Sukhan Kim

New selective inhibitors of calcium-activated chloride channels – T16Ainh-A01, CaCCinh-A01 and MONNA – what do they inhibit?

T16Ainh‐A01, CaCCinh‐A01 and MONNA are identified as selective inhibitors of the TMEM16A calcium‐activated chloride channel (CaCC). The aim of this study was to examine the chloride‐specificity of these compounds on isolated resistance arteries in the presence and absence (±) of extracellular chloride.

Endothelial alkalinisation inhibits gap junction communication and endothelium-derived hyperpolarisations in mouse mesenteric arteries

Gap junctions are important for coordination and transfer of signals between cells. Signals initiated in the vascular endothelium can spread through myoendothelial gap junctions and cause relaxation of coupled vascular smooth muscle cells. The cellular level of acidity is important for control of vascular function. The mechanisms linking disturbed acid–base balance to changes in vascular tone have not been understood in detail. We show that intracellular alkalinisation of endothelial cells in resistance arteries inhibits myoendothelial coupling and endothelium‐dependent vasorelaxation. Although hyperpolarisations are generated in the endothelial cells under alkaline conditions, they are not transferred to the smooth muscle cells. Similarly, dye transfer between endothelial cells is inhibited during intracellular alkalinisation. These results support the suggestion that intracellular pH is important for control of gap junction conductivity and intercellular communication. We describe a potential new molecular mechanism for development of vascular dysfunction in pathologies (e.g. diabetes, hypertension) involving altered myoendothelial signalling.

Intracellular acidification alters myogenic responsiveness and vasomotion of mouse middle cerebral arteries.

Intracellular pH (pHi) in the vascular wall modulates agonist-induced vasocontractile and vasorelaxant responses in mesenteric arteries, whereas effects on myogenic tone have been unsettled. We studied the role of Na+,HCO3− cotransporter NBCn1 in mouse isolated middle cerebral arteries and the influence of pHi disturbances on myogenic tone. Na+,HCO3− cotransport was abolished in arteries from NBCn1 knockout mice and steady-state pHi ∼0.3 units reduced compared with wild-type mice. Myogenic tone development was low under control conditions but increased on treatment with the NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME). This effect of L-NAME was smaller in arteries from NBCn1 knockout than wild-type mice. Myogenic tone with L-NAME present was significantly lower in arteries from NBCn1 knockout than wild-type mice and was abolished by rho-kinase inhibitor Y-27632. The arteries displayed vasomotion, and this rhythmic contractile pattern was also attenuated in arteries from NBCn1 knockout mice. No differences in membrane potential or intracellular [Ca2+] were seen between arteries from NBCn1 knockout and wild-type mice. We propose that NO production and rho-kinase-dependent Ca2+ sensitivity are reduced at low pHi in pressurized mouse middle cerebral arteries. This likely impedes the ability to adjust to changes in perfusion pressure and regulate cerebral blood flow.

Voltage‐gated sodium channels contribute to action potentials and spontaneous contractility in isolated human lymphatic vessels

Initial studies have described the electrical properties of lymphatic smooth muscle cells from different animal species and the ion channels contributing to these properties. However, there is a translational gap to the human situation where studies on human tissue are lacking. Voltage‐gated sodium channels are essential for the generation of action potentials, and thus spontaneous contractions in human lymphatic vessels, but not noradrenaline‐induced contractions. A sodium channel opener elicited contractions as a result of calcium influx via voltage‐gated calcium channels and the sodium–calcium exchanger. Pharmacological characterization and the mRNA expression profile indicate that Nav1.3 is the most prevalent sodium channel. These results provide support for an important role of sodium channels in spontaneous human lymphatic vessel electrical activity and contractility.

Perivascular tissue inhibits rho‐kinase‐dependent smooth muscle Ca2+ sensitivity and endothelium‐dependent H2S signalling in rat coronary arteries

Local regulation of vascular resistance adjusts coronary blood flow to metabolic demand, although the mechanisms involved are not comprehensively understood We show that heart tissue surrounding rat coronary arteries releases diffusible factors that regulate vasoconstriction and relaxation Perivascular tissue reduces rho‐kinase‐dependent smooth muscle Ca2+ sensitivity and constriction of coronary arteries to serotonin, the thromboxane analogue U46619 and the α1‐adrenergic agonist phenylephrine Endothelium‐dependent relaxation of coronary arteries in response to cholinergic stimulation is inhibited by perivascular tissue as a result of reduced endothelial Ca2+ responses and attenuated H2S‐dependent signalling These results establish cellular mechanisms by which perivascular heart tissue can modify local vascular tone and coronary blood flow

Human lymphatic vessel contractile activity is inhibited in vitro but not in vivo by the calcium channel blocker nifedipine

We studied the effects of antihypertensive calcium channel blockers on CaV1.2, the predominantly expressed L‐type calcium channel in the largest human lymphatic vessel, the thoracic duct. Human lymphatic collecting vessels, both large and small, are highly‐sensitive in vitro to calcium channel blockers; exposure to these drugs inhibits endogenous lymphatic contractile activity and action potentials and diminishes noradrenaline‐induced phasic contractions. In vivo administration of calcium channel blocker nifedipine to healthy volunteers did not reduce lymphatic contractile activity despite all subjects achieving nifedipine plasma concentrations comparable with those observed to affect contractile function in vitro. These results indicate that calcium channel blocker‐related oedema is unlikely to be exacerbated by an off‐target effect of the drugs diminishing lymphatic pumping and fluid removal.

