Hypersensitivity to BKCa channel opening in persistent post-traumatic headache.

BACKGROUND: Large conductance  calcium-activated potassium (BKCa) channels have been implicated in the neurobiological underpinnings of migraine. Considering the clinical similarities between migraine and persistent post-traumatic headache (PPTH), we aimed to examine whether MaxiPost (a BKCa channel opener) could induce migraine-like headache in persons with PPTH. METHODS: This is a randomized double-blind, placebo-controlled, two-way crossover study from September 2023 to December 2023. Eligible participants were adults with PPTH after mild traumatic brain injury who reported having no personal history of migraine. The randomized participants received a single dose of either MaxiPost (0.05 mg/min) or placebo (isotonic saline) that was infused intravenously over 20 minutes. The two experiment sessions were scheduled at least one week apart to avoid potential carryover effects. The primary endpoint was the induction of migraine-like headache after MaxiPost as compared to placebo within 12 hours of drug administration. The secondary endpoint was the area under the curve (AUC) values for headache intensity scores between MaxiPost and placebo over the same 12-hour observation period. RESULTS: Twenty-one adult participants (comprising 14 females and 7 males) with PPTH were enrolled and completed both experiment sessions. The proportion of participants who developed migraine-like headache was 11 (52%) of 21 participants after MaxiPost infusion, in contrast to four (19%) participants following placebo (P = .02). Furthermore, the median headache intensity scores, represented by AUC values, were higher following MaxiPost than after placebo (P < .001). CONCLUSIONS: Our results indicate that BKCa channel opening can elicit migraine-like headache in persons with PPTH. Thus, pharmacologic blockade of BKCa channels might present a novel avenue for drug discovery. Additional investigations are nonetheless needed to confirm these insights and explore the therapeutic prospects of BKCa channel blockers in managing PPTH. GOV IDENTIFIER: NCT05378074.

Functional and Histological Changes in Umbilical Artery and Myometrium Isolated from IUGR Complicated Pregnancies.

Objective: To investigate the relaxation responses mediated by L-type Ca2+ channels and big-conductance Ca2+-activated K+ (BKCa) channels and histological changes in the human umbilical artery (HUA) and myometrium smooth muscle isolated from pregnancies complicated with intrauterine growth restriction (IUGR).Methods: The muscle reactivity and the histology of the smooth muscle of the HUA and myometrium retrieved from 14 women with IUGR and 14 controls were investigated by the isolated tissue bath and immunohistochemical method.Results: In HUA, the maximum relaxation responses and pD2 values of nifedipine and NS11021 (BKCa channel opener) were significantly increased and significant histopathological changes are observed in the IUGR group.Conclusions: The pathogenesis of IUGR might be associated with the impairment in the functional responses of L-type Ca2+ channels and BKCa channels in HUA smooth muscle. The increased staining of myometrium and UC with HIF-1α in IUGR may indicate apoptosis, histological damage, and impaired fetal growth.

Moderate Ethanol-Preconditioning Offers Ischemic Tolerance Against Focal Cerebral Ischemic/Reperfusion: Role of Large Conductance Calcium-Activated Potassium Channel.

