Protective effects of insulin during ischemia-reperfusion injury in hamster cheek pouch microcirculation.
OBJECTIVE
The effects of insulin (0.18 nM-0.18 microM) 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 microM) were investigated after inhibition of tyrosine kinase (TK), nitric oxide synthase (NOS), protein kinase C (PKC), phosphatidylinositol 3-kinase and K+(ATP) 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 microm venular length, with the lower and higher concentration, respectively). Nitric oxide synthase inhibition by N(G)-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 microm of venular length, with higher concentration). Blockade of K+(ATP) 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 microm 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+(ATP) channels and increase in nitric oxide release.
Colantuoni
A
A
Department of Neuroscience, Federico II University Medical School, Naples, Italy. colantuo@unina.it
Lapi
D
D
Paterni
M
M
Marchiafava
P L
PL
D000470:Alkaloids; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D053119:Benzophenanthridines; D016227:Benzoquinones; D002199:Capillary Permeability / Q000187:drug effects; D002610:Cheek; D006224:Cricetinae; D005905:Glyburide / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D047029:Lactams, Macrocyclic; D008297:Male; D008647:Mesocricetus; D008833:Microcirculation / Q000187:drug effects; D009061:Mouth Mucosa / Q000098:blood supply*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D010617:Phenanthridines / Q000494:pharmacology; D015221:Potassium Channels / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011809:Quinones / Q000494:pharmacology; D015427:Reperfusion Injury / Q000517:prevention & control*; D017828:Rifabutin / Q000031:analogs & derivatives; D000077191:Wortmannin
D000470:Alkaloids; D000730:Androstadienes; D053119:Benzophenanthridines; D016227:Benzoquinones; D007328:Insulin; D047029:Lactams, Macrocyclic; D010617:Phenanthridines; D015221:Potassium Channels; D011809:Quinones; D017828:Rifabutin; C020754:herbimycin; C016299:chelerythrine; D011493:Protein Kinase C; D005905:Glyburide; D019331:NG-Nitroarginine Methyl Ester; D000077191:Wortmannin