Mauricio P. Boric

Microvascular transport and endothelial cell alterations preceding skeletal muscle damage in ischemia and reperfusion injury

We determined the leakage of macromolecules using FITC-dextran-150 as a tracer and measured the extent of no-reflow phenomenon by video field analysis. The cremaster muscle of anesthetized rats was fashioned as a single layer, splayed on a lucite chamber and suffused with bicarbonate solution at 35 degrees C. After a 1 hour period of baseline data collection, ischemia was produced by cross-clamping the cremasteric vascular pedicle for periods of 30 minutes and 2 hours in separate experiments. Macromolecular leakage was visualized after reinstitution of perfusion. Leakage occurred at postcapillary venules 15 to 50 micron in diameter and quickly spread to the interstitium. The magnitude of leakage decreased as a function of time with continuous buffer suffusion, but remained higher than in the control period. No reflow occurred in approximately 30 percent of the muscle microvasculature upon reperfusion. The no-reflow values at 30 minute and 2 hour periods of ischemia were significantly different from the control values but were not from each other. Electron micrographs demonstrated endothelial cell swelling and migration of leukocytes and normal myocytes after 1 hour of reperfusion following 2 hours of ischemia. Our results demonstrate that permeability changes, occurrence of no reflow, and leukocyte migration precede the onset of damage to skeletal muscle in ischemia and reperfusion injury.

Endothelial nitric oxide synthase regulates microvascular hyperpermeability in vivo

Nitric oxide (NO) is an important regulator of blood flow, but its role in permeability is still challenged. We tested in vivo the hypotheses that: (a) endothelial nitric oxide synthase (eNOS) is not essential for regulation of baseline permeability; (b) eNOS is essential for hyperpermeability responses in inflammation; and (c) molecular inhibition of eNOS with caveolin‐1 scaffolding domain (AP‐Cav) reduces eNOS‐regulated hyperpermeability. We used eNOS‐deficient (eNOS−/−) mice and their wild‐type control as experimental animals, platelet‐activating factor (PAF) at 10−7m as the test pro‐inflammatory agent, and integrated optical intensity (IOI) as an index of microvascular permeability. PAF increased permeability in wild‐type cremaster muscle from a baseline of 2.4 ± 2.2 to a peak net value of 84.4 ± 2.7 units, while the corresponding values in cremaster muscle of eNOS−/− mice were 1.0 ± 0.3 and 15.6 ± 7.7 units (P < 0.05). Similarly, PAF increased IOI in the mesentery of wild‐type mice but much less in the mesentery of eNOS−/− mice. PAF increased IOI to comparable values in the mesenteries of wild‐type mice and those lacking the gene for inducible NOS (iNOS). Administration of AP‐Cav blocked the microvascular hyperpermeability responses to 10−7m PAF. We conclude that: (1) baseline permeability does not depend on eNOS; (2) eNOS and NO are integral elements of the signalling pathway for the hyperpermeability response to PAF; (3) iNOS does not affect either baseline permeability or hyperpermeability responses to PAF; and (4) caveolin‐1 inhibits eNOS regulation of microvascular permeability in vivo. Our results establish eNOS as an important regulator of microvascular permeability in inflammation.

ACh-induced endothelial NO synthase translocation, NO release and vasodilatation in the hamster microcirculation in vivo

Studies in cultured cells show that activation of endothelial nitric oxide (NO) synthase (eNOS) requires the dissociation of this enzyme from its inhibitory association with caveolin‐1 (Cav‐1), and perhaps its translocation from plasma membrane caveolae to other cellular compartments. We investigated the hypothesis that in vivo NO‐dependent vasodilatation is associated with the translocation of eNOS from the cell membrane. To this end, we applied ACh topically (10‐100 μm for 10 min) to the hamster cheek pouch microcirculation and measured NO production, blood flow and vessel diameter, and assessed subcellular eNOS distribution by Western blotting. Baseline NO production was 54.4 ± 5.2 pmol min−1 (n= 16). ACh increased NO release, caused arteriolar and venular dilatation and elevated microvascular flow. These responses were inhibited by NG‐nitro‐L‐arginine (30 μm). The maximal increase in NO production induced by 10 μm and 100 μm ACh was 45 ± 20 % and 111 ± 33 %, respectively; the corresponding blood flow increases were 50 ± 10 % and 130 ± 24 %, respectively (n= 4‐6). Both responses followed a similar time course, although increases in NO preceded flow changes. In non‐stimulated tissues, eNOS was distributed mainly in the microsomal fraction. ACh‐induced vasodilatation was associated with eNOS translocation to the cytosolic and Golgi‐enriched fractions. After 1.5, 3.0 or 6.0 min of application, 10 μm ACh decreased the level of membrane‐bound eNOS by ‐13 ± 4 %, ‐60 ± 4 % and ‐19 ± 17 %, respectively; at the same time points, 100 μm ACh reduced microsomal eNOS content by ‐38 ± 9 %, ‐61 ± 16 % and ‐40 ± 18 %, respectively (n= 4‐5). In all cases, microsomal Cav‐1 content did not change. The close ACh concentration dependence and the concomitance between eNOS subcellular redistribution and NO release support the concept that eNOS translocation from the plasma membrane is part of an activation mechanism that induces NO‐dependent vasodilatation in vivo.

Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo.

To assess the hypothesis that gap junctions (GJs) participate on leukocyte-endothelium interactions in the inflammatory response, we compared leukocyte adhesion and transmigration elicited by cytokine stimulation in the presence or absence of GJ blockers in the hamster cheek pouch and also in the cremaster muscle of wild-type (WT) and endothelium-specific connexin 43 (Cx43) null mice (Cx43e(-/-)). In the cheek pouch, topical tumor necrosis factor-alpha (TNF-alpha; 150 ng/ml, 15 min) caused a sustained increment in the number of leukocytes adhered to venular endothelium (LAV) and located at perivenular regions (LPV). Superfusion with the GJ blockers 18-alpha-glycyrrhetinic acid (AGA; 75 microM) or 18-beta-glycyrrhetinic acid (50 microM) abolished the TNF-alpha-induced increase in LAV and LPV; carbenoxolone (75 microM) or oleamide (100 microM) reduced LAV by 50 and 75%, respectively, and LPV to a lesser extent. None of these GJ blockers modified venular diameter, blood flow, or leukocyte rolling. In contrast, glycyrrhizin (75 microM), a non-GJ blocker analog of AGA, was devoid of effect. Interestingly, when AGA was removed 90 min after TNF-alpha stimulation, LAV started to rise at a similar rate as in control. Conversely, application of AGA 90 min after TNF-alpha reduced the number of previously adhered cells. In WT mice, intrascrotal injection of TNF-alpha (0.5 microg/0.3 ml) increased LAV (fourfold) and LPV (threefold) compared with saline-injected controls. In contrast to the observations in WT animals, TNF-alpha stimulation did not increase LAV or LPV in Cx43e(-/-) mice. These results demonstrate an important role for GJ communication in leukocyte adhesion and transmigration during acute inflammation in vivo and further suggest that endothelial Cx43 is key in these processes.

Rise in endothelium-derived NO after stimulation of rat perivascular sympathetic mesenteric nerves

To evaluate whether sympathetic activity induces nitric oxide (NO) production, we perfused the rat arterial mesenteric bed and measured luminally accessible norepinephrine (NE), NO, and cGMP before, during, and after stimulation of perivascular nerves. Electrical stimulation (1 min, 30 Hz) raised perfusion pressure by 97 +/- 7 mmHg, accompanied by peaks of 23 +/- 3 pmol NE, 445 +/- 48 pmol NO, and 1 pmol cGMP. Likewise, perfusion with 10 microM NE induced vasoconstriction coupled to increased NO and cGMP release. Electrically elicited NO release depended on stimulus frequency and duration. Endothelium denudation with saponin abolished the NO peak without changing NE release. Inhibition of NO synthase with 100 microM N(omega)-nitro-L-arginine reduced basal NO and cGMP release and blocked the electrically stimulated and exogenous NE-stimulated NO peak while enhancing vasoconstriction. Blocking either sympathetic exocytosis with 1 microM guanethidine or alpha1-adrenoceptors with 30 nM prazosin abolished the electrically evoked vasoconstriction and NO release. alpha2-Adrenoceptor blockade with 1 microM yohimbine reduced both vasoconstriction and NO peak while increasing NE release. In summary, sympathetically released NE induces vasoconstriction, which triggers a secondary release of endothelial NO coupled to cGMP production.To evaluate whether sympathetic activity induces nitric oxide (NO) production, we perfused the rat arterial mesenteric bed and measured luminally accessible norepinephrine (NE), NO, and cGMP before, during, and after stimulation of perivascular nerves. Electrical stimulation (1 min, 30 Hz) raised perfusion pressure by 97 ± 7 mmHg, accompanied by peaks of 23 ± 3 pmol NE, 445 ± 48 pmol NO, and 1 pmol cGMP. Likewise, perfusion with 10 μM NE induced vasoconstriction coupled to increased NO and cGMP release. Electrically elicited NO release depended on stimulus frequency and duration. Endothelium denudation with saponin abolished the NO peak without changing NE release. Inhibition of NO synthase with 100 μM N ω-nitro-l-arginine reduced basal NO and cGMP release and blocked the electrically stimulated and exogenous NE-stimulated NO peak while enhancing vasoconstriction. Blocking either sympathetic exocytosis with 1 μM guanethidine or α1-adrenoceptors with 30 nM prazosin abolished the electrically evoked vasoconstriction and NO release. α2-Adrenoceptor blockade with 1 μM yohimbine reduced both vasoconstriction and NO peak while increasing NE release. In summary, sympathetically released NE induces vasoconstriction, which triggers a secondary release of endothelial NO coupled to cGMP production.

