17 beta-estradiol inhibits T-type calcium channels in mouse spermatogenic cells

OBJECTIVE: To investigate the effect of 17 beta-estradiol (E2) on T-type calcium currents (ICaT) in spermatogenic cells. METHODS: Ca2+ currents were obtained in acutely dissociated mouse spermatogenic cells by the whole-cell patch clamp technique and the effects of E2 on ICaT were observed. RESULTS: E2 at the concentrations of 1, 10 and 100 micromol/L significantly inhibited ICaT in the mouse spermatogenic cells, with the KC50 value of 8.89 micromol/L and the inhibition rates of (13.48 +/- 1.86) %, (28.98 +/- 2.70) and (52.93 +/- 3.42)%, respectively (n = 6, P < 0.05). E2 of 100 micromol/L significantly changed the activation and inactivation of ICaT: the half activation potential (Va 1/2) and the activation steepness factor (Ka) from ( -48.94 +/- 0.22) mV and (5.19 +/- 0.19) mV to (-54.34 +/- 1.02) mV and (6.02 +/- 0.84) mV ( n = 5, P < 0.05) , and the half inactivation potential (Vi 1/2) and the inactivation steepness factor (Ki) from (-56.51 +/- 0.13) mV and (3.36 +/- 0.11) mV to (-61.78 +/- 0.50) mV and (4.25 +/- 0.37) mV, respectively (n = 5, P < 0.05). CONCLUSION: E2 has a significant inhibitory effect on ICaT in mouse spermatogenic cells in a con-centration-dependent manner.     

T型钙通道与疼痛

疼痛信号的传递依赖于伤害感受性神经元内的电压依赖性钙通道。N型钙通道作为传递疼痛信号的重要成员一直备受关注，近年来，发现T型钙通道在外周及CNS都参与疼痛信号的处理。抑制DRG神经元内的T型钙通道可减轻疼痛，而敲除CNS内的T型钙通道则可促进疼痛。因此，T型钙通道有望成为疼痛治疗的又一靶点。     

Progesterone accelerates the onset of capacitation in mouse sperm via T-type calcium channels

In previous conventional reports sperm and seminal plasma were not well separated and therefore it was highly probable that isoform (CK-BB) of seminal plasma was determined together with that of sperm. The authors developed a method to purify sperm specimen free from seminal plasma. Human sperm was purified by means of the continuous-step density gradient centrifugation with an inner column and the subsequent modified swim-up procedure. CK isoforms in the purified sperm and the seminal plasma were analyzed by ion exchange chromatography and electrophoresis. The purified sperm contained mitochondrial CK-MiMi as well as cytosolic CK, of which isoform was almost occupied by CK-MM but CK-BB was not detected. On the other hand, CK isoform in the seminal plasma was detected as CK-BB alone. CK activity in human sperm and seminal plasma were 2.0 +/- 0.7 x 10(-6) IU/10(6) sperm and 661 +/- 360 IU/L, respectively. CK activity in the seminal plasma gave no correlation with the semen parameters.     

