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Age-related changes in the contribution of BKCa channels and NO to acetylcholine-induced dilation of pial arteries were studied in normotensive (Wistar–Kyoto) and spontaneously hypertensive rats (SHRs). Using intravital microphotography (×470), responses of pial arteries to acetylcholine chloride (ACh, 10−7 M, 5 min) with and without BKCa channel blockade by tetraethylammonium chloride (TEA, 2 mМ) and nitric oxide (NO) synthesis by L-NAME (10−3 M) were comparatively evaluated in WKY rats and SHRs aged 4 and 20 months. The evaluation criteria were the number and degree of arterial dilations arising in response to ACh after application of inhibitors, with the latter criterion being evaluated, in turn, by measuring the erythrocyte flow width in three individual groups of arteries: small (40 µm). It was established that in WKY rats aging leads to diminish the role of BKCa channels in ACh-induced dilation of all pial arteries, no matter the caliber. Similar processes were observed in the pial vascular bed of young SHRs. It appears that changes in the role of BKCa channels in vascular dilatatory responses in aging WKY rats and young SHRs are largely associated with the impairment of NO synthesis and changes in the role of NO-mediated mechanisms of vasodilation. Aging in SHRs is accompanied by increasing contribution of the NO-dependent mechanism to the regulation of ACh-induced dilatatory responses of small-caliber arteries.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science, Research and Arts Baden-Württemberg (MWK-BW Sonderlinie Medizin) Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence. One of the important indicators for AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to such changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in their expression and activity associated with AF. Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or with sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC-signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel and its pharmacology indicated presence of ‘big potassium channels’, BKCa. In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of stretch-induced BKCa current. No co-immunoprecipitation of Piezo1 and BKCa was detected. Human atrial fibroblasts express functional Piezo1 and BKCa channels. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data show the presence and activity of Piezo1 and BKCa in human atrial fibroblasts and suggest an interplay between the two in the absence of direct physical interactions.

Conventional treatments have failed to improve the prognosis of heart failure with preserved ejection fraction (HFpEF) patients. Thus, the purpose of this study was to determine the therapeutic efficacy of chronic interval exercise training (IT) on large-conductance Ca2+-activated K+ (BKCa) channel-mediated coronary vascular function in heart failure. We hypothesized that chronic interval exercise training would attenuate pressure overload-induced impairments to coronary BKCa channel-mediated function. A translational large-animal model with cardiac features of HFpEF was used to test this hypothesis. Specifically, male Yucatan miniswine were divided into three groups ( n = 7/group): control (CON), aortic banded (AB)-heart failure (HF), and AB-interval trained (HF-IT). Coronary blood flow, vascular conductance, and vasodilatory capacity were measured after administration of the BKCa channel agonist NS-1619 both in vivo and in vitro in the left anterior descending coronary artery and isolated coronary arterioles, respectively. Skeletal muscle citrate synthase activity was decreased and left ventricular brain natriuretic peptide levels increased in HF vs. CON and HF-IT animals. A parallel decrease in NS-1619-dependent coronary vasodilatory reserve in vivo and isolated coronary arteriole vasodilatory responsiveness in vitro were observed in HF animals compared with CON, which was prevented in the HF-IT group. Although exercise training prevented BKCa channel-mediated coronary vascular dysfunction, it did not change BKCa channel α-subunit mRNA, protein, or cellular location (i.e., membrane vs. cytoplasm). In conclusion, these results demonstrate the viability of chronic interval exercise training as a therapy for central and peripheral adaptations of experimental heart failure, including BKCa channel-mediated coronary vascular dysfunction. NEW & NOTEWORTHY Conventional treatments have failed to improve the prognosis of heart failure with preserved ejection fraction (HFpEF) patients. Our findings show that chronic interval exercise training can prevent BKCa channel-mediated coronary vascular dysfunction in a translational swine model of chronic pressure overload-induced heart failure with relevance to human HFpEF.

This study aimed to investigate the expression and function of BKCa channels in the Sphincter of Oddi (SO) in a rabbit model of hypercholesterolemia (HC). New Zealand white rabbits were randomly divided into 2 groups: the control group was fed standard chow (n = 18) whereas the high-cholesterol group was fed cholesterol-enriched chow containing 1.5% cholesterol (n = 18). The serum cholesterol level was significantly greater in the HC groups than in the control group, but there was no significant difference in body weight between the control and HC groups. Although the total protein expression of BKCa α- and β1-subunit was not significantly different between the control and HC groups, the Tyr-phosphorylation of BKCa α-subunit was significantly decreased in the HC group than in the control group. In addition, hypercholesterolemia significantly increased Acetylcholine (ACh)-induced contraction of the SO rings. Pretreatment with 30 μM NS1619, a BKCa channel agonist, significantly reduced ACh-induced c...

