Thymosin β4 impeded murine stem cell proliferation with an intact cardiovascular differentiation / 华中科技大学学报（医学）（英德文版）

Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.

Decrease of extracellular pH modulates the whole cell voltage-gated potassium currents in rat pulmonary artery smooth muscle cells / 中国应用生理学杂志

<p><b>AIM</b>To study the modulation of extracellular pH on the voltage-gated potassium currents (I(Kv)) in isolated pulmonary artery smooth muscle cells (PASMCs).</p><p><b>METHODS</b>I(Kv) was recorded using whole-cell patch clamp technique under the external solutions with different pH. The electrophysiological characteristics of I(Kv) were then analyzed.</p><p><b>RESULTS</b>(1) As compared to the normoxic group, I(K), decreased under acidic condition. When the extracellular pH were 7.0, 6.5, 6.0, the peak currents at a potential of +60 mV were inhibited by 16.93% +/- 2.47% (P < 0.01), 33.03% +/- 2.13% (P < 0.01), 41.59% +/- 6.53% (P < 0.01) respectively, and the current-voltage relationship (I/V) curve shifted to the right. (2) When the extracellular pH was 7.0, 6.5, 6.0, the voltage-depended Gk-Em was shifted to the direction of positive and the activation was sped up.</p><p><b>CONCLUSION</b>The results suggest that with the development of hypoxic pulmonary vasoconstriction (HPV), extracellular pH may take part in the modulation of Kv channels partly, then make the cell depolarized and decrease the Kv currents, this will lead to open the L-type calcium channel and contract the pulmonary artery smooth muscle. It may be one of the mechanisms that hypoxic leads to HPV and finally accelerate the development of HPV.</p>

Action of beta-amyloid peptide₁₋₄₀ on I(HVA) and its modulation by ginkgolide B / 生理学报

Whole-cell patch clamp recording was used to investigate the action of beta-amyloid peptide(1-40) (Abeta(1-40)) on high voltage-activated calcium channel current (I(HVA)) in acutely isolated hippocampal CA1 pyramidal neurons in rats and observe its modulation by ginkgolide B (GB). Drug was applied by extracellular bath or adding in the pipette solution, and its effect was determined by comparing the amplitude of I(HVA) before and after the drug application. Bath application of aggregated Abeta(1-40) at concentrations of 0.01~30 mumol/L increased the amplitude of I(HVA) in a dose-dependent manner by (5.43+/-3.01)% (n=8, P>0.05), (10.49+/-4.13) % (n=11, P>0.05), (40.69+/-8.01) % (n=16, P<0.01), (58.32+/-4.85) % (n=12, P<0.01), and (75.45+/-5.81) % (n=6, P<0.01), respectively, but had no effect on the I-V curve of I(HVA); fresh Abeta(1-40) almost had no effect on I(HVA) (n=5, P>0.05). L-type calcium channel antagonist nifedipine abolished the increase of I(HVA)by Abeta(1-40). The increase of I(HVA) by Abeta(1-40) (1.0 mumol/L) was enhanced to (66.19+/-5.74) % (P<0.05) by 8-Br-cAMP (membrane permeable analogue of cAMP) and to (73.21+/-6.90) % (P<0.05) by forskolin, an adenylyl cyclase (AC) agonist, and reduced to (20.08+/-2.18) % (P<0.05) by H-89, cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) antagonist. GB effectively inhibited the increase of I(HVA) by Abeta(1-40). The results indicate that Abeta(1-40) leads to an intracellular calcium overload by increasing I(HVA) via AC-cAMP-PKA. This may be one of the mechanisms for its neurotoxicity. GB can prevent neurons from neurotoxicity by inhibiting abnormal calcium influx caused by Abeta(1-40).

Investigation on spontaneous electrical activity of murine embryonic heart using microelectrode arrays / 生理学报

In our studies, we have applied a novel tool, microelectrode arrays (MEA), to investigate the electrophysiological properties of murine embryonic hearts in vitro. The electrical signals were recorded from the areas of the heart adhering to the 60 MEA electrodes, being called field potentials (FPs). As an extracelluar recording, the waveform of the FP appeared similar to a reversed action potential obtained from single cell by whole cell current clamp and the FP duration was comparable with the action potential duration. To study propagation of spontaneous electrical activity, we have compared the occurrence time of FPs recorded from different electrodes. It is shown that there was already an apparent A-V delay [(50.21+/-9.7) ms] at day 9.5 post coitum (E9.5) when heart was still tubular-like and atrium and ventricle were not separated anatomically, while occurence of FP at different electrodes of ventricular area were almost synchronous. Further, we looked into the modulation of spontaneous electrical activity during cardiac development: at E9.5 of embryonic development, 1 mumol/L of isoproterenol (Iso) increased beating frequency by (34.04+/-7.31)%, shortened the A-V delay by (20.00+/-6.44) % and prolonged FP duration. In contrast, 1 mumol/L of carbachol (CCh) slowed down beating frequency by (42.32+/-5.36) %, A-V conduction by (26.00+/-4.81) % and shortened FP duration; however at late stage (E16.5), the regulatory effect of Iso and CCh was strengthened. Therefore we conclude that cardiac conduction system is already established at E9.5 when the four-chambered heart is not formed yet and the regulation of spontaneous activity by sympathetic and para-sympathetic system is gradually matured during cardiac development.

