IKCa Channels Research Articles

Role of cGMP and EDHF Pathways in Hydrogen Sulfide‐induced Vasorelaxation in the Human Artery

BackgroundEndogenous H2S generated by mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT) in endothelium as a physical vasodilator and blood pressure regulator may play an important protective role in the pathogenesis of coronary heart disease. The present study aimed to investigate the role of cGMP and endothelium‐derived hyperpolarizing factor (EDHF) in H2S‐induced vasorelaxation in the human internal mammary artery (IMA).MethodsIn the human IMA segments from 54 patients undergoing coronary artery bypass grafting, the acetylcholine (ACh)‐induced relaxation and the effect of CAT inhibitor (aminooxyacetic acid [AOAA] and L‐aspartate) and KCa channel blocker or KATP channel blocker were studied in a myograph. Enzyme‐linked Immunosorbent Assay for PDE5A and western blotting analysis for eNOS and p‐eNOSser1177 were performed. Methylene blue assay was used to quantify the contents of H2S.ResultsThe maximal relaxation induced by ACh was significantly attenuated by AOAA or L‐aspartate, Iberiotixin (BKCa channel blocker) (P<0.01), or IKCa channel blocker plus SKCa channel blocker (TRAM‐34+Apamin) (P<0.01), or KATP channel blocker (Glibenclamide) (P<0.01). The H2S donor NaHS increased eNOS phosphorylation at its activating site S1177 and down‐regulated PDE5A activity. Incubation with ACh for 4 hours significantly increased the H2S concentration.ConclusionsWe for the first time demonstrated in the human IMA that endogenous H2S could be produced under the stimulation of ACh and that CAT is one of the key enzymes in production of H2S in the endothelial cell of the IMA. H2S may activate eNOS to produce NO that activates PKG and may also inhibit PDE5A activity; both enhance the cGMP pathway‐mediated relaxation. Further, H2S as a potential EDHF directly stimulates KCa channels to induce vasodilatation. These findings may provide new therapeutical targets for the treatment of vasospasm of coronary artery bypass grafting vessels and provide a pharmacological basis for the development of new vasodilator drugs.Support or Funding InformationSupported by grants from the National Natural Science Foundation of China (81170148 & 81641017), Zhejiang Provincial Natural Science Foundation (LY15H020008), & Tianjin Binhai Key Platform for Creative Research Program (2012‐BK110004), Binhai New Area Health Bureau (2012BWKZ008, 2016BWKY007, 2016BWKZ003).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vasoconstrictor stimulus determines the functional contribution of myoendothelial feedback to mesenteric arterial tone.

In isolated resistance arteries, endothelial modulation of vasoconstrictor responses to α1 -adrenoceptor agonists occurs via a process termed myoendothelial feedback: localized inositol trisphosphate (InsP3 )-dependent Ca2+ transients activate intermediate conductance Ca2+ -activated K+ (IKCa ) channels, hyperpolarizing the endothelial membrane potential to limit further reductions in vessel diameter. We demonstrate that IKCa channel-mediated myoendothelial feedback limits responses of isolated mesenteric arteries to noradrenaline and nerve stimulation, but not to the thromboxane A2 mimetic U46619 or to increases in intravascular pressure. In contrast, in the intact mesenteric bed, although responses to exogenous noradrenaline were limited by IKCa channel-mediated myoendothelial feedback, release of NO and activation of endothelial small conductance Ca2+ -activated K+ (SKCa ) channels in response to increases in shear stress appeared to be the primary mediators of endothelial modulation of vasoconstriction. We propose that (1) the functional contribution of myoendothelial feedback to arterial tone is determined by the nature of the vasoconstrictor stimulus, and (2) although IKCa channel-mediated myoendothelial feedback may contribute to local control of arterial diameter, in the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction. Constriction of isolated resistance arteries in response to α1 -adrenoceptor agonists is limited by reciprocal engagement of inhibitory endothelial mechanisms via myoendothelial feedback. In the current model of feedback, agonist stimulation of smooth muscle cells results in localized InsP3 -dependent Ca2+ transients that activate endothelial IKCa channels. The subsequent hyperpolarization of the endothelial membrane potential then feeds back to the smooth muscle to limit further reductions in vessel diameter. We hypothesized that the functional contribution of InsP3 -IKCa channel-mediated myoendothelial feedback to limiting arterial diameter may be influenced by the nature of the vasoconstrictor stimulus. To test this hypothesis, we investigated the functional role of myoendothelial feedback in modulating responses of rat mesenteric resistance arteries to the adrenoceptor agonist noradrenaline, the thromboxane A2 mimetic U46619, increases in intravascular pressure and stimulation of perivascular sympathetic nerves. In isolated arteries, responses to noradrenaline and stimulation of sympathetic nerves, but not to U46619 and increases in intravascular pressure, were modulated by IKCa channel-dependent myoendothelial feedback. In the intact mesenteric bed perfused under conditions of constant flow, responses to exogenous noradrenaline were modulated by myoendothelial feedback, but shear stress-induced release of NO and activation of endothelial SKCa channels appeared to be the primary mediators of endothelial modulation of vasoconstriction to agonists and nerve stimulation. Thus, we propose that myoendothelial feedback may contribute to local control of diameter within arterial segments, but at the level of the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction.