Glomerular autacoids stimulated by bradykinin regulate efferent arteriole tone

BACKGROUND: We have shown that when efferent arterioles are perfused retrograde to avoid the influence of vasoactive autacoids released by the glomerulus, bradykinin causes dilatation via release of cytochrome p450 (cp450) metabolites, probably epoxyeicosatrienoic acids (EETs). Here we tested the hypothesis that the glomerulus releases cyclooxygenase (COX) and cp450 metabolites. These eicosanoids, acting as vasopressor and vasodepressor autacoids, control efferent arteriole resistance downstream from the glomerulus. METHODS: Rabbit efferent arterioles were perfused orthograde through the glomerulus from the end of the afferent arteriole to determine whether bradykinin induces the release of glomerular autacoids that influence efferent arteriole resistance. Efferent arterioles were preconstricted with norepinephrine, and increasing doses of bradykinin were added to the perfusate in the presence or absence of COX and cp450 inhibitors. RESULTS: When efferent arterioles were perfused orthograde through the glomerulus, bradykinin at 10 nmol/L caused significant and reproducible dilatation; diameter increased from 8.0 +/- 0.5 to 12.6 +/- 0.4 microm (P < 0.05). This effect was not modified by a nitric oxide synthase (NOS) inhibitor. In the presence of indomethacin, a COX inhibitor, bradykinin-induced dilatation was almost completely blocked (from 8.0 +/- 0.5 to 9.3 +/- 0.6 microm). This blockade was completely reversed by 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), a specific antagonist of the vasoconstrictor cp450 metabolite 20-hydroxyeicosatetraenoic acid (20-HETE); diameter increased from 6.6 +/- 0.7 to 13.2 +/- 0.5 microm. To test the hypothesis that this dilatation was due to EETs, a specific inhibitor of EET synthesis, N-methylsulphonyl-6-(2-proparglyloxyphenyl)hexanamide (MS-PPOH), was added to the arteriolar perfusate. In the presence of indomethacin and 20-HEDE, bradykinin caused dilatation and this effect was completely blocked by MS-PPOH (1 microm) (from 7.6 +/- 0.6 to 7.3 +/- 0.5 microm). CONCLUSIONS: We concluded that in response to bradykinin, the glomerulus releases COX metabolites (probably prostaglandins) that have a vasodilator effect. When COXs are inhibited, the vasoconstrictor 20-HETE released by the glomerulus is able to oppose the vasodilator effect of bradykinin. This vasodilator effect is mediated by EETs released by the glomerulus and/or the efferent arteriole and does not involve nitric oxide. The balance between these opposing effects of various eicosanoids controls efferent arteriole resistance downstream from the glomerulus.     

Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels

Mechanisms by which bone microdamage triggers repair response are not completely understood. It has been shown that calcium efflux ([Ca^2 +]_E) occurs from regions of bone undergoing microdamage. Such efflux has also been shown to trigger intracellular calcium signaling ([Ca^2 +]_I) in MC3T3-E1 cells local to damaged regions. Voltage-gated calcium channels (VGCCs) are implicated in the entry of [Ca^2 +]_E to the cytoplasm. We investigated the involvement of VGCC in the extracellular calcium induced intracellular calcium response (ECIICR). MC3T3-E1 cells were subjected to one dimensional calcium efflux from their basal aspect which results in an increase in [Ca^2 +]_I. This increase was concomitant with membrane depolarization and it was significantly reduced in the presence of Bepridil, a non-selective VGCC inhibitor. To identify specific type(s) of VGCC in ECIICR, the cells were treated with selective inhibitors for different types of VGCC. Significant changes in the peak intensity and the number of [Ca^2 +]_I oscillations were observed when L-type and T-type specific VGCC inhibitors (Verapamil and NNC55-0396, respectively) were used. So as to confirm the involvement of L- and T-type VGCC in the context of microdamage, cells were seeded on devitalized notched bone specimen, which were loaded to induce microdamage in the presence and absence of Verapamil and NNC55-0396. The results showed significant decrease in [Ca^2 +]_I activity of cells in the microdamaged regions of bone when L- and T-type blockers were applied. This study demonstrated that extracellular calcium increase in association with damage depolarizes the cell membrane and the calcium ions enter the cell cytoplasm by L- and T-type VGCCs.     

Angiotensin II-mediated constriction of afferent and efferent arterioles involves T-type Ca2+ channel activation

