Calcium Channel Binding Research Articles

Exploring the potential therapeutic benefits of 7-methoxy coumarin for neuropathy pain: an in vivo, in vitro, and in silico approach.

7-Methoxycoumarin (7-MC) is well recognized for its anti-inflammatory and anti-nociceptive actions. Its capacity to lessen neuropathic pain hasn't been documented yet. Hence the impact of 7-MC on vincristine-induced peripheral neuropathic pain in rodents was investigated. The investigation also looked at the impact of 7-MC in reducing neuropathic pain via voltage-gated calcium channels and phospholipase enzyme inhibition using pertinent in vitro and in silico methods. Vincristine (0.1mg/kg, i.p., daily) was administered continuously for 7 days to induce peripheral neuropathic pain in mice, with cold allodynia and thermal hyperalgesia and evaluated on the 8th day using the acetone bubble test and hot water tail immersion test. In order to derive the mechanistic approach for ameliorating neuropathic pain, the role of 7-MC in the inhibition of the phospholipase enzyme, gene expression studies on voltage-gated calcium channels using mouse BV2 microglial cells and in silico studies for its calcium channel binding affinity were also performed. The test compounds reduced vincristine-induced cold allodynia and thermal hyperalgesia in mice in a dose-dependent experiments. In vitrostudies on phospholipase inhibition by 7-MC showed an IC50 of 27.08µg/ml and down-regulated the gene expression of calcium channels in the BV2 microglial cell line. In silico docking scores for 7-MCwere higher than the standard drug gabapentin. The compound 7-MC has shown promise in alleviating vincristine-induced peripheral neuropathicin mice. Studies conducted in parallel, both in silico and in vitro have demonstrated that 7-MC effectively reduces neuropathic pain. This pain reduction is achieved through two mechanisms: inhibiting the phospholipase enzyme and blocking voltage-gated calcium channels.

Pitavastatin and Lovastatin Exhibit Calcium Channel Blocking Activity Which Potentiate Vasorelaxant Effects of Amlodipine: A New Futuristic Dimension in Statin's Pleiotropy.

Background and Objectives: We have recently reported that Fluvastatin, Atorvastatin, Simvastatin and Rosuvastatin have calcium channel antagonistic activities using rabbits' intestinal preparations. The current study is focused on the effects of Pitavastatin and Lovastatin for possible inhibition of vascular L-Type calcium channels, which may have vasorelaxant effect(s). Combined effects of Pitavastatin and Lovastatin in the presence of Amlodipine were also tested for vasorelaxation. Materials and Methods: Possible relaxing effects of Pitavastatin and Lovastatin on 80 mM Potassium chloride (KCL)-induced contractions and on 1 µM norepinephrine (N.E)-induced contractions were studied in isolated rabbit's aortic strips preparations. Relaxing effects on 80 mM KCL-induced vascular contractions were further verified by constructing Calcium Concentration Response Curves (CCRCs), in the absence and presence of three different concentrations of Pitavastatin and Lovastatin using CCRCs as negative control. Verapamil was used as a standard drug that has L-Type calcium channel binding activity. In other series of experiments, we studied drug interaction(s) among Pitavastatin, Lovastatin, and amlodipine. Results: The results of this study imply that Lovastatin is more potent than Pitavastatin for having comparatively lower EC50 (7.44 × 10-5 ± 0.16 M) in intact and (4.55 × 10-5 ± 0.10 M) in denuded aortae for KCL-induced contractions. Lovastatin amplitudes in intact and denuded aortae for KCL-induced contractions were, respectively, 24% and 35.5%; whereas amplitudes for Pitavastatin in intact and denuded aortae for KCL-induced contractions were 34% and 40%, respectively. A left shift in the EC50 values for the statins was seen when we added amlodipine in EC50 (Log Ca++ M). Right shift for CCRCs state that Pitavastatin and Lovastatin have calcium channel antagonistic effects. Lovastatin in test concentration (6.74 × 10-7 M) produced a right shift in relatively lower EC50 (-2.5 ± 0.10) Log Ca++ M as compared to Pitavastatin, which further confirms that lovastatin is relatively more potent. The right shift in EC50 resembles the right shift of Verapamil. Additive effect of Pitavastatin and Lovastatin was noted in presence of amlodipine (p < 0.05). Conclusions: KCL (80 mM)-induced vascular contractions were relaxed by Pitavastatin and Lovastatin via inhibitory effects on L-Type voltage-gated calcium channels. Lovastatin and Pitavastatin also relaxed Norepinephrine (1 µM)-induced contractions giving an insight for involvement of dual mode of action of Pitavastatin and Lovastatin.

