This study was designed to investigate whether calcium-activated potassium channels of small (SK Ca or K Ca 2) and intermediate (IK Ca or K Ca 3.1) conductance activated by 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) are involved in both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in large and small rat mesenteric arteries. Segments of rat superior and small mesenteric arteries were mounted in myographs for functional studies. NO was recorded using NO microsensors. SK Ca and IK Ca channel currents and mRNA expression were investigated in human umbilical vein endothelial cells (HUVECs), and calcium concentrations were investigated in both HUVECs and mesenteric arterial endothelial cells. In both superior (ϳ1093 m) and small mesenteric (ϳ300 m) arteries, NS309 evoked endothelium-and concentration-dependent relaxations. In superior mesenteric arteries, NS309 relaxations and NO release were inhibited by both N G ,N G -asymmetric dimethyl-L-arginine (ADMA) (300 M), an inhibitor of NO synthase, and apamin (0.5 M) plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 M), blockers of SK Ca and IK Ca channels, respectively. In small mesenteric arteries, NS309 relaxations were reduced slightly by ADMA, whereas apamin plus an IK Ca channel blocker almost abolished relaxation. Iberiotoxin did not change NS309 relaxation. HUVECs expressed mRNA for SK Ca and IK Ca channels, and NS309 induced increases in calcium, outward current, and NO release that were blocked by apamin and TRAM-34 or charybdotoxin. These findings suggest that opening of SK Ca and IK Ca channels leads to endothelium-dependent relaxation that is mediated mainly by NO in large mesenteric arteries and by EDHF-type relaxation in small mesenteric arteries. NS309-induced calcium influx appears to contribute to the formation of NO.

BACKGROUND AND PURPOSEThe intermediate conductance calcium/calmodulin-regulated K + channel KCa3.1 produces hyperpolarizing K + currents that counteract depolarizing currents carried by transient receptor potential (TRP) channels, and provide the electrochemical driving force for Cl − and fluid movements. We investigated whether a deficiency in KCa3.1 (KCa3.1) protects against fatal pulmonary circulatory collapse in mice after pharmacological activation of the calcium-permeable TRP subfamily vanilloid type 4 (TRPV4) channels. EXPERIMENTAL APPROACHAn opener of TRPV4 channels, GSK1016790A, was infused in wild-type (wt) and KCa3.1 −/− mice; haemodynamic parameters, histology and pulmonary vascular reactivity were measured; and patch clamp was performed on pulmonary arterial endothelial cells (PAEC). KEY RESULTSIn wt mice, GSK1016790A decreased right ventricular and systemic pressure leading to a fatal circulatory collapse that was accompanied by increased protein permeability, lung haemorrhage and fluid extravasation. In contrast, KCa3.1 −/− mice exhibited a significantly smaller drop in pressure to GSK1016790A infusion, no haemorrhage and fluid water extravasation, and the mice survived. Moreover, the GSK1016790A-induced relaxation of pulmonary arteries of KCa3.1 −/− mice was significantly less than that of wt mice. GSK1016790A induced TRPV4 currents in PAEC from wt and KCa3.1 −/− mice, which co-activated KCa3.1 and disrupted membrane resistance in wt PAEC, but not in KCa3.1 −/− PAEC. CONCLUSIONS AND IMPLICATIONSOur findings show that a genetic deficiency of KCa3.1 channels prevented fatal pulmonary circulatory collapse and reduced lung damage caused by pharmacological activation of calcium-permeable TRPV4 channels. Therefore, inhibition of KCa3.1channels may have therapeutic potential in conditions characterized by abnormal high endothelial calcium signalling, barrier disruption, lung oedema and pulmonary circulatory collapse.

These novel PDE1 inhibitors induce vasodilation and lower BP, suggesting a potential use of these vasodilators in the treatment of hypertension and vasospasm.

