Dihydropyridines,phenylalkylamines and benzothiazepines block N-, P/Q- and R-type calcium currents

We compared the effects of representative members of three major classes of cardiac L-type channel antagonists, i.e. dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines (BTZs) on high-voltage-activated (HVA) Ca²⁺ channel currents recorded from a holding potential of –100 mV in rat ventricular cells, mouse sensory neurons and rat motoneurons. Nimodipine (DHP), verapamil (PAA) and diltiazem (BTZ) block the cardiac L-type Ca²⁺ channel current (EC₅₀: 1 M, 4 M and 40 M, respectively). At these concentrations, the drugs could also inhibit HVA Ca²⁺ channel currents in both sensory and motor neurons. Large blocking effects (> 50%) could be observed at 2–10 times these concentrations. The -conotoxin-GVIA-sensitive (-CTx-GVIA, N-type), -agatoxin-IVA-sensitive (-Aga-IVA, P- and Q-types) and non-L-type -CTx-GVIA-, -Aga-IVA-insensitive (R-types) currents accounted for more than 90% of the global current. Furthermore, our data showed that CTx-GVIA and -Aga-IVA spare L-type currents and have only additive blocking effects on neuronal HVA currents. We conclude that DHPs, PAAs and BTZs have substantial inhibitory effects on neuronal non-L-type Ca²⁺ channels. Inhibitions occur at concentrations that are not maximally active on cardiac L-type Ca²⁺ channels.     

Short-term potentiation of mEPSCs requires N-, P/Q- and L-type Ca2+ channels and mitochondria in the supraoptic nucleus

The glutamatergic synapses of the supraoptic nucleus display a unique activity-dependent plasticity characterized by a barrage of tetrodotoxin-resistant miniature EPSCs (mEPSCs) persisting for 5–20 min, causing postsynaptic excitation. We investigated how this short-term synaptic potentiation (STP) induced by a brief high-frequency stimulation (HFS) of afferents was initiated and maintained without lingering presynaptic firing, using in vitro patch-clamp recording on rat brain slices. We found that following the immediate rise in mEPSC frequency, STP decayed with two-exponential functions indicative of two discrete phases. STP depends entirely on extracellular Ca²⁺ which enters the presynaptic terminals through voltage-gated Ca²⁺ channels but also, to a much lesser degree, through a pathway independent of these channels or reverse mode of the plasma membrane Na⁺–Ca²⁺ exchanger. Initiation of STP is largely mediated by any of the N-, P/Q- or L-type channels, and only a simultaneous application of specific blockers for all these channels attenuates STP. Furthermore, the second phase of STP is curtailed by the inhibition of mitochondrial Ca²⁺ uptake or mitochondrial Na⁺–Ca²⁺ exchanger. mEPSCs amplitude is also potentiated by HFS which requires extracellular Ca²⁺. In conclusion, induction of mEPSC-STP is redundantly mediated by presynaptic N-, P/Q- and L-type Ca²⁺ channels while the second phase depends on mitochondrial Ca²⁺ sequestration and release. Since glutamate influences unique firing patterns that optimize hormone release by supraoptic magnocellular neurons, a prolonged barrage of spontaneous excitatory transmission may aid in the induction of respective firing activities.     

Acute and chronic alterations in calcium homeostasis in 3-nitropropionic acid-treated human NT2-N neurons

