Expression Of BKCa Research Articles

BKCa channels are involved in spontaneous and lipopolysaccharide-stimulated uterine contraction in late gestation mice†.

The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) is one of the most abundant potassium channels in the myometrium. Previous work conducted by our group has identified a link between inflammation, BKCa channels and excitability of myometrial smooth muscle cells. Here, we investigate the role of BKCa channels in spontaneous and lipopolysaccharide (LPS)-stimulated uterine contraction to gain a better understanding of the relationship between the BKCa channel and uterine contraction in basal and inflammatory states. Uteri of C57BL/6J mice on gestational day 18.5 (GD18.5) were obtained and either fixed in formalin or used immediately for tension recording or isolation of primary myocytes for patch-clamp. Paraffin sections were used for immunofluorescenctdetection of BKCa and Toll-like receptor (TLR4). For tension recordings, LPS was administered to determine its effect on uterine contractions. Paxilline, a BKCa inhibitor, was used to dissect the role of BKCa in uterine contraction in basal and inflammatory states. Finally, patch-clamp recordings were performed to investigate the relationship between LPS, the BKCa channel and membrane currents in mouse myometrial smooth muscle cells (mMSMCs). We confirmed the expression of BKCa and TLR4 in the myometrium of GD18.5 mice and found that inhibiting BKCa channels with paxilline suppressed both spontaneous and LPS-stimulated uterine contractions. Furthermore, application of BKCa inhibitors (paxilline or iberiotoxin) after LPS inhibited BKCa channel activity in mMSMCs. Moreover, pretreatment with BKCa inhibitor or the TLR4 inhibitor suppressed LPS-activated BKCa currents. Our study demonstrates that BKCa channels are involved in both basal and LPS-stimulated uterine contraction in pregnant mice.

Aerobic exercise improves BKCa channel-mediated vasodilation in diabetic vascular smooth muscle via AMPK/Nrf2/HO-1 pathway.

This study aims to investigate the effect of aerobic exercise training on BKCa channel in diabetic vascular smooth muscle and explore the underlying mechanism. Control m/m mice and diabetic db/db mice were randomly assigned to sedentary groups (W and D) and exercise training groups (WE and DE). Mice in exercise groups underwent training sessions lasting for 12weeks, with a speed of 12m/min for 60min, five times per week. The thoracic aorta was extracted isolated and examined for measurement of vascular structure, global levels of reactive oxygen species (ROS), vasodilation, and protein expression. Rat thoracic aorta vascular smooth muscle cells (USMCs) were cultured, and siRNA transfection was conducted to detect whether AMPK contributed to the regulation. ROS level and protein expression were measured. Compared with control mice, diabetic mice had a larger vascular medium thickness, impaired BKCa-mediated vasodilation, a higher level of ROS, and a lower expression of BKCa α, BKCa β1, Nrf2, p-Nrf2, p-Nrf2/Nrf2, HO-1, and p-AMPK/AMPK. Exercise training increased the expression of BKCa α, Nrf2, p-Nrf2, p-Nrf2/Nrf2, HO-1, and p-AMPK/AMPK. AMPK deletion led to lower ROS levels and expression of BKCa α, β1, Nrf2, and HO-1 in USMCs cultured in high glucose conditions. BKCa channel protein expression reduction in VSMCs contributes to vasodilation and vascular remodeling dysfunction in diabetes mellitus. Aerobic exercise can promote the expression of BKCa channel and improve BKCa-mediated vascular dysfunction in diabetic VSMCs through AMPK/Nrf2/HO-1 signaling pathway.

Estrogen increases the expression of BKCa and impairs the contraction of colon smooth muscle via upregulation of sphingosine kinase 1.

