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      Protective Effects of Insulin during Ischemia-Reperfusion Injury in Hamster Cheek Pouch Microcirculation


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          Objective: The effects of insulin (0.18 n M–0.18 µ M) on reduced capillary perfusion, microvascular permeability increase and leukocyte adhesion induced by ischemia-reperfusion injury were investigated in the hamster cheek pouch microcirculation. To gain insight into the insulin’s mechanism of action, the effects of its higher concentration (0.18 µ M) were investigated after inhibition of tyrosine kinase (TK), nitric oxide synthase (NOS), protein kinase C (PKC), phosphatidylinositol 3-kinase and K<sup>+</sup><sub>(ATP)</sub> channels, alone or in combination. Two concentrations for each inhibitor were used. Methods: Microcirculation was visualized by fluorescence microscopy. Perfused capillary length, microvascular permeability, leukocyte adhesion to venular walls, vessel diameter and capillary red blood cell velocity were assessed by computer-assisted methods. Measurements were made at baseline (B), after 30 min of ischemia (I), and after 30 min of reperfusion (R). Results: In control animals, perfused capillary length decreased by 63 ± 5% of baseline at R. Microvascular permeability increased at I and R, while leukocyte adhesion was most pronounced in V1 postcapillary venules at R. Insulin dose-dependently preserved capillary perfusion at R (–28 ± 6 and –15 ± 6% of baseline), but was unable to prevent the increase in permeability at I (0.25 ± 0.05 and 0.29 ± 0.06 Normalized Grey Levels, NGL) and R (0.49 ± 0.10 and 0.53 ± 0.09 NGL), according to the concentrations. Adhesion of leukocytes was observed mostly in V3 venules at R (9 ± 2 and 10 ± 2/100 µm venular length, with the lower and higher concentration, respectively). Nitric oxide synthase inhibition by N<sup>G</sup>-nitro- L-arginine-methyl ester prior to insulin did not affect capillary perfusion at R (–18 ± 3% of baseline with higher concentration), but prevented permeability increase (0.20 ± 0.04 NGL, according to higher concentration) and reduced leukocyte adhesion in V3 venules at R (1.5 ± 1.0/100 µm of venular length, with higher concentration). Blockade of K<sup>+</sup><sub>(ATP)</sub> channels by glibenclamide prior to insulin decreased perfused capillary length at R (–58 ± 6% of baseline with higher concentration), attenuated leakage at R (0.30 ± 0.04 NGL, according to higher concentration) and caused leukocyte adhesion mainly in V1 venules at R (9.0 ± 1.5/100 µm of venular length, with higher concentration). Inhibition of either TK, PKC or phosphatidylinositol 3-kinase did not affect microvascular responses to insulin. Simultaneous inhibition of TK and NOS did not increase protection. Conclusions: Insulin prevents ischemia-reperfusion injury by promoting capillary perfusion through an apparent activation of K<sup>+</sup><sub>(ATP)</sub> channels and increase in nitric oxide release.

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          Most cited references 20

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          Insulin and PIP3 activate PKC-zeta by mechanisms that are both dependent and independent of phosphorylation of activation loop (T410) and autophosphorylation (T560) sites.

