L型Ca²⁺チャネル β_2a サブユニットはdibutyryl-cAMPによるCa²⁺電流増加に関与する

抄録

To find the different role of β subunit isoforms (β_2a and β₃) in the regulation of L-type Ca²⁺ channel by intracellular cAMP, we measured the Ca²⁺ channel current (I_Ca(L)) in wild type, β_2a- and β₃-overexpressed vascular smooth muscle (VSM) cell lines (A7r5, β_2a/A7r5 and β₃/A7r5). I_Ca(L) was recorded by whole-cell voltage clamp using a patch-clamp technique. 5 mM BaCl₂ was used as a charge carrier to record I_Ca(L). The β subunits and phosphodiesterase (PDEs) mRNA expressions were quantitated by the comparative RT-PCR method. Intracellular cAMP concentration was measured by chemiluminescent ELISA assay. Current-voltage relationships revealed that the current density of I_Ca(L) did not differ among the three groups. After 3 min-perfusion of 3 mM dibutyryl-cAMP (db-cAMP), the peak amplitude of I_Ca(L) was significantly decreased in A7r5, but was significantly increased in β_2a/A7r5. There was no change in β₃/A7r5. As compared with A7r5 cell line, type 3B and 4D PDE mRNA expressions were increased in β_2a/A7r5 cell lines and basal intracellular cAMP concentrations were decreased in β_2a/A7r5 and β₃/A7r5cell lines. Forskolin (100 μM)-induced elevation of intracellular cAMP was only seen in β_2a/A7r5 cell line. We further tested the responsiveness of I_Ca(L) to db-cAMP in β_2a and/or β₃-knock down A7r5 cells using shRNA expression vectors. [J Physiol Sci. 2008;58 Suppl:S71]