BK Channelopathies and KCNMA1-Linked Disease Models.

Novel KCNMA1 variants, encoding the BK K+ channel, are associated with a debilitating dyskinesia and epilepsy syndrome. Neurodevelopmental delay, cognitive disability, and brain and structural malformations are also diagnosed at lower incidence. More than half of affected individuals present with a rare negative episodic motor disorder, paroxysmal nonkinesigenic dyskinesia (PNKD3). The mechanistic relationship of PNKD3 to epilepsy and the broader spectrum of KCNMA1-associated symptomology is unknown. This review summarizes patient-associated KCNMA1 variants within the BK channel structure, functional classifications, genotype-phenotype associations, disease models, and treatment. Patient and transgenic animal data suggest delineation of gain-of-function (GOF) and loss-of-function KCNMA1 neurogenetic disease, validating two heterozygous alleles encoding GOF BK channels (D434G and N999S) as causing seizure and PNKD3. This discovery led to a variant-defined therapeutic approach for PNKD3, providing initial insight into the neurological basis. A comprehensive clinical definition of monogenic KCNMA1-linked disease and the neuronal mechanisms currently remain priorities for continued investigation.

Acute, pro-contractile effects of prorenin on rat mesenteric arteries.

Prorenin and the prorenin receptor ((P)RR) are important, yet controversial, members of the renin-angiotensin-aldosterone system. The ((P)RR) is expressed throughout the body, including the vasculature, however, the direct effect of prorenin on arterial contractility is yet to be determined. Within rat mesenteric arteries, immunostaining and proximity ligation assays were used to determine the interacting partners of (P)RR in freshly isolated vascular smooth muscle cells (VSMCs). Wire myography examined the functional effect of prorenin. Simultaneous changes in [Ca2+ ]i and force were recorded in arteries loaded with Fura-2AM. Spontaneously transient outward currents were recorded via perforated whole-cell patch-clamp configuration in freshly isolated VSMCs. We found that the (P)RR is located within a distance of less than 40 nm from the V-ATPase, caveolin-1, ryanodine receptors, and large conductance Ca2+ -activated K+ channels (BKCa ) in VSMCs. [Ca2+ ]i imaging and isometric tension recordings indicate that 1 nM prorenin enhanced α1-adrenoreceptor-mediated contraction, associated with an increased number of Ca2+ waves, independent of voltage-gated Ca2+ channels activation. Incubation of VSMCs with 1 nM prorenin decreased the amplitude and frequency of spontaneously transient outward currents and attenuated BKCa -mediated relaxation. Inhibition of the V-ATPase with 100 nM bafilomycin prevented prorenin-mediated inhibition of BKCa -derived relaxation. Renin (1 nM) had no effect on BKCa -mediated relaxation. In conclusion, prorenin enhances arterial contractility by inhibition of BKCa and increasing intracellular Ca2+ release. It is likely that this effect is mediated through a local shift in pH upon activation of the (P)RR and stimulation of the V-ATPase.

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, SM contraction and expression of BKCa , SphK1, sphingosine-1-phosphate receptor (S1PR) were analyzed after the treatment with vehicle, BSA-E2, E2, and E2 receptor antagonist. The role of BKCa , SphK1, and S1PR in E2-induced SM dysmotility was investigated in rat colonic SMCs. The effect of SphK1 on SM contraction 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 SM and a delayed colonic transit. The treatment with E2 significantly upregulated the expression of BKCa , SphK1, S1PR1, and S1PR2, but not S1PR3, in colon SM and SMCs. Inhibition of BKCa , SphK1, S1PR1, and S1PR2 expression attenuated the effect of E2 on Ca2+ mobilization in rat colon SMCs. WT mice 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 SM through 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.

Influence of atherosclerosis on the molecular expression of the TRPC1/BK signal complex in the aortic smooth muscles of mice.