Disrupted placental vitamin D metabolism and calcium signaling in gestational diabetes and pre-eclampsia patients.

Gestational diabetes (GDM) and pre-eclampsia (PE) represents the unrecognized risk factors for reduced bone content in neonates. The present study is planned to explore the components of vitamin D metabolism and calcium transport in placenta of GDM and PE cases and its effect on the neonatal bone mass determination using bone densitometry system. We have collected serum and placenta tissues from GDM (n = 20), PE (n = 20), and healthy pregnancies (n = 20). In the present study, we found mRNA expression of oxidative stress markers, vitamin D metabolic components and calcium channels, calcium channel binding proteins, plasma membrane calcium ATPase, ATP synthase and Ca2+ release genes; Ryanodine receptors genes were assessed by quantitative real-time PCR (qRT-PCR) in placental tissue of GDM, PE, and healthy pregnancies. We observed high level of oxidative stress in both GDM and PE placenta compared to normal pregnancies. CYP2R1 and VDR mRNA expression was significantly downregulated and upregulation of CYP27B1 and CYP24A1 in GDM and PE compared with healthy cases. Similarly, calcium transporters were downregulated in GDM and PE placental tissues. In addition, CYP24A1, VDR, CaBP28K, TRPV5 and PMCA3 mRNA expression were correlated with BMC of neonates. Oxidative stress is probably relevant to disrupted vitamin D homeostasis and calcium transport in the placenta of GDM and PE cases. The altered regulatory mechanism of CYP24A1 and VDR could indicates more pronounced serum 25(OH)D reduction. Additionally, reduced BMC in the neonates of these cases might be as consequences of modified CYP24A1, VDR, CaBP28K, TRPV5 and PMCA3 mRNA expression.

P/Q-Type Calcium Channel Antibodies and Late Life First Break Psychosis: A Case Report