The mechanism underlying moderate ethanol (EtOH)-preconditioning (PC) against ischemic brain injury remains unclear. We evaluated the role of large conductance calcium-sensitive potassium (BKCa) channels in EtOH-PC. Almost one hundred and ninety normal adult SD rats (8 to 10 weeks, 320-350 g) were enrolled in this study. Ischemic/reperfusion (I/R) brain injury was induced in rats by middle cerebral artery occlusion for 2 h followed by reperfusion for 24 h. EtOH or the BKCa channel opener, NS11021, was administered 24 h before I/R with or without pre-treatment with the BKCa channel blocker, paxilline. Infarct volumes were measured by tissue staining and imaging, and neurological functions were assessed by a scoring system. The expression of BKCa channel subunit α was detected by Western blotting, and cell apoptosis was assessed using staining. Prior (24 h) administration of ethanol that produced a peak plasma concentration of ~ 45 mg/dl in rats would offer neuroprotection after cerebral I/R. In addition, the expression of BKCa channel α-subunit was significantly increased 24 h after EtOH-PC (n = 10; control: 2.00 ± 0.09, EtOH: 1.00 ± 0.06; P < 0.5). Compared to I/R, EtOH-PC enhanced the expression of BKCa channel α-subunit both in the penumbra (n = 10; 24 h: I/R: 1.25 ± 0.10, EtOH-PC + I/R: 1.99 ± 0.12; P < 0.01; 4 h: I/R: 1.03 ± 0.03, EtOH-PC + I/R: 1.49 ± 0.05; P < 0.001) and infarct core (n = 10; 4 h: I/R: 1.04 ± 0.04, EtOH-PC + I/R: 1.42 ± 0.05; P < 0.001), improved the neurological function (n = 10; I/R: 14.00 (12.75-15.00), EtOH-PC + I/R: 7.00 (4.75-8.25); P < 0.001), attenuated the apoptosis (n = 10; I/R: 26.80 ± 0.69, EtOH-PC + I/R: 8.46 ± 0.31; P < 0.001), and decreased the infarct volume (n = 10; I/R: 244.00 ± 26.24, EtOH-PC + I/R: 70.09 ± 14.69; P < 0.001) after experimental cerebral I/R. These changes were reversed by paxilline administration. The moderate EtOH-PC protects against I/R-induced brain damage dependent on the upregulation BKCa channels.

BKCa channel participates in insulin-induced lipid deposition in adipocytes by increasing intracellular calcium.

Storing energy in the form of triglyceride (TG) is one of the basic functions of adipose tissue. Large-conductance calcium-activated potassium channels (BKCa channels) are expressed in adipose tissue and adipocyte-specific BKCa deficiency resists obesity in mice, but the role of BKCa channels in lipid deposition and the underlying mechanisms have not been elucidated. In the present study, we generated BKCa knockout (KO) rats and performed a transcriptome analysis of adipose tissue. We found that the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, which is important for lipid deposition, exhibited the most notable reduction among various signaling pathways in BKCa KO rats compared to wild-type rats. Insulin-induced TG deposition, glucose uptake, and Akt (Ser473) phosphorylation were significantly reduced in cultured adipocytes differentiated from adipose-derived stem cells of BKCa KO rats. Furthermore, we found that the insulin-induced increase of intracellular calcium resulting from extracellular calcium influx was significantly impaired in BKCa KO adipocytes. Finally, insulin activated BKCa currents through PI3K, which was independent of Akt and intracellular calcium. The results of this study suggested that BKCa channels participate in the insulin signaling pathway and promote TG deposition by increasing extracellular calcium influx in adipocytes.

The extracellular loop of the auxiliary ß1-subunit is involved in the regulation of BKCa channel mechanosensitivity.

The large conductance Ca2+-activated potassium (BKCa) channel is activated by stretch. The stress-regulated exon (STREX) in α-subunits is known to affect the mechanosensitivity of BKCa channels. However, in human colonic smooth muscle cells (HCoSMCs), we found that the α-subunits without STREX (ZERO-BK) and ß1-subunits could be detected yet the cells were mechanosensitive. Whether the ß1-subunit is involved in the regulation of BKCa mechanosensitivity is unclear. In the present study, ZERO-BK and ß1-subunits were individually expressed or coexpressed in HEK293 cells and cell-attached patch-clamp techniques were used to measure BKCa currents defining mechanosensitivity. Single-channel patch-clamp recordings from HEK293 cells cotransfected with ZERO-BK and ß1-subunits showed stretch sensitivity, but there was no mechanosensitivity in HEK293 cells transfected only with ZERO-BK. We also showed that expression of the ß1-subunit could increase mechanosensitivity of the STREX-type α-subunits (STREX-BK). To identify the domain in ß1-subunits responsible for affecting stretch sensitivity, we expressed ß1- LoopDel (chimeric ß1-subunits without the extracellular loop) or ß1- C TermDel (chimeric ß1-subunits without COOH terminus) with ZERO-BK in HEK293 cells. The data showed that deletion of the extracellular loop but not the COOH terminus of ß1-subunits virtually abolished the mechanosensitivity. These results suggest that the extracellular loop of the ß1-subunit is involved in the regulation of BKCa channel mechanosensitivity and that role is independent of STREX.

