Large-conductance KCa Channels Research Articles

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.

A biophysically detailed computational model of urinary bladder small DRG neuron soma.

Bladder small DRG neurons, which are putative nociceptors pivotal to urinary bladder function, express more than a dozen different ionic membrane mechanisms: ion channels, pumps and exchangers. Small-conductance Ca2+-activated K+ (SKCa) channels which were earlier thought to be gated solely by intracellular Ca2+ concentration ([Ca]i) have recently been shown to exhibit inward rectification with respect to membrane potential. The effect of SKCa inward rectification on the excitability of these neurons is unknown. Furthermore, studies on the role of KCa channels in repetitive firing and their contributions to different types of afterhyperpolarization (AHP) in these neurons are lacking. In order to study these phenomena, we first constructed and validated a biophysically detailed single compartment model of bladder small DRG neuron soma constrained by physiological data. The model includes twenty-two major known membrane mechanisms along with intracellular Ca2+ dynamics comprising Ca2+ diffusion, cytoplasmic buffering, and endoplasmic reticulum (ER) and mitochondrial mechanisms. Using modelling studies, we show that inward rectification of SKCa is an important parameter regulating neuronal repetitive firing and that its absence reduces action potential (AP) firing frequency. We also show that SKCa is more potent in reducing AP spiking than the large-conductance KCa channel (BKCa) in these neurons. Moreover, BKCa was found to contribute to the fast AHP (fAHP) and SKCa to the medium-duration (mAHP) and slow AHP (sAHP). We also report that the slow inactivating A-type K+ channel (slow KA) current in these neurons is composed of 2 components: an initial fast inactivating (time constant ∼ 25-100 ms) and a slow inactivating (time constant ∼ 200-800 ms) current. We discuss the implications of our findings, and how our detailed model can help further our understanding of the role of C-fibre afferents in the physiology of urinary bladder as well as in certain disorders.

Probucol prevents the attenuation of β2-adrenoceptor-mediated vasodilation of retinal arterioles in diabetic rats.

Probucol is an antihyperlipidemic drug with potent antioxidant properties. Oxidative stress plays an important role in the pathogenesis of diabetic retinopathy. In this study, we aimed to investigate the protective effects of probucol against diabetes-induced retinal vascular dysfunction in a rat model of diabetes. Diabetes was induced by a combination of streptozotocin treatment and D-glucose feeding, and retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles. The vasodilator effect of salbutamol, a β2-adrenoceptor agonist, on retinal arterioles was significantly diminished 2weeks after the induction of diabetes. In non-diabetic rats, vasodilator responses to salbutamol were significantly reduced after an intravitreal injection of iberiotoxin, a blocker of large-conductance KCa (BKCa) channels. However, this effect was not observed in diabetic rats. Probucol had no significant effect on salbutamol-induced changes in diameter of retinal arterioles in non-diabetic rats, whereas it could prevent the attenuation of retinal vasodilator response to salbutamol in diabetic rats. These results suggest that the reduced function of BKCa channels is involved in the attenuation of β2-adrenoceptor-mediated retinal vasodilation in diabetic rats. Probucol preserves the BKCa channel function in retinal arterioles under diabetic conditions; therefore, it may show beneficial effects on diabetic retinopathy by preventing or slowing the impairment of the retinal circulation in patients with diabetes mellitus.

CNTF-Treated Astrocyte Conditioned Medium Enhances Large-Conductance Calcium-Activated Potassium Channel Activity in Rat Cortical Neurons.

Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM's effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM's effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM's effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p<0.05). CNTF-ACM produced a significant increase in BKα1 and BKβ3 expression (p<0.05) but had no significant effect upon SK2 or SK3 expression (p>0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p>0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p>0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons.

