IKCa Channels Research Articles

N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine induces endothelium-dependent hyperpolarization-mediated vasorelaxation via store-operated calcium entry mechanism in healthy and intestinal inflammatory mice

Although the store-operated Ca2+ entry (SOCE) plays a critical role in maintaining Ca2+ homeostasis in vascular endothelial cells (VECs), its role in regulating endothelium-dependent hyperpolarization (EDH)-mediated vasorelaxation is largely unknown. Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) are the most common gastrointestinal disorders with no effective cures. The present study applied N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) as a Ca2+ chelator in the endoplasmic reticulum (ER) to study the SOCE/EDH-mediated vasorelaxation of micro-arteries and their involvements in the pathogenesis of IBD and IBS. Human submucosal arterioles and the second-order branch of 6–8 weeks male C57BL/6 mouse mesenteric arterioles were used, and TPEN-induced vasorelaxation was recorded by Danish DMT520A microvascular measuring system. The mice were fed water with 2.5 % dextran sulfate sodium for 7 days to induce mouse model of ulcerative colitis, and water avoidance stress was used to induce mouse model of IBS. The statistical significance of differences in the means of experimental groups was determined using a t-test for two groups or one-way ANOVA for more than two groups. TPEN concentration-dependently induced vasorelaxation of human colonic submucosal arterioles and the second-order branch of murine mesenteric arteries in endothelium-dependent manner. TPEN-induced vasorelaxation was much greater in the arteries pre-constricted by noradrenaline than those by high K+. While TPEN-induced vasorelaxation was unaffected by inhibitors of NO and PGI2, it was significantly inhibited by the selective inhibitors of IKCa and SKCa channels but was potentiated by their activator. Moreover, TPEN-induced vasorelaxation was attenuated by selective inhibitors of NCX, NKA, SOCE, STIM translocation and Orai transportation. Finally, TPEN-induced vasorelaxation via SOCE/EDH was impaired in colitic mice but remained intact in IBS mice. Interestingly, TPEN could rescue vagus neurotransmitter ACh-induced vasorelaxation that was impaired in IBS mice. Therefore, since TPEN-induced SOCE/EDH-mediated vasorelaxation of mesenteric arteries is well-preserved to be able to rescue ACh-induced vasorelaxation impaired in IBS, TPEN has therapeutic potentials for IBS.

The Roles of Potassium Channels and Nitric oxide in the Modulation of Apelin induced-relaxation in Isolated Diabetic Rat Aorta

Diabetes is associated with endothelial dysfunction, which impairs blood vesselscapacity to maintain vascular tone. Apelin is an adipocyte-produced relaxing factor that has endothelium-dependent and nitric oxide (NO)-mediated vasorelaxant effects. The current study investigated how streptozotocin (STZ)-induced type one diabetes modulates the mechanisms involved in aortic vascular response to apelin, focusing on the role of potassium channels and endothelial derived relaxing factors (EDRF). In this study, precontracted rat thoracic aortic segments were pre-incubated with the NO inhibitor, cyclooxygenase (COX) inhibitor and potassium channels blockers including: non-selective calcium-activated potassium channel, big conductance calcium-activated potassium channels (BKca), intermediate conductance calcium activated potassium channels (IKca), delayed inward rectifier potassium channels (Kir), adenosine triphosphates-sensitive potassium channels (KATP) and voltage sensitive potassium channels (Kv) blockers, then cumulative concentrations of apelin were applied to each group in both non-diabetic and diabetic conditions. The statistical analysis between diabetic and non-diabetic groups revealed that endothelial impairment induced by diabetes in rat thoracic aorta remarkably attenuated the vascular responses to apelin. An important new finding in this study was that almost all potassium channels blockers noticeably P<0.001 increased apelin efficacy with relatively no changes in the peptide potency. However, in diabetic aortic segments, the non-selective Kca, BKca blockers, NO inhibitor and COX inhibitor reversed vascular responses to apelin, but Kir, KATP and Kv blockers significantly reduced vascular responses to apelin in comparison to control rats. It is worth noting that diabetes did not only alter the peptide potency in these experimental groups but also significantly increased the maximum responses when Kca and BKca blockers preincubated. In conclusion, our findings shed light on the mechanisms behind diabetes-induced aortic artery hypo-reactivity to apelin which involve the inhibition of endothelial NO synthase activities and decreased contribution of Kca, BKca, IKca, Kv, Kir and KATP channels.

