scholarly journals Enhanced large conductance K+ channel activity contributes to the impaired myogenic response in the cerebral vasculature of Fawn Hooded Hypertensive rats

2014 ◽  
Vol 306 (7) ◽  
pp. H989-H1000 ◽  
Author(s):  
Mallikarjuna R. Pabbidi ◽  
Olga Mazur ◽  
Fan Fan ◽  
Jerry M. Farley ◽  
Debebe Gebremedhin ◽  
...  
Keyword(s):  
Congenic Strain ◽  
Cerebral Arteries ◽  
Bk Channel ◽  
Chromosome 1 ◽  
Brown Norway ◽  
K Channel ◽  
Myogenic Response ◽  
Voltage Sensitivity ◽  
Channel Activity ◽  
Open Probability

Recent studies have indicated that the myogenic response (MR) in cerebral arteries is impaired in Fawn Hooded Hypertensive (FHH) rats and that transfer of a 2.4 megabase pair region of chromosome 1 (RNO1) containing 15 genes from the Brown Norway rat into the FHH genetic background restores MR in a FHH.1BN congenic strain. However, the mechanisms involved remain to be determined. The present study examined the role of the large conductance calcium-activated potassium (BK) channel in impairing the MR in FHH rats. Whole-cell patch-clamp studies of cerebral vascular smooth muscle cells (VSMCs) revealed that iberiotoxin (IBTX; BK inhibitor)-sensitive outward potassium (K+) channel current densities are four- to fivefold greater in FHH than in FHH.1BN congenic strain. Inside-out patches indicated that the BK channel open probability ( NP o) is 10-fold higher and IBTX reduced NP o to a greater extent in VSMCs isolated from FHH than in FHH.1BN rats. Voltage sensitivity of the BK channel is enhanced in FHH as compared with FHH.1BN rats. The frequency and amplitude of spontaneous transient outward currents are significantly greater in VSMCs isolated from FHH than in FHH.1BN rats. However, the expression of the BK-α and -β-subunit proteins in cerebral vessels as determined by Western blot is similar between the two groups. Middle cerebral arteries (MCAs) isolated from FHH rats exhibited an impaired MR, and administration of IBTX restored this response. These results indicate that there is a gene on RNO1 that impairs MR in the MCAs of FHH rats by enhancing BK channel activity.

Abstract 485: Enhanced Large Conductance Ca+2 -sensitive K+ Channels (bk) Activity Impairs the Myogenic Response in the Cerebral Vasculature of Fawn Hooded Hypertensive (fhh) Rat

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Mallikarjuna R Pabbidi ◽  
Jerry Farley ◽  
Debebe Gebremedhin ◽  
David R. Harder ◽  
Richard J. Roman
Keyword(s):  
Congenic Strain ◽  
Single Channel ◽  
Cerebral Arteries ◽  
Fold Increase ◽  
Bk Channel ◽  
Chromosome 1 ◽  
K Channel ◽  
Myogenic Response ◽  
Whole Cell Patch Clamp ◽  
Current Densities

Our recent studies have revealed that the myogenic response in cerebral arteries and autoregulation of cerebral blood flow is impaired in FHH and that transfer of a 2.4 Mb region of chromosome 1 from BN into FHH.1 BN congenic strain restores these responses. The present study examined the role of large conductance calcium activated potassium (BK) channel in altering the myogenic response in FHH rats. Whole-cell patch-clamp of cerebral vascular smooth muscle cells (VSMC) revealed a 4.6 fold increase in outward potassium (K) channel current densities (pA/pF) in FHH rats compared to FHH.1 BN congenic strain. Iberiotoxin (IBTX -a selective BK channel inhibitor) sensitive current densities are significantly greater in FHH rats compared with the FHH.1 BN congenic strain (FHH rats: +40mV; pre IBTX 43.1 ± 7.2, after IBTX 11.8 ± 2.2 pA/pF versus pre IBTX 5.6 ± 1 pA/pF and after IBTX 4.1 ± 0.6 pA/pF in the FHH.1 BN congenic strain). In excised patches, the BK channel exhibited similar single-channel slope conductance for FHH and the FHH.1 BN rats (208.9 pS versus 208.7 pS). However, the open channel probability (NP o ) was ~10 fold higher in FHH rats than in FHH.1 BN rats (1μM free (Ca +2 ) i : +40mV: FHH; 0.8 ± 0.04 versus 0.08 ± 0.004 in FHH.1 BN rats). Voltage and Ca 2+ sensitivity of the BK channel is similar in cerebral VSMC isolated from FHH and FHH.1 BN rats. Middle cerebral arterioles (MCA) isolated from FHH rats increased in diameter from 142 ± 16 to 157 ± 19 μm when pressure was increased from 40 to 140 mmHg. In contrast, the diameter of the MCA decreased by 49% in the FHH.1 BN congenic strain from 127 ± 16 to 65 ± 13 μm. Pharmacological block of BK channel by IBTX (100nM) restored myogenic response in FHH rats but had no effect in FHH.1 BN rats. These results indicate that the impaired myogenic response of the cerebral vessels in FHH rats is mediated via a gene and mechanism that enhances BK channel activity.

