Novel Roles for Kv7 Channels in Shaping Histamine-Induced Contractions and Bradykinin-Dependent Relaxations in Pig Coronary Arteries.

Voltage-gated Kv7 channels are inhibited by agonists of Gq-protein-coupled receptors, such as histamine. Recent works have provided evidence that inhibition of vascular Kv7 channels may trigger vessel contractions. In this study, we investigated how Kv7 activity modulates the histamine-induced contractions in “healthy” and metabolic syndrome (MetS) pig right coronary arteries (CAs). We performed isometric tension and immunohistochemical studies with domestic, lean Ossabaw, and MetS Ossabaw pig CAs. We found that neither the Kv7.2/Kv7.4/Kv7.5 activator ML213 nor the general Kv7 inhibitor XE991 altered the tension of CA rings under preload, indicating that vascular Kv7 channels are likely inactive in the preloaded rings. Conversely, ML213 potently dilated histamine-pre-contracted CAs, suggesting that Kv7 channels are activated during histamine applications and yet partially inhibited by histamine. Immunohistochemistry analysis revealed strong Kv7.4 immunostaining in the medial and intimal layers of the CA wall, whereas Kv7.5 immunostaining intensity was strong in the intimal but weak in the medial layers. The medial Kv7 immunostaining was significantly weaker in MetS Ossabaw CAs as compared to lean Ossabaw or domestic CAs. Consistently, histamine-pre-contracted MetS Ossabaw CAs exhibited attenuated ML213-dependent dilations. In domestic pig CAs, where medial Kv7 immunostaining intensity was stronger, histamine-induced contractions spontaneously decayed to ~31% of the peak amplitude within 4 minutes. Oppositely, in Ossabaw CAs, where Kv7 immunostaining intensity was weaker, the histamine-induced contractions were more sustained. XE991 pretreatment significantly slowed the decay rate of histamine-induced contractions in domestic CAs, supporting the hypothesis that increased Kv7 activity correlates with a faster rate of histamine-induced contraction decay. Alternatively, XE991 significantly decreased the amplitude of bradykinin-dependent dilations in pre-contracted CAs. We propose that in CAs, a decreased expression or a loss of function of Kv7 channels may lead to sustained histamine-induced contractions and reduced endothelium-dependent relaxation, both risk factors for coronary spasm

Mechanisms underlying capsaicin effects in canine coronary artery: implications for coronary spasm

AIMS: The TRPV1, transient receptor potential vanilloid type 1, agonist capsaicin is considered to be beneficial for cardiovascular health because it dilates coronary arteries through an endothelial-dependent mechanism and may slow atheroma progression. However, recent reports indicate that high doses of capsaicin may constrict coronary arterioles and even provoke myocardial infarction. Thus far, the mechanisms by which TRPV1 activation modulates coronary vascular tone remain poorly understood. This investigation examined whether there is a synergistic interplay between locally acting vasoconstrictive pro-inflammatory hormones (autacoids) and capsaicin effects in the coronary circulation. METHODS AND RESULTS: Experiments were performed in canine conduit coronary artery rings and isolated smooth muscle cells (CASMCs). Isometric tension measurements revealed that 1-10 μM capsaicin alone did not affect resting tension of coronary artery rings. In contrast, in endothelium-intact rings pre-contracted with a Gq/11-coupled FP/TP (prostaglandin F/thromboxane) receptor agonist, prostaglandin F2α (PGF2α; 10 μM), capsaicin first induced transient dilation that was followed by sustained contraction. In endothelium-denuded rings pre-contracted with PGF2α or thromboxane analogue U46619 (1 μM, a TP receptor agonist), capsaicin induced only sustained contraction. Blockers of the TP receptor or TRPV1 significantly inhibited capsaicin effects, but these were still observed in the presence of 50 μM nifedipine and 70 mM KCl. Capsaicin also potentiated 20 mM KCl-induced contractions. Fluorescence imaging experiments in CASMCs revealed that the Gq/11-phospholipase C (PLC)-protein kinase C (PKC) and Ca(2+)-PLC-PKC pathways are likely involved in sensitizing CASMC TRPV1 channels. CONCLUSION: Capsaicin alone does not cause contractions in conduit canine coronary artery; however, pre-treatment with pro-inflammatory prostaglandin-thromboxane agonists may unmask capsaicin's vasoconstrictive potential

