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Objective The mechanisms of docosahexaenoic acid (DHA) protecting the cardiovascular system have not yet been clarified. This study was to investigate the vasorelaxative effect of 13,14-epoxy docosapentaenoic acid (13,14-EpDPE) on coronary arterioles in normal rats and its action mechanisms.Methods We isolated coronary artery smooth muscle cells (CASMCs) from normal rats by enzyme digestion, examined the open probabilities of the large conductance calcium-activated potassium (BK) channels in inside-out single channel configuration in the presence of different concentrations (0, 1, 10 and 100 pmol/L) of 13,14-EpDPE, and recorded the BK currents with the patch clamp in whole cell configuration. Then we assessed the coronary arterial relaxation by measuring dilatory responses to 13,14-EpDPE in pre-contracted tissues with or without pre-treatment with iberiotoxin.Results In the presence of 0, 1, 10 and 100 pmol/L of 13,14-EpDPE, the open probabilities of the BK channels were 0.25±0.03, 0.34±0.03, 0.44±0.06 and 0.85±0.16 (n=6), respectively, significantly increased at 100 pmol/L as compared with 0, 1 and 10 pmol/L (P<0.05). The BK channels were activated by 13,14-EpDP in a concentration-dependent manner and its half-effect concentration was (15.94±1.21) pmol/L. The current density was increased from (58.27±16.35) to (95.94±23.00) pA/pF (P=0.002) after 10 pmol/L 13,14-EpDP perfusion when the stimulation voltage was 100 mV. 13,14-EpDPE dilated the isolated coronary arterioles in a dose-dependent manner, and its effects were abolished after pre-treatment with iberiotoxin (100 nM).Conclusion 13,14-EpDPE can dilate coronary arterioles by activating BK channels in CASMCs, which might be one of the mechanisms underlying its protective effect on the cardiovascular system.
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Background Diabetic retinopathy (DR) is a common microvascular complications of the retina,retinal vascular smooth muscle cells of large conductance calcium-activated potassium channels (BK) is a major factor in regulating vasomotor and hemodynamic.Currently,functional changes of BK channel in retinal artery smooth muscle cells (RASMCs) and its role in DR were rarely reported.Objective This study was to investigate the early vascular damage mechanisms in DR by detecting the changes of BK channels current,calcium concentration and open probability (NP0) of BK channel with different calcium concentration in RASMCs of normal and diabetic rats.Method Fifty SPF SD 8-12 weeks old rats were randomly divided into normal control group and diabetic model group.Forty diabetic rats was intraperitoneally injected with 60 mg/kg streptozotocin to form type 1 diabetic model,10 rats (the normal control group) were injected sodium citrate solution with the same manner.Fluorescent probe was applied to detect calcium concentration in rat RASMCs;RASMCs were isolated by using enzyme digestion,and BK-channel electric currents and calcium concentrations in the RASMCs were measured by whole-cell patch clamp technique and fluorescence assay,respectively.The NP0 of BK channel was measured by single patch clamp technique.Results Diabetic models were successfully established in 36 rats with the success rate 90％.When stimulation voltage is greater than 60 mV,the current density of BK channel in RASMCs of diabetic model group decreased;when stimulating voltage was 100 mV,the BK channel currents of RASMCs in the normal control group and diabetic model group were (100±23) PA/PF and (50 ± 7) PA/PF,the difference was statistically significant (t =19.80,P ＜ 0.05).After adding specific BK channel blocker African scorpion toxin 100 nmol,the BK channel current in the normal control group significantly reduced,and that in the diabetes model group was not significantly changed;the calcium ion concentrations in RASMCs were (123±11)nmol/L and (255± 10)nmol/L in the normal control group and diabetic model group,the difference was statistically significant (t =32.50,P＜0.05).When stimulation voltage was 60 mV,with increasing calcium ion concentration,the NP0 of BK channel increased (F =15.28,P＜0.05).Conclusions The electric current and NP0 of BK-channel are obviously reduced and the calcium concentration is evidently elevated in RASMCs of diabetic rats,suggesting that the abnormal of BK-channel is probably one of the important causes of retinal artery abnormal contraction in diabetic rats.
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[ ABSTRACT] AIM: To discuss the relevance between the pathogenesis of diabetic gastroparesis and the large-conductance calcium-activated potassium channels ( BKCa ) in gastric smooth muscle cells.METHODS:The SD rats were randomly divided into control group and model group.The gastric smooth muscle cells of the SD rats were enzymatically iso-lated in a low calcium solution containing papain.The current was recorded by patch clamp single channel recording tech-nique.The expression of KCNMA and KCNMB1 were observed by the method of immunohistochemistry.RESULTS:The value of BKCa single channel conductance was (220.10 ±10.90) pS;the channels had distinct voltage dependent and cal-cium dependent characteristics.In outside-out patch (Vm =+30 mV), the activation of BKCa was blocked by 200 nmol/L IbTX completely.Compared with control group, the open probability and amplitude of current in model group significant-ly increased, while the mean open time and mean close time significantly decreased.Compared with control group, the ex-pression of KCNMB1 in model group was significantly increased.CONCLUSION: Up-regulation of β1-subunit and in-crease in BKCa functional activities may be associated with diabetes gastroparesis in rats.
