Role of Sigma-1 Receptor in Cocaine-Induced Endothelial Cell Damage

Background :Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. We showed that chronic cocaine use is associated with markers of endothelial dysfunction (ED) (Sáez et al. Thromb Res 2011; 128:18). The hallmark of ED is a change to a prothrombotic and proadhesive phenotype associated with a decreased bioavailability of nitric oxide (NO). Several experimental studies have shown that cocaine induces ED in vitro and in vivo; however, the receptor and the signaling pathways involved in the interaction of cocaine with endothelial cells (EC) are unknown. Sigma-1 receptor (sigma-1R) belongs to the family of nonopioid sigma receptors that regulate a variety of cellular functions in neuronal, glial and peripheral cells and are involved in pathological conditions such as neurodegenerative diseases, drug addiction, smooth muscle cell contraction and ischemia. Cocaine binding to sigma-1R has been demonstrated in microglial cell lines, where is located in the lipid raft microdomains of the endoplasmic reticulum being translocated into the plasma membrane upon activation. The effect of cocaine on sigma-1R expression and function in systemic endothelial cells EC are largely unknown. We hypothesize that activation of sigma-1R in EC and its downstream signaling pathway may be involved in the pathogenesis of cocaine-associated endothelial dysfunction, a key phenomenon in the onset and progression of atherosclerosis.

Objectives:The main aim of this study was to demonstrate the presence of sigma-1R in EC and its role in cocaine-induced EC damage/activation.

Methods:Human umbilical vein endothelial cells (HUVECs) were cultured under standard conditions and exposed to cocaine (10μM) or vehicle in the presence or absence of BD1047 (sigma-1 receptor antagonist). Sigma-1R was demonstrated by western blotting using rabbit-anti-OPRS1 polyclonal antibody. Sub-cellular distribution of sigma-1R and von Willebrand factor (VWF) bound to the membrane were studied by indirect immunofluorescence (IF) microscopy. The production of NO was determined by fluorometric assay using DAF-2DA (5μM) and results expressed as arbitrary fluorescence units (AFU). Platelet adhesion under static conditions was assessed by co-incubation of resting platelets with cultured HUVEC; platelets and EC were identified and quantified by IF microscopy using anti-glycoprotein Ib and anti-VE-cadherin antibodies, respectively.

Results:HUVEC expressed sigma-1R protein as demonstrated by Western blot analysis. By IF the receptor showed a perinuclear pattern in untreated HUVEC. Treatment with cocaine resulted in translocation to the cytoplasm and suggest co-localization with actin. These changes were abrogated completely by pre-incubation of the cells with BD1047. HUVEC treated with cocaine exhibited an increase in VWF deposited on the cell membrane with the formation of strings, phenomena that were inhibited by incubation with BD1047. With respect to platelet adhesion, cocaine induced a significant increase in the number of platelets adhered to HUVEC as compared with the controls (46±24 versus 13.3±7 platelets per cell, respectively), which was drastically reduced by BD1047 (10.5± 6.3 platelets per cell). NO production in HUVEC exposed to cocaine was significantly reduced with respect to cells treated with vehicle (10.7±0.67 vs 19.5± 6.4 nM/ml, P=0.002), the use of BD1047 only partially restored NO production (12.1±2.5 nM/ml).

Conclusions: Our results show that in cultured HUVEC expressed sigma 1-R with a sub-cellular distribution and changes upon cell activation similar to those observed in other cell systems. The blockade of sigma-1R inhibited significantly the pro-adhesive and pro-thrombotic changes observed in HUVEC exposed to cocaine, which support the notion that the receptor may play an important role in cocaine-induce endothelial cell dysfunction. These findings have implications for the management of drug addiction, since sigma-1R antagonists may provide a novel therapeutic intervention to protect the endothelium from the deleterious effects associated with cocaine abuse.