<h3>Introduction</h3> Paraneoplastic Syndrome, described in the 1940s, a phenomenon of cancer presenting with multiple different manifestations, not directly related to tumor itself, has had an overdue recognition with these conditions still being studied and researched, especially when it comes to Paraneoplastic Syndromes presenting with Neuropsychiatric Disorders (Lorraine C. Pelosof, 2010). In the psychiatric and neurological literature, there is slow yet gradual recognition of new onset psychosis in patients with no prior history of such, which are attributable to autoantibodies of different types. In some psychiatric literature, this presentation is termed as autoimmune psychosis (Thomas A Pollak, 2020). Currently known autoantigens include NMDAR, Gamma-Aminobutyric Acid Receptor- B (GABABR), Gamma-Aminobutyric Acid Receptor- A (GABAAR), components of voltage gated potassium channel (VGKC) complex, dopamine D2 receptor (D2R)(Nese Sinmaz, 2015). Autoimmune N-methyl-D-aspartate (NMDA) receptor encephalitis has been of particular interest to psychiatrists with it being the most studied paraneoplastic syndrome, as 77% of affected patients first present to psychiatry with a broad range of symptoms (Jennifer L.Kruse, 2015). Very few and limited data have been found on the association between voltage gated calcium channel (P/Q type) and psychosis. In this poster, we describe a case of an individual presenting with late onset first episode of psychosis, with positive P/Q channel antibodies. <h3>Methods</h3> Previous studies and reviews over the past 10 years were reviewed using PubMed, Medline and Google Scholar. Search terms were "psychosis", "autoimmune psychosis", "autoantibodies and psychosis" "geriatric psychosis" "P/Q-type Calcium channel" "Paraneoplastic syndrome" "very late onset psychosis". We reviewed available literature with regards to first episode of psychosis in old age and possible association with P/Q-type voltage gated calcium channel. <h3>Results</h3> DiscussionVery late onset psychosis is delineated as onset of psychosis after the age of 60 years (Howard, 2000). Prevalence of late life psychosis is 23%, however 60% of new onset psychosis in older individuals are of secondary etiology (Cohen, 2015). Psychosis in the elderly individuals could be primarily due to Very Late Onset Schizophrenia Like Psychosis (VLOSLP) vs secondary causes (Delirium, Dementia, Drugs/illicit substances/toxin, Medical conditions). In the discussed case, the patient presented with first episode of psychosis at the age of 70. The extensive workup was positive for P/Q-type Calcium channel binding antibody. Per literature review, the positive predictive value for an autoimmune neurological diagnosis (Lambert-Eaton syndrome or diverse encephalomyeloneuropathic phenotypes) among patients with positive values for P/Q -type calcium channel antibodies < 0.10 nM is 19%. The positive predictive value for a cancer diagnosis (diverse types) among patients positive for P/Q-type calcium channel antibody is 21%; approximately 18% are historical neoplasms, and 3% are detected prospectively (Zalewski NL, 2016). A spectrum of psychiatric autoimmunity beyond NMDA-R IgG is still under-recognized (Jennifer L.Kruse, 2015). In a retrospective case control study conducted by Dr. Jennifer L.Kruse in Mayo clinic, out of the 213 adult psychiatric inpatient 3 patients were found to have positive P/Q-type Calcium channel, who manifested with delusional parasitosis, mood disorder with psychotic features, and Major Depressive Disorder with Psychotic features (Jennifer L.Kruse, 2015). Another international case study found genetic association between voltage gated Calcium channel and Schizophrenia (Wen Li1, 2018). While these studies indicate the potential association between voltage gated Calcium channel and psychosis, limited case studies and research could be found on the association, extent and elaboration of the aforementioned. <h3>Conclusions</h3> While there is progressively enriched literature on psychosis due to Paraneoplastic syndrome, specifically Anti NMDA receptor encephalitis, there is not much mention of correlation between P/Q-type calcium channel binding antibodies and psychosis. Cases with rare presentations as above warrant elaborate workup. Along with the abovementioned etiologies, given the increased incidence of psychosis due to autoimmune antibodies, addition of full autoimmune antibody panel should be considered. Shared clinical experience, research and prospective clinical studies will direct developing guidelines for evaluation, management and prognostic indicators of Paraneoplastic Syndromes with Neuropsychiatric symptoms, particularly due to autoimmunity against voltage gated calcium channel (P/Q-type). <h3>This research was funded by</h3> There are no financial conflicts of interest to disclose

S100B, GFAP, UCH-L1 and NSE as predictors of abnormalities on CT imaging following mild traumatic brain injury: a systematic review and meta-analysis of diagnostic test accuracy.

Biomarkers such as calcium channel binding protein S100 subunit beta (S100B), glial fibrillary acidic protein (GFAP), ubiquitin c-terminal hydrolase L1 (UCH-L1) and neuron-specific enolase (NSE) have been proposed to aid in screening patients presenting with mild traumatic brain injury (mTBI). As such, we aimed to characterise their accuracy at various thresholds. MEDLINE, SCOPUS and EMBASE were searched, and articles reporting the diagnostic performance of included biomarkers were eligible for inclusion. Risk of bias was assessed using the QUADAS-II criteria. A meta-analysis was performed to assess the predictive value of biomarkers for imaging abnormalities on CT. A total of 2939 citations were identified, and 38 studies were included. Thirty-two studies reported data for S100B. At its conventional threshold of 0.1μg/L, S100B had a pooled sensitivity of 91% (95%CI 87-94) and a specificity of 30% (95%CI 26-34). The optimal threshold for S100B was 0.72μg/L, with a sensitivity of 61% (95% CI 50-72) and a specificity of 69% (95% CI 64-74). Nine studies reported data for GFAP. The optimal threshold for GFAP was 626pg/mL, at which the sensitivity was 71% (95%CI 41-91) and specificity was 71% (95%CI 43-90). Sensitivity of GFAP was maximised at a threshold of 22pg/mL, which had a sensitivity of 93% (95%CI 73-99) and a specificity of 36% (95%CI 12-68%). Three studies reported data for NSE and two studies for UCH-L1, which precluded meta-analysis. There is evidence to support the use of S100B as a screening tool in mild TBI, and potential advantages to the use of GFAP, which requires further investigation.

