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OBJECTIVE@#To investigate the mechanisms through which myocyte large-conductance Ca-activated K (BK) channels mediate the vasodilation effects of melatonin on cerebral arteries (CAs).@*METHODS@#Middle cerebral arteries (MCA) were obtained from 8-week-old male Wistar rats after anaesthetized. Middle cerebral arterial smooth muscle cells were enzymatically isolated. Whole cell recording mode of patch clamp technique was used to measure the current density of BK channel and voltage-gated potassium (K) channel before and after adding melatonin. Currents density of melatonin on BK channels with melatonin receptor inhibitor 2-phenyl-N-acetyl (luzindole) was recorded using whole cell recording mode and open probability (Po) was recorded using single-channel attached recording mode. The conductance (G) and average open time (To) and off time (Tc) of the BK channel were detected before and after the addition of melatonin in the internal-outward mode.@*RESULTS@#① Melatonin markedly increased the whole-cell BK channel current density but not the voltage-gated potassium (K) channel current density. ② Luzindole (1 μmol/L) greatly suppressed melatonin-induced increase of BK channel current density. ③ The Po of BK channel was significantly increased by melatonin (100 μmol/L) under cell attached recording mode, which was markedly inhibited by luzindole (1 μmol/L). ④ In inside-outside recording mode, melatonin (1 μmol/L, 100 μmol/L) reduced both To and Tc of BK channel, and Tc was reduced much more than To.@*CONCLUSIONS@#Melatonin mediates vasodilation of MCA through the activation of BK channels both melatonin receptor dependent and independent mode.
Subject(s)
Animals , Male , Rats , Melatonin , Middle Cerebral Artery , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , Patch-Clamp Techniques , Potassium Channels, Calcium-Activated , Rats, WistarABSTRACT
Large-conductance Ca2+-activated K+ (BKCa) channels, activated by stress, exhibit mechanosensitivity and are involved in stress-regulated cellular function. For different types of cells, they will have different expression and activity changes due to their BKCa channels responding to different stress patterns. Correspondingly, the mechanism underling channel activation behaves differently, which is activated by the elevation in Ca2+ concentration or changes in membrane bilayer and cytoskeleton. In this review, the research progress in mechanosensitivity of BKCachannels was summarized from 3 aspects, including its molecular structure basis, manifestation and stress activation mechanism.
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Objective To investigate the effect of alizarin on BKCa channel beta subunit and electrophysiological characteristics in interlobar renal artery of SHR.Methods Tail artery pressure measurement instrument,pressure myograph system,whole cell patch clamp technique and Western blot were used to measure the change of systolic pressure of SHR,renal interlobar arterial diameter,single vascular smooth muscle cell electrophysiological characteristics and expression of the BKCa channel beta subunit before and after alizarin intervention,respectively.Results (1) SHR systolic pressure was decreased after alizarin intervention (P ＜ 0.01).(2) ibTX inhibited alizarin-mediated renal interlobar arterial relaxation (P ＜ 0.01).(3) Alizarin enhanced BKCa channel-mediated outward currents (P ＜ 0.05).(4) Alizarin up-regulated BKCa channel beta subunits after alizarin intervention (P ＜ 0.01).Conclusion Alizarin reduced SHR systolic pressure and relaxed interlobar renal artery by enhancement BKCa currents via mediation of the function of beta subunits.