Diabetes modifies the role of prostanoids and potassium channels which regulate the hypereactivity of the rabbit renal artery to BNP.

Diabetic nephropathy is associated with increased risk of cardiovascular disease. B-type natriuretic peptide (BNP) plays an important role in cardiovascular pathophysiology and therapeutics. The aim of the present study was to investigate the influence of experimental diabetes on the mechanisms that regulate the relaxant response of the rabbit renal artery to BNP. Arterial relaxations to BNP were enhanced in diabetic rabbits. Indomethacin enhanced BNP-induced relaxation in control rabbits but showed no effect in diabetic rabbits. BNP-induced release of thromboxane A2 or prostacyclin was not different in both groups of animals. Iberiotoxin had no effect on relaxations to BNP in both groups of animals. Charybdotoxin displaced to the right the concentration-response curve to BNP in both group of animals, and inhibited BNP-induced relaxation only in diabetic rabbits. Glibenclamide did not modify the BNP-induced relaxations in control rabbits, but inhibited it in diabetic rabbits. These results suggest that diabetes induces hypereactivity of the rabbit renal artery to BNP by mechanisms that at least include (1) a reduced vasoconstrictor influence of arachidonic acid metabolites via cyclooxygenase 2, which is not related with changes in thromboxane A2 and prostacyclin release from the arterial wall and (2) a selectively increased modulatory activity of KATP and endothelial IKCa channels.

Endothelial-dependent dilation following chronic hypoxia involves TRPV4-mediated activation of endothelial BK channels

Following chronic hypoxia (CH), the systemic vasculature exhibits blunted vasoconstriction due to endothelial-dependent hyperpolarization (EDH). Previous data demonstrate that subsequent to CH, EDH-mediated vasodilation switches from a reliance on SKca and IKca channels to activation of the endothelial BKca channels (eBK). The mechanism by which endothelial cell stimulation activates eBK channels following CH is not known. We hypothesized that following CH, EDH-dependent vasodilation involves a TRPV4-dependent activation of eBK channels. ACh induced concentration-dependent dilation in pressurized gracilis arteries from both normoxic and CH rats. Inhibition of TRPV4 (RN-1734) attenuated the ACh response in arteries from CH rats but had no effect in normoxic animals. In the presence of L-NNA and indomethacin, TRPV4 blockade attenuated ACh-induced vasodilation in arteries from CH rats. ACh elicited endothelial TRPV4-mediated Ca2+ events in arteries from both groups. GSK1016790A (GSK101, TRPV4 agonist) elicited vasodilation in arteries from normoxic and CH rats. In arteries from normoxic animals, TRAM-34/apamin abolished the dilation to TRPV4 activation, whereas luminal iberiotoxin had no effect. In CH rats, only administration of all three Kca channel inhibitors abolished the dilation to TRPV4 activation. Using Duolink®, we observed co-localization between Cav-1, TRPV4, and BK channels in gracilis arteries and in RAECs. Disruption of endothelial caveolae with methyl-β-cyclodextrin significantly decreased ACh-induced vasodilation in arteries from both groups. In gracilis arteries, endothelial membrane cholesterol was significantly decreased following 48 h of CH. In conclusion, CH results in a functional coupling between muscarinic receptors, TRPV4 and Kca channels in gracilis arteries.

