Regulation of intracellular Ca^2＋ and calcineurin by NO／PKG in proliferation of vascular smooth muscle cells

AIM: To determine whether Ca2+/calcineurin mediated the inhibitory effects of nitric oxide /cGMP-dependent protein kinase (NO/PKG) on the proliferation of vascular smooth muscle cells (VSMC). METHODS: Proliferation and viability of primary VSMC from rat aorta were measured using [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay and acridine orange and ethidium bromide staining, respectively. Cytosolic Ca2+ was determined by Fluo-3/AM. Calcineurin protein and its activity were assayed using immunoblotting and free inorganic phosphate analysis, respectively. RESULTS: (+/-)-S-nitroso-N-acetyl-penicillamine (SNAP) and Sp-8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Sp-8-pCPT-cGMPS) decreased phenylephrine (PE)-induced proliferation of VSMC by 27.3% and 36.6%, respectively, but Rp-8-[(4-chlorophenyl)thio]-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS) increased PE-induced proliferation of VSMC. SNAP, Sp-8-pCPT-cGMPS, and Rp-8-pCPT-cGMPS did not affect the viability of VSMC. Calcineurin protein was decreased by 63.1% and its activity was decreased by 59.7% in smooth muscle cells (SMC) pretreated with verapamil (Ver) and then stimulated by PE. In SMC pretreated with Ver, the absorbance of cells stimulated by PE decreased by 22.0% and was further inhibited by the additional treatment of SNAP and Sp-8-pCPT-cGMPS. In SMC pretreated with cyclosporin A (CsA), the absorbance of cells stimulated by PE decreased by 36.7%, but could not be further altered by the additional treatment of SNAP, Sp-8-pCPT-cGMPS, and Rp-8-pCPT-cGMPS. In addition, Ver inhibited PE-induced intracellular Ca2+ variations, which could be further inhibited by SNAP and Sp-8-pCPT-cGMPS, but not by Rp-8-pCPT-cGMPS. Moreover, the increase in calcineurin activity induced by PE was inhibited by SNAP and Sp-8-pCPT-cGMPS, but was promoted by Rp-8-pCPT-cGMPS. Conclusion: NO/PKG regulates calcineurin activity via the modulation of intracellular Ca2+ concentration, and thus partially inhibits the proliferation of VSMC without affecting their viability.     

Opioid receptor antagonists increase [Ca^2＋]i in rat arterial smooth muscle cells in hemorrhagic shock

INTRODUCTION Circulation hypotension induced by hemorrhagicshock is one of the main causes of death after severewounds or trauma. When the hemorrhagic shock hasdeveloped to a decompensatory stage, it is difficult toreverse the hypotension using usual vasoconstrictor,such as norepinephrine. One of the explanations ofhypotension is due to the hyposensitivity of arterialsmooth muscle cells (SMC) to vascular constrictorstimuli[1,2]. Previous studies showed that the vascularhyporesponse was r…     

Sympathetically evoked Ca^2＋ signaling in arterial smooth muscle

The sympathetic nervous system plays an essential role in the control of total peripheral vascular resistance and blood flow, by controlling the contraction of small arteries. Perivascular sympathetic nerves release ATP, norepinephrine (NE) and neuropeptide Y. This review summarizes our knowledge of the intracellular Ca2+ signals that are activated by ATP and NE, acting respectively on P2X1 and alpha1-adrenoceptors in arterial smooth muscle. Each neurotransmitter produces a unique type of post-synaptic Ca2+ signal and associated contraction. The neural release of ATP and NE is thought to vary markedly with the pattern of nerve activity, probably reflecting both pre- and post-synaptic mechanisms. Finally, we show that Ca2+ signaling during neurogenic contractions activated by trains of sympathetic nerve fiber action potentials are in fact significantly different from that elicited by simple bath application of exogenous neurotransmitters to isolated arteries (a common experimental technique), and end by identifying important questions remaining in our understanding of sympathetic neurotransmission and the physiological regulation of contraction of small arteries.     

