β‐Adrenergic receptor stimulation of L‐type Ca²⁺ channels in rabbit portal vein myocytes involves both αs and βγ G protein subunits

Abstract

Previous studies have shown that purified G protein αs and βγ subunits stimulate vascular L‐type Ca2+ channels through protein kinase A and C (PKA and PKC), respectively. The present study tested whether activation of endogenous G proteins via β‐adrenergic receptor binding also stimulates vascular Ca2+ channels through both Gαs and Gβγ and the subsequent activation of PKA and PKC. Peak Ba2+ current (IBa) in freshly isolated rabbit portal vein smooth muscle cells was significantly increased by bath application of 0.5 μm isoproterenol (isoprenaline; ISO) when measured using the whole‐cell patch clamp method (53 ± 3 % increase, n= 15). Stimulation of IBa by ISO was partially reversed by a PKA inhibitor, KT 5720, or a PKC inhibitor, calphostin C, and completely blocked when cells were pretreated with both KT 5720 and calphostin C. Dialysis of cells with polyclonal antibody to Gαs significantly reduced but did not completely eliminate ISO‐induced stimulation of IBa. The remaining stimulation was abolished by calphostin C. Dialysis of cells with a polyclonal antibody to Gβ also significantly reduced ISO‐induced stimulation and the remaining stimulation was abolished by KT 5720. Dialysis of cells with both antibodies completely prevented the stimulation of IBa by ISO. ISO‐induced stimulation of IBa was reversed by ICI‐118,551, a specific β2‐adrenoceptor antagonist, but not by CGP 20712A, a specific β1‐adrenoceptor antagonist. In addition, the β2‐adrenoceptor agonist zinterol significantly increased peak IBa while the β1‐adrenoceptor agonist dobutamine and β3‐adrenoceptor agonist BRL 37344A had little effect on peak IBa. These data suggest that β‐adrenergic receptor stimulation of vascular L‐type Ca2+ channels involves both αs and βγ G‐protein subunits, which exert their effects through PKA and PKC, respectively.

Sphingosine 1-phosphate evokes calcium signals in C2C12 myoblasts via Edg3 and Edg5 receptors

“…However, it is tempting to speculate that it could be represented by L-type calcium channels which are expressed in C # C "# myoblasts (L. Formigli, F. Francini, E. Meacci, M. Vassalli, D. Nosi, F. Quercioli, B. Tiribilli, C. Bencini, C. Piperio, P. Bruni and S. Zecchi Orlandini, unpublished work). This hypothesis is supported by the recent observations that PKC-dependent activation of L-type calcium channels occurs in cardiac and portal vein myocytes [27,28].…”

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

“…In previous studies, we have shown that this antibody distinguishes between the G␤␥ and G␣s effects associated with adrenergic ␤ 2a receptors in portal vein myocytes. 3 Dialysis of cells with G␤ antibody (10 g/mL) entirely blocked the stimulatory effects of ACh (nϭ7; Figures 1E and 1F). …”

“…3 Inward currents were measured using an Axopatch-1D patch-clamp amplifier, digitized with a 16-bit analog to digital converter (Model DIGIDATA 1320A, Axon Instruments), and controlled by pClamp8 (Axon Instruments). Ba 2ϩ currents (I Ba ) in myocytes were measured using both the dialyzed whole cell and the perforated patch configurations.…”

“…This action involves both G-protein subunits, ie, effects of G␣s are mediated by protein kinase A (PKA) and effects of G␤␥ are mediated by protein kinase C (PKC). 3,4 The G␤␥ subunit and PKC are also required for AT 1 receptor stimulation of Cav1.2b currents in rat portal vein myocytes. 5,6 In these studies, the immediate downstream mediator of G␤␥ was found to be phosphatidylinositol-3 kinase␥ (PI3K␥).…”

Conditional Stimulation of Type V and VI Adenylyl Cyclases by G Protein βγ Subunits

“…Because ␤-adrenergic receptors have not been shown to couple to G proteins other than G s or G i , it would appear that the endogenous G␤␥ subunits that participate in isoproterenol-mediated stimulation of hACVI are most likely derived from activation of G s , implying that both ␣ and ␤␥ subunits of G s are required for a full stimulatory response from hACVI. This is akin to the role of both the ␣ and ␤␥ subunits of G s in regulation of L-type calcium channels in response to isoproterenol (49).…”