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, induced ATP depletion and both necrosis and apoptosis in human NT2-N neurons. Necrosis occurred predominantly within the first two days, and increased in a dose-dependent fashion with the concentration of 3-NP, whereas apoptosis was observed after 24 h or later at a similar rate in 0.1 mM and 5 mM 3-NP. We focused our efforts on intracellular calcium homeostasis during the first 48 h in 1 mM 3-NP, a period during which 10% of the neurons died by necrosis and 3% by apoptosis. All NT2-N neurons showed a stereotyped [Ca(2+)](i) rise, from 48+/-2 to 140+/-12 nM (mean +/-S.E.M.), during the first 2 h in 3-NP. Despite severe ATP depletion, however, [Ca(2+)](i) remained above 100 nM in only 17% and 25% of the NT2-N neurons after 24 and 48 h in 3-NP, respectively, indicating that most neurons were able to recover from acute [Ca(2+)](i) rise, and suggesting that chronic [Ca(2+)](i) dysregulation is a better indicator of subsequent necrosis. Blockade of N-methyl-D-aspartate-glutamate receptor by MK-801 substantially ameliorated 3-NP-induced ATP depletion, subsequent chronic [Ca(2+)](i) elevation, and survival. Moreover, xestospongin C, an inhibitor of endoplasmic reticulum Ca(2+) release, enhanced the capacity of NT2-N neurons to maintain [Ca(2+)](i) homeostasis and resist necrosis while subjected to sustained energy deprivation. As far as we know, this report is the first to employ human neurons to study the pathophysiology of 3-NP neurotoxicity.     

Voltage-sensitive calcium channels in a human small-cell lung cancer cell line

Pancrazio , J.J., Oie , H.K. & Kim , Y.I. 1992. Voltage-sensitive calcium channels in a human small-cell lung cancer cell line. Acta Physiol Scand 144 , 463468. Received 1 October 1 991 , accepted 24 October 1991. ISSN 0001–6772. Departments of Biomedical Engineering and Neurology, University of Virginia Health Sciences Center, and the National Cancer Institute, Navy Medical Oncology Branch, Naval Hospital Bethesda, USA. Utilizing the whole-cell patch-clamp method we assessed the Ca²⁺ current (I_ca) in well-established cell lines from human small-cell carcinoma (SCC) of the lung, NCI-H209 and NCI-H187. The Ca²⁺ current was readily observed in H209 tumour cells (90% of the cells tested), whereas H187 tumour cells only occasionally expressed Ca^z+ channels (26% of the cells tested). H209 Ca²⁺ current was evoked by potentials greater than -30 mV and exhibited partial inactivation over the duration of a 40 ms command potential. This inward current was unchanged by alteration of the holding potential from - 80 to - 40 mV and the activation phase of the Ca²⁺ current was best fitted by Hodgkin-Huxley m(t)² kinetics. H209 Ca²⁺ current was reduced over 80% by verapamil (100 μM), whereas w-conotoxin (5 μM) appeared to be without effect. In contrast, H209 Ca²⁺ current was rapidly abolished by nifedipine (10 μm), strongly suggesting the presence of L-type Ca²⁺ channels. Voltage-gated Ca²⁺ channels may be important to the secretion of ectopic hormones and the etiology and pathogenesis of Lambert-Eaton syndrome, an autoimmune disorder of the motor nerve terminal in which autoantibodies directed against voltage-gated Ca²⁺ channels are produced.     

Effects of ablation of N- and R-type Ca(2+) channels on pain transmission

Recently several mutant mouse lines lacking neuronal voltage-dependent Ca(2+) channels (VDCCs) have been established by the use of gene targeting in embryonic stem cells. Pain-related behaviors in Ca(v)2.2 (alpha(1B)) and Ca(v)2.3 (alpha(1E)) knockout mice were studied to gain further insight into the mechanism of pain transmission, where VDCCs are thought to play important roles. We review here the data from these recent studies. Ca(v)2.3-/- mice showed normal responses to acute painful stimuli, and reduced responses to the somatic inflammatory pain stimuli. Ca(v)2.3+/- mice exhibited reduced symptoms of visceral inflammatory pain. Ca(v)2.3-/- mice showed abnormal behavior related to the descending antinociceptive mechanism activated by the intraperitoneal injection of acetic acid. Ca(v)2.2-/- mice showed variable acute nociceptive responses depending on the mutant lines. However, all the lines of Ca(v)2.2-/- mice exhibited reduced responses in the phase 2 of the formalin test, suggesting a suppression of inflammatory pain. Furthermore Ca(v)2.2-/- mice showed markedly reduced neuropathic pain symptoms after spinal nerve ligation. Impaired antinociception, similar to that seen in the Ca(v)2.3-/- mice, was also observed in the Ca(v)2.2-/- mice. Therefore, it is suggested that these mutant mice could provide novel models to delineate the nociceptive and antinociceptive mechanisms.     