Estrogen (E2) may impair the contraction of colonic smooth muscle (SM) leading to constipation. Large conductance Ca2+ -activated K+ channels (BKCa ) are widely expressed in the smooth muscle cells (SMCs) contributing to hyperpolarization and relaxation of SMCs. Sphingosine kinase 1 (SphK1) is known to influence the expression of BKCa . We aimed to elucidate the potential underlying molecular mechanism of BKCa and SphK1 that may influence E2-induced colonic dysmotility. In ovariectomized rats, SMcontraction and expression of BKCa , SphK1, sphingosine-1-phosphate receptor (S1PR) were analyzed after the treatment with vehicle, BSA-E2, E2, and E2receptor antagonist. The role of BKCa , SphK1, and S1PR in E2-induced SMdysmotility was investigated in rat colonic SMCs. The effect of SphK1 on SMcontraction as well as on the expression of BKCa and S1PR was analyzed in SphK1 knock-out mutant mice and wild-type (WT) mice treated with or without E2. The E2-treated group exhibited a weak contraction of colonic SMand a delayed colonic transit. The treatment with E2 significantly upregulated the expression of BKCa , SphK1, S1PR1, and S1PR2, but not S1PR3, in colon SMand SMCs. Inhibition of BKCa , SphK1, S1PR1, and S1PR2 expression attenuated the effect of E2 on Ca2+ mobilization in rat colon SMCs. WTmice treated with E2 showed impaired gastrointestinal motility and enhanced expression of BKCa , S1PR1, and S1PR2 compared with those without E2 treatment. Conversely, in SphK1 knock-out mice treated with E2, these effects were partially reversed. E2 increased the release of S1P which in turn could have activated S1PR1 and S1PR2. Loss of SphK1 attenuated the effect of E2 on the upregulation of S1PR1 and S1PR2 expression. These findings indicated that E2 impaired the contraction of colon SMthrough activation of BKCa via the upregulation of SphK1 and the release of S1P. In the E2-induced BKCa upregulation, S1PR1 and S1PR2 might also be involved. These results may provide further insights into a therapeutic target and optional treatment approaches for patients with constipation.

The effect of large-conductance calcium-activated potassium channels on the migration of pericytes in the mice of senile cochlear stria vascularis

Objective: To investigate whether large conductance calcium-activated potassium channel (BK(Ca)) was involved in the migration of pericytes (PC) in the mice of senile cochlear stria vascularis capillaries PC. Methods: C57BL/6J mice were divided into 3-month (n=10) and 12-month groups (n=10). Auditory brainstem response (ABR) was used to test the hearing threshold of each group. The immunofluorescence was used to detect the expression changes of osteopontin (OPN) and β-BK(Ca) channels on cochlear stria vascularis PC. The morphological changes of perivascular cells in cochlea were observed by transmission electron microscope (TEM). Cell experiment: The PC, which were in the stria vascularis of the cochlea were primary cultured and identified. A cell senile model was made with D-gal. The appropriate intervention concentration of low galactose (D-gal) was determined by CCK8. β-galactosidase (SA-β-gal) staining was used to evaluate the cell decrept level. The change of BK(Ca) channels current on PC were recorded by whole cell patch clamp technique. The expression of BK(Ca) channels on PC was detected by immunofluorescence. The migration and invasion ability of two groups were detected by using Scratch test and Transwell. The levels of OPN and β-BK(Ca) channels were detected by Western blot. SPSS 22.0 software was used to analyze the data. Results: The ABR threshold in the 12-month group was higher than 3-month group (t=12.66, P<0.01). In the 12-month group, the expression of β-BK(Ca) channel was lower and the expression of OPN was increased (t=14.64, P<0.01; t=20.73, P<0.01). In TEM, cochlear stria vascularis PC were tightly connected to endothelial cells in 3-month group, while PC were loosely connected to endothelial cells or PC soma were separated from the capillary in 12-month group. Cell experiment: The positive rate of PC in the primary cultured cochlear stria vascularis is above 95%. Compared with the SA-β-gal stained cells in the control group, the positive rate of 15 mg/ml D-gal intervention PC was 85% (t=36.90, P<0.01). Whole cell patch clamp BK(Ca) channels current decreased in the D-gal group compared with the young group PC (t=12.18, P<0.05). The OPN expression in the senile group was higher than control group (t=16.30, P<0.01), while the β-BK(Ca) channels expression was decreased (t=11.98, P<0.01; t=15.72, P<0.05), and migration ability raised (t=7.91, P<0.01;t=7.59, P<0.01). After intervened of BK(Ca) channels specific blocker IBTX in the D-gal group, the expression of OPN and migration were increased (t=4.26, P<0.05; t=5.88, P<0.01; t=21.97, P<0.01). Conclusion: PC migration capacity were increased during the senile period, and the expression of β-BK(Ca) channel was decreased. The administration of IBTX, a specific blocker of BK(Ca) channel, at the cell level could increase the migration capacity, suggesting that BK(Ca) might be involved in the migration of PC in the stria vascularis of the aging cochlea.