β-adrenergic Receptor Blocker ICI 118,551 Selectively Increases Intermediate-Conductance Calcium-Activated Potassium Channel (IKCa )-Mediated Relaxations in Rat Main Mesenteric Artery.

Endothelial IKCa and/or SKCa channels play an important role in the control of vascular tone by participating in endothelium-dependent relaxation. Whether β-AR antagonists, mainly used in hypertension, affect endothelial KCa channel function is unknown. In this study, we examined the effect of the β2-AR antagonist and inverse agonist ICI 118,551 on the IKCa /SKCa channel activity by assessing functional relaxation responses to several agonists that stimulate these channels. Mesenteric arterial rings isolated from male Sprague Dawley mounted to organ baths. Acetylcholine elicited IKCa - and SKCa -mediated relaxations that were abolished by TRAM-34 and apamin, respectively. ICI 118,551, which did not dilate the arteries per se, increased the IKCa -mediated relaxations, whereas SKCa -mediated relaxations remained unaltered. Same potentiating effect was also detected on the IKCa -mediated relaxations to carbachol and A23187, but not to NS309. Neither acetylcholine-induced nitric oxide-mediated relaxations nor SNP relaxations changed with ICI 118,551. The PKA inhibitor KT-5720, the selective β2-AR agonist salbutamol, the selective β2-AR antagonist butoxamine, the non-selective β-AR antagonist propranolol, and the inverse agonists carvedilol or nadolol failed to affect the IKCa -mediated relaxations. ICI 118,551-induced increase was not reversed by salbutamol or propranolol as well. Besides, low potassium-induced relaxations in endothelium-removed arteries remained the same in the presence of ICI 118,551. These data demonstrate a previously unrecognized action of ICI 118,551, the ability to potentiate endothelial IKCa channel-mediated vasodilation, through a mechanism independent of β2-AR antagonistic or inverse agonistic action. Instead, the enhancement of acetylcholine relaxation seems likely to occur by a mechanism secondary to endothelial calcium increase.

Possible mechanisms of diverse spaceflight effects on endothelial function in different vascular beds

Possible mechanisms of diverse spaceflight effects on endothelial function in different vascular beds

Impaired coronary arteriolar function after cardioplegic-ischemia/reperfusion in pig with metabolic syndrome

Introduction Metabolic syndrome (MetS) is associated with inactivation of coronary endothelial small/intermediate (SK Ca /IK Ca ) conductance calcium-activated potassium channels and dysregulation of coronary arteriolar endothelial function in animals and humans. We investigated the effects of cardioplegia-ischemia/reperfusion (I/R) and NS309 pretreatment on the in-vitro coronary arteriolar responses to endothelium-dependent vasodilators substance P and ADP in pigs with or without MetS. Case description The MetS pigs were developed by feeding with a hyper-caloric, fat/cholesterol diet and the control animals fed with a regular diet for 12 weeks (n=8/group). Coronary arterioles (90-180 micrometers in diameter) were dissected from the harvested left ventricle tissue sample of pigs with and without MetS. The changes in diameter were measured with video microscopy. Microvessel was perfused in the presence or absence of selective SK Ca /IK Ca activator NS309 (10 -5 M). The in-vitro coronary arterioles were then subjected to 60 minutes of cardioplegia-hypoxia (15°C) and 60 minutes of re-oxygenation. Results and Conclusions At the end of reperfusion, the microvessel was treated with the endothelium-dependent vasodilators substance P and ADP. The relaxation responses to the substance P and ADP after cardioplegia-I/R were significantly decreased in MetS vessels versus control (Lean), respectively (P Ca /IK Ca activator NS309 (10 -5 M) significantly improved the recovery of coronary endothelial function showing increased response to substance P and ADP as compared with no pretreatment alone (P Take home message This study demonstrates that cardioplegic-ischemia/reperfusion impairs endothelial function and inactivation of endothelial SK Ca /IK Ca channels of the coronary microcirculation in the setting of metabolic syndrome.