Aims Regulation of vascular tone by 3′,5′-cyclic adenosine monophosphate (cAMP) involves many effectors including the large conductance, Ca2+-activated, K+ (BKCa) channels. In arteries, cAMP is mainly hydrolyzed by type 3 and 4 phosphodiesterases (PDE3, PDE4). Here, we examined the specific contribution of BKCa channels to tone regulation by these PDEs in rat coronary arteries, and how this is altered in heart failure (HF). Methods and results Concomitant application of PDE3 (cilostamide) and PDE4 (Ro-20-1724) inhibitors increased BKCa unitary channel activity in isolated myocytes from rat coronary arteries. Myography was conducted in isolated, U46619-contracted coronary arteries. Cilostamide (Cil) or Ro-20-1724 induced a vasorelaxation that was greatly reduced by iberiotoxin (IBTX), a BKCa channel blocker. Ro-20-1724 and Cil potentiated the relaxation induced by the β-adrenergic agonist isoprenaline (ISO) or the adenylyl cyclase activator L-858051 (L85). IBTX abolished the effect of PDE inhibitors on ISO but did not on L85. In coronary arteries from rats with HF induced by aortic stenosis, contractility and response to acetylcholine were dramatically reduced compared with arteries from sham rats, but relaxation to PDE inhibitors was retained. Interestingly, however, IBTX had no effect on Ro-20-1724- and Cil-induced vasorelaxations in HF. Expression of the BKCa channel α-subunit, of a 98 kDa PDE3A and of a 80 kDa PDE4D were lower in HF compared with sham coronary arteries, while that of a 70 kDa PDE4B was increased. Proximity ligation assays demonstrated that PDE3 and PDE4 were localized in the vicinity of the channel. Conclusion BKCa channels mediate the relaxation of coronary artery induced by PDE3 and PDE4 inhibition. This is achieved by co-localization of both PDEs with BKCa channels, enabling tight control of cAMP available for channel opening. Contribution of the channel is prominent at rest and on β-adrenergic stimulation. This coupling is lost in HF.

Previous studies suggest that the large-conductance Ca2+-activated K+ (BKCa) channel and Rho-kinase play major roles in the control of urinary bladder tone. Here, we investigated their involvement in β-adrenoceptor (AR)-mediated relaxation of rat and human bladder. Concentration-response curves of isoprenaline and mirabegron-induced bladder relaxation were generated against passive tension and KCl- and carbachol-induced tone, in the absence or presence of the BKCa channel inhibitor iberiotoxin (100 nM) or the Rho-kinase inhibitor Y27,632 (1 μM). Myosin light chain (MLC) phosphorylation was studied by Western blot. In rat, iberiotoxin only slightly altered isoprenaline- and mirabegron-induced relaxation against KCl-induced tone but attenuated relaxation by both agonists against carbachol-induced tone. Y27,632 enhanced isoprenaline- or mirabegron-induced relaxation only against carbachol-induced tone. In humans, iberiotoxin slightly enhanced relaxation by both agonists against carbachol-induced pre-contraction. Y27,632 did not change isoprenaline-induced relaxation but enhanced that by mirabegron. Under passive tension, MLC phosphorylation was markedly reduced by both β-AR agonists, an effect insensitive to Y27,632. In the presence of carbachol, both β-AR agonists increased MLC phosphorylation, an effect reduced by Y27,632 only in the presence of 1 μM carbachol. These results indicate that the extent of BKCa channel and Rho-kinase involvement in relaxation induced by β-AR agonists depends on pre contractile stimulus and species. Electronic supplementary material The online version of this article (doi:10.1007/s00210-015-1128-z) contains supplementary material, which is available to authorized users.

Background Activation of large conductance calcium-activated potassium (BKCa) channels is cardioprotective for ischemic injury and can enhance vasorelaxation. Rottlerin has recently been identified as a potent BKCa activator. We demonstrated that rottlerin improves cardiac function and increases coronary flow when used as a cardioplegia additive in rat and mouse models of cardioplegic arrest and reperfusion. In this study we examined the effectiveness and specificity of the putative BKCa activator rottlerin on vascular reactivity in response to specific contractile and dilatory agonists. Methods Aortic rings from wild-type (wt) and BKCa knock-out (KO) mice were mounted in a tissue bath with force transducers. The vasodilatory effect of rottlerin was evaluated after pre-constriction with U46619. Dose responses to the contractile agonists U46619 and phenylephrine (PE), and vasodilation responses to rottlerin, hydrogen sulfide (H2S), and sodium nitroprusside (SNP) were performed after pretreatment with rottlerin. Similar studies were performed in pig coronary vessels. Results The BKCa KO mouse aortic rings exhibited spontaneous contraction and had greater contractile responses to U46619 and reduced vasodilation to SNP compared with wt mice. The wt and KO responses to phenylephrine were similar. Rottlerin dose dependently dilated wild-type vessels, but not in BKCa KO animals. Pretreatment with rottlerin caused depressed U46619 responses, but had no effect on PE, SNP, or H2S-mediated responses. However, pig coronary vessels pretreated with rottlerin exhibited reduced contractile responses and enhanced nitric oxide-dependent dilation. Conclusions Rottlerin directly causes vasodilation through BKCa channel dependent mechanisms. The BKCa channel activator pretreatment enhances vasodilatory responses and impairs specific vasoconstrictive agonists.

