Acyl-ghrelin administration increases food intake, body weight, and blood glucose. In contrast, mice lacking ghrelin or ghrelin receptors (GHSRs) exhibit life-threatening hypoglycemia during starvation-like conditions, but do not consistently exhibit overt metabolic phenotypes when given ad libitum food access. These results, and findings of ghrelin resistance in obese states, imply nutritional state dependence of ghrelin's metabolic actions. Here, we hypothesized that liver-enriched antimicrobial peptide-2 (LEAP2), a recently characterized endogenous GHSR antagonist, blunts ghrelin action during obese states and postprandially. To test this hypothesis, we determined changes in plasma LEAP2 and acyl-ghrelin due to fasting, eating, obesity, Roux-en-Y gastric bypass (RYGB), vertical sleeve gastrectomy (VSG), oral glucose administration, and type 1 diabetes mellitus (T1DM) using humans and/or mice. Our results suggest that plasma LEAP2 is regulated by metabolic status: its levels increased with body mass and blood glucose and decreased with fasting, RYGB, and in postprandial states following VSG. These changes were mostly opposite of those of acyl-ghrelin. Furthermore, using electrophysiology, we showed that LEAP2 both hyperpolarizes and prevents acyl-ghrelin from activating arcuate NPY neurons. We predict that the plasma LEAP2/acyl-ghrelin molar ratio may be a key determinant modulating acyl-ghrelin activity in response to body mass, feeding status, and blood glucose.

Growth hormone secretagogue receptor (GHSR) 1a is the only molecularly identified receptor for ghrelin, mediating ghrelin-related effects on eating, body weight and blood glucose control, among others. The expression pattern of GHSR within the brain has been assessed previously using several neuroanatomical techniques. However, inherent limitations to these techniques and the lack of reliable anti-GHSR antibodies and reporter rodent models that identify GHSR-containing neurons have prevented a more comprehensive functional characterization of ghrelin-responsive neurons. Here, we have systematically characterized the brain expression of an enhanced green fluorescence protein (eGFP) transgene controlled by the Ghsr promoter in a recently-reported GHSR reporter mouse. Expression of eGFP in coronal brain sections was compared with GHSR mRNA expression detected in the same sections by in situ hybridization histochemistry. eGFP-immunoreactivity was detected in several areas including the prefrontal cortex, insular cortex, olfactory bulb, amygdala and hippocampus, which showed no or low GHSR mRNA expression. In contrast, eGFP expression was low in several midbrain regions and in several hypothalamic nuclei – particularly the arcuate nucleus– where robust GHSR mRNA expression has been well-characterized. eGFP expression in several brainstem nuclei showed high to moderate degrees of co-localization with GHSR mRNA labeling. Further quantitative PCR and electrophysiological analyses of eGFP-labeled hippocampal cells confirmed faithful expression of eGFP within GHSR-containing, ghrelin-responsive neurons. In summary, the GHSR-eGFP reporter mouse model may be a useful tool to study GHSR function – particularly within the brainstem and hippocampus– however, it underrepresents GHSR expression in nuclei within the hypothalamus and midbrain.

