Gorm Nielsen

Pharmacological activation of KCa3.1/KCa2.3 channels produces endothelial hyperpolarization and lowers blood pressure in conscious dogs.

BACKGROUND AND PURPOSE In rodents, the endothelial KCa channels, KCa3.1 and KCa2.3, have been shown to play a crucial role in initiating endothelium‐derived hyperpolarizing factor (EDHF) vasodilator responses. However, it is not known to what extent these channels are involved in blood pressure regulation in large mammals, which would also allow us to address safety issues. We therefore characterized canine endothelial KCa3.1 and KCa2.3 functions and evaluated the effect of the KCa3.1/KCa2.3 activator SKA‐31 on blood pressure and heart rate in dogs.

Opening of Small and Intermediate Calcium-Activated Potassium Channels Induces Relaxation Mainly Mediated by Nitric-Oxide Release in Large Arteries and Endothelium-Derived Hyperpolarizing Factor in Small Arteries from Rat

This study was designed to investigate whether calcium-activated potassium channels of small (SKCa or KCa2) and intermediate (IKCa or KCa3.1) 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. SKCa and IKCa 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 (∼1093 μm) and small mesenteric (∼300 μm) arteries, NS309 evoked endothelium- and concentration-dependent relaxations. In superior mesenteric arteries, NS309 relaxations and NO release were inhibited by both NG,NG-asymmetric dimethyl-l-arginine (ADMA) (300 μM), an inhibitor of NO synthase, and apamin (0.5 μM) plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 μM), blockers of SKCa and IKCa channels, respectively. In small mesenteric arteries, NS309 relaxations were reduced slightly by ADMA, whereas apamin plus an IKCa channel blocker almost abolished relaxation. Iberiotoxin did not change NS309 relaxation. HUVECs expressed mRNA for SKCa and IKCa 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 SKCa and IKCa 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.

Improvement of endothelium-dependent vasodilations by SKA-31 and SKA-20, activators of small- and intermediate-conductance Ca2+-activated K+-channels

Aim:  Endothelial membrane hyperpolarization mediated by KCa3.1 and KCa2.3 channels has been demonstrated to initiate endothelium‐derived hyperpolarizing factor (EDHF)‐type vasodilations. Moreover, pharmacological potentiation of KCa3.1/KCa2.3 channels has been suggested to improve EDHF‐type vasodilations. Herein, we determined whether the KCa3.1/KCa2.3 activator SKA‐31 and its derivative SKA‐20 improve endothelial dysfunction in KCa3.1−/− and NOS3−/− mice.

A Role for the RNA Chaperone Hfq in Controlling Adherent-Invasive Escherichia coli Colonization and Virulence

Adherent-invasive Escherichia coli (AIEC) has been linked with the onset and perpetuation of inflammatory bowel diseases. The AIEC strain LF82 was originally isolated from an ileal biopsy from a patient with Crohns disease. The pathogenesis of LF82 results from its abnormal adherence to and subsequent invasion of the intestinal epithelium coupled with its ability to survive phagocytosis by macrophages once it has crossed the intestinal barrier. To gain further insight into AIEC pathogenesis we employed the nematode Caenorhabditis elegans as an in vivo infection model. We demonstrate that AIEC strain LF82 forms a persistent infection in C. elegans, thereby reducing the host lifespan significantly. This host killing phenotype was associated with massive bacterial colonization of the nematode intestine and damage to the intestinal epithelial surface. C. elegans killing was independent of known LF82 virulence determinants but was abolished by deletion of the LF82 hfq gene, which encodes an RNA chaperone involved in mediating posttranscriptional gene regulation by small non-coding RNAs. This finding reveals that important aspects of LF82 pathogenesis are controlled at the posttranscriptional level by riboregulation. The role of Hfq in LF82 virulence was independent of its function in regulating RpoS and RpoE activity. Further, LF82Δhfq mutants were non-motile, impaired in cell invasion and highly sensitive to various chemical stress conditions, reinforcing the multifaceted function of Hfq in mediating bacterial adaptation. This study highlights the usefulness of simple non-mammalian infection systems for the identification and analysis of bacterial virulence factors.

Activation of endothelial and epithelial KCa2.3 calcium-activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles

Small (KCa2) and intermediate (KCa3.1) conductance calcium‐activated potassium channels (KCa) may contribute to both epithelium‐ and endothelium‐dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we investigated the expression of KCa2.3 and KCa3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries.

Amplification of EDHF-type vasodilatations in TRPC1-deficient mice

BACKGROUND AND PURPOSE TRPC1 channels are expressed in the vasculature and are putative candidates for intracellular Ca2+ handling. However, little is known about their role in endothelium‐dependent vasodilatations including endothelium‐derived hyperpolarizing factor (EDHF) vasodilatations, which require activation of Ca2+‐activated K+ channels (KCa). To provide molecular information on the role of TRPC1 for KCa function and the EDHF signalling complex, we examined endothelium‐dependent and independent vasodilatations, KCa currents and smooth muscle contractility in TRPC1‐deficient mice (TRPC1‐/‐).

Pulmonary hypertension in wild type mice and animals with genetic deficit in KCa2.3 and KCa3.1 channels.

Objective In vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension. Approach and Result Male wild type and KCa3.1−/−/KCa2.3T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The KCa3.1−/−/KCa2.3T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype. Conclusion Despite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of KCa2.3/KCa3.1 activators for the treatment of pulmonary hypertension.

Alterations of N-3 Polyunsaturated Fatty Acid-Activated K2P Channels in Hypoxia-Induced Pulmonary Hypertension

Polyunsaturated fatty acid (PUFA)‐activated two‐pore domain potassium channels (K2P) have been proposed to be expressed in the pulmonary vasculature. However, their physiological or pathophysiological roles are poorly defined. Here, we tested the hypothesis that PUFA‐activated K2P are involved in pulmonary vasorelaxation and that alterations of channel expression are pathophysiologically linked to pulmonary hypertension. Expression of PUFA‐activated K2P in the murine lung was investigated by quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR), immunohistochemistry (IHC), by patch clamp (PC) and myography. K2P‐gene expression was examined in chronic hypoxic mice. qRT‐PCR showed that the K2P2.1 and K2P6.1 were the predominantly expressed K2P in the murine lung. IHC revealed protein expression of K2P2.1 and K2P6.1 in the endothelium of pulmonary arteries and of K2P6.1 in bronchial epithelium. PC showed pimozide‐sensitive K2P‐like K+‐current activated by docosahexaenoic acid (DHA) in freshly isolated endothelial cells as well as DHA‐induced membrane hyperpolarization. Myography on pulmonary arteries showed that DHA induced concentration‐dependent instantaneous relaxations that were resistant to endothelial removal and inhibition of NO and prostacyclin synthesis and to a cocktail of blockers of calcium‐activated K+ channels but were abolished by high extracellular (30 mM) K+‐concentration. Gene expression and protein of K2P2.1 were not altered in chronic hypoxic mice, while K2P6.1 was up‐regulated by fourfold. In conclusion, the PUFA‐activated K2P2.1 and K2P6.1 are expressed in murine lung and functional K2P‐like channels contribute to endothelium hyperpolarization and pulmonary artery relaxation. The increased K2P6.1‐gene expression may represent a novel counter‐regulatory mechanism in pulmonary hypertension and suggest that arterial K2P2.1 and K2P6.1 could be novel therapeutic targets.