          Activation of protein kinase C-zeta (PKC-zeta) by insulin requires phosphatidylinositol (PI) 3-kinase-dependent increases in phosphatidylinositol-3,4,5-(PO(4))(3) (PIP(3)) and phosphorylation of activation loop and autophosphorylation sites, but actual mechanisms are uncertain. Presently, we examined: (a) acute effects of insulin on threonine (T)-410 loop phosphorylation and (b) effects of (i) alanine (A) and glutamate (E) mutations at T410 loop and T560 autophosphorylation sites and (ii) N-terminal truncation on insulin-induced activation of PKC-zeta. Insulin acutely increased T410 loop phosphorylation, suggesting enhanced action of 3-phosphoinositide-dependent protein kinase-1 (PDK-1). Despite increasing in vitro autophosphorylation of wild-type PKC-zeta and T410E-PKC-zeta, insulin and PIP(3) did not stimulate autophosphorylation of T560A, T560E, T410A/T560E, T410E/T560A, or T410E/T560E mutant forms of PKC-zeta; thus, T560 appeared to be the sole autophosphorylation site. Activating effects of insulin and/or PIP(3) on enzyme activity were completely abolished in T410A-PKC-zeta, partially compromised in T560A-PKC-zeta, T410E/T560A-PKC-zeta, and T410A/T560E-PKC-zeta, and largely intact in T410E-PKC-zeta, T560E-PKC-zeta, and T410E/T560E-PKC-zeta. Activation of the T410E/T560E mutant suggested a phosphorylation-independent mechanism. As functional correlates, insulin effects on epitope-tagged GLUT4 translocation were compromised by expression of T410A-PKC-zeta, T560A-PKC-zeta, T410E/T560A, and T410A/T560E-PKC-zeta but not T410E-PKC-zeta, T560E-PKC-zeta, or T410E/T560E-PKC-zeta. Insulin, but not PIP(3), activated truncated, pseudosubstrate-lacking forms of PKC-zeta and PKC-lambda by a wortmannin-sensitive mechanism, apparently involving PI 3-kinase/PDK-1-dependent phosphorylations but independent of PIP(3)-dependent conformational activation. Our findings suggest that insulin, via PIP(3), provokes increases in PKC-zeta enzyme activity through (a) PDK-1-dependent T410 loop phosphorylation, (b) T560 autophosphorylation, and (c) phosphorylation-independent/conformational-dependent relief of pseudosubstrate autoinhibition.
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            Matrix metalloproteinases regulate neutrophil-endothelial cell adhesion through generation of endothelin-1[1-32].

            We recently reported that matrix metalloproteinase 2 (MMP-2, gelatinase A) cleaves big endothelin 1 (ET-1), yielding the vasoactive peptide ET-1[1-32]. We tested whether ET-1[1-32] could affect the adhesion of human neutrophils to coronary artery endothelial cells (HCAEC). ET-1[1-32] rapidly down-regulated the expression of L-selectin and up-regulated expression of CD11b/CD18 on the neutrophil surface, with EC50 values of 1-3 nM. These actions of ET-1[1-32] were mediated via ETA receptors and did not require conversion of ET-1[1-32] into ET-1 by neutrophil proteases, as revealed by liquid chromatography and mass spectroscopy. Moreover, ET-1[1-32] evoked release of neutrophil gelatinase B, which cleaved big ET-1 to yield ET-1[1-32], thus revealing a positive feedback loop for ET-1[1-32] generation. Up-regulation of CD11b/CD18 expression and gelatinase release was tightly associated with activation of extracellular signal-regulated kinase (Erk). Stimulation of Erk activity was due to activation of Ras, Raf-1, and MEK (MAPK kinase). ET-1[1-32] also produced slight increases in the expression of ICAM-1 and E-selectin on HCAEC, and markedly enhanced beta2 integrin-dependent adhesion of neutrophils to activated HCAEC. These results are the first indication that gelatinolytic MMPs via cleavage of big ET-1 to yield ET-1[1-32] activate neutrophils and promote leukocyte-endothelial cell adhesion and, consequently, neutrophil trafficking into inflamed tissues.
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              Microvascular measurements by video image shearing and splitting.


                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                February 2005
                28 January 2005
                : 42
                : 1
                : 55-66
                aDepartment of Neuroscience, ‘Federico II’ University Medical School, Naples, bDepartment of Physiology and Biochemistry, University of Pisa, and cCNR Institute of Clinical Physiology, Pisa, Italy
                83092 J Vasc Res 2005;42:55–66
                © 2005 S. Karger AG, Basel

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                Page count
                Figures: 5, Tables: 5, References: 36, Pages: 12
                Research Paper


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