Formaldehyde (FA) has been linked to the detrimental cardiovascular effects. Here, we explored the effects and mechanisms of FA on rat aortas both in vivo and in vitro. The results presented that FA evidently lowered the blood pressures of rats. The expression levels of BKCa subunits α and β1 and iNOS of the aortas were up-regulated by FA in vivo. However, FA markedly reduced the levels of Cav1.2 and Cav1.3, which are the subunits of L-Ca2+ channel. Furthermore, the contents of NO, cGMP and iNOS in the aortas were augmented by FA. To further confirm these findings, the mechanisms accredited to these effects were examined in vitro. The data showed that FA contracted the isolated aortic rings at low concentrations ( 500 μM). The FA-induced vasoconstriction at low concentrations was blocked partly by an inhibitor of ACE. The relaxation caused by FA at high concentrations was attenuated by the inhibitors of NO-cGMP pathway, L-Ca2+ channel and BKCa channel, respectively. Similarly, the expression of iNOS was strongly enhanced by FA in vitro. The effects of FA on the aortic rings with endothelium were significantly greater than those on the rings without endothelium. Our results indicate that the vasoconstriction of FA at low concentrations might be partially pertinent to endothelin, and the FA-caused vasorelaxation at high concentrations is possibly associated with the NO-cGMP pathway, L-Ca2+ channel and BKCa channel. This study will improve our understanding of the pathogenic mechanisms for FA-related cardiovascular diseases.

s Background Cold is an important factor contributing to the high incidence of hypertension. Large conductance calcium-activated potassium (BKCa) channels modulate vascular smooth muscle tone. The objective of this study is to determine whether BKCa channels in vascular smooth muscle cells (VSMC) play a role in the development of hypertension during chronic exposure to cold. Methods Forty-eight rats were divided into cold-treated group and control group randomly, and then, rats of cold-treated group were exposed to 4±1 °C (4 h/day) and rats of another group were kept at 25±1 °C. Results Compared to control group, systolic blood pressure of the rats exposed to cold for 2, 4, 6, 8 weeks were increased by 7.14%, 20.42%, 40.54%, 28.74%, respectively. The BKCa current and BKCa peak current density in aortic VSMC increased significantly as the time of exposure to cold went until the 6th week exposure to cold. At the 6th weeks, the BKCa peak current density in the cold exposed group compared to the control group were increased 345.21% (n=6, P Conclusion Our data showed that BKCa channels have apparent change with the development of hypertension during repeated cold exposure, suggesting that BKCa channel could be thought as a new target for prevention and treating cold-induced hypertension.

Exposure to perinatal hypoxia results in alteration of the adult pulmonary circulation, which is linked among others to alterations in K(+) channels in pulmonary artery (PA) smooth muscle cells. In particular, large conductance Ca(2+)-activated K(+) (BK(Ca)) channels protein expression and activity were increased in adult PA from mice born in hypoxia compared with controls. We evaluated long-term effects of perinatal hypoxia on the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway-mediated activation of BK(Ca) channels, using isoproterenol, forskolin, and dibutyryl-cAMP. Whole-cell outward current was higher in pulmonary artery smooth muscle cells from mice born in hypoxia compared with controls. Spontaneous transient outward currents, representative of BK(Ca) activity, were present in a greater proportion in pulmonary artery smooth muscle cells of mice born in hypoxia than in controls. Agonists induced a greater relaxation in PA of mice born in hypoxia compared with controls, and BK(Ca) channels contributed more to the cAMP/PKA-mediated relaxation in case of perinatal hypoxia. In summary, perinatal hypoxia enhanced cAMP-mediated BK(Ca) channels activation in adult murine PA, suggesting that this pathway could be a potential target for modulating adult pulmonary vascular tone after perinatal hypoxia.

We have previously reported the electrophysiological properties of sarcolemmal stretch-activated BKCa (SAKCA) channels cloned from cultured chick embryonic ventricular myocytes. However, the role of BKCa channels in the electrophysiology of the more mature heart is not clear. We have investigated the effects on the BKCa current of axial stretch in post-hatch ventricular myocytes. Whole-cell currents of ventricular myocytes isolated from 2-week-old chicks were recorded using the patch-clamp technique, while the cells were either held at resting length or stretched to cause a 10% increase in sarcomere length using a pair of carbon fibres attached to opposite ends of the cell. Stretch did not affect whole-cell currents immediately after the stretch was applied. However, sustained stretch for 3 min significantly increased outward currents. This stretch-induced change was reversed by applying 10 nm iberiotoxin, a specific BKCa channel blocker, or a Na+–Ca2+-free environment. These results were reproduced in a computer simulation study. The present study is the first report about the sarcolemmal BKCa current from post-hatch ventricular myocytes. The present results suggest that axial stretch activates BKCa channels via a stretch-induced increase in the cytosolic Na+ concentration followed by an increased Ca2+ influx.

