Ciliary Beat Frequency: Proceedings and Recommendations from a Multi-laboratory Ring Trial Using 3-D Reconstituted Human Airway Epithelium to Model Mucociliary Clearance.

The use of reconstituted human airway (RHuA) epithelial tissues to assess functional endpoints is highly relevant in respiratory toxicology, but standardised methods are lacking. In June 2015, the Institute for In Vitro Sciences (IIVS) held a technical workshop to evaluate the potential for standardisation of methods, including ciliary beat frequency (CBF). The applicability of a protocol suggested in the workshop was assessed in a multi-laboratory ring study. This report summarises the findings, and uses the similarities and differences identified between the laboratories to make recommendations for researchers in the absence of a validated method. Two software platforms for the assessment of CBF were used - Sisson-Ammons Video Analysis (SAVA; Ammons Engineering, Clio, MI, USA) and ciliaFA (National Institutes of Health, Bethesda, MD, USA). Both were utilised for multiple read temperatures, one objective strength (10×) and up to four video captures per tissue, to assess their utility. Two commercial RHuA tissue cultures were used: MucilAir™ (Epithelix, Geneva, Switzerland) and EpiAirway™ (MatTek, Ashland, MA, USA). IL-13 and procaterol were used to induce CBF-specific responses as positive controls. Further testing addressed the impact of tissue acclimation duration, the number of capture fields and objective strengths on baseline CBF readings. Both SAVA and ciliaFA reliably collected CBF data. However, ciliaFA failed to generate accurate CBF measurements above ∼10 Hz. The positive controls were effective, but were subject to inter-laboratory variability. CBF endpoints were generally uniform across replicate tissues, objective strengths and laboratories. Longer tissue acclimation increased the percentage active area, but had minimal impact on CBF. Taken together, these findings support the development and validation of a standardised CBF measurement protocol.

Cytotoxicity, oxidative stress, and inflammatory response of smokeless tobacco extracts and cytotoxicity of combustible cigarette whole smoke in a 3D oral organotypic buccal cell model.

Oral disease is frequently associated with viral and environmental exposures and oral hygiene. The use of tobacco is a risk factor in the development of oral disease. Cytotoxicity, inflammatory response, and oxidative stress have been reported to have a role in the development of oral disease. These three endpoints were evaluated in a 3D human oral buccal model, EpiOral™, following exposure to CORESTA reference smokeless tobacco products (CRPs) and cigarette whole smoke. CRPs for Swedish style snus (CRP1), moist snuff (CRP2), and dry snuff (CRP3) were each extracted in complete artificial saliva (CAS) with a ratio of 300 mg CRP to 1 mL of CAS. Each of the CRP extracts (15-300 mg/ml) were applied to the apical side of a 3D organotypic buccal cell model for 24 or 48 h continuously, then cytotoxicity (LDH), oxidative stress (8-isoprostane), and inflammatory response (IP10, IL-1α, and IL-8) were measured. Experiments with 3R4F cigarettes were conducted by exposing the buccal tissues to whole smoke for a maximum of 2.5 h. Cytotoxicity (MTT) was measured 24 h post-exposure. Exposure of buccal tissues to whole smoke from a cigarette induced a dose-dependent cytotoxic response. In contrast, the CRP extracts elicited minimal cytotoxicity (<15%) when compared to CAS (vehicle control), but time- and dose-dependent effects on oxidative stress and inflammatory response were observed. Collectively, these data demonstrate that a 3D organotypic buccal human model may be used to assess biological mechanisms (MOAs) involved in the development of oral disease following exposure to smokeless tobacco products and may be applicable for differentiation between tobacco product categories.

Apoptotic cell death in rat lung following mustard gas inhalation.