Voltage-sensitive calcium channels in a human small-cell lung cancer cell line.

Utilizing the whole-cell patch-clamp method we assessed the Ca2+ current (ICa) in well-established cell lines from human small-cell carcinoma (SCC) of the lung, NCI-H209 and NCI-H187. The Ca2+ current was readily observed in H209 tumour cells (90% of the cells tested), whereas H187 tumour cells only occasionally expressed Ca2+ channels (26% of the cells tested). H209 Ca2+ current was evoked by potentials greater than -30 mV and exhibited partial inactivation over the duration of a 40 ms command potential. This inward current was unchanged by alteration of the holding potential from -80 to -40 mV and the activation phase of the Ca2+ current was best fitted by Hodgkin-Huxley m(t)2 kinetics. H209 Ca2+ current was reduced over 80% by verapamil (100 microM), whereas w-conotoxin (5 microM) appeared to be without effect. In contrast, H209 Ca2+ current was rapidly abolished by nifedipine (10 microM), strongly suggesting the presence of L-type Ca2+ channels. Voltage-gated Ca2+ channels may be important to the secretion of ectopic hormones and the etiology and pathogenesis of Lambert-Eaton syndrome, an autoimmune disorder of the motor nerve terminal in which autoantibodies directed against voltage-gated Ca2+ channels are produced.     

N- and P/Q-type Ca2+ channels in adrenal chromaffin cells

Ca2+ is the most ubiquitous second messenger found in all cells. Alterations in [Ca2+]i contribute to a wide variety of cellular responses including neurotransmitter release, muscle contraction, synaptogenesis and gene expression. Voltage-dependent Ca2+ channels, found in all excitable cells (Hille 1992), mediate the entry of Ca2+ into cells following depolarization. Ca2+ channels are composed of a large pore-forming subunit, called the alpha1 subunit, and several accessory subunits. Ten different alpha1 subunit genes have been identified and classified into three families, Ca(v1-3) (Dunlap et al. 1995, Catterall 2000). Each alpha1 gene produces a unique Ca2+ channel. Although chromaffin cells express several different types of Ca2+ channels, this review will focus on the Cav(2.1) and Cav(2.2) channels, also known as P/Q- and N-type respectively (Nowycky et al. 1985, Llinas et al. 1989b, Wheeler et al. 1994). These channels exhibit physiological and pharmacological properties similar to their neuronal counterparts. N-, P/Q and to a lesser extent R-type Ca2+ channels are known to regulate neurotransmitter release (Hirning et al. 1988, Horne & Kemp 1991, Uchitel et al. 1992, Luebke et al. 1993, Takahashi & Momiyama 1993, Turner et al. 1993, Regehr & Mintz 1994, Wheeler et al. 1994, Wu & Saggau 1994, Waterman 1996, Wright & Angus 1996, Reid et al. 1997). N- and P/Q-type Ca2+ channels are abundant in nerve terminals where they colocalize with synaptic vesicles. Similarly, these channels play a role in neurotransmitter release in chromaffin cells (Garcia et al. 2006). N- and P/Q-type channels are subject to many forms of regulation (Ikeda & Dunlap 1999). This review pays particular attention to the regulation of N- and P/Q-type channels by heterotrimeric G-proteins, interaction with SNARE proteins, and channel inactivation in the context of stimulus-secretion coupling in adrenal chromaffin cells.     

Differential facilitation of N- and P/Q-type calcium channels during trains of action potential-like waveforms

Inhibition of presynaptic voltage-gated calcium channels by direct G-protein βγ subunit binding is a widespread mechanism that regulates neurotransmitter release. Voltage-dependent relief of this inhibition (facilitation), most likely to be due to dissociation of the G-protein from the channel, may occur during bursts of action potentials. In this paper we compare the facilitation of N- and P/Q-type Ca²⁺ channels during short trains of action potential-like waveforms (APWs) using both native channels in adrenal chromaffin cells and heterologously expressed channels in tsA201 cells. While both N- and P/Q-type Ca²⁺ channels exhibit facilitation that is dependent on the frequency of the APW train, there are important quantitative differences. Approximately 20 % of the voltage-dependent inhibition of N-type I _Ca was reversed during a train while greater than 40 % of the inhibition of P/Q-type I _Ca was relieved. Changing the duration or amplitude of the APW dramatically affected the facilitation of N-type channels but had little effect on the facilitation of P/Q-type channels. Since the ratio of N-type to P/Q-type Ca²⁺ channels varies widely between synapses, differential facilitation may contribute to the fine tuning of synaptic transmission, thereby increasing the computational repertoire of neurons.     

