Naltrindole Attenuates Superoxide Release in Polymorphonuclear Leukocytes by a Novel Mechanism Devoid of Opioid Receptors

Introduction: Previously, naltrindole (NTI), known as a delta opioid receptor antagonist, produced robust cardioprotective effects (e.g ~ 80% reduction of infarct size) in both ex-vivo (2.5-5 μM) and in-vivo (3.75-7.5 mg/kg; ~ 100-200 μM serum concentration) rat myocardial ischemia-reperfusion (MIR) models compared to control hearts that received H 2 O-vehicle or naloxone (NX), a broad-spectrum opioid receptor antagonist. Therefore, we planned to test if NTI elicits cardioprotective effects via a novel mechanism of action (MOA) devoid of opioid receptors. To investigate this hypothesis, we conducted experiments using KB-R7943 (KB) as a positive control, known to minimize intracellular calcium (Ca 2+ ) via inhibition of the reverse mode sodium (Na + )/Ca 2+ exchanger in phorbol 12-myristate 13-acetate (PMA) induced polymorphonuclear leukocytes (PMNs) superoxide (SO) release. PMNs do not express opioid receptors. NX was used as a negative control. We predict that NTI will diminish PMA-induced PMN SO release similarly to KB, while NX will have no effect compared to vehicle controls without compromising cell viability. Methods: PMNs were isolated from anesthetized male Sprague Dawley rats (~ 400g) and incubated (5x10 6 ) for 15 min at 37°C in the presence or absence (dH 2 O vehicle control) of NTI 10-200 µM, KB 5-20 µM, and NX 100-200 µM. PMN SO release was calculated by the change in absorbance at 550 nm over 420 sec via ferricytochrome c reduction after PMA stimulation (100 nM). 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 200 μM (n=10, 0.196±0.06, p<0.05) and 100 µM (n=7, 0.461±0.08, p<0.05) compared to control (n=22, 0.731±0.07). KB also showed a significant decrease in PMN SO release at 20 µM (n=7, 0.507±0.07), 10 µM (n=7, 0.552±0.07), and 5 µM (n=5, 0.615±0.11) compared to control (n=10, 0.908±0.07). NX 100/200 µM (n=3, 0.593±0.06) was not significant compared to control. Cell viability was statistically similar (80-90±5%) among all study groups. Conclusion: NTI (100 μM, 200 μM) and KB 5-20 µM exhibited concentration-dependent effects in reducing PMN SO release, suggesting that the novel effect of NTI is likely mediated through a reduction in intracellular Ca 2+ . Moreover, it indicates that NTI diminishes PMN SO release through a mechanism independent of delta opioid receptor antagonism, corroborated by its effects observed in MIR injury experiments when compared to KB and NX. 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. This study was funded by the Philadelphia College of Osteopathic Medicine, Department of Biomedical Sciences, the Center for Chronic Diseases of Aging and Young Therapeutics. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.