To investigate apoptosis as a mechanism of sulfur mustard (SM) inhalation injury in animals, we studied different caspases (caspase-8, -9, -3, and -6) in the lungs from a ventilated rat SM aerosol inhalation model. SM activated all four caspases in cells obtained from bronchoalveolar lavage fluid (BALF) as early as 6 h after exposure. Caspase-8, which is known to initiate the extrinsic Fas-mediated pathway of apoptosis, was increased fivefold between 6 and 24 h, decreasing to the unexposed-control level at 48 h. The initiator, caspase-9, in the intrinsic mitochondrial pathway of apoptosis as well as the executioner caspases, caspase-3 and -6, all peaked (P < 0.01) at 24 h; caspase-3 and -6 remained elevated, but caspase-9 decreased to unexposed-control level at 48 h. To study further the Fas pathway, we examined soluble as well as membrane-bound Fas ligand (sFas-L and mFas-L, respectively) and Fas receptor (Fas-R) in both BALF cells and BALF. At 24 h after SM exposure, sFas-L increased significantly in both BALF cells (P < 0.01) and BALF (P < 0.05). However, mFas-L increased only in BALF cells between 24 and 48 h (P < 0.1 and P < 0.001, respectively). Fas-R increased only in BALF cells by 6 h (P < 0.01) after SM exposure. Apoptosis in SM-inhaled rat lung specimens was also confirmed by both immunohistochemical staining using cleaved caspase-3 and -9 antibodies and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining as early as 6 h in the proximal trachea and bronchi, but not before 48 h in distal airways. These findings suggest pathogenic mechanisms at the cellular and molecular levels and logical therapeutic target(s) for SM inhalation injury in animals.

Mustard Gas Inhalation Injury: Therapeutic Strategy.

Mustard gas (sulfur mustard [SM], bis-[2-chloroethyl] sulfide) is a vesicating chemical warfare agent and a potential chemical terrorism agent. Exposure of SM causes debilitating skin blisters (vesication) and injury to the eyes and the respiratory tract; of these, the respiratory injury, if severe, may even be fatal. Therefore, developing an effective therapeutic strategy to protect against SM-induced respiratory injury is an urgent priority of not only the US military but also the civilian antiterrorism agencies, for example, the Homeland Security. Toward developing a respiratory medical countermeasure for SM, four different classes of therapeutic compounds have been evaluated in the past: anti-inflammatory compounds, antioxidants, protease inhibitors and antiapoptotic compounds. This review examines all of these different options; however, it suggests that preventing cell death by inhibiting apoptosis seems to be a compelling strategy but possibly dependent on adjunct therapies using the other drugs, that is, anti-inflammatory, antioxidant, and protease inhibitor compounds.

Differentiated NSC-34 cells as an in vitro cell model for VX.

The US military has placed major emphasis on developing therapeutics against nerve agents (NA). Current efforts are hindered by the lack of effective in vitro cellular models to aid in the preliminary screening of potential candidate drugs/antidotes. The development of an in vitro cellular model to aid in discovering new NA therapeutics would be highly beneficial. In this regard, we have examined the response of a differentiated hybrid neuronal cell line, NSC-34, to the NA VX. VX-induced apoptosis of differentiated NSC-34 cells was measured by monitoring the changes in caspase-3 and caspase-9 activity post-exposure. Differentiated NSC-34 cells showed an increase in caspase-3 activity in a manner dependent on both time (17-23 h post-exposure) and dose (10-100 nM). The maximal increase in caspase-3 activity was found to be at 20-h post-exposure. Caspase-9 activity was also measured in response to VX and was found to be elevated at all concentrations (10-100 nM) tested. VX-induced cell death was also observed by utilizing annexin V/propidium iodide flow cytometry. Finally, VX-induced caspase-3 or -9 activities were reduced with the addition of pralidoxime (2-PAM), one of the current therapeutics used against NA toxicity, and dizocilpine (MK-801). Overall the data presented here show that differentiated NSC-34 cells are sensitive to VX-induced cell death and could be a viable in vitro cell model for screening NA candidate therapeutics.