Nitric oxide, endothelium-derived hyperpolarizing factor, and smooth muscle-dependent mechanisms contribute to magnesium-dependent vascular relaxation in mouse arteries.

Magnesium (Mg2+ ) is a vasorelaxant. The underlying physiological mechanisms driving this vasorelaxation remain unclear. Studies were designed to test the hypothesis that multiple signaling pathways including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in endothelial cells as well as Ca2+ antagonization and TRPM7 channels in vascular smooth muscle cells mediate Mg2+ -dependent vessel relaxation. To uncover these mechanisms, force development was measured exvivo in aorta rings from mice using isometric wire myography. Concentration responses to Mg2+ were studied in intact and endothelium-denuded aortas. Key findings were confirmed in second-order mesenteric resistance arteries perfused exvivo using pressure myography. Effects of Mg2+ on NO formation were measured in Chinese Hamster Ovary (CHO) cells, isolated mesenteric vessels, and mouse urine. Mg2+ caused a significant concentration-dependent relaxation of aorta rings. This relaxation was attenuated significantly in endothelium-denuded aortas. The endothelium-dependent portion was inhibited by NO and cGMP blockade but not by cyclooxygenase inhibition. Mg2+ stimulated local NO formation in CHO cells and isolated mesenteric vessels without changing urinary NOx levels. High extracellular Mg2+ augmented acetylcholine-induced relaxation. SKCa and IKCa channel blockers apamin and TRAM34 inhibited Mg2+ -dependent relaxation. The endothelium-independent relaxation in aorta rings was inhibited by high extracellular Ca2+ . Combined blockade of NO, SKCa , and IKCa channels significantly reduced Mg2+ -dependent dilatation in mesenteric resistance vessels. In mouse conductance and resistance arteries Mg2+ -induced relaxation is contributed by endothelial NO formation, EDHF pathways, antagonism of Ca2+ in smooth muscle cells, and additional unidentified mechanisms.

M2 muscarinic receptors negatively modulate cell migration in human glioblastoma cells

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.

A single coiled-coil domain mutation in hIKCa channel subunits disrupts preferential formation of heteromeric hSK1:hIKCa channels.

The expression of IKCa (SK4) channel subunits overlaps with that of SK channel subunits, and it has been proposed that the two related subunits prefer to co-assemble to form heteromeric hSK1:hIKCa channels. This implicates hSK1:hIKCa heteromers in physiological roles that might have been attributed to activation of SK channels. We have used a mutation approach to confirm formation of heterometric hSK1:hIKCa channels. Introduction of residues within hSK1 that were predicted to impart sensitivity to the hIKCa current blocker TRAM-34 changed the pharmacology of functional heteromers. Heteromeric channels formed between wildtype hIKCa and mutant hSK1 subunits displayed a significantly higher sensitivity and maximum block to addition of TRAM-34 than heteromers formed between wildtype subunits. Heteromer formation was disrupted by a single point mutation within one COOH-terminal coiled-coil domain of the hIKCa channel subunit. This mutation only disrupted the formation of hSK1:hIKCa heteromeric channels, without affecting the formation of homomeric hIKCa channels. Finally, the Ca2+ gating sensitivity of heteromeric hSK1:hIKCa channels was found to be significantly lower than the Ca2+ gating sensitivity of homomeric hIKCa channels. These data confirmed the preferred formation of heteromeric channels that results from COOH-terminal interactions between subunits. The distinct sensitivity of the heteromer to activation by Ca2+ suggests that heteromeric channels fulfil a distinct function within those neurons that express both subunits.

Ca2+ -activated K+ channels regulate cell volume in human glioblastoma cells.