This investigation was designed to examine the hypothesis that impaired function of coronary microvascular large-conductance Ca2+-activated K+ (BKCa) channels in metabolic syndrome (MetS) significantly attenuates the balance between myocardial oxygen delivery and metabolism at rest and during exercise-induced increases in myocardial oxygen consumption (MV̇o2). Studies were conducted in conscious, chronically instrumented Ossabaw swine fed a normal maintenance diet (11% kcal from fat) or an excess calorie atherogenic diet (43% kcal from fat, 2% cholesterol, 20% kcal from fructose) that induces many common features of MetS. Data were collected under baseline/resting conditions and during graded treadmill exercise before and after selective blockade of BKCa channels with penitrem A (10 μg/kg iv). We found that the exercise-induced increases in blood pressure were significantly elevated in MetS swine. No differences in baseline cardiac function or heart rate were noted. Induction of MetS produced a parallel downward shift in the relationship between coronary venous Po2 and MV̇o2 ( P < 0.001) that was accompanied by a marked release of lactate (negative lactate uptake) as MV̇o2 was increased with exercise ( P < 0.005). Inhibition of BKCa channels with penitrem A did not significantly affect blood pressure, heart rate, or the relationship between coronary venous Po2 and MV̇o2 in lean or MetS swine. These data indicate that BKCa channels are not required for local metabolic control of coronary blood flow under physiological (lean) or pathophysiological (MetS) conditions. Therefore, diminished function of BKCa channels does not contribute to the impairment of myocardial oxygen-supply demand balance in MetS.

Large-conductance calcium and voltage-dependent potassium (BK(Ca)) channel is an important determinant of vascular tone. It is activated by hydrogen peroxide (H(2)O(2)) which occurs in various physiological and pathological processes. However, the regulation mechanism is not fully understood. In the present study, the mSlo in the presence or absence of hbeta1 were cotransfected with the PTEN(wt), PTEN(C124S), PTEN(G129E) in HEK 293 cells. Typical BK(Ca) channel currents could be recorded in cell-attached configurations. We found that PTEN(wt) reduced the H(2)O(2)-induced BK(Ca) channel activation during the initial 10 min treatment. In contrast, coexpression with catalytically inactive PTEN(C124S)/PTEN(G129E) mutants that lack lipid phosphatase activity produced no regulation on the H(2)O(2)-induced BK(Ca) channel activation. These results demonstrated that PTEN regulated the H(2)O(2)-induced BK(Ca) channel activation through phosphatidylinositol 3-phosphatse. However, the inhibitory effect of PTEN on the H(2)O(2)-induced BK(Ca) channel activation was attenuated when cells were treated with H(2)O(2) at concentrations higher than 100 microM or at 100 microM for long-term treatment. In addition, the p-AKT expression level in PTEN(wt) overexpressing cells was lower than that in control cells, and the increase of cytoplasmic free calcium concentration ([Ca(2+)](i)) induced by H(2)O(2) was also inhibited. These findings may elucidate a new mechanism for H(2)O(2)-induced BK(Ca) channel activation and provide some evidences for the role of PTEN on vasodilation induced by H(2)O(2).

The normal physiological contraction of the urinary bladder, which is required for voiding, is predominantly mediated by muscarinic receptors, primarily the M-3 subtype, with the M-2 subtype providing a secondary backup role. Bladder relaxation, which is required for urine storage, is mediated by beta-adrenoceptors, in most species involving a strong beta(3)-component. An excessive stimulation of contraction or a reduced relaxation of the detrusor smooth muscle during the storage phase of the micturition cycle may contribute to bladder dysfunction known as the overactive bladder. Therefore, interference with the signal transduction of these receptors may be a viable approach to develop drugs for the treatment of overactive bladder. The prototypical signaling pathway of M-3 receptors is activation of phospholipase C (PLC), and this pathway is also activated in the bladder. Nevertheless, PLC apparently contributes only in a very minor way to bladder contraction. Rather, muscarinic-receptor-mediated bladder contraction involves voltage-operated Ca2+ channels and Rho kinase. The prototypical signaling pathway of beta-adrenoceptors is an activation of adenylyl cyclase with the subsequent formation of cAMP. Nevertheless, cAMP apparently contributes in a minor way only to beta-adrenoceptor-mediated bladder relaxation. BKCa channels may play a greater role in beta-adrenoceptor-mediated bladder relaxation. We conclude that apart from muscarinic receptor antagonists and beta-adrenoceptor agonists, inhibitors of Rho kinase and activators of BKCa channels may have potential to treat an overactive bladder

