Correction to: Abnormal Membrane Localization of α2 Isoform of Na,K-ATPase in m. soleus of Dysferlin-Deficient Mice.

The third author's name should read: V. V. Matchkov.

Rebaudioside A directly stimulates insulin secretion from pancreatic beta cells: a glucose-dependent action via inhibition of ATP-sensitive K-channels.

Recently, we showed that rebaudioside A potently stimulates the insulin secretion from isolated mouse islets in a dose-, glucose- and Ca(2+)-dependent manner. Little is known about the mechanisms underlying the insulinotropic action of rebaudioside A. The aim of this study was to define the signalling system by which, rebaudioside A acts. Isolated mouse islets were used in the cAMP[(125)I] scintillation proximity assay to measure total cAMP level, and in a luminometric method to measure intracellular ATP and ADP concentrations. Conventional and permeabilized whole-cell configuration of the patch-clamp technique was used to verify the effect of rebaudioside A on ATP-sensitive K(+)-channels from dispersed single beta cells from isolated mouse islets. Insulin was measured by radioimmunoassay from insulinoma MIN6 cells. In the presence of 16.7 mM glucose, the addition of the maximally effective concentration of rebaudioside A (10(-9) M) increased the ATP/ADP ratio significantly, while it did not change the intracellular cAMP level. Rebaudioside A (10(-9) M) and stevioside (10(-6) M) reduced the ATP-sensitive potassium channel (K(ATP)) conductance in a glucose-dependent manner. Moreover, rebaudioside A stimulated the insulin secretion from MIN6 cells in a dose- and glucose-dependent manner. In conclusion, the insulinotropic effect of rebaudioside A is mediated via inhibition of ATP-sensitive K(+)-channels and requires the presence of high glucose. The inhibition of ATP-sensitive K(+)-channels is probably induced by changes in the ATP/ADP ratio. The results indicate that rebaudioside A may offer a distinct therapeutic advantage over sulphonylureas because of less risk of causing hypoglycaemia.

The α2 isoform Na,K-ATPase modulates contraction of rat mesenteric small artery via cSrc-dependent Ca2+ sensitization.

AIMS: The Na,K-ATPase is involved in a large number of regulatory activities including cSrc-dependent signalling. Upon inhibition of the Na,K-ATPase with ouabain, cSrc activation is shown to occur in many cell types. This study tests the hypothesis that acute potentiation of agonist-induced contraction by ouabain is mediated through Na,K-ATPase-cSrc signalling-dependent sensitization of vascular smooth muscle cells to Ca2+ . METHODS: Agonist-induced rat mesenteric small artery contraction was examined in vitro under isometric conditions and in vivo in anaesthetized rats. Arterial wall tension and [Ca2+ ]i in vascular smooth muscle cells were measured simultaneously. Changes in cSrc and myosin phosphatase targeting protein 1 (MYPT1) phosphorylation were analysed by Western blot. Protein expression was examined with immunohistochemistry. The α1 and α2 isoforms of the Na,K-ATPase were transiently downregulated by siRNA transfection in vivo. RESULTS: Ten micromolar ouabain, but not digoxin, potentiated contraction to noradrenaline. This effect was not endothelium-dependent. Ouabain sensitized smooth muscle cells to Ca2+ , and this was associated with increased phosphorylation of cSrc and MYPT1. Inhibition of tyrosine kinase by genistein, PP2 or pNaKtide abolished the potentiating effect of ouabain on arterial contraction and Ca2+ sensitization. Downregulation of the Na,K-ATPase α2 isoform made arterial contraction insensitive to ouabain and tyrosine kinase inhibition. CONCLUSION: Data suggest that micromolar ouabain potentiates agonist-induced contraction of rat mesenteric small artery via Na,K-ATPase-dependent cSrc activation, which increases Ca2+ sensitization of vascular smooth muscle cells by MYPT1 phosphorylation. This mechanism may be critical for acute control of vascular tone.

KV 7 channels are involved in hypoxia-induced vasodilatation of porcine coronary arteries.

BACKGROUND AND PURPOSE: Hypoxia causes vasodilatation of coronary arteries, but the underlying mechanisms are poorly understood. We hypothesized that hypoxia reduces intracellular Ca(2+) concentration ([Ca(2+)](i)) by opening of K channels and release of H2S. EXPERIMENTAL APPROACH: Porcine coronary arteries without endothelium were mounted for measurement of isometric tension and [Ca(2+)](i), and the expression of voltage-gated K channels K(V)7 channels (encoded by KCNQ genes) and large-conductance calcium-activated K channels (K(Ca)1.1) was examined. Voltage clamp assessed the role of K(V)7 channels in hypoxia. KEY RESULTS: Gradual reduction of oxygen concentration from 95 to 1% dilated the precontracted coronary arteries and this was associated with reduced [Ca(2+)](i) in PGF(2α) (10 µM)-contracted arteries whereas no fall in [Ca(2+)](i) was observed in 30 mM K-contracted arteries. Blockers of ATP-sensitive voltage-gated potassium channels and K(Ca)1.1 inhibited hypoxia-induced dilatation in PGF2α -contracted arteries; this inhibition was more marked in the presence of the K(v)7 channel blockers, XE991 and linopirdine, while a K(V)7.1 blocker, failed to change hypoxic vasodilatation. XE991 also inhibited H2S- and adenosine-induced vasodilatation. PCR revealed the expression of K(V)7.1, K(V)7.4, K(V)7.5 and K(Ca)1.1 channels, and K(Ca)1.1, K(V)7.4 and K(V)7.5 were also identified by immunoblotting. Voltage clamp studies showed the XE991-sensitive current was more marked in hypoxic conditions. CONCLUSION: The K(V)7.4 and K(V)7.5 channels, which we identified in the coronary arteries, appear to have a major role in hypoxia-induced vasodilatation. The voltage clamp results further support the involvement of K(V)7 channels in this vasodilatation. Activation of these K(V)7 channels may be induced by H2S and adenosine.