Glioblastoma (GBM), the most lethal form of brain tumors, bases its malignancy on the strong ability of its cells to migrate and invade the narrow spaces of healthy brain parenchyma. Cell migration and invasion are both critically dependent on changes in cell volume and shape driven by the transmembrane transport of osmotically important ions such as K+ and Cl- . However, while the Cl- channels participating in cell volume regulation have been clearly identified, the precise nature of the K+ channels involved is still uncertain. Using a combination of electrophysiological and imaging approaches in GBM U87-MG cells, we found that hypotonic-induced cell swelling triggered the opening of Ca2+ -activated K+ (KCa ) channels of large and intermediate conductance (BKCa and IKCa , respectively), both highly expressed in GBM cells. The influx of Ca2+ mediated by the hypotonic-induced activation of mechanosensitive channels was found to be a key step for opening both the BKCa and the IKCa channels. Finally, the activation of both KCa channels mediated by mechanosensitive channels was found to be essential for the development of the regulatory volume decrease following hypotonic shock. Taken together, these data indicate that KCa channels are the main K+ channels responsible for the volume regulation in U87-MG cells.

Metformin relaxes rat thoracic aorta via nitric oxide, AMPK, potassium channels, and PKC.

The present research aimed to identify the functional effects and underlying mechanisms of metformin on the rat thoracic aorta. Thoracic aorta segments of Wistar Albino rats were put in the chambers of an isolated tissue bath system. The resting tone was adjusted to 1 g. Following the equilibration time, potassium chloride or phenylephrine was used to contract the vascular segments. The vessel segments were cumulatively treated with metformin (10-7-10-3 M) when a steady contraction was achieved. The described experimental approach was repeated after incubations with signaling pathway inhibitors and selective blockers of potassium channels to identify the effect mechanisms of metformin. Metformin had a potent vasorelaxant effect in a concentration-dependent way (P<0.001). After the endothelium was removed, the vasorelaxant effect level of metformin was significantly reduced. The level of vasorelaxant effect of metformin was increased by the maintenance of perivascular adipose tissue. Following administrations of L-NAME, methylene blue, compound C, BIM-I, and potassium channel blockers, the level of vasodilatory action of metformin was significantly reduced (P<0.001). According to the results of this investigation, metformin significantly relaxes the thoracic aorta segments of rats. Metformin-mediated vasorelaxation involves the activation of numerous subtypes of potassium channels, including BKCa, IKCa, Kv, Kir, and K2p channels, as well as endothelium-dependent processes, including AMPK and eNOS/NO/sGS signaling pathways. Moreover, metformin-induced vasorelaxation is mediated through PVAT activation and the PKC signaling pathway.

Functional cooperation between IKCa and TRPC1 channels regulates serum-induced vascular smooth muscle cell proliferation via mediating Ca2+ influx and ERK1/2 activation.

The increased proliferation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of vascular diseases. The intermediate conductance calcium-activated potassium (IKCa ) channel plays a critical role in VSMC proliferation by raising the intracellular calcium concentration ([Ca2+ ]i ), but the underlying mechanism is still not unclear. Here we investigated the cooperation between IKCa and transient receptor potential canonical 1 (TRPC1) channels in mediating extracellular Ca2+ entry, which in turn activates downstream Ca2+ signalling in the regulation of VSMC proliferation using serum-induced cell proliferation model. Serum-induced cell proliferation was accompanied with up-regulation of IKCa expression and an increase in [Ca2+ ]i . Serum-induced cell proliferation and increase in [Ca2+ ]i were suppressed by IKCa inhibition with TRAM-34 or IKCa knockdown. Serum-induced cell proliferation was strongly reduced by the removal of extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA-AM and, additionally, by TRPC1 knockdown. Moreover, the increase in [Ca2+ ]i induced by serum or by IKCa activation with 1-EBIO was attenuated by TRPC1 knockdown. Finally, serum induced ERK1/2 activation, which was attenuated by treatment with TRAM-34 or BAPTA-AM, as well as TRPC1 knockdown. Consistently, serum-induced cell proliferation was suppressed by ERK1/2 inhibition with PD98059. Taken together, these results suggest that the IKCa and TRPC1 channels cooperate in mediating Ca2+ influx that activates the ERK1/2 pathway to promote cell proliferation, thus providing new mechanistic insights into VSMC proliferation.