Long-chain fatty dihydropyridines: Docking calcium channel studies and antihypertensive activity

AimsFrom the synthesis of 43 lipophilic dihydropyridines, the aim of this study was to verify whether the new dihydropyridines have calcium channel affinity using coupling studies and to determine antihypertensive and antioxidant properties, as well as toxicology and toxicity nifedipine and three new compounds, were chosen from the previous results. Materials and methodsThe animals were treated for 56 days, 28 days with N (ω) -nitro-L-arginine methyl ester to induce hypertension, and then treated for another 28 days with the new di- hydropyridine and the standard drug nifedipine. Throughout the treatment the animals had their blood pressure measured and their heart rate checked by pletysmography. After treatment the animals were euthanised, blood samples were collected for creatine kinase and urea analysis, and the brain, heart and liver were collected for oxidative status analysis (quantification of reactive oxygen species, total antioxidant capacity, and lipid peroxidation). Key findingsCompounds 2c, and 9a, and nifedipine significantly reduced blood pressure to control group levels. The tachycardia caused by the induction of hypertension was reversed by 2c and 9a compounds. Regarding oxidative stress analyzes, the compounds that had the best performances were also 2c and 9a. Overall the results demonstrate that two of the three new dihydropyridines tested demonstrated performance equal to or superior to the standard drug nifedipine. SignificanceIn this study, for the first time, docking was applied to analyse 43 fatty dihydropyridines regarding their calcium channel binding. Afterwards, three fatty dihydropyridines were chosen and their antihypertensive and antioxidant properties.

Effects of IgG anti-GM1 monoclonal antibodies on neuromuscular transmission and calcium channel binding in rat neuromuscular junctions.

Guillain-Barré syndrome is a type of acute inflammatory neuropathy that causes ataxia and is associated with the IgG anti-GM1 antibody. However, the pathogenic role of the IgG anti-GM1 antibody and calcium channels in neuromuscular junctions (NMJs) remains unclear. Thus, the aim of the present study was to investigate the effects of the IgG anti-GM1 monoclonal antibody (mAb) on spontaneous muscle action potentials (SMAPs), and the effects of calcium channel blockers, in a rat spinal cord-muscle co-culture system. In addition, the binding of IgG anti-GM1 mAb to calcium channels was investigated in the rat hemidiaphragm. The frequency of SMAPs in the innervated muscle cells was acutely inhibited by the IgG anti-GM1 mAb; however, this effect was blocked by the N-type calcium channel blocker, ω-conotoxin GVIA (30 nM). Furthermore, the P/Q-type calcium channel blocker, ω-agatoxin IVA (10 nM), was found to partially block the IgG anti-GM1 mAb-induced inhibitory effect in the spinal cord-muscle co-culture system. Immunohistochemical analysis of the rat hemidiaphragm indicated that IgG anti-GM1 mAb binding overlapped with anti-Cav2.2 (α1B) antibody binding in the nerve terminal. In addition, IgG anti-GM1 mAb binding partially overlapped with anti-Cav2.1 (α1A) antibody binding. Thus, the results demonstrated that the IgG anti-GM1 mAb binds to calcium channels in the nerve terminals of NMJs. Therefore, the inhibitory effect of IgG anti-GM1 mAb on SMAPs may involve N-type and P/Q-type calcium channels in motor nerve terminals at the NMJ.