Atherosclerosis (AS) is one a disease that seriously endangers human health. Previous studies have demonstrated that transient receptor potential channel-1 (TRPC1)/large conductance Ca2+ activated K+ channel (BK) signal complex is widely distributed in arteries. Therefore, it was hypothesized that TRPC1-BK signal complex may be a new target for the treatment of AS-related diseases. Apolipoprotein E-/- (ApoE-/-) mice were used to establish an atherosclerotic animal model in the present study, and the association between AS and the TRPC1-BK signal complex was examined. The present study aimed to compare the differences in the expression levels of mRNAs and proteins of the TRPC1-BK signal complex expressed in the aortic vascular smooth muscle tissue, between mice with AS and control mice. There were 10 mice in each group. Reverse transcription PCR, western blotting and immunohistochemistry were used to detect the differences in the mRNA and protein expression levels of TRPC1, BKα (the α subunit of BK) and BKß1 (the ß1 subunit of BK). The mRNA expression level of TRPC1 in AS model mice was significantly higher compared with that in the control group (P<0.05). However, the mRNA expression levels of BKα and BKß1 were lower compared with those in the controls (both P<0.01). The mice in the ApoE-/- group successfully developed AS. In this group, the protein expression level of TRPC1 was significantly higher than that in the control group (P<0.01), while the protein expression levels of BKα and BKß1 were lower compared with those in the control group (P<0.01 and P<0.05, respectively). Collectively, it was identified that the protein and mRNA expression levels of the TRPC1/BK signal complex in the aortic vascular smooth muscle tissue could be influenced by the development of AS in mice. Hence, the TRPC1/BK signal complex may be a potential therapeutic target for the prevention and treatment of AS-related complications in the future.

Kynurenine Relaxes Arteries of Normotensive Women and Those With Preeclampsia.

[Figure: see text].

Heme is required for carbon monoxide activation of mitochondrial BKCa channel.

Carbon monoxide (CO) is an endogenously synthesized gaseous mediator and is involved in the regulation of numerous physiological processes. Mitochondria, in which hemoproteins are abundant, are among the targets for CO action. Large-conductance calcium-activated (mitoBKCa) channels in the inner mitochondrial membrane share multiple biophysical similarities with the BKCa channels of the plasma membrane and could be a potential target for CO. To test this hypothesis, the activity of the mitoBKCa channels in human astrocytoma U-87 MG cell mitochondria was assessed with the patch-clamp technique. The effects of CO-releasing molecules (CORMs), such as CORM-2, CORM-401, and CORM-A1, were compared to the application of a CO-saturated solution to the mitoBKCa channels in membrane patches. The applied CORMs showed pleiotropic effects including channel inhibition, while the CO-containing solution did not significantly modulate channel activity. Interestingly, CO applied to the mitoBKCa channels, which were inhibited by exogenously added heme, stimulated the channel. To summarize, our findings indicate a requirement of heme binding to the mitoBKCa channel for channel modulation by CO and suggest that CORMs might have complex unspecific effects on mitoBKCa channels.

Prenatal hypoxia inhibited propionate-evoked BK channels of mesenteric artery smooth muscle cells in offspring.

As a common complication of pregnancy, gestational hypoxia has been shown to predispose offspring to vascular dysfunction. Propionate, one of short-chain fatty acids, exerts cardioprotective effects via reducing blood pressure. This study examined whether prenatal hypoxia impaired propionate-stimulated large-conductance Ca2+ -activated K+ (BK) channel activities in vascular smooth muscle cells (VSMCs) of offspring. Pregnant rats were exposed to hypoxia (10.5% oxygen) and normoxia (21% oxygen) from gestational day 7-21. At 6 weeks of age, VSMCs in mesenteric arteries of offspring were analysed for BK channel functions and gene expressions. It was shown firstly that propionate could open significantly BK single channel in VSMCs in a concentration-dependent manner. Antagonists of G protein ßγ subunits and inositol trisphosphate receptor could completely suppress the activation of BK by propionate, respectively. Gαi/o and ryanodine receptor were found to participate in the stimulation on BK. Compared to the control, vasodilation and increments of BK NPo (the open probability) evoked by propionate were weakened in the offspring by prenatal hypoxia with down-regulated Gßγ and PLCß. It was indicated that prenatal hypoxia inhibited propionate-stimulated BK activities in mesenteric VSMCs of offspring via reducing expressions of Gßγ and PLCß, in which endoplasmic reticulum calcium release might be involved.

[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.

Downregulation of large conductance calcium-activated potassium channels in paraventricular nucleus contributes to sympathoexcitation in rats with chronic heart failure / 中华心血管病杂志

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.

