Mibefradil is a Ca 2ϩ channel antagonist that inhibits both Ttype and high-voltage-activated Ca 2ϩ channels. We previously showed that block of high-voltage-activated channels by mibefradil occurs through the production of an active metabolite by intracellular hydrolysis. In the present study, we modified the structure of mibefradil to develop a nonhydrolyzable analog, (1S, 2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride , that exerts a selective inhibitory effect on T-type channels. The acute IC 50 of NNC 55-0396 to block recombinant ␣ 1 G T-type channels in human embryonic kidney 293 cells was ϳ7 M, whereas 100 M NNC 55-0396 had no detectable effect on high-voltage-activated channels in INS-1 cells. NNC 55-0396 did not affect the voltage-dependent activation of T-type Ca 2ϩ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca 2ϩ current was partially relieved by membrane hyperpolarization and enhanced at a high-stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca 2ϩ 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 Ca 2ϩ currents, arguing against a cytoplasmic route of action. After incubating cells from an insulin-secreting cell line (INS-1) with NNC 55-0396 for 20 min, mass spectrometry did not detect the mibefradil metabolite that causes L-type Ca 2ϩ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of Ltype Ca 2ϩ channels, thus rendering it more selective to T-type Ca 2ϩ channels.Voltage-gated Ca 2ϩ channels are transmembrane proteins involved in the regulation of cellular excitability and intracellular Ca 2ϩ signaling. Calcium channels have been divided into various categories based on functional and pharmacological criteria. High-voltage-activated (HVA) channels, which have been further subdivided into L-, N-, P/Q-, and R-types, require strong depolarizations for activation, whereas lowvoltage-activated or T-type channels activate over a much more negative voltage range and exhibit unique inactivation and deactivation kinetics (Armstrong and Matteson, 1985;Catterall, 1998;Perez-Reyes, 1998). The main structural component of the voltage-gated calcium channel is the ␣ 1

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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.

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

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 24 hr 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.

BackgroundImmortalized 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.ResultsWe 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.ConclusionTaken 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.

Infants develop hypertrophic cardiomyopathy in~30% of diabetic pregnancies. We have characterized the effects of glucose on voltage-gated T-type Ca 2ϩ channels and intracellular free calcium concentration, [Ca 2ϩ ] i in neonatal rat cardiomyocytes. We found that T-type Ca 2ϩ 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. Diabetes is one of the most common medical complications seen in pregnancy. Approximately 3-10% of pregnant women have disorders of glucose intolerance (1). This disease affects the fetal cardiovascular system in many ways, both directly and indirectly. The risk for congenital heart disease in infants of diabetic mothers (IDM) is reported to be 5 times higher than in the general population (2). It has been estimated that 30% of IDM have cardiomegaly, only a few of which develop congestive heart failure (1). Diabetic hypertrophic cardiomyopathy has been associated with poor maternal glucose regulation; however, recent studies indicate that cardiac hypertrophy can be attributed to a more complex mechanism possibly including Ca 2ϩ overload (3-5), which may result from an elevated expression of voltage gated Ca 2ϩ channels.Two well-documented voltage gated Ca 2ϩ channels found in fetal and neonatal hearts are the L-type and T-type Ca 2ϩ channels (6 -8). The L-type Ca 2ϩ channels become more abundant as some organisms develop into adulthood (8 -10); however, T-type Ca 2ϩ channels are down-regulated or disappear in adult animals (6,(11)(12)(13)(14). The density of T-type Ca 2ϩ channels is predominant only in pacemaker cells of the sinoatrial node and the Purkinje fibers (15,16). The physiologic role of the T-type Ca 2ϩ channel has been postulated to be involved in the cell cycle during proliferation (17); therefore, we expect to see an increase in T-type Ca 2ϩ channel activity in glucoseinduced neonatal hypertrophic cardiomyopathy secondary to cell proliferation.We hypothesized that T-type calcium currents would be increased in response to high glucose. We found that this indeed was the case and that many of the effects of high glucose were prevented by manipulations that reduced T-type calcium currents. METHODSCell preparation. Neonatal ventricular myocytes were prepared from the hearts of 4-d-old Charles River rats using a previously described protocol Received April 20, 2004; accepted September 3, 2004

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, LC-type, and LD-type) in rat pancreatic ␤-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 glucoseinduced changes in calcium. glucose desensitization; insulinoma cells; diabetes HIGH-VOLTAGE-ACTIVATED (HVA) CALCIUM CHANNELS are membrane-spanning proteins that are involved in the regulation of intracellular calcium in many different cell types (14). The HVA calcium channel family is composed of seven different genes (A-F, S) that encode the various ␣1-pore-forming subunits (7,22). Calcium entry through these channels plays a key role in a variety of physiological responses, including membrane excitability and modulation of transmitter or hormone release. However, the differences in tissue distribution and efficacy with which HVA channels stimulate vesicular secretion vary among the channel classes (20). In pancreatic ␤-cells, the expression of many HVA calcium channels' mRNA has been demonstrated (5,13,27,30,33,39,46,58,64,68). These include the ␣1A (Cav2.1), ␣1B (Cav2.2), ␣1C (Cav1.2), ␣1D (Cav1.3), and ␣1E (Cav2.3). However, only a few studies have been published to show HVA calcium channel protein expression in pancreatic ␤-cells. Expression of the ␣1C protein has been found in mouse islets cells, whereas the ␣1D protein has been shown to be expressed in normal (42) and obese mouse islet cells (69) and in rat islet ␤-cells (28). Protein expression of ␣1A, ␣1B, and ␣1E has also been found in rat ␤-cells (33,60,63).Even though many types of HVA channels have been identified in pancreatic ␤-cells, it is generally accepted that HVA calcium channels sensitive to dihydropyridines (DHPs, L-type) contribute to most of the ␤-cell calcium current and subsequent insulin secretion (9, 26). However, it is unclear as to which L-type isoform (Cav1.2 or Cav1.3) is directly linked with insulin secretion. Cav...