4-Hydroxy-2-nonenal attenuates β2-adrenoceptor-mediated vasodilation of rat retinal arterioles

4-Hydroxy-2-nonenal (4-HNE) is a major reactive aldehyde formed by lipid peroxidation, and it plays an important role in the pathogenesis of several vascular diseases, including diabetes mellitus. In this study, we examined the effects of 4-HNE on the vasodilatory mechanisms of rat retinal arterioles. The retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles in the fundus images. Intravitreal injection of 4-HNE significantly prevented the vasodilation of retinal arterioles induced by the β2-adrenoceptor agonist salbutamol but not the nitric oxide donor (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3). Iberiotoxin, an inhibitor of large-conductance KCa (BKCa) channels, significantly reduced the salbutamol-induced vasodilation of retinal arterioles. The vasodilator effect of BMS-191011, a BKCa channel opener, on retinal arterioles was significantly attenuated by 4-HNE. These results suggest that 4-HNE attenuates retinal vasodilator responses to β2-adrenoceptor agonists through the impairment of the BKCa pathway. The direct effect of 4-HNE on retinal blood vessels may, therefore, contribute to the retinal vascular dysfunction observed in patients with diabetes mellitus.

Inhibitory actions by ibandronate sodium, a nitrogen-containing bisphosphonate, on calcium-activated potassium channels in Madin-Darby canine kidney cells.

The nitrogen-containing bisphosphonates used for management of the patients with osteoporosis were reported to influence the function of renal tubular cells. However, how nitrogen-containing bisphosphates exert any effects on ion currents remains controversial. The effects of ibandronate (Iban), a nitrogen-containing bisphosphonate, on ionic channels, including two types of Ca2+-activated K+ (KCa) channels, namely, large-conductance KCa (BKCa) and intermediate-conductance KCa (IKCa) channels, were investigated in Madin–Darby canine kidney (MDCK) cells. In whole-cell current recordings, Iban suppressed the amplitude of voltage-gated K+ current elicited by long ramp pulse. Addition of Iban caused a reduction of BKCa channels accompanied by a right shift in the activation curve of BKCa channels, despite no change in single-channel conductance. Ca2+ sensitivity of these channels was modified in the presence of this compound; however, the magnitude of Iban-mediated decrease in BKCa-channel activity under membrane stretch with different negative pressure remained unchanged. Iban suppressed the probability of BKCa-channel openings linked primarily to a shortening in the slow component of mean open time in these channels. The dissociation constant needed for Iban-mediated suppression of mean open time in MDCK cells was 12.2 μM. Additionally, cell exposure to Iban suppressed the activity of IKCa channels, and DC-EBIO or 9-phenanthrol effectively reversed its suppression. Under current-clamp configuration, Iban depolarized the cells and DC-EBIO or PF573228 reversed its depolarizing effect. Taken together, the inhibitory action of Iban on KCa-channel activity may contribute to the underlying mechanism of pharmacological or toxicological actions of Iban and its structurally similar bisphosphonates on renal tubular cells occurring in vivo.

Ginsenoside Re enhances small-conductance Ca2 +-activated K+ current in human coronary artery endothelial cells

AimsGinsenosides, active components in ginseng, have been shown to increase nitric oxide (NO) production in aortic endothelial cells. This effect was reversed by tetraethylammonium (TEA) inhibition of endothelial Ca2+-activated K+ (KCa) channels. The objectives of this study, therefore, were to test 1) whether vasorelaxing ginsenoside Re could affect KCa current, an important regulator of NO production, in human coronary artery endothelial cells (HCAECs); and 2) whether small-conductance KCa (SKCa) channel was the channel subtype involved. Main methodsIonic currents of cultured HCAECs were studied using whole-cell patch clamp technique. Key findingsGinsenoside Re dose-dependently increased endothelial outward currents, with an EC50 of 408.90±1.59nM, and a maximum increase of 36.20±5.62% (mean±SEM; p<0.05). Apamin, an SKCa channel inhibitor, could block this effect, while La3+, a nonselective cation channel (NSC) blocker, could not. When NSC channel, inward-rectifier K+ channel, intermediate-, and large-conductance KCa channels were simultaneously blocked, ginsenoside Re could still increase outward currents significantly (35.49±4.22%; p<0.05); this effect was again abolished by apamin. Repeating the experiments when Cl− channel was additionally blocked gave similar results. Finally, we demonstrated that ginsenoside Re could hyperpolarize HCAECs; this effect was reversed by apamin. These data clearly indicate that ginsenoside Re increased HCAEC outward current via SKCa channel activation, and NSC channel was not involved. SignificanceThis is the first report to demonstrate that ginsenoside Re could increase SKCa channel activity in HCAECs. This can be a mechanism mediating ginseng's beneficial actions on coronary vessels.