Mitochondrial Ca2+-activated large-conductance K+ (BKCa) channels are thought to provide protection during ischemic insults in the heart. Rottlerin (mallotoxin) has been implicated as a potent BKCa activator. The purpose of this study was twofold: 1) to investigate the efficacy of BKCa channel activation as a cardioprotective strategy during ischemic cardioplegic arrest and reperfusion (CP/R) and 2) to assess the specificity of rottlerin for BKCa channels. Wild-type (WT) and BKCa knockout (KO) mice were subjected to an isolated heart model of ischemic CP/R. A mechanism of rottlerin-induced cardioprotection was also investigated using H9c2 cells subjected to in vitro CP/reoxygenation and assessed for mitochondrial membrane potential and reactive oxygen species (ROS) production. CP/R decreased left ventricular developed pressure, positive and negative first derivatives of left ventricular pressure, and coronary flow (CF) in WT mice. Rottlerin dose dependently increased the recovery of left ventricular function and CF to near baseline levels. BKCa KO hearts treated with or without 500 nM rottlerin were similar to WT CP hearts. H9c2 cells subjected to in vitro CP/R displayed reduced mitochondrial membrane potential and increased ROS generation, both of which were significantly normalized by rottlerin. We conclude that activation of BKCa channels rescues ischemic damage associated with CP/R, likely via effects on improved mitochondrial membrane potential and reduced ROS generation.

Bradykinin-induced activation of the pulmonary endothelium triggers a nitric oxide (NO)- signaling pathway. Pathway activation causes vasodilation and is critical to the fetal transition at birth, ...

The properties of K+ channels in these cells were studied using patch-clamp methods. Two channels, with conductances of 165+/-13 pS (n=6) and 30+/-1 pS (n=3), were identified in single-channel experiments. In cell-attached patches the reversal potentials were -67+/-8 and -74+/-2 mV for the large and small conductance channel, respectively, suggesting that both channels are K+-selective. The large conductance channel was also shown to be K+-selective in inside-out patches. The open probability (P(o)) of this channel was increased at depolarizing potentials and by increasing intracellular Ca2+ concentration ([Ca2+]i). These properties suggest that the large conductance channel is a 'maxi' Ca2+-activated K+ channel (BK(Ca)). The small conductance channel was not observed in inside-out patches. Carbachol (CCh; 10(-5) M) activated the BK(Ca) channel, but not the small conductance channel, in cell-attached patches. CCh also caused a dose-dependent increase in [Ca2+]i measured by fura-2 in microspectrofluorimetric studies, with a half-maximal response at approximately 3x10(-6) M. Neither isoproterenol (10(-5) M) nor substance P (10(-6) M) affected K+-channel activity or [Ca2+]i. In whole-cell experiments, CCh caused an increase in outward current. Charybdotoxin (10(-7) M), a BK(Ca) blocker, inhibited a large component of the CCh-induced current. A large component of the charybdotoxin-insensitive current may be carried by Ca2+-activated Cl- channels, which were also observed in human parotid acinar cells. The results indicate that BK(Ca) channels make a significant contribution to the whole-cell conductance in human parotid acinar cells.

Large-conductance Ca(2+)-activated K(+) (BKCa) channels provide a negative feedback that regulates vascular tone in the brain circulation. This study investigated the effects of aerobic exercise on gating properties of BKCa channels in rat cerebral artery.Rats were subjected to moderate-intensity exercise at low (EX-3d/w) and high (EX-5d/w) training volume on a motor-driven treadmill, and compared with age-matched sedentary animals (SED). Inside-out (I/O) patch clamp recording was performed to measure gating properties of the BKCa channel.Aerobic exercise induced a reduction in heart rate and body weight in both training groups. Exercise increased the channel activity, which was more pronounced in EX-5d/w than that in EX-3d/w group. Kinetic analysis revealed that (1) the contribution of short open states was elevated and the duration of both short and long open states were extended by exercising in EX-3d/w; (2) Ex-3d/w had no significant change on conformation of close states; (3) EX-3d/w increased the mean open time without changing mean closed time; (4) EX-5d/w increased both the contribution and duration of long open states; (5) EX-5d/w increased channel mean open time while decreased mean closed time.The results suggest that regular aerobic exercise may enhance BKCa channel activity in cerebral arterial myocytes by changing its biophysical properties, and the electrical remolding induced by exercise may be training volume-dependent.

