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Intravital microscopic techniques were used to examine the mechanisms underlying bradykinin-induced leukocyte/endothelial cell adhesive interactions (LECA) and venular protein leakage (VPL) in single postcapillary venules of the rat mesentery. The effects of bradykinin superfusion to increase LECA and VPL were prevented by coincident topical application of either a bradykinin-B2receptor antagonist, a cell-permeant superoxide dismutase (SOD) mimetic or antioxidant, or inhibitors of cytochrome P-450 epoxygenase (CYPE) or protein kinase C (PKC) but not by concomitant treatment with either SOD, a mast cell stabilizer, or inhibitors of nitric oxide synthase, cyclooxygenase, xanthine oxidase, NADPH oxidase, or platelet-activating factor. Immunoneutralizing P-selectin or intercellular adhesion molecule-1 (ICAM-1) completely prevented bradykinin-induced leukocyte adhesion and emigration but did not affect VPL. On the other hand, stabilization of F-actin with phalloidin prevented bradykinin-induced leukocyte emigration and VPL but did not alter leukocyte adhesion. These data indicate that bradykinin induces LECA in rat mesenteric venules via a B2-receptor-initiated, CYPE-, oxidant- and PKC-mediated, P-selectin- and ICAM-1-dependent mechanism. Bradykinin also produced VPL, an effect that was initiated by stimulation of B2receptors and involved CYPE and PKC activation, oxidant generation, and cytoskeletal reorganization but was independent of leukocyte adherence and emigration.

Long-term ethanol consumption at low to moderate levels exerts cardioprotective effects in the setting of ischemia and reperfusion (I/R). The aims of this study were to determine whether 1) a single orally administered dose of ethanol [ethanol preconditioning (EtOH-PC)] would induce a biphasic temporal pattern of protection (early and late phases) against the inflammatory responses to I/R and 2) adenosine and nitric oxide (NO) act as initiators of the late phase of protection. Ethanol was administered as a bolus to C57BL/6 mice at a dose that achieved a peak plasma concentration of ∼45 mg/dl 30 min after gavage and returned to control levels within 60 min of alcohol ingestion. The superior mesenteric artery was occluded for 45 min followed by 60 min of reperfusion beginning 10 min or 1, 2, 3, 4, or 24 h after ethanol ingestion, and the numbers of fluorescently labeled rolling and firmly adherent (stationary) leukocytes in single postcapillary venules of the small intestine were quantified using intravital microscopic approaches. I/R induced marked increases in leukocyte rolling and adhesion, effects that were attenuated by EtOH-PC 2–3 h before I/R (early phase), absent when assessed after 10 min, 1 h, and 4 h of ethanol ingestion, with an even more powerful late phase of protection reemerging when I/R was induced 24 h later. The anti-inflammatory effects of late EtOH-PC were abolished by treatment with adenosine deaminase, an adenosine A2(but not A1) receptor antagonist, or a NO synthase (NOS) inhibitor during the period of EtOH-PC. Preconditioning with an adenosine A2(but not an A1) receptor agonist in lieu of ethanol 24 h before I/R mimicked the protective actions of late phase EtOH-PC. Like EtOH-PC, the effect of preconditioning with an adenosine A2receptor agonist was abrogated by coincident NOS inhibition. These findings suggest that EtOH-PC induces a biphasic temporal pattern of protection against the proinflammatory effects of I/R. In addition, our observations are consistent with the hypothesis that the late phase of EtOH-PC is triggered by NO formed secondary to adenosine A2receptor-dependent activation of NOS during the period of ethanol exposure.

We and others have previously demonstrated that intestinal ischemia-reperfusion (I/R) is associated with a large increase in oxidant production that contributes to microvascular barrier disruption in the small bowel. It has been suggested that the bulk of tissue damage during reperfusion can be attributed to adherent, activated neutrophils. From these observations, we hypothesized that pretreatment with PR-39, an endogenous neutrophil antibacterial peptide that is also a potent inhibitor of the neutrophil NADPH oxidase, would prevent postischemic oxidant production and the development of oxidant-dependent sequelae to I/R such as increased venular protein leakage. To test this postulate, oxidant production, venular protein leakage, leukocyte adhesion, and leukocyte emigration were monitored during reperfusion in control (no ischemia) rat mesenteric venules and in mesenteric venules subjected to I/R alone or PR-39 + I/R. Treatment with a single intravenous bolus injection of PR-39 (administered at a dose to achieve an initial blood concentration of 5 μM) abolished I/R-induced leukocyte adhesion and emigration in vivo. In vitro studies indicated that PR-39 prevents platelet-activating factor-induced neutrophil chemotaxis as well as phorbol myristate acetate (PMA)-stimulated intercellular adhesion molecule-1 expression by cultured endothelial cells. PR-39 pretreatment of rat neutrophils also blocked PMA-stimulated neutrophil adhesion to activated endothelial monolayers. In vivo, I/R was associated with a marked and progressive increase in oxidant production and venular protein leakage during reperfusion, effects that were abolished by PR-39 treatment. The results of this study indicate that PR-39 completely abolishes postischemic leukocyte adhesion and emigration. The time course for inhibition of oxidant production by PR-39 suggests that its antiadhesive properties account for this effect of the peptide. PR-39 may thus be therapeutically useful for prevention of neutrophil adhesion and activation during the postischemic inflammatory response.