NS11021, a novel opener of large-conductance Ca(2+)-activated K(+) channels, enhances erectile responses in rats.

BACKGROUND AND PURPOSE: Large-conductance Ca(2+)-activated K(+) channels (BK(Ca)), located on the arterial and corporal smooth muscle, are potential targets for treatment of erectile dysfunction (ED). This study investigated whether NS11021 (1-(3,5-Bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea), a novel opener of BK(Ca) channels, relaxes erectile tissue in vitro and enhances erectile responses in intact rats. The effects were compared with sildenafil, an inhibitor of phosphodiesterase type 5. EXPERIMENTAL APPROACH: Patch clamp was used to record whole cell current in rat isolated corpus cavernosum smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs). Isometric tension was measured in intracavernous arterial rings and corpus cavernosum strips isolated from rats and men, and simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension were performed in intracavernous arteries. Erectile response was measured in anaesthetized rats. KEY RESULTS: In patch clamp recordings, NS11021 increased currents sensitive to the selective BK(Ca) channel blocker, iberiotoxin (IbTX) in SMCs, but did not modulate K(+) current in HUVECs. NS11021 reduced [Ca(2+)](i) and tension in penile arteries. IbTX inhibited the vasorelaxation induced by NS11021 and sildenafil in human erectile tissue. NS11021 and sildenafil but not vehicle increased erectile responses in anaesthetized rats, an effect which was abolished after pretreatment with tetraethylammonium. CONCLUSIONS AND IMPLICATIONS: NS11021 leads to relaxation of both intracavernous arteries and corpus cavernosum strips primarily through opening of BK(Ca) channels. It is also effective in facilitating erectile responses in anaesthetized rats. These results suggest a potential for use of BK(Ca) openers in the treatment of ED.

Vasomotion has chloride-dependency in rat mesenteric small arteries.

The possibility that Ca(2+)-activated Cl(-) conductances (CaCCs) contribute to oscillations in vascular tone (vasomotion) is tested in isolated mesenteric small arteries from rats where cGMP independent (I (Cl(Ca))) and cGMP-dependent (I (Cl(Ca,cGMP))) chloride conductances are important. The effect of anion substitution and Cl(-) channel blockers on noradrenaline (NA)-stimulated tension in isometrically mounted mesenteric arteries and for chloride conductance of smooth muscle cells isolated from these arteries were assessed electrophysiologically. Cl(-) (o) replacement with aspartate blocked vasomotion while 36mM SCN(-) (o) (substituted for Cl(-)) was sufficient to inhibit vasomotion. Oscillations in tone, membrane potential, and [Ca(2+)](i) disappeared with 36mM SCN(-). DIDS and Zn(2+) blocked I (Cl(Ca,cGMP)) but not I (Cl(Ca)). Vasomotion was not sensitive to DIDS and Zn(2+), and DIDS and Zn(2+) induce vasomotion in arteries without endothelium. The vasomotion in the presence of DIDS and Zn(2+) was sensitive to 36mM SCN(-) (o). The anion substitution data indicate that Cl(-) is crucial for the V (m) and [Ca(2+)](i) oscillations underlying vasomotion. The Cl(-) channel blocker data are consistent with both CaCCs being important.

An experimental study and mathematical simulation of adrenergic control of hindlimb vessels in rats after 3-week tail suspension.

Adrenoreactivity of rat hindlimb vessels was studied in experiments with constant-pressure saline perfusion. An original mathematical model was applied to evaluate the mechanism of changes in vascular tone regulation. A 3-week suspension resulted in decreased responses to sympathetic nerve stimulation, as well as to exogenous noradrenaline, the latter effect being registered when the pressure level was close to normal. Mathematical simulation indicated that long-term suspension induces both structural and functional changes in the vascular bed of the hind limbs, one of which is a disorder in the myogenic mechanisms of vascular tone regulation. In suspended rats, suppression of the myogenic response can be one of the reasons for decreased vessel reactivity to constrictor stimuli and, consequently, for disturbances in blood flow regulation in skeletal muscles of the hind limbs.