Oxidized low-density lipoproteins induce apoptosis in vascular smooth muscle cells

目的：研究氧化型低密度脂蛋白（ｏｘＬＤＬ）诱导血管平滑肌细胞凋亡．方法：梯度超速离心分离血浆ＬＤＬ，以ＣｕＳＯ４１０μｍｏｌ·Ｌ－１氧化，观察ｏｘＬＤＬ对培养兔胸主动脉平滑肌细胞的损伤作用．Ｈｏｅｃｈｓｔ３３２５８荧光染色观察形态学改变，抽提细胞ＤＮＡ进行琼脂糖凝胶电泳．结果：ｏｘＬＤＬ３００ｍｇ·Ｌ－１与ＶＳＭＣ共温育２４ｈ诱导典型的凋亡形态学变化和ＤＮＡ降解，但天然低密度脂蛋白无此作用．当ｏｘＬＤＬ为４００ｍｇ·Ｌ－１或温育时间延至４８ｈ，上述变化更加明显．硫酸葡聚糖２０ｍｇ·Ｌ－１和ＢＨＴ５０μｍｏｌ·Ｌ－１对此作用无影响．ＬＰＣ１２５μｍｏｌ·Ｌ－１无诱导凋亡作用．结论：ｏｘＬＤＬ诱导血管平滑肌细胞凋亡，氧自由基和ＬＰＣ不参与这一过程．     

牛磺酸对H2O2引起的大鼠血管平滑肌细胞核[Ca^2＋]及细胞浆[Ca^2＋]变化的影响

目的 毒性剂量H2O2可引起细胞坏死或凋亡,此过程是否与H2O2引起的核[Ca^2 ][Ca^2 ]n变化有关未见报导。本文研究牛磺酸对H2O2引起的血管平滑肌细胞（VSMC）[Ca^2 ]n与胞浆[Ca^2 ][Ca^2 ]c）的变化影响。方法 应用激光共聚焦显微镜对负载Fluo-3的培养在大鼠VSMC的[Ca^2 ]n及[Ca^2 ]c变化进行研究。结果 在0．5％H2O2作用下,[Ca^2 ]n及[Ca^2 ]c持续升高,用抗氧化细胞保护剂－牛磺酸20mmol.L^-1预处理细胞,可降低H2O2引起的[Ca^2 ]n升幅（P＜0．05）,但不影响[Ca^2 ]c升幅（P＞0．05）,表明[Ca^2 ]n变化与[Ca^2 ]c变化对牛磺酸反应性存在差异。结论 VSMC的核Ca^2 调控与胞浆Ca^2 调控各自具有一定的独立性。牛磺酸对H2O2引起的大鼠VSMC核Ca^2 的变化具有保护作用。     

Neferine inhibits angiotensin Ⅱ-stimulated proliferation in vascular smooth muscle cells through heme oxygenase-1

Aim:

To explore the effect of neferine on angiotensin II (Ang II)-induced vascular smooth muscle cell (VSMC) proliferation.

Methods:

Human umbilical vein smooth muscle cells (HUVSMCs) were used. Cell proliferation was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis. Heme oxygenase (HO)-1 protein expression was tested by Western blot analysis. Extracellular signal-regulated protein kinase 1/2 (ERK1/2) activation was determined by using immunoblotting.

