Mitochondria are central players in the pathophysiology of ischemia-reperfusion. of adenosine preconditioning (i.e., when 5-HD administration brackets adenosine perfusion) , but mitoKATP opening is required during the phase (we.e., when 5-HD administration precedes ischemia) [26C29]. Open in a separate window Number 1 GPCR-mediated signaling to mitochondriaOccupation of the GPCR prospects to activation of PI3-kinase, phosphorylation of phosphatidylinositol bisphosphate, and activation of the phosphatidylinositol-dependent kinases (PDKs) . PDKs then phosphorylate Akt, which initiates the remainder of the cytosolic signaling pathway: endothelial nitric oxide synthase (eNOS) is phosphorylated, leading to production of NO. NO stimulates guanylyl cyclase, and the cGMP produced activates protein kinase G (PKG) , which causes mitoKATP opening [62, 130C132]. Protection afforded by all of the trigger substances is blocked by PKC inhibitors, and PKC, probably PKC, is thought to be a common target of cardioprotective signaling . It has been difficult to localize the critical PKC, because multiple PKC isoforms participate in cardioprotection . In ouabain signaling, PKC acts proximally in conjunction with EGFR transactivation . The adenosine A1 receptor is believed to directly stimulate PLC and PLD to activate PKC . These PKCs are cytosolic. As discussed below, two PKC isoforms regulate mitoKATP and MPT at the level of the inner mitochondrial membrane . Thus, the physiological effect of PKC activation depends entirely on its location and not on its biochemistry, which appears to be invariant. 2.2 Non-GPCR pathways of protection – digitalis Cardiac glycosides are classic inhibitors of the plasma membrane Na+/K+-ATPase, but this enzyme also has important non-canonical functions that are triggered by digitalis. Thus, ouabain interaction with the Na, K-ATPase activates src kinase, causing formation of a binary receptor that phosphorylates and assembles other proteins into signaling modules lorcaserin HCl pontent inhibitor that transmit signals to intracellular compartments [33, 34]. Ouabain signaling has been shown to depend on mitoKATP opening and mitochondrial ROS production . Ouabain is cardioprotective in rat heart [31, 36, 37], and this cardioprotection is blocked by the mitoKATP blocker 5-hydroxydecanoate (5-HD), the ROS scavenger N-2-mercaptopropionylglycine (MPG), and the src kinase inhibitor PP2 . It is interesting to note that, whereas inhibition of the pump and consequent increase in intracellular Na+ and Ca2+ is required for positive inotropy, ouabain cardioprotection occurs at doses (about 10 M in rat) that do not produce significant enzyme inhibition  or increased contractility [31, 36, 37]. These distinctions further emphasize the dissociation of the pumping and signaling functions of Na,K-ATPase. Ouabain cardioprotection does not depend on guanylyl cyclase or PKG activities, showing that this signaling pathway differs from that triggered by GPCR agonists . Ouabain-induced inotropy also requires mitoKATP opening and ROS production [36, 38]. The rat heart Na,K-ATPase exhibits a low sensitivity to cardiac glycosides; however, we have observed qualitatively similar phenomena in the ouabain-sensitive rabbit heart. Thus, cardioprotection occurs at lower ouabain doses than those required for inotropy, and both cardioprotection and inotropy require mitoKATP opening (S. Pierre, unpublished data). lorcaserin HCl pontent inhibitor 3. From receptor to mitochondria by signalosomes We propose that cardioprotective signals are transmitted to mitochondria by signalosomes, which are vesicular, multimolecular signaling complexes that are assembled in caveolae and deliver signals to the mitochondrial outer membrane (MOM) . A diagram of the signalosome hypothesis is shown in Fig. 2. Rabbit polyclonal to ZC3H12D Open up in another window Shape 2 Signal transmitting by signalosomeIt can be proposed that discussion lorcaserin HCl pontent inhibitor of bradykinin (and additional GPCR agonists) or ouabain using their plasma membrane receptors induces development of the vesicular caveolar signaling system (signalosome) that phosphorylates receptor (R1 and R2) for the mitochondrial external membrane (Mother). The terminal kinase from the bradykinin signalosome can be PKG. The terminal kinases from the ouabain signalosome are Src and PKC kinase. Pursuing phosphorylation of mother receptor, the sign can be transmitted over the intermembrane space to activate PKC for the mitochondrial internal membrane. PKC, subsequently opens mitoKATP with a phosphorylation response . 3.1 Rationale Signaling cascades like the one portrayed in Fig. 1 must occur and precisely rapidly. We consider it unlikely that these spatio-temporal requirements can be met by random diffusional collisions. Cytosolic proteins are extensively hydrated, and the organization of this water causes a phase separation from bulk cytosolic water. Minimization of the phase boundary, in turn, causes proteins to coalesce within.