Bharath K. Mani

Differential effects of selective cyclooxygenase-2 inhibitors on vascular smooth muscle ion channels may account for differences in cardiovascular risk profiles.

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 (Kv7) 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 endothelium-denuded 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.

Neuroanatomical characterization of a growth hormone secretagogue receptor-green fluorescent protein reporter mouse

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 by 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 colocalization 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 for studying GHSR function, particularly within the brainstem and hippocampus; however, it underrepresents GHSR expression in nuclei within the hypothalamus and midbrain. J. Comp. Neurol. 522:3644–3666, 2014.

Activation of vascular KCNQ (Kv7) potassium channels reverses spasmogen‐induced constrictor responses in rat basilar artery

BACKGROUND AND PURPOSE Cerebral 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.

Opposite regulation of KCNQ5 and TRPC6 channels contributes to vasopressin-stimulated calcium spiking responses in A7r5 vascular smooth muscle cells

Physiologically relevant concentrations of [Arg(8)]-vasopressin (AVP) induce repetitive action potential firing and Ca(2+) spiking responses in the A7r5 rat aortic smooth muscle cell line. These responses may be triggered by suppression of KCNQ potassium currents and/or activation of non-selective cation currents. Here we examine the relative contributions of KCNQ5 channels and TRPC6 non-selective cation channels to AVP-stimulated Ca(2+) spiking using patch clamp electrophysiology and fura-2 fluorescence measurements in A7r5 cells. KCNQ5 or TRPC6 channel expression levels were suppressed by short hairpin RNA constructs. KCNQ5 knockdown resulted in more positive resting membrane potentials and induced spontaneous action potential firing and Ca(2+) spiking. However physiological concentrations of AVP induced additional depolarization and increased Ca(2+) spike frequency in KCNQ5 knockdown cells. AVP activated a non-selective cation current that was reduced by TRPC shRNA treatment or removal of external Na(+). Neither resting membrane potential nor the AVP-induced depolarization was altered by knockdown of TRPC6 channel expression. However, both TRPC6 shRNA and removal of external Na(+) delayed the onset of Ca(2+) spiking induced by 25pM AVP. These results suggest that suppression of KCNQ5 currents alone is sufficient to excite A7r5 cells, but AVP-induced activation of TRPC6 contributes to the stimulation of Ca(2+) spiking.

Vascular KCNQ (Kv7) potassium channels as common signaling intermediates and therapeutic targets in cerebral vasospasm

Abstract: Cerebral vasospasm after subarachnoid hemorrhage (SAH) is characterized by prolonged severe constriction of the basilar artery, which often leads to ischemic brain damage. Locally elevated concentrations of spasmogenic substances induce persistent depolarization of myocytes in the basilar artery, leading to continuous influx of calcium (Ca2+) through voltage-sensitive Ca2+ channels and myocyte contraction. Potassium (K+) channel openers may have therapeutic utility to oppose membrane depolarization, dilate the arteries, and reduce ischemia. Here, we examined the involvement of vascular Kv7 K+ channels in the pathogenesis of cerebral vasospasm and tested whether Kv7 channel openers are effective therapeutic agents in a rat model of SAH. Patch-clamp experiments revealed that 3 different spasmogens (serotonin, endothelin, and vasopressin) suppressed Kv7 currents and depolarized freshly isolated rat basilar artery myocytes. These effects were significantly reduced in the presence of a Kv7 channel opener, retigabine. Retigabine (10 &mgr;M) also significantly blocked L-type Ca2+ channels, reducing peak inward currents by >50%. In the presence of a selective Kv7 channel blocker, XE991, the spasmogens did not produce additive constriction responses measured using pressure myography. Kv7 channel openers (retigabine or celecoxib) significantly attenuated basilar artery spasm in rats with experimentally induced SAH. In conclusion, we identify Kv7 channels as common targets of vasoconstrictor spasmogens and as candidates for therapeutic intervention for cerebral vasospasm.

An eGFP-expressing subpopulation of growth hormone secretagogue receptor cells are distinct from kisspeptin, tyrosine hydroxylase, and RFamide-related peptide neurons in mice

Ghrelin acts on the growth hormone secretagogue receptor (GHSR) in the brain to elicit changes in physiological functions. It is associated with the neural control of appetite and metabolism, however central ghrelin also affects fertility. Central ghrelin injection in rats suppresses luteinizing hormone (LH) concentrations and pulse frequency. Although ghrelin suppresses LH and regulates kisspeptin mRNA in the anteroventral periventricular/periventricular nucleus (AVPV/PeN), there is no neuroanatomical evidence linking GHSR neural circuits to kisspeptin neurons. In this study, we first determined coexpression of GHSR and GnRH neurons using a GHSR-eGFP reporter mouse line. Using dual-label immunohistochemistry, we saw no coexpression. GHSR-eGFP expressing cells were present in the AVPV/PeN and over 90% of these expressed estrogen receptor-α (ERα). Despite this, we observed no evidence of GHSR-eGFP/kisspeptin coexpressing neurons in the AVPV/PeN. To further examine the phenotype of GHSR-eGFP cells in the AVPV/PeN, we determined coexpression with tyrosine hydroxylase (TH) and showed virtually no coexpression in the AVPV/PeN (<2%). We also observed no coexpression of GHSR-eGFP and RFamide-related peptide-3 (RFRP3) neurons in the dorsomedial hypothalamic nucleus. Importantly, we observed that approximately half of the GHSR-eGFP cells in the AVPV coexpressed Ghsr mRNA (as determined by in situ hybridization) so these data should be interpreted accordingly. Although ghrelin influences the hypothalamic reproductive axis, our data using a GHSR-eGFP reporter suggests ghrelin regulates neurons expressing ERα but does not directly act on GnRH, kisspeptin, TH, or RFRP3 neurons, as little or no GHSR-eGFP coexpression was observed.

Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents

Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-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+ 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 α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-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 PKA-dependent 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.

Altered ghrelin secretion in mice in response to diet-induced obesity and Roux-en-Y gastric bypass

The current study examined potential mechanisms for altered circulating ghrelin levels observed in diet-induced obesity (DIO) and following weight loss resulting from Roux-en-Y gastric bypass (RYGB). We hypothesized that circulating ghrelin levels were altered in obesity and after weight loss through changes in ghrelin cell responsiveness to physiological cues. We confirmed lower ghrelin levels in DIO mice and demonstrated elevated ghrelin levels in mice 6 weeks post-RYGB. In both DIO and RYGB settings, these changes in ghrelin levels were associated with altered ghrelin cell responsiveness to two key physiological modulators of ghrelin secretion – glucose and norepinephrine. In DIO mice, increases in ghrelin cell density within both the stomach and duodenum and in somatostatin-immunoreactive D cell density in the duodenum were observed. Our findings provide new insights into the regulation of ghrelin secretion and its relation to circulating ghrelin within the contexts of obesity and weight loss.

Role of calcium and EPAC in norepinephrine-induced ghrelin secretion.

Ghrelin is an orexigenic hormone secreted principally from a distinct population of gastric endocrine cells. Molecular mechanisms regulating ghrelin secretion are mostly unknown. Recently, norepinephrine (NE) was shown to enhance ghrelin release by binding to β1-adrenergic receptors on ghrelin cells. Here, we use an immortalized stomach-derived ghrelin cell line to further characterize the intracellular signaling pathways involved in NE-induced ghrelin secretion, with a focus on the roles of Ca(2+) and cAMP. Several voltage-gated Ca(2+) channel (VGCC) family members were found by quantitative PCR to be expressed by ghrelin cells. Nifedipine, a selective L-type VGCC blocker, suppressed both basal and NE-stimulated ghrelin secretion. NE induced elevation of cytosolic Ca(2+) levels both in the presence and absence of extracellular Ca(2+). Ca(2+)-sensing synaptotagmins Syt7 and Syt9 were also highly expressed in ghrelin cell lines, suggesting that they too help mediate ghrelin secretion. Raising cAMP with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated ghrelin secretion, although such a cAMP-mediated effect likely does not involve protein kinase A, given the absence of a modulatory response to a highly selective protein kinase A inhibitor. However, pharmacological inhibition of another target of cAMP, exchange protein-activated by cAMP (EPAC), did attenuate both basal and NE-induced ghrelin secretion, whereas an EPAC agonist enhanced basal ghrelin secretion. We conclude that constitutive ghrelin secretion is primarily regulated by Ca(2+) influx through L-type VGCCs and that NE stimulates ghrelin secretion predominantly through release of intracellular Ca(2+). Furthermore, cAMP and its downstream activation of EPAC are required for the normal ghrelin secretory response to NE.

Ghrelin as a Survival Hormone

Ghrelin administration induces food intake and body weight gain. Based on these actions, the ghrelin system was initially proposed as an antiobesity 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. Here we review 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.