Results:

Pre-incubation of HUVSMCs with neferine (0.1, 0.5, 1.0, and 5.0 μmol/L) significantly inhibited Ang II-induced cell proliferation in a concentration-dependent manner and neferine 5.0 μmol/L increased HO-1 expression by 259% compared with control. The antiproliferative effect of neferine was significantly attenuated by coapplication of zinc protoporphyrin IX (ZnPP IX, an HO-1 inhibitor) with neferine. Ang II-enhanced ERK1/2 phosphorylation was markedly reversed by neferine. By inhibiting HO-1 activity with ZnPP IX, the inhibitive effect of neferine on ERK1/2 phosphorylation was significantly attenuated. Cobalt-protoporphyrin (CoPP), an HO-1 inducer, significantly decreased Ang II-induced ERK1/2 phosphorylation and inhibited Ang II-induced cell proliferation. The ERK1/2 pathway inhibitor PD98059 significantly blocked Ang II-enhanced ERK1/2 phosphorylation and inhibited cell proliferation.

Conclusion:

These findings suggest that neferine can inhibit Ang II-induced HUVSMC proliferation by upregulating HO-1, leading to the at least partial downregulation of ERK1/2 phosphorylation.     

Effects of docosahexaenoic acid on large-conductance Ca^2＋-activated K^＋ channels and voltage-dependent K^＋ channels in rat coronary artery smooth muscle cells

Aim： To investigate the effects of docosahexaenoic acid （DHA） on large-conductance Ca^2＋-activated K^＋ （BKca） channels and voltage-dependent K^＋ （Kv） channels in rat coronary artery smooth muscle cells （CASMCs）.
Methods： Rat CASMCs were isolated by an enzyme digestion method. BKCa and Kv currents in individual CASMCs were recorded by the patch-clamp technique in a whole-cell configuration at room temperature. Effects of DHA on BKCa and Kv channels were observed when it was applied at 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L. Results： When DHA concentrations were greater than 10 μmol/L, BKCa currents increased in a dose-dependent manner. At a testing potential of ＋80 mV, 6.1%±0.3%, 76.5%±3.8%, 120.6%±5.5%, 248.0%±12.3%, 348.7%±17.3%, 374.2%±18.7%, 432.2%±21.6%, and 443.1%±22.1% of BKCr currents were increased at the above concentrations, respectively. The half-effective concentration （EC50） of DHA on BKca currents was 37.53±1.65 μmol/L. When DHA concentrations were greater than 20 μmol/L, Kv currents were gradually blocked by increasing concentrations of DHA. At a testing potential of ＋50 mV, 0.40%±0.02%, 1.37%±0.06%, 11.80%±0.59%, 26.50%±1.75%, 56.50%±2.89%, 73.30%±3.66%, 79.70%±3.94%, and 78.1%±3.91% of Kv currents were blocked at the different concentrations listed above, respectively. The EC50 of DHA on Kv currents was 44.20±0.63 μmol/L.
Conclusions： DHA can activate BKca channels and block Kv channels in rat CASMCs, and the EC50 of DHA for BKca channels is lower than that for Kv channels; these findings indicate that the vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BKCa channels.     

Proliferation of aortic smooth muscle cells and renin-angiotensin system in SHR rats

目的：探讨ＳＨＲ大鼠主动脉平滑肌细胞（ＡＳＭＣ）异常增殖和肾素血管紧张素系统（ＲＡＳ）的关系．方法：测定血管紧张素Ｉ（Ａｎｇ）、卡托普利（Ｃａｐ）、沙拉新（Ｓａｒ）对培养的ＳＨＲ、ＷＫＹＡＳＭＣ增殖和Ａｎｇ、血管紧张素转化酶（ＡＣＥ）的影响．结果：Ａｎｇ在２％血清培养基中可刺激ＳＨＲＡＳＭＣ增生．ＳＨＲＡＳＭＣ分裂增殖能力比ＷＫＹ强，ＳＨＲＡＳＭＣＲＡＳ处于高功能状态．Ｃａｐ长期（４周）干预显著抑制ＳＨＲＡＳＭＣ异常增殖和Ａｎｇ、ＡＣＥ活性，Ｓａｒ长期干预同样抑制ＳＨＲＡＳＭＣ的增殖和ＡＣＥ活性，但Ａｎｇ水平反而升高．Ｃａｐ短期（２４小时）干预不影响两种大鼠ＡＳＭＣＲＡＳ．结论：Ｃａｐ和Ｓａｒ长期干预通过减少ＳＨＲＡＳＭＣＡｎｇ生成或阻断Ａｎｇ和特异受体结合，抑制其异常增殖．     