BKCa Channels as Targets for Cardioprotection.

The large-conductance calcium- and voltage-activated K+ channel (BKCa) are encoded by the Kcnma1 gene. They are ubiquitously expressed in neuronal, smooth muscle, astrocytes, and neuroendocrine cells where they are known to play an important role in physiological and pathological processes. They are usually localized to the plasma membrane of the majority of the cells with an exception of adult cardiomyocytes, where BKCa is known to localize to mitochondria. BKCa channels couple calcium and voltage responses in the cell, which places them as unique targets for a rapid physiological response. The expression and activity of BKCa have been linked to several cardiovascular, muscular, and neurological defects, making them a key therapeutic target. Specifically in the heart muscle, pharmacological and genetic activation of BKCa channels protect the heart from ischemia-reperfusion injury and also facilitate cardioprotection rendered by ischemic preconditioning. The mechanism involved in cardioprotection is assigned to the modulation of mitochondrial functions, such as regulation of mitochondrial calcium, reactive oxygen species, and membrane potential. Here, we review the progress made on BKCa channels and cardioprotection and explore their potential roles as therapeutic targets for preventing acute myocardial infarction.

BKCa compensates impaired coronary vasoreactivity through RhoA/ROCK pathway in hind-limb unweighted rats.

Previous studies have demonstrated cardiac and vascular remodeling induced by microgravity exposure. Yet, as the most important branch of vasculatures circulating the heart, the coronary artery has been seldomly studied about its adaptations under microgravity conditions. Large-conductance Ca2+-activated potassium channel (BKCa) and the Ras homolog family member A (RhoA)/Rho kinase (ROCK) pathway play key roles in control of vascular tone and mediation of microgravity-induced vascular adjustments. Therefore, we investigated the adaptation of coronary vasoreactivity to simulated microgravity and the role of BKCa and the RhoA/ROCK pathway in it. Four-week-old hind-limb unweighted (HU) rats were adopted to simulate effects of microgravity. Right coronary artery (RCA) constriction was measured by isometric force recording. The activity and expression of BKCa and the RhoA/ROCK pathway were examined by Western blot, patch-clamp recordings, and immunoprecipitation. We found HU significantly decreased RCA vasoconstriction to KCl, serotonin, and U-46619, but increased protein expression and current densities of BKCa, inhibition of which by iberiotoxin (IBTX) further decreased RCA vasoconstriction (P < 0.05). Expression of RhoA and ROCK as well as active RhoA and phosphorylation of myosin light chain (MLC) at Ser19 and MLC phosphatase target-1 at Thr696 were significantly increased by HU, and ROCK inhibitor Y-27632 exerted greater suppressing effect on HU RCA vasoconstriction than that of control (P < 0.05). BKCa opener NS1619 increased HU RCA vasoconstriction, which was blocked by both RhoA and ROCK inhibitor, similar to the effect of IBTX. These results indicate that HU impairs coronary vasoconstriction but enhances BKCa activity acting as a protective mechanism avoiding excessive decrease of coronary vasoreactivity through activation of the RhoA/ROCK pathway.-Wu, Y., Yue, Z., Wang, Q., Lv, Q., Liu, H., Bai, Y., Li, S., Xie, M., Bao, J., Ma, J., Zhu, X., Wang, Z. BKCa compensates impaired coronary vasoreactivity through RhoA/ROCK pathway in hind-limb unweighted rats.

Su1575 – Estrogen Increases the Expression of BkCA and Impairs the Contraction of Colon Smooth Muscle via Up-Regulation of SPHK1

Su1575 – Estrogen Increases the Expression of BkCA and Impairs the Contraction of Colon Smooth Muscle via Up-Regulation of SPHK1

Vasodilatory effect of formaldehyde via the NO/cGMP pathway and the regulation of expression of KATP, BKCa and L-type Ca2+ channels