S-Nitrosation of β-Catenin and p120 Catenin A Novel Regulatory Mechanism in Endothelial Hyperpermeability

Rationale:Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of endothelial nitric oxide synthase (eNOS) to cytosol and stimulation of eNOS-derived nitric oxide signaling cascade. The mechanisms by which nitric oxide signaling regulates permeability at adherens junctions are still incompletely understood. Objective:We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. Methods and Results:We measured PAF-stimulated SNO of β-catenin and p120-catenin (p120) in 3 cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein), and postcapillary venular endothelial cells (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of β-catenin and p120 at the adherens junction and with hyperpermeability. Tumor necrosis fact...Rationale: Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of endothelial nitric oxide synthase (eNOS) to cytosol and stimulation of eNOS-derived nitric oxide signaling cascade. The mechanisms by which nitric oxide signaling regulates permeability at adherens junctions are still incompletely understood. Objective: We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. Methods and Results: We measured PAF-stimulated SNO of &bgr;-catenin and p120-catenin (p120) in 3 cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein), and postcapillary venular endothelial cells (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of &bgr;-catenin and p120 at the adherens junction and with hyperpermeability. Tumor necrosis factor-&agr; increased nitric oxide production and caused similar increase in SNO as PAF. To ascertain the importance of eNOS subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced SNO of &bgr;-catenin and p120 and significantly diminished association between these proteins in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Inhibitors of nitric oxide production and of SNO blocked PAF-induced SNO and hyperpermeability, whereas inhibition of the cGMP pathway had no effect. Mass spectrometry analysis of purified p120 identified cysteine 579 as the main S-nitrosated residue in the region that putatively interacts with vascular endothelial-cadherin. Conclusions: Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability.

Neuropeptide Y induced inhibition of noradrenaline release in rat hypothalamus: role of receptor subtype and nitric oxide

We aimed at characterizing the receptor subtype and the signaling pathway involved in the inhibitory effect of neuropeptide Y on the release of endogenous noradrenaline from rat hypothalamus. Slices of hypothalamus were stimulated with two trains of electrical pulses, and the release of noradrenaline and nitric oxide was measured. The electrical stimulation of hypothalamic slices induced a consistent release of both endogenous noradrenaline and NO. Neuropeptide Y inhibited concentration dependently the stimulated noradrenaline release. Similarly, agonists for neuropeptide Y Y1, Y2 and Y5 receptors inhibited noradrenaline release, albeit with a potency lower than neuropeptide Y. GW1229, a selective neuropeptide Y Y1 receptor antagonist counteracted the effect of neuropeptide Y, but not that of PYY-(3-36), an agonist active at neuropeptide Y Y5 and Y2 receptors. These results indicate that the inhibitory effect of neuropeptide Y is likely mediated by several receptor subtypes, including neuropeptide Y Y1, Y5 and possibly Y2 receptors. One microM NPY significantly enhanced NO release induced by the electrical stimulation. NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, abolished NO release and blocked the inhibitory effect of neuropeptide Y on noradrenaline release. We conclude that nitric oxide participates in the signaling pathway of neuropeptide Y in the rat hypothalamus.

