J Korean Med Sci.  2011 Feb;26(2):290-296. 10.3346/jkms.2011.26.2.290.

Morphine Postconditioning Attenuates ICAM-1 Expression on Endothelial Cells

• Min TJ
• Kim JI
• Kim JH
• Noh KH
• Kim TW
• Kim WY
• Lee YS
• Park YC

• KMID: 1782121
• DOI: http://doi.org/10.3346/jkms.2011.26.2.290

Abstract

The purpose of this study is to determine 1) whether morphine postconditiong (MPostC) can attenuate the intercellular adhesion molecules-1 (ICAM-1) expression after reoxygenation injury and 2) the subtype(s) of the opioid receptors (ORs) that are involved with MPostC. Human umbilical vein endothelial cells (HUVECs) were subjected to 6 hr anoxia followed by 12 hr reoxygenation. Three morphine concentrations (0.3, 3, 30 microM) were used to evaluate the protective effect of MPostC. We also investigated blockading the OR subtypes' effects on MPostC by using three antagonists (a micro-OR antagonist naloxone, a kappa-OR antagonist nor-binaltorphimine, and a delta-OR antagonist naltrindole) and the inhibitor of protein kinase C (PKC) chelerythrine. As results, the ICAM-1 expression was significantly reduced in the MPostC (3, 30 microM) groups compared to the control group at 1, 6, 9, and 12 hours reoxygenation time. As a consequence, neutrophil adhesion was also decreased after MPostC. These effects were abolished by coadministering chelerythrine, nor-binaltorphimine or naltrindole, but not with naloxone. In conclusion, it is assumed that MPostC could attenuate the expression of ICAM-1 on endothelial cells during reoxygenation via the kappa and delta-OR (opioid receptor)-specific pathway, and this also involves a PKC-dependent pathway.

Keyword

Morphine; Postconditioning; Reperfusion injury; Humans; Umblical Veins; Endothelial Cells; Cell Culture

MeSH Terms

Animals
Benzophenanthridines/pharmacology
Endothelial Cells/cytology/*drug effects/*metabolism
Endothelium, Vascular/cytology
Humans
Intercellular Adhesion Molecule-1/genetics/*metabolism
Morphine/*pharmacology
Naloxone/pharmacology
Naltrexone/analogs & derivatives/pharmacology
Narcotic Antagonists/pharmacology
Narcotics/*pharmacology
Protein Isoforms/metabolism
Protein Kinase C/antagonists & inhibitors/metabolism
Receptors, Opioid/metabolism
Reperfusion Injury/*metabolism
Signal Transduction/physiology
Umbilical Veins/cytology

Figure

• 

  Fig. 1 Experimental protocol. MPostC represents morphine postconditioning.

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  Fig. 2 Attenuation of the ICAM-1 protein expression in the HUVEC cells by MPostC. (A) The intercellular adhesion molecules-1 (ICAM-1) expression in the HUVECs is compared between the morphine postconditioning (MPostC) groups and the control group after 6 hr anoxia. The numbers of viable cells was 1 × 105 and the cell viability was 92%. The groups were divided to the control group and the 0.3, 3, and 30 µM MPostC groups. The mean fluorescence index (MFI) from each group was recorded at 0, 1, 3, 6, 9, and 12 hr. The valus are the mean ± SD of 6 experiments. *P < 0.05. (B) Phenotypical graph of the HUVECs. Flow cytometry analysis was done to characterize the ICAM-1 expressions on the HUVECs. PE Mouse Anti-Human CD54 monoclonal antibody was used to detect the ICAM-1 expression. The isotype antibody was used as the negative control (bold). The values were measured at 6 hr reperfusion time.

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  Fig. 3 Ratio of adhesion neutrophils to ECs. The ratio of adhesion neutrophils to ECs was measured at 6 hr reoxygenation. Baseline meant the value of the control group at 0 hr reoxygenation. The valus are the mean ± SD of 6 experiments. *is P < 0.05.

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  Fig. 4 Attenuation of the ICAM-1 mRNA level in the HUVEC cells by MPostC. qRT-PCR was performed to measure the ICAM-1 mRNA levels with using SYBR Premix Ex Taq. The relative gene expression levels were calculated as ratios by using β-actin for normalization. The value of the 0 hr control was baseline and it was calculated as a ratio of 1, and the others were recalculated as ratios relevant to a ratio of 1. All the values were compared to the value of the control group at 6 hr reoxygenation. The values are the mean ± SD of 6 experiments. *P < 0.05.

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  Fig. 5 Selective OR antagonists reverse the attenuation of the ICAM expression induced by MPostC. The intercellular adhesion molecule-1 (ICAM-1) expression was measured in the 3 µM morphine postconditioned cells in the presence of various concentrations of selective blockers. The mean florescence index of ICAM-1 was measured at 6 hr reoxygenation time. The dosages of each of the blockers were increased five folds at each time. The value of the control group at 0 hr reoxygenation was baseline and it was calculated as a ratio of 1, and the others were recalculated as ratios relevant to a ratio of 1. All the values were compared to the value of the 3 µM MpostC. The valus are the mean ± SD of 6 experiments. *P < 0.05.

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