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Objective To study the effects of hydrogen dioxide (oxygen free radical donator) and vitamin C (oxygen free radical scavenger) on the electric current of large conductance calcium -activated potassium channels (BKCa channels) in isolated outer hair cells in aging guinea pigs .Methods Acute enzyme was used to isolat outer hair cells of aging guinea pigs ,in which of BKCa channel's electric current was observed and recorded by whole-cell recording mode of patch -clamp .After recording the stable and normal electric current of BKCa channels ,added H2 O2 dilution (0 .2 mmol/L) 40 μl in the 2 ml chambers within freshly isolated outer hair cells so that the concen‐tration of H2 O2 in the balneum would be 4 μmol /L .The groups(n=5) received individually vitamin C solution (5 mg/ml) 0 ,10 ,20 ,40μl in the 2 ml chambers within freshly isolated outer hair cells so that the concentration of vi‐tamin C in the balneum would be 0 ,25 ,50 ,100 μg /ml ,observing and recording the effects of different concentration of vitamin C to electric current of BKCa channels .Results ①In the w hole-cell mode of patch -clamp ,the rapid activation and non-deactivation electric current with a string of large amplitude was recorded ,above -40~ -30 mV activation voltage .The electric current increased with the increasing membrane potential .The amplitude in‐creased continuously and performed characteristics of outward rectification .When the concentration of IbTX was 100 nmol /L ,the activity of the channel was completely blocked and confirmed BKCa channel's electric current .②Medication within three minutes ,when VT was +50 mV ,the BKCa channels'the maximum peak current densities of 4 μmol /L H2 O2 group rose from 22 .09 ± 0 .27 PA /PF to 43 .53 ± 1 .09 PA/PF ,amplification was 97 .06% .In H2 O2 4 μmol /L + vitamin C with different concentrations as 25 ,50 ,100 μg/ml groups ,the BKCa channels'elec‐tric current performed about concentration-dependent inhibition ,and electric current's amplitude and peak current density decreased with the increasing concentration of vitamin C ,the I-V curves were reduced .However ,this still could not be recovered to the normal levels .Conclusion The oxygen free radical /BKCa exists in the process .The vitamin C as oxygen free radical scavenger can reverse the process to a large extent .
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Objective To investigate the role of large conductance calcium-activated potassium channels (BK) in neuronal Ca2+ overload following traumatic brain injury (TBI). Methods Neuronal cells of C57BL/6 mouse cortex were collected and cultured. Patch-clamp technique was applied to investigate the changes of intracellular free calcium [Ca2+] i and firing frequency of neuronal action potentials in rest condition or evoked by 100 pA electric current lasting 500 ms after perfusion of Iberiotoxin ( 100 nmmol/L), a BK specific blocker. The cells were divided randomly into experimental group ( plus Iberiotoxin) and control group. Extracellular solution of cultured neurons was further perfused with KCl (20 mmol/L) to induce elevation of [Ca2 +]i and influence of Iberiotoxin ( 100 nmol/L) on amplitude of [Ca2 +] i elevation was determined. Results No significant changes of neuronal spontaneous action potential frequency and [Ca2 +]i were observed in rest condition after perfusion of Iberiotoxin (P＞0.05).However, when evoked by electric current, the frequency of action potential was (10.4 ± 3.0) Hz,which was increased to ( 13.8 ± 3.7 ) Hz after perfusion of Iberiotoxin, with statistical difference (P＜0.05 ). The [Ca2 +] i level was ( 14.21 ± 16.98 ) nmol/Lbefore perfusion with Iberiotoxin but was increased to (44.07 ± 34. 4) nmol/L after perfusion of Iberiotoxin (P ＜ 0.05 ). Extracellular high concentration of KCl increased [Ca2 +] i of neurons, while perfudion of Iberiotoxin further elevated [Ca2 +]i (P ＜ 0.05).Conclusion BK may play an important role in the regulation of neuronal [Ca2+] i and in neuronal Ca2+overload following TBI.
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Objective Large-conductance culcium-activated potassium (BKCa) channel modulates vascular smooth muscle tone. In the present study, we tested the hypothesis that salt, one of the factors which significantly influence bleed pressure (BP), can regulate BKCa activity and then elevate blood pressure. Methods Male Spragne-Dawley rats aged 6 weeks were randomized into high salt diet group (HS) and control group, fed with high salt diet (containing 5% NaCi) and standard rat chow (containing 0.4% NaCl) respectively for 16 weeks. Tail systolic blood pressure (SBP), body weight (BW) and 24-hour urinary output were tested every 4 weeks. Content of urinary Na+ was detected using flame spectrophotometrical method. At the end of 16 weeks, all the rats were killed, the mesenteric arteries were obtained, and single mesenteric smooth muscle cells were isolated at once. The resting membrane potential (Em), the total potassium currents and the currents after perfusion with TEA solution of the cells were all recorded by whole cell patch clamp. The transcriptions of BKCa channel α and β1 sobunits in mesenteric arterial vascular smooth muscle cells (VSMC) of each group were calculated by real-time RT-PCR. Results There was no difference in SBP and BW at each stage between control group and HS group; the urinary Na+ level in HS animals was elevated significantly after 4 weeks.The negative values of Em in HS group VSMCs were reduced compared with these in the control group. Transcriptions of β1 subanit of BKCa channels were decreased in HS group, but α subunit transcriptions did not differ between the two groups. Whole cell potassium currents did not differ hetween HS and control groups, but BKCa currents of HS group VSMCs were lower than these of control group ones. Conclusion Even without elevating SBP, salt-loading can still modulate the expression and activity of BKCa channel in the mesenteric arterial VSMC and elevate vascular tone.