Mechanisms of [Ca2+]i elevation following P2X receptor activation in the guinea-pig small mesenteric artery myocytes

BackgroundThere is growing evidence suggesting involvement of L-type voltage-gated Ca²⁺ channels (VGCCs) in purinergic signaling mechanisms. However, detailed interplay between VGCCs and P2X receptors in intracellular Ca²⁺ mobilization is not well understood. This study examined relative contribution of the Ca²⁺ entry mechanisms and induced by this entry Ca²⁺ release from the intracellular stores engaged by activation of P2X receptors in smooth muscle cells (SMCs) from the guinea-pig small mesenteric arteries.MethodsP2X receptors were stimulated by the brief local application of αβ-meATP and changes in [Ca²⁺]_i were monitored in fluo-3 loaded SMCs using fast x-y confocal Ca²⁺ imaging. The effects of the block of L-type VGCCs and/or depletion of the intracellular Ca²⁺ stores on ab-meATP-induced [Ca²⁺]_i transients were analyzed.ResultsOur analysis revealed that Ca²⁺ entry via L-type VGCCs is augmented by the Ca²⁺-induced Ca²⁺ release significantly more than Ca²⁺ entry via P2X receptors, even though net Ca²⁺ influxes provided by the two mechanisms are not significantly different.ConclusionsThus, arterial SMCs upon P2X receptor activation employ an effective mechanism of the Ca²⁺ signal amplification, the major component of which is the Ca²⁺ release from the SR activated by Ca²⁺ influx via L-type VGCCs. This signaling pathway is engaged by depolarization of the myocyte membrane resulting from activation of P2X receptors, which, being Ca²⁺ permeable, per se form less effective Ca²⁺ signaling pathway. This study, therefore, rescales potential targets for therapeutic intervention in purinergic control of vascular tone.     

肺动脉高压模型大鼠肺动脉平滑肌细胞内钙的改变

目的分析肺动脉高压时肺动脉平滑肌细胞Ryanod-ine受体[Ca2+]i释放功能的改变。方法腹腔注射野百合碱建立大鼠肺动脉高压模型,原代培养肺动脉平滑肌细胞,Fura-2/AM负载培养细胞,荧光测钙技术测量Ryanodine受体激动剂对[Ca2+]i变化的影响。结果10nmol.L-1Ry-anodine使对照组[Ca2+]i平均增加(93.31±12.41)nmol.L-1,使PAH组[Ca2+]i平均增加(141.71±13.59)nmol.L-1。两组样本[Ca2+]i增加的数值差异有显著性(P<0.01);10mmol.L-1Caffine使对照组[Ca2+]i平均增加(149.02±13.02)nmol.L-1,使PAH大鼠PASMC的[Ca2+]i平均增加(191.2±21.26)nmol.L-1,两组样本[Ca2+]i数值的变化差异有显著性(P<0.01)。结论肺动脉高压大鼠原代培养的肺动脉平滑肌细胞对Ryanodine受体激动剂的敏感性增强,提示肺动脉高压时Ryanodine受体释放[Ca2+]i的功能发生了异常改变。     