Endothelial cell dysfunction and vessel stiffening are associated with a worsened prognosis in diabetic patients with cardiovascular diseases. The present study hypothesized that sex impacts endothelial dysfunction and structural changes in arteries from diabetic mice. In diabetic (db/db) and normoglycaemic (db/db+) mice, the mechanical properties were investigated in pressurized isolated left anterior descending coronary arteries and aorta segments that were subjected to tensile testing. Functional studies were performed on wire-mounted vascular segments. The male and female db/db mice were hyperglycaemic and had markedly increased body weight. In isolated aorta segments without the contribution of smooth muscle cells, load to rupture, viscoelasticity, and collagen content were decreased suggesting larger distensibility of the arterial wall in both male and female db/db mice. In male db/db aorta segments with smooth muscle cell contribution, lumen diameter was smaller and the passive stretch-tension curve was leftward-shifted, while they were unaltered in female db/db aorta segments versus control db/db+ mice. In contrast to female db/db mice, coronary arteries from male db/db mice had altered stress-strain relationships and increased distensibility. Transthoracic echocardiography revealed a dilated left ventricle with unaltered cardiac output, while aortic flow velocity was decreased in male db/db mice. Impairment of acetylcholine relaxation was aggravated in aorta from female db/db compared to control and male db/db mice, while impairment of sodium nitroprusside relaxations was only observed in aorta from male db/db mice. The remodeling in the coronary arteries and aorta suggests an adaptation of the arterial wall to the reduced flow velocity with sex-specific differences in the passive properties of aorta and coronary arteries. The findings of less distensible arteries and more pronounced endothelial dysfunction in female compared to male diabetic mice may have implications for the observed higher incidence of macrovascular complications in diabetic women.

Endothelial cell dysfunction and fibrosis are associated with worsening of the prognosis in patients with cardiovascular disease. Pirfenidone has a direct antifibrotic effect, but vasodilatation may also contribute to the effects of pirfenidone. Therefore, in a first study we investigated the mechanisms involved in the relaxant effect of pirfenidone in rat intrapulmonary arteries and coronary arteries from normal mice. Then in a second study, we investigated whether pirfenidone restores endothelial function in the aorta and mesenteric arteries from diabetic animals. From 16–18-week old normal male C57BL/6 mice and normoglycemic (db/db+), and type 2 diabetic (db/db) male and female mice, arteries were mounted in microvascular isometric myographs for functional studies, and immunoblotting was performed. In rat pulmonary arteries and mouse coronary arteries, pirfenidone induced relaxations, which were inhibited in preparations without endothelium. In mouse coronary arteries, pirfenidone relaxation was inhibited in the presence of a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine (L-NOARG), a blocker of large-conductance calcium-activated potassium channels (BKCa), iberiotoxin, and a blocker of KV7 channels, XE991. Patch clamp studies in vascular smooth muscle revealed pirfenidone increased iberiotoxin-sensitive current. In the aorta and mesenteric small arteries from diabetic db/db mice relaxations induced by the endothelium-dependent vasodilator, acetylcholine, were markedly reduced compared to db/db + mice. Pirfenidone enhanced the relaxations induced by acetylcholine in the aorta from diabetic male and female db/db mice. An opener of KV7 channels, flupirtine, had the same effect as pirfenidone. XE991 reduced the effect of pirfenidone and flupirtine and further reduced acetylcholine relaxations in the aorta. In the presence of iberiotoxin, pirfenidone still increased acetylcholine relaxation in aorta from db/db mice. Immunoblotting for KV7.4, KV7.5, and BKCa channel subunits were unaltered in aorta from db/db mice. Pirfenidone failed to improve acetylcholine relaxation in mesenteric arteries, and neither changed acetylcholine-induced transient decreases in blood pressure in db/db+ and db/db mice. In conclusion, pirfenidone vasodilates pulmonary and coronary arteries. In coronary arteries from normal mice, pirfenidone induces NO-dependent vasodilatation involving BKCa and KV7 channels. Pirfenidone improves endothelium-dependent vasodilatation in aorta from diabetic animals by a mechanism involving voltage-gated KV7 channels, a mechanism that may contribute to the antifibrotic effect of pirfenidone.