scholarly journals Elevated K+ channel activity opposes vasoconstrictor response to serotonin in cerebral arteries of the Fawn Hooded Hypertensive rat

2017 ◽  
Vol 49 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Mallikarjuna R. Pabbidi ◽  
Richard J. Roman
Keyword(s):  
Calcium Concentration ◽  
Cerebral Arteries ◽  
Cytosolic Calcium ◽  
Small Region ◽  
Bk Channel ◽  
Chromosome 1 ◽  
Myogenic Response ◽  
Channel Activity ◽  
Cytosolic Calcium Concentration ◽  
Vasoconstrictor Response

Previous studies suggest that middle cerebral arteries (MCAs) of Fawn Hooded Hypertensive (FHH) rats exhibit impaired myogenic response and introgression of a small region of Brown Norway chromosome 1 containing 15 genes restored the response in FHH.1BN congenic rat. The impaired myogenic response in FHH rats is associated with an increase in the activity of the large conductance potassium (BK) channel in vascular smooth muscle cells (VSMCs). The present study examined whether the increased BK channel function in FHH rat alters vasoconstrictor response to serotonin (5-HT). Basal myogenic tone and spontaneous myogenic response of the MCA was attenuated by about twofold and about fivefold, respectively in FHH compared with FHH.1BN rats. 5-HT (0.1 μM)-mediated vasoconstriction was about twofold lower, and inhibition of the BK channel increased the vasoconstrictor response by about threefold in FHH compared with FHH.1BN rats. 5-HT (3 μM) decreased BK channel and spontaneous transient outward currents in VSMCs isolated from FHH.1BN but had no effect in FHH rats. 5-HT significantly depolarized the membrane potential in MCAs of FHH.1BN than FHH rats. Blockade of the BK channel normalized 5-HT-induced depolarization in MCAs of FHH rats. The 5-HT-mediated increase in cytosolic calcium concentration was significantly reduced in plateau phase in the VSMCs of FHH relative to FHH.1BN rats. These findings suggest that sequence variants in the genes located in the small region of FHH rat chromosome 1 impairs 5-HT-mediated vasoconstriction by decreasing its ability to inhibit BK channel activity, depolarize the membrane and blunt the rise in cytosolic calcium concentration.

Abstract 616: Diminished K Channel Inhibition Impairs Serotonin-Mediated Vasoconstriction in the Middle Cerebral Arteries From the Fawn Hooded Hypertensive Rat

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Mallikarjuna R Pabbidi ◽  
Richard Roman
Keyword(s):  
Cerebral Arteries ◽  
Cytosolic Calcium ◽  
Bk Channel ◽  
Chromosome 1 ◽  
Brown Norway ◽  
K Channel ◽  
Channel Function ◽  
Channel Inhibition ◽  
Middle Cerebral Arteries ◽  
Vessel Response

