NNC 55-0396 [(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]: a new selective inhibitor of T-type calcium channels.
ABSTRACT Mibefradil is a Ca2+ channel antagonist that inhibits both T-type and high-voltage-activated Ca2+ 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 (NNC 55-0396), that exerts a selective inhibitory effect on T-type channels. The acute IC(50) of NNC 55-0396 to block recombinant alpha(1)G T-type channels in human embryonic kidney 293 cells was approximately 7 microM, whereas 100 microM 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 Ca2+ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca2+ 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 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. 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 Ca2+ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channels, thus rendering it more selective to T-type Ca2+ channels.
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ABSTRACT: Voltage-gated Ca2+ (CaV) channels are transmembrane proteins comprising three subfamilies named CaV1, CaV2 and CaV3. The CaV3 channel subfamily groups the low-voltage activated Ca2+ channels (LVA or T-type) a significant role in regulating neuronal excitability. CaV3 channel activity may lead to the generation of complex patterns of action potential firing such as the postinhibitory rebound (PIR). In the adult spinal cord, these channels have been found in dorsal horn interneurons where they control physiological events near the resting potential and participate in determining excitability. In motoneurons, CaV3 channels have been found during development, but their functional expression has not yet been reported in adult animals. Here, we show evidence for the presence of CaV3 channel-mediated PIR in motoneurons of the adult turtle spinal cord. Our results indicate that Ni2+ and NNC55-0396, two antagonists of CaV3 channel activity, inhibited PIR in the adult turtle spinal cord. Molecular biology and biochemical assays revealed the expression of the CaV3.1 channel isotype and its localization in motoneurons. Together, these results provide evidence for the expression of CaV3.1 channels in the spinal cord of adult animals and show also that these channels may contribute to determine the excitability of motoneurons.PLoS ONE 09/2014; 9(9):e108187. · 3.53 Impact Factor
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ABSTRACT: Several cellular mechanisms contribute to the neuroendocrine differentiation of prostate cancer cells, including exposure to sodium butyrate (NaBu), a naturally occurring salt of the short chain fatty acid n-butyric acid. NaBu belongs to a class of histone deacetylase inhibitors, with potential anticancer function. T-type calcium channel expression constitutes an important route for calcium influx in tumor cells that may trigger changes in cell proliferation and differentiation. In this work we investigated the role NaBu on the differentiation of lymph node carcinoma of the prostate (LNCaP) cells and its effect on T-type Ca(2+) channel expression. NaBu stimulates the morphological and molecular differentiation of LNCaP cells. Stimulation of LNCaP cells with NaBu evokes a significant increase in the expression of the Cav3.2T-type channel subunits. Furthermore, the increased Cav3.2 expression promotes membrane insertion of T-type Ca(2+) channels capable of generating fast inactivating Ca(2+) currents, sensitive to 100μM Ni(2+) ions. Inhibition of T-type Ca(2+) channel function reduces the outgrowth of neurite-like processes in LNCaP cells. NaBu-evoked expression of T-type Ca(2+) channels is also involved in the regulation of cell viability. Inhibition of T-type Ca(2+) channels causes a significant reduction in the viability of LNCaP cells treated with 1mM NaBu, suggesting that Ca(2+) influx via T-type channels can promote cell proliferation. However, increased expression of T-type Ca(2+) channels enhanced the cytotoxic effect of thapsigargin and paclitaxel on cell proliferation. These findings demonstrate that NaBu stimulates T-type Ca(2+) channel expression, thereby regulating both the morphological differentiation and growth of prostate cancer cells. Copyright © 2014. Published by Elsevier B.V.European journal of pharmacology. 12/2014; 749.
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ABSTRACT: Mitral cells express low-voltage activated Cav3.3 channels on their distal apical tuft dendrites (McKay et al., 2006; Johnston and Delaney, 2010). They also discharge Na ϩ -dependent dendritic action potentials and release glutamate from these dendrites. Around resting membrane potentials, between Ϫ65 and Ϫ50 mV, Cav3.x channels are a primary determinant of cytoplasmic [Ca 2ϩ ]. In this study using C57 mice, we present evidence that subthreshold Cav3.x-mediated Ca 2ϩ influx modulates action potential evoked transmitter release and directly drives asynchronous release from distal tuft dendrites. Presynaptic hyperpolarization and selective block of Cav3.x channels with Z941 (Tringham et al., 2012) reduce mitral-to-mitral EPSP amplitude, increase the coefficient of variation of EPSPs, and increase paired-pulse ratios, consistent with a reduced probability of transmitter release. Both hyperpolarization and Cav3.x channel blockade reduce steady-state cytoplasmic [Ca 2ϩ ] in the tuft dendrite without reducing action potential evoked Ca 2ϩ influx, suggesting that background [Ca 2ϩ ] modulates evoked release. We demonstrate that Cav3.x-mediated Ca 2ϩ influx from even one mitral cell at membrane potentials between Ϫ65 and Ϫ50 mV is sufficient to produce feedback inhibition from periglomerular neurons. Deinactivation of Cav3.x channels by hyperpolarization increases T-type Ca 2ϩ influx upon repolarization and increases feedback inhibition to produce subthreshold modulation of the mitral–periglomerular reciprocal circuit.Journal of Neuroscience 10/2014; 34(42):14032-14045. · 6.75 Impact Factor