Inhibition of platelet aggregation by activation of platelet intermediate conductance Ca2+‐activated potassium channels

BackgroundWithin the vasculature platelets and endothelial cells play crucial roles in hemostasis and thrombosis. Platelets, like endothelial cells, possess intermediate conductance Ca2+‐activated K+ (IKCa) channels and generate nitric oxide (NO). Although NO limits platelet aggregation, the role of IKCa channels in platelet function and NO generation has not yet been explored. ObjectivesWe investigated whether IKCa channel activation inhibits platelet aggregation, and per endothelial cells, enhances platelet NO production. MethodsPlatelets were isolated from human volunteers. Aggregometry, confocal microscopy, and a novel flow chamber model, the Quartz Crystal Microbalance (QCM) were used to assess platelet function. Flow cytometry was used to measure platelet NO production, calcium signaling, membrane potential, integrin αIIb/β3 activation, granule release, and procoagulant platelet formation. ResultsPlatelet IKCa channel activation with SKA‐31 inhibited aggregation in a concentration‐dependent manner, an effect reversed by the selective IKCa channel blocker TRAM‐34. The QCM model along with confocal microscopy demonstrated that SKA‐31 inhibited platelet aggregation under flow conditions. Surprisingly, IKCa activation by SKA‐31 inhibited platelet NO generation, but this could be explained by a concomitant reduction in platelet calcium signaling. IKCa activation by SKA‐31 also inhibited dense and alpha‐granule secretion and integrin αIIb/β3 activation, but maintained platelet phosphatidylserine surface exposure as a measure of procoagulant response. ConclusionsPlatelet IKCa channel activation inhibits aggregation by reducing calcium‐signaling and granule secretion, but not by enhancing platelet NO generation. IKCa channels may be novel targets for the development of antiplatelet drugs that limit atherothrombosis, but not coagulation.

Contribution of IKCa Channels to Dilation of Pial Arteries in young Rats after Ischemia/Reperfusion

Contribution of IKCa Channels to Dilation of Pial Arteries in young Rats after Ischemia/Reperfusion

IKCa channels control breast cancer metabolism including AMPK-driven autophagy

Ca2+-activated K+ channels of intermediate conductance (IK) are frequently overexpressed in breast cancer (BC) cells, while IK channel depletion reduces BC cell proliferation and tumorigenesis. This raises the question, of whether and mechanistically how IK activity interferes with the metabolic activity and energy consumption rates, which are fundamental for rapidly growing cells. Using BC cells obtained from MMTV-PyMT tumor-bearing mice, we show that both, glycolysis and mitochondrial ATP-production are reduced in cells derived from IK-deficient breast tumors. Loss of IK altered the sub-/cellular K+- and Ca2+- homeostasis and mitochondrial membrane potential, ultimately resulting in reduced ATP-production and metabolic activity. Consequently, we find that BC cells lacking IK upregulate AMP-activated protein kinase activity to induce autophagy compensating the glycolytic and mitochondrial energy shortage. Our results emphasize that IK by modulating cellular Ca2+- and K+-dynamics contributes to the remodeling of metabolic pathways in cancer. Thus, targeting IK channel might disturb the metabolic activity of BC cells and reduce malignancy.