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Objective: Large-conductance calcium-activated potassium (BKCa) channel modulates vascular smooth muscle tone. In the present study, we tested the hypothesis that salt, one of the factors which significantly influence blood pressure (BP), can regulate BKCa activity and then elevate blood pressure. Methods: Male Sprague-Dawley rats aged 6 weeks were randomized into high salt diet group (HS) and control group, fed with high salt diet (containing 5% NaCl) and standard rat chow (containing 0.4% NaCl) respectively for 16 weeks. Tail systolic blood pressure (SBP), body weight (BW) and 24-hour urinary output were tested every 4 weeks. Content of urinary Na+ was detected using flame spectrophotometrical method. At the end of 16 weeks, all the rats were killed, the mesenteric arteries were obtained, and single mesenteric smooth muscle cells were isolated at once. The resting membrane potential (Em), the total potassium currents and the currents after perfusion with TEA solution of the cells were all recorded by whole cell patch clamp. The transcriptions of BKCa channel α and β1 subunits in mesenteric arterial vascular smooth muscle cells (VSMC) of each group were calculated by real-time RT-PCR. Results: There was no difference in SBP and BW at each stage between control group and HS group; the urinary Na+ level in HS animals was elevated significantly after 4 weeks. The negative values of Em in HS group VSMCs were reduced compared with those in the control group. Transcriptions of β1 subunit of BKCa channels were decreased in HS group, but α subunit transcriptions did not differ between the two groups. Whole cell potassium currents did not differ between HS and control groups, but BK Ca currents of HS group VSMCs were lower than those of control group ones. Conclusion: Even without elevating SBP, salt-loading can still modulate the expression and activity of BKCa channel in the mesenteric arterial VSMC and elevate vascular tone.
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AIM: To evaluate the effects of atorvastatin on large-conductance calcium-activated potassium channel(BKCa,MaxiK) of arteria mesenterica minor smooth muscle cells in spontaneously hypertensive rats.METHODS: Twelve male spontaneously hypertensive rats(SHR) aged 9 weeks were randomly divided into atorvastatin treatment group(ATV group,n=6) and distilled water group(DW group,n=6),and 6 Wistar-Kyoto rats were as normal control group(n=6).Atorvastatin and appropriate distilled water were administered to rats in ATV group(50 mg?kg-1?d-1) for 10 weeks by intragastric administration.The changes of abdominal aortic blood pressure were observed and the contents of TC,TG,LDL-C in serum were measured before and after treatment.The arterial mesenterica smooth muscle cell potassium current were recorded using whole cell patch clamp.The BKCa membrane capacitance and its current densitys were detected after the BKCa was blocked using tetraethylammonium.RESULTS: The abdominal aorta blood pressure in ATV group was much lower than that in DW group[(171?8) mm Hg vs(190?10) mm Hg,P
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OBJECTIVE: Tyrosine kinase inhibitors may be useful in the management of cerebral vasospasm. It has not yet reported whether potassium channel plays a role in tyrosine kinase inhibitors-induced vascular relaxation of cerebral artery. This study is undertaken to clarify the role of potassium channel in tyrosine kinase inhibitors-induced vascular relaxation, and to investigate the effect of tyrosine kinase inhibitors on outward potassium currents in freshly isolated smooth muscle cells from rat basilar artery. METHODS: The isolation of rat basilar smooth muscle cells was performed by special techniques. The whole cell currents were recoreded by whole cell patch clamp technique in freshly isolated smooth muscle cells from rat basilar artery. RESULTS: In present study, genistein(n=10), tyrphostin A-23(n=10), A-25(n=10) 30microM into bath solution increased the amplitude of the outward K+ current which was completely blocked by large conductance calcium-activated potassium channel(BK(Ca)) blocker, iberiotoxin(0.1microM), and calcium chelator, BAPTA, in whole cell mode. In contrast, diadzein 30microM(n=10), inactive analogue of genistein, did not increase the amplitude of the outward K+ current. CONCLUSION: Our results suggest tyrosine kinase inhibitors such as genistein, tyrphostin A-23 and A-25 increase the BK(Ca) channel activity in cerebral basilar smooth muscle cells, thereby contributing to the relaxation of cerebral artery.