Differential targeting and signalling of voltage-gated T-type Cav 3.2 and L-type Cav 1.2 channels to ryanodine receptors in mesenteric arteries.

KEY POINTS: In arterial smooth muscle, Ca2+ sparks are elementary Ca2+ -release events generated by ryanodine receptors (RyRs) to cause vasodilatation by opening maxi Ca2+ -sensitive K+ (BKCa ) channels. This study elucidated the contribution of T-type Cav 3.2 channels in caveolae and their functional interaction with L-type Cav 1.2 channels to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs). Our data demonstrate that L-type Cav 1.2 channels provide the predominant Ca2+ pathway for the generation of Ca2+ sparks in murine arterial VSMCs. T-type Cav 3.2 channels represent an additional source for generation of VSMC Ca2+ sparks. They are located in pit structures of caveolae to provide locally restricted, tight coupling between T-type Cav 3.2 channels and RyRs to ignite Ca2+ sparks. ABSTRACT: Recent data suggest that T-type Cav 3.2 channels in arterial vascular smooth muscle cells (VSMCs) and pits structure of caveolae could contribute to elementary Ca2+ signalling (Ca2+ sparks) via ryanodine receptors (RyRs) to cause vasodilatation. While plausible, their precise involvement in igniting Ca2+ sparks remains largely unexplored. The goal of this study was to elucidate the contribution of caveolar Cav 3.2 channels and their functional interaction with Cav 1.2 channels to trigger Ca2+ sparks in VSMCs from mesenteric, tibial and cerebral arteries. We used tamoxifen-inducible smooth muscle-specific Cav 1.2-/- (SMAKO) mice and laser scanning confocal microscopy to assess Ca2+ spark generation in VSMCs. Ni2+ , Cd2+ and methyl-ß-cyclodextrin were used to inhibit Cav 3.2 channels, Cav 1.2 channels and caveolae, respectively. Ni2+ (50 µmol L-1 ) and methyl-ß-cyclodextrin (10 mmol L-1 ) decreased Ca2+ spark frequency by ∼20-30% in mesenteric VSMCs in a non-additive manner, but failed to inhibit Ca2+ sparks in tibial and cerebral artery VSMCs. Cd2+ (200 µmol L-1 ) suppressed Ca2+ sparks in mesenteric arteries by ∼70-80%. A similar suppression of Ca2+ sparks was seen in mesenteric artery VSMCs of SMAKO mice. The remaining Ca2+ sparks were fully abolished by Ni2+ or methyl-ß-cyclodextrin. Our data demonstrate that Ca2+ influx through CaV 1.2 channels is the primary means of triggering Ca2+ sparks in murine arterial VSMCs. CaV 3.2 channels, localized to caveolae and tightly coupled to RyR, provide an additional Ca2+ source for Ca2+ spark generation in mesenteric, but not tibial and cerebral, arteries.

Interplay among distinct Ca2+ conductances drives Ca2+ sparks/spontaneous transient outward currents in rat cerebral arteries.