Gestational hypoxia up-regulates protein kinase C and inhibits calcium-activated potassium channels in ovine uterine arteries.

Objective: The present study tested the hypothesis that gestational hypoxia up-regulates protein kinase C (PKC) and inhibits calcium-activated potassium channels (KCa)-mediated relaxations of uterine arteries in pregnancy. Study design: Uterine arteries were isolated from nonpregnant (NPUA) and pregnant (PUA) (~140 day gestation) sheep maintained at either sea level or high altitude (3,820 m for 110 days, PaO2: 60 mmHg). Contractions of uterine arteries were determined. Key findings: In normoxic PUA, selective inhibition of large-conductance KCa (BK) channels significantly enhanced PKC activator phorbol 12, 13-dibutyrate (PDBu)-induced contractions. This effect was abrogated by chronic hypoxia in gestation. Unlike BK channels, inhibition of small-conductance KCa (SK) channels had no significant effect on PDBu-mediated contractions. In normoxic PUA, activation of both BK with NS1619 or SK with NS309 produced concentration-dependent relaxations, which were not altered by the addition of PDBu. However, in uterine arteries treated with chronic hypoxia (10.5% O2 for 48 h), both NS1619- and NS309-induced relaxations were significantly attenuated by PDBu. In NPUAs, inhibition of BK channels significantly enhanced PDBu-induced contractions in both normoxic and hypoxic animals. Conclusion: The results suggest that in the normoxic condition BK inhibits PKC activity and uterine vascular contractility, which is selectively attenuated by chronic hypoxia during gestation. In addition, hypoxia induces PKC-mediated inhibition of BK and SK activities and relaxations of uterine arteries in pregnancy.

The Intermediate Conductance Calcium-activated Potassium Channel KCa3.1 Regulates Vascular Smooth Muscle Cell Proliferation via Controlling Calcium-dependent Signaling

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF β-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

Iberiotoxin-sensitive and -insensitive BK currents in Purkinje neuron somata

Purkinje cells have specialized intrinsic ionic conductances that generate high-frequency action potentials. Disruptions of their Ca or Ca-activated K (KCa) currents correlate with altered firing patterns in vitro and impaired motor behavior in vivo. To examine the properties of somatic KCa currents, we recorded voltage-clamped KCa currents in Purkinje cell bodies isolated from postnatal day 17-21 mouse cerebellum. Currents were evoked by endogenous Ca influx with approximately physiological Ca buffering. Purkinje somata expressed voltage-activated, Cd-sensitive KCa currents with iberiotoxin (IBTX)-sensitive (>100 nS) and IBTX-insensitive (>75 nS) components. IBTX-sensitive currents activated and partially inactivated within milliseconds. Rapid, incomplete macroscopic inactivation was also evident during 50- or 100-Hz trains of 1-ms depolarizations. In contrast, IBTX-insensitive currents activated more slowly and did not inactivate. These currents were insensitive to the small- and intermediate-conductance KCa channel blockers apamin, scyllatoxin, UCL1684, bicuculline methiodide, and TRAM-34, but were largely blocked by 1 mM tetraethylammonium. The underlying channels had single-channel conductances of ∼150 pS, suggesting that the currents are carried by IBTX-resistant (β4-containing) large-conductance KCa (BK) channels. IBTX-insensitive currents were nevertheless increased by small-conductance KCa channel agonists EBIO, chlorzoxazone, and CyPPA. During trains of brief depolarizations, IBTX-insensitive currents flowed during interstep intervals, and the accumulation of interstep outward current was enhanced by EBIO. In current clamp, EBIO slowed spiking, especially during depolarizing current injections. The two components of BK current in Purkinje somata likely contribute differently to spike repolarization and firing rate. Moreover, augmentation of BK current may partially underlie the action of EBIO and chlorzoxazone to alleviate disrupted Purkinje cell firing associated with genetic ataxias.