Effects of IgM Anti-Galnac-GD1a Monoclonal Antibody on Neuromuscular Transmission and Calcium Channel Binding in the Rat Neuromuscular Junction

Guillain-Barre syndrome with antibodies against ganglioside N-acetylgalactosaminyl GD1a (GalNAc-GD1a) is characterized by a rapid onset of predominantly distal pure motor neuropathy. However, the pathogenic role of anti- GalNAc-GD1a antibody and calcium channels in neuromuscular junctions (NMJs) remains unclear. We investigated the effects of IgM anti-GalNAc-GD1a monoclonal antibody (IgM anti-GalNAc-GD1a mAb) on spontaneous muscle action potentials (SMAP) in a rat spinal cord–muscle co-culture system and the localization of IgM anti-GalNAc- GD1a mAb and calcium channel binding in the rat hemi-diaphragm. Immunohistochemistry of the rat hemidiaphragm showed that IgM anti-GalNAc-GD1a mAb binding overlapped with anti-neurofilament 200 antibody and α- bungarotoxin staining, demonstrating that IgM anti-GalNAc-GD1a mAb was localized at the motor nerve terminal. Moreover, IgM anti-GalNAc-GD1a mAb binding overlapped with anti-Cav2.1 antibody in the nerve terminal. We suggest that the inhibitory effect of IgM anti-GalNAc-GD1a mAb on SMAP is related to the GalNAc-GD1a epitope on P/Q-type calcium channels in motor nerve terminals at NMJs.

Copy number variation in schizophrenia in Sweden.

Schizophrenia (SCZ) is a highly heritable neuropsychiatric disorder of complex genetic etiology. Previous genome-wide surveys have revealed a greater burden of large, rare copy number variations (CNVs) in SCZ cases and identified multiple rare recurrent CNVs that increase risk of SCZ although with incomplete penetrance and pleiotropic effects. Identification of additional recurrent CNVs and biological pathways enriched for SCZ CNVs requires greater sample sizes. We conducted a genome-wide survey for CNVs associated with SCZ using a Swedish national sample (4719 cases and 5917 controls). High-confidence CNV calls were generated using genotyping array intensity data, and their effect on risk of SCZ was measured. Our data confirm increased burden of large, rare CNVs in SCZ cases as well as significant associations for recurrent 16p11.2 duplications, 22q11.2 deletions and 3q29 deletions. We report a novel association for 17q12 duplications (odds ratio=4.16, P=0.018), previously associated with autism and mental retardation but not SCZ. Intriguingly, gene set association analyses implicate biological pathways previously associated with SCZ through common variation and exome sequencing (calcium channel signaling and binding partners of the fragile X mental retardation protein). We found significantly increased burden of the largest CNVs (>500 kb) in genes present in the postsynaptic density, in genomic regions implicated via SCZ genome-wide association studies and in gene products localized to mitochondria and cytoplasm. Our findings suggest that multiple lines of genomic inquiry--genome-wide screens for CNVs, common variation and exonic variation--are converging on similar sets of pathways and/or genes.

N-type Calcium Channel Binding Antibodies in a Patient with Paraneoplastic Cerebellar Degeneration in Association with Non-Hodgkins Lymphoma: A Case Report

N-type Calcium Channel Binding Antibodies in a Patient with Paraneoplastic Cerebellar Degeneration in Association with Non-Hodgkins Lymphoma: A Case Report

ChemInform Abstract: Highly Regioselective Synthesis of N‐3 Organophosphorous Derivatives of 3,4‐Dihydropyrimidin‐2(1H)‐ones and Their Calcium Channel Binding Studies.

AbstractA series of novel dihydropyrimidine derivatives bearing diaminophosphinyl (III), phosphonate moieties (Vc) or phosphacycles (VII) at N‐3 are prepared by regioselective reactions between phosphoryl dichlorides (II) and various nucleophiles.

Highly regioselective synthesis of N-3 organophosphorous derivatives of 3,4-dihydropyrimidin-2(1H)-ones and their calcium channel binding studies

A series of novel N-3 substituted 3,4-dihydropyrimidin-2(1H)-ones derivatives bearing diaminophosphinyl, phosphonate and phosphorous containing heterocycles were obtained from 3,4-dihydropyrimidinones (DHPMs) in a regioselective manner through an efficient reaction protocol, tolerant to substitutional variation at the key diversity positions around the DHPM core. None of the representative compounds screened for calcium channel blocking activity was found to have significant activity compared to nifedipine.