Ischemia and reperfusion (I/R) induces neutrophil infiltration in skeletal muscle that is localized to the ischemic region. To transmigrate at ischemic regions, granulocytes must first arrest in the postcapillary venular segment of the microcirculation. Initially, leukocytes roll along the endothelium of these venules, a weak adhesive interaction that is mediated by the selectins (L-, E-, and P-selectin). Leukocyte rolling functions to slow the neutrophil during its transit through the microcirculation, thereby allowing it to monitor its local environment for the presence of activating factors arising from the ischemic tissues. When activated, the rolling granulocyte is rendered capable of forming the stronger adhesive interactions that allow the cell to become arrested in postcapillary venules in the ischemic region. These adhesive interactions are mediated by a leukocyte glycoprotein complex designated CD11/CD18 and intercellular adhesion molecule-1 (ICAM-1) expressed on endothelial cells. The stationary neutrophil uses the gradient in concentration of soluble chemoattractants liberated from ischemic tissues as a directional cue to move from the vascular to extravascular compartment, being guided in its transit across the endothelium by interactions with platelet endothelial cell adhesion molecule-1 (PECAM-1), an adhesive molecule localized to the interendothelial cleft. This paper reviews current understanding of the mechanisms underlying the establishment of leukocyte/endothelial cell interactions in postischemic skeletal muscle in terms of specific adhesion molecules that participate in neutrophil sequestration after I/R. Discovery of the molecular determinants of neutrophil/endothelial cell adhesion has uncovered potential mechanisms whereby agents exhibiting anti-adhesive properties may act. The micronized purified flavonoid fraction (450 mg diosmin, 50 mg hesperidin) prevents I/R-induced leukocyte adhesion in skeletal muscle. This anti-adhesive effect appears to be mediated at least in part by inhibition of induced expression of ICAM-1.

We investigated the influence of aging on cardiac baroreflex function during dynamic exercise in seven young (22 ± 1 yr) and eight older middle-aged (59 ± 2 yr) healthy subjects. Carotid-cardiac baroreflex function was assessed at rest and during moderate-intensity steady-state cycling performed at 50% heart rate reserve (HRR). Five-second pulses of neck pressure and neck suction from +40 to −80 Torr were applied to determine the operating point gain (GOP) and maximal gain (GMAX) of the full carotid-cardiac baroreflex function curve and examine baroreflex resetting during exercise. At rest, mean arterial pressure (MAP) and heart rate were similar between the younger and older subjects. In contrast, the resting GOP and GMAX were significantly lower in the older subjects. The increase in MAP from rest to exercise was greater in the older subjects (Δ +20 ± 2 older vs. Δ +6 ± 3 younger mmHg; P < 0.001). However, the GOP was similar in both groups during exercise because of a reduction in the younger subjects. In contrast, GMAX was unchanged from rest and therefore remained lower in older subjects (−0.19 ± 0.05 older vs. −0.42 ± 0.05 younger beats·min−1·mmHg−1; 50% HRR; P < 0.001). Furthermore, exercise resulted in an upward and rightward resetting of the cardiac baroreflex function curve in both groups. Collectively, these findings suggest that the cardiac baroreflex function curve appropriately resets during exercise in older subjects but operates at a reduced GMAX primarily because of age-related reductions in carotid-cardiac control manifest at rest.

