Signaling Mechanisms That Induce the Adaptive Transformation to a Protected Phenotype in the Microcirculation after Ethanol Ingestion

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The fact that the protected or preconditioned state that is induced by ethanol ingestion does not become apparent until it is removed from the tissues implies that the alcohol induces biochemical adaptations within the affected cells that render them resistant to the deleterious effects of I/R. Moreover, the differences in the time course for development and magnitudes of protection of the acute versus delayed phases of the anti-inflammatory phenotype induced by ethanol suggest that they may rely on different mechanisms.

The acute phase of protection induced by ethanol ingestion appears to arise as a result of the effect of ethanol to increase the concentration of adenosine in extracellular fluid, an effect related to the ability of the alcohol to inhibit the nucleoside transporter in cell membranes. This concept is based on the observation that the anti-inflammatory effects of superfusing the mesentery with ethanol at 45mg/dL for 10 minutes followed by a 10-minute washout period prior to I/R are prevented by coincident treatment with adenosine deaminase or an adenosine A1/A3, but not an A2, receptor antagonist. Moreover, the acute-phase beneficial actions of ethanol in the microcirculation are mimicked by treatment with adenosine A1 receptor agonists. It appears that activation of specific protein kinase C isoforms and mitochondrial ATP-sensitive potassium (KATP) channels may play a role as downstream signaling elements in producing the protected phenotype. These results suggest that acute ethanol exposure induces ischemic tolerance by a mechanism similar to that described for early phase (or classical) ischemic preconditioning. However, one major difference in these two forms of cardioprotection appear to involve their respective mechanisms for increasing interstitial fluid adenosine concentrations. That is, decreased washout of adenosine occurs secondary to the reductions in blood flow during the bouts of preconditioning ischemia, whereas acute ethanol exposure inhibits the nucleoside transporter in cell membranes, thereby preventing adenosine reuptake.

Increased tissue adenosine levels are also required to trigger the development of the late-phase anti-inflammatory phenotype (Figure 2). However, adenosine A2 receptor occupancy is required for this process, an important mechanistic distinction as the downstream signaling elements activated by adenosine A1/A3 versus A2 receptors are very different. These findings suggest that the acute versus chronic phases of protection may involve downstream phophotidylinositol-3-kinase (PI3K)/protein kinase B (Akt)- versus adenylyl cyclase/cAMP/protein kinase A-dependent signaling mechanisms, respectively. Indeed, adenylyl cyclase or protein protein kinase A blockade, but not PI3K inhibitors, prevents the late phase of ethanol preconditioning. In addition, administration of cell-permeant

NADPH Oxidase AdenĀ°sme i

Adenosine A2-Receptor Activation

Adenylyl Cyclase ^ATP

cAMP

Protein Kinase A i eNOS Activation

Superoxide + Nitric Oxide + Superoxide i

Peroxynitrite

Anti-inflammatory Phenotype

Figure 2 Factors involved in the triggering or initiation of the antiinflammatory phenotype exhibited by postcapillary venules 12 to 24 hours after ethanol ingestion (late phase of ethanol preconditioning). See text for explanation.

cAMP analogs or adenylyl cyclase activators (e.g., isoproterenol, forskolin) mimics the postischemic antiinflammatory effects of late-phase ethanol preconditioning.

Because ethanol enhances both basal and flow-stimulated nitric oxide synthase (NOS) activity and nitric oxide (NO) production in vivo and in cultured endothelial cells, it has been suggested that production of this gaseous monoxide during the period of ethanol exposure may serve as an important triggering element for the late phase of ethanol preconditioning. Support for this concept is derived from four lines of evidence. First, administration of NOS antagonists just prior to, but not 1 hour after, ethanol administration on day 1 abolishes the antiadhesive effects of late EtOH-PC on day 2. The latter observation supports the concept that NO plays an important role in instigating the development of the anti-inflammatory phenotype that becomes apparent 18 to 24 hours later. Second, plasma levels of nitrite/nitrate, a marker for NO production, are increased during the period of ethanol exposure. Third, tissues pre-treated with NO donors in lieu of ethanol develop an anti-inflammatory phenotype 24 hours after administration. Finally, the anti-inflammatory phenotype induced by

Xanthine Oxidase ethanol does not appear in mice that are genetically deficient in endothelial nitric oxide synthase (eNOS). This last finding not only provides the fourth line of evidence supporting a role for NO as a triggering element in ethanol preconditioning, but also indicates that the eNOS isoform is essential for the development of the anti-inflammatory phenotype in response to ethanol.

