Signaling pathway underlying stimulation of L-type Ca²⁺ channels in rabbit portal vein myocytes by recombinant Gβγ subunits

Abstract

(Callaghan B, Koh SD, and Keef KD, Circ Res 94: 626 -633, 2004) have shown that voltage-dependent L-type Ca 2ϩ channels (Cav) in portal vein myocytes are enhanced when muscarinic M2 receptors are activated with ACh. Current stimulation was coupled to the G protein subunit G␤␥ along with the downstream mediators phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC), and c-Src. The present study was designed to determine whether the same second messenger pathway could be identified when exogenous recombinant G␤␥ subunits are introduced into cells. Smooth muscle myocytes were freshly isolated from rabbit portal vein, and Cav currents were recorded by using the patch-clamp technique. Dialysis of cells with recombinant G␤␥ (50 nM) significantly increased Cav currents (141%). Nifedipine (1 M) reduced both control and stimulated currents by ϳ90%. The enhancement of current by G␤␥ was equivalent to that produced by ACh (142%), whereas the PKC activator phorbol 12,13-dibutyrate (PdBu) gave rise to greater current stimulation (192%). Current stimulation with G␤␥, ACh, and PdBu were not associated with changes in the voltage dependence of activation or inactivation. The PI3K inhibitor LY-294002 (20 M) reduced peak currents by 32% in cells dialyzed with G␤␥, whereas the inactive analog LY-303511 resulted in a small but significant reduction in current (12%). The c-Src inhibitor PP2 (1 M) also significantly reduced currents (34%), whereas the inactive analog PP3 was without effect. These data provide further evidence for the hypothesis that G␤␥ leads to stimulation of Cav currents in rabbit portal vein myocytes via a signaling pathway that includes PI3K, PKC, and c-Src. vascular calcium channel; G protein ␤␥ subunits; tyrosine kinase THE ACTIVITY of voltage-dependent L-type Ca 2ϩ channels (Cav) can be modified by a number of hormones and mediators coupled to G protein-coupled receptors (GPCR) (1,12,27). G proteins are heterotrimers consisting of G␣ and G␤␥ subunits. Receptor activation of G proteins causes GDP replacement with GTP and separation of G␣ and G␤␥ subunits, which can target different effectors (20). In previous studies, we and others have shown that endogenous G␤␥ coupled to either G i , G s , or G13, as well as exogenous G␤␥ dialyzed into cells, leads to stimulation of Cav currents in portal vein myocytes (6,18,37,42,43).G␤␥ has been shown to activate some isoforms of phosphatidylinositol-3-kinase (PI3K; Ref. 4), and substantial evidence has accumulated to suggest that G␤␥ stimulation of Cav in portal vein is coupled to activation of PI3K␥ (6, 26). We and others have also proposed that a downstream mediator of PI3K after muscarinic M2 receptor activation of G␤␥ is PKC (6, 39), which in turn can give rise to activation of c-Src (6). c-Src also appears to mediate stimulation of Cav currents with M2 receptors stimulation in rabbit colonic myocytes (13), integrin ␣5␤1-mediated stimulation of Cav currents in cremaster arteriolar myocytes (41), and PDGF receptor stimulation in rabbit ear artery (40). In the ...

Hormonal signaling and signal pathway crosstalk in the control of myometrial calcium dynamics

“…In contrast, L-type current was inhibited by oxytocin in pregnant rat myometrial cells [49]. In other smooth muscles, L-type channels can be stimulated by GPCR pathways involving Gβγ, PI-3K and PKC [50]. Oxytocin has been implicated in the opening of other cation channels (consistent with SRCE) and of Ca 2+ -activated Cl − channels in myometrium, leading indirectly to depolarization and opening of voltage-dependent Ca 2+ channels [51].…”

Kinase-dependent activation of voltage-gated Ca2+channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia

“…Precedent for voltage-independent activation of VDCCs is abundant in studies conducted in cardiomyocytes and smooth muscle from systemic vascular beds (9, 13, 18 -20, 29, 32, 61) and appears to be mediated primarily by phosphorylation of VDCCs, leading to changes in channel activity independent of E m . Rho kinase (70), PKC (9,13,20,40,61), and/or tyrosine kinases, including Src (14,27,32,41), have been shown to activate VDCCs, although PKC-and tyrosine kinase-dependent inhibition of channel activity has also been reported (53,65). We found that inhibition of each of these kinases could inhibit the ET-1-induced change in [Ca 2ϩ ] i , suggesting that all were involved in the response.…”

“…The mechanisms responsible for the E m -independent activation of VDCCs are unclear. Studies (9,10,40,45,61,70) have shown that activation could be achieved through phosphorylation of the channel, possibly by Rho kinase and protein kinase C (PKC), both of which can be activated by ET-1.…”

Hemoglobin Neurotoxicity is Attenuated by Inhibitors of the Protein Kinase CK2 Independent of Heme Oxygenase Activity

“…These include inhibition of voltage-dependent calcium and potassium channels (El-Kholy et al, 2003; Callaghan et al, 2006), NF-kappaB binding activity (Kim et al, 2005), inducible nitric oxide synthase expression (Kim et al, 2005), and cell proliferation (Kristof et al, 2005). In addition, it sensitizes tumor cells to drug-induced apoptosis by increasing hydrogen peroxide production via a PI3K-independent mechanism (Poh and Pervaiz, 2005).…”