BACKGROUND/AIMS: Previous studies have shown that L-type Ca2+ channel (LCC) blockers prevent the afferent arteriolar (AA) vasoconstriction elicited by angiotensin II (Ang II), but do not influence its vasoconstrictor effect on efferent arterioles (EA). The present study tested the hypothesis that Ang II-mediated constriction of AA and EA involves T-type Ca2+ channel (TCC) activation, which may mediate Ca2+ entry responsible for Ang II-induced EA and possibly AA constriction. METHODS: Video-microscopic measurements of vascular dimensions were performed on isolated blood-perfused juxtamedullary nephrons from Sprague-Dawley rats. Single AA or EA were visualized and superfused with solutions containing Ang II alone or with a TCC blocker, pimozide, or a LCC blocker, diltiazem. RESULTS: Pimozide at 10 micromol/l significantly dilated EA (19.7 +/- 1.4%) as well as AA (24.8 +/- 3.6%). In response to superfusion with Ang II at concentrations of 0.1, 1.0 and 10.0 nmol/l, AA diameter decreased significantly by 15.2 +/- 1.7, 23.3 +/- 3.2 and 36.1 +/- 3.4% and EA diameter also decreased significantly by 11.9 +/- 1.7, 19.6 +/- 2.8 and 31.0 +/- 2.6%, respectively. Pimozide (10 micromol/l) markedly blunted AA (4.6 +/- 1.2, 7.5 +/- 0.6 and 7.9 +/- 1.2%) and EA (2.2 +/- 0.6, 5.4 +/- 1.5 and 7.7 +/- 1.3%) diameter responses to Ang II. Diltiazem (10 micromol/l) significantly dilated AA (26.8 +/- 2.2%), and prevented Ang II-mediated constriction of AA. In contrast, diltiazem did not dilate EA (3.3 +/- 0.6%) and failed to inhibit the Ang II-induced EA vasoconstriction; however, the vasoconstriction was reversed by the subsequent addition of pimozide (5 micromol/l). CONCLUSION: This study provides further functional evidence for TCC channels in the regulation of AA and EA indicating that Ang II-mediated arteriolar constriction may involve activation of TCC in both AA and EA. TCC may play an important role in mediating Ca2+ entry responsible for Ang-induced EA and AA constriction. The role of TCC in mediating Ang II-constrictor actions on EA may be of particular significance because LCC are not normally functional in these vessels.     

T型钙通道Cav3.2在紫杉醇致神经痛觉过敏中的调制作用

背景 Cav3.2是T型钙通道的一种亚型,在脊髓背根神经节中分布广泛.研究表明:低电压激活的T型钙离子(Ca2+)通道参与神经病理痛的形成.Car3.2在慢性疼痛,尤其是在化疗药紫杉醇诱发的痛觉过敏中的作用已成为研究关注的热点.目的 阐明T型钙通道Cav3.2在紫杉醇诱发神经痛觉过敏中的作用.内容 就Car3.2的电生理特性、参与疼痛的可能机制、与不同疼痛间的关系等方面进行阐述.趋向 T型钙通道Cav3.2亚型在痛觉过敏的发生发展过程中发挥着重要作用.充分了解Cav3.2的生理作用,阐明其参与紫杉醇诱发的痛觉过敏的可能机制,可为化疗药所致痛觉过敏的防治提供思路.     

G proteins regulate calcium channels in the luminal membranes of the rabbit nephron

The filtered calcium (Ca2+) is reabsorbed by the luminal membrane of the proximal and distal nephron. Ca2+ enters cells across apical plasma membranes along a steep electrochemical gradient, through Ca2+ channels. Regulation by various hormones implies several steps, including binding of these hormones to the basolateral membrane, interaction with G proteins, liberation of messengers, activation of kinases and finally opening of the channels at the opposite pole of the cells. In the present study, we examined whether the Ca2+ entry through the luminal membranes of proximal and distal tubules is also regulated by G proteins, by a membrane-limited process. Luminal membranes were purified from rabbit proximal and distal tubule suspensions, and their vesicles were loaded with GTPgammas or the carrier. Then, the 45Ca2+ uptake by these membrane vesicles was measured in the presence and absence of 100 mM NaCl. In the absence of Na+, intravesicular GTPgammas significantly enhanced 0.5 mM Ca2+ uptake by the proximal membrane vesicles from 0.53 +/- 0.06 to 0.72 +/- 0.06 pmol/microg/10 s (p < 0.05). In the presence of Na+, however, this effect disappeared. In the distal tubules, intravesicular GTPgammas increased 0.5 mM Ca2+ uptake in the absence (from 0.57 +/- 0.02 to 0.79 +/- 0.02 pmol/microg/10 s, p < 0.02) and in the presence (from 0.36 +/- 0.03 to 0.55 +/- 0.03 pmol/microg/10 s, p < 0.02) of Na+. The action of GTPgammas, when present, was dose dependent with a half-maximal effect at 20 microM. The distal luminal membrane is the site of two Ca2+ channels with different kinetics parameters. GTPgammas increased the Vmax value of the low-affinity component exclusively, in the presence as in the absence of Na+. Finally, Ca2+ uptake by the membranes of the two segments was differently influenced by toxins: cholera toxin slightly stimulated transport by the proximal membrane, but had no influence on the distal membrane, whereas pertussis toxin decreased the cation uptake by the distal tubule membrane exclusively. We conclude that the nature of Ca2+ channels differs in the proximal and distal luminal membranes: Ca2+ channels present in the proximal tubule and the low-affinity Ca2+ channels present in the distal tubule membranes are directly regulated by Gs and Gi proteins respectively, whereas the high-affinity Ca2+ channel in the distal tubule membrane is insensitive to any of them.     