Endothelin reduces microvascular blood flow by acting on and venules of the hamster cheek pouch

Superfusion of the cheek pouch with 0.1–10 nM endothelin (E) produced a concentration-related reduction in the clearance of 22Na+ used as an indicator of microvascular plasma flow. The median effective concentration was about 2 nM. The time course of E action was also concentration related. Superfusion with 10 nM E for 10 min caused a greater than 80% reduction in 22Na+ clearance: the rate at which the action of E started was significantly faster than the rate at which its action ended. Recovery did not exceed 70% even though the tissue was superfused with drug-free buffer for 90 min. The E-induced reduction in 22Na+ clearance was associated with vasoconstriction, as determined by intravital microscopy. Arterioles of 4th branching order were more sensitive to E action than arterioles of 1st or 2nd order; however, the constriction lasted considerably longer in the latter vessels. E-induced venular constriction followed a pattern analogous to that of arterioles of the same category, with the exception that the finer venules responded the least. Pretreatment of the cheek pouch with 300 nM nifedipine diminished but did not abolish the 1 nM E-induced reduction in 22Na+ clearance. and the recovery of clearance upon E washout was not accelerated by nifedipine.

GW1229, a novel neuropeptide Y Y1 receptor antagonist, inhibits the vasoconstrictor effect of neuropeptide Y in the hamster microcirculation

We studied the effect of GW1229, a novel neuropeptide Y Y1 receptor antagonists, on the vasoconstriction induced by neuropeptide Y and structurally related analogs in the hamster cheek pouch microcirculation. Changes in arteriolar diameter and microvascular conductance were assessed by intravital microscopy and measurement of sodium22 clearance. GW1229 did not affect basal vascular conductance but inhibited, concentration dependently, the reduction in arteriolar diameter and vascular conductance induced by 100 nM neuropeptide Y. GW1229 also counteracted the vasoconstrictor effect of 100 nM [Leu31,Pro34]neuropeptide Y, and that of 300 nM neuropeptide Y-[(13-36). In contrast, GW1229 had no effect on the vasoconstriction induced by noradrenaline. We conclude that the vasoconstrictor effect on neuropeptide Y in the hamster cheek pouch is mediated by neuropeptide Y Y1 receptors. The maintenance of physiological tone in this vascular bed does not involve the participation of endogenous neuropeptide Y.

Neuropeptide Y is a vasoconstrictor and adrenergic modulator in the hamster microcirculation by acting on neuropeptide Y1 and Y2 receptors

The microvascular effects of neuropeptide Y, and two analogs with preferential affinity for different neuropeptide Y receptor subtypes, were assessed by intravital microscopy on the hamster cheek pouch. The interaction of neuropeptide Y and its analogs with noradrenaline was also studied. Superfusion with 0.1-300 nM neuropeptide Y caused a concentration-dependent reduction in microvascular conductance that was paralleled by reductions in arteriolar and venular diameters. These effects of neuropeptide Y were equipotent with noradrenaline, but slower to develop and longer-lasting than that of noradrenaline. Neuropeptide Y did not affect permeability to macromolecules, as measured by extravasation of fluorescent dextran. The neuropeptide Y Y1 receptor agonist, [Leu31,Pro34]neuropeptide Y, mimicked neuropeptide Y with similar potency but shorter duration, while neuropeptide Y-(13-36), a neuropeptide Y Y2 receptor agonist, was at least 10-fold less potent than neuropeptide Y to induce a delayed and prolonged reduction in microvascular conductance. The joint superfusion of 1 nM neuropeptide Y plus 0.1 mu M noradrenaline did not cause synergism, nor even summation of effects, but reduced the contractile effect of noradrenaline. No synergism was observed after a 10 min priming with 1 nM neuropeptide Y, followed by its joint application with 0.1 mu M noradrenaline, but a significant vasodilation and hyperemia ensued upon stopping noradrenaline application. Priming with 1 nM [Leu31,Pro34]neuropeptide Y prolonged noradrenaline vasoconstriction without evidence of hyperemia. In contrast, priming with 1 nM neuropeptide Y-(13-36) significantly antagonized noradrenaline vasoconstriction. These findings indicate that both neuropeptide Y receptor subtypes are present in arterioles and venules of the hamster, and suggest that their activation with neuropeptide Y induces a rapid (Y1 receptor subtype activation) and a delayed (Y2 receptor subtype activation) vasocontractile response. The interaction with noradrenaline is complex, without evidence for synergism, but neuropeptide Y Y2 receptor activation seems to antagonize noradrenaline and/or to facilitate auto-regulatory vasodilation after the catecholamine-induced vasoconstriction.