KEY POINTS: Distinct Ca2+ channels work in a coordinated manner to grade Ca2+ spark/spontaneous transient outward currents (STOCs) in rat cerebral arteries. The relative contribution of each Ca2+ channel to Ca2+ spark/STOC production depends upon their biophysical properties and the resting membrane potential of smooth muscle. Na+ /Ca2+ exchanger, but not TRP channels, can also facilitate STOC production. ABSTRACT: Ca2+ sparks are generated in a voltage-dependent manner to initiate spontaneous transient outward currents (STOCs), events that moderate arterial constriction. In this study, we defined the mechanisms by which membrane depolarization increases Ca2+ sparks and subsequent STOC production. Using perforated patch clamp electrophysiology and rat cerebral arterial myocytes, we monitored STOCs in the presence and absence of agents that modulate Ca2+ entry. Beginning with CaV 3.2 channel inhibition, Ni2+ was shown to decrease STOC frequency in cells held at hyperpolarized (-40 mV) but not depolarized (-20 mV) voltages. In contrast, nifedipine, a CaV 1.2 inhibitor, markedly suppressed STOC frequency at -20 mV but not -40 mV. These findings aligned with the voltage-dependent profiles of L- and T-type Ca2+ channels. Furthermore, computational and experimental observations illustrated that Ca2+ spark production is intimately tied to the activity of both conductances. Intriguingly, this study observed residual STOC production at depolarized voltages that was independent of CaV 1.2 and CaV 3.2. This residual component was insensitive to TRPV4 channel modulation and was abolished by Na+ /Ca2+ exchanger blockade. In summary, our work highlights that the voltage-dependent triggering of Ca2+ sparks/STOCs is not tied to a single conductance but rather reflects an interplay among multiple Ca2+ permeable pores with distinct electrophysiological properties. This integrated orchestration enables smooth muscle to grade Ca2+ spark/STOC production and thus precisely tune negative electrical feedback.

Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels.

Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10-100µM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.

Maternal nutrient restriction during pregnancy impairs an endothelium-derived hyperpolarizing factor-like pathway in sheep fetal coronary arteries.

The mechanisms underlying developmental programming are poorly understood but may be associated with adaptations by the fetus in response to changes in the maternal environment during pregnancy. We hypothesized that maternal nutrient restriction during pregnancy alters vasodilator responses in fetal coronary arteries. Pregnant ewes were fed a control [100% U.S. National Research Council (NRC)] or nutrient-restricted (60% NRC) diet from days 50 to 130 of gestation (term = 145 days); fetal tissues were collected at day 130. In coronary arteries isolated from control fetal lambs, relaxation to bradykinin was unaffected by nitro-l-arginine (NLA). Iberiotoxin or contraction with KCl abolished the NLA-resistant response to bradykinin. In fetal coronary arteries from nutrient-restricted ewes, relaxation to bradykinin was fully suppressed by NLA. Large-conductance, calcium-activated potassium channel (BKCa) currents did not differ in coronary smooth muscle cells from control and nutrient-restricted animals. The BKCa openers, BMS 191011 and NS1619, and 14,15-epoxyeicosatrienoic acid [a putative endothelium-derived hyperpolarizing factor (EDHF)] each caused fetal coronary artery relaxation and BKCa current activation that was unaffected by maternal nutrient restriction. Expression of BKCa-channel subunits did not differ in fetal coronary arteries from control or undernourished ewes. The results indicate that maternal undernutrition during pregnancy results in loss of the EDHF-like pathway in fetal coronary arteries in response to bradykinin, an effect that cannot be explained by a decreased number or activity of BKCa channels or by decreased sensitivity to mediators that activate BKCa channels in vascular smooth muscle cells. Under these conditions, bradykinin-induced relaxation is completely dependent on nitric oxide, which may represent an adaptive response to compensate for the absence of the EDHF-like pathway.

Stimulation of the calcium-sensing receptor induces relaxations of rat mesenteric arteries by endothelium-dependent and -independent pathways via BKCa and KATP channels.