Uterine myometrial cells are maintained in a quiescent state during pregnancy, in part due to hyperpolarization of the membrane by the large-conductance Ca2+-activated K+ (BKCa) channel. During labor, uterine contractility is increased and BKCa current is suppressed, however, expression of BKCa is increased, suggesting that multiple mechanisms regulate BKCa channel function. We have focused on the roles of an auxiliary β1-subunit and two N-terminal α-subunit BKCa variants, whose peptide sequences start at different methionines, MANG and MDAL. We found that whereas MDAL expression was equivalent in all samples, MANG was expressed at a higher level in myometrium from term laboring women than from term non-laboring or non-pregnant women. To examine the possibility that differential expression of these variants reduces BKCa channel activity at the onset of labor, we expressed the MANG and MDAL variants, with or without the β1-subunit, in a heterologous system and recorded currents using patch-clamp. Although whole-cell voltage-gated currents were similar between variants, the β1-subunit enhanced Ca2+ activation of MDAL but not MANG. Inside-out single-channel recordings confirmed that MANG currents were not modulated by β1. Our finding that β1 co-immunoprecipitated with both MDAL and MANG variants suggests that the longer N-terminus of MANG does not affect the interaction between BKCa α- and β1-subunits, but instead inhibits the functional modulation of the channel by β1. These observations suggest that BKCa variants are differentially regulated by auxiliary subunits to regulate myometrial contractility during pregnancy and labor.

Excess activation and expression of large-conductance Ca(2+)-activated K(+) channels (BKCa channels) may be an important mechanism for delayed neuronal death after cerebral ischemia/reperfusion injury. Electroacupuncture can regulate BKCa channels after cerebral ischemia/reperfusion injury, but the precise mechanism remains unclear. In this study, we established a rat model of cerebral ischemia/reperfusion injury. Model rats received electroacupuncture of 1 mA and 2 Hz at Shuigou (GV26) for 10 minutes, once every 12 hours for a total of six times in 72 hours. We found that in cerebral ischemia/reperfusion injury rats, ischemic changes in the cerebral cortex were mitigated after electroacupuncture. Moreover, BKCa channel protein and mRNA expression were reduced in the cerebral cortex and neurological function noticeably improved. These changes did not occur after electroacupuncture at a non-acupoint (5 mm lateral to the left side of Shuigou). Thus, our findings indicate that electroacupuncture at Shuigou improves neurological function in rats following cerebral ischemia/reperfusion injury, and may be associated with down-regulation of BKCa channel protein and mRNA expression. Additionally, our results suggest that the Shuigou acupoint has functional specificity.

The effect of tetraethylammonium in a dose of 1 mM (inhibiting functional activity and preventing opening of BKCa channels) was least pronounced during restraint stress. The influence of this agent was more significant in animals exposed to 14-day hyperglycemia alone or in combination with restraint stress. Therefore, hyperglycemia and stress (individual or combined exposure) significantly inhibit functional activity of BKCa channels in smooth muscles of the coronary vessels. Our results suggest that the development of hyperglycemia is realized via pathogenetic mechanisms of vascular injury in the heart (similarly to stress conditions). Permanent increase in blood glucose level and 6-h immobilization probably induces nonspecific post-stress abnormalities in channel function.

We recently described that carbon monoxide (CO) participated in the regulation of rhythmic respiration in medullary slices. The present study was undertaken to further assess whether the large-conductance calcium-activated potassium channels (BK(Ca) channels) are involved in the CO-mediated central regulation of respiratory rhythm in medullary slices. The rhythmic discharge of hypoglossal rootlets of medullary slices of neonatal rats was recorded. We observed that blocking BK(Ca) channels could partially abolish the effects of CO on the rhythmic bursts of hypoglossal rootlets. With whole-cell patch-clamp recording technique, we further observed that CO could reversibly augment potassium current density of the neurons in the rostral ventrolateral medulla. The CO-induced increase in potassium current was entirely blocked by the pretreatment of slices with BK(Ca) channels blocker; whereas blockade of CO generation with zinc protoporphyrin-IX produced an opposite response. Altogether, these data indicate that BK(Ca) channels of the neurons in neonatal rostral ventrolateral medulla could be activated by CO and involved in CO-mediated central regulation of respiratory rhythm in medullary slices.

Exposure to chronic hypoxia (CH) results in blunted myogenic and agonist-induced tone in systemic arteries that persists upon return to normoxia. This impaired vasoconstrictor reactivity is reverse...