Formaldehyde (FA), a well-known toxic gas molecule similar to nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S), is widely produced endogenously via numerous biochemical pathways, and has a number of physiological roles in the biosystem. We attempted to investigate the vasorelaxant effects of FA and their underlying mechanisms. We found that FA induced vasorelaxant effects on rat aortic rings in a concentration-dependent manner. The NO/cyclic guanosine 5′ monophosphate (cGMP) pathway was up-regulated when the rat aortas were treated with FA. The expression of large-conductance Ca2+-activated K+ (BKCa) channel subunits α and β of the rat aortas was increased by FA. Similarly, the levels of ATP-sensitive K+ (KATP) channel subunits Kir6.1 and Kir6.2 were also up-regulated when the rat aortas were incubated with FA. In contrast, levels of the L-type Ca2+ channel (LTCC) subunits, Cav1.2 and Cav1.3, decreased dramatically with increasing concentrations of FA. We demonstrated that the regulation of FA on vascular contractility may be via the up-regulation of the NO/cGMP pathway and the modulation of ion channels, including the upregulated expression of the KATP and BKCa channels and the inhibited expression of LTCCs. Further study is needed to explore the in-depth mechanisms of FA induced vasorelaxation.

BKCa participates in E2 inducing endometrial adenocarcinoma by activating MEK/ERK pathway

BackgroundThe large-conductance, voltage-gated, calcium (Ca (2+))-activated potassium channel (BKCa) plays an important role in regulating Ca (2+) signaling and cell physiological function, and is aberrantly expressed in some types of cancers. The present study focuses on identifying the oncogenic potential and clinical significance of BKCa in endometrial adenocarcinoma, as well as exploring the mechanistic relevance by 17β -estradiol (E2) inducing aberrant activation of MEK1/2 and ERK1/2 via BKCa.MethodsThe expression of BKCa, ERK1/2 and p-ERK1/2 were examined by immunohistochemical staining in 263 cases, including 185 primary types I endometrial cancer tissues, 38 atypical endometrial hyperplasia tissues and 40 normal endometrium tissues. Cell growth, cycle, apoptosis rate, migration and invasion was separately tested in Ishikawa cells using siRNA-BKCa and/or E2 treatment, as well as the expression of these interested proteins by western blot analysis.ResultsWe showed that expression of BKCa is significantly elevated in 185 types I endometrial adenocarcinoma tissues compared to those of the normal endometrium and atypical endometrial hyperplasia tissues. Furthermore, in vitro observations revealed that down-regulation of BKCa expression inhibited cell growth by both enhancing apoptosis and blocking G1/S transition, suppressed cell migration and invasion in Ishakiwa cells, and decreased the expression of p-MEK1/2 and p-ERK1/2. Additionally, RNAi-mediated knockdown of BKCa attenuated the increased cellular growth and invasion, as well as the elevated expression of p-MEK1/2 and p-ERK1/2 proteins, induced by E2 stimulation. More importantly, the aberrant expression of BKCa and p-ERK1/2 were closely related with poor prognostic factors in type I endometrial cancer, and up-regulated expression of p-ERK1/2 was significantly associated with shorter disease-free survival (DFS) and overall survival (OS) and was an independent prognostic factor in type I endometrial cancer patients.ConclusionOur results demonstrated that BKCa and the key downstream effectors p-ERK1/2 could be involved in important signaling pathways in initiation and development of endometrial adenocarcinoma and may provide a new therapeutic approach for women with endometrial cancer.

PL-015 Aerobic exercise increases BKCa channel expression to enhance tracheal smooth muscle relaxation in a murine asthma model

Objective Increasing evidence has shown that moderate-intensity aerobic exercise training reduces airway hyperresponsiveness (AHR) in patients with asthma. However, the mechanisms underlying exercise-induced improvements in smooth muscle contractility have not been fully elucidated. Large-conductance Ca2+-activated K+ channels (BKCa) are expressed broadly on smooth muscle cells and play an important role in the regulation of smooth muscle contraction. We tested the hypothesis that exercise training increases the contribution of BKCa channel to tracheal smooth muscle relaxation in in ovalbumin (OVA)-challenged asthmatic rats.&#x0D; Methods Rats were sensitized/challenged with OVA or saline and exercised at a moderate intensity 5 times/week for 4 weeks. Tracheal smooth muscle contractility was tested. Membrane potential of primary cultured tracheal smooth muscle cells was measured. In addition, western immunoblotting was performed to study the expression levels of BKCa channel protein.&#x0D; Results The contraction of rat airway smooth muscle induced by carbachol was significantly increased with asthma and exercise training reversed this alteration. Application of BKCa channel agonist, NS1619, induced tracheal smooth muscle relaxation. NS1619-induced relaxation was decreased in asthmatic rats, however exercise training significantly increased NS1619-induced relaxation. In primary cultured smooth muscle cells, NS1619-induced membrane potential was reduced with asthma and this alteration was diminished after exercise training. Additionally, western blotting revealed that the protein expression of BKCa was reduced in asthmatic group and aerobic exercise significantly improved BKCa expression.&#x0D; Conclusions The present study reveals that aerobic exercise training increases BKCa expression on tracheal smooth muscle, which partly underlies the beneficial effect of exercise on improving airway smooth muscle relaxation in asthma.