Long-term spironolactone treatment reduces coronary TRPC expression, vasoconstriction, and atherosclerosis in metabolic syndrome pigs

Coronary transient receptor potential canonical (TRPC) channel expression is elevated in metabolic syndrome (MetS). However, differential contribution of TRPCs to coronary pathology in MetS is not fully elucidated. We investigated the roles of TRPC1 and TRPC6 isoforms in coronary arteries of MetS pigs and determined whether long-term treatment with a mineralocorticoid receptor inhibitor, spironolactone, attenuates coronary TRPC expression and associated dysfunctions. MetS coronary arteries exhibited significant atherosclerosis, endothelial dysfunction, and increased histamine-induced contractions. Immunohistochemical studies revealed that TRPC6 immunostaining was significantly greater in the medial layer of MetS pig coronary arteries compared to that in Lean pigs, whereas little TRPC6 immunostaining was found in atheromas. Conversely, TRPC1 immunostaining was weak in the medial layer but strong in MetS atheromas, where it was predominantly localized to macrophages. Spironolactone treatment significantly decreased coronary TRPC expression and dysfunctions in MetS pigs. In vivo targeted delivery of the dominant-negative (DN)-TRPC6 cDNA to the coronary wall reduced histamine-induced calcium transients in the MetS coronary artery medial layer, implying a role for TRPC6 in mediating calcium influx in MetS coronary smooth muscles. Monocyte adhesion was increased in Lean pig coronary arteries cultured in the presence of aldosterone; and spironolactone antagonized this effect, suggesting that coronary mineralocorticoid receptor activation may regulate macrophage infiltration. TRPC1 expression in atheroma macrophages was associated with advanced atherosclerosis, whereas medial TRPC6 upregulation correlated with increased histamine-induced calcium transients and coronary contractility. We propose that long-term spironolactone treatment may be a therapeutic strategy to decrease TRPC expression and coronary pathology associated with MetS

K_v7.4 and K_v7.5 immunostaining and function in Ossabaw pig CAs.

<p><b>A-D</b>, Sample isometric tension recordings and summary data illustrating different decay rates of histamine-induced contractions observed in domestic and Ossabaw CA rings (Domestic: n = 7; Lean: n = 7; MetS: n = 5). <b>E</b> shows the pattern of K_v7.4 and K_v7.5 protein distribution across the conduit coronary artery cross-sectional segments from Ossabaw pigs. <b>F and G</b>, Summary data for K_v7.4 and K_v7.5 protein distribution. The ratio of the background-corrected intensities of media-intimal layer and the adventitia layer staining was calculated to quantify the relative expression levels of the K_v7 proteins (Domestic: n = 3; Lean: n = 4; MetS: n = 3). <b>H</b> shows the summary data illustrating 10 μM ML213 dilatory effects in histamine pre-contracted rings from Ossabaw lean and MetS pigs (Lean: n = 5; MetS: n = 6).</p

Effects of K_v7 modulators on pig CAs.

<p>Sample isometric tension measurement traces and statistical analysis data are shown. 70 mM KCl-induced contractions were regularly assessed to monitor the general reactivity of the tested CA rings. Neither 10 μM XE991 (<b>A,</b> n = 6) nor 10 μM ML213 (<b>B,</b> n = 6) affected the 2–3 g preloaded CA ring tension. <b>C</b>, 10 μM ML213 potently dilated the pig CA pre-contracted with histamine (50 μM), whereas XE991 (10 μM) reversed the dilatory effect of ML213 (n = 4). <b>D</b>. Histamine concentration-dependently induced CA contractions. The shown isometric tension recording represents a time-matched control for “E.” The second histamine application induced a contractions with a smaller amplitude and slower onset kinetics (n = 4). <b>E</b> shows that the histamine-induced contractions exhibit slower kinetics of decay in the presence of 10 μM XE991 (n = 8). <b>F,</b> Comparison of 10 μM ML213-induced dilations of histamine-pre-contracted CA rings in the presence and the absence of 10 μM XE991. <b>G.</b> Comparison of normalized forces of contractions (F_peak) in the presence and absence of 10 μM XE991 (summary data for “D” and “E”). The maximal force of the second histamine-induced contraction was normalized to the maximal force of the first histamine-induced contraction. <b>H</b>. Comparison of decay rates of histamine-induced contractions in the absence and presence of 10 μM XE991. <b>I</b>. Comparison of time to peak values for the indicated groups. <b>J</b>. A sample trace illustrating that 10 μM XE991 can completely restore the magnitude of histamine-induced contractions even when there was no apparent contractile response to a preceding histamine application. 10 μM nifedipine was added to the bath to assess the contribution of voltage-gated Ca²⁺ channels in mediating the contraction. <b>K</b>. Summary of data that are shown in “J” (n = 4).</p

Immunostaining of K_v7 channels in the CA.