Context: β3-Adrenoreceptor modulation in human myometrium during pregnancy is linked functionally to myometrial inhibition. Maxi-K+ channels (BKCa) play a significant role in modulating cell membrane potential and excitability. Objective: This study was designed to investigate the potential involvement of BKCa channel function in the response of human myometrium to β3-adrenoceptor activation. Design: Single and whole-cell electrophysiological BKCa channel recordings from freshly dispersed myocytes were obtained in the presence and absence of BRL37344, a specific β3-adrenoreceptor agonist. The in vitro effects of BRL37344 on isolated myometrial contractions, in the presence and absence of the specific BKCa channel blocker, iberiotoxin (IbTX), were investigated. Setting: The study was carried out at the Clinical Science Institute. Patients or Other Participants: Myometrial biopsies were obtained at elective cesarean delivery. Intervention: No intervention was applied. Main Outcome Measures: Open state probability of single channel recordings, whole cell currents, and myometrial contractile activity were measured. Results: Single-channel recordings identified the BKCa channel as a target of BRL37344. BRL37344 significantly increased the open state probability of this channel in a concentration-dependent manner (control 0.031 ± 0.004; 50 μm BRL37344 0.073 ± 0.005 (P < 0.001); and 100 μm BRL37344 0.101 ± 0.005 (P < 0.001). This effect was completely blocked after preincubation of the cells with 1 μm bupranolol, a nonspecific β-adrenoreceptor blocker, or 100 nm SR59230a, a specific β3-adrenoreceptor antagonist. In addition, BRL37344 increased whole-cell currents over a range of membrane potentials, and this effect was reversed by 100 nm IbTX. In vitro isometric tension studies demonstrated that BRL37344 exerted a significant concentration-dependent relaxant effect on human myometrial tissue (P < 0.05), and preincubation of these strips with IbTX attenuated this effect on both spontaneous and oxytocin-induced contractions (44.44 and 57.84% at 10−5m, respectively). Conclusions: These findings outline that activation of the BKCa channel may explain the potent uterorelaxant effect of β3-adrenoreceptor agonists.

Objectives Cold is an important factor contributing to the high incidence of hypertension. Large conductance calcium-activated potassium (BK Ca ) channels modulate vascular smooth muscle tone. The objective of this study is to determine whether BK Ca channels in vascular smooth muscle cells (VSMC) play a role in the development of hypertension during chronic exposure to cold. Methods Forty-eight rats were divided into cold-treated group and control group randomly, and then, rats of cold-treated group were exposed to 4 ± 1° (4 h/day) and rats of another group were kept at 25 ± 1°. Results Compared to control group, systolic blood pressure of the rats exposed to cold for 2, 4, 6, 8 weeks were increased by 7.14%, 20.42%, 40.54%, 28.74%, respectively. The BK Ca current and BK Ca peak current density in aortic VSMC increased significantly as the time of exposure to cold went until the 6th week exposure to cold. At the 6 th weeks, the BK Ca peak current density in the cold exposed group compared to the control group were increased 345.21% (n = 6, P Ca channel α-subunit mRNA in the cold-treated rats was higher than control (P Conclusions Our data showed that BK Ca channels have apparent change with the development of hypertension during repeated cold exposure, suggesting that BK Ca channel could be thought as a new target for prevention and treating cold-induced hypertension.

Objectives Cold is an important factor contributing to the high incidence of hypertension. In order to explore the mechanisms of cold-induced hypertension. This study observed whether Large conductance calcium-activated potassium channel (BK Ca ) on vascular smooth muscle cells (VSMC) played a role in the development of hypertension during chronic exposure to cold. Methods Forty-eight rats were divided into cold-treated group and control group randomly, then, one subgroup of the cold-treated and one subgroup of control rats were sacrificed at 2, 4, 6 and 8 weeks. Results Systolic blood pressure started to rise after 2 weeks of cold exposure and continued to increase, reaching a maximal level by the 6th week of exposure to cold, and then pressure begin to decrease slowly throughout the remainder of the experiment. The BK Ca current and BK Ca peak current density in aortic VSMC increased significantly as the time of exposure to cold went until the 6th week exposure to cold. Furthermore, the expression of L-type Cav1.2 channelα 1 C subunit mRNA in the cold-treated rats were higher than those in the control rats. Conclusions Cold stress increased expression of L-type Cav1.2 channelα 1 C subunit mRNA, indicate that cytoplasmic Ca 2+ concentration increased with cold-treated extended at 6 weeks in the beginning, resulting in increasing blood pressure. BK Ca channel currents increased in this progress as the negative-feedback regulators of vascular tone. Our results indicated that activation of BK Ca channel could reduce blood pressure (7th and 8th week) in hypertensive subjects.

Inthelastdecadesseveralinvestigatorshavesuggestedtheassociationofandrogenswithhypertension. Recently, some studies have shown that the incidence of cardiovascular diseases is increased in men with low testosterone levels, suggesting a protective role of androgens. Hypertension is one of the mostcommonproblemsinpregnancythatcomplicates5-10 %ofpregnancies.Anincreaseofmorbidity wasobservedinbabiesfrompregnantwomenwithhypertension.However,thepathogenesisremains unclearandthislimitstheabilitytopreventandtreatthispathology.Thebeneficialeffectsofandrogens for vascular system are associated with their ability to cause vasorelaxation. Inhuman vessels, this non genomiceffectofandrogensappeartobeduetoactivationoflargeconductancecalcium-activatedpotassiumchannels(BKCa)andvoltagegatedpotassiumchannels(KV)whichisinducedbycGMPincrease andaconsequentactivationofcGMP-dependentproteinkinase(PKG).Ontheotherhand,thegenomic effects of androgens concerning ionic channels are almost unknown. Our previous studies suggested that androgens increase the expression of BKCa channels and decrease expression of L-type calcium channels (LTCC). The aim of this work was to analyze the genomic effects of androgens on theexpressionofotherproteinsinvolvedintheregulationofvascularcontractility,suchassolubleguanylate cyclase (sGC), the natriuretic peptide receptor-A (NPRA) and PKG. This study also aimed to compare the expression levels of these proteins in human umbilical artery (HUA) from normotensive andhypertensivepregnantwomen.ToachievethesegoalsrealtimePCRwasperformedusingsmooth