Mechanisms of vasorelaxation induced by oleoylethanolamide in the rat small mesenteric artery

The actions of the anandamide-like mono-unsaturated fatty acid oleoylethanolamide (OEA) were first linked to satiety and control of food intake and recently reported to relax resistance vessels. This study characterizes its vasorelaxant mechanisms. Vasorelaxation to OEA were assessed in third order branches of rat superior mesenteric artery using a wire myograph. The roles of the endothelium, KCa channels, perivascular sensory nerves, NO, cannabinoid receptors, and the phospholipase C (PLC)/inositol trisphosphate (InsP3) and RhoA/ROCK signalling pathways, were assessed. OEA caused concentration- and endothelium-dependent vasorelaxation (pEC50=6.7±0.1, Rmax=93.1±2.5%). L-NAME greatly reduced the response (residual relaxation of only 24.6±12.8%). Capsaicin and pertussis toxin significantly reduced the vasorelaxation. Precontraction with KCl abolished the response. TRAM-34 had no effect, but both iberiotoxin and apamin+charybdotoxin markedly shifted the OEA concentration–response curve to the right (∼5-fold). O-1918 but not rimonabant attenuated the vasorelaxation. Both the CB1 receptor antagonist, AM251 and the CB2 receptor antagonist, AM630, given alone or in combination, reduced the response to OEA. Inhibition of PLC by U73122, ROCK by Y-27632 and antagonism of inositol trisphosphate (InsP3) receptors by 2-APB abolished OEA vasorelaxation. OEA vasorelaxation involves an endothelial site of action but not the known cannabinoid receptors. It involves Ca2+ released from InsP3-sensitive endothelial stores by mechanisms involving RhoA kinase and phospholipase C. It is likely that the released Ca2+ causes NO generation and opening of mainly large-conductance KCa channels. This study demonstrates a possible novel endothelial target that might be important in the control of regional blood flow induced by this lipid molecule.

Effects of ginsenoside on large-conductance KCa channels in human corporal smooth muscle cells

Ginseng was known to be an effective natural product that enhances penile erection. However, the precise biological function and mechanisms of action of ginseng with regard to erectile function remain unknown. The principal objective of this study was to identify ginsenoside (principal molecular ingredients of ginseng)-induced activation of large-conductance K(Ca) channel in human corporal smooth muscle cells, and to determine ginseng's mechanism of action on penile erection. Electrophysiological studies using cultured human corporal smooth muscle cells were conducted. We evaluated the effects of total ginsenosides (TGS) and ginsenoside Rg3 on large-conductance K(Ca) channel by determining whole-cell currents and single-channel activities. There was an increase in outward current dependent on TGS concentration (at +60 mV, 1 μg ml(-1); 168.3±59.3%, n=6, P<0.05, 10 μg ml(-1); 173.2±36.8%, n=4, P<0.05, 50 μg ml(-1); 295.3±62.3%, n=19, P<0.001, 100 μg ml(-1); and 462.3±97.1%, n=5, P<0.001) and Rg3 concentration (at +60 mV, 1 μM (0.78 μg ml(-1)); 222.8±64.8%, n=11, P<0.0001, 10 μM; 672.6±137.1%, n=10, P<0.0001, 50 μM; and 1713.3±234.7%, n=15, P<0.001) in the solution that was blocked completely by tetraethylammonium (TEA). Channel opening in cell-attached mode and channel activity in the inside-out membrane patches was also increased significantly by 50 μg of TGS or 10 μM of Rg3. The results of this study suggested that the activation of large-conductance K(Ca) channels by ginsenoside could be one mechanism of ginsenoside-induced relaxation in corporal smooth muscle.

