Naltrindole inhibits phorbol 12-myristate 13-acetate-induced superoxide release by a novel mechanism related to reduced intracellular calcium levels in polymorphonuclear leukocytes

Philadelphia, PA

Introduction:

We recently showed that naltrindole (NTI) 100-200µM attenuated phorbol 12-myristate 13-acetate (PMA) induced polymorphonuclear leukocytes (PMNs) superoxide (SO) release by a novel mechanism of action (MOA) devoid of opioid receptors. We hypothesize that NTI’s novel MOA is a reduction of intracellular calcium (Ca2+). To test this hypothesis, we conducted PMA-induced PMN SO release using KB-R7943(KB). KB is known to reduce intracellular Ca2+ via inhibition of reverse mode Na+/Ca2+ exchanger and is used as a positive control to attenuate PMA-induced PMN SO release. Naloxone (NX), a broad-spectrum opioid receptor antagonist, is used as a negative control and should have no effect on PMA-induced PMN SO release. Therefore, we predict that NTI will attenuate PMA-induced PMN SO release in a similar manner as KB, whereas NX should have no effect on PMN SO release compared to vehicle controls.

Methods:

Male Sprague Dawley (SD) rat PMNs (5x106) were incubated for 15 min at 37oC in the presence or absence (dH2O vehicle control) of NTI (10μM, 50μM, 100 μM or 200μM), 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 (100nM). The cell viability was determined microscopically by 0.2% trypan blue exclusion at the end of the PMN SO release 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 (n=10, 0.196±0.06, p

Conclusion:

NTI (100 μM, 200 μM) and KB 5-20 µM showed concentration-dependent effects on reducing PMN SO release. The novel effect is most likely mediated via a reduction in intracellular Ca2+ and suggests that NTI reduces 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.

Funding

This study was funded by the Philadelphia College of Osteopathic Medicine, Department of Biomedical Sciences, Division of Research, and the Center for Chronic Diseases of Aging and Young Therapeutics, LLC.