家兔动脉血管平滑肌细胞培养方法的改进

目的提高家兔血管平滑肌细胞(vascular smooth mus-cle cell,VSMC)体外培养的成功率。方法以传统的主动脉平滑肌细胞培养方法中的组织块贴壁法为基础,从动物选择、培养基配制、血管取材、组织块的大小和接种间距、培养液的量及换液量、细胞传代的时机等多个重要环节进行改进;用倒置显微镜、电镜对培养细胞进行形态学观察,并用平滑肌细胞特异性α-肌动蛋白(α-actin)单克隆抗体免疫组织化学方法进行鉴定。结果台盼蓝检查传代细胞的存活率大于97%;光镜下见培养细胞平行排列呈长梭形及"峰、谷"样结构特征;免疫组织化学染色鉴定培养细胞中VSMC的纯度为97%;电镜技术观察到VSMC完整的超微结构。结论此培养方法能提高动脉平滑肌细胞原代培养的成功率,传代后血管平滑肌细胞生物学特征的稳定性好,可作为研究血管平滑肌细胞生物学行为的有效模型。     

血管平滑肌和内皮细胞Ca2+内流机制及其与Cl-通道的关系

血管平滑肌和内皮细胞的Ｃａ２＋内流机制不同,前者是兴奋性细胞,Ｃａ２＋内流通过电压依赖性（ＶＤＣ）和非电压依赖性Ｃａ２＋通道;后者是非兴奋性细胞,Ｃａ２＋内流主要通过非ＶＤＣ途径。Ｃｌ－通道参与了这两种细胞的Ｃａ２＋调控,平滑肌细胞Ｃｌ－通道开放导致细胞膜去极化,促进ＶＤＣ开放,Ｃａ２＋内流增加;而内皮细胞Ｃｌ－通道开放导致细胞膜超极化,使Ｃａ２＋进入细胞内的电化学趋势增加,胞外Ｃａ２＋经非ＶＤＣ途径内流增加。目前对血管平滑肌和内皮细胞Ｃｌ－通道的分型、特性和功能还不清楚。     

Characterization of action potential-triggered [Ca2+]i transients in single smooth muscle cells of guinea-pig ileum

1. To characterize increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) associated with discharge of action potentials, membrane potential and [Ca²⁺]_i were simultaneously recorded from single smooth muscle cells of guinea-pig ileum by use of a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques.
2. A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca²⁺]_i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca²⁺]_i transients with an almost constant amplitude.
3. Nicardipine (1 μM) and La³⁺ (10 μM), blockers of voltage-dependent Ca²⁺ channels (VDCCs), abolished both the action potential discharge and the [Ca²⁺]_i transient.
4. Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca²⁺-activated K⁺ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca²⁺]_i transients.
5. Thapsigargin (1 μM), an inhibitor of SR Ca²⁺-ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential-triggered [Ca²⁺]_i transients without affecting action potentials. In addition, in voltage-clamped cells, the drug had little effect on the voltage step-evoked Ca²⁺ current but exerted a similar effect on its concomitant rise in [Ca²⁺]_i to that on the action potential-triggered [Ca²⁺]_i transient.
6. Similar action potential-triggered [Ca²⁺]_i transients were induced by brief exposures to high-K⁺ solution. They were not decreased, but rather increased, after depletion of intracellular Ca²⁺ stores by a combination of ryanodine (30 μM) and caffeine (10 mM) through an open-lock of Ca²⁺-induced Ca²⁺ release (CICR)-related channels.
7. The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca²⁺]_i. Intracellular Ca²⁺ stores have a role, especially in accelerating the falling phase of the action potential-triggered [Ca²⁺]_i transients by replenishing cytosolic Ca²⁺. No evidence was provided for the involvement of CICR in the action potential-triggered [Ca²⁺]_i transient.

