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      The Role of L- and T-Type Calcium Channels in Local and Remote Calcium Responses in Rat Mesenteric Terminal Arterioles

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          Background/Aims: The roles of intercellular communication and T-type versus L-type voltage-dependent Ca<sup>2+</sup> channels (VDCCs) in conducted vasoconstriction to local KCl-induced depolarization were investigated in mesenteric arterioles. Methods: Ratiometric Ca<sup>2+</sup> imaging (R) using Fura-PE3 with micro-ejection of depolarizing KCl solution and VDCC blockers, and immunohistochemical and RT-PCR techniques were applied to isolated rat mesenteric terminal arterioles (n = 71 from 47 rats; intraluminal diameter: 24 ± 1 μm; length: 550–700 μm). Results: Local application of KCl (at 0 μm) led to local (ΔR = 0.54) and remote (ΔR = 0.17 at 500 μm) increases in intracellular Ca<sup>2+</sup>. Remote Ca<sup>2+</sup> responses were inhibited by the gap junction uncouplers carbenoxolone and palmitoleic acid. Ca<sub>V</sub>1.2, Ca<sub>V</sub>3.1 and Ca<sub>V</sub>3.2 channels were immunolocalized in vascular smooth muscle cells and Ca<sub>V</sub>3.2 in adjacent endothelial cells. Local and remote Ca<sup>2+</sup> responses were inhibited by bath application of L- and T-type blockers [nifedipine, NNC 55-0396 and R(–)-efonidipine]. Remote Ca<sup>2+</sup> responses (500 μm) were not affected by abolishing Ca<sup>2+</sup> entry at an intermediate position on the arterioles (at 200–300 μm) using micro-application of VDCC blockers. Conclusion: Both L- and T-type channels mediate Ca<sup>2+</sup> entry during conducted vasoconstriction to local KCl in mesenteric arterioles. However, these channels do not participate in the conduction process per se.

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          Most cited references 25

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          Abnormal coronary function in mice deficient in alpha1H T-type Ca2+ channels.

          Calcium ion (Ca2+) influx through voltage-gated Ca2+ channels is important for the regulation of vascular tone. Activation of L-type Ca2+ channels initiates muscle contraction; however, the role of T-type Ca2+ channels (T-channels) is not clear. We show that mice deficient in the alpha1H T-type Ca2+ channel (alpha(1)3.2-null) have constitutively constricted coronary arterioles and focal myocardial fibrosis. Coronary arteries isolated from alpha(1)3.2-null arteries showed normal contractile responses, but reduced relaxation in response to acetylcholine and nitroprusside. Furthermore, acute blockade of T-channels with Ni2+ prevented relaxation of wild-type coronary arteries. Thus, Ca2+ influx through alpha1H T-type Ca2+ channels is essential for normal relaxation of coronary arteries.
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            Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels.

            In contrast to other kinds of voltage-gated Ca2+ channels, the underlying molecular basis of T-type and R-type channels is not well-understood. To facilitate comparisons with cloned Ca2+ channel subunits, we have carried out a systematic analysis of the properties of T-type currents in undifferentiated NG108-15 cells and R-type currents in cerebellar granule neurons. Marked differences were found in their biophysical and pharmacological features under identical recording conditions. T-type channels became activated at potentials approximately 25 mV more negative than R-type channels; however, T-type channels required potentials approximately 15 mV less negative than R-type channels to be available. Accordingly, T-type channels display a much larger overlap between the curves describing inactivation and activation, making them more suitable for generating sustained Ca2+ entry in support of secretion or pacemaker activity. In contrast, R-type channels are not equipped to provide a steady current, but are very capable of supplying transient surges of Ca2+ influx. In response to a series of increasingly strong depolarizations T-type and R-type Ca2+ channels gave rise to very different kinetic patterns. T-type current records crossed each other in a characteristic pattern not found for R-type currents. These biophysical distinctions were independent of absolute membrane potential and were, therefore, complementary to the conventional categorization of T- and R-type Ca2+ channels as low- and high-voltage activated. R-type channels deactivated approximately eight-fold more quickly than T-type channels, with clear consequences for the generation of divalent cation influx during simulated action potentials. Pharmacological comparisons revealed additional contrasts. R-type current was responsive to block by omega-Aga IIIA but not nimodipine, while the opposite was true for T-type current. Both channel types were potently inhibited by the non-dihydropyridine compound mibefradil. In all respects examined, R-type currents were similar to currents derived from expression of the alpha1E subunit whereas T-type currents were not.
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              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.

              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.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                February 2009
                02 September 2008
                : 46
                : 2
                : 138-151
                aDivision of Renal and Vascular Research, Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark; bDepartment of Physiology, Fukuoka University School of Medicine, and cDepartment of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; dLoyola University Medical Center, Maywood, Ill., USA
                151767 J Vasc Res 2009;46:138–151
                © 2008 S. Karger AG, Basel

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                Page count
                Figures: 9, Tables: 2, References: 42, Pages: 14
                Research Paper


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