Stimulation of the calcium-sensing receptor (CaSR) induces both vasoconstrictions and vasorelaxations but underlying cellular processes remain unclear. This study investigates expression and effect of stimulating the CaSR by increasing external Ca2+ concentration ([Ca2+ ]o ) on contractility of rat mesenteric arteries. Immunofluorescence studies showed expression of the CaSR in perivascular nerves, vascular smooth muscle cells (VSMCs), and vascular endothelium cells. Using wire myography, increasing [Ca2+ ]o from 1 to 10 mM induced vasorelaxations which were inhibited by the calcilytic Calhex-231 and partially dependent on a functional endothelium. [Ca2+ ]o -induced vasorelaxations were reduced by endothelial NO synthase (eNOS, L-NAME) and large conductance Ca2+ -activated K+ channels (BKCa , iberiotoxin), with their inhibitory action requiring a functional endothelium. [Ca2+ ]o -induced vasorelaxations were also markedly inhibited by an ATP-dependent K+ channel (KATP ) blocker (PNU37883), which did not require a functional endothelium to produce its inhibitory action. Inhibitor studies also suggested contributory roles for inward rectifying K+ channels (Kir ), Kv7 channels, and small conductance Ca2+ -activated K+ channels (SKCa ) on [Ca2+ ]o -induced vasorelaxations. These findings indicate that stimulation of the CaSR mediates vasorelaxations involving multiple pathways, including an endothelium-dependent pathway involving NO production and activation of BKCa channels and an endothelium-independent pathway involving stimulation of KATP channels.

Oxidative and Nitrous Stress Underlies Vascular Malfunction Induced by Ionizing Radiation and Diabetes.

Oxidative stress results from the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in quantities exceeding the potential activity of the body's antioxidant system and is one of the risk factors for the development of vascular dysfunction in diabetes and exposure to ionizing radiation. Being the secondary products of normal aerobic metabolism in living organisms, ROS and RNS act as signaling molecules that play an important role in the regulation of vital organism functions. Meanwhile, in high concentrations, these compounds are toxic and disrupt various metabolic pathways. The various stress factors (hyperglycemia, gamma-irradiation, etc.) trigger free oxygen and nitrogen radicals accumulation in cells that are capable to damage almost all cellular components including ion channels and transporters such as Na+/K+-ATPase, BKCa, and TRP channels. Vascular dysfunctions are governed by interaction of ROS and RNS. For example, the reaction of ROS with NO produces peroxynitrite (ONOO-), which not only oxidizes DNA, cellular proteins, and lipids, but also disrupts important signaling pathways that regulate the cation channel functions in the vascular endothelium. Further increasing in ROS levels and formation of ONOO- leads to reduced NO bioavailability and causes endothelial dysfunction. Thus, imbalance of ROS and RNS and their affect on membrane ion channels plays an important role in the pathogenesis of vascular dysfunction associated with various disorders.

Activation of lysophosphatidic Acid receptor is coupled to enhancement of ca(2+)-activated potassium channel currents.

The calcium-activated K(+) (BKCa) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. Ca(2+) is the main regulator of BKCa channel activation. The BKCa channel contains two high affinity Ca(2+) binding sites, namely, regulators of K(+) conductance, RCK1 and the Ca(2+) bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular Ca(2+) levels through diverse G proteins such as Gαq/11, Gαi, Gα12/13, and Gαs and the related signal transduction pathway. In the present study, we examined LPA effects on BKCa channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated BKCa channel activation was also attenuated by the PLC inhibitor U-73122, IP3 inhibitor 2-APB, Ca(2+) chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated BKCa channel activation. The present study indicates that LPA-mediated activation of the BKCa channel is achieved through the PLC, IP3, Ca(2+), and PKC pathway and that LPA-mediated activation of the BKCa channel could be one of the biological effects of LPA in the nervous and vascular systems.

Involvement of Potassium Channel Signalling in Migraine Pathophysiology.