In vitro permeation of nicotine and tobacco specific nitrosamines from smokeless tobacco product extracts in a 3D buccal tissue model.

Tobacco product use is a risk factor in the development of oral cancer, although epidemiology studies show this risk is far less with smokeless tobacco product use than cigarette smoking. While smokeless tobacco contains harmful and potentially harmful constituents (HPHCs), the oral permeation of HPHCs in oral tobacco products is not completely understood. To improve the understanding, three different extract concentrations of the CORESTA reference products (CRP) for snus (CRP1.1) and moist snuff (CRP2.1) were applied to cellular tissue derived from two donors of EpiOral™ model, a 3D human buccal model, and permeation of nicotine and tobacco-specific nitrosamines (TSNAs) were measured over two hours. Permeation of 0.15% caffeine in complete artificial saliva and cell viability were also measured. Results showed that a consistent and concentration dependent cumulative permeation of nicotine and TSNAs was observed with high percent recovery in all conditions. A high degree of sensitivity was seen for all analytes, with minimal cytotoxicity for both CRPs. The data presented here show the EpiOral™ model is fit-for-purpose to evaluate the permeation of nicotine and TSNAs in nicotine-containing snus and moist snuff oral tobacco.

Evaluation of Cytotoxicity and Oxidative Stress of Whole Aerosol from Vuse Alto ENDS Products.

Assessment of in vitro cytotoxicity is an important component of tobacco product toxicological evaluations. However, current methods of regulatory testing involve exposing monolayer cell cultures to various preparations of aerosols from cigarettes or other emerging products such as electronic nicotine delivery systems (ENDS), which are not representative of human exposure. In the present study, a whole aerosol (WA) system was used to expose lung epithelial cultures (2D and 3D) to determine the potential of six Vuse Alto ENDS products that varied in nicotine content (1.8%, 2.4%, and 5%) and flavors (Golden Tobacco, Rich Tobacco, Menthol, and Mixed Berry), along with a marketed ENDS and a marked cigarette comparator to induce cytotoxicity and oxidative stress. The WA from the Vuse Alto ENDS products was not cytotoxic in the NRU and MTT assays, nor did it activate the Nrf2 reporter gene, a marker of oxidative stress. In summary, Vuse Alto ENDS products did not induce cytotoxic or oxidative stress responses in the in vitro models. The WA exposures used in the 3D in vitro models described herein may be better suited than 2D models for the determination of cytotoxicity and other in vitro functional endpoints and represent alternative models for regulatory evaluation of tobacco products.

In vitro toxicological evaluation of glo menthol and non-menthol heated tobacco products.

Heated tobacco products (HTPs) are non-combustible, inhaled tobacco products that generate an aerosol with fewer and lower levels of toxicants, with a potential to reduce risk relative to cigarette smoking. Here, we assessed in vitro toxicological effects of three menthol (glo neo neoCLICK, neo Smooth Menthol and Fresh Menthol) and one non-menthol (neo Smooth Tobacco) variants of glo HTP, along with market comparators for cigarettes and HTPs. Limited chemical characterization of the study products revealed significantly lower levels of acetaldehyde, acrolein, crotanaldehyde and formaldehyde in test samples from HTPs than those from cigarettes. The glo HTPs were non-mutagenic in the bacterial reverse mutagenesis assay. Although, the whole aerosol exposures of glo HTPs were classified as genotoxic in the in vitro micronucleus assay, and cytotoxic in the NRU (monolayer) and MTT (3 dimensional EpiAirway™ tissues) assays, the cigarette comparators were the most toxic study products in each of these assessments. Further, glo HTPs elicited oxidative stress responses only at the highest dose tested, whereas the cigarette comparators were potent inducers of oxidative stress at substantially lower doses in the EpiAirway tissues. The comparator (non-glo) HTP results were similar to the glo HTPs in these assays. Thus, the glo HTPs exhibit substantially lower toxicity compared to cigarettes.

