Inhibition of rabbit platelet phosphodiesterase activity and aggregation by calcium channel blockers

Rabbit platelet cyclic AMP phosphodiesterase is inhibited by the three calcium channel blockers nifedipine, diltiazem, and verapamil with IC50's of 100 microM, 100 microM and 420 microM, respectively. Also, platelet aggregation induced by 4 microM ADP is inhibited by those compounds. Verapamil is the most potent aggregation inhibitor with an IC50 of 260 microM while diltiazem and nifedipine have IC50's of 630 microM and 840 microM, respectively. All three compounds display a maximum inhibition of 80-85%. Diltiazem and PGD2 potentiate the antiaggregatory activity of each other in that the inhibitions occurring in the presence of the combination of the two (at varying concentrations) exceed the calculated sums of the inhibitions produced by each alone. On the other hand, the antiaggregatory activities of verapamil or nifedipine, are additive with that of PGD2 in that no significant differences exist between the observed inhibitions produced by the combinations and the calculated summed values of the individual inhibitions. Our data suggest, therefore, that in addition to lowering intracellular calcium ions, which are required for platelet aggregation, the three calcium channel blockers inhibit the breakdown of cyclic AMP thereby promoting antiaggregation.     

Inhibitory effect of calcium channel blockers on proliferation of human glioma cells in vitro

The effects of 2 specific calcium channel blockers, verapamil and nimodipine, on the proliferation of human glioma tumour cells were investigated in vitro. Tumour tissues for primary cell cultures were obtained bioptically from 3 patients with the histopathological diagnosis of glioblastoma. The [3H]-thymidine incorporation into glioma tumour cells DNA was used as a sensitive index of the cell proliferation. It was found that verapamil (10(-4)-10(-5) M) and nimodipine (10(-4)-10(-6) M) significantly inhibited the [3H]-thymidine uptake in a dose-related manner. The inhibitory effect of both calcium channel antagonists was reversed by simultaneous addition of calcium chloride (5 x 10(-3) M). These results indicate that verapamil and nimodipine may exert an antiproliferative effect on glioma cells growth acting through a blockade of specific voltage-dependent calcium channels.     

Use of calcium channel blockers after stroke is not associated with poor outcome

Background Control of hypertension is essential for the secondary prevention of stroke. Although several trials have assessed the role of calcium channel blockers (CCBs) in the acute stroke, few address their safety and efficacy in secondary prevention. The recovery process after stroke requires neurite outgrowth, which may be dependent on activation of calcium channels and NMDA receptors. We asked whether treatment of hypertension using CCBs is safe during the recovery of patients following stroke and whether it contributes to their functional outcome. Methods The Registry of the Canadian Stroke Network provided access to information from 1545 patients with ischemic stroke. Primary outcome variables were mortality and functional outcome, which was assessed using the Stroke Impact Scale-16. Results Patients discharged on CCB had a 2.5% 6-month mortality rate compared with 5.5% in those who were not on CCB at discharge (OR 0.38, 95% CI 0.17−0.88). There was no change in 6-month mortality with respect to treatment with ACE-I, B-blockers or diuretics at discharge. Patients that were admitted on CCB had improved SIS-16 at 6-months if they were also discharged on CCB, as compared with patients who had their CCB discontinued (73.8 veas 66.8, p = 0.032). Conclusions CCB treatment at the time of discharge did not impede functional recovery, and was associated with reduced mortality and improved SIS-16 at 6 months. for the Investigators of the Registry of the Canadian Stroke Network. Received in revised form: 24 January 2006     

A meta-analysis of treating acute traumatic brain injury with calcium channel blockers