We sought to determine the mechanisms whereby brief administration of bradykinin (bradykinin preconditioning, BK-PC) before prolonged ischemia followed by reperfusion (I/R) prevents postischemic microvascular dysfunction. Intravital videomicroscopic approaches were used to quantify I/R-induced leukocyte/endothelial cell adhesive interactions and microvascular barrier disruption in single postcapillary venules of the rat mesentery. I/R increased the number of rolling, adherent, and emigrated leukocytes and enhanced venular albumin leakage, effects that were prevented by BK-PC. The anti-inflammatory effects of BK-PC were largely prevented by concomitant administration of a B2-receptor antagonist but not by coincident B1receptor blockade, nitric oxide (NO) synthase inhibition, or cyclooxygenase blockade. However, NO synthase blockade during reperfusion after prolonged ischemia was effective in attenuating the anti-inflammatory effects of BK-PC. Pan protein kinase C (PKC) inhibition antagonized the beneficial effects of BK-PC but only when administered during prolonged ischemia. In contrast, specific inhibition of the conventional PKC isotypes failed to alter the effectiveness of BK-PC. These results indicate that bradykinin can be used to pharmacologically precondition single mesenteric postcapillary venules to resist I/R-induced leukocyte recruitment and microvascular barrier dysfunction by a mechanism that involves B2receptor-dependent activation of nonconventional PKC isotypes and subsequent formation of NO.

Ischemic preconditioning (IPC) prevents the deleterious effects of prolonged ischemia and reperfusion (I/R). Because leukocyte infiltration is required to produce the microvascular dysfunction induced by I/R in the small intestine, and P-selectin-dependent leukocyte rolling is a requisite step in this process, we hypothesized that IPC would attenuate postischemic P-selectin expression. To address this postulate, P-selectin expression was evaluated in nonischemic (control) rat jejunum and in rat jejunum subjected to I/R alone (20 min ischemia/60 min reperfusion), or IPC (5 min ischemia/10 min reperfusion) + I/R using a dual radiolabeled monoclonal antibody approach. I/R was associated with a sevenfold increase in jejunal P-selectin expression, an effect that was completely abolished by IPC. Exposing the bowel to adenosine deaminase or an adenosine A1, but not an A2, receptor antagonist during the period of preconditioning ischemia or to selective PKC antagonists during prolonged ischemia prevented the beneficial effect of IPC to limit I/R-induced P-selectin expression. Our data indicate that P-selectin expression is a novel downstream effector target of the adenosine-initiated, PKC-dependent, anti-inflammatory signaling pathway in IPC.

Although a number of recent reports indicate that bradykinin attenuates ischemia- reperfusion (I/R)-induced tissue injury, the mechanisms underlying its protective actions are not fully understood. However, because bradykinin induces endothelial nitric oxide (NO) production and NO donors have been shown to attenuate postischemic leukocyte adhesion, endothelial barrier disruption, and tissue injury, we hypothesized that bradykinin may act to reduce I/R-induced tissue injury by preventing leukocyte recruitment and preserving microvascular barrier function. To address this postulate, we used intravital videomicroscopic approaches to quantify leukocyte-endothelial cell interactions and microvascular barrier function in single postcapillary venules in the rat mesentery. Reperfusion after 20 min of ischemia significantly decreased wall shear rate and leukocyte rolling velocity, increased the number of rolling, adherent, and emigrated leukocytes, and disrupted the microvascular barrier as evidenced by enhanced venular albumin leakage. Superfusion of the mesentery with bradykinin (10 nM) during I/R significantly reduced these deleterious effects of I/R. Although these inhibitory effects of bradykinin were not affected by cyclooxygenase blockade with indomethacin (10 μM), coadministration with NO synthase ( Nω-nitro-l-arginine methyl ester, 10 μM) or bradykinin B2-receptor (HOE-140, 1 μM) antagonists abolished the protective actions of bradykinin. Plasma NO concentration was measured in the mesenteric vein and was significantly decreased after I/R, an effect that was prevented by bradykinin treatment. These results indicate that bradykinin attenuates I/R-induced leukocyte recruitment and microvascular dysfunction by a mechanism that involves bradykinin B2-receptor-dependent NO production.

The results of several recent studies indicate that bradykinin protects tissues against the deleterious effects of ischemia-reperfusion (I/R). However, other studies indicate that bradykinin can act as a proinflammatory agent, inducing P-selectin expression, the formation of chemotactic stimuli, and endothelial barrier disruption. In the present study, we used intravital microscopic techniques to examine the dose-dependent effects of bradykinin on leukocyte-endothelial cell interactions, the formation of platelet-leukocyte aggregates, and venular hemodynamics in rat mesentery in an attempt to explain these divergent findings. Superfusion of the mesentery with low concentrations of bradykinin (≤10−7 M) increased venular erythrocyte velocity ( V RBC) without increasing the number of adherent leukocytes, whereas higher concentrations (≥10−6M) decreased V RBC, increased the number of platelet-leukocyte aggregates, and induced leukocyte adhesion in single postcapillary venules. The formation of platelet-leukocyte aggregates and increased leukocyte adhesion induced by high-dose bradykinin were attenuated by administration of a B2-receptor (HOE-140) or a platelet-activating factor (PAF, WEB-2086) antagonist. Thus these adhesive interactions induced by high-dose bradykinin appear to be mediated by a mechanism that is dependent on B2-receptor activation and the formation of PAF or PAF-like lipids. The effects of bradykinin on venular V RBC and blood flow were also concentration dependent, with low doses producing nitric oxide-mediated vasodilation, whereas high doses decreased V RBC by a mechanism that is PAF independent.