Na-K-ATPase regulates intercellular communication in the vascular wall via cSrc kinase-dependent connexin43 phosphorylation

Communication between vascular smooth muscle cells (VSMCs) is dependent on gap junctions and is regulated by the Na-K-ATPase. The Na-K-ATPase is therefore important for synchronized VSMC oscillatory activity, i.e., vasomotion. The signaling between the Na-K-ATPase and gap junctions is unknown. We tested here the hypothesis that this signaling involves cSrc kinase. Intercellular communication was assessed by membrane capacitance measurements of electrically coupled VSMCs. Vasomotion in isometric myograph, input resistance, and synchronized [Ca2+]i transients were used as readout for intercellular coupling in rat mesenteric small arteries in vitro. Phosphorylation of cSrc kinase and connexin43 (Cx43) were semiquantified by Western blotting. Micromole concentration of ouabain reduced the amplitude of norepinephrine-induced vasomotion and desynchronized Ca2+ transients in VSMC in the arterial wall. Ouabain also increased input resistance in the arterial wall. These effects of ouabain were antagonized by inhibition of tyrosine phosphorylation with genistein, PP2, and by an inhibitor of the Na-K-ATPase-dependent cSrc activation, pNaKtide. Moreover, inhibition of cSrc phosphorylation increased vasomotion amplitude and decreased the resistance between cells in the vascular wall. Ouabain inhibited the electrical coupling between A7r5 cells, but pNaKtide restored the electrical coupling. Ouabain increased cSrc autophosphorylation of tyrosine 418 (Y418) required for full catalytic activity whereas pNaKtide antagonized it. This cSrc activation was associated with Cx43 phosphorylation of tyrosine 265 (Y265). Our findings demonstrate that Na-K-ATPase regulates intercellular communication in the vascular wall via cSrc-dependent Cx43 tyrosine phosphorylation.

4‐Aminopyridine: a pan voltage‐gated potassium channel inhibitor that enhances Kv7.4 currents and inhibits noradrenaline‐mediated contraction of rat mesenteric small arteries

Kv7.4 and Kv7.5 channels are regulators of vascular tone. 4‐Aminopyridine (4‐AP) is considered a broad inhibitor of voltage‐gated potassium (KV) channels, with little inhibitory effect on Kv7 family members at mmol concentrations. However, the effect of 4‐AP on Kv7 channels has not been systematically studied. The aim of this study was to investigate the pharmacological activity of 4‐AP on Kv7.4 and Kv7.5 channels and characterize the effect of 4‐AP on rat resistance arteries.

Murine breast cancer feed arteries are thin-walled with reduced α 1A -adrenoceptor expression and attenuated sympathetic vasocontraction

BackgroundPerfusion of breast cancer tissue limits oxygen availability and metabolism but angiogenesis inhibitors have hitherto been unsuccessful for breast cancer therapy. In order to identify abnormalities and possible therapeutic targets in mature cancer arteries, we here characterize the structure and function of cancer feed arteries and corresponding control arteries from female FVB/N mice with ErbB2-induced breast cancer.MethodsWe investigated the contractile function of breast cancer feed arteries and matched control arteries by isometric myography and evaluated membrane potentials and intracellular [Ca2+] using sharp electrodes and fluorescence microscopy, respectively. Arterial wall structure is assessed by transmission light microscopy of arteries mounted in wire myographs and by evaluation of histological sections using the unbiased stereological disector technique. We determined the expression of messenger RNA by reverse transcription and quantitative polymerase chain reaction and studied receptor expression by confocal microscopy of arteries labelled with the BODIPY-tagged α1-adrenoceptor antagonist prazosin.ResultsBreast cancer feed arteries are thin-walled and produce lower tension than control arteries of similar diameter in response to norepinephrine, thromboxane-analog U46619, endothelin-1, and depolarization with elevated [K+]. Fewer layers of similarly-sized vascular smooth muscle cells explain the reduced media thickness of breast cancer arteries. Evidenced by lower media stress, norepinephrine-induced and thromboxane-induced tension development of breast cancer arteries is reduced more than is explained by the thinner media. Conversely, media stress during stimulation with endothelin-1 and elevated [K+] is similar between breast cancer and control arteries. Correspondingly, vascular smooth muscle cell depolarizations and intracellular Ca2+ responses are attenuated in breast cancer feed arteries during norepinephrine but not during endothelin-1 stimulation. Protein expression of α1-adrenoceptors and messenger RNA levels for α1A-adrenoceptors are lower in breast cancer arteries than control arteries. Sympathetic vasocontraction elicited by electrical field stimulation is inhibited by α1-adrenoceptor blockade and reduced in breast cancer feed arteries compared to control arteries.ConclusionThinner media and lower α1-adrenoceptor expression weaken contractions of breast cancer feed arteries in response to sympathetic activity. We propose that abnormalities in breast cancer arteries can be exploited to modify tumor perfusion and thereby either starve cancer cells or facilitate drug and oxygen delivery during chemotherapy or radiotherapy.

Smooth muscle Ca2+ sensitization causes hypercontractility of middle cerebral arteries in mice bearing the familial hemiplegic migraine type 2 associated mutation:

Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2+/−G301R) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca2+ and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2+/−G301R and WT showed similar contractile responses. The Atp1a2+/−G301R MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca2+ change was smaller than in WT. The enhanced constriction of Atp1a2+/−G301R MCA was associated with increased cSrc activation, stronger sensitization to [Ca2+]i and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2+/−G301R mice had reduced resting blood flow through MCA in comparison with WT mice. FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca2+, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.