The role of ВK&lt;sub&gt;Са&lt;/sub&gt; and IK&lt;sub&gt;Са&lt;/sub&gt; channels in H&lt;sub&gt;2&lt;/sub&gt;S-induced dilatation of pial arteries in rats after nephrectomy

BACKGROUND. Chronic kidney disease (CKD) is accompanied by the development of endothelial dysfunction, leading to a decrease in arterial reactivity to vasoactive agents. Uremia causes a change in the dilatation of arteries in various vascular regions, incl. and arteries of the pial membrane of the brain. The action of hydrogen sulfide (H2S), which can induce relaxation of smooth muscle cells of blood vessels, is currently considered a possible route of vasoprotection in various diseases, particularly, in CKD. THE AIM. To evaluate the role of calcium-activated potassium channels of large (BKCa) and intermediate (IKCa) conductance in H2S-induced dilatation of pial arteries in nephrectomized (NE) rats. MATERIAL AND METHODS. In Wistar rats nephrectomy (NE) was performed by resection of 5/6 of the renal tissue mass. Sham-operated (LO) animals served as control. The reaction of the pial arteries of the sensomotor cortex of NE and control SO rats to the application of H2S under physiological conditions and against the background of the use of BKCa channel blockers – tetraethylammonium (TEA) and IKCa – channels – TRAM-34. RESULTS. 4 months after NE, the application of H2S led to the dilatation of a smaller number of pial arteries (1.4 – 1.7 times) compared with SO rats. The preliminary exposure to TEA led to a decrease in the number of pial arteries responding by dilatation to the action of H2S in NE and SO rats. Against the background of the action of TRAM-34, the number of dilated arteries decreased under the action of H2S in SO rats, while in NE rats it practically did not change. CONCLUSION. Under physiological conditions, dilatation of the pial arteries in rats under the action of H2S is realized (at least in part) through the activation of the BKCa and IKCa channels of the membrane of endothelial and smooth muscle cells. Uremia, caused by nephrectomy, leads to impairment of the mechanism of dilatation of pial arteries, mediated by activation of calcium-activated potassium channels intermediate conductance apparently due to dysfunction of endothelial cells.

Involvement of small-conductance Ca2+-activated K+ (SKCa2) channels in spontaneous Ca2+ oscillations in rat pinealocytes

Melatonin secretion from the pineal glands regulates circadian rhythms in mammals. Melatonin production is decreased by an increase in cytosolic Ca2+ concentration following the activation of nicotinic acetylcholine receptors in parasympathetic systems. We previously reported that pineal Ca2+ oscillations were regulated by voltage-dependent Ca2+ channels and large-conductance Ca2+-activated K+ (BKCa) channels, which inhibited melatonin production. In the present study, the contribution of small- and intermediate-conductance Ca2+-activated K+ (SKCa and IKCa) channels to the regulation of spontaneous Ca2+ oscillations was examined in rat pinealocytes. The amplitude and frequency of spontaneous Ca2+ oscillations were increased by a SKCa channel blocker (100 nM apamin), but not by an IKCa channel blocker (1 μM TRAM-34). On the other hand, they were decreased by a SKCa channel opener (100 μM DCEBIO), but not by an IKCa channel opener (1 μM DCEBIO). Expression analyses using quantitative real-time PCR, immunocytochemical staining, and Western blotting revealed that the SKCa2 channel subtype was abundantly expressed in rat pinealocytes. Moreover, the enhanced amplitude of Ca2+ oscillations in the presence of apamin was further increased by a BKCa channel blocker (1 μM paxilline). These results suggest that the activity of SKCa2 channels regulates cytosolic Ca2+ signaling and melatonin production during parasympathetic activation in pineal glands.

Post‐menopausal Cerebral Microvascular Alterations in a Mouse Model of Alzheimer’s Disease