Charactarization techniques of particular proteins in cerebellar cortex using acoustic impedance microscope

Two-dimensional acoustic impedance imaging is useful for observation of living organs without invasion. Because acoustic impedance is proportional to sonic speed and density, organelle having larger density, e.g. nucleus, showed higher impedance. We have proved that cerebellar cortical layers and Purjinje cell bodies were identified using the acoustic impedance microscopy. In order to visualize the distribution of specific functional proteins in acoustic imaging, we proposed direct or complex-including heavy metal treatment to elevate the density and acoustic impedance of a particular protein. Heavy metal binding was useful for acoustic impedance imaging; however, metal binding to a protein molecule was not always specific. To observe the distribution of wanted molecules, we investigated two types of heavy metal binding materials; one was p-cymene ruthenium (Ru)-binding calcium channel binding peptides, and another was cadmiun nanocrystal binding antibodies, QdotTM. We could observe the characterized acoustic area by both p-cymene Ru-binding peptide treatment and Qdot treatment, whereas specimen plate was required hydrophobic condition to stabilize acoustic impedance. We suggest that a metal-binding reagent would be useful for specialization of an acoustic imaging.

T1305 A Novel Type of Therapeutic Potential in a Traditional Herbal Medicine Kakkonto Against Gastrointestinal Anaphylaxis By Reducing Mucosal Mast Cell-Surface Expression of FCɛRi

Racemic verapamil, primarily a cardiovascular agent, has been widely used off-label in patients with irritable bowel syndrome (IBS). Initial observations of its usefulness followed recognition of a high incidence of constipation with use in cardiovascular conditions. The enantiomers of verapamil are known to differ in cardiovascular potency, the S-isomer being much more potent than the R-isomer. In addition we found the S-isomer to be equiactive in relaxing vascular and colonic smooth muscle but the R-isomer to be 5-times more potent in relaxing colonic than vascular muscle. This selectivity led us to develop R-verapamil (Rezular) as a gut selective treatment in IBS and we have shown doses from 60mg/day to 240mg/day to greatly improve symptoms in non-constipation IBS patients. To better understand the mechanism by which Rverapamil improved the symptoms of IBS, we undertook an in-vitro screen of binding of R-verapamil to 147 receptors/receptor sub-types. Specific ligand binding was initially assessed using 10x-5 M verapamil and if there was greater than 50% inhibition of control specific binding, then binding at 8 different concentrations was tested and IC50 values (concentration for half-maximal inhibition of control specific binding (x10 -7M)) calculated. The therapeutic plasma concentration range of free R-verapamil was conservatively set at 0.1-3x10-7 M. Within this range R-verapamil showed affinity for 3 receptors: melatonin (MT1)(IC50 0.6), 5-HT2b (IC50 1.1) and L-type calcium channel (IC50 2.4). In addition compared with S-verapamil, R-verapamil showed stereoselectivity (x40)for MT1 binding , whereas S-verapamil showed stereoselectivity (x 3)for L-type calcium channel binding. R-Verapamil was selective for 5-HT2b relative to other 5-HT receptor sub types and affinity was low for 5-HT3(IC 50 3,400) or 5-HT4(>100) receptors.It was also highly selective for MT1(IC50 0.6) versus MT2 (IC50 >100)receptors. We conclude that Rverapamil most likely exerts its therapeutic effects in IBS via a previously unrecognized mechanism involving combined effects at melatonin receptors, serotonin receptors and Ltype calcium channels.