Rapid versus slow ascending vasodilatation: intercellular conduction versus flow-mediated signalling with tetanic versus rhythmic muscle contractions.

In response to exercise, vasodilatation ascends from downstream arterioles into upstream feed arteries (FAs). We hypothesized that the signalling events underlying ascending vasodilatation variy with the intensity and duration of skeletal muscle contraction. In the gluteus maximus muscle of C57BL/6 mice, brief tetanic contraction evoked rapid onset vasodilatation (ROV) (<1s) throughout the resistance network. Selective damage to endothelium midway between FAs and primary arterioles eliminated ROV only in FAs. Blocking SKCa and IKCa channels attenuated ROV, implicating hyperpolarization as the underlying signal. During rhythmic twitch contractions, slow onset vasodilatation (10-15s) in FAs remained intact following loss of ROV and was eliminated following nitric oxide synthase inhibition. Tetanic contraction initiates hyperpolarization that conducts along endothelium into FAs. Rhythmic twitch contractions stimulate FA endothelium to release nitric oxide in response to elevated shear stress secondary to metabolic dilatation of arterioles. Complementary endothelial signalling pathways for ascending vasodilatation ensure increased oxygen delivery to active skeletal muscle. In response to exercise, vasodilatation initiated within the microcirculation of skeletal muscle ascends the resistance network into upstream feed arteries (FAs) located external to the tissue. Ascending vasodilatation (AVD) is essential for reducing FA resistance that otherwise restricts blood flow into the microcirculation. In the present study, we tested the hypothesis that signalling events underlying AVD vary with the intensity and duration of muscle contraction. In the gluteus maximus muscle of anaesthetized male C57BL/6 mice (aged 3-4months), brief tetanic contraction (100Hz for 500ms) evoked rapid onset vasodilatation (ROV) in FAs that peaked within 4s. By contrast, during rhythmic twitch contractions (4Hz), slow onset vasodilatation (SOV) of FAs began after ∼10s and plateaued within 30s. Selectively damaging the endothelium with light-dye treatment midway between a FA and its primary arteriole eliminated ROV in the FA along with conducted vasodilatation of the FA initiated on the arteriole using ACh microiontophoresis. Superfusion of SKCa and IKCa channel blockers UCL 1684+TRAM 34 attenuated ROV, implicating endothelial hyperpolarization as the underlying signal. Nevertheless, the SOV of FAs during rhythmic contractions persisted until inhibition of nitric oxide synthase with Nω -nitro-l-arginine methyl ester. Thus, ROV of FAs reflects hyperpolarization of downstream arterioles that conducts along the endothelium into proximal FAs. By contrast, SOV of FAs reflects the local production of nitric oxide by the endothelium in response to luminal shear stress, which increases secondary to arteriolar dilatation downstream. Thus, AVD ensures increased oxygen delivery to active muscle fibres by reducing upstream resistance via complementary signalling pathways that reflect the intensity and duration of muscle contraction.

The Natural Flavone Acacetin Blocks Small Conductance Ca2+-Activated K+ Channels Stably Expressed in HEK 293 Cells.

The natural flavone acacetin inhibits several voltage-gated potassium currents in atrial myocytes, and has anti-atrial fibrillation (AF) effect in experimental AF models. The present study investigates whether acacetin inhibits the Ca2+-activated potassium (KCa) currents, including small conductance (SKCa1, SKCa2, and SKCa3), intermediate conductance (IKCa), and large-conductance (BKCa) channels stably expressed in HEK 293 cells. The effects of acacetin on these KCa channels were determined with a whole-cell patch voltage-clamp technique. The results showed that acacetin inhibited the three subtype SKCa channel currents in concentration-dependent manner with IC50 of 12.4 μM for SKCa1, 10.8 μM for SKCa2, and 11.6 μM for SKCa3. Site-directed mutagenesis of SKCa3 channels generated the mutants H490N, S512T, H521N, and A537V. Acacetin inhibited the mutants with IC50 of 118.5 μM for H490N, 275.2 μM for S512T, 15.3 μM for H521N, and 10.6 μM for A537V, suggesting that acacetin interacts with the P-loop helix of SKCa3 channel. However, acacetin at 3–10 μM did not decrease, but induced a slight increase of BKCa (+70 mV) by 8% at 30 μM. These results demonstrate the novel information that acacetin remarkably inhibits SKCa channels, but not IKCa or BKCa channels, which suggests that blockade of SKCa by acacetin likely contributes to its anti-AF property previously observed in experimental AF.

