Muscarinic M2 Receptor Stimulation of Cav1.2b Requires Phosphatidylinositol 3-Kinase, Protein Kinase C, and c-Src

Abstract

Abstract-This study investigated regulation of L-type calcium channels (Cav1.2b) by acetylcholine (ACh) in rabbit portal vein myocytes. Whole-cell currents were recorded using 5 mmol/L barium as charge carrier. ACh (10 mol/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 mol/L). The classical and novel protein kinase C (PKC) antagonist calphostin C (50 nmol/L) abolished ACh responses, whereas the classical PKC antagonist Gö6976 (200 nmol/L) had no effect. ACh responses were also abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 mol/L), by the c-Src inhibitor PP2 (10 mol/L) (but not the inactive analogue PP3), and by dialyzing cells with an antibody to the G-protein subunit G␤␥. 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 mol/L) increased currents by 74%. This effect was abolished by calphostin C and reduced by Gö6976. The PDBu response was also reduced by PP2, and the PP2-insensitive component was blocked by Gö6976. In summary, these data suggest that ACh enhances Cav1.2b currents via M2 receptors that couple sequentially to G␤␥, 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.

Ryanodine receptor subtype 2 encodes Ca2+ oscillations activated by acetylcholine via the M2 muscarinic receptor/cADP-ribose signalling pathway in duodenum myocytes

“…In vascular myocytes, the function of RYR2 and RYR1 subtypes in Ca 2+ release has been studied by using antisense oligonucleotides targeting the RYR subtypes (Coussin et al, 2000). With the same antisense strategy, we showed that, in duodenum myocytes, RYR2 was the trigger for ACh-induced Ca 2+ oscillations whereas RYR1 participated Journal of Cell Science 118 (10) Although activation of M2 muscarinic receptors is classically known to inhibit adenylyl cyclase activity (Peralta et al, 1988) or to modify membrane potential by inhibiting Ca 2+ -activated K + channel (Kotlikoff et al, 1992), it has been recently proposed that M2 muscarinic receptors may induce Ca 2+ signals by activation of the cADPR pathway (White et al, 2003) or by stimulation of a voltage-dependent Ca 2+ channel (Ca v 1.2b) via the phosphatidylinositol 3-kinase/PKC pathway (Callaghan et al, 2004). Activation of the cADPR pathway by ACh in duodenum myocytes is shown by: (1) inhibition of Ca 2+ oscillations by application of the cADPR competitive antagonist (8Br-cADPR), (2) inhibition of ACh-induced Ca 2+ oscillations by inhibitors of ADP-ribosyl cyclase (ZnCl 2 , anti-CD38 antibody) and (3) detection of ADP-ribosyl cyclase activity by fluorescence experiments as the enzyme cyclizes NGD+ (non fluorescent) to produce cGDPR, a fluorescent compound (Graeff et al, 1994).…”

Oxidative Inactivation of the Lipid Phosphatase Phosphatase and Tensin Homolog on Chromosome Ten (PTEN) as a Novel Mechanism of Acquired Long QT Syndrome

“…This is not without precedent, because PDK1 is known to activate several other substrates in addition to Akt, including different PKC isoforms, p70-S6K kinases, p90-RSK kinases, SGK kinases, and even PKA (39). In fact, PI3K-dependent stimulation of calcium currents has been linked to PKC activation in both skeletal and smooth muscle myocytes (31,32). Similarly, we found that As 2 O 3 effects were blunted by PKC inhibitors.…”

“…Similarly, in myocytes isolated from rabbit portal vein, PI3K-dependent stimulation of L-type calcium currents requires downstream PKC activity (32). Consequently, we tested two PKC inhibitors for their ability to modulate As 2 O 3 -dependent increases in L-type calcium currents.…”

Identification of a Novel Vasoconstrictor Peptide Specific for the Systemic Circulation

“…Because of the relatively slow on-kinetics of the LNPinduced response, we explored the potential involvement of principal intracellular signaling pathways, such as phosphatidylinositol 3-kinase and PKC, which are known to modulate L-type Ca 2ϩ channels in vessels. 22 Although the phosphatidylinositol 3-kinase inhibitor LY294002 had no impact on vasoconstriction by LNP (10 mol/L; nϭ4; data not shown), PKC inhibition led to a reduction of LNP-induced vasoconstriction and inhibition of action potential generation in VSMCs. These findings are in agreement with earlier reports, showing that inhibition of PKC can prevent activation of L-type Ca 2ϩ channels, resulting in a reduction of vascular tone.…”