Cognitive decline, as seen in Alzheimer’s disease (AD), is a growing public health concern and is linked to decreased cerebral blood flow, particularly in women after menopause. Impaired cerebrovascular function precedes the onset of AD, possibly due to reduced endothelial function in parenchymal arterioles, the bottlenecks of the cerebral microcirculation, although exact mechanisms by menopause remain elusive. The goal of this study was to determine whether menopause impairs endothelial function in parenchymal arterioles of wild‐type (WT) and 5x‐FAD mouse model of AD. Menopause was induced by the 4‐vinylcyclohexene diepoxide (VCD) model, which induces gradual ovarian failure. Cerebral parenchymal arterioles were isolated and studied by pressure myography. Data are means ± SEM. In WT mice, menopause caused a significant increase in myogenic tone (myogenic tone: 24.73 ± 1.2 vs. 34 ± 2.6%, vehicle vs VCD, n = 5 / 8; p&lt;0.05), as well as a small but significant reduction in resting lumen diameter (32.68 ± 1.3 vs. 28.40 ± 2.1 µm, vehicle vs VCD, n = 5 / 8; p&lt;0.05). We then tested endothelial function, focusing on K+ channels, namely small and intermediate Ca2+‐activated K+ channels (SKCa / IKCa) and inwardly rectifying K+ channels (KIR2). Menopause did not affect arteriolar dilation to a cumulative concentration‐response curve of the SKCa/ IKCa activator NS‐309 (at 1 µM, vasodilation: 23.49 ± 11.5 vs. 17.80 ± 1.7%, vehicle vs VCD, n = 3 / 6). Similarly, KIR2 function was unchanged (vasodilation: 16.06 ± 6.6 vs. 16.21 ± 2.9%, vehicle vs VCD, n = 4 / 8). In the 5x‐FAD mice, menopause did not affect myogenic tone (myogenic tone: 23.49 ± 2.2 vs. 28 3.2%, vehicle vs VCD, n = 10 / 7), although there was a significant decrease in resting lumen diameter (39.16 ± 2.6 vs. 27.79 ± 2.0 µm, vehicle vs VCD, n = 10 / 7 p&lt;0.05). Arterioles from menopausal 5x‐FAD mice showed a significantly blunted response to NS‐309 (at 1 µM, vasodilation: 14.96 ± 2.9 vs. 9.42 ± 1.4%, vehicle vs VCD, n = 7 / 4, p&lt;0.05), without differences in KIR2‐induced dilation (vasodilation: 16.02 ± 1.3 vs. 13.69 ± 2.3%, vehicle vs VCD n = 9 / 6). In conclusion, we found that in WT mice, menopause leads to changes in myogenic tone without affecting K+‐dependent dilation. However, in 5x‐FAD mice, menopause impairs endothelial SKCa/IKCa channels independent of KIR2 function or myogenic tone. These findings identify new targets modulated by menopause in the cerebral microvasculature of WT and AD mice, which may contribute to cognitive dysfunction.

3-Hydroxyphenylacetic Acid: A Blood Pressure-Reducing Flavonoid Metabolite.

Regular intake of polyphenol-rich food has been associated with a wide variety of beneficial health effects, including the prevention of cardiovascular diseases. However, the parent flavonoids have mostly low bioavailability and, hence, their metabolites have been hypothesized to be bioactive. One of these metabolites, 3-hydroxyphenylacetic acid (3-HPAA), formed by the gut microbiota, was previously reported to exert vasorelaxant effects ex vivo. The aim of this study was to shed more light on this effect in vivo, and to elucidate the mechanism of action. 3-HPAA gave rise to a dose-dependent decrease in arterial blood pressure when administered i.v. both as a bolus and infusion to spontaneously hypertensive rats. In contrast, no significant changes in heart rate were observed. In ex vivo experiments, where porcine hearts from a slaughterhouse were used to decrease the need for laboratory animals, 3-HPAA relaxed precontracted porcine coronary artery segments via a mechanism partially dependent on endothelium integrity. This relaxation was significantly impaired after endothelial nitric oxide synthase inhibition. In contrast, the blockade of SKCa or IKCa channels, or muscarinic receptors, did not affect 3-HPAA relaxation. Similarly, no effects of 3-HPAA on cyclooxygenase nor L-type calcium channels were observed. Thus, 3-HPAA decreases blood pressure in vivo via vessel relaxation, and this mechanism might be based on the release of nitric oxide by the endothelial layer.

Calcium-Dependent Ion Channels and the Regulation of Arteriolar Myogenic Tone.

