Muscarinic M2 receptor stimulation of Cav1.2b requires phosphatidylinositol 3-kinase, protein kinase C, and c-Src

被引:39
作者
Callaghan, B [1 ]
Koh, SD [1 ]
Keef, KD [1 ]
机构
[1] Univ Nevada, Dept Cell Biol & Physiol, Reno, NV 89557 USA
关键词
smooth muscle; L-type calcium channels; kinase; patch clamp;
D O I
10.1161/01.RES.0000118248.17466.B7
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
This study investigated regulation of L- type calcium channels ( Cav 1.2b) by acetylcholine ( ACh) in rabbit portal vein myocytes. Whole- cell currents were recorded using 5 mmol/ L barium as charge carrier. ACh ( 10 mumol/ L) increased peak currents by 40%. This effect was not reversed by the selective muscarinic M3 receptor antagonist 4- DAMP ( 100 nmol/ L) but was blocked by the M2 receptor antagonist methoctramine ( 5 mumol/ L). The classical and novel protein kinase C ( PKC) antagonist calphostin C ( 50 nmol/ L) abolished ACh responses, whereas the classical PKC antagonist Go6976 ( 200 nmol/ L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3- kinase ( PI3K) inhibitor LY294002 ( 20 mumol/ L), by the c- Src inhibitor PP2 ( 10 mumol/ L) ( but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G- protein subunit Gbetagamma. Cells dialyzed with c- Src had significantly greater currents than control cells. Current enhancement persisted in the presence of LY294002, suggesting that c- Src is downstream of PI3K. Phorbol 12,13- dibutyrate ( PDBu, 0.1 mumol/ L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Go6976. The PDBu response was also reduced by PP2, and the PP2- insensitive component was blocked by Go6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to Gbetagamma, PI3K, a novel PKC, and c- Src. PDBu stimulates the novel PKC/ c- Src pathway along with a second pathway that is independent of c- Src and involves a classical PKC.
引用
收藏
页码:626 / 633
页数:8
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