A₁ adenosine receptor-mediated PKC and p42/p44 MAPK signaling in mouse coronary artery smooth muscle cells

Abstract

The A(1) adenosine receptor (A(1)AR) is coupled to G(i)/G(o) proteins, but the downstream signaling pathways in smooth muscle cells are unclear. This study was performed in coronary artery smooth muscle cells (CASMCs) isolated from the mouse heart [A(1)AR wild type (A(1)WT) and A(1)AR knockout (A(1)KO)] to delineate A(1)AR signaling through the PKC pathway. In A(1)WT cells, treatment with (2S)-N(6)-(2-endo-norbornyl)adenosine (ENBA; 10(-5)M) increased A(1)AR expression by 150%, which was inhibited significantly by the A(1)AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (10(-6)M), but not in A(1)KO CASMCs. PKC isoforms were identified by Western blot analysis in the cytosolic and membrane fractions of cell homogenates of CASMCs. In A(1)WT and A(1)KO cells, significant levels of basal PKC-alpha were detected in the cytosolic fraction. Treatment with the A(1)AR agonist ENBA (10(-5)M) translocated PKC-alpha from the cytosolic to membrane fraction significantly in A(1)WT but not A(1)KO cells. Phospholipase C isoforms (betaI, betaIII, and gamma(1)) were analyzed using specific antibodies where ENBA treatment led to the increased expression of PLC-betaIII in A(1)WT CASMCs while having no effect in A(1)KO CASMCs. In A(1)WT cells, ENBA increased PKC-alpha expression and p42/p44 MAPK (ERK1/2) phospohorylation by 135% and 145%, respectively. These effects of ENBA were blocked by Gö-6976 (PKC-alpha inhibitor) and PD-98059 (p42/p44 MAPK inhibitor). We conclude that A(1)AR stimulation by ENBA activates the PKC-alpha signaling pathway, leading to p42/p44 MAPK phosphorylation in CASMCs.

Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle

“…Ultimately, we demonstrated that the relationship between MAP kinase and PKC is more complex than originally hypothesized as MAP kinase is not regulated by PKC through direct phosphorylation, rather PKC indirectly regulates MAP kinase activity via activation of MAP kinase phosphatase 1 (MKP-1, also known as dual-specificity protein phosphatase 1), a phosphatase that dephosphorylates tyrosine, serine, and threonine residues (40,51). MKP-1 is present in most cell types, but, important to this present study, it is expressed in arterial smooth muscle (2,14,15,38). Moreover, we also show a negative feedback regulatory mechanism between MAP ki- nase and MKP-1 that may serve to tightly regulate MAP kinase activity.…”

“…, a requirement for MAP kinase activation (2,37,43). The increase of active MAP kinase, which translocates to the nucleus when activated, in response to histamine activation, was not augmented in the presence of Bis, but abolished with U0126 (Fig.…”

“…PKC inhibits myosin light chain phosphatase activity by activating PKC-potentiated myosin phosphatase inhibitor 17 (CPI-17), resulting in increased levels of myosin light chain phosphorylation (47,62). MAP kinase and its activator MEK have been shown to be activated via PKCmediated phosphorylation in various cell types (2,21,37,43). Furthermore, MAP kinase and PKC have been shown to translocate to the surface membrane upon phenylephrine-induced smooth muscle contraction (61).…”

Mechanisms involved in the adenosine-induced vasorelaxation to the pig prostatic small arteries

“…In fact, A 1 receptors protect against the injury caused by myocardial ischemia and reperfusion, by inhibiting adenylyl cyclase and by activating K ATP channels [23]. The A 1 receptor-mediated contractile effect has been reported in mouse aorta and coronary arteries [24,25]. A role for the phospholipase C (PLC)-protein kinase C (PKC) system has been suggested in adenosine A 1 receptor-mediated contraction of coronary vascular smooth muscle has also been demonstrated [25].…”

“…The A 1 receptor-mediated contractile effect has been reported in mouse aorta and coronary arteries [24,25]. A role for the phospholipase C (PLC)-protein kinase C (PKC) system has been suggested in adenosine A 1 receptor-mediated contraction of coronary vascular smooth muscle has also been demonstrated [25]. Other studies have shown that adenosine A 1 receptor enhances PKC expression in porcine coronary arteries [26].…”