Reduced role of large conductance KCa channels in the response to noradrenaline after ischemia‐reperfusion in goat cerebral arteries

Vascular calcium‐activated potassium (KCa) channels seem to play a role in the regulation of cerebral blood flow. To analyze their functional state after cerebral ischemia‐reperfusion, the left middle cerebral artery (MCA) of anesthetized goats was subjected to 120‐min occlusion followed by 60‐min reperfusion. Isolated segments from the left (ischemic) and right (control) MCA exhibited constriction to noradrenaline (10−8‐10−4 M, in the presence of β‐adrenoceptor blockade) that was greater in ischemic arteries. The large‐conductance KCa channel inhibitor Iberiotoxin (10−7 M) enhanced the contraction to noradrenaline in both control and ischemic arteries, but this effect was significantly lower in ischemic (21±12 %) than in control vessels (72±18 %, p&lt;0.05). TRAM‐34 (2×10−8 M, intermediate‐conductance KCa channel inhibitor) or Apamin (10−6 M, small‐conductance KCa channel inhibitor) did not modify the noradrenaline effect in both types of arteries. The NO synthesis inhibitor L‐NAME (10−4 M) potentiated the response to noradrenaline similarly in control and ischemic arteries. These results suggest that the response to noradrenaline is enhanced after ischemia‐reperfusion due, in part, to impaired activation of large‐conductance KCa channels. NO might not be accounting for the adrenergic activation of K channels. (Supported by Ministerio de Ciencia e Innovación grant Nº SAF2008‐01410).

Endothelial SKCa and IKCa Channels Regulate Brain Parenchymal Arteriolar Diameter and Cortical Cerebral Blood Flow

Calcium-sensitive potassium (K(Ca)) channels have been shown to modulate the diameter of cerebral pial arteries; however, little is known regarding their roles in controlling cerebral parenchymal arterioles (PAs). We explored the function and cellular distribution of small-conductance (SK(Ca)) and intermediate-conductance (IK(Ca)) K(Ca) channels and large-conductance K(Ca) (BK(Ca)) channels in endothelial cells (ECs) and smooth muscle cells (SMCs) of PAs. Both SK(Ca) and IK(Ca) channels conducted the outward current in isolated PA ECs (current densities, ~20 pA/pF and ~28 pA/pF at +40 mV, respectively), but these currents were not detected in PA SMCs. In contrast, BK(Ca) currents were prominent in PA SMCs (~154 pA/pF), but were undetectable in PA ECs. Pressurized PAs constricted to inhibition of SK(Ca) (~16%) and IK(Ca) (~16%) channels, but were only modestly affected by inhibition of BK(Ca) channels (~5%). Blockade of SK(Ca) and IK(Ca) channels decreased resting cortical cerebral blood flow (CBF) by ~15%. NS309 (6,7-dichloro-1H-indole-2,3-dione3-oxime), a SK(Ca)/IK(Ca) channel opener, hyperpolarized PA SMCs by ~27 mV, maximally dilated pressurized PAs, and increased CBF by ~40%. In conclusion, these data show that SK(Ca) and IK(Ca) channels in ECs profoundly modulate PA tone and CBF, whereas BK(Ca) channels in SMCs only modestly influence PA diameter.

SKCa and IKCa channels are involved in epithelium‐dependent relaxation of rat bronchioles

We investigated whether Ca2+‐activated K+ channels of small (SKCa) and intermediate (IKCa) conductance are present, and in bronchioles can be modulated by an opener, NS309 (6,7‐dichloro‐1H‐indole‐2,3‐dione 3‐oxime). Immunohistochemical studies were conducted, and segments of rat bronchioles (847 ± 25 μm, n=51) were mounted in microvascular myographs for isometric tension recordings. The effect of NS309 was compared to that in pulmonary arteries. Immunoreaction for IKCa and SKCa3 was found in rat bronchiole epithelium. In 5‐HT (1 µM)‐contracted rat bronchioles, NS309 (0.01‐10 µM) and a β2‐adrenoceptor agonist, salbutamol, induced concentration‐ and epithelium‐dependent relaxations. An inhibitor of cyclooxygenase, indomethacin (3 µM), a NO‐synthase inhibitor, L‐NNA (100 µM) and a CYP2C9 inhibitor, sulphaphenazole (100 µM), did not reduce NS309 relaxation. In contrast to a blocker of large‐conductance KCa (BKCa) channels, iberiotoxin (0.1 µM), a blocker of IKCa and BKCa channels, charybdotoxin (0.07 µM) and a blocker of SKCa channels, apamin (0.5 μM), reduced salbutamol and NS309‐induced relaxation. The study suggests that IKCa and SKCa3 channels are located in rat bronchiole epithelium, and that activation of SKCa and IKCa channels by NS309 leads to epithelium‐derived hyperpolarizing factor (EpDHF)‐type relaxation of bronchioles analogue to the EDHF‐type relaxation in rat pulmonary arteries.