Migraine is a primary headache disorder ranked as the leading cause of years lived with disability among individuals younger than 50 years. The aetiology of migraine is complex and might involve several molecules of different signalling pathways. Emerging evidence implicates potassium channels, predominantly ATP-sensitive potassium (KATP) channels and large (big) calcium-sensitive potassium (BKCa) channels in migraine attack initiation. Basic neuroscience revealed that stimulation of potassium channels activated and sensitized trigeminovascular neurons. Clinical trials showed that administration of potassium channel openers caused headache and migraine attack associated with dilation of cephalic arteries. The present review highlights the molecular structure and physiological function of KATP and BKCa channels, presents recent insights into the role of potassium channels in migraine pathophysiology, and discusses possible complementary effects and interdependence of potassium channels in migraine attack initiation.

Bidirectional TRP/L Type Ca2+ Channel/RyR/BKCa Molecular and Functional Signaloplex in Vascular Smooth Muscles.

TRP channels are expressed both in vascular myocytes and endothelial cells, but knowledge of their operational mechanisms in vascular tissue is particularly limited. Here, we show for the first time the biphasic contractile reaction with relaxation followed by a contraction in response to TRPV4 agonist, GSK1016790A, in a rat pulmonary artery preconstricted with phenylephrine. Similar responses were observed both with and without endothelium, and these were abolished by the TRPV4 selective blocker, HC067047, confirming the specific role of TRPV4 in vascular myocytes. Using selective blockers of BKCa and L-type voltage-gated Ca2+ channels (CaL), we found that the relaxation phase was inducted by BKCa activation generating STOCs, while subsequent slowly developing TRPV4-mediated depolarisation activated CaL, producing the second contraction phase. These results are compared to TRPM8 activation using menthol in rat tail artery. Activation of both types of TRP channels produces highly similar changes in membrane potential, namely slow depolarisation with concurrent brief hyperpolarisations due to STOCs. We thus propose a general concept of bidirectional TRP-CaL-RyR-BKCa molecular and functional signaloplex in vascular smooth muscles. Accordingly, both TRPV4 and TRPM8 channels enhance local Ca2+ signals producing STOCs via TRP-RyR-BKCa coupling while simultaneously globally engaging BKCa and CaL channels by altering membrane potential.

Effects of KCa channels on biological behavior of trophoblasts.

The Ca2+-activated potassium (KCa) channels are involved in many cellular functions, but their roles in trophoblasts are unclear. This study aimed to clarify the effects of KCa channels on the biological behavior of trophoblasts. The localization and expression of the three types of KCa channels, including large-conductance KCa channels (BKCa), intermediate-conductance KCa channels (IKCa), and small-conductance KCa channels (SKCa), were detected in human chorionic villi taken from pregnant women between 5 and 8 weeks of gestation (n = 15) and HTR-8/SVneo cells. The effects of KCa channels on proliferation, apoptosis, and migration of HTR-8/SVneo cells were examined by using the activators or inhibitors of KCa channels. Results showed that KCa channels were mainly localized on the membrane and in the cytoplasm of trophoblasts in human chorionic villi and HTR-8/SVneo cells. The proliferation and migration of HTR-8/SVneo cells were inhibited by activating KCa channels. Apoptosis of trophoblasts was promoted through activating BKCa channels but was not affected by neither activating nor inhibiting IKCa and SKCa channels. This study substantiated the abovementioned biological roles of KCa channels in trophoblast cells, which is fundamental to further research on whether dysfunction of KCa channels is involved in the pathogenesis of pregnancy-related complications.

Apelin Does Not Impair Coronary Artery Relaxation Mediated by Nitric Oxide-Induced Activation of BKCa Channels.

