids and in situ chromosomal hybridization, we have now mapped the alpha 1-AR gene to chromosome 5q32----q34, the same position as beta 2-AR, and the beta 1-AR gene to chromosome 10q24----q26, the region where alpha 2-AR is located. In mouse, both alpha 2- and beta 1-AR genes were assigned to chromosome 19, and the alpha 1-AR locus was localized to chromosome 11. Pulsed field gel electrophoresis has shown that the alpha 1- and beta 2-AR genes in humans are within 300 kilobases (kb) and the distance between the alpha 2- and beta 1-AR genes is less than 225 kb. The proximity of these two pairs of AR genes and the sequence similarity that exists among all the ARs strongly suggest that they are evolutionarily related. Moreover, they likely arose from a common ancestral receptor gene and subsequently diverged through gene duplication and chromosomal duplication to perform their distinctive roles in mediating the physiological effects of catecholamines. The AR genes thus provide a paradigm for understanding the evolution of such structurally conserved yet functionally divergent families of receptor molecules.

Impaired formation of beta-adrenergic receptor-nucleotide regulatory protein complexes in pseudohypoparathyroidism.

Decreased activity of the guanine nucleotide regulatory protein (N) of the adenylate cyclase system is present in cell membranes of some patients with pseudohypoparathyrodism (PHP-Ia) whereas others have normal activity of N (PHP-Ib). Low N activity in PHP-Ia results in a decrease in hormone (H)-stimulatable adenylate cyclase in various tissues, which might be due to decreased ability to form an agonist-specific high affinity complex composed of H, receptor (R), and N. To test this hypothesis, we compared beta-adrenergic agonist-specific binding properties in erythrocyte membranes from five patients with PHP-Ia (N = 45% of control), five patients with PHP-Ib (N = 97%), and five control subjects. Competition curves that were generated by increasing concentrations of the beta-agonist isoproterenol competing with [125I]pindolol were shallow (slope factors less than 1) and were computer fit to a two-state model with corresponding high and low affinity for the agonist. The agonist competition curves from the PHP-Ia patients were shifted significantly (P less than 0.02) to the right as a result of a significant (P less than 0.01) decrease in the percent of beta-adrenergic receptors in the high affinity state from 64 +/- 22% in PHP-Ib and 56 +/- 5% in controls to 10 +/- 8% in PHP-Ia. The agonist competition curves were computer fit to a "ternary complex" model for the two-step reaction: H + R + N in equilibrium HR + N in equilibrium HRN. The modeling was consistent with a 60% decrease in the functional concentration of N, and was in good agreement with the biochemically determined decrease in erythrocyte N protein activity. These in vitro findings in erythrocytes taken together with the recent observations that in vivo isoproterenol-stimulated adenylate cyclase activity is decreased in patients with PHP (Carlson, H. E., and A. S. Brickman, 1983, J. Clin. Endocrinol. Metab. 56:1323-1326) are consistent with the notion that N is a bifunctional protein interacting with both R and the adenylate cyclase. It may be that in patients with PHP-Ia a single molecular and genetic defect accounts for both decreased HRN formation and decreased adenylate cyclase activity, whereas in PHP-Ib the biochemical lesion(s) appear not to affect HRN complex formation.

Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways.

Organisms in the wild develop with varying food availability. During periods of nutritional scarcity, development may slow or arrest until conditions improve. The ability to modulate developmental programs in response to poor nutritional conditions requires a means of sensing the changing nutritional environment and limiting tissue growth. The mechanisms by which organisms accomplish this adaptation are not well understood. We sought to study this question by examining the effects of nutrient deprivation on Caenorhabditis elegans development during the late larval stages, L3 and L4, a period of extensive tissue growth and morphogenesis. By removing animals from food at different times, we show here that specific checkpoints exist in the early L3 and early L4 stages that systemically arrest the development of diverse tissues and cellular processes. These checkpoints occur once in each larval stage after molting and prior to initiation of the subsequent molting cycle. DAF-2, the insulin/insulin-like growth factor receptor, regulates passage through the L3 and L4 checkpoints in response to nutrition. The FOXO transcription factor DAF-16, a major target of insulin-like signaling, functions cell-nonautonomously in the hypodermis (skin) to arrest developmental upon nutrient removal. The effects of DAF-16 on progression through the L3 and L4 stages are mediated by DAF-9, a cytochrome P450 ortholog involved in the production of C. elegans steroid hormones. Our results identify a novel mode of C. elegans growth in which development progresses from one checkpoint to the next. At each checkpoint, nutritional conditions determine whether animals remain arrested or continue development to the next checkpoint.