Calcium-Activated Potassium Channels Contribute to Human Coronary Microvascular Dysfunction After Cardioplegic Arrest

Cardioplegic arrest (CP) followed by reperfusion after cardiopulmonary bypass induces coronary microvascular dysfunction. We investigated the role of calcium-activated potassium (K(Ca)) channels in this dysfunction in the human coronary microvasculature. Human atrial tissue was harvested before CP from a nonischemic segment and after CP from an atrial segment exposed to hyperkalemic cold blood CP (mean CP time, 58 minutes) followed by 10-minute reperfusion. In vitro relaxation responses of precontracted arterioles (80 to 180 mum in diameter) in a pressurized no-flow state were examined in the presence of K(Ca) channel activators/blockers and several other vasodilators. We also examined expression and localization of K(Ca) channel gene products in the coronary microvasculature using reverse transcriptase-polymerase chain reaction, immunoblot, and immunofluorescence photomicroscopy. Post-CP reperfusion relaxation responses to the activator of intermediate and small conductance K(Ca) channels (IK(Ca)/SK(Ca)), NS309 (10(-5) M), and to the endothelium-dependent vasodilators, substance P (10(-8) M) and adenosine 5diphosphate (10(-5) M), were significantly reduced compared with pre-CP responses (P<0.05, n=8/group). In contrast, relaxation responses to the activator of large conductance K(Ca) channels (BK(Ca)), NS1619 (10(-5) M), and to the endothelium-independent vasodilator, sodium nitroprusside (10(-4) M), were unchanged pre- and post-CP reperfusion (n=8/group). Endothelial denudation significantly diminished NS309-induced vasodilatation and abolished substance P- or adenosine 5 diphosphate-induced relaxation (P<0.05), but had no effect on relaxation induced by either NS1619 or sodium nitroprusside. The total polypeptide levels of BK(Ca), IK(Ca), and SK(Ca) and the expression of IK(Ca) mRNA were not altered post-CP reperfusion. Cardioplegic arrest followed by reperfusion after cardiopulmonary bypass causes microvascular dysfunction associated with and likely in part due to impaired function of SK(Ca) and IK(Ca) channels in the coronary microcirculation. These results suggest novel mechanisms of endothelial and smooth muscle microvascular dysfunction after cardiac surgery.

Activation of endogenous GABAA channels on airway smooth muscle potentiates isoproterenol-mediated relaxation

Reactive airway disease predisposes patients to episodes of acute smooth muscle mediated bronchoconstriction. We have for the first time recently demonstrated the expression and function of endogenous ionotropic GABA(A) channels on airway smooth muscle cells. We questioned whether endogenous GABA(A) channels on airway smooth muscle could augment beta-agonist-mediated relaxation. Guinea pig tracheal rings or human bronchial airway smooth muscles were equilibrated in organ baths with continuous digital tension recordings. After pretreatment with or without the selective GABA(A) antagonist gabazine (100 muM), airway muscle was contracted with acetylcholine or beta-ala neurokinin A, followed by relaxation induced by cumulatively increasing concentrations of isoproterenol (1 nM to 1 muM) in the absence or presence of the selective GABA(A) agonist muscimol (10-100 muM). In separate experiments, guinea pig tracheal rings were pretreated with the large conductance K(Ca) channel blocker iberiotoxin (100 nM) after an EC(50) contraction with acetylcholine but before cumulatively increasing concentrations of isoproterenol (1 nM to 1 uM) in the absence or presence of muscimol (100 uM). GABA(A) activation potentiated the relaxant effects of isoproterenol after an acetylcholine or tachykinin-induced contraction in guinea pig tracheal rings or an acetylcholine-induced contraction in human endobronchial smooth muscle. This muscimol-induced potentiation of relaxation was abolished by gabazine pretreatment but persisted after blockade of the maxi K(Ca) channel. Selective activation of endogenous GABA(A) receptors significantly augments beta-agonist-mediated relaxation of guinea pig and human airway smooth muscle, which may have important therapeutic implications for patients in severe bronchospasm.