Postexposure application of Fas receptor small-interfering RNA to suppress sulfur mustard-induced apoptosis in human airway epithelial cells: implication for a therapeutic approach.

Sulfur mustard (SM) is a vesicant chemical warfare and terrorism agent. Besides skin and eye injury, respiratory damage has been mainly responsible for morbidity and mortality after SM exposure. Previously, it was shown that suppressing the death receptor (DR) response by the dominant-negative Fas-associated death domain protein prior to SM exposure blocked apoptosis and microvesication in skin. Here, we studied whether antagonizing the Fas receptor (FasR) pathway by small-interfering RNA (siRNA) applied after SM exposure would prevent apoptosis and, thus, airway injury. Normal human bronchial/tracheal epithelial (NHBE) cells were used as an in vitro model with FasR siRNA, FasR agonistic antibody CH11, and FasR antagonistic antibody ZB4 as investigative tools. In NHBE cells, both SM (300 µM) and CH11 (100 ng/ml) induced caspase-3 activation, which was inhibited by FasR siRNA and ZB4, indicating that SM-induced apoptosis was via the Fas response. FasR siRNA inhibited SM-induced caspase-3 activation when added to NHBE cultures up to 8 hours after SM. Results using annexin V/propidium iodide-stained cells showed that both apoptosis and necrosis were involved in cell death due to SM; FasR siRNA decreased both apoptotic and necrotic cell populations. Bronchoalveolar lavage fluid (BALF) of rats exposed to SM (1 mg/kg, 50 minutes) revealed a significant (P < 0.05) increase in soluble Fas ligand and active caspase-3 in BALF cells. These findings suggest an intervention of Fas-mediated apoptosis as a postexposure therapeutic strategy with a therapeutic window for SM inhalation injury and possibly other respiratory diseases involving the Fas response.

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid.

BACKGROUND: Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells. RESULTS: We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium (Ca2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 µM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG. CONCLUSION: Taken together, retinoic acid treated cells had improved morphological features as well as neuronal characteristics and functions; thus, these retinoic acid differentiated BE(2)-M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity.

Part two: an unblinded, parallel, randomized study to assess nicotine pharmacokinetics of four Vuse Solo ENDS flavors in smokers.

We report the findings from a randomized, parallel study designed to evaluate nicotine pharmacokinetics (PK) following 10 min of ad libitum use of electronic nicotine delivery system (ENDS) in four flavor variants. Subjects were randomized an investigational product (IP) and blood samples were collected for PK assessments during a test session. Primary endpoints were baseline-adjusted values of maximum plasma nicotine concentration (Cmax) and area under the nicotine concentration-vs-time curve up to 60 min (AUCnic0-60). Baseline-adjusted mean Cmax ranged from 6.53 to 8.21 ng/mL, and mean AUCnic0-60 ranged from 206.87 to 263.52 ng min/mL for all ENDS IPs. Results of geometric mean Cmax and AUCnic0-60 values were within 95% confidence intervals (CI) among the ENDS IP flavor variants tested.

Characterization of smoke and aerosol deliveries from combustible cigarettes, heated tobacco products and electronic nicotine delivery systems in the Vitrocell® Mammalian 6/48 exposure module.