Celecoxib, rofecoxib, and diclofenac are clinically used cyclooxygenase-2 (COX-2) inhibitors, which have been under intense scrutiny because long-term rofecoxib (Vioxx; Merck, Whitehouse Station, NJ) treatment was found to increase the risk of adverse cardiovascular events. A differential risk profile for these drugs has emerged, but the underlying mechanisms have not been fully elucidated. We investigated the effects of celecoxib, rofecoxib, and diclofenac on ionic currents and calcium signaling in vascular smooth muscle cells (VSMCs) using patch-clamp techniques and fura-2 fluorescence and on arterial constriction using pressure myography. Celecoxib, but not rofecoxib or diclofenac, dramatically enhanced KCNQ (K v 7) potassium currents and suppressed L-type voltage-sensitive calcium currents in A7r5 rat aortic smooth muscle cells (native KCNQ currents or overexpressed human KCNQ5 currents) and freshly isolated rat mesenteric artery myocytes. The effects of celecoxib were concentration-dependent within the therapeutic concentration range, and were reversed on washout. Celecoxib, but not rofecoxib, also inhibited calcium responses to vasopressin in A7r5 cells and dilated intact or endotheliumdenuded rat mesenteric arteries. A celecoxib analog, 2,5-dimethyl-celecoxib, which does not inhibit COX-2, mimicked celecoxib in its enhancement of vascular KCNQ5 currents, suppression of L-type calcium currents, and vasodilation. We conclude that celecoxib inhibits calcium responses in VSMCs by enhancing KCNQ5 currents and suppressing L-type calcium currents, which ultimately reduces vascular tone. These effects are independent of its COX-2 inhibitory actions and may explain the differential risk of cardiovascular events in patients taking different drugs of this class.Celecoxib (Celebrex; Pfizer, New York, NY) and rofecoxib (Vioxx; Merck, Whitehouse Station, NJ) are nonsteroidal anti-inflammatory drugs (NSAIDs) that selectively inhibit cyclooxygenase-2 (COX-2). They were introduced to the market in 1999 and rapidly became the most frequently prescribed new drugs in the United States. These drugs are used clinically to treat pain and inflammation. COX-1 and COX-2 convert arachidonic acid into prostaglandin H 2 , which is further converted to a variety of prostanoids, including prostaglandins, thromboxanes, and prostacyclins. Thromboxane A 2 , a product of COX-1 activity in platelets, promotes vasoconstriction, smooth muscle proliferation, and platelet aggregation. In contrast, prostacyclin generated by COX-2 in the blood vessel walls promotes vasodilatation and inhibition of platelet aggregation. As analgesic/anti-inflammatory agents, COX-2 inhibitors were considered to be an improvement over less selective COX-1/COX-2 inhibitors because they prevent the generation of prostaglandins involved in inflammation and pain while sparing some beneficial effects of COX-1-generated prostanoids. However, these drugs have been under intense scrutiny since 2004, when Vioxx was voluntarily withdrawn from the market because of a r...

Ghrelin administration induces food intake and body weight gain. Based on these actions, the ghrelin system was initially proposed as an anti-obesity target. Subsequent studies using genetic mouse models have raised doubts about the role of the endogenous ghrelin system in mediating body weight homeostasis or obesity. However, this is not to say that the endogenous ghrelin system is not important metabolically or otherwise. This manuscript reviews an emerging concept in which the endogenous ghrelin system serves an essential function during extreme nutritional and psychological challenges to defend blood glucose, protect body weight, avoid exaggerated depression, and ultimately allow survival.

No abstract

Kv7 (KCNQ) channels, formed as homo-or heterotetramers of Kv7.4 and Kv7.5 a-subunits, are important regulators of vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K 1 currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific a-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo-or heterotetrameric channels in A7r5 cells. Stimulation of Ga scoupled b-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2-to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKAdependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 .. Kv7.4/Kv7.5 . Kv7.4.

No abstract

BACKGROUND AND PURPOSECerebral vasospasm is the persistent constriction of large conduit arteries in the base of the brain. This pathologically sustained contraction of the arterial myocytes has been attributed to locally elevated concentrations of vasoconstrictor agonists (spasmogens). We assessed the presence and function of KCNQ (Kv7) potassium channels in rat basilar artery myocytes, and determined the efficacy of Kv7 channel activators in relieving spasmogen-induced basilar artery constriction. EXPERIMENTAL APPROACHExpression and function of Kv7 channels in freshly isolated basilar artery myocytes were evaluated by reverse transcriptase polymerase chain reaction and whole-cell electrophysiological techniques. Functional responses to Kv7 channel modulators were studied in intact artery segments using pressure myography. KEY RESULTSAll five mammalian KCNQ subtypes (KCNQ1-5) were detected in the myocytes. Kv currents were attributed to Kv7 channel activity based on their voltage dependence of activation (V0.5~-34 mV), lack of inactivation, enhancement by flupirtine (a selective Kv7 channel activator) and inhibition by 10,10-bis(pyridin-4-ylmethyl)anthracen-9-one (XE991; a selective Kv7 channel blocker). XE991 depolarized the myocytes and constricted intact basilar arteries. Celecoxib, a clinically used anti-inflammatory drug, not only enhanced Kv7 currents but also inhibited voltage-sensitive Ca 2+ currents. In arteries pre-constricted with spasmogens, both celecoxib and flupirtine were more effective in dilating artery segments than was nimodipine, a selective L-type Ca 2+ channel blocker. CONCLUSIONS AND IMPLICATIONSKv7 channels are important determinants of basilar artery contractile status. Targeting the Kv7 channels using flupirtine or celecoxib could provide a novel strategy to relieve basilar artery constriction in patients with cerebral vasospasm. LINKED ARTICLES