口虾蛄提取物对人鼻咽癌细胞P53、COX-2和VEGF表达的影响

目的: 研究口虾蛄提取物(EOS)对人低分化鼻咽癌细胞系(CNE-2Z)中P53、环氧化酶2(COX-2)和血管内皮生长因子(VEGF)表达的影响及意义。方法: 不同浓度EOS(0 mg/L、125 mg/L、250 mg/L和500 mg/L)作用CNE-2Z细胞24 h后,Western blotting检测P53蛋白表达,免疫细胞化学法及RT-PCR法分别检测COX-2 、VEGF蛋白和mRNA的表达。结果: CNE-2Z细胞中P53蛋白、COX-2和VEGF蛋白和mRNA表达均随EOS作用浓度增高而逐渐降低,呈量效关系(P<0.01);且COX-2 和VEGF表达量呈正相关(P<0.05),COX-2 和P53表达量亦呈正相关(P<0.05)。结论: EOS可能通过抑制CNE-2Z细胞中P53蛋白的表达,干扰COX-2和VEGF的表达而发挥其抗肿瘤作用的。     

Expression of complement regulators and receptors on human NT2-N neurons--effect of hypoxia and reoxygenation

Complement activation can cause tissue damage in cerebral stroke by the release of biologically potent activation products and impaired function of regulatory proteins. We investigated the constitutive and hypoxia-reoxygenation-dependent expression of complement receptor 1 (CD35), membrane cofactor protein (CD46), decay-accelerating factor (CD55), protectin (CD59), and complement C3a and C5a receptors (C3aR and C5aR) on human NT2-N neurons. The effect of hypoxia-reoxygenation on C3d-deposition on neurons and endothelial cells was also investigated. NT2-N neurons were examined by cellular enzyme-linked immunosorbent assay and immunofluorescence microscopy. Endothelial cells were examined by flow cytometry. Three hours 1% or 0.1% hypoxia and 21h reoxygenation with 50% AB-serum were used to investigate the effect of hypoxia-reoxygenation on regulators and C3d-deposition. NT2-N neurons expressed significant amounts of CD59 (Clone H19/Clone BRIC229: p=0.000006/p=0.000003), CD46 (p=0.00006), CD55 (p=0.003) and C3aR (p=0.00003). CD35 and C5aR were not significantly expressed. There were no effects of hypoxia-reoxygenation on any of the regulators or receptors after 1% hypoxia and reoxygenation. However, CD55 (p=0.02) was down-regulated after 0.1% hypoxia and subsequent reoxygenation with AB-serum. There were no difference observed in the C3d-deposition during hypoxia-reoxygenation in either neurons or endothelial cells. In conclusion, human NT2-N neurons constitutively express C3aR, CD46, CD55 and, in particular, CD59. The cells may respond to locally produced C3a and, at the same time, be well protected against complement attack. Although severe hypoxia-reoxygenation may down-regulate CD55 expression, it does not seem to influence C3d-deposition.     

