Neurochemistry International 96 (2016) 13e23 Contents lists available at ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/nci Neuropharmacological effects of Phoneutria nigriventer venom on astrocytes Catarina Raposo^ a, 1, Ulrika Bjorklund€ b, Evanguedes Kalapothakis c,Bjorn€ Biber d, * Maria Alice da Cruz-Ho€fling a, Elisabeth Hansson b, a Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), 13 083-970 Campinas, SP, Brazil b Department of Clinical Neuroscience, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, SE 413 45 Gothenburg, Sweden c Department of General Biology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), 31270-901 Belo Horizonte, MG, Brazil d Department of Anaesthesiology and Intensive Care Medicine, Institute of Clinical Sciences, The Sahlgrenska Academy, University of Gothenburg, SE 413 45, Gothenburg, Sweden article info abstract Article history: Bites from genus Phoneutria (Ctenidