Alison J. Davis

Expression and function of the K+ channel KCNQ genes in human arteries

BACKGROUND AND PURPOSE KCNQ‐encoded voltage‐gated potassium channels (Kv7) have recently been identified as important anti‐constrictor elements in rodent blood vessels but the role of these channels and the effects of their modulation in human arteries remain unknown. Here, we have assessed KCNQ gene expression and function in human arteries ex vivo.

Downregulation of Kv7.4 Channel Activity in Primary and Secondary Hypertension

BACKGROUND Voltage-gated potassium (K(+)) channels encoded by KCNQ genes (Kv7 channels) have been identified in various rodent and human blood vessels as key regulators of vascular tone; however, nothing is known about the functional impact of these channels in vascular disease. We ascertained the effect of 3 structurally different activators of Kv7.2 through Kv7.5 channels (BMS-204352, S-1, and retigabine) on blood vessels from normotensive and hypertensive animals. METHODS AND RESULTS Precontracted thoracic aorta and mesenteric artery segments from normotensive rats were relaxed by all 3 Kv7 activators, with potencies of BMS-204352=S-1>retigabine. We also tested these agents in the coronary circulation using the Langendorff heart preparation. BMS-204352 and S-1 dose dependently increased coronary perfusion at concentrations between 0.1 and 10 μmol/L, whereas retigabine was effective at 1 to 10 μmol/L. In addition, S-1 increased K(+) currents in isolated mesenteric artery myocytes. The ability of these agents to relax precontracted vessels, increase coronary flow, or augment K(+) currents was impaired considerably in tissues isolated from spontaneously hypertensive rats (SHRs). Of the 5 KCNQ genes, only the expression of KCNQ4 was reduced (≈3.7 fold) in SHRs aorta. Kv7.4 protein levels were ≈50% lower in aortas and mesenteric arteries from spontaneously hypertensive rats compared with normotensive vessels. A similar attenuated response to S-1 and decreased Kv7.4 were observed in mesenteric arteries from mice made hypertensive by angiotensin II infusion compared with normotensive controls. CONCLUSIONS In 2 different rat and mouse models of hypertension, the functional impact of Kv7 channels was dramatically downregulated.

Expression profile and protein translation of TMEM16A in murine smooth muscle

Recently, overexpression of the genes TMEM16A and TMEM16B has been shown to produce currents qualitatively similar to native Ca(2+)-activated Cl(-) currents (I(ClCa)) in vascular smooth muscle. However, there is no information about this new gene family in vascular smooth muscle, where Cl(-) channels are a major depolarizing mechanism. Qualitatively similar Cl(-) currents were evoked by a pipette solution containing 500 nM Ca(2+) in smooth muscle cells isolated from BALB/c mouse portal vein, thoracic aorta, and carotid artery. Quantitative PCR using SYBR Green chemistry and primers specific for transmembrane protein (TMEM) 16A or the closely related TMEM16B showed TMEM16A expression as follows: portal vein > thoracic aorta > carotid artery > brain. In addition, several alternatively spliced variant transcripts of TMEM16A were detected. In contrast, TMEM16B expression was very low in smooth muscle. Western blot analysis with different antibodies directed against TMEM16A revealed a number of products with a consistent band at ∼120 kDa, except portal vein, where an 80-kDa band predominated. TMEM16A protein was identified in the smooth muscle layers of 4-μm-thick slices of portal vein, thoracic aorta, and carotid artery. In isolated myocytes, fluorescence specific to a TMEM16A antibody was detected diffusely throughout the cytoplasm, as well as near the membrane. The same antibody used in Western blot analysis of lysates from vascular tissues also recognized an ∼147-kDa mouse TMEM16A-green fluorescent protein (GFP) fusion protein expressed in HEK 293 cells, which correlated to a similar band detected by a GFP antibody. Patch-clamp experiments revealed that I(ClCa) generated by transfection of TMEM16A-GFP in HEK 293 cells displayed remarkable similarities to I(ClCa) recorded in vascular myocytes, including slow kinetics, steep outward rectification, and a response similar to the pharmacological agent niflumic acid. This study shows that TMEM16A expression is robust in murine vascular smooth muscle cells, consolidating the view that this gene is a viable candidate for the native Ca(2+)-activated Cl(-) channel in this cell type.

Reduced KCNQ4-Encoded Voltage-Dependent Potassium Channel Activity Underlies Impaired β-Adrenoceptor–Mediated Relaxation of Renal Arteries in Hypertension

KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to &bgr;-adrenoceptor–mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein expression of KCNQ1, KCNQ3, KCNQ4, KCNQ5, and Kv7.1, Kv7.4, and Kv7.5 in rat renal artery. Isoproterenol produced concentration-dependent relaxation of precontracted renal arteries and increased Kv7 channel currents in isolated smooth muscle cells. Application of the Kv7 blocker linopirdine attenuated isoproterenol-induced relaxation and current. Isoproterenol-induced relaxations were also reduced in arteries incubated with small interference RNAs targeted to KCNQ4 that produced a ≈60% decrease in Kv7.4 protein level. Relaxation to isoproterenol and the Kv7 activator S-1 were abolished in arteries from spontaneously hypertensive rats, which was associated with ≈60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to &bgr;-adrenoceptor–mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired &bgr;-adrenoceptor pathway in spontaneously hypertensive rats. These findings may provide a novel pathogenic link between arterial dysfunction and hypertension.

Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension

Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca(2+) levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca(2+)-activated Cl- channels (Cl(Ca)), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl(Ca) channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca(2+)-activated Cl(-) current [I(Cl(Ca))] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I(Cl(Ca)) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl(Ca) channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 μM), an l-type Ca(2+) channel blocker, and NFA (30 or 100 μM, with or without 10 μM indomethacin to inhibit cyclooxygenases) or T16A(Inh)-A01 (10 μM), TMEM16A/Cl(Ca) channel inhibitors, than that of control animals. In conclusion, augmented Cl(Ca)/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease.

Potent vasorelaxant activity of the TMEM16A inhibitor T16Ainh‐A01

T16Ainh‐A01 is a recently identified inhibitor of the calcium‐activated chloride channel TMEM16A. The aim of this study was to test the efficacy of T16Ainh‐A01 for inhibition of calcium‐activated chloride channels in vascular smooth muscle and consequent effects on vascular tone.

Cholesterol depletion alters amplitude and pharmacology of vascular calcium-activated chloride channels

AIMS Calcium-activated chloride channels (CACCs) share common pharmacological properties with Kcnma1-encoded large conductance K(+) channels (BK(Ca) or K(Ca)1.1) and it has been suggested that they may co-exist in a macromolecular complex. As K(Ca)1.1 channels are known to localize to cholesterol and caveolin-rich lipid rafts (caveolae), the present study investigated whether Ca(2+)-sensitive Cl(-) currents in vascular myocytes were affected by the cholesterol depleting agent methyl-beta-cyclodextrin (M-betaCD). METHODS AND RESULTS Calcium-activated chloride and potassium currents were recorded from single murine portal vein myocytes in whole cell voltage clamp. Western blot was undertaken following sucrose gradient ultracentrifugation using protein lysates from whole portal veins. Ca(2+)-activated Cl(-) currents were augmented by 3 mg mL(-1) M-betaCD with a rapid time course (t(0.5) = 1.8 min). M-betaCD had no effect on the bi-modal response to niflumic acid or anthracene-9-carboxylate but completely removed the inhibitory effects of the K(Ca)1.1 blockers, paxilline and tamoxifen, as well as the stimulatory effect of the K(Ca)1.1 activator NS1619. Discontinuous sucrose density gradients followed by western blot analysis revealed that the position of lipid raft markers caveolin and flotillin-2 was altered by 15 min application of 3 mg mL(-1) M-betaCD. The position of K(Ca)1.1 and the newly identified candidate for CACCs, TMEM16A, was also affected by M-betaCD. CONCLUSION These data reveal that CACC properties are influenced by lipid raft integrity.

Are bisphosphonates effective in the treatment of osteoarthritis pain? A meta-analysis and systematic review.

Objective Osteoarthritis (OA) is the most common form of arthritis worldwide. Pain and reduced function are the main symptoms in this prevalent disease. There are currently no treatments for OA that modify disease progression; therefore analgesic drugs and joint replacement for larger joints are the standard of care. In light of several recent studies reporting the use of bisphosphonates for OA treatment, our work aimed to evaluate published literature to assess the effectiveness of bisphosphonates in OA treatment. Methods Literature databases were searched from inception to the 30th June 2012 for clinical trials of bisphosphonates to treat OA pain. Data was appraised and levels of evidence determined qualitatively using best evidence synthesis from the Cochrane Collaboration. The two largest studies were conducted with risedronate in the treatment of knee OA, for which meta-analyses were performed for pain and functional outcomes. Results Our searches found 13/297 eligible studies, which included a total of 3832 participants. The trials recruited participants with OA of the hand (n = 1), knee (n = 8), knee and spine (n = 3), or hip (n = 1). Our meta-analysis of the two largest knee studies using risedronate 15 mg showed odds ratios favouring placebo interventions for the Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain (1.73), WOMAC function (2.03), and WOMAC stiffness (1.82). However, 8 trials (61.5%) reported that bisphosphonates improve pain assessed by VAS scores and 2 (38.5%) reported significant improvement in WOMAC pain scores compared to control groups. Conclusions There is limited evidence that bisphosphonates are effective in the treatment of OA pain. Limitations of the studies we analysed included the differences in duration of bisphosphonate use, the dose and route of administration and the lack of long-term data on OA joint structure modification post-bisphosphonate therapy. Future more targeted studies are required to appreciate the value of bisphosphonates in treating osteoarthritis pain. Trial Registration PROSPERO Register CRD42012002541

Pharmacological dissection of Kv7.1 channels in systemic and pulmonary arteries

BACKGROUND AND PURPOSE The aim of this study was to characterize the functional impact of KCNQ1‐encoded voltage‐dependent potassium channels (Kv7.1) in the vasculature.

Effective contractile response to voltage-gated Na+ channels revealed by a channel activator

This study investigated the molecular identity and impact of enhancing voltage-gated Na(+) (Na(V)) channels in the control of vascular tone. In rat isolated mesenteric and femoral arteries mounted for isometric tension recording, the vascular actions of the Na(V) channel activator veratridine were examined. Na(V) channel expression was probed by molecular techniques and immunocytochemistry. In mesenteric arteries, veratridine induced potent contractions (pEC(50) = 5.19 ± 0.20, E(max) = 12.0 ± 2.7 mN), which were inhibited by 1 μM TTX (a blocker of all Na(V) channel isoforms, except Na(V)1.5, Na(V)1.8, and Na(V)1.9), but not by selective blockers of Na(V)1.7 (ProTx-II, 10 nM) or Na(V)1.8 (A-80347, 1 μM) channels. The responses were insensitive to endothelium removal but were partly (~60%) reduced by chemical destruction of sympathetic nerves by 6-hydroxydopamine (2 mM) or antagonism at the α1-adrenoceptor by prazosin (1 μM). KB-R7943, a blocker of the reverse mode of the Na(+)/Ca(2+) exchanger (3 μM), inhibited veratridine contractions in the absence or presence of prazosin. T16A(inh)-A01, a Ca(2+)-activated Cl(-) channel blocker (10 μM), also inhibited the prazosin-resistant contraction to veratridine. Na(V) channel immunoreactivity was detected in freshly isolated mesenteric myocytes, with apparent colocalization with the Na(+)/Ca(2+) exchanger. Veratridine induced similar contractile effects in the femoral artery, and mRNA transcripts for Na(V)1.2 and Na(V)1.3 channels were evident in both vessel types. We conclude that, in addition to sympathetic nerves, NaV channels are expressed in vascular myocytes, where they are functionally coupled to the reverse mode of Na(+)/Ca(2+) exchanger and subsequent activation of Ca(2+)-activated Cl(-) channels, causing contraction. The TTX-sensitive Na(V)1.2 and Na(V)1.3 channels are likely involved in vascular control.