Heteromeric TRPV4/TRPC1 channels mediate calcium-sensing receptor-induced nitric oxide production and vasorelaxation in rabbit mesenteric arteries

Stimulation of calcium-sensing receptors (CaSR) by increasing the external calcium concentration (Ca2+]o) induces endothelium-dependent vasorelaxation through nitric oxide (NO) production and activation of intermediate Ca2+-activated K+ currents (IKCa) channels in rabbit mesenteric arteries. The present study investigates the potential role of heteromeric TRPV4-TRPC1 channels in mediating these CaSR-induced vascular responses. Immunocytochemical and proximity ligation assays showed that TRPV4 and TRPC1 proteins were expressed and co-localised at the plasma membrane of freshly isolated endothelial cells (ECs). In wire myography studies, increasing [Ca2+]o between 1 and 6mM induced concentration-dependent relaxations of methoxamine (MO)-induced pre-contracted tone, which were inhibited by the TRPV4 antagonists RN1734 and HC067047, and the externally-acting TRPC1 blocking antibody T1E3. In addition, CaSR-evoked NO production in ECs measured using the fluorescent NO indicator DAF-FM was reduced by RN1734 and T1E3. In contrast, [Ca2+]o-evoked perforated-patch IKCa currents in ECs were unaffected by RN1734 and T1E3. The TRPV4 agonist GSK1016790A (GSK) induced endothelium-dependent relaxation of MO-evoked pre-contracted tone and increased NO production, which were inhibited by the NO synthase inhibitor L-NAME, RN1734 and T1E3. GSK activated 6pS cation channel activity in cell-attached patches from ECs which was blocked by RN1734 and T1E3. These findings indicate that heteromeric TRPV4-TRPC1 channels mediate CaSR-induced vasorelaxation through NO production but not IKCa channel activation in rabbit mesenteric arteries. This further implicates CaSR-induced pathways and heteromeric TRPV4-TRPC1 channels in regulating vascular tone.

Involvement of dysregulated IKCa and SKCa channels in preeclampsia

IntroductionExcessive constriction of placental chorionic plate arteries (CPAs) may be associated with preeclampsia (PE). Nitric oxide (NO) as well as intermediate and small Ca2+-activated K+ channels (IKCa and SKCa) plays vital roles in vasodilation of CPAs. We hypothesized that dysregulated IKCa and SKCa channels may be involved in the pathogenesis of PE mediated by the impaired NO system on CPAs. MethodsThe location of IKCa and SKCa channels, activities of NO synthases (NOS), and expression levels of these molecules were studied on CPAs from 30 normal pregnancies and 30 PE. The vasodilating function of CPAs was measured under openers or blockers of IKCa/SKCa channels in the presence or absence of NO donor or inhibitor. ResultsIKCa and SKCa channels were located both on endothelium and on smooth muscles of CPAs and the expressions of them were downregulated in PE women comparing to those in normal pregnant women. The protein expressions of endothelial NOS (eNOS) and inducible NOS (iNOS) were downregulated on CPAs in PE accompanied by decreased activity of eNOS. Notably, the vasodilatory functions mediated by IKCa/SKCa channels and by NO were aberrant on preeclamptic CPAs. In addition, IKCa and SKCa channels were responsible for nitric oxide (NO)-attributable vasorelaxation and activity modulation of NO synthases. ConclusionsThis study provides evidence that dysregulated IKCa and SKCa channels might contribute to fetal pathogenesis of PE through direct promotion of vascular constriction of CPAs and through affecting functions of NO and activities of NOS.