Arterioles in the peripheral microcirculation regulate blood flow to and within tissues and organs, control capillary blood pressure and microvascular fluid exchange, govern peripheral vascular resistance, and contribute to the regulation of blood pressure. These important microvessels display pressure-dependent myogenic tone, the steady state level of contractile activity of vascular smooth muscle cells (VSMCs) that sets resting arteriolar internal diameter such that arterioles can both dilate and constrict to meet the blood flow and pressure needs of the tissues and organs that they perfuse. This perspective will focus on the Ca2+-dependent ion channels in the plasma and endoplasmic reticulum membranes of arteriolar VSMCs and endothelial cells (ECs) that regulate arteriolar tone. In VSMCs, Ca2+-dependent negative feedback regulation of myogenic tone is mediated by Ca2+-activated K+ (BKCa) channels and also Ca2+-dependent inactivation of voltage-gated Ca2+ channels (VGCC). Transient receptor potential subfamily M, member 4 channels (TRPM4); Ca2+-activated Cl− channels (CaCCs; TMEM16A/ANO1), Ca2+-dependent inhibition of voltage-gated K+ (KV) and ATP-sensitive K+ (KATP) channels; and Ca2+-induced-Ca2+ release through inositol 1,4,5-trisphosphate receptors (IP3Rs) participate in Ca2+-dependent positive-feedback regulation of myogenic tone. Calcium release from VSMC ryanodine receptors (RyRs) provide negative-feedback through Ca2+-spark-mediated control of BKCa channel activity, or positive-feedback regulation in cooperation with IP3Rs or CaCCs. In some arterioles, VSMC RyRs are silent. In ECs, transient receptor potential vanilloid subfamily, member 4 (TRPV4) channels produce Ca2+ sparklets that activate IP3Rs and intermediate and small conductance Ca2+ activated K+ (IKCa and sKCa) channels causing membrane hyperpolarization that is conducted to overlying VSMCs producing endothelium-dependent hyperpolarization and vasodilation. Endothelial IP3Rs produce Ca2+ pulsars, Ca2+ wavelets, Ca2+ waves and increased global Ca2+ levels activating EC sKCa and IKCa channels and causing Ca2+-dependent production of endothelial vasodilator autacoids such as NO, prostaglandin I2 and epoxides of arachidonic acid that mediate negative-feedback regulation of myogenic tone. Thus, Ca2+-dependent ion channels importantly contribute to many aspects of the regulation of myogenic tone in arterioles in the microcirculation.

Physiological role of K+ channels in irisin-induced vasodilation in rat thoracic aorta

Irisin, an exercise-induced myokine, has been shown to have a peripheral vasodilator effect. However, little is known about the mechanisms underlying its effects. In this study, it was aimed to investigate the vasoactive effects of irisin on rat thoracic aorta, and the hypothesis that voltage-gated potassium (KV) channels, ATP-sensitive potassium (KATP) channels, small-conductance calcium-activated potassium (SKCa) channels, large-conductance calcium-activated potassium (BKCa) channels, intermediate-conductance calcium-activated potassium (IKCa) channels, inward rectifier potassium (Kir) channels, and two-pore domain potassium (K2P) channels may have roles in these effects. Isometric contraction-relaxation responses of isolated thoracic aorta rings were measured with an organ bath model. The steady contraction was induced with both 10−5 M phenylephrine and 45 mM KCl, and then the concentration-dependent responses of irisin (10-9-10-6 M) were examined. Irisin exerted the vasorelaxant effects in both endothelium-intact and -denuded aortic rings at concentrations of 10-8, 10-7, and 10-6 M (p < 0.001). Besides, KV channel blocker 4-aminopyridine, KATP channel blocker glibenclamide, SKCa channel blocker apamin, BKCa channel blockers tetraethylammonium and iberiotoxin, IKCa channel blocker TRAM-34, and Kir channel blocker barium chloride incubations significantly inhibited the irisin-induced relaxation responses. However, incubation of K2P TASK-1 channel blocker anandamide did not cause a significant decrease in the relaxation responses of irisin. In conclusion, the first physiological findings were obtained regarding the functional relaxing effects of irisin in rat thoracic aorta. Furthermore, this study is the first to report that irisin-induced relaxation responses are associated with the activity of KV, KATP, SKCa, BKCa, IKCa, and Kir channels.