The rapid development associated with Next Generation Tobacco Products (NGTP) has necessitated the development of high throughput methodologies to test their genotoxic potential in vitro when compared to conventional cigarette smoke (CS). An assessment of two Vitrocell® Mammalian 6/48 exposure modules in three independent experiments was made by comparing results from multiple dosimetric techniques applied to aerosol generated from 3R4F Kentucky Reference cigarettes, commercially available electronically heated tobacco product (eHTP) and Electronic Nicotine Delivery System (ENDS) using the Vitrocell® VC10®. Real-time aerosol particle concentration was assessed by means of light scattering photometers and expressed as area under the curve (∑AUC). Nicotine concentrations were determined analytically by LC/MS. Humectant amount and distribution was assessed for eHTP and ENDS by the quantification of free glycerol in a phosphate buffered saline (PBS) trap, whereas total particulate matter (TPM) was assessed in the 3R4F cigarettes by the fluorescence of the particulate at 485 nm in anhydrous dimethyl sulfoxide (DMSO) trap within the exposure. Dose was adjusted by means of the addition of ambient air to dilute the whole smoke/aerosol in L/min and sampled into the system at a rate of 5 mL/min. Dilution of CS ranged from 8.0 to 0.5 L/min and for the eHTP and ENDS ranged from 4 to 0 L/min (undiluted). Dosimetric analysis of the system showed good concordance within replicates (p-values ranged from p = 0.3762 to p = 0.8926) and showed that the Vitrocell® Mammalian 6/48 is a viable means for genotoxic assessment of aerosol generated from both conventional cigarettes and NGTP. Results demonstrate the need to tailor dosimetry approaches to different aerosols due to variations in the physio-chemical composition, with a multi-dosimetry approach recommended.

Investigation of multiple whole smoke dosimetry techniques using a VITROCELL®VC10® smoke exposure system.

The Vitrocell® VC10® smoke exposure system offers multiple platforms for air liquid interface (ALI) and air agar interface (AAI) exposure that mimic in vivo conditions for assessing toxicological impact of whole smoke using in vitro assays. The aim of this study was to investigate and compare multiple dosimetry techniques that may be employed during combustible cigarette whole smoke exposure using the Vitrocell® VC10® smoking robot. The following techniques were assessed: (1) quartz crystal microbalances (QCMs), (2) aerosol photometers (using area under curve, AUC), and (3) fluorescence of anhydrous dimethyl sulfoxide (DMSO)-captured smoke constituents. Results showed that each of the dosimetry techniques was able to distinguish different levels of whole smoke airflow in a concentration-related manner. When compared to each other, the three techniques showed a high level of consistency and all were considered efficient tools in quantifying dose during an exposure, although higher variation was observed at the higher airflows tested. Overall, the dosimetry tools investigated here provide effective measures of the whole smoke concentrations tested during the exposure.

Development of a quantitative method for assessment of dose in in vitro evaluations using a VITROCELL® VC10® smoke exposure system.

The assessment of potential cytotoxicity or genotoxicity of combustible tobacco products has historically been performed using partitioned exposures (i.e. total particulate matter [TPM], gas vapor phase [GVP]) rather than whole smoke. The VITROCELL® VC10® smoke exposure system offers multiple platforms for air liquid interface (ALI) or air agar interface (AAI) exposure to mimic in vivo-like conditions for assessing the toxicological impact of whole smoke using in vitro assays (e.g. cytotoxicity, mutagenicity and DNA modifications). The aims of this study were to investigate dosimetry during whole smoke exposure in the VITROCELL® VC10® smoking robot using quartz crystal microbalances (QCMs) and to support the use of photometers for concurrent assessment of 'dose' during whole smoke exposures. QCM results showed consistent deposition across different exposure chambers, between dilution bars, experiments and modules. Higher levels of variation were noted at higher airflows (i.e., >8 L/min). Dosimetry assessed using photometers also showed a high level of consistency between experiments, with no notable impact on deposition on the QCM when the photometers were placed 'in-line' between the dilution bar and the exposure module. However, the use of photometers alone may be not be sufficient to estimate deposition; the predictability of the data-generated equation was poor. Further development of dosimetry methodology and information for use in validated in vitro biological test methods is needed to facilitate on-going aerosol-based research and relative assessment.

Selective blockade of T-type Ca2+ channels suppresses human breast cancer cell proliferation.