Endothelium-dependent vasorelaxant effect of procyanidin B2 on human internal mammary artery

The aim of the present study was to investigate and characterize vasorelaxant effect of procyanidin B2 on human internal mammary artery (HIMA) as one of the mechanisms of its protective effect against vascular risk. Procyanidin B2 induced strong concentration-dependent relaxation of HIMA rings pre-contracted by phenylephrine. Pretreatment with L-NAME, a NO synthase inhibitor, hydroxocobalamin, a NO scavenger, and ODQ, an inhibitor of soluble guanylate cyclase, significantly inhibited procyanidin B2-induced relaxation of HIMA, while indomethacin, a cyclooxygenase inhibitor, considerably reduced effects of low concentrations. Among K+ channel blockers, iberiotoxin, a selective blocker of large conductance Ca2+-activated K+ channels (BKCa), abolished procyanidin B2-induced relaxation, glibenclamide, a selective ATP-sensitive K+(KATP) channels blocker, induced partial inhibition, while 4-aminopyridine, a blocker of voltage-gated K+(KV) channels, and TRAM-34, an inhibitor of intermediate-conductance Ca2+-activated K+(IKCa) channels, slightly reduced maximal relaxation of HIMA. Further, procyanidin B2 relaxed contraction induced by phenylephrine in Ca2+-free Krebs solution, but had no effect on contraction induced by caffeine. Finally, thapsigargin, a sarcoplasmic reticulum Ca2+-ATPase inhibitor, significantly reduced relaxation of HIMA produced by procyanidin B2. These results demonstrate that procyanidin B2 produces endothelium-dependent relaxation of HIMA pre-contracted by phenylephrine. This effect is primarily the result of an increased NO synthesis and secretion by endothelial cells and partially of prostacyclin, although it involves activation of BKCa and KATP, as well as KV and IKCa channels in high concentrations of procyanidin B2.

Intermediate-Conductance-Ca2-Activated K Channel IKCa1 Is Upregulated and Promotes Cell Proliferation in Cervical Cancer

BackgroundAccumulating data point to intermediate-conductance calcium-activated potassium channel (IKCa1) as a key player in controlling cell cycle progression and proliferation of human cancer cells. However, the role that IKCa1 plays in the growth of human cervical cancer cells is largely unexplored.Material/MethodsIn this study, Western blot analysis, immunohistochemical staining, and RT-PCR were first used for IKCa1protein and gene expression assays in cervical cancer tissues and HeLa cells. Then, IKCa1 channel blocker and siRNA were employed to inhibit the functionality of IKCa1 and downregulate gene expression in HeLa cells, respectively. After these treatments, we examined the level of cell proliferation by MTT method and measured IKCa1 currents by conventional whole-cell patch clamp technique. Cell apoptosis was assessed using the Annexin V-FITC/Propidium Iodide (PI) double-staining apoptosis detection kit.ResultsWe demonstrated that IKCa1 mRNA and protein are preferentially expressed in cervical cancer tissues and HeLa cells. We also showed that the IKCa1 channel blocker, clotrimazole, and IKCa1 channel siRNA can be used to suppress cervical cancer cell proliferation and decrease IKCa1 channel current. IKCa1 downregulation by specific siRNAs induced a significant increase in the proportion of apoptotic cells in HeLa cells.ConclusionsIKCa1 is overexpressed in cervical cancer tissues, and IKCa1 upregulation in cervical cancer cell linea enhances cell proliferation, partly by reducing the proportion of apoptotic cells.

DCEBIO facilitates myogenic differentiation via intermediate conductance Ca2+ activated K+ channel activation in C2C12 myoblasts

Membrane hyperpolarization is suggested to be a trigger for skeletal muscle differentiation. We investigated whether DCEBIO, an opener of the small/intermediate conductance Ca2+ activated K+ (SKCa/IKCa) channels, increase myogenic differentiation in C2C12 skeletal myoblasts. DCEBIO significantly increased myotube formation, protein expression level of myosin heavy chain II, and mRNA expression level of myogenin in C2C12 myoblasts cultured in differentiation medium. DCEBIO induced myotube formation and hyperpolarization were reduced by the IKCa channel blocker TRAM-34, but not by the SKCa channel blocker apamin. These findings show that DCEBIO increases myogenic differentiation by activating IKCa channels.