The purpose of this systematic review was to evaluate and meta-analyse the current evidence for the use of calcium channel blockers (CCBs) in the treatment of acute traumatic brain injury (TBI) and traumatic subarachnoid haemorrhage (tSAH). A systematic search of clinical trials.gov, Cochrane library databases, EMBASE, MEDLINE, Web of science search and WHO trial registry, plus hand-searching of grey literature, was undertaken in March 2013. Two reviewers independently extracted the data using a pre-defined data extraction form. RevMan 5 software was used to synthesise data and calculate the risk ratio (RR) based on event rates as well as the 95% confidence interval (CI). Finally, nine RCTs with a total of 2182 patients were included. Meta-analysis showed that there was no difference between CCBs and control groups for rates of mortality (n = 1337, 5 RCTs, RR 0.93 CI 0.77–1.12). In a subgroup tSAH analysis, the difference was not significant (n = 389, 2 RCTs, RR 0.73 CI 0.53–1.02). There were slightly fewer unfavourable outcomes in the treatment group, but the difference was not statistically significant (n = 2101, 8 RCTs, RR 0.90 CI 0.76–1.08). In the subgroup tSAH analysis, again, the difference did not reach statistical significance (n = 1074, 5 RCTs, RR 0.95 CI 0.73–1.24). It seems that larger, well-designed RCTs are necessary in order to ascertain any clinical benefit CCBs may or may not have for the treatment of acute TBI.     

AMPA receptor activation induces association of G-beta protein with the alpha subunit of the sodium channel in neurons

Glutamatergic transmission is mediated by ionotropic receptors that directly gate cationic channels and metabotropic receptors that are coupled to second messenger generating systems and to ionic channels via heterotrimeric guanine-nucleotide binding- (G) proteins. This distinction cannot be made for the ionotropic receptor subclass activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), which has been shown to be physically associated with the alpha-subunit of Gi1 protein and activates this G-protein. Here, we report that, in addition to a Ca2+ influx, AMPA induces the mobilization of Ca2+ from the mitochondrial pool by reversing the mitochondrial Na+/Ca2+ exchanger in mouse neurons in primary culture. Both processes required the activation of tetrodotoxin-sensitive Na+ channels. AMPA receptor activation modified the gating properties of the Na+ channel, independently of the AMPA current, suggesting a G-protein-mediated process. Indeed, co-immunoprecipitation experiments indicated that AMPA receptor activation induced the association of Gbeta with the alpha-subunit of the Na+ channel. These results suggest that, in addition to its ionic channel function, the AMPA receptor is coupled to Na+ channels through G-proteins and that this novel metabotropic function is involved in the control of neuronal excitability.     

Variable effect of calcium channel blockers on the decremental response in experimental autoimmune myasthenia gravis

We tested the effect of intravenous administration of verapamil and nimodipine on the decremental response in rabbits with experimental autoimmune myasthenia. Nimodipine produced an immediate augmentation of the decremental response to 3-Hz nerve stimulation, which lasted about 30 min. In contrast, verapamil caused marked amelioration of the decrement beginning 30 min after injection. Our findings are consistent with previous reports suggesting that verapamil has a presynaptic effect of enhanced acetylcholine release at the neuromuscular junction. Since evaluation of a drug effect in vivo in animals with experimental autoimmune myasthenia gravis may be more pertinent to its effect on patients with myasthenia gravis (MG), verapamil might prove to be safer in MG than nimodipine. However, due to the additional effects of calcium channel blockers, the safety of their use in myasthenia gravis cannot be inferred from the experimental results. © 1994 John & Sons, Inc.     

Muscle sodium channel inactivation defect in paramyotonia congenita with the thr1313met mutation

Mutations of the skeletal muscle sodium (Na) channel have been reported in families with paramyotonia congenita (PC), an autosomal dominant disorder with cold and/or exercise induced stiffness and myotonia. Functional consequences of specific Na channel mutations responsible for PC have not been described. Patch clamp recording of single Na channels were made in cultured myotubes at 22 and 34°C from a PC patient with the thr1313met mutation. Cell-attached and outside-out recordings of mutant PC channels contained long duration and late openings. The mean open time was increased and the ensemble average showed a prolonged inward Na current. This membrane depolarization could cause repetitive action potentials and the clinical syndrome.     

Expression of sodium channel subtypes during development in rat skeletal muscle.