Effects of WNK3 kinase on regulation of large-conductance calcium-activated potassium channels and its mechanisms / 中华肾脏病杂志

Objective To investigate the effects of WNK3 kinase on the regulation of large-conductance calcium-activated potassium channels (Maxi K channels) on African green monkey kidney fibroblast-like cells (Cos-7 cells) and its mechanisms.Methods (1) Cos-7 cells were transfected with 0,0.6,1.2,1.8 μg WNK3 plasmid+0.5 μg Maxi K plasmid.The total protein expression of Maxi K channel and the phosphorylation of mitogen-activated protein kinase (MAPK) extracellular regulated kinase-1 and-2 (ERK1/2) were detected by Western blotting.(2) Cos-7 cells were divided into the control group (2.5 μg Maxi K plasmid) and the experimental group (2.5 μg WNK3 plasmid+2.5 μg Maxi K plasmid).Cell surface biotinylation was used to investigate the cell surface protein expression of Maxi K channel in Cos-7 cells.Immunoprecipitation and Western blotting were used to detect the ubiquitination of Maxi K channel protein.(3) WNK3 kinase was knocked down by WNK3 siRNA.The lysosomal degradation pathway was blocked by the proton pump inhibitor (Baf-A1).Cos-7 cells were divided into Maxi K+negative control siRNA group,Maxi K+WNK3 siRNA group and Maxi K+WNK3 siRNA+Baf-A1 group.The protein expression of Maxi K channel protein was detected by Western blotting.Results (1) Compared with those in 0 μg WNK3 plasmid groups,in 0.6,1.2,1.8 μg WNK3 plasmid groups the total protein expression of the Maxi K channel increased and the phosphorylation level of MAPK ERK1/2 reduced on a dose-dependent manner (all P ＜ 0.01).(2)Compared with those in the control group,the total protein expression and cell surface membrane protein expression of the Maxi K channel increased in the experimental group (P ＜ 0.01),while the ubiquitination of the Maxi K channel protein reduced (P ＜ 0.01).(3) Compared with the Maxi K +negative control siRNA group,the expression of Maxi K protein reduced in the Maxi K+WNK3 siRNA group (P ＜ 0.01),but did not change in the Maxi K+WNK3 siRNA + Bar-A1 group (P ＞ 0.05).The expression of Maxi K protein in Maxi K+WNK3 siRNA+Baf-A1 group was higher than that in Maxi K+WNK3 siRNA group (P ＜ 0.01).Conclusions WNK3 kinase inhibits the lysosomal degradation pathway of Maxi K channel protein by reducing the ubiquitination of Maxi K channel,and promotes the expression of Maxi K channel protein in cells and on cell membrane.These effects may be achieved by suppressing MAPK ERK1/2 signal transduction pathway.

Molecular Mechanisms Underlying Renin-Angiotensin-Aldosterone System Mediated Regulation of BK Channels.

Large-conductance calcium-activated potassium channels (BK channels) belong to a family of Ca2+-sensitive voltage-dependent potassium channels and play a vital role in various physiological activities in the human body. The renin-angiotensin-aldosterone system is acknowledged as being vital in the body's hormone system and plays a fundamental role in the maintenance of water and electrolyte balance and blood pressure regulation. There is growing evidence that the renin-angiotensin-aldosterone system has profound influences on the expression and bioactivity of BK channels. In this review, we focus on the molecular mechanisms underlying the regulation of BK channels mediated by the renin-angiotensin-aldosterone system and its potential as a target for clinical drugs.

T-type voltage-gated Ca2+ channels do not contribute to the negative feedback regulation of myogenic tone in murine superior epigastric arteries.

T-type voltage-gated Ca2+ channels (CaV3.2 VGCC) have been hypothesized to control spontaneous transient outward currents (STOCs) through large-conductance Ca2+-activated K+ channels (BKCa), and contribute to the negative-feedback regulation of myogenic tone. We tested this hypothesis in superior epigastric arteries (SEAs) isolated from male C57BL/6 mice. SEAs were isolated and enzymatically dissociated to obtain single smooth muscle cells (SMCs) for whole-cell recording of paxilline-sensitive (PAX, 1 µmol/L) STOCs at -30 mV, or cannulated and studied by pressure myography (80 cm H2O, 37°C). The CaV3.2 blocker Ni2+ (30 µmol/L) had no effect on STOC amplitude (20.1 ± 1.7 pA vs. 20.6 ± 1.7 pA; n = 12, P = 0.6), but increased STOC frequency (0.79 ± 0.15 Hz vs. 1.21 ± 0.22 Hz; n = 12, P = 0.02). Although Ni2+ produced concentration-dependent constriction of isolated, pressurized SEAs (logEC50 = -5.8 ± 0.09; Emax = 72 ± 5% constriction), block of BKCa with PAX had no effect on vasoconstriction induced by 30 µmol/L Ni2+ (in the absence of PAX = 66 ± 4% constriction vs. in the presence of 1 µmol/L PAX = 65 ± 4% constriction; n = 7, P = 0.06). In contrast to Ni2+, the nonselective T-type blocker, mibefradil, produced only vasodilation (logEC50 = -6.9 ± 0.2; Emax = 74 ± 8% dilation), whereas the putative T-type blocker, ML218, had no significant effect on myogenic tone between 10 nmol/L and 10 µmol/L (n = 6-7, P = 0.59). Our data do not support a role for CaV3.2 VGCC in the negative-feedback regulation of myogenic tone in murine SEAs and suggest that Ni2+ may constrict SEAs by means other than block of CaV3.2 VGCC.

