2-Arachidonylglyceryl ether and abnormal cannabidiol-induced vascular smooth muscle relaxation in rabbit pulmonary arteries via receptor-pertussis toxin sensitive G proteins-ERK1/2 signaling.

The receptor(s) used by cannabinoids to relax vascular smooth muscle is unknown. Here, we investigated the effects of 2-arachidonylglyceryl ether (2-AG ether), a metabolically stable endocannabinoid, and abnormal cannabidiol (abn-CBD) on relaxation of permeabilized pulmonary arterial strips monitored with force, and on extracellular signal-regulated mitogen-activated protein kinases (ERK1/2) phosphorylation in permeabilized vascular smooth muscle cells using immunoblotting. We found that 2-AG ether and abn-CBD caused relaxation and increased phosphorylation of ERK1/2. 2-AG ether effects were completely abolished by N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A), and partially blocked by (-)-1.3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol (O-1918). In contrast, abn-CBD effects were completely abolished by O-1918, and only partially blocked by AM251, and SR141716A. Both 2-AG ether and abn-CBD effects were partially blocked by pertussis toxin, an inhibitor of Gi/o proteins. PD98059, an inhibitor of mitogen activated protein kinase kinase (MEK), completely abolished the relaxation, but only partially blocked the increased phosphorylation of ERK1/2 by 2-AG ether. In contrast, abn-CBD-induced relaxation was partially blocked and the increased phosphorylation of ERK1/2 was abolished by PD98059. These findings suggest that 2-AG ether and abn-CBD-induced vascular smooth muscle relaxation are mediated by the cannabinoid CB1 receptor, and the abn-CBD receptor, respectively, and are modulated by cross-talk between the receptors. These responses occur mainly by coupling to pertussis toxin sensitive G proteins, but also, in part independent of these G proteins, which have been classically thought to initiate MEK/ERK1/2 signaling to relax vascular smooth muscle.

Role of protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and mitogen-activated protein kinases in volatile anesthetic-induced relaxation in newborn rabbit pulmonary artery.

BACKGROUND: This study examined the responsiveness of skinned pulmonary arteries from newborn rabbit to volatile anesthetics and the role of protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the downstream effectors, mitogen-activated protein kinases (ERK1/2 and p38). METHODS: Pulmonary arterial strips from 9- to 12-day-old rabbits were mounted on force transducers and treated with saponin ("skinned" strips). The skinned strips were activated by pCa 6.3 until force reached a steady state (control). Isoflurane or halothane was then administered. The result (test) was expressed as a percentage of the control. Inhibitors included bisindolylmaleimide (Ca2+-dependent and -independent PKC), Gö6976 (Ca2+-dependent PKC), CKIINtide (CaMKII), KN-93 (CaMKII), PD98059 (MEK/ERK1/2), and SB203580 (p38). RESULTS: The anesthetics dose-dependently decreased pCa-induced force (4-32% for 1-5% isoflurane; 17-76% for 1-3% halothane). The inhibitors of PKC (bisindolylmaleimide and Gö6976) and MEK/ERK1/2 (PD98059) completely prevented the relaxation induced by 3% isoflurane and partially prevented that induced by 2% and 3% halothane with the same effective inhibitor concentrations. In contrast, the effective concentration of CaMKII inhibitors was a direct function of the anesthetic concentration for different inhibitors (KN-93 for isoflurane and CKIINtide for halothane), and that of the p38 inhibitor (SB20358) was a direct function of both anesthetics. CONCLUSIONS: In Ca2+-clamped skinned pulmonary arterial strips from newborn rabbits, the anesthetics induce relaxation, which is prevented by the PKC inhibitors MEK/ERK/12, CaMKII, and p38. It is proposed that the anesthetic-induced relaxation is via cPKC/MEK/ERK1/2 and CaMKII/p38 pathways and, in addition, via CaMKII-p/MLCK-p(-)/MLC-p(-) for halothane.

Ca(2+)-calmodulin-dependent protein kinase II plays a major role in halothane-induced dose-dependent relaxation in the skinned pulmonary artery.