Apelin-APJ receptor signaling regulates vascular tone in cerebral and peripheral arteries. We recently reported that apelin inhibits BKCa channel function in cerebral arteries, resulting in impaired endothelium-dependent relaxations. In contrast, apelin causes endothelium-dependent relaxation of coronary arteries. However, the effects of apelin on BKCa channel function in coronary arterial myocytes have not yet been explored. We hypothesized that apelin-APJ receptor signaling does not have an inhibitory effect on coronary arterial BKCa channels and hence does not alter nitric oxide (NO)-dependent relaxation of coronary arteries. Patch clamp recording was used to measure whole cell K+ currents in freshly isolated coronary smooth muscle cells. Apelin had no effect on the increases in current density in response to membrane depolarization or to NS1619 (a BKCa channel opener). Moreover, apelin did not inhibit NO/cGMP-dependent relaxations that required activation of BKCa channels in isolated coronary arteries. Apelin-APJ receptor signaling caused a marked increase in intracellular Ca2+ levels in coronary arterial smooth muscle cells, but failed to activate PI3-kinase to increase phosphorylation of Akt protein. Collectively, these data provide mechanistic evidence that apelin has no inhibitory effects on BKCa channel function in coronary arteries. The lack of inhibitory effect on BKCa channels makes it unlikely that activation of APJ receptors in coronary arteries would adversely affect coronary flow by creating a vasoconstrictive environment. It can be expected that apelin or other APJ receptor agonists in development will not interfere with the vasodilator effects of endogenous BKCa channel openers.

Endothelium-derived hydrogen sulfide acts as a hyperpolarizing factor and exerts neuroprotective effects via activation of large-conductance Ca2+ -activated K+ channels.

BACKGROUND AND PURPOSE: Endothelium-derived hyperpolarizing factor (EDHF) has been suggested as a therapeutic target for vascular protection against ischaemic brain injury. However, the molecular entity of EDHF and its action on neurons remains unclear. This study was undertaken to demonstrate whether the hydrogen sulfide (H2 S) acts as EDHF and exerts neuroprotective effect via large-conductance Ca2+ -activated K+ (BKCa /KCa 1.1) channels. EXPERIMENTAL APPROACH: The whole-cell patch-clamp technology was used to record the changes of BKCa currents in rat neurons induced by EDHF. The cerebral ischaemia/reperfusion model of mice and oxygen-glucose deprivation/reoxygenation (OGD/R) model of neurons were used to explore the neuroprotection of EDHF by activating BKCa channels in these neurons. KEY RESULTS: Increases of BKCa currents and membrane hyperpolarization in hippocampal neurons induced by EDHF could be markedly inhibited by BKCa channel inhibitor iberiotoxin or endothelial H2 S synthase inhibitor propargylglycine. The H2 S donor, NaHS-induced BKCa current and membrane hyperpolarization in neurons were also inhibited by iberiotoxin, suggesting that H2 S acts as EDHF and activates the neuronal BKCa channels. Besides, we found that the protective effect of endothelium-derived H2 S against mice cerebral ischaemia/reperfusion injury was disrupted by iberiotoxin. Importantly, the inhibitory effect of NaHS or BKCa channel opener on OGD/R-induced neuron injury and the increment of intracellular Ca2+ level could be inhibited by iberiotoxin but enhanced by co-application with L-type but not T-type calcium channel inhibitor. CONCLUSION AND IMPLICATIONS: Endothelium-derived H2 S acts as EDHF and exerts neuroprotective effects via activating the BKCa channels and then inhibiting the T-type calcium channels in hippocampal neurons.

Regulatory mechanisms of mitochondrial BKCa channels.

The mitochondrial BKCa channel (mitoBKCa) is a splice variant of plasma membrane BKCa (Maxi-K, BKCa, Slo1, KCa1.1). While a high-resolution structure of mitoBKCa is not available yet, functional and structural studies of the plasma membrane BKCa have provided important clues on the gating of the channel by voltage and Ca2+, as well as the interaction with auxiliary subunits. To date, we know that the control of expression of mitoBKCa, targeting and voltage-sensitivity strongly depends on its association with its regulatory ß1-subunit, which overall participate in the control of mitochondrial Ca2+-overload in cardiac myocytes. Moreover, novel regulatory mechanisms of mitoBKCa such as ß-subunits and amyloid-ß have recently been proposed. However, major basic questions including how the regulatory BKCa-ß1-subunit reaches mitochondria and the mechanism through which amyloid-ß impairs mitoBKCa channel function remain to be addressed.