The endogenous lipid N-arachidonoyl glycine is hypotensive and nitric oxide-cGMP-dependent vasorelaxant

N-arachidonoyl glycine (NAGLY), is the endogenous lipid that activates the G protein-couple receptor 18 (GPR18) with vasodilatory activity in resistance arteries. This study investigates its hemodynamic effects and mechanisms of vasorelaxation. Hemodynamic effects of NAGLY in rats were assessed using a Biopac system and its vascular responses were assessed using a wire myograph. NAGLY (1mg/kg) decreased blood pressure by 69.4±5.5% and reduced renal blood flow by 88±12% and the effects were not sensitive to inhibition by O-1918 (3mg/kg). In resistant vessels, NAGLY (1–30µM) induced concentration- and endothelium-dependent vasorelaxation and the effect was inhibited by the nitric oxide synthase inhibitor, L-NAME (300µM), a cGMP synthase inhibitor, ODQ (10µM), the antagonists of “endothelial anandamide” receptor, rimonabant (3µM) and O-1918 (10µM) and the inhibitor of Na+/Ca2+ exchanger (NCX), KB-R7943 (10µM). On the other hand, NAGLY-induced vasorelaxation was not affected by CID 16020046 (GPR55 antagonist), AM 251 (cannabinoid CB1 receptor antagonist), AM 630 (cannabinoid CB2 receptor antagonist), capsazepine (TRPV1 antagonist), indomethacin (cyclooxygenase inhibitor), TRAM34 (IKCa channel blocker), iberiotoxin (BKCa channel blocker) and GW9662 (PPARɤ antagonist). At low concentrations of carbachol, NAGLY potentiated carbachol-induced vasorelaxation. NAGLY is an endothelium-dependent vasodilator and hypotensive lipid. The vasorelaxation is predominantly via activation of nitric oxide-cGMP pathway and NCX and probably mediated by the "endothelial anandamide” receptor, while the hypotensive effect of NAGLY appears not to involve the anandamide receptor. NAGLY also potentiates carbachol-induced vasorelaxation, the mechanism of which might involve stimulation of NO release.

PHARMACOLOGICAL CHARACTERIZATION OF THE FUNCTIONAL ROLE OF CALCIUM-ACTIVATED POTASSIUM CHANNELS IN PLATELETS

In arteries, stimulation of endothelial cell small (SKCa) and intermediate (IKCa) conductance calcium-activated potassium channels provides a negative-feedback mechanism to limit agonist-induced vasoconstriction. Additionally, endothelial cell KCa channels in conjunction with nitric oxide (NO) mediate vasodilation in response to agonists and physical stimuli such as increases in blood flow. Platelets, like endothelial cells, possess KCa channels and have the capability to generate NO via endothelial nitric oxide synthase (eNOS). NO is known to limit platelet aggregation but the role of KCa channels in platelet function and NO-generation has not been explored. Our objective was to pharmacologically characterize SKCa and IKCa channel function in platelets, and to investigate their role in platelet NO production. Our hypothesis was that pharmacological activation of KCa channels would inhibit platelet aggregation and enhance platelet NO production.

Altered calcium influx of peripheral Th2 cells in pediatric Crohn's disease: infliximab may normalize activation patterns.

ObjectiveCrohn's disease is a chronic inflammation of the gastrointestinal tract with an abnormal immune phenotype. We investigated how intracellular calcium kinetics of Th1 and Th2 lymphocytes alter upon specific inhibition of Kv1.3 and IKCa1 channels in pediatric Crohn's disease.Study designBlood was taken from 12 healthy and 29 Crohn's disease children. Of those, 6 were switched to infliximab and re-sampled after the 4th infliximab treatment. Intracellular calcium levels were monitored using flow cytometry in the presence or absence of specific inhibitors of Kv1.3 and IKCa1 potassium channels.ResultsIn Crohn's disease treated with standard therapy, calcium response during activation was higher than normal in Th2 cells. This was normalized in vitro by inhibition of Kv1.3 or IKCa1 potassium channels. After the switch to infliximab, potassium channel function and expression in Th2 lymphocytes were comparable to those in Th1 cells.ConclusionThese results may indicate that potassium channels are potential immune modulatory targets in Crohn's disease.