Background It has been hypothesised that down-regulation of the potassium BK Ca channel function at term is involved in myometrial activation at labour onset. However, there are conflicting reports on the contribution of this channel to uterine contractility. Our previous data suggests that BK channels do not modulate spontaneous contractility of human and rat myometrium in vitro. The BK Ca activator NS1619, which can relax myometrial tissue, has been reported to mediate effects via ERG channels in other tissues. Our aim was to examine the effect of a more specific BK Ca channel activator NS11021 versus NS1619 in non-pregnant and late pregnant mouse myometrium. Methods Longitudinal myometrial strips obtained from non-pregnant (oestrous) and late pregnant (day 17–18) C57/BL6 mice were used for isometric tension recording; activity measured as mean integral tension. NS1619 (n=6–9), NS11021 (n=6–7) and its inactive analogue NS13558 (n=4–6) versus vehicle controls (n=3–12). Results NS11021 (10 M) and NS13558 (10 M) had no effect on spontaneous myometrial contractions. NS1619 (10 M) had a small but non-significant effect on myometrial contractility from non-pregnant mice, but at 30 M concentrations induced a significant decrease in spontaneous contractility compared to vehicle control (260.5% vs 137.3%, p Conclusions NS11021 has no effect on spontaneous myometrial contractions from non-pregnant or late pregnant mice. The effect of NS1619, seen in this and previous studies, are more likely to have been mediated by an action of NS1619 on ERG channels.

Lysophospholipids are known to serve as intra- and extracellular messengers affecting many physiological processes. Lysophosphatidylinositol (LPI), which is produced in endothelial cells, acts as an endogenous agonist of the orphan receptor, G protein-coupled receptor 55 (GPR55). Stimulation of GPR55 by LPI evokes an intracellular Ca(2+) rise in several cell types including endothelial cells. In this study, we investigated additional direct, receptor-independent effects of LPI on endothelial large-conductance Ca(2+) and voltage-gated potassium (BK(Ca)) channels. Electrophysiological experiments in the inside-out configuration revealed that LPI directly affects the BK(Ca) channel gating properties. This effect of LPI strictly depended on the presence of Ca(2+) and was concentration-dependent, reversible, and dual in nature. The modulating effects of LPI on endothelial BK(Ca) channels correlated with their initial open probability (Po): stimulation at low Po (0.3) and inhibition at high Po levels (0.3). In the whole-cell configuration, LPI in the pipette facilitated membrane hyperpolarization in response to low (0.1-2 μM) histamine concentrations. In contrast, LPI counteracted membrane hyperpolarization in response to supramaximal cell stimulation with histamine. These results highlight a novel receptor-independent and direct bidirectional modulation of BK(Ca) channels by LPI on endothelial cells. We conclude that LPI via this mechanism serves as an important modulator of endothelial electrical responses to cell stimulation.

Matthias E. Werner is with the Division of Cardiovascular and Endocrine Science, School of Medicine, University of Manchester, Manchester M13 9NT, UK -- Andrea L. Meredith is with the Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD 21201, USA -- Richard W. Aldrich is with the Section of Neurobiology, 1 University Station C7000, The University of Texas at Austin, Austin, TX 78712, USA -- Mark T. Nelson is with the Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405, USA

We have previously reported the electrophysiological properties of stretch-activated BKCa (SAKCA) channels cloned from cultured chick embryonic ventricular myocytes. However, the physiological role of SAKCA channels in the post-hatch heart in situ is not clear. We have investigated the effects on the SAKCA current of cell length changes, applied axially using a pair of carbon fibers attached to opposite ends of an isolated ventricular myocyte of a 2 week-old chick. Whole-cell currents were recorded using the patch-clamp technique, while the cells were either held at resting length, or stretched to cause a 10% increase in sarcomere length. Stretch did not affect whole-cell currents immediately after the stretch was applied. However, sustained stretch for 3 minutes significantly increased outward currents. This stretch-induced change was reversed by applying 10 nM Iberiotoxin, a specific BKCa channel blocker, or a Na+/Ca2+ free environment. These results were reproduced in a computer simulation study, suggesting that stretch does not activate SAKCA channels directly, but does so in a secondary manner via a stretch-induced increase in the cytosolic Na+ concentration followed by an increased Ca2+ influx.