Effects of docosahexenoic acid on large conductance Ca+-activated K+ channels in retinal smooth muscle cells / 中华眼底病杂志

Objective To investigate the effects ofdocosahexenoic acid (DHA) on large conductance Ca2+-activated K+ (BK) channels in normal retinal artery smooth muscle cells (RASMCs).Methods Cultured human RASMCs (6 th-8 th generations) were used to patch clamp experiment.The open probabihties (NP0) in BK channels with different concentrations (0.0,1.0,3.0,5.0,7.5,10.0 μmol/L) of DHA were recorded by patch clamp technique in single channel configuration.RASMCs were intervened by different concentrations (0.0,1.0,5.0 μmol/L) of DHA as control group,low and high doses of DHA groups,respectively.The protein expressions of β subunit of BK channels in RASMCs from three groups were measured by Western blot.Results The NP0 of BK channels were 0.044 4±0.001 2,0.081 2±0.004 2,0.209 0±0.006 1,0.310 5±0.005 3,0.465 0±0.007 8 and 0.497 7±0.014 5 with perfusate of 0.0,1.0,3.0,5.0,7.5,10.0 μtmol/L DHA.DHA activated BK channels in a dose-dependent manner (F=2.621,P＜0.05).There was no significant difference in the protein expression of control group,low and high doses of DHA groups (F=1 1.657,P＞0.05).Conclusion DHA can directly activate BK channels,no increasing in subunit expression of BK channels.

[Construction and identification of BKCaαsubunit expression plas-mid with double labeling of Flag and GFP].

OBJECTIVE: This study aimed to construct a large conductance calcium activated potassium channel α (BKCa) subunit plasmid with two tags by the overlapping PCR technique to set up a steady base for future ion channel study. METHODS: Based on the existing coding BKCa channel α subunit expression plasmid pcDNA3.1-hSlo, we constructed a double-tag expression plasmid, namely, pcDNA3.1-Flag-hSlo-GFP (Flag-hSlo-GFP). RESULTS: Flag tag was inserted into the S1-S2 extracellular loop of BKCa channel α subunit, and GFP tag was connected to the C-terminus of BKCa channel α subunit. Sequence of the constructed plasmid was confirmed successful. CONCLUSIONS: The expression plasmid Flag-hSlo-GFP was constructed successfully with overlapping PCR. Overlapping PCR is a valuable method for amplifying long size genes.

Cognitive recovery by chronic activation of the large-conductance calcium-activated potassium channel in a mouse model of Alzheimer's disease.

We previously showed that activity of the large conductance calcium-activated potassium (Big-K; BK) channels is suppressed in 3xTg Alzheimer disease (AD) model mice. However, its behavioral significance is not known. In the present report, ventricular injection of the BK channel activator isopimaric acid (ISO) was conducted to examine whether BK channel activation ameliorates cognition in 3xTg mice. The novel object recognition (NOR) test revealed that chronic injection of ISO improved non-spatial memory in 3xTg mice. In the Morris water maze, the probe test demonstrated an improved spatial memory after ISO injection. Electrophysiological underpinnings of the ISO effect were then examined in slices obtained from the mice after behavior. At hippocampal CA1 synapses, the basic synaptic transmission was abnormally elevated and long-term potentiation (LTP) was partially suppressed in 3xTg mice. These were both recovered by ISO treatment. We then confirmed suppressed BK channel activity in 3xTg mice by measuring the half-width of evoked action potentials. This was also recovered by ISO treatment. We previously showed that the recovery of BK channel activity accompanies reduction of neuronal excitability in pyramidal cells. Here again, pyramidal cell excitability, as assessed by calculating the frequency of evoked spikes, was elevated in the 3xTg mouse and was normalized by ISO. ELISA experiments demonstrated an ISO-induced reduction of Aß1-42 content in hippocampal tissue in 3xTg mice. The present study thus suggests a potential therapeutic utility of BK channel activators for AD.