Calcium influx kinetics, and the features of potassium channels of peripheral lymphocytes in primary Sjögren’s syndrome

ObjectiveThe transient increase of the cytoplasmic free calcium level plays a key role in the process of lymphocyte activation. Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx and present a possible target for selective immunomodulation. DesignCase–control study. Subjects and methodsWe took peripheral blood samples from 8 healthy individuals and 15 primary Sjögren’s syndrome (pSS) patients. We evaluated calcium influx kinetics following activation in peripheral T lymphocytes. We also assessed the sensitivity of T lymphocytes to specific inhibition of the Kv1.3 and IKCa1 potassium channels, and the Kv1.3 channel expression. ResultsThe basal cytoplasmatic calcium levels were lower in both Th1 and Th2 lymphocytes in pSS compared to controls. The peak of calcium influx in lymphocytes isolated from pSS patients is reached later, indicating that they respond more slowly to stimulation compared to controls. In healthy individuals, the inhibition of the IKCa1 channel decreased calcium influx in Th2 and CD4 cells to a lower extent than in Th1 and CD8 cells. On the contrary, the inhibition of Kv1.3 channels resulted in a larger decrease of calcium entry in Th2 and CD4 than in Th1 and CD8 cells. In the pSS group, neither of the inhibitors induced alteration in calcium influx. Expression of Kv1.3 channels on CD4, Th2 and CD8 lymphocytes in pSS was significantly higher compared to controls. ConclusionThe altered expression and specific inhibition of potassium channels seem to be related to altered calcium influx kinetics in pSS which distinguish pSS either from healthy controls or other systemic autoimmune diseases.

Assessing the role of IKCa channels in generating the sAHP of CA1 hippocampal pyramidal cells

ABSTRACTOur previous work reported that KCa3.1 (IKCa) channels are expressed in CA1 hippocampal pyramidal cells and contribute to the slow afterhyperpolarization that regulates spike accommodation in these cells. The current report presents data from single cell RT-PCR that further reveals mRNA in CA1 cells that corresponds to the sequence of an IKCa channel from transmembrane segments 5 through 6 including the pore region, revealing the established binding sites for 4 different IKCa channel blockers. A comparison of methods to internally apply the IKCa channel blocker TRAM-34 shows that including the drug in an electrode from the onset of an experiment is unviable given the speed of drug action upon gaining access for whole-cell recordings. Together the data firmly establish IKCa channel expression in CA1 neurons and clarify methodological requirements to obtain a block of IKCa channel activity through internal application of TRAM-34.

Contribution of K(+) channels to endothelium-derived hypolarization-induced renal vasodilation in rats in vivo and in vitro.

We investigated the mechanisms behind the endothelial-derived hyperpolarization (EDH)-induced renal vasodilation in vivo and in vitro in rats. We assessed the role of Ca(2+)-activated K(+) channels and whether K(+) released from the endothelial cells activates inward rectifier K(+) (Kir) channels and/or the Na(+)/K(+)-ATPase. Also, involvement of renal myoendothelial gap junctions was evaluated in vitro. Isometric tension in rat renal interlobar arteries was measured using a wire myograph. Renal blood flow was measured in isoflurane anesthetized rats. The EDH response was defined as the ACh-induced vasodilation assessed after inhibition of nitric oxide synthase and cyclooxygenase using L-NAME and indomethacin, respectively. After inhibition of small conductance Ca(2+)-activated K(+) channels (SKCa) and intermediate conductance Ca(2+)-activated K(+) channels (IKCa) (by apamin and TRAM-34, respectively), the EDH response in vitro was strongly attenuated whereas the EDH response in vivo was not significantly reduced. Inhibition of Kir channels and Na(+)/K(+)-ATPases (by ouabain and Ba(2+), respectively) significantly attenuated renal vasorelaxation in vitro but did not affect the response in vivo. Inhibition of gap junctions in vitro using carbenoxolone or 18α-glycyrrhetinic acid significantly reduced the endothelial-derived hyperpolarization-induced vasorelaxation. We conclude that SKCa and IKCa channels are important for EDH-induced renal vasorelaxation in vitro. Activation of Kir channels and Na(+)/K(+)-ATPases plays a significant role in the renal vascular EDH response in vitro but not in vivo. The renal EDH response in vivo is complex and may consist of several overlapping mechanisms some of which remain obscure.