BACKGROUND: Previously, the authors have shown in Ca(2+)-clamped skinned arterial strips that protein kinase C (PKC) plays a role in 3% halothane- or isoflurane-increased force. PKC in the pulmonary artery and Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) in the femoral artery have been implicated in isoflurane-induced relaxation. For this study, the authors used clinical concentrations of halothane to examine the role of PKC and CaMKII in the halothane-induced biphasic effect on contraction in skinned pulmonary arterial strips. METHODS: Rabbit pulmonary arterial strips were mounted on force transducers and treated with saponin to make the sarcolemma permeable ("skinning"). Skinned strips were activated by low Ca(2+) (pCa 6.3) buffered with 7 mm EGTA, or the PKC activator phorbol-12,13-dibutyrate (PDBu, 1 microm) until force reached a steady state (control). Halothane (1, 2, and 3%) was administered, and the force was observed at peak and 15 min (test results). Ca(2+) ionophore (A23187, 10 microm) and inhibitors were preincubated in a relaxing solution and present in subsequent contracting solutions. Inhibitors were bisindolylmaleimide and Gö6976 for PKC, and KN-93 and the inhibitor protein (CKIINtide) for CaMKII. RESULTS: Halothane (1-3%) dose-dependently caused an initial increase (18-35%) and a subsequent decrease (48-68%) in pCa 6.3-induced force. Bisindolylmaleimide, 3 and 10 microm, completely blocked the increase in force at 2% and 3% halothane, respectively. CKIINtide, 0.1 microm, reduced the force at 3% halothane. The decrease in force at 1% and 2% halothane was partially prevented by 0.01 microm bisindolylmaleimide, and at 1, 2, and 3% halothane by 0.01, 0.1, and 1 microm CKIINtide, respectively. At 3% halothane, the increased force was abolished by A23187. In PDBu-induced force, 3% halothane-induced relaxation was also partially prevented by lower concentrations of KN-93 and CKIINtide. CONCLUSIONS: In skinned pulmonary arterial strips, the dose-dependent increase in force by halothane is associated with PKC activation, and that of decrease is associated with CaMKII activation.

Role of PKC in isoflurane-induced biphasic contraction in skinned pulmonary arterial strips.

BACKGROUND: Activation or inhibition of protein kinase C (PKC) has been implicated in the anesthetic-induced contraction or relaxation of different types of arteries. In skinned pulmonary arterial strips, the initial halothane-induced contraction has been attributed to PKC activation, but the subsequent relaxation has not. Whether isoflurane has a similar biphasic effect is not known. This study examined the role of PKC and its isoforms in the effect of isoflurane on pulmonary artery. METHODS: Rabbit pulmonary arterial strips were mounted on force transducers and treated with saponin to make the sarcolemma permeable ("skinned" strips). Skinned strips were activated by low Ca(2+) (pCa 6.5 or pCa 6.3 buffered with 7 mm EGTA) or the PKC activator phorbol-12,13-dibutyrate (1 microm) until force reached a steady state (control). Various concentrations of isoflurane (test) were administered, and force was observed at time intervals up to 60 min. The PKC inhibitors (bisindolylmaleimide and Go6976 from 0.1 to 10 microm) were preincubated in a relaxing solution and the subsequent contracting solutions. The results were expressed as a percentage of control, with P < 0.05 considered significant for statistical comparison between the tests and time controls. RESULTS: In a dose-dependent fashion, isoflurane (1-5%) initially increased (5-40%) and then decreased (3-70%) the Ca(2+)- or phorbol-12,13-dibutyrate (pCa 6.7 buffer)-activated force. The increased in force caused by isoflurane was partially reduced by 3 and 10 microm bisindolylmaleimide, but not by Go6976. Isoflurane-induced relaxation was partially prevented by both inhibitors at 0.1 and 0.3 microm. CONCLUSIONS: Isoflurane causes biphasic effects in skinned pulmonary arterial strips that may be in part mediated by different isoforms of PKC.