Naltrindole Attenuates Phorbol 12-myristate 13-acetate Induced Superoxide Release In Polymorphonuclear Leukocytes Independently Of Delta Opioid Receptor Antagonism

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 controls subjected to ischemia-reperfusion (I/R) injury (p<0.05). In ex vivo isolated rat I/R 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 PMN SO release via a calcium handling mechanism, a similar mechanism that explained cardioprotective effects of NTI pretreatment mitigating ischemic hypercontracture due to an increase in intracellular calcium. Rat PMNs (5x10-6) were incubated for 15 min at 37 C in the presence or absence (dH2O vehicle controls) of NTI (10μM, 50μM, or 200μM). PMN SO release was calculated by the change in absorbance at 550 nm over 420 sec via ferricytochrome c reduction after phorbol 12-myristate-13 acetate (PMA) stimulation (100nM). 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. NTI significantly decreased PMN SO release from 30 sec to 420 sec. Changes in final absorbance of NTI 200μM (0.244±0.07, n=8, p<0.05) and 50μM (0.293±0.06, n=4, p<0.05), but not NTI 10μM (0.412±0.07, n=5), were significant compared to controls (0.487±0.12, n=9). Cell viability was not significantly different compared to PMA across all NTI concentrations. These results suggest that NTI reduces I/R injury and PMN SO release, by a means independent of delta opioid receptor antagonism. Future studies will assess NTI SO attenuation in human umbilical vein endothelial cells. We will use different concentrations to investigate optimal concentration response.