We have measured the expression of T-type Ca2+ channel mRNA in breast cancer cell lines (MCF-7 (ERalpha+) using Western blot and quantitative real-time PCR (Q-RT-PCR). These results revealed that the MCF-7 cells express both alpha1G and alpha1H isoforms of T-type Ca2+ channels. In order to further clarify the role of T-type Ca2+ channels in proliferation, we tested the effects of a selective T-type Ca2+ channel inhibitor NNC-55-0396 on cellular proliferation. MCF-7 (ERalpha+) cellular proliferation was inhibited by the compound. In contrast, NNC-55-0396 at same concentration had no effect on the proliferation of MCF-10A cells, a non-cancer breast epithelial cell line. We also found that message expression of the T-type Ca2+ channels were only expressed in rapidly growing non-confluent cells but not in the cytostatic confluent cells. Knocking down the expression of T-type Ca2+ channels with siRNA targeting both alpha1G and alpha1H resulted in growth inhibition as much as 45%+/-5.0 in MCF-7 cells as compared to controls. In conclusion, our results suggest that T-type Ca2+ channel antagonism/silencing may reduce cellular proliferation in mitogenic breast cells.

Conceptual approaches for treatment of phosgene inhalation-induced lung injury.

Toxic industrial chemicals are used throughout the world to produce everyday products such as household and commercial cleaners, disinfectants, pesticides, pharmaceuticals, plastics, paper, and fertilizers. These chemicals are produced, stored, and transported in large quantities, which poses a threat to the local civilian population in cases of accidental or intentional release. Several of these chemicals have no known medical countermeasures for their toxic effects. Phosgene is a highly toxic industrial chemical which was used as a chemical warfare agent in WWI. Exposure to phosgene causes latent, non-cardiogenic pulmonary edema which can result in respiratory failure and death. The mechanisms of phosgene-induced pulmonary injury are not fully identified, and currently there is no efficacious countermeasure. Here, we provide a proposed mechanism of phosgene-induced lung injury based on the literature and from studies conducted in our lab, as well as provide results from studies designed to evaluate survival efficacy of potential therapies following whole-body phosgene exposure in mice. Several therapies were able to significantly increase 24h survival following an LCt50-70 exposure to phosgene; however, no treatment was able to fully protect against phosgene-induced mortality. These studies provide evidence that mortality following phosgene toxicity can be mitigated by neuro- and calcium-regulators, antioxidants, phosphodiesterase and endothelin receptor antagonists, angiotensin converting enzymes, and transient receptor potential cation channel inhibitors. However, because the mechanism of phosgene toxicity is multifaceted, we conclude that a single therapeutic is unlikely to be sufficient to ameliorate the multitude of direct and secondary toxic effects caused by phosgene inhalation.

Sulfur mustard induces apoptosis in lung epithelial cells via a caspase amplification loop.

Sulfur mustard (SM [bis-(2-chloroethyl) sulfide]) is a chemical warfare agent that causes skin blisters presumably due to DNA alkylation and cross-links. We recently showed that SM also induces apoptotic death in cultured normal human bronchial/tracheal epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. In this process, caspases-8 and -3, but not caspase-9, were strongly activated; this suggests a death receptor pathway for apoptosis. We now show that rat lungs were induced to undergo apoptosis in vivo following exposure of rats to SM by inhalation. Further study of the mechanism of apoptosis due to SM was performed with cultured NHBE cells and SAEC using tetrapeptide inhibitors of caspases-3, and -8. Inhibition of caspase-8 drastically reduced the activation of caspase-3 and almost eliminated that of caspase-9. Moreover, caspase-3 inhibition markedly reduced the activation of caspase-8 and also almost completely inhibited activation of caspase-9. These results suggest a death receptor pathway of apoptosis that utilizes a feedback amplification mechanism involving an activated death receptor complex that leads to the activation of caspase-9 via a caspase-3 pathway. These results may be important for the design of inhibitors of these pathways for therapeutic intervention to attenuate SM injury in respiratory tract lesions.