Different signal molecules involved in the muscarinic modulation of pacemaker current I(f) on the heart of mouse embryo in different developmental stages / 生理学报

We isolated mouse embryonic cardiomyocytes derived from timed-pregnant females at different periods and used patch-clamp technique to investigate the muscarinic cholinergic modulation of pacemaker current I(f) in different developmental stages. In early development stage (EDS), muscarinic agonist carbachol (CCh) significantly decreased the magnitude of the pacemaker current I(f) but had no effect in late development stage (LDS). Forskolin (a direct adenylate cyclase activator) and IBMX (a non-selective phosphodiesterase inhibitor) increased I(f) in both EDS and LDS cells. Interestingly, although both forskolin and IBMX increased basal I(f), their effects on CCh-inhibited I(f) were different. Forskolin did not reverse the inhibitory action of CCh until intermediate development stage (IDS). In contrast, IBMX reversed the inhibitory action of CCh on I(f) in EDS but not in IDS. It is suggested that a decrease in intracellular cAMP is a possible mechanism for CCh to modulate I(f). During the EDS and IDS CCh controls the cytoplasmic cAMP level by different pathways: In EDS, CCh modulates I(f) possibly by activating PDE which accelerates the breakdown of cAMP, but in IDS possibly by inhibiting adenylate cyclase (AC) which then reduces the synthesis of cAMP.

Extracellular calcium modulates the whole cell potassium currents in Deiters cells isolated from guinea pig cochlea / 生理学报

To study the modulatory effect of extracellular calcium on the whole cell K(+) currents (I(K)) in isolated Deiters cells, the whole cell K(+) currents were recorded when Deiters cells bathed in normal physiological solutions and calcium-free saline, respectively. The electrophysiological characteristics of I(K) currents were then analyzed with the patch clamp technique. Removing extracellular calcium significantly enhanced the amplitude of the I(K) currents, which increased by 70.2% at +50 mV test pulse. The chord conductance, measured at -30 mV test pulse, also significantly increased from (3.31-/+3.08) ns (n=42) in the normal solutions to (10.81-/+6.01) ns (n=42) in the calcium-free solutions, whereas, the zero current potential of the I(K) currents remained unchanged. In calcium-free solutions, the reversal potential of the I(K) currents was shifted to the direction of hyperpolarization, which was very close to the equilibrium K(+) potential based on the Nernst equation. In addition, both the steady state activation curve and the half activation potential, with the averaged value at (-10.13-/+5.64) mV (n=42), were shifted to the negative. However, the tendency for activation (slope conductances) was the same as that in the normal solutions. Interestingly, both the I-V and the G-V functions deduced from the calcium-inhibited K(+) currents in Deiters cells were "S" shape, implying that at least two different kinds of K(+) conductance were involved in this calcium-inhibited K(+) currents. In summary, we hypothesize that there are two mechanisms for this modulation: one is that the I(K) channels in Deiters cells containing a specific calcium sensitive domain, by which extracellular calcium modulates the structure of the K(+) channels and then the I(K) currents; the other is a novel double gated K(+) channel or an ionotropic receptor coupled to K(+) channels or a new subtype of outward K(+) channels. Removing extracellular calcium activates this novel conductance and then modulates the I(K) currents. These results indicate that a decrease in extracellular calcium not only facilitates the efflux of K(+) out of Deiters cells but also accelerates the repolorization by enhancing the I(K) currents, which in turn can effectively buffer the K(+) concentration around the outer hair cells and maintain the resting membrane potential of Deiters cells.

Chronic intermittent hypoxia decreases acute hypoxic inhibition of voltage-gated potassium channel in rat pulmonary arterial smooth muscle cells / 生理学报

For determination the ionic mechanisms of the hypoxic acclimatization at the level of channels, male Spradue-Dawley rats were divided into two groups: control normoxic group and chronic intermittent hypoxic group [O2 concentration: (10 +/-0.5)%, hypoxia 8 h a day]. Using whole cell patch-clamp technique, voltage-gated potassium channel currents (IK(V)) were recorded in freshly isolated pulmonary arterial smooth muscle cells (PASMCs) of rat with acute isolated method. The effect of acute hypoxia on IK(V) of PASMCs from chronic intermittent hypoxia group was investigated to offer some basic data for clarifying the ionic mechanisms of the hypoxic acclimatization. The results showed: (1) In control normoxic group, after acute hypoxia free-Ca(2+) solution, the resting membrane potential (Em) of PASMCs was depolarized significantly from -47.2+/-2.6 mV to -26.7+/-1.2 mV, and the IK(V) of PASMCs was decreased significantly from 153.4+/-9.5 pA/pF to 70.1+/-0.6 pA/pF, the peak current percent inhibition was up to (57.6+/-3.3)% at +60 mV, and current-voltage relationship curve shifted to the right. (2) In chronic intermittent hypoxic group, the IK(V) of PASMCs was decreased significantly by exposure to intermittent hypoxia in a time-dependent manner, appeared to start on day 10 and continued to day 30 (the longest time tested) of hypoxia, and current-voltage relationship curve shifted to the right in a time-dependent manner. (3) Compared with the control normoxic group, the percent IK(V) inhibition by acute hypoxia was significantly attenuated in the chronic intermittent hypoxia group and this inhibition effect declined with time exposure to hypoxia. The results suggest that K(V) inhibition was significantly attenuated by chronic intermittent hypoxia, and this effect may be a critical mechanism of the body hypoxic acclimatization.