In vitro naltrindole mitigates phorbol 12-myristate 13-acetate induced superoxide release in polymorphonuclear leukocytes independent of delta opioid receptor antagonism

Philadelphia, PA

Introduction:

Naltrindole (NTI) is a selective delta opioid receptor antagonist that recently has been shown to be cardioprotective in both in vivo and ex vivo studies. In vivo, NTI showed cardioprotective effects that were dose-dependent significantly reducing infarct size up to 82% compared to control (p<0.05). In ex vivo isolated rat hearts, NTI significantly reduced infarct size compared to controls (p<0.01). NTI restored cardiac left ventricular end diastolic pressure to near baseline values compared to controls or other general opioid receptor antagonists in ex vivo hearts, suggesting a tissue salvaging mechanism independent of opioid delta receptor antagonism. Polymorphonuclear leukocytes (PMNs) do not express opioid receptors. In this study, we investigated NTI attenuation of PMN superoxide (SO) release in vitro. We hypothesized that NTI would attenuate SO release in PMNs via a calcium handling mechanism, a similar mechanism that explained cardioprotective effects of NTI pretreatment that mitigated ischemic hypercontracture that is due to an increase in intracellular calcium.

Methods:

Rat PMNs (5x 106) were incubated for 15 min at 37oC in the presence or absence (dH2O vehicle control) of NTI (10μM, 50μM, or 200 μM). PMN SO release was calculated by the change in absorbance at 550 nm over 420 sec via ferricytochrome c reduction after phorbal 12-myristate-13 acetate (PMA) stimulation (100nM). The cell viability was determined microscopically by 0.2% trypan blue exclusion at the end of the assay. Data were analyzed using ANOVA Fisher’s PLSD post-hoc test.

Results:

NTI significantly decreased PMN SO release at 420 sec at 200 μM (0.244±0.07, n=8, p<0.05) and 50μM (0.293±0.06, n=4, p<0.05), compared to control (0.487±0.12, n=9). NTI 10μM (0.412±0.07, n=5) was not significant compared to control. Cell viability was not significantly different compared to PMA across all NTI concentrations.

Discussion:

These results suggest that NTI reduces I/R injury and PMN SO release by a mechanism independent of delta opioid receptor antagonism. Future studies will assess the effects of NTI on ROS attenuation in human umbilical vein endothelial cells. We will use different concentrations to investigate optimal concentration response.

Support or Funding Information

This research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine. Current research license is supported by Young Therapeutics, LLC.