Towards selective antagonists of T-type calcium channels: design, characterization and potential applications of NNC 55-0396.

NNC 55-0396 is a structural analog of mibefradil (Ro 40-5967) that inhibits both T-type and high-voltage-activated (HVA) Ca2+ channels with a higher selectivity for T-type Ca2+ channels. The inhibitory effect of mibefradil on HVA Ca2+ channels can be attributed to a hydrolyzed metabolite of the drug: the methoxy acetate side chain of mibefradil is removed by intracellular enzymes, thus it forms (1S,2S)-2-(2-(N-[(3-benzoimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl hydroxy dihydrochloride (dm-mibefradil), which causes potent inhibition of HVA Ca2+ currents. By replacing the methoxy acetate chain of mibefradil with cyclopropanecarboxylate, a more stable analog was developed (NNC 55-0396). The acute IC50 of NNC 55-0396 to block recombinant Cav3.1 T-type channels expressed in HEK293 cells is approximately 7 muM, whereas 100 microM NNC 55-0396 has no detectable effect on high voltage-activated currents in INS-1 cells. Block of T-type Ca2+ current was partially reduced by membrane hyperpolarization and was enhanced at high stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channel channels, thus rendering it more selective to T-type Ca2+ channels.

Role of high-voltage-activated calcium channels in glucose-regulated beta-cell calcium homeostasis and insulin release.

High-voltage-activated (HVA) calcium channels are known to be the primary source of calcium for glucose-stimulated insulin secretion. However, few studies have investigated how these channels can be regulated by chronically elevated levels of glucose. In the present study, we determined the level of expression of the four major HVA calcium channels (N-type, P/Q-type, L(C)-type, and L(D)-type) in rat pancreatic beta-cells. Using quantitative real-time PCR (QRT-PCR), we found the expression of all four HVA genes in rat insulinoma cells (INS-1) and in primary isolated rat islet cells. We then determined the role of each channel in insulin secretion by using channel-selective antagonists. Insulin secretion analysis revealed that N- and L-type channels are both involved in immediate glucose-induced insulin secretion. However, L-type was preferentially coupled to secretion at later time points. P/Q-type channels were not found to play a role in insulin secretion at any stage. It was also found that long-term exposure to elevated glucose increases basal calcium in these cells. Interestingly, chronically elevated glucose decreased the mRNA expression of the channels involved with insulin secretion and diminished the level of stimulated calcium influx in these cells. Using whole cell patch clamp, we found that N- and L-type channel currents increase gradually subsequent to lower intracellular calcium perfusion, suggesting that these channels may be regulated by glucose-induced changes in calcium.

T-type Ca2+ channels are involved in high glucose-induced rat neonatal cardiomyocyte proliferation.

Infants develop hypertrophic cardiomyopathy in approximately 30% of diabetic pregnancies. We have characterized the effects of glucose on voltage-gated T-type Ca2+ channels and intracellular free calcium concentration, [Ca2+]i in neonatal rat cardiomyocytes. We found that T-type Ca2+ channel current density increased significantly in primary culture neonatal cardiac myocytes that were treated with 25 mM glucose for 48 h when compared with those that were treated with 5 mM glucose. High-glucose treatment also caused a higher Ca2+ influx elicited by 50 mM KCl in the myocytes. KCl-induced Ca2+ influx was attenuated when nickel was present. Real-time PCR studies demonstrated that mRNA levels of both alpha1G (Ca(v)3.1) and alpha1H (Ca(v)3.2) T-type Ca2+ channels were elevated after high-glucose treatment. High-glucose also significantly increased ventricular cell proliferation as well as the proportion of cells in the S-phase of the cell cycle; both effects were reversed by nickel or mibefradil. These results indicate that high glucose causes a rise in [Ca2+]i in neonatal cardiac myocytes by a mechanism that is associated with the regulation of the T-type Ca2+ channel activity.