病毒介导的P53低表达HepG2细胞株的建立

目的构建干扰P53的逆转录病毒载体,建立稳定低表达P53的HepG2细胞株。方法合成干扰P53基因的寡核苷酸序列插入pMSCV-hyg-U6质粒,转化受体菌DH5α,经酶切测序鉴定后瞬时转染HepG2细胞,通过半定量RT-PCR方法检测P53 mRNA表达水平评估干扰效果;选择干扰效果最好的重组质粒转染PT67细胞进行病毒包装,获得逆转录病毒颗粒感染HepG2细胞,通过Hygromycin筛选获得多个细胞克隆,Western blot检测P53蛋白的表达情况;选择P53表达水平最低的细胞克隆。通过5-氟尿嘧啶(5-FU)诱导凋亡实验,检测P53低表达的HepG2细胞p53通路介导的细胞凋亡情况。结果经酶切和测序验证成功构建重组病毒载体;瞬时转染HepG2细胞后P53 mRNA表达下调;病毒颗粒感染HepG2细胞后P53的mRNA、蛋白表达下调;用5-FU处理后P53低表达HepG2细胞凋亡水平明显低于对照。结论成功构建了干扰P53的逆转录病毒载体,建立了P53低表达HepG2细胞株,明显阻断了P53通路依赖的细胞凋亡。     

Na/P(i) cotransporter ( Npt2) gene disruption increases duodenal calcium absorption and expression of epithelial calcium channels 1 and 2

Mice homozygous for the disrupted type-II Na/P(i) cotransporter gene ( Npt2(-/-)) exhibit hypophosphataemia, increased serum concentration of 1,25-dihydroxyvitamin D (1,25-(OH)(2)D) and calcium (Ca) and elevated urinary Ca excretion. To determine whether the hypercalcaemia and hypercalciuria are secondary to 1,25-(OH)(2)D-stimulated intestinal Ca absorption, we examined the effect of Npt2 gene disruption on serum Ca and urinary Ca excretion after an overnight fast, and on duodenal Ca absorption. We also compared the duodenal expression of the epithelial Ca channels, ECaC1 and ECaC2, and calbindinD(9K) mRNAs, relative to that of beta-actin mRNA, in Npt2(+/+) and Npt2(-/-) mice. Both serum Ca and urine Ca/creatinine were significantly decreased in Npt2(-/-) mice after an overnight fast and were no longer different from that in wild-type mice. Absorption of (45)Ca from isolated duodenal segments in vivo and (45)Ca appearing in the plasma were significantly increased in Npt2(-/-) compared with Npt2(+/+) mice. In addition, the duodenal abundance of ECaC1, ECaC2 and calbindinD(9K) mRNAs was significantly elevated in mutant mice relative to that in wild-type mice. In contrast, both duodenal Ca absorption and ECaC1 and ECaC2 mRNA abundance were lower in mice with X-linked hypophosphataemia ( Hyp) than in normal littermates. In summary, we provide evidence for increased duodenal Ca absorption in Npt2(-/-) mice and suggest a role for ECaC1, ECaC2 and calbindinD(9K) in mediating this response.     

Expression of recombinant calcium channels support secretion in a mouse pheochromocytoma cell line

We have characterized a recently established mouse pheochromocytoma cell line (MPC 9/3L) as a useful model for studying neurotransmitter release and neuroendocrine secretion. MPC 9/3L cells express many of the proteins involved in Ca2+-dependent neurotransmitter release but do not express functional endogenous Ca2+-influx pathways. When transfected with recombinant N-type Ca2+ channel subunits alpha1B,beta2a,alpha2delta (Cav2.2), the cells expressed robust Ca2+ currents that were blocked by omega-conotoxin GVIA. Activation of N-type Ca2+ currents caused rapid increases in membrane capacitance of the MPC 9/3L cells, indicating that the Ca2+ influx was linked to exocytosis of vesicles similar to that reported in chromaffin or PC12 cells. Synaptic protein interaction (synprint) sites, like those found on N-type Ca2+ channels, are thought to link voltage-dependent Ca2+ channels to SNARE proteins involved in synaptic transmission. Interestingly, MPC 9/3L cells transfected with either LC-type (alpha1C, beta2a, alpha2delta, Cav1.2) or T-type (alpha1G, beta2a, alpha2delta, Cav3.1) Ca2+ channel subunits, which do not express synprint sites, expressed appropriate Ca2+ currents that supported rapid exocytosis. Thus MPC 9/3L cells provide a unique model for the study of exocytosis in cells expressing specific Ca2+ channels of defined subunit composition without complicating contributions from endogenous channels. This model may help to distinguish the roles that different Ca2+ channels play in Ca2+-dependent secretion.