不同浓度褪黑激素对新生大鼠海马锥体神经元电压门控性Ca2+通道的影响

目的 探讨不同浓度褪黑激素对新生大鼠海马锥体神经元电压门控性ca2通道的影响.方法 原代培养新生Wistar大鼠(出生时问<12 h)海马锥体神经元7～12 d.配制褪黑激素溶液,终浓度依次为1 nmol/L、10 nmol/L、100 nmol/L、1 μmol/L、10μmol/L、100μmol/L和1 mmol/L.选择胞体清晰、光晕良好、轴突明显的锥体神经元,采用膜片钳全细胞记录模式观察Ca2+通道的基本电生理特点,记录不同浓度褪黑激素作用下Ca2+电流,分析Ca+2通道动力学特性,并计算Ca2+电流变化率.结果 不同浓度褪黑激素均可快速、可逆性地增强Ca2+电流,10 nmol/L和100 nmol/L褪黑激素使Ca2+电流激活曲线向超极化方向移动,其余浓度褪黑激素对Ca2+电流激活曲线的动力学特性无影响.与100 mol/L褪黑激素比较,其余浓度褪黑激素作用后Ca2+电流变化率降低(P<0.05或0.01);与Iμmol/L褪黑激素比较,10 μmol/L、100μmol/L和1 mmol/L褪黑激素作用后Ca2+电流变化率降低(P<0.05).结论 l nmol/L～1 mmol/L褪黑激素均可增强体外培养的新生大鼠海马锥体神经元Ca2+电流,100 nmol/L褪黑激素作用最强.     

Reactivity of gomerular afferent and efferent arterioles in renal hypertension.

Renal tissue from neonatal hamsters was grafted into the cheek pouch of adult hamsters. Renal hypertension was induced in 28 hamsters, and sham operation was performed in 27. When blood pressure became elevated in hypertensive hamsters (10 to 12 days), the renal microcirculation of both groups was evaluated by direct microscopy. Wall and luminal diameter and vascular responses to norepinephrine (NE) and angiotensin II (AII) were determined on glomerular afferent and efferent arterioles and on the corresponding cheek pouch arterioles. In the hypertensive hamsters, the luminal diameter of the afferent arteriole increased, as did the wall/lumen ratio of the efferent arteriole. Although all vessels responded to direct application of NE and AII in both groups, the response of the afferent to AII and the response of the efferent to both NE and AII was greater in the hypertensive hamsters as compared to the sham-operated hamsters. These results indicate that during the development of renal hypertension structural alterations of glomerular vessels and the selective vascular responses to vasoactive agents would lead to an increased glomerular capillary pressure.     

