Analysis of Intracellular Ca2+ fluctuation and of inflammation markers in astrocytes co-cultured with brain endothelial cells, after exposure to Phoneutria nigriventer spider venom

Abstract

The blood-brain barrier (BBB) restricts the access of substances to the central nervous system (CNS). Among the cellular components of the BBB the astrocytes have been specially studied since these glial cells induce and maintain the barrier phenotype typical of the brain endothelium. Moreover, the astrocytes are influenced by endothelium signaling and respond to changes in the neural microenvironment by regulating the inflammatory processes, neuronal activation and pain through mechanisms that involve Ca2+ mediated signaling. Nitric oxide (NO) donors increase the Ca2+ intracellular concentrations in astrocytes and endothelial cells through the soluble guanylate cyclase activation and consequently through the production of cyclic GMP (cGMP) (NO-cGMP pathway). The venom of Phoneutria nigriventer spider (PNV) or some of its isolated components have been shown to be important modulators of ion channels and neurotransmitter release. They produce neuroinflammation and BBB permeability and are inducers of pain and activators of specific neuronal pathways in rats. Cultured astrocytes when incubated with PNV produce inflammation mediators such as cytokines and NO. Therefore, it is hypothesized that the astrocytes are modulators of the PNV effects through a cGMP- and Ca2+ intracellular fluctuations-dependent mechanism. Interestingly, it has been demonstrated that opioids, specially m-opioid agonists, exert a role in the control of neuroinflammatory activity in astrocytes. It is possible that opioid agonists may stabilize the mechanisms involved in the neuroinflammation and pain induced by PNV, through the modulation of intracellular Ca2+ oscillation. In this work, we will investigate the effect of PNV in astrocytes co-cultured with endothelial cells to simulate the in vivo BBB physical organization. The intracellular Ca2+ mobilization, the production of pro-inflammatory cytokines and the expression of stress fibers and connexin-43, the main protein of gap junctions in astrocytes, will be investigated in astrocyte-endothelial co-cultured cells incubated with PNV with or without NO-GMPc pathway modulators. Moreover, the ability of some synthetic agonists of opioid receptors in maintaining the cell physiologic balance, after damage induced by the venom will be also investigated. (AU)