Fawn Hooded Hypertensive (FHH) rat exhibit impaired pressure-mediated myogenic response (MR) in the middle cerebral arteries (MCAs) that is associated with an increase in large conductance potassium (BK) channel function. Introgression of 2.4 Mbp region of BN (Brown Norway) chromosome 1 into FHH rats (FHH.1BN) restored MR and BK channel function. The present study assessed the hypothesis that FHH rats also exhibit impaired serotonin (5-HT)-mediated vasoconstriction due to diminished BK channel inhibition compared to FHH.1BN rats. We used pressure myography and patch-clamp to measure vessel response and K channel function respectively and Fluo 4 method to measure cytosolic calcium. Basal myogenic tone of MCAs as measured by change in diameter from 22oC to 37oC temperature was ~2.6 fold lower in FHH rats compared to FHH.1BN rats (FHH, 8.1 ± 2% and FHH.1BN, 21.7 ± 3%, n = 4; p<0.05). Vascular smooth muscle cells (VSMCs) of FHH rats have significantly more negative membrane potentials (Em) (–32 ± 1 mV; n=9) compared to FHH.1BN rats (-16 ± 2mV; n=10). 5-HT-mediated vasoconstriction was lower in MCAs isolated from FHH rats compared to FHH.1BN rats (5-HT: 1μM; FHH, 36 ± 5%; FHH.1BN rats, 58 ± 9%; n = 4; p<0.05). 5-HT-mediated BK channel inhibition was less in FHH rats (3μM 5-HT: FHH, 8 ± 3%; FHH.1BN, 39 ± 4%; n = 4; p<0.05). 5-HT did not affect VSMC membrane potential in FHH rats compared to FHH.1BN rats (delta change in Em: FHH, -2 ± 2mV; n = 4; FHH.1BN, -9 ± 1 mV; n = 5; p<0.05). 5-HT-mediated increase in calcium fluorescence (F/Fo) during plateau phase was blunted in the VSMCs isolated from FHH rats compared to FHH.1BN rats (FHH: 1 + 0.01% Vs 1.22 + 0.02% in FHH.1BN (3 rats)). Finally, inhibition of BK channel restored 5-HT-mediated vasoconstriction in MCAs of FHH rats but did not affect FHH.1BN vessels (5-HT + Paxilline; FHH, 2.6 ± 0.6 fold; FHH.1BN, 1.4 ± 0.4 fold; n = 5; p<0.05). In conclusion, mutation in the genes located in 2.4 Mbp region of FHH rats disrupts 5-HT-mediated BK channel inhibition that prevents the raise in [Ca2+]i and this may contribute to impaired 5HT-mediated vasoconstriction.

Abstract 127: Identification of a Region of Rat Chromosome 1 That Impairs the Myogenic Response and Cerebral Blood Flow Autoregulation in Fawn Hooded Hypertensive Rats.

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Mallikarjuna R Pabbidi ◽  
Julio Juncos ◽  
Marija Renic ◽  
Hurtis J. Tullos ◽  
Jozeph Lazar ◽  
...  
Keyword(s):  
Blood Flow ◽  
Cerebral Blood Flow ◽  
Infarct Size ◽  
Congenic Strain ◽  
Bk Channel ◽  
Chromosome 1 ◽  
Brown Norway ◽  
Myogenic Response ◽  
Congenic Strains ◽  
Channel Current

This study examined the effect of transfer of a 2.4 Mbp region of Brown Norway (BN) rat Chr 1 into the Fawn Hooded Hypertensive (FHH) genetic background on autoregulation (AR) of cerebral blood flow (CBF) and the myogenic response of isolated middle cerebral arteries (MCA). Autoregulation of CBF measured by laser Doppler flowmetry was poor in FHH rats (AR index (AI), 0.8±0.1) and in FHH.1BN congenic strains which excluded the critical region (AR-: AI, 0.9± 0.1). In contrast, autoregulation of CBF was completely restored by transfer of the region of BN Chr 1 between 258.8 to 261.2 Mbp in AR+ FHH.1BN congenic strains (AI, 0.3 ± 0.1). The diameter of MCA of FHH rats and AR- congenic strains increased by ∼10% (140 ± 1 to 157± 2 μm), when transmural pressure was increased from 40 to 140 mmHg. In contrast, the diameter of the MCA in AR+ congenic strain fell from 127 ± 2 to 65 ± 1 μm. Whole-cell patch-clamp of cerebral VSM cells revealed a 4.3-fold increase in BK channel current densities at depolarized potentials in FHH versus AR+ rats (105 ± 2 versus 24 ± 4 pA/pF at +80mV). Using single channel analysis we found that the increase in BK channel current was largely due to a marked increase in the NPo of BK channel in FHH as compared to AR+ rats (0.9 ± 0.1 and 0.2 ± 0.1 at +80mV). To explore the significance of the impaired myogenic response, we compared changes in CBF and infarct size following transient occlusion and reperfusion of the MCA in FHH rats and the AR- and AR+ congenic strains. Occlusion of MCA reduced CBF similarly in all the strains. However, the hyperemic response following reperfusion in FHH and AR- strains was significantly greater and more prolonged than that seen in AR+ rats (AR-: 173 ± 1%, 45 min versus AR+: 124 ± 5%, 15 min). Moreover, infarct size and edema formation was significantly greater in the AR- congenic strain (39 ± 3 % and 12 ± 2 %) in comparison to that seen in the AR+ strain (28 ± 2 %; 7 ± 1%). These results indicate that there is a gene that plays a critical role in the regulation of the myogenic response of the cerebral vasculature by altering BK channel activity in the critical 2.4 Mb region of Chr 1 containing just 15 genes and that transfer of this region from BN to FHH rats restores autoregulation of CBF, vascular reactivity and reduces infarct size following ischemia/reperfusion injury.