Calmodulin Antagonist W-7 Enhances Intermediate Conductance Ca2+- Sensitive Basolateral Potassium Channel (IKCa) Activity in Human Colonic Crypts

Intermediate conductance potassium (IKCa) channels are exquisitively Ca2+ sensitive, intracellular Ca2+ regulating channel activity by complexing with calmodulin (CaM), which is bound to the cytosolic carboxyl tail. Although CaM antagonists might be expected to decrease IKCa channel activity, the effect of W-7 in human T lymphocytes are conflicting. We therefore evaluated the effect of W-7 on basolateral IKCa channels in human colonic crypt cells. Intact crypts obtained from normal human colonic biopsies by Ca2+ chelation were used for patch clamp studies of basolateral IKCa channels in the cell-attached configuration. IKCa channel activity was studied when the bath Ca2+ concentration was changed from 1.2 mmol/L to 100 μmol/L and back to 1.2 mmol/L, as well as from 100 μmol/L to 1.2 mmol/L and back to 100 μmol/L, both in the absence and presence of 25 μmol/L W-7. Decreasing bath Ca2+ from 1.2 mmol/L to 100 μmol/L decreased IKCa channel activity reversibly in the absence of W-7, whereas there was a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. In separate experiments, increasing bath Ca2+ from 100 μmol/L to 1.2 mmol/L increased IKCa channel activity reversibly in the absence of W-7, whereas there was again a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. We, therefore, propose that W-7 has a specific stimulatory effect on basolateral IKCa channel activity, despite its ability to inhibit Ca2+/CaM-mediated, IKCa channel-dependent Cl− secretion in human colonic epithelial cells.Graphic

Stimulation of vascular smooth muscle cell proliferation by stiff matrix via the IKCa channel-dependent Ca2+ signaling.

Vascular stiffening, an early and common characteristic of cardiovascular diseases (CVDs), stimulates vascular smooth muscle cell (VSMC) proliferation which reciprocally accelerates the progression of CVDs. However, the mechanisms by which extracellular matrix stiffness accompanying vascular stiffening regulates VSMC proliferation remain largely unknown. In the present study, we examined the role of the intermediate-conductance Ca2+ -activated K+ (IKCa ) channel in the matrix stiffness regulation of VSMC proliferation by growing A7r5 cells on soft and stiff polydimethylsiloxane substrates with stiffness close to these of arteries under physiological and pathological conditions, respectively. Stiff substrates stimulated cell proliferation and upregulated the expression of the IKCa channel. Stiff substrate-induced cell proliferation was suppressed by pharmacological inhibition using TRAM34, an IKCa channel blocker, or genetic depletion of the IKCa channel. In addition, stiff substrate-induced cell proliferation was also suppressed by reducing extracellular Ca2+ concentration using EGTA or intracellular Ca2+ concentration using BAPTA-AM. Moreover, stiff substrate induced activation of extracellular signal-regulated kinases (ERKs), which was inhibited by treatment with TRAM34 or BAPTA-AM. Stiff substrate-induced cell proliferation was suppressed by treatment with PD98059, an ERK inhibitor. Taken together, these results show that substrates with pathologically relevant stiffness upregulate the IKCa channel expression to enhance intracellular Ca2+ signaling and subsequent activation of the ERK signal pathway to drive cell proliferation. These findings provide a novel mechanism by which vascular stiffening regulates VSMC function.

Involvement of Mitochondrial Intermediate Conductance Ca2+-Activated K+ Channels in DCEBIO-Induced C2C12 Skeletal Muscle Cell Hypertrophy

Differentiation of skeletal muscle stem cells to myofibers is a critical step for muscle maintenance and repair. Although potassium channels play pivotal roles in myogenic differentiation, the effect of potassium channel openers on myogenic differentiation is unclear. Our study examined the change of differentiation rate of muscle stem cells and mass of differentiated myotubes induced by DCEBIO, a small molecule opener of intermediate conductance Ca2+-activated K+ channel (IKCa channel) which abundantly expressed in skeletal muscle stem cells.