This study contrasts the developmental patterns of expression of 2 subtypes of the voltage-dependent sodium channel in rat muscle that are differentiated by their immunoreactivity with monoclonal antibodies raised to the purified muscle sodium channel protein. One subtype is found in the transverse tubular (T) system of slow twitch fibers as well as the plasma membrane of fast and slow twitch fibers in the anterior tibial and soleus muscles. The second is present in the plasma membrane in all fibers of both muscles. The transverse tubular subtype exhibits 2 immunocytochemical staining patterns within muscle fibers, reticular and homogeneous, which may represent labeling of the developing T tubular system and of a cytoplasmic pool of alpha subunits of the sodium channel respectively. The reticular pattern eventually disappears in fast twitch fibers but persists into the adult stage in slow twitch fibers. The homogeneous pattern is also seen with antibodies to the plasma membrane subtype and disappears in early development as immunoreactivity to both subtypes gradually appears in the surface membrane. A reticular pattern is never seen with the plasma membrane subtype. The factors that modulate the expression of these subtypes is unknown.     

Calcium-induced damage of skeletal muscle fibers is markedly reduced by calcium channel blockers

Summary Vascular perfusion of rat hind limbs with a Ca²⁺-free physiological solution containing ethylenediaminetetraacetate, when followed by a physiological solution with normal concentration of Ca²⁺, caused a marked rise of creatine kinase (CK) in the venous effluent. When calcium channel blockers were present in the perfusing solutions, no rise of CK occurred. On histological sampling of perfused muscles, CK rise was roughly correlated with muscle fiber damage of the appropriate muscles. Removal of calcium from the plasmalemma of muscle fibers appears to prevent closure of calcium channels, making the muscle fibers susceptible to a deleterious influx of extracellular calcium. This influx can be prevented by the presence of calcium channel blockers in the perfusates.Supported by the Medical Research Council of Canada, the Muscular Dystrophy Association of Canada and the Gildemeister Foundation     

Predicting the impact of sodium channel mutations in human brain disease

Genetic alteration of the sodium channel provides a remarkable opportunity to understand how epilepsy and its comorbidities arise from a molecular disease of excitable membranes, and a chance to create a better future for children with epileptic encephalopathy. In a single cell, the channel reliably acts as a voltage‐sensitive switch, enabling axon impulse firing, whereas at a network level, it becomes a variable rheostat for regulating dynamic patterns of neuronal oscillations, including those underlying cognitive development, seizures, and even premature lethality. Despite steady progress linking genetic variation of the channels with distinctive clinical syndromes, our understanding of the intervening biologic complexity underlying each of them is only just beginning. More research on the functional contribution of individual channel subunits to specific brain networks and cellular plasticity in the developing brain is needed before we can reliably advance from precision diagnosis to precision treatment of inherited sodium channel disorders.     

Effect of proteolysis on the activity of the sodium channel in isolated lobster nerve membrane

The effect of enzymatic proteolysis on structural and functional properties of the isolated lobster nerve membrane was investigated. The membranes were treated with different amounts of either trypsin or unspecific protease. Sodium channel activity was determined by measuring the veratridine-tetrodotoxin-sensitive sodium influx in proteoliposomes prepared with nerve membrane and soybean lipids. The changes in the membrane proteins were followed by electrophoresis in polyacrylamide gradient gels. From the densitometric scan of the gels the relative area for each protein was obtained, and the ratio of enzyme-treated to control areas was evaluated. Under a similar degree of proteolysis catalyzed either by trypsin or by unspecific protease, the sensitive sodium influx is not affected by trypsin, whereas it is about 60% diminished by the unspecific protease. In this condition the zones corresponding to molecular weights of 240,000 and 166,000 daltons appear modified in the electrophoretic gels by both enzymes. The 117,000-dalton range is modified only by the unspecific protease. Increasing trypsin concentration diminishes sodium influx about 60%; and the 240,000-, 166,000-, and 117,000-dalton zones appear modified. A further increase of the protease concentration totally abolishes the sensitive sodium influx and modifies practically all of the membrane proteins. The present results indicate the rather high sensitivity of the membrane sodium channel activity to proteolytic action, and show that the membrane sites that respond to veratridine appear to be highly affected by proteolysis. In contrast, the tetrodotoxin receptor retains its binding capacity even after treatment of the membrane with protease concentrations 1,000 times higher than those affecting the sensitive sodium influx [Benzer and Raftery, 1972; Villegas et al, 1973].     