Tu1267 - S1P Increases the Expression of BkCa and Impairs Ca2+ Mobilization in Colonic Smooth Muscle Cells via the PKCΔ/JNK Pathway

Tu1267 - S1P Increases the Expression of BkCa and Impairs Ca2+ Mobilization in Colonic Smooth Muscle Cells via the PKCΔ/JNK Pathway

Downregulation of large conductance calcium-activated potassium channels in paraventricular nucleus contributes to sympathoexcitation in rats with chronic heart failure

Objective: To elucidate the association between large conductance calcium-activated potassium channels (BKCa) in the paraventricular hypothalamic nucleus (PVN) and sympathetic outflow in rats with chronic heart failure (CHF) . Methods: Male Wistar rats (6-7 weeks old) were randomized to sham operated group and CHF group (coronary artery ligation) . Two weeks after operation, BKCa inhibitor Iberiotoxin (IBTX) was infused into PVN by osmotic minipumps, rats were divided into following groups: sham+aCSF, CHF+aCSF, sham+low dose IBTX (0.125 nmol/nl) , CHF+low dose IBTX, sham+moderate dose IBTX (1.25 nmol/nl) , CHF+moderate dose IBTX, sham+ high dose IBTX (12.5 nmol/nl) , and CHF+high dose IBTX (n=6 each) . Additional rats were grouped as follows: sham+vehicle, sham+KCNMB4 knockdown (by rAAV2-KCNMB4 shRNA virus injection in PVN) , CHF+vehicle, CHF+ KCNMB4 knockdown group (n=6 each) . The cardiac function was determined by echocardiography, left ventricular hemodynamics were measured invasively, renal sympathetic nerve activity (RSNA) was recorded at 6 weeks after coronary artery ligation or sham operation. The contents of norepinephrine (NE) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in plasma were determined by enzyme-linked immunosorbent assay. The protein and mRNA expression of KCNMB4 in PVN were measured by immunofluorescence staining, Western blot, and real-time PCR, mRNA expression of BKCa in PVN was detected by real-time PCR. Results: Compared with the sham operation group, the cardiac function of the heart failure group was significantly reduced (P<0.05) , and the plasma NE and the serum NT-proBNP were significantly elevated (P<0.05) . The protein and mRNA expression of KCNMB4 in PVN were obviously down-regulated in CHF rats (P<0.05) . After perfusion of IBTX or KCNMB4 knockdown by microinjection of rAAV2-KCNMB4 shRNA virus,right ventricular weight/body weight and lung weight/body weight ratio as well as left ventricular end-diastolic diameter were increased and left ventricular ejection fraction was decreased (all P<0.05) , the sympathetic driving indexes was increased in sham rats, changes of these parameters further aggravated in CHF rats (P<0.05) . KCNMB4 knockdown further downregulated protein expression in PVN of CHF rats. Conclusion: Downregulation and blunted function of BKCa in PVN may contribute to sympathoexcitation and deterioration of cardiac function in rats with chronic heart failure.