Effects of New 4-Aryl-1,4-Dihydropyridines and 4-Arylpyridines on Drug Efflux Mediated by Multidrug Resistance-Associated Protein 1

The purpose of this study is to evaluate the effects of newly synthesized 4-aryl-1,4-dihydropyridine and pyridines on drug efflux mediated by multidrug resistance-associated protein 1 (MRP1, ABCC1). These compounds were designed to maximize inhibition of P-glycoprotein and minimize calcium channel binding activity, based on structure modifications of niguldipine. A [3H]vinblastine accumulation study was conducted in human small cell lung cancer H69AR (overexpressing MRP1) and wild type H69 cells. Five out of 16 dihydropyridines and 6 out of 9 pyridines were found to significantly increase the intracellular accumulation of vinblastine in resistant H69AR cells (p<0.01) at a concentration of 2.5 microM. Daunomycin accumulation studies, determined using a flow cytometric assay, were also performed in H69AR and human pancreatic adenocarcinoma Panc-1 cells and the results were highly correlated with those obtained from the [3H]vinblastine accumulation studies. Four compounds, which significantly increased vinblastine accumulation, were tested for their effect on daunomycin cytotoxicity in H69AR cells and found to significantly decrease the IC50 of daunomycin, confirming the accumulation study results. Compounds were also tested for their effect on intracellular glutathione (GSH) concentrations, a cosubstrate for MRP1-mediated efflux in H69AR and Panc-1 cells. No significant changes in the intracellular GSH level were observed in H69AR cells after treatment with these test compounds. However, following a 2-hr and 24-hr incubation with a dihydropyridine compound, Im, and its pyridine derivative IIm, there was a small (approximately 20%) but statistically significant decrease in intracellular GSH in Panc-1 cells. Our results indicate that some dihydropyridine and pyridine compounds in our series could inhibit MRP1-mediated transport and that GSH modulation plays a minor, if any, role in this effect.

NEW 4-ARYL-1,4-DIHYDROPYRIDINES AND 4-ARYLPYRIDINES AS P-GLYCOPROTEIN INHIBITORS

Efflux of cytotoxic agents mediated by P-glycoprotein is believed to be an important mechanism of multidrug resistance, which remains a serious limitation to successful chemotherapy in cancers such as metastatic breast cancer. A series of 4-aryl-1,4-dihydropyridines and corresponding aromatized 4-arylpyridines have been synthesized based on structure modifications of niguldipine to enhance multidrug resistance reversal activity, while minimizing calcium channel binding. Thirty new compounds were characterized. [(3)H]Vinblastine accumulation studies indicated that at a concentration level of 3 muM, 15 of 18 4-aryl-1,4-dihydropyridines and all 4-arylpyridines can successfully restore intracellular accumulation of vinblastine in a resistant human breast adenocarcinoma cell line, MCF-7/adr, which overexpresses P-glycoprotein. The most potent compounds led to an approximately 15-fold increase of vinblastine accumulation. All of the test compounds that significantly increased vinblastine accumulation in MCF/adr cells were able to substantially reduce IC(50) values of daunomycin and increase its cytotoxicity in MCF-7/adr-resistant cells, confirming the results of the vinblastine accumulation studies. Calcium channel binding assays for these newly synthesized compounds were conducted using rat cerebral cortex membrane. All but eight compounds demonstrated negligible calcium channel binding over the concentration range from 15 to 2500 nM. The results demonstrate that the newly synthesized series of 1,4-dihydropyridines and pyridines represent P-glycoprotein modulators with negligible calcium channel blocking activity.

Characterization of Calcium Channel Binding

Voltage-dependent calcium channels are expressed in a variety of tissues including heart, muscles and brain. Saturation binding of a radioligand to the calcium channel is commonly used to characterize the expression level of the channel protein. Compound competition binding assay is a conventional screening method to determine the affinity of unlabeled compounds for the channel protein. This unit provides detailed experimental methods for two types of radioligand binding assays using [³H]PN200-100 and [¹²⁵I](conotoxin MVIIA. Voltage-dependent calcium channels are expressed in a variety of tissues including heart, muscles and brain.