Flow modulation of pressure-sensitive tone in rat pial arterioles: role of the endothelium

BACKGROUND: Cerebral arteriolar tone is modulated in response to changes in transmural pressure and luminal flow. The effect of flow on the relation between pressure and diameter has not been fully evaluated in these vessels. This study was conducted to investigate this interaction and to determine the role of the endothelium in mediating it. METHODS: Rat pial arterioles from the territory of the posterior cerebral artery were mounted in a perfusion myograph. In some arterioles, the endothelium was removed by air perfusion. Diameters were recorded at pressures from 20 to 200 mmHg in the presence and absence of flow (10 microl/min). The response to flow (0-30 microl/min) was recorded at 60 and 120 mmHg. RESULTS: In the absence of flow, endothelium-intact arterioles demonstrated tone at distending pressures between 40 and 140 mmHg. In the presence of flow, tone did not develop until pressure exceeded 100 mmHg, and the vessels remained active at pressures up to 200 mmHg. Endothelium-denuded arterioles developed tone at the same pressure when perfused as when unperfused, but perfused vessels were able to maintain active tone at higher pressures. At 60 mmHg, flow caused dilation if the endothelium was intact and constriction if it had been removed. At 120 mmHg, flow caused constriction. Endothelium-dependent flow-relaxation was inhibited by N(G)-nitro-L-arginine methyl ester (10(-5) M) and abolished by indomethacin (10(-5) M). CONCLUSION: Flow inhibits the development of pial arteriolar tone at low intraluminal pressures through endothelium-dependent mechanisms. Conversely, perfusion extends the upper limit of the myogenically regulated pressure range through endothelium-independent activation of arteriolar smooth muscle contraction.     

Anandamide decreases glomerular filtration rate through predominant vasodilation of efferent arterioles in rat kidneys

For determining the effects of anandamide (ANA) on renal hemodynamics and microcirculation, a clearance study was performed in Sprague-Dawley rats that received injections of ANA in doses of 15, 150, and 1500 pmol/kg. At doses up to 150 pmol/g, ANA significantly decreased GFR and increased renal blood flow (RBF) without affecting mean arterial pressure (MAP). In the presence of the cannabinoid type 1 (CB1) receptor antagonist AM251, only the 15-pmol/kg dose significantly increased GFR and RBF without altering MAP, with higher doses having no effect on GFR, RBF, or MAP. By contrast, AM281, which antagonizes cannabinoid receptors nonselectively, inhibited the GFR, RBF, and MAP responses to ANA. The arteriolar responses to ANA were also assessed in vitro by the blood-perfused juxtamedullary nephron technique. Higher doses of ANA significantly increased the diameter of both afferent and efferent arterioles, whereas lower doses elicited predominant efferent arteriolar dilation. AM251 attenuated the afferent arteriolar response to ANA and inhibited the efferent arteriolar response to ANA, whereas AM281 inhibited the responses in both arterioles. The CB1 receptor mRNA was expressed in afferent arterioles, and immunohistochemical staining demonstrated the presence of CB1 receptors in both afferent and efferent arterioles. These results suggest that ANA causes afferent arteriolar dilation via both CB1 and non-CB1 receptors and greater efferent arteriolar dilation via CB1 receptors, resulting in a decreased GFR and an increased RBF without affecting MAP.     