Afferent Arteriolar Dilation to 11, 12‐EET Analogs Involves PP2A Activity and Ca2+‐Activated K+ Channels

The epoxygenase metabolite, 11, 12-epoxyeicosatrienoic acid (11, 12-EET), has renal vascular actions. 11, 12-EET analogs have been developed to determine the structure activity relationship for 11, 12-EET and as a tool to investigate signaling mechanisms responsible for afferent arteriolar dilation. We hypothesized that 11, 12-EET mediated afferent arteriolar dilation involves increased phosphoprotein phosphatase 2A (PP2A) and large-conductance calcium activated K+ (KCa) channels. We evaluated the chemically and/or metabolically table 11, 12-EET analogs: 11, 12-EET-N-methylsulfonimide (11, 12-EET-SI), 11-nonyloxy-undec-8(Z)-enoic acid (11, 12-ether-EET-8-ZE), and 11, 12-trans-oxidoeicosa-8(Z)-eonoic acid (11, 12-tetra-EET-8-ZE). Afferent arteriolar responses were assessed. Activation of KCa channels by 11, 12-EET analogs were established by single cell channel recordings in renal myocytes. Assessment of renal vascular responses revealed that 11, 12-EET analogs increased afferent arteriolar diameter. Vasodilator responses to 11, 12-EET analogs were abolished by K+ channel or PP2A inhibition. 11, 12-EET analogs activated renal myocyte large-conductance KCa channels. 11, 12-EET analogs increased cAMP by 2-fold and PP2A activity increased 3-8 fold in renal myocytes. PP2A inhibition did not significantly affect the 11, 12-EET analog mediated increase in cAMP and PP2A increased renal myocyte KCa channel activity to a much greater extent than PKA. These data support the concept that 11, 12-EET utilizes PP2A dependent pathways to activate large-conductance KCa channels and dilate the afferent arteriole.

Smooth muscle sparklet Cav channels defined: 1.2 is the number

Systemic blood pressure and tissue blood flow are regulated by the contractile status of smooth muscle cells located within the wall of small arteries and arterioles. One important signal that regulates arterial smooth muscle cell contractility is the intracellular calcium ion (Ca2+) concentration. Although Ca2+ was once considered to act only as a global intracellular signaling element, research performed over the past decade has revealed Ca2+ to act in a far more dynamic and versatile manner than previously thought possible.

17Beta-Estradiol Inhibits Calcium-Activated Potassium Channel Expressions in Rat Whole Bladder

Purpose:To investigate the effect of estrogen on the expression of calcium-activated potassium (KCa) channels in an overactive bladder rat model. To this end, mRNA and protein levels of KCa channel subtypes in the bladder of ovariectomized rats were measured by reverse transcription polymerase chain reaction and western blotting, respectively.Methods:Ten-week-old female Sprague-Dawley rats were divided randomly into 3 groups: sham-operated control group (n=11), ovariectomy group (n=11), and the group treated with estrogen after ovariectomy (n=12). Rats in the last group were subcutaneously injected with 17β-estradiol (50 μg/kg) every other day for 2 weeks, whereas rats in the other 2 groups received vehicle (soybean oil) alone. Two weeks after treatment, the whole bladder was excised for mRNA and protein measurements.Results:Protein levels of the large-conductance KCa (BK) channels in the ovariectomy group were 1.5 folds higher than those in the sham-operated control group. However, the protein levels of the other KCa channel subtypes did not change significantly upon bilateral ovariectomy. Treatment with 17β-estradiol after ovariectomy restored BK channel protein levels to the control value. In contrast, BK channel mRNA levels were not significantly affected by either ovariectomy alone or 17β-estradiol treatment. The small-conductance KCa type 3 channel (SK3) mRNA and protein levels decreased to 75% of control levels upon 17β-estradiol treatment.Conclusions:These results suggest that 17β-estradiol may influence urinary bladder function by modulating BK and SK3 channel expression.