Extra Virgin Olive Oil Phenols Vasodilate Rat MesentericResistance Artery via Phospholipase C (PLC)-CalciumMicrodomains-Potassium Channels (BKCa) Signals.

Recent evidence suggests that the reason Extra Virgin Olive Oil (EVOO) lowers blood pressure and reduces the risk of developing hypertension is partly due to minor components of EVOO, such as phenols. However, little is still known about the mechanism(s) through which EVOO phenols mediate anti-hypertensive effects. The aim of the present study was to investigate the mechanisms of action of EVOO phenols on mesenteric resistance arteries. A pressure myograph was used to test the effect of EVOO phenols on isolated mesenteric arteries in the presence of specific inhibitors of: 1) BKca channels (Paxillin, 10-5 M); 2) L-type calcium channels (Verapamil, 10-5 M); 3) Ryanodine receptor, RyR (Ryanodine, 10-5 M); 4) inositol 1,4,5-triphosphate receptor, IP3R, (2-Aminoethyl diphenylborinate, 2-APB, 3 × 10-3 M); 5) phospholipase C, PLC, (U73122, 10-5 M), and 6) GPCR-Gαi signaling, (Pertussis Toxin, 10-5 M). EVOO phenols induced vasodilation of mesenteric arteries in a dose-dependent manner, and this effect was reduced by pre-incubation with Paxillin, Verapamil, Ryanodine, 2-APB, U73122, and Pertussis Toxin. Our data suggest that EVOO phenol-mediated vasodilation requires activation of BKca channels potentially through a local increase of subcellular calcium microdomains, a pivotal mechanism on the base of artery vasodilation. These findings provide novel mechanistic insights for understanding the vasodilatory properties of EVOO phenols on resistance arteries.

Glabridin Relaxes Vascular Smooth Muscles by Activating BKCa Channels and Inhibiting Phosphodiesterase in Human Saphenous Vein.

The aim of the current study was to investigate the pharmacological activity of glabridin on the isolated human saphenous vein (SV) and explore the underlying mechanisms. Samples of patients' SVs were removed during bypass surgery, and 4-mm lengths of the vessels were placed in Krebs solution at +4°C and hung in an isolated organ bath to assess their contraction/relaxation responses. The contraction/relaxation responses were recorded to observe if the cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway mediates the relaxant effect of glabridin after treatment with blockers like ODQ (a guanylate cyclase inhibitor), KT5823 (a PKG inhibitor), isobutylmethylxanthine [IBMX, a phosphodiesterase (PDE) inhibitor], and cantharidin [Cant, a myosin light-chain phosphatase (MLCP) inhibitor]. Moreover, nitric oxide (NO), cGMP, and PKG levels in SV tissues were determined by ELISA after incubation with glabridin, N(ω)-nitro-L-arginine methyl ester (L-Name, a NO synthetase inhibitor), phenylephrine (PE), ODQ, IBMX, and KT5823. The results showed that glabridin relaxed the vascular smooth muscle of human SV pretreated with PE in a dose-dependent manner, which was independent of the endothelium. The vasorelaxant effect of glabridin was only inhibited by iberiotoxin (IbTX), Cant, and KT5823. Glabridin increased cGMP and PKG levels in SV homogenates, whereas it did not alter the NO level. The enhancing effects of cGMP and PKG levels by glabridin were abolished by ODQ and KT5823. In conclusion, glabridin has a vasorelaxant effect, which is associated with the activation of BKCa channels and inhibition of PDE.