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Objective To investigate the protective effects of isopimaric acid ( ISO), the BKCa channel activator, on cognitive function and synaptic plasticity in APP/PS1 mice. Methods Alzet osmotic pump was loaded with ISO or DMSO only and assembled with ALZET Brain Infusion Kit III. The cannula was implanted into the lateral ventricle of 4-month-old male APP/PS1 mice or matched wild type ( WT) mice. Two weeks later, open field test and Morris water maze were conducted. Paired-pulse facilitation ( PPF) and TBS-induced long-term potentiation ( LTP ) were recorded in CA1 region of hippocampus. Results The open field test showed that there was no significant difference among the four groups in spontaneous activities and vertical plane movement distance within 30 minutes. Floor plane movement distance was significantly greater in APP/PS1+DMSO group than that in WT+DMSO group(P<0.05) . Compared with the WT+DMSO group, APP/PS1+DMSO group had significantly longer escape latency from the third to fifth day and lower percentage of time spent in the target quadrant ((43.27±3.24)% vs (34.19±2.56)%) and the number of crossing through the platform ((4.25±0.66)times vs (1.93±0.33)times)(P<0.05). Compared with the APP/PS1+DMSO group, the APP/PS1+ISO group had significantly shorter escape latency from the fourth to fifth day and higher percentage of time spent in the target quadrant ((46.16±3.51)%) and the number of crossing through the platform ((3.41±0.34) times) (P<0.05). PPF in APP/PS1+DMSO group significantly reduced compared with that in WT+DMSO group at 30-50ms interstimulus interval(P<0.05). PPF in APP/PS1+ISO group((224.50±13.79)%) was significantly augment compared with APP/PS1+DMSO ((174.99 ±6.68)%) group at 40 ms interstimulus interval (P<0.05). The LTP at 60 min post-TBS was significantly smaller in the APP/PS1+DMSO group ((135.19±1.32)%) than that in the WT+DMSO group ((172.17± 4.15)%)(P<0.001). The LTP of the APP/PS1+ISO group((160.48±1.19)%) became significantly in-creased compared with that in the APP/PS1+DMSO group(P<0.001).Conclusion BKCa channel activator ISO improve the learning and memory function of APP/PS1 mice by promoting PPF and increasing LTP to recover synaptic plasticity in the hippocampus.
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Objective To investigate the early onset of learning and memory function of 4-month-old APP/PS1/Tau Alzheimer' s disease (3×Tg-AD) model mice and explore the pathogenesis of AD in early stage through evaluating neuron excitability and BKCa channel activity in cingulate cortex pyramidal cells.Methods Ten 4-month-old male 3×Tg-AD mice and matched ten wild type (WT) mice.Behavior was tested with the novel object recognition task to observe the ability of learning and memory.Whole-cell patch-clamp recordings were performed to assess the excitability of cingulate cortex pyramidal cells in terms of resting membrane potential and frequencies of spikes evoked by current injection.A train of five pulses of depolarizing currents were injected at 100 Hz to assess the spike width,which was used as an index for BKCa channel activity.Results Compared with the WT group (0.72±0.03),the novel object recognition index significantly decreased in 3 × Tg-AD group (0.55 ± 0.04) (P =0.004).Compared to the WT group((-66.03±0.43) mV),the resting membrane potential in cingulate cortex neurons of 3×Tg-AD group((-62.31±0.54)mV) was significantly depolarized(P=0.000).In contrast to WT group,the action potential firing frequencies evoked by depolarizing current injections were higher in neurons from 3×Tg-AD group(P=0.000),demonstrating that excitability of cingulate cortex neurons was elevated by intracellular Aβ.Spikes were broader in the 3×Tg-AD group than those in the WT group(P＜0.01).Suppression of BKCa channels in cingulate cortex neurons from the 3×Tg-AD group was confirmed on the basis of the spike half-width,since BKCa channels affect the descending phase of spikes.Conclusion Compared to WT mice,4-month-old 3×Tg-AD mice are impaired in learning and memory.The suppression of BKCa channels by intracellular Aβ leads to increase of excitability in cingulate cortex pyramidal cells.
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Potassium channels in human skin fibroblast have been studied as a possible site of Alzheimer disease pathogenesis. Fibroblasts in Alzheimer disease show alterations in signal transduction pathway such as changes in Ca2+ homeostasis and/or Ca2+-activated kinases, phosphatidylinositol cascade, protein kinase C activity, cAMP levels and absence of specific K+ channel. However, little is known so far about electrophysiological and pharmacological characteristics of large-conductance Ca2+-activated K+ (BKCa) channel in human fibroblast (CRL-1474). In the present study, we found Iberiotoxin- and TEA-sensitive outward rectifying oscillatory current with whole-cell recordings. Single channel analysis showed large conductance K+ channels (106 pS of chord conductance at +40 mV in physiological K+ gradient). The 106 pS channels were activated by membrane potential and [Ca2+]i, consistent with the known properties of BKCa channels. BKCa channels in CRL-1474 were positively regulated by adenylate cyclase activator (10microM forskolin), 8-Br-cyclic AMP (300microM) or 8-Br-cyclic GMP (300microM). These results suggest that human skin fibroblasts (CR-1474) have typical BKCa channel and this channel could be modulated by c-AMP and c-GMP. The electrophysiological characteristics of fibroblasts might be used as the diagnostic clues for Alzheimer disease.