Abstract MP05: Knockdown of Gamma-adducin Expression Impairs the Myogenic Response of the Cerebral and Renal Arterioles

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Fan Fan ◽  
Mallikarjuna R Pabbidi ◽  
Ying Ge ◽  
Richard J Roman
Keyword(s):  
Cerebral Arteries ◽  
Bk Channel ◽  
Chromosome 1 ◽  
Causal Role ◽  
Myogenic Response ◽  
Dependent Manner ◽  
Channel Activity ◽  
Transfected Cells ◽  
293 Cells ◽  
Vasoconstrictor Response

We recently identified a region on chromosome 1 containing 15 genes that rescues the impaired myogenic response in the afferent arteriole (Af-art) and the development of renal injury in Fawn Hood Hypertensive (FHH) rats. We also found an inactivating K572Q mutation in the gamma-Adducin (Add3) gene in FHH rats in this region that may play a causal role. To test this hypothesis, the present study examed the effect of knockdown of the expression of Add3 with DsiRNA on the myogenic response of renal and cerebral arterioles. A newly designed 27-mer Add3 DsiRNA blocked the expression of the Add3 protein in 293 cells and reduced Add3 mRNA expression in a dose-dependent manner up to 75% in cultured middle cerebral arteries (MCA). The inner diameter of MCA and renal Af-art 36 hours after co-transfection of Add3 DsiRNA dilated by 9 ± 2% and 4 ± 3%, respectively, in response to an elevation in transmural pressure from 60 to 140 mmHg. In contrast, these vessels still constricted normally by 11± 2% and 10 ± 1% in vessels transfected with scrambled siRNA. Add3 DsiRNA had no effect on the vasoconstrictor response of the Af-art to NE (10-7 M). The large conductance calcium sensitive potassium (BK) current was 5-fold higher in vascular smooth muscle cells (VSMC) freshly isolated from the MCA that were transfected with Add3 DsiRNA as indicated by the appearance of SiGLO red fluorescence compared with that seen in non-transfected cells. Administration of IBTX normalized the elevated BK channel current recorded from VSMC transfected with Add3 DsiRNA, but it had little effect in non-transfected cells. These results indicate that knockdown of the expression of Add3 impairs the myogenic response of both MCA and Af-art and it is associated with an elevation in BK channel activity. It also supports the hypothesis that the K572Q mutation of Add3 found in FHH rats may play a causal role in the impaired myogenic response and autoregulation of renal and cerebral blood flow by elevating BK channel activity in VSMC.

scholarly journals Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

2014 ◽  
Vol 306 (5) ◽  
pp. C460-C470 ◽  
Author(s):  
Kiril L. Hristov ◽  
Amy C. Smith ◽  
Shankar P. Parajuli ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Regulation ◽  
Pkc Activation

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility.

scholarly journals Knockdown of Add3 impairs the myogenic response of renal afferent arterioles and middle cerebral arteries

2017 ◽  
Vol 312 (6) ◽  
pp. F971-F981 ◽  
Author(s):  
Fan Fan ◽  
Mallikarjuna R. Pabbidi ◽  
Ying Ge ◽  
Longyang Li ◽  
Shaoxun Wang ◽  
...  
Keyword(s):  
Vascular Reactivity ◽  
Vascular Dysfunction ◽  
Cerebral Arteries ◽  
Chromosome 1 ◽  
Brown Norway ◽  
Myogenic Response ◽  
Norway Rat ◽  
Vasoconstrictor Response ◽  
Brown Norway Rat ◽  
Interfering Rna