ABSENCE OF LARGE CONDUCTANCE CALCIUM AND VOLTAGE ACTIVATED POTASSIUM CHANNEL CAUSES CARDIAC DYSFUNTION

Expression and activation of BKCa encoded by a Kcnma1 gene, is known to play a direct role in cardioprotection against ischemia-reperfusion injury by modulating mitochondrial function, and in regulating the heart rate. However, the role of BKCa in cardiac function is not yet deciphered Cardiac

Expression and alteration of BKCa channels in the sphincter of Oddi's from rabbits with hypercholesterolemia

ABSTRACTThis study aimed to investigate the expression and function of BKCa channels in the Sphincter of Oddi (SO) in a rabbit model of hypercholesterolemia (HC). New Zealand white rabbits were randomly divided into 2 groups: the control group was fed standard chow (n = 18) whereas the high-cholesterol group was fed cholesterol-enriched chow containing 1.5% cholesterol (n = 18). The serum cholesterol level was significantly greater in the HC groups than in the control group, but there was no significant difference in body weight between the control and HC groups. Although the total protein expression of BKCa α- and β1-subunit was not significantly different between the control and HC groups, the Tyr-phosphorylation of BKCa α-subunit was significantly decreased in the HC group than in the control group. In addition, hypercholesterolemia significantly increased Acetylcholine (ACh)-induced contraction of the SO rings. Pretreatment with 30 μM NS1619, a BKCa channel agonist, significantly reduced ACh-induced contraction of the SO rings in HC rabbits. Moreover, pretreatment with 100 μM Na3OV4, a protein tyrosine phosphatase inhibitor, significantly reduced ACh-induced contraction of the SO rings in HC rabbits, whereas it significantly increased upon pretreating with 10 μM Genistein, a tyrosine kinase inhibitor. Whole-cell patch clamp recordings showed that BKCa current density was significantly lower in SOSMCs from HC group than that from control group. Our findings suggest that hypercholesterolemia-induced downregulation of BKCa channel, and Tyr-phosphorylation of BKCa α-subunit may contribute to the hyperresponsiveness of the SO ring in HC rabbits.

Exercise Prevents Upregulation of RyRs-BKCa Coupling in Cerebral Arterial Smooth Muscle Cells From Spontaneously Hypertensive Rats.

Regular exercise is an effective nonpharmacological means of preventing and controlling hypertension. However, the molecular mechanisms underlying its effects remain undetermined. The hypothesis that hypertension increases the functional coupling of large-conductance Ca(2+)-activated K(+) (BKCa) channels with ryanodine receptors in spontaneously hypertensive rats (SHR) as a compensatory response to an increase in intracellular Ca(2+) concentration in cerebral artery smooth muscle cells was assessed here. It was further hypothesized that exercise training would prevent this increase in functional coupling. SHR and Wistar-Kyoto (WKY) rats were randomly assigned to sedentary groups (SHR-SED and WKY-SED) and exercise training groups (SHR-EX and WKY-EX). Cerebral artery smooth muscle cells displayed spontaneous transient outward currents at membrane potentials more positive than -40 mV. The amplitude of spontaneous transient outward currents together with the spontaneous Ca(2+) sparks in isolated cerebral artery smooth muscle cells was significantly higher in SHR-SED than in WKY-SED. Moreover, hypertension displayed increased whole-cell BKCa, voltage-gated Ca(2+) channel, but decreased KV currents in cerebral artery smooth muscle cells. In SHRs, the activity of the single BKCa channel increased markedly, and the protein expression of BKCa (β1, but not α-subunit) also increased, but KV1.2 decreased significantly. Exercise training ameliorated all of these functional and molecular alterations in hypertensive rats. These data indicate that hypertension leads to enhanced functional coupling of ryanodine receptors-BKCa to buffer pressure-induced constriction of cerebral arteries, which attributes not only to an upregulation of BKCa β1-subunit function but also to an increase of Ca(2+) release from ryanodine receptors. However, regular aerobic exercise efficiently prevents augmented coupling and so alleviates the pathological compensation and restores cerebral arterial function.

Decreased Role for BKCa Channels in Endothelium‐Dependent Relaxation of Nitrate Tolerant Coronary Arteries