Role of large conductance calcium-activated potassium channels in vascular hyporesponsiveness in rats with obstructive jaundice / 中华麻醉学杂志

Objective To evaluate the role of large conductance calcium-activated potassium (BKCa) channels in vascular hyporesponsiveness in rats with obstructive jaundice.Methods Eighteen male Sprague-Dawley rats,weighing 180-200 g,were randomly divided into 3 groups (n =6 each) using a random number table:control group (group C),sham operation group (group S),and bile duct ligation group (group BDL).Obstructive jaundice was produced by common bile duct ligation.At 7 days after surgery,blood samples were collected for determination of the levels of serum total bilirubin (TBL),direct bilirubin (DBL),indirect bilirubin (IBL),alanine aminotransferase (ALT),and aspartate aminotransferase (AST).Thoracic aortic rings were prepared,and the endothelium was removed.The aortic rings were sequentially perfused with different concentrations of norepinephrine (NE) and sodium nitroprusside (SNP),and the maximum amplitude of contraction and dilatation of aortic rings was recorded.The aortic rings were then perfused with BKCa channel blocker Chtx with the final concentration of 10 7 mol/L,followed by perfusion with different concentrations of NE and SNP again,and the maximum amplitude of contraction and dilatation of aortic rings was recorded under each concentration.The percentage of maximum contraction and dilatation (maximum amplitude after Chtx administration÷maximum amplitude before Chtx administration× 100％) was calculated.Results Compared with C and S groups,the levels of TBL,DBL,IBL,ALT and AST in serum were significantly increased,the maximum amplitude of NE-induced contraction of aortic rings was decreased,and the percentage of the maximum NE-induced dilatation of aortic rings was increased,the maximum amplitude of SNP-induced contraction of aortic rings was increased,and the percentage of the maximum SNP-induced dilatation of aortic rings was decreased in group BDL.Conclusion Excessivc opening of BKCa channels may be involved in the mechanism of vascular hyporesponsiveness in rats with obstructive jaundice.

The quantitative determination of BKCa channels in placental arteriole smooth muscle cells of hypertensive disorder complicating pregnancy / 重庆医学

Objective To observe the change of protein levels of large conductance calcium activated potassium channel (BK‐Ca) in placental arteriole smooth muscle cells from hypertensive disorder complicating pregnancy and discuss its role .Methods Western blot analysis was used to examine protein expression of α subunit andβ1 subunit of BKCa channels in placental arteriole smooth muscle cells from hypertensive disorder complicating pregnancy .Results The relative protein expression level of α‐subunit in the HDCP group was 1 .001 2 ± 0 .169 8(n=15) ,and the NT group was 1 .028 2 ± 0 .180 6 (n=15) .There was no significant differences between the two groups (P> 0 .05);the relative protein expression level of β1 subunit in the HDCP group was 0 .418 1 ± 0 .080 8 (n=15) ,and the NT group was 1 .616 8 ± 0 .012 6 (n=15) ,theβ1‐subunit protein expression levels of HDCP group were significantly lower (P<0 .05) .Conclusion The protein expression ofβ1‐subunit ,but notα‐subunit ,was reduced in pla‐cental arteriole smooth muscle cells from hypertensive disorder complicating pregnancy .Therefore ,BKCa channel activity may have been involved in hypertensive disorder complicating pregnancy ;and the abnormal expression ofβ1 subunit maybe an important basis in hypertensive disorder complicating pregnancy .

Specific respiratory neuron types have increased excitability that drive presympathetic neurones in neurogenic hypertension.