Effects of short-term chemical ablation of the GIP receptor on insulin secretion, islet morphology and glucose homeostasis in mice

Glucosedependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by endocrine Kcells in response to nutrient absorption. In this study we have utilized a specific and enzymatically stable GIP receptor antagonist, (Pro(3))GIP, to evaluate the contribution of endogenous GIP to insulin secretion and glucose homeostasis in mice. Daily injection of (Pro(3))GIP (25 nmol/kg body weight) for 11 days had no effect on food intake or body weight. Nonfasting plasma glucose concentrations were significantly raised (p

Chemical ablation of gastric inhibitory polypeptide receptor action by daily (Pro3) GIP administration improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure in obesity-diabetes.

Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide [GIP]) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. In this study, we investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-related diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult ob/ob mice received once-daily intraperitoneal injections of saline vehicle or (Pro3)GIP over an 11-day period. Nonfasting plasma glucose levels and the overall glycemic excursion (area under the curve) to a glucose load were significantly reduced (1.6-fold; P <0.05) in (Pro3)GIP-treated mice compared with controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P <0.05) and insulin (1.5-fold; P <0.05) responses to feeding. These changes were associated with significantly enhanced (1.6-fold; P <0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP reduced pancreatic insulin content (1.3-fold; P <0.05) and partially corrected the obesity-related islet hypertrophy and ß-cell hyperplasia of ob/ob mice. These comprehensive beneficial effects of (Pro3)GIP were reversed 9 days after cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes.

Characterisation and biological activity of Glu(3) amino acid substituted GIP receptor antagonists

Glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal hormone, which regulates insulin release and glucose homeostasis, but is rapidly inactivated by enzymatic N-terminal truncation. Here we report the enzyme resistance and biological activity of several Glu(3) -substituted analogues of GIP namely; (Ala(3))GIP, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))GIP. Only (Lys(3))- GIP demonstrated moderately enhanced resistance to DPP-IV (p <0.05 to p <0.01) compared to native GIP. All analogues demonstrated a decreased potency in cAMP production (EC50 1.47 to 11.02 nM; p <0.01 to p <0.001) with (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated cAMP production (p <0.05). In BRIN-BD11 cells, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))- GIP did not stimulate insulin secretion with both (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated insulin secretion (p <0.05). Injection of each GIP analogue together with glucose in oblob mice significantly increased the glycaemic excursion compared to control (p <0.05 to p <0.001). This was associated with lack of significant insulin responses. (Ala(3))GIP, (Phe(3))GIP and (Tyr(3))GIP, when administered together with GIP, significantly reduced plasma insulin (p <0.05 top <0.01) and impaired the glucose-lowering ability (p <0.05 to p <0.01) of the native peptide. The DPP-IV resistance and GIP antagonism observed were similar but less pronounced than (Pro(3))GIP. These data demonstrate that position 3 amino acid substitution of GIP with (Ala(3)), (Phe(3)), (Tyr(3)) or (Pro(3)) provides a new class of functional GIP receptor antagonists. (C) 2007 Elsevier Inc. All rights reserved.

Incretin hormone mimetics and analogues in diabetes therapeutics

The incretin hormones glucagon-like peptide-I (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are physiological gut peptides with insulin-releasing and extrapancreatic glucoregulatory actions. Incretin analogues/mimetics activate GLP-I or GIP receptors whilst avoiding physiological inactivation by dipeptidyl peptidase 4 (DPP-4), and they represent one of the newest classes of antidiabetic drug. The first clinically approved GLP-1 mimetic for the treatment of type-2 diabetes is exenatide (Byetta/exendin) which is administered subcutaneously twice daily. Clinical trials of liraglutide, a GLP-1 analogue suitable for once-daily administration, are ongoing. A number of other incretin molecules are at earlier stages of development. This review discusses the various attributes of GLP-1 and GIP for diabetes treatment and summarises current clinical data. Additionally, it explores the therapeutic possibilities offered by preclinical agents, such as non-peptide GLP-1 mimetics, GLP-1/glucagon hybrid peptides, and specific GIP receptor antagonists.