We have reported that the myogenic response of the renal afferent arteriole (Af-art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow are impaired in Fawn-Hooded Hypertensive (FHH) rats. Transfer of a region of chromosome 1 containing γ-adducin (Add3) from the Brown Norway rat rescued the vascular dysfunction and the development of renal disease. To examine whether Add3 is a viable candidate gene altering renal and cerebral hemodynamics in FHH rats, we knocked down the expression of Add3 in rat Af-arts and MCAs cultured for 36-h using a 27-mer Dicer-substrate short interfering RNA (DsiRNA). Control Af-arts constricted by 10 ± 1% in response to an elevation in pressure from 60 to 120 mmHg but dilated by 4 ± 3% when treated with Add3 DsiRNA. Add3 DsiRNA had no effect on the vasoconstrictor response of the Af-art to norepinephrine (10−7 M). Add3 DsiRNA had a similar effect on the attenuation of the myogenic response in the MCA. Peak potassium currents were threefold higher in smooth muscle cells isolated from Af-arts or MCAs transfected with Add3 DsiRNA than in nontransfected cells isolated from the same vessels. This is the first study demonstrating that Add3 plays a role in the regulation of potassium channel function and vascular reactivity. It supports the hypothesis that sequence variants in Add3, which we previously identified in FHH rats, may play a causal role in the impaired myogenic response and autoregulation in the renal and cerebral circulation.

Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

2001 ◽  
Vol 281 (6) ◽  
pp. C1769-C1775 ◽  
Author(s):  
Guillermo J. Pérez ◽  
Adrian D. Bonev ◽  
Mark T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Laser Scanning ◽  
Cerebral Arteries ◽  
Bk Channels ◽  
Bk Channel ◽  
Hill Coefficient ◽  
Channel Activity ◽  
Acetoxymethyl Ester ◽  
Open Probability ◽  
Apparent Dissociation Constant

The goal of the present study was to test the hypothesis that local Ca2+ release events (Ca2+ sparks) deliver high local Ca2+concentration to activate nearby Ca2+-sensitive K+ (BK) channels in the cell membrane of arterial smooth muscle cells. Ca2+ sparks and BK channels were examined in isolated myocytes from rat cerebral arteries with laser scanning confocal microscopy and patch-clamp techniques. BK channels had an apparent dissociation constant for Ca2+ of 19 μM and a Hill coefficient of 2.9 at −40 mV. At near-physiological intracellular Ca2+ concentration ([Ca2+]i; 100 nM) and membrane potential (−40 mV), the open probability of a single BK channel was low (1.2 × 10−6). A Ca2+spark increased BK channel activity to 18. Assuming that 1–100% of the BK channels are activated by a single Ca2+ spark, BK channel activity increases 6 × 105-fold to 6 × 103-fold, which corresponds to ∼30 μM to 4 μM spark Ca2+ concentration. 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid acetoxymethyl ester caused the disappearance of all Ca2+sparks while leaving the transient BK currents unchanged. Our results support the idea that Ca2+ spark sites are in close proximity to the BK channels and that local [Ca2+]i reaches micromolar levels to activate BK channels.

scholarly journals 14-3-3γ, a novel regulator of the large-conductance Ca2+-activated K+ channel

2020 ◽  
Vol 319 (1) ◽  
pp. F52-F62
Author(s):  
Shan Chen ◽  
Xiuyan Feng ◽  
Xinxin Chen ◽  
Zhizhi Zhuang ◽  
Jia Xiao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Specific Inhibitor ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Protein Ubiquitination ◽  
293 Cells

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.

Peroxynitrite reversibly inhibits Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

2000 ◽  
Vol 278 (6) ◽  
pp. H1883-H1890 ◽  
Author(s):  
Anna K. Brzezinska ◽  
Debebe Gebremedhin ◽  
William M. Chilian ◽  
Balaraman Kalyanaraman ◽  
Stephen J. Elliott
Keyword(s):  
Single Channel ◽  
Cerebral Arteries ◽  
Ionic Current ◽  
Inhibitory Effect ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Cell Current

Peroxynitrite (ONOO−) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO−bioactivity, the present study examined the effect of ONOO− on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K+ current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca2+-activated K+ (BK) channels, and ONOO− (10–100 μM) were determined. At a pipette potential of +50 mV, ONOO− inhibited 39% of iberiotoxin-sensitive current. ONOO− was selective for iberiotoxin-sensitive current, whereas decomposed ONOO− had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K+solutions. Unitary currents were sensitive to increases in internal Ca2+ concentration, consistent with activity due to BK channels. Internal ONOO− dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO− could be overcome by reduced glutathione. Glutathione, added after ONOO−, restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO− on membrane K+ current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO− has the potential to alter cerebral vascular tone by inhibiting BK channel activity.

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