A major aspect of hypertension is excessive sympathetic activity but the reasons for this remain elusive. Sympathetic tone is increased in the spontaneously hypertensive (SH) rat reflecting, in part, enhanced respiratory-sympathetic coupling. We aimed to identify which respiratory cells might have altered properties. Using the working heart-brain stem preparation, we monitored simultaneously sympathetic and respiratory nerve activity in combination with intracellular recordings of physiologically characterized medullary presympathetic or respiratory neurons. In SH rats, respiratory modulation of both inspiratory and postinspiratory phases of sympathetic activity was larger relative to Wistar rats. An additional burst of sympathetic activity in the preinspiratory phase was also present in SH rats. After synaptic isolation of rostral medullary presympathetic neurons, there was no difference in their excitability compared with neurons in Wistar rats. Rather, both pre-Bötzinger preinspiratory and Bötzinger postinspiratory neurons had increased neuronal excitability in SH rats relative to Wistar rats; this was attributed to higher input resistance/reduced leak current in preinspiratory neurons and reduced calcium activated potassium conductance in postinspiratory neurons. Thus, the respiratory network of the SH rat is reconfigured to a pattern dominated by heightened excitability of preinspiratory and postinspiratory neurons. These neurons both provide augmented excitatory synaptic drive to rostral medullary presympathetic neurons contributing to excessive sympathetic nerve activity associated with hypertension in the in situ SH rat. Our data indicate selective modulation of potassium conductances in 2 subsets of respiratory neurons contributing to neurogenic hypertension.

Role of large-conductance Ca2+-activated K+ channels and protein kinase G in ketamine-induced isolated tracheal smooth muscle relaxation in rats with asthma / 中华麻醉学杂志

Objective To investigate the role of large-conductance Ga2+-activated K+ (BKCa) channels and protein kinase G (PKG) in ketamine-induced isolated tracheal smooth muscle relaxation in rats with asthma.Methods Healthy Sprague-Dawley rats,weighing 250-300 g,were used in this study.Asthma was induced with egg albumin.Thirty-six tracheal rings of 15 rats in which asthma model was successfully established were randomly divided into 3 groups (n =12 each):ketamine treatment group (group AK),IBTX (BKCa channel blocker) +ketamine treatment group (group AKI),and KT-58232 (PKG inhibitor) + ketamine treatment group (group AKK).Tracheal rings were suspended in an organ bath filled with oxygenated Kreb's solution at 36.5-37.5 ℃.In group AK,the tracheal rings were precontracted with acetyleholine 0.1 mmol/L,and the rings were then exposed to ketamine 0.4 g/L for 15 min.In group AKI,before acetyleholine and ketamine were added to the solution,the rings were pretreated with IBTX 3μmol/L for 30 min.In group AKK,before acetyleholine and ketamine were added to the solution,the rings were pretreated with KT-5823 2μmol/L for 30 min.The tension of rings was measured by using a force-displacement transducer.Results The amplitude of relaxation of isolated tracheal smooth muscle was significantly decreased in groups AKI and AKK as compared with group AK (P ＜ 0.05).Conclusion Ketamine induces isolated tracheal smooth muscle relaxation through activating BKCa channels and PKG signaling pathway in rats with asthma.

Alterations in Calcium-Activated Potassium Channel Expressions in Human Prostate Cancer / 대한비뇨기과학회지

PURPOSE: Recent studies have shown that potassium (K+) and sodium channels are involved in prostate cell growth. However, a great many of the studies have been done in prostate cancer cell lines and there are only scant studies on prostate cancer and benign prostatic hypertrophy (BPH) tissue. The present study was aimed to evaluate the alterations of the calcium-activated K+ channel (KCa) expression in prostate cancer, and to compare them with the expression profiles in human BPH tissue to understand their potential role in the progression of prostate cancer. MATERIALS AND METHODS: The prostate tissues obtained from radical prostatectomy (n=10) and transurethral resection of the prostate (n=18) were quickly frozen in liquid nitrogen for the RNA measurements. The protein and mRNA levels of the KCa subtypes and connexins were measured by performing immunoblot analysis and reverse-transcription polymerase chain reaction, respectively. RESULTS: The mRNA levels of type 2 (SK2) and type 3 (SK3) small-conductance and large-conductance (BK) KCas in the prostate cancer tissues were decreased more than 50% compared with those in the BPH samples. In addition, the BK and SK2 protein levels in prostate cancer were also significantly lower than those in the BPH. As reported previously, the connexin 26 and 43 transcript signals in the prostate cancer were significantly reduced compared with those in the BPH samples. CONCLUSIONS: These results suggest that the impaired expression of KCas may have a role in tumor progression via aberrant and uncontrolled prostate cell growth.