Prolonged exposure to nitroglycerin (NTG) leads to tolerance and impaired endothelium‐dependent vasodilation. Causes of endothelial dysfunction in nitrate tolerant arteries are well studied, but little is known about mechanisms that underlie the responses to endothelium‐derived mediators in nitrate tolerant blood vessels. Here we evaluated the role of large conductance, calcium‐activated K channels (BKCa) in acetylcholine (ACh)‐induced relaxation of isolated coronary arteries. Rats (Sprague‐Dawley, 250–300g) were treated with or without transdermal NTG patches (0.6 mg/hr) for 3 days. Isolated coronary arterial rings (diameter: 150–300 μm) were mounted in a multi‐wire myograph for isometric tension recording. Relaxation responses were determined in rings contracted with 5‐HT (0.1–1μM). In some experiments, arterial segments were incubated with the selective BKCa channel inhibitor, iberiotoxin (IBTx, 100 nM), for 20 min prior to contraction. Expression of BKCa channel proteins was determined by Western blot. Nitrate tolerance was confirmed by the impaired relaxation response to NTG (1 nM – 100 μM) observed in rings from rats treated with NTG patches (pD2= 7.28 ± 0.1 vs 6.45 ± 0.2, control vs NTG patch‐treated, respectively; p&lt;0.05). Relaxations to the endothelium‐dependent vasodilator, ACh (1 nM – 10 μM) were impaired in nitrate tolerant arteries (pD2 = 7.01 ± 0.1 vs 6.52 ± 0.1; control vs tolerant, respectively; n=8; p&lt;0.05). Incubation with the eNOS inhibitor, nitro‐l‐arginine (30 μM), or the guanylyl cyclase inhibitor, ODQ (10 μM), markedly inhibited the response to ACh in control and tolerant arteries, consistent with the release of endothelium‐derived NO in ACh‐induced relaxation of both control and tolerant arteries. Similarly, IBTx markedly reduced the response to ACh in control and tolerant arteries; however, IBTx was a more effective inhibitor of ACh‐induced relaxation in control arteries. Relaxations to 8‐Br‐cGMP (100 nM – 300 μM), a stable, cell‐permeable cGMP analog, were inhibited by IBTx in control arteries (pD2 = 5.37 ± 0.2 vs 4.21 ± 0.2; in the absence and presence of IBTx, respectively; n=4; p&lt;0.05), but not in tolerant arteries (pD2 = 4.45 ± 0.1 vs 4.03 ± 0.1; in the absence and presence of IBTx; n=3; p&gt;0.05). Protein expression of BKCa α‐subunits and β‐subunits was decreased by more than 40% in nitrate tolerant arteries (n=4–5; p&lt;0.05). Taken together, the data indicate that, under control conditions, BKCa channels play a pivotal role in endothelium‐dependent relaxation to ACh in rat coronary arteries. In nitrate tolerant coronary arteries, the response to ACh is impaired and the contribution of BKCa channels to ACh‐induced relaxation is diminished.Support or Funding InformationSupported in part by grant HL098896 from the National Institutes of Health

Lisinopril alters contribution of nitric oxide and K(Ca) channels to vasodilatation in small mesenteric arteries of spontaneously hypertensive rats.

To investigate lisinopril effect on the contribution of nitric oxide (NO) and K(Ca) channels to acetylcholine (ACh)-induced relaxation in isolated mesenteric arteries of spontaneously hypertensive rats (SHRs). Third branch mesenteric arteries isolated from lisinopril treated SHR rats (20 mg/kg/day for ten weeks, SHR-T) or untreated (SHR-UT) or normotensive WKY rats were mounted on tension myograph and ACh concentration-response curves were obtained. Westernblotting of eNOS and K(Ca) channels was performed. ACh-induced relaxations were similar in all groups while L-NMMA and indomethacin caused significant rightward shift only in SHR-T group. Apamin and TRAM-34 (SK(Ca) and IK(Ca) channels blockers, respectively) significantly attenuated ACh-induced maximal relaxation by similar magnitude in vessels from all three groups. In the presence of L-NMMA, indomethacin, apamin and TRAM-34 further attenuated ACh-induced relaxation only in SHR-T. Furthermore, lisinopril treatment increased expression of eNOS, SK(Ca) and BK(Ca) proteins. Lisinopril treatment increased expression of eNOS, SK(Ca), BK(Ca) channel proteins and increased the contribution of NO to ACh-mediated relaxation. This increased role of NO was apparent only when EDHF component was blocked by inhibiting SK(Ca) and IK(Ca) channels. Such may suggest that in mesenteric arteries, non-EDHF component functions act as a reserve system to provide compensatory vasodilatation if (and when) hyperpolarization that is mediated by SK(Ca) and IK(Ca) channels is reduced.

Different expressions of large-conductance Ca2+-activated K+ channels in the mouse renal cortex and hippocampus during postnatal development.