Isoform-selective effects of isoflurane on voltage-gated Na+ channels

BACKGROUND: Voltage-gated Na channels modulate membrane excitability in excitable tissues. Inhibition of Na channels has been implicated in the effects of volatile anesthetics on both nervous and peripheral excitable tissues. The authors investigated isoform-selective effects of isoflurane on the major Na channel isoforms expressed in excitable tissues. METHODS: Rat Nav1.2, Nav1.4, or Nav1.5 alpha subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage clamp recording. The effects of isoflurane on Na current activation, inactivation, and recovery from inactivation were analyzed. RESULTS: The cardiac isoform Nav1.5 activated at more negative potentials (peak INa at -30 mV) than the neuronal Nav1.2 (0 mV) or skeletal muscle Nav1.4 (-10 mV) isoforms. Isoflurane reversibly inhibited all three isoforms in a concentration- and voltage-dependent manner at clinical concentrations (IC50 = 0.70, 0.61, and 0.45 mm, respectively, for Nav1.2, Nav1.4, and Nav1.5 from a physiologic holding potential of -70 mV). Inhibition was greater from a holding potential of -70 mV than from -100 mV, especially for Nav1.4 and Nav1.5. Isoflurane enhanced inactivation of all three isoforms due to a hyperpolarizing shift in the voltage dependence of steady state fast inactivation. Inhibition of Nav1.4 and Nav1.5 by isoflurane was attributed primarily to enhanced inactivation, whereas inhibition of Nav1.2, which had a more positive V1/2 of inactivation, was due primarily to tonic block. CONCLUSIONS: Two principal mechanisms contribute to Na channel inhibition by isoflurane: enhanced inactivation due to a hyperpolarizing shift in the voltage dependence of steady state fast inactivation (Nav1.5 approximately Nav1.4 > Nav1.2) and tonic block (Nav1.2 > Nav1.4 approximately Nav1.5). These novel mechanistic differences observed between isoforms suggest a potential pharmacologic basis for discrimination between Na channel isoforms to enhance anesthetic specificity.     

Interleukin-10 prevents loss of tone of rat skeletal muscle arterioles exposed to endotoxin

BACKGROUND: The anti-inflammatory cytokine interleukin-10 (IL-10) is known to inhibit the development of septic shock in animal models. This study was conducted to investigate the effect of IL-10 on the loss of vascular tone during exposure to endotoxin. Unlike numerous proinflammatory cytokines, the effects of IL-10 at the level of the microvasculature have not been previously studied. MATERIALS AND METHODS: First-order rat cremasteric arterioles (n = 27) were placed in an isolated vessel preparation and allowed to achieve spontaneous tone. An opened segment of thoracic aorta was then placed upstream from the arteriole in the superfusion line. The resistance arteriole of this in-series model, in contrast to the isolated arteriole alone, demonstrates a significant loss of tone when exposed to endotoxin. Following 1 h of equilibration in the presence or absence of IL-10 (20 ng/ml), the aorta and arteriole were then superfused with 2.5 microg/ml endotoxin or physiologic buffer for 60 min and serial arteriolar diameter measurements were recorded. Group 1 was exposed to endotoxin only, Group 2 was a time control, and Group 3 was pretreated with IL-10 prior to endotoxin exposure, while Group 4 was a control pretreated with IL-10 only. RESULTS: After the 60-min equilibration period there were no differences among the four groups in arteriolar tone. At t = 120 min, the percentage of tone in the control group was 43.6 +/- 3% (mean +/- SEM) and this was not changed by treatment with IL-10 (47.0 +/- 7% tone). Endotoxin alone caused arteriolar tone to fall to 31.4 +/- 3% (P < 0.05). However, endotoxin applied to arterioles pretreated with IL-10 was without effect (47.5 +/- 2%). CONCLUSIONS: Resistance arterioles pretreated with IL-10 maintain vascular tone during endotoxin exposure. We conclude that IL-10 pretreatment prevents loss of vascular tone of isolated arterioles exposed to endotoxin.     

Constriction velocities of renal afferent and efferent arterioles of mice are not related to SMB expression