Developmental and cellular expression pattern of epithelial sodium channel alpha, beta and gamma subunits in the inner ear of the rat

Endolymphatic ion composition in the adult inner ear is characterized by high K(+) and low Na(+) concentration. This unique ion composition is essential for proper functioning of sensory processing. Although a lot has been learned in recent years about molecules involved in K(+) transport in inner ear, the molecules involved in Na(+) transport are only beginning to emerge. The epithelial Na(+) channel (ENaC) is a highly selective Na(+) channel that is expressed in many Na(+)-reabsorbing tissues. The aim of our study was to investigate whether ENaC is expressed in inner ear of rats and could account for Na(+) reabsorption from endolymph. We detected mRNA for the three channel-forming subunits (alpha, beta and gamma ENaC) in cochlea, vestibular system and endolymphatic sac. mRNA abundance increased during the first 12 days of life in cochlea and vestibular system, coinciding with decreasing Na(+) concentration in endolymph. Expression was strongest in epithelial cells lining scala media, most notably Claudius' cells. As these cells are characterized by a very negative resting potential they would be ideally suited for reabsorption of Na(+). mRNA abundance in endolymphatic sac decreased during the first 6 days of life, suggesting that ENaC might be implicated in reabsorption of endolymph in the endolymphatic sac of neonatal animals. Together, our results suggest that the epithelial Na+ channel is a good candidate for a molecule involved in Na(+) homeostasis in inner ear.     

电压门控钠通道与相关调制剂相互作用的分子机制

电压门控钠通道（VGSC）在神经元及大多可兴奋性细胞动作电位的形成和传播过程中扮演着极为重要的角色，它也是许多特异性天然动植物神经毒素作用的靶器，这些毒素可调节靶通道的各种功能活性，包括通道的电导，激活和失活化相等，进而影响电信号产生与传导过程，使动物麻痹，甚至死亡，本文从分子水平简介相关神经毒素与VGSC靶受体之间相互识别与作用的分子机制。     

Effect of orphanin FQ and morphine on sodium channel current in somatosensory area of rat cerebral cortex

BACKGROUND: Some experiments have demonstrated that injecting orphanin FQ (OFQ) into lateral ventricle, which can obviously decrease the pain threshold. It is indicated that OFQ is an anti-opiate substance. However, whether OFQ has effects on sensory neuron ion channel in cerebral cortex needs to be further studied. OBJECTIVE: To investigate the effects of OFQ, morphine or their combination on sodium channel current of somatosensory neurons in rat cerebral cortex. DESIGN: Repeated measurement trial. SETTING: Department of Physiology, Harbin Medical University. MATERIALS: Fifty healthy Wistar rats, aged 12–16 days, of either gender, were provided by the Experimental Animal Center, Second Hospital Affiliated to Harbin Medical University. OFQ was purchased from Sigma-Aldrich Company, and morphine was provided by the Shenyang First Pharmaceutical Factory. PC2C patch clamp amplifier and LabmasterTL1were purchased from Yibo Life Science Instrument Co.,Ltd. of Huazhong University of Science and Techgnology. METHODS: This experiment was carried out in the Department of Physiology (provincial laboratory), Harbin Medical University between January 2005 and May 2006. Cortical neurons were acutely isolated from rats, and prepared into cell suspension following culture. ①Sodium channel current of somatosensory neurons in rat cerebral cortex was recorded before and after administration by whole-cell Patch clamp technique after 50 nmol/L OFQ being added to extracellular fluid. ②The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex was recorded before and after administration by the same method after 20 μmol/L morphine being added to extracellular fluid, and then the change of sodium channel current was recorded after 50 nmol/L OFQ being added. MAIN OUTCOME MEASURES: The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex following the administration of OFQ, morphine separately or their combination.. RESULTS: ①The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex was significantly lower after administration of 50 nmol/L OFQ than before at the clampe of the voltage of –30 mV (P < 0.05). ②The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex was significantly lower after administration of 20 μmol/L morphine than before at the clampe of the voltage of –30 mV (P < 0.05). The sodium channel current recovered to –（2 345.24±174.18）pA after 50 nmol/L OFQ was administrated. There were significant differences in the amplitude of Na+ channel current between two interventions (P < 0.05). CONCLUSION: Morphine and OFQ can respectively reduce the amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex, and OFQ can reverse the effect of morphine partly. It is indicated that OFQ can produce antiopioid activity in the central nervous system by influencing sodium channel current.     