BACKGROUNDBoth endothelial cell dysfunction and vessel stiffness is associated with worsening of the prognosis in patients with cardiovascular diseases. In the present study, we investigated the effect of the antifibrotic drug, pirfenidone on vascular tone and whether it restores endothelial function in arteries from diabetic animals. METHODS Wild type mice and 18-20 week old normoglycaemic (db/+) and type 2 diabetic (db/db) female and male mice were sacrificed by cervical dislocation. Aorta, coronary and mesenteric small arteries were isolated and mounted in microvascular myographs for isometric tension studies. RESULTSIn coronary arteries contracted with U46619, pirfenidone induced concentration-dependent relaxations that were reduced by an inhibitor of nitric oxide (NO) synthase, by high extracellular potassium, and blockers of voltage-gated and calciumactivated K channels, XE991, tetraethylammonium, and iberiotoxin. Body weights and blood glucose levels were doubled in male and female db/db versus db/+ mice. In aorta, coronary and mesenteric small arteries from male and female db/db mice, relaxations induced by the endothelium-dependent vasodilator, acetylcholine, were significantly reduced compared to arteries from db/+ mice. In aorta segments pirfenidone and an opener of KV7 channels, flurpirtine enhanced the relaxations induced by acetylcholine. A blocker of KV7 channels, XE991 reduced the effect of both pirfenidone and flurpirtine and further reduced acetylcholine relaxations in aorta. In contrast, in mesenteric small arteries the potentiating effect of pirfenidone was absent despite impaired endothelium-dependent vasodilation to acetylcholine in arteries from diabetic db/db mice. CONCLUSIONS The present findings suggest that pirfenidone is a direct vasodilator and that it improves endothelium-dependent vasodilatation by a mechanism involving K channels in large arteries from diabetic animals. Thus, at relevant therapeutic concentrations the antifibrotic drug pirfenidone may restore endothelial function.

IntroductionActivation of endothelial small conductance calcium‐activated K+ channels (KCa2.3 or SK3) and intermediate conductance calcium‐activated K+ channels (IK or KCa3.1) leads to vascular relaxation. Our previous studies have shown that KCa2.3 down‐regulation diminishes erectile function; this channel it is the most expressed KCa channel in the endothelium of corpus cavernosum. In the present study we hypothesized that KCa2.3 channel function is altered in erectile tissue of type 2 diabetic animals.Materials and MethodsErectile function was measured in diabetic db/db mice and was compared to heterozygous control animals. Corpus cavernosum strips were mounted for isometric tension recording, and they were processed for qPCR or Western Blot. The current research was performed in accordance to FASEB Statement of Principles for the use of Animal in Research and Education.ResultsIn anesthetized diabetic db/db mice erectile function was diminished compared to control animals. Concentration‐dependent contractions to noradrenaline were increased in corpus cavernosum strips from db/db compared to strips from control mice. Apamin, a blocker of KCa2 channels inhibited acetylcholine relaxation in corpus cavernosum from db/db and control animals, being less effective in db/db compared to control animals. NS309 (0.5 μM), an activator of KCa2 and KCa3.1 channels, potentiated concentration‐response curves for acetylcholine in corpus cavernosum from control, but this was not the case in corpus cavernosum from db/db mice. SNP relaxation was similar in corpus cavernosum from db/db and control animals. KCa2.3 gene and protein expression was increased in corpus cavernosum from db/db mice, nevertheless protein expressed in aorta was unchanged.ConclusionsOur results suggest that in type 2 diabetes KCa2.3 channel expression is unchanged in the systemic circulation, and upregulated in erectile tissue. In erectile tissue noradrenaline contraction is increased and the effect of NS309 reduced which may be related to impaired KCa2.3 channel function. These changes may contribute to impairment of erectile function in diabetes.Support or Funding InformationAarhus University, Novo Nordisk Foundation and the Danish Research Council.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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