Intravital microscopy was used to determine whether ischemic preconditioning (IPC; 5 min ischemia and 10 min reperfusion) would attenuate leukocyte adhesion and emigration induced by subsequent prolonged ischemia (60 min) and reperfusion (60 min) (I/R) in murine cremaster muscle and whether adenosine produced during IPC and/or reperfusion contributed to these beneficial effects. I/R elicited a marked increase in the number of adherent and emigrated leukocytes compared with the nonischemic control muscles, an effect that was largely prevented by IPC. Superfusion of the cremaster with adenosine deaminase only during IPC or only during 60-min reperfusion attenuated the inhibitory effect of IPC on postischemic leukocyte adhesion and emigration. However, the beneficial effects of IPC were mimicked in cremaster muscles preconditioned with adenosine (topical application for 10 min beginning 20 min before the onset of prolonged ischemia). Similar results were obtained in experiments in which adenosine was topically applied to the cremaster only during the 60-min reperfusion period. Our findings suggest that the ability of IPC to attenuate postischemic leukocyte adhesion and emigration may be mediated by adenosine released during IPC and during reperfusion after prolonged ischemia.

The purpose of this study was to determine whether fructose-1,6-diphosphate (FDP) or adenosine (Ado), administered at the onset of reperfusion, would prevent ischemia/reperfusion (I-R)-induced leukocyte adherence and microvascular dysfunction in skeletal muscle. Changes in vascular permeability and tissue neutrophil content were assessed by measurement of the solvent drag reflection coefficient (delta) for total plasma proteins and muscle myeloperoxidase (MPO) activity, respectively, in continuously perfused, isolated canine gracilis muscles and in muscles subjected to I-R alone, I-R + FDP, and I-R + Ado. To determine whether FDP or Ado would attenuate leukocyte-endothelial cell adhesive interactions induced by I-R, leukocyte adherence and emigration were assessed in postischemic mouse cremaster muscles, using intravital microscopy in the presence and absence of FDP or Ado during reperfusion. I-R was associated with a marked increase in microvascular permeability and muscle MPO activity relative to nonischemic controls. These increases were attenuated by FDP and Ado. I-R also increased the number of adherent and emigrated leukocytes relative to control. I-R-induced leukocyte adherence and emigration were significantly attenuated by either FDP or Ado. These results indicate that FDP and Ado attenuate postischemic microvascular barrier dysfunction in skeletal muscle by a mechanism that may be related to their ability to inhibit leukocyte adhesion and emigration.

Ischemic preconditioning (IPC) refers to a phenomenon in which a tissue is rendered resistant to the deleterious effects of prolonged ischemia and reperfusion by prior exposure to brief, repeated periods of vascular occlusion. The purposes of this study were to determine whether IPC would reduce the extent of capillary no-reflow in postischemic skeletal muscle and whether the protective effect of IPC was due to activation of ATP-sensitive potassium (KATP) channels. To address the first aim, capillary perfusion was assessed in vascularly isolated canine gracilis muscles subjected to 4.5 h of continuous perfusion, 4 h of ischemia followed by 30 min of reperfusion (I-R), and IPC (4 periods of 5 min ischemia followed by 5 min reperfusion) before I-R. I-R was associated with a reduction in the number of patent capillaries per fiber (0.6 +/- 0.1) relative to nonischemic control muscles (2.5 +/- 0.1), an effect that was attenuated by IPC (1.3 +/- 0.1 patent capillaries fiber). A role for KATP channels in the protective effect of IPC is supported by the observation that administration of a KATP channel antagonist (glibenclamide) 10 min before induction of IPC abolished the protective effect of preconditioning (0.6 +/- 0.1 patent capillaries/fiber). On the other hand, treatment of nonpreconditioned muscles with a KATP channel agonist (pinacidil) mimicked the protection afforded by IPC (1.2 +/- 0.1 patent capillaries/fiber). Moreover, the protective effect of pinacidil treatment was reversed by prior administration of glibenclamide (0.5 +/- 0.1 patient capillaries/fiber). These data indicate that IPC improves postischemic capillary perfusion by a mechanism that involves activation of KATP channels.