Role of the sixth transmembrane segment of domain IV of the cockroach sodium channel in the action of sodium channel blocker insecticides

Sodium channel blocker insecticides (SCBIs), such as indoxacarb and metaflumizone, are a new class of insecticides with a mechanism of action different from those of other insecticides that target sodium channels. SCBIs block sodium channels in a manner similar to local anesthetics (LAs) such as lidocaine. Several residues, particularly F1579 and Y1586, in the sixth transmembrane segment (S6) of domain IV (IV) of rat Na_v1.4 sodium channels are required for the action of LAs and SCBIs and may form part of overlapping receptor sites. However, the binding site for SCBIs in insect sodium channels remains undefined. We used site-directed mutagenesis, the Xenopus laevis oocyte expression system, and the two-electrode voltage clamp technique to study the effects on SCBI activity of mutating F1817 and Y1824 (analogous to those residues identified in mammalian sodium channels) to alanine, in the voltage-sensitive sodium channel of the German cockroach, Blattella germanica. The mutant channels showed no effect or a marked increase in channel sensitivity to both DCJW (the active metabolite of indoxacarb) and metaflumizone. Thus, it appeared that although the F1817 residue plays a role in the action of SCBIs and that both residues are involved in LA activity in mammalian sodium channels, neither F1817 nor Y1824 are integral determinants of SCBI binding on insect sodium channels. Our results suggest that the receptor site of SCBIs on insect sodium channels may be significantly different from that on mammalian sodium channels.     

肌阵挛-站立不能性癫痫的临床特征及其电压门控性钠通道α1亚基基因突变检测

目的 探讨肌阵挛-站立不能性癫痫(MAE)的临床特点、药物疗效及可能的分子遗传学机制.方法 根据2001年国际抗癫痫联盟癫痫综合征分类标准对自2006年至2008年在广州医学院第二附属医院神经内科就诊的MAE患者进行诊断,收集患者的临床资料及外周血DNA,采用高效液相色谱分析和直接测序法对电压门控性钠通道α1亚基(5CN1A)基因突变进行筛查,并对其临床治疗情况随访1年以上.结果 共收集10例MAE患者,其中散发8例,有热性惊厥或癫痫家族史者2例;起病年龄介于5～39个月间;有多种全面性发作形式;2例曾出现癫痫持续状态;起病后精神发育迟滞者7例.对其中8例患者进行SCN1A基因突变筛查,均未发现突变.丙戊酸、氯硝安定和左乙拉西坦疗效最好,部分患者托吡酯和拉莫三嗪治疗亦有效.结论 MAE是少见的癫痫综合征,分子遗传学机制不明,丙戊酸、氯硝安定和左乙拉西坦治疗有效,但预后较差.     

Genomewide association study in cervical dystonia demonstrates possible association with sodium leak channel

Dystonia is a common movement disorder. A number of monogenic causes have been identified. However, the majority of dystonia cases are not explained by single gene defects. Cervical dystonia is one of the commonest forms without genetic causes identified. This pilot study aimed to identify large effect‐size risk loci in cervical dystonia. A genomewide association study (GWAS) was performed. British resident cervical dystonia patients of European descent were genotyped using the Illumina‐610‐Quad. Comparison was made with controls of European descent from the Wellcome Trust Case Control Consortium using logistic regression algorithm from PLINK. SNPs not genotyped by the array were imputed with 1000 Genomes Project data using the MaCH algorithm and minimac. Postimputation analysis was done with the mach2dat algorithm using a logistic regression model. After quality control measures, 212 cases were compared with 5173 controls. No single SNP passed the genomewide significant level of 5 × 10^?8 in the analysis of genotyped SNP in PLINK. Postimputation, there were 5 clusters of SNPs that had P value <5 × 10^?6, and the best cluster of SNPs was found near exon 1 of NALCN, (sodium leak channel) with P = 9.76 × 10^?7. Several potential regions were found in the GWAS and imputation analysis. The lowest P value was found in NALCN. Dysfunction of this ion channel is a plausible cause for dystonia. Further replication in another cohort is needed to confirm this finding. We make this data publicly available to encourage further analyses of this disorder. © 2013 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.