Large-conductance Ca(+)-activated K(+) (BKCa) channels are widely distributed in a variety of cells and play a pivotal and specific role in many pathophysiological conditions. However, the function of BK(Ca) channels in the kidney cortex and hippocampus during the postnatal development has not received attention. In this study, to elucidate the role of BK(Ca) channels during the development, it is essential to establish the location and quantitation of expression of BK(Ca). The expressions of BK(Ca) were detected in the kidney and hippocampus on postnatal days (P) 1, 3, 5, 7, 14, 21, 28, and 49 by immunohistochemical and Western blot analysis. Our results showed that expressions of BK(Ca) channels were found in tubules and corpuscles at all time points. The expression was also observed at all developmental stages of the renal corpuscles, such as comma-shaped body, S-shaped body, renal corpuscles of stage III, and renal corpuscles of stage IV. During the development, the expression of BK(Ca) channels was decreased and the most prominent change of BK(Ca) protein level appeared between P14 and P21. In contrast, BK(Ca) channels were expressed in all regions of the hippocampus at every time point with the level increasing during the early development (P1 to P14). The findings of the present study suggest that BKCa channels play an important role during the postnatal development in both the renal cortex and hippocampus.

Effects of sodium metabisulfite on the expression of BKCa, KATP, and L-Ca2+ channels in rat aortas in vivo and in vitro

Sodium metabisulfite (SMB) is most commonly used as the preservative in many food preparations and drugs. So far, few studies about its negative effects were reported. The purpose of this study was to investigate the effect of SMB on the expression of big-conductance Ca2+-activated K+ (BKCa), ATP-sensitive K+ (KATP), and L-type calcium (L-Ca2+) channels in rat aorta in vivo and in vitro. The results showed that the mRNA and protein levels of the BKCa channel subunits α and β1 of aorta in rats were increased by SMB in vivo and in vitro. Similarly, the expression of the KATP channel subunits Kir6.1, Kir6.2, and SUR2B were increased by SMB. However, SMB at the highest concentration significantly decreased the expression of the L-Ca2+ channel subunits Cav1.2 and Cav1.3. These results suggest that SMB can activate BKCa and KATP channels by increasing the expression of α, β1, and Kir6.1, Kir6.2, SUR2B respectively, while also inhibit L-Ca2+ channels by decreasing the expression of Cav1.2 and Cav1.3 of aorta in rats. The molecular mechanism of SMB-induced vasorelaxant effect might be related to the expression changes of BKCa, KATP, and L-Ca2+ channels subunits. Further work is needed to determine the relative contribution of each channel in SMB-mediated vasorelaxant effect.

Effects of gaseous sulfur dioxide and its derivatives on the expression of KATP, BKCa and L-Ca2+ channels in rat aortas in vitro

Epidemiological investigations have revealed that sulfur dioxide (SO2) exposure is linked to cardiovascular diseases. Our previous study indicated that the vasorelaxant effect of SO2 might be partly related to ATP-sensitive K+ (KATP), big-conductance Ca2+-activated K+ (BKCa) and L-type calcium (L-Ca2+) channels. The present study was designed to further investigate the effects of gaseous SO2 and its derivatives on the gene and protein expression of these channels in the rat aortas in vitro. The results showed that the mRNA and protein levels of the KATP channel subunits Kir6.1, Kir6.2 and SUR2B of the rat aortas in SO2 and its derivatives groups were higher than those in control group. Similarly, the expression of the BKCa channel subunits α and β1 was increased by SO2 and its derivatives. However, SO2 and its derivatives at 1500μM significantly decreased the expression of the L-Ca2+ channel subunits Cav1.2 and Cav1.3. Histological examination of the rat aorta tissues showed moderate injury of tunica media induced by SO2 and its derivatives at 1500μM. These results suggest that SO2 and its derivatives can activate the KATP and BKCa channels by increasing the expression of Kir6.1, Kir6.2, SUR2B and α, β1, respectively, while also inhibiting the L-Ca2+ channels by decreasing the expression of Cav1.2 and Cav1.3 of the rat aortas. The molecular mechanism of the vasorelaxant effect of SO2 and its derivatives might be related to the expression changes of KATP, BKCa and L-Ca2+ channel subunits, which may play a role in the pathogenesis of SO2-associated cardiovascular diseases.