     

Anomalous response to potassium in vascular smooth muscle cells of human saphenous vein

1.--We have examined the relationship between the resting membrane potential (E(m)) and the concentration of the external ions, K(+), Cl(-) and Ca(2+), as well as the effects of K(+) on active force generation in human saphenous veins. 2.--As measured with sharp glass microelectrodes, the E(m) of vascular muscle cells was -76.0 +/- 7.0 mV (mean +/- SD; n = 328). Raising the concentration of external potassium ([K(+)](e)) from 4.2 to 20, 40, 80, 120 or 150 mm produced an incremental depolarization, revealing a maximal slope factor of 15 mV per 10-fold increase. 3.--Oubain (1.0 microm) did not have any effect on E(m) (-79.0 +/- 8.0 mV; n = 80). Replacement of external Cl(-) with propionate resulted in significant (P < 0.05) depolarization (E(m): -65.5 +/- 7.5 mV; n = 40). In Cl(-)-free buffer containing 80 mm K(+), E(m) depolarized to -52.0 +/- 6.7 mV (n = 45) compared with -64.7 +/- 6.5 mV (n = 55) (P < 0.05) measured in buffer containing 80 mm [K(+)](e) and Cl(-) 138.7 mm. Removal of Ca(2+) did not significantly modify the depolarizing response to K(+) 80 mm: E(m), -68.2 +/- 4.9 mV (n = 42) vs.-64.7 +/- 6.5 mV (n = 55) in the presence of Ca(2+). 4.--Despite their small size, changes in E(m) correlated closely with force generation in buffer containing high K(+), approximately 3.62 mN force being generated per mV of change in E(m). 5.--These data demonstrate that, in human saphenous smooth muscle cells, (i) the magnitude of depolarization induced by raising [K(+)](e) deviates considerably from the theoretical values predicted by the Goldman-Hodgkin-Katz equations, (ii) Cl(-) appears to contribute to the maintenance of E(m), and (iii) electromechanical coupling has a low threshold.     

Effects of berbamine on ATP-induced [Ca2+]i mobilization in cultured vascular smooth muscle cells and cardiomyocytes.

AIM: To study the effects of berbamine (Ber) on [Ca2+]i homeostasis induced by adenosine triphosphate (ATP) in vascular smooth muscle cells (VSMC) of rabbits and cardiomyocytes of rats. METHODS: Both cell types were cultured and loaded with Fura 3-AM. [Ca2+]i was measured by fluorescent intensity (FI) in each cell with confocal microscopy. RESULTS: (1) ATP 30 mumol.L-1 elevated [Ca2+]i in VSMC and cardiomyocytes, FI values reached 660 +/- 258 and 1058 +/- 252 from 250 +/- 84 and 218 +/- 76 at 19 s +/- 5 s and 11.8 s +/- 2.4 s, but FI in nucleus was not changed in VSMC. (2) Ber 30 mumol.L-1 did not affect the resting FI in both cell types, but prolonged the time to peak (P < 0.01) and reduced the FI elevated by ATP (P < 0.01), but not completely inhibited even at 100 mumol.L-1. (3) In D-Hanks' solution or in the presence of egtazic acid (EGTA) 3 mmol.L-1, the inhibitory effect of Ber was not seen (P > 0.05). (4) All effects of Ber on ATP-induced [Ca2+]i mobilization were similar to those of Ver 10 mumol.L-1. CONCLUSION: In VSMC and cardiomyocytes, ATP-induced CA2+ influx was inhibited by Ber and Ver, while the Ca2+ release was not.     

糖尿病大鼠冠状动脉平滑肌细胞体外培养方法的探讨

目的探讨糖尿病大鼠冠状动脉平滑肌细胞体外培养方法,建立糖尿病大鼠冠状动脉平滑肌细胞模型,为糖尿病性冠心病的研究奠定基础。方法建立糖尿病大鼠模型,用酶消化法体外培养冠状动脉血管平滑肌细胞。结果糖尿病大鼠造模成功后分离冠状动脉,用酶消化法培养糖尿病大鼠血管平滑肌细胞,24h更换培养液液,培养7~10d细胞重叠生长达多层,高低起伏呈“峰—谷”状。细胞α-actin免疫组织化学染色鉴定为平滑肌细胞。结论糖尿病大鼠血管平滑肌细胞增殖速度快,培养条件要求严格,在形态学上与正常大鼠平滑肌细胞相同。