Although the factors responsible for increasing eNOS activity in late EtOH-PC (or any form of preconditioning for that matter) are unknown, the observations that NOS inhibition was as effective as adenosine deaminase or adenosine A2 receptor blockade in abolishing the protective effects of late EtOH-PC suggest that adenosine and NO may serve as sequential triggering elements in the signaling cascade that induces the development of the protected phenotype rather than acting as independent initiators of this preconditioned state. Indeed, the findings that (1) NOS inhibition prevents late preconditioning induced by coadministration of an adenosine A2 receptor agonist; (2) the development of the late-phase anti-inflammatory phenotype in response to NO donors is not prevented by adenosine A2 receptor antagonists; and (3) NO donors, but not adenosine A2 receptor agonists, induce the development of a preconditioned state in eNOS knockout animals support the concept that ethanol triggers the development of an anti-inflammatory state by a mechanism involving adenosine A2 receptor-dependent eNOS activation. This notion is supported by the observations that ligation of adenosine A2 receptors increases the activity of cAMP-dependent kinase (PKA), which in turn activates eNOS by phosphorylating Ser-1177. Moreover, adenosine stimulates l-arginine transport and NO biosynthesis by activation of A2 receptors on human umbilical vein endothelial cells.

Another well-known effect of ethanol is to increase the generation of reactive oxygen species, including superoxide and the hydroxyethyl radical. Although they are generally considered to exert deleterious effects in biologic systems, it is becoming increasingly apparent that reactive oxygen species may participate in a number of normal physiologic phenomena by serving as second messengers in transmembrane signaling processes. Indeed, administration of a cell-permeant superoxide dismutase mimetic, MnTBAP, coincident with ethanol prevents the postischemic antiadhe-sive effects that become apparent 24 hours after ingestion of the alcohol. Moreover, exposing postcapillary venules to a superoxide generating system (hypoxanthine/xanthine oxidase) 24 hours prior to I/R mimicked the antiadhesive effects produced by antecedent ethanol exposure. Additional support for the concept that oxidants may participate in triggering the development of the anti-inflammatory phenotype in response to antecedent ethanol ingestion is derived from studies directed at elucidating their source of production. Inhibition of either xanthine oxidase or NADPH oxidase alone attenuated the antiadhesive effects of ethanol preconditioning by 50 percent, whereas concomitant inhibition of both oxidant-producing enzymes effectively prevented the development of the protected phenotype. The latter studies indicate that xanthine oxidase and NADPH oxidase are important enzymatic sources of the reactive oxygen species that trigger entrance into the anti-inflammatory phenotype displayed by postcapillary venules exposed to ethanol 24 hours prior to I/R.

As noted earlier, there is evidence implicating nitric oxide, formed secondary to adenosine A2-receptor-dependent activation of endothelial nitric oxide synthase, in the beneficial actions of antecedent ethanol ingestion. This raises the possibility that NO produced during the period of ethanol preconditioning initiates the protective effects of late EtOH-PC by a mechanism that involves its interaction with xanthine oxidase- and/or NAD(P)H oxidase-derived oxidants. This is an important issue because we have obtained preliminary evidence implicating isoform-selective protein kinase C (PKC) translocation and activation as an obligatory downstream signaling element in late EtOH-PC (unpublished observations). However, NO and NO-releasing agents reversibly inactivate PKC. On the other hand, peroxynitrite, which is formed by the interaction of NO with superoxide, not only induces PKC activation, but has been implicated as a trigger for the beneficial actions of other forms of preconditioning including that induced by antecedent exposure to brief ischemia.

Although the mediators of late phase ethanol preconditioning are unclear, the time course required for its development suggests that the appearance of the protected phenotype 18 to 24 hours after ethanol ingestion requires the formation of new gene products capable of producing anti-inflammatory agents. In this regard, it is tempting to speculate that ethanol might enhance the expression of cyclooxygenase-2 (COX-2), inducible NOS (iNOS), or heme oxygenase-1 (HO-1), which in turn generate prostacy-clin and other eicosanoids, NO, and carbon monoxide, respectively, all of which produce robust antiadhesive effects in postcapillary venules. In addition to carbon monoxide, HO-1 also generates the powerful antioxidants bilirubin and biliverdin, which may act to prevent the formation of oxidant-dependent chemoattractants during I/R. It is also possible that ethanol may exploit the antiadhesive properties of adenosine as a mediator of the preconditioned state secondary to enhanced production or decreased salvaging (via activation of 5'-nucleotidase or inhibition of adenosine kinase, respectively) of the nucleoside in post-ischemic tissues.

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