<p><b>A</b>, 10 μM ML277, an opener of K_v7.1 channels, did not affect the tension in histamine-pre-contracted domestic pig CA rings. A sample trace is shown. <b>B and C</b>, Shown are sample isometric tension recordings illustrating that a K_v7.4 activator diclofenac (200 μM) causes greater dilations of histamine-pre-contracted rings than a K_v7.5 activator UCL2077 (50 μM). <b>D</b>, Summary data for “A-C” (ML213: n = 12; Diclofenac: n = 5; ML277, n = 8; UCL2077: n = 5). In these experiments, we selected those domestic pig CA rings that exhibited the slower decay rate of histamine-induced contractions. <b>E and F,</b> Images illustrating typical immunohistochemical staining patterns for K_v7.4 and K_v7.5 isoforms in the domestic pig CA wall.</p

Functional role of K_v7 channels in the endothelium.

<p><b>A,</b> Sample isometric tension measurement traces obtained in intact (black trace) and denuded (grey trace) domestic pig CA rings are shown. The insert shows that both intact and endothelium-denuded CA rings were equally dilated by ML213 (10 μM, n = 3). PGF_2α stands for Prostaglandin F_2α (10 μM). <b>B,</b> A sample trace illustrating that the bradykinin-induced dilation is reduced in the presence of XE991 (10 μM) in a histamine-pre-contracted CA ring (10 nM bradykinin: 41.3±7.3% for DMSO vs. 23.5±5.73% for XE991; 100 nM bradykinin: 74.1±5. 8% for DMSO vs. 52.7±7.7% for XE991, n = 8). <b>C</b>, The concentration-response curves for bradykinin-induced dilations of histamine-pre-contracted rings obtained in the presence or absence of XE991 (10 μM). <b>D</b>, A sample trace illustrating the effect of XE991 (10 μM) on bradykinin-induced dilations in a 30 mM KCl-pre-contracted RCA ring. XE991 reduced bradykinin-induced relaxations. <b>E</b>, The summary for the data shown in the panel D. BK stands for bradykinin. Coronary artery rings were treated with 1 μM BK (n = 6) and 10 μM BK (n = 7).</p

Diagram summarizing the roles of K_v7 channels in the pig right coronary artery.

<p>It appears that a balance between K_v7 channel activation and inhibition contributes to sculpturing the shape of histamine-induced CA contractile responses. Histamine acts on the histamine H1 receptor (H1R) expressed in CA smooth muscle cells. This results in a phospholipase C (PLC)-dependent stimulation of receptor-operated and store-operated channels (ROC/SOC) mediating cation influx that depolarizes the smooth muscle cells. In turn, smooth muscle cell depolarization activates voltage-gated Ca²⁺ channels that leads to massive Ca²⁺ influx into the smooth muscle cells with a consecutive smooth muscle contraction and further depolarization. Such stronger depolarization stimulates K_v7 activity repolarizing smooth muscles and weakening coronary artery ring contractions. Conversely, histamine-dependent inhibition of K_v7 channels via a PLC-dependent mechanism leads to an increased smooth muscle cell depolarization and strengthens ring contractions. In endothelium, bradykinin-dependent ROC/SOC activation also results in Na⁺ and Ca²⁺ influx that is driven by the gradient of these cations and negative potential inside endothelial cells. However, such cation influx depolarizes endothelial cells reducing the driving force. Activation of endothelial K_v7 channels likely enhances endothelium-dependent dilatory responses in the pig right coronary artery by maintaining more negative potential inside endothelial cells that results in greater Ca²⁺ influx known to stimulate Ca²⁺-dependent nitric oxide synthase (eNOS) causing increased NO production.</p

Effects of ML213 on KCl-pre-contracted CAs.

<p><b>A,</b> Sample isometric tension recoding illustrating the effects of 10 μM ML213 on the rings pre-constricted with histamine or 30 mM KCl. <b>B</b>, Summary data for ML213 effects in pre-contracted CA rings.</p