Vatanidipine hydrochloride: a new long-lasting antihypertensive agent

Vatanidipine is a novel dihydropyridine (DHP)-type calcium channel blocker with slow-onset pharmacological actions, which are probably due to both its slow uptake into vascular tissues and resistance in its approach to the calcium channel binding site. Vatanidipine once incorporated into vascular tissues is not easily released, even by repeated washing, thus resulting in a long-lasting action of the agent. A slow-onset and long-lasting hypotensive action was observed in various experimental hypertensive models. Clinical trials using human subjects with essential hypertension indicated that vatanidipine exerts an antihypertensive effect with a slow onset and long duration. In spite of its potent hypotensive effect, the incidence of adverse effects by vatanidipine administration has been reported to be lower than that in cases of nitrendipine. In addition to its vasodilatory effects, vatanidipine efficiently suppressed noradrenaline release from sympathetic nerve endings, thus suggesting this agent exhibits a beneficial effect in the treatment of hypertensive patients, in which the reflex activation of peripheral sympathetic nerves is unfavourable to antihypertensive therapy. In a double-blind study, vatanidipine did not show reflex tachycardia, despite producing a potent and long-lasting hypotensive effect, in contrast to the administration of nitrendipine. In an animal study, vatanidipine exhibited a protective effect against cerebrovascular lesions, through a mechanism independent of its hypotensive effect. In addition, a renoprotective effect was also observed in experimental hypertensive models. In cholesterol-fed rabbits, vatanidipine exerted an anti-atherosclerotic action, which is probably attributable to the inhibitory action of the agent on low-density lipoprotein oxidation. In essential hypertensive patients, the plasma levels of cholesterol and triglyceride decreased after vatanidipine treatment, thus suggesting that this agent may have a therapeutic potential in preventing such vascular diseases as atherosclerosis. Taken together, vatanidipine appears to be a novel and useful antihypertensive agent, which can both prevent target-organ damage and reduce cardiovascular morbidity and mortality.

Mechanism of vascular relaxation by thaligrisine: Functional and binding assays

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca 2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with α-adrenoceptors we examined the effect of the alkaloid on [ 3H]-(+)- cis diltiazem, [ 3H]-nitrendipine and [ 3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC 50=8.9±2.9 μM, n=5) and in tail artery (IC 50=3.04±0.3 μM, n=6) or noradrenaline induced contraction in rat aorta (IC 50=23.0±0.39 μM, n=9) and in tail artery (IC 50=3.8±0.9 μM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca 2+-free solution (IC 50 = 13.3 μM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC 50 = 7.7 μM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [ 3H]-prazosin than for [ 3H]-(+)- cis-diltiazem binding sites, with Ki values of 0.048±0.007 μM and 1.5±1.1 μM respectively. [ 3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [ 3H]-prazosin binding site than [ 3H]-(+)- cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an α 1-adrenoceptor antagonist and as a Ca 2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca 2+-stores sensitive to noradrenaline, by blocking Ca 2+-entry through voltage-dependent Ca 2+-channels.

Allosteric modulation of [ 3H]gabapentin binding by ruthenium red

Gabapentin is an anticonvulsant with an unknown mechanism of action. However, it has been proposed that gabapentin acts by binding to voltage-gated calcium channels. To further characterize the interaction of gabapentin with its endogenous binding site in cerebral cortex, we tested for competitive and allosteric interactions between [ 3H]gabapentin and a variety of calcium channel binding ligands. Most ligands for voltage- or ligand-gated calcium channels (verapamil, the ω-conotoxins MVIIC and GVIA, ryanodine, caffeine, capsaicin, MK-801) had no significant effect on [ 3H]gabapentin binding. However, ruthenium red, a relatively nonselective calcium channel ligand, was found to robustly modulate [ 3H]gabapentin binding. Ruthenium red slowed the association and dissociation kinetics of [ 3H]gabapentin while increasing the number of detectable binding sites. Spermine and MgCl 2, which also bind to calcium channels and modulate [ 3H]gabapentin binding, were found to act in a similar manner. These findings support the contention that the principal endogenous binding site for gabapentin is a calcium channel; they characterize the nature of the allosteric interaction of spermine, MgCl 2 and ruthenium red with this binding site; and they suggest possible mechanisms by which gabapentin may modulate calcium channel function and ultimately produce therapeutic actions.