BACKGROUND: Constriction of renal arterioles contributes significantly to the control of perfusion and glomerular filtration. Afferent but not efferent arterioles express smooth muscle myosin heavy chain B (SMB) (with a 5'-insert of seven amino acids). The aim of the present study was to investigate (1) the constriction characteristics of afferent and efferent arterioles under physiologic load and (2) whether expression of SMB may causally contribute to these constriction characteristics. METHODS: We compared constriction parameters [constriction amplitude, maximal rate of constriction velocity ("dc/dt(max)"), and time to half-maximal constriction (t(1/2)) of in vitro perfused renal afferent and efferent arterioles of wild-type (smb(+/+)] and homozygous SMB knockout [smb(-/-)] mice upon stimulation with angiotensin II (Ang II) (10(-8) mol/L) and potassium chloride (KCl) (100 mmol/L). SMB expression was investigated by double-labeling immunofluorescence. RESULTS: Contraction amplitude and dc/dt(max) of mouse afferent arterioles upon Ang II stimulation were significantly greater compared to efferent arterioles. However, constriction amplitudes, dc/dt(max), and t(1/2) of afferent as well as efferent arterioles upon Ang II stimulation were similar in smb(+/+) and smb(-/-) mice. Constriction amplitudes upon KCl stimulation of afferent arterioles were similar in both smb(+/+) and smb(-/-) mice. Furthermore, KCl-induced dc/dt(max) and t(1/2) of afferent arterioles were similar in both smb(+/+) and smb(-/-) mice. SMB expression could be detected in afferent but not efferent arterioles in smb(+/+) mice. No SMB expression in either arteriole could be observed in smb(-/-) mice. CONCLUSION: Our results suggest that the presence of different alternatively 5'-spliced smooth muscle-myosin heavy chain (SM-MHC) isoforms does not dominate the different contractile features of physiologically loaded renal afferent or efferent arterioles.     

T-型钙通道在利多卡因致神经母细胞瘤SH-SY5Y细胞损伤中的作用

目的 探讨T-型钙通道在利多卡因致神经母细胞瘤SH-SY5Y细胞损伤中的作用.方法 SH-SY5Y细胞离体培养后,采用随机数字表法,将SH-SY5Y细胞随机分为4组,每组66孔,正常对照组(C组):SH-SY5Y细胞继续培养24 h;米贝地尔+SH-SY5Y细胞组(M组):培养液中加入T-型钙通道阻断剂米贝地尔,终浓度5 μmol/L,孵育24 h;利多卡因+SH-SY5Y细胞组(L组):培养液中加入利多卡因,终浓度10 mmol/L,孵育24 h;米贝地尔+利多卡因+SH-SYSY细胞组(ML组):培养液中加入米贝地尔和利多卡因,终浓度分别为5 μmol/L、10 mmol/L,孵育24 h.于开始培养或孵育时(T0)、培养或孵育1、6、12和24 h时测定SH-SY5Y细胞活力和凋亡率,孵育24 h时观察SH-SY5Y细胞形态学.结果 与C组比较,L组和ML组SH-SY5Y细胞活力降低,凋亡率升高(P＜0.05),M组差异无统计学意义(P＞0.05);与M组比较,L组和ML组SH-SY5Y细胞活力降低,凋亡率升高(P＜0.05);与L组比较,ML组SH-SYSY细胞活力升高,凋亡率降低(P＜0.05).L组SH-SY5Y细胞形态学发生改变,ML组SH-SY5Y细胞形态学改变较L组减轻.结论 T-型钙通道参与了利多卡因导致的神经细胞损伤.

Abstract：

Objective To investigate the role of T-type calcium channel in lidocaine-induced neuronal cytotoxicity . Methods SH-SYSY cell line was a gift from cell biology laboratory of our medical university. The cells were cultured in DMEM liquid culture medium at 37℃ in incubator filled with 5% CO2 , and randomly divided into 4 groups ( n = 66 each) : control group (group C)and M, L and ML groups were exposed to 5 μmol/L mibefradil (a T-type calcium channel blocker), 10 mmol/L lidocaine and 5 μmoL/L mibefradil + 10 mmol/L lidocaine for 24 h. Cell morphology was examined by electronic microscopy at 24 h of drug exposure. Cell viability (by MTT) and neuronal apoptosis (by flow cytometry) were detected immediately before and at 1, 6, 12 and 24 h of exposure to mibefradil or/and lidocaine.Results In C and M groups, the cells demonstrated dendritic protrusions, enlarged nerve processes and dense lattice. After being exposed to lidocaine for 24 h, the dendritic protrusions disappeared,the cells decreased in size, shrinked and became round; the cell viability was significantly decreased while the neuronal apoptosis increased. The lidocaine-induced changes were significantly attenuated by co-incubation with mibefradil. ConclusionT-type calcium channel is involved in lidocaine-induced neuronal cytotoxicity.