Metabolic stability, receptor binding, cAMP generation, insulin secretion and antihyperglycaemic activity of novel N-terminal Glu(9)-substituted analogues of glucagon-like peptide-1

Glucagonlike peptide-1(7 36)amide (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. Rapid removal of the Nterminal dipeptide, His7-Ala8, by the ubiquitous enzyme dipeptidyl peptidase IV (DPP IV) curtails the biological activity of GLP-1. Chemical modifications or substitutions of GLP-1 at His7 or Ala8 improve resistance to DPPIV action, but this often reduces potency. Little attention has focused on the metabolic stability and functional activity of GLP-1 analogues with amino acid substitution at Glu9, adjacent to the DPP IV cleavage site. We generated three novel Glu9-substituted GLP-1 analogues, (Pro9)GLP-1, (Phe9)GLP-1 and (Tyr9)GLP-1 and show for the first time that Glu9 of GLP-1 is important in DPP IV degradation, since replacing this amino acid, particularly with proline, substantially reduced susceptibility to degradation. All three novel GLP-1 analogues showed similar or slightly enhanced insulinotropic activity compared with native GLP-1 despite a moderate 4 10-fold reduction in receptor binding and cAMP generation. In addition, (Pro9)GLP 1 showed significant ability to moderate the plasma glucose excursion and increase circulating insulin concentrations in severely insulin resistant obese diabetic (ob/ob) mice. These observations indicate the importance of Glu9 for the biological activity of GLP-1 and susceptibility to DPP IVmediated degradation.

Degradation, receptor binding, insulin secreting and antihyperglycaemic actions of palmitate-derivatised native and Ala(8)-substituted GLP-1 analogues

The hormone glucagonlike peptide-1(736)amide (GLP-1) is released in response to ingested nutrients and acts to promote glucosedependent insulin secretion ensuring efficient postprandial glucose homeostasis. Unfortunately, the beneficial actions of GLP-1 which give this hormone many of the desirable properties of an antidiabetic drug are short lived due to degradation by dipeptidylpeptidase IV (DPP IV) and rapid clearance by renal filtration. In this study we have attempted to extend GLP-1 action through the attachment of palmitoyl moieties to the epsilon-amino group in the side chain of the Lys(26) residue and to combine this modification with substitutions of the Ala(8) residue, namely Val or aminobutyric acid (Abu). In contrast to native GLP-1, which was rapidly degraded, [Lys(pal)(26)]GLP-1, [Abu(8),Lys(pal)(26)]GLP-1 and [Val(8),Lys(pal)(26)]GLP-1 all exhibited profound stability during 12 h incubations with DPP IV and human plasma. Receptor binding affinity and the ability to increase cyclic AMP in the clonal beta-cell line BRIN-BD11 were decreased by 86- to 167-fold and 15- to 62-fold, respectively compared with native GLP-1. However, insulin secretory potency tested using BRIN-BD11 cells was similar, or in the case of [Val(8),Lys(pal)(26)]GLP-1 enhanced. Furthermore, when administered in vivo together with glucose to diabetic (ob/ob) mice, [Lys(pal)(26)]GLP-1, [Abu(8),Lys(pal)(26)]GLP-1 and [Val8,Lys(pal)26]GLP-1 did not demonstrate acute glucoselowering or insulinotropic activity as observed with native GLP-1. These studies support the potential usefulness of fatty acid linked analogues of GLP-1 but indicate the importance of chain length for peptide kinetics and bioavailability.

Evidence that the major degradation product of glucose-dependent insulinotropic polypeptide, GIP(3-42), is a GIP receptor antagonist in vivo

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) is rapidly degraded in the circulation by dipeptidyl peptidase IV forming the N-terminally truncated peptide GIP(3-42). The present study examined the biological activity of this abundant circulating fragment peptide to establish its possible role in GIP action. Human GIP and GIP(3-42) were synthesised by Fmoc solid-phase peptide synthesis, purified by HPLC and characterised by electrospray ionisation-mass spectrometry. In GIP receptor-transfected Chinese hamster lung fibroblasts, GIP(3-42) dose dependently inhibited GIP-stimulated (10(-7) M) cAMP production (up to 75.4 +/-5.4%; P

Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase

Oestrogen produces diverse biological effects through binding to the oestrogen receptor (ER)(1). The ER is a steroid hormone nuclear receptor, which, when bound to oestrogen, modulates the transcriptional activity of target genes(2). Controversy exists, however, concerning whether ER has a role outside the nucleus(3), particularly in mediating the cardiovascular protective effects of oestrogen(4). Here we show that the ER isoform, ER alpha, binds in a ligand-dependent manner to the p85 alpha regulatory subunit of phosphatidylinositol-3-OH kinase (PI(3)K). Stimulation with oestrogen increases ER alpha-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). Recruitment and activation of PI(3)K by ligand-bound ERa are independent of gene transcription, do not involve phosphotyrosine adapter molecules or src-homology domains of p85 alpha, and extend to other steroid hormone receptors. Mice treated with oestrogen show increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury. This vascular protective effect of oestrogen was abolished in the presence of PI(3)K or eNOS inhibitors. Our findings define a physiologically important non-nuclear oestrogen-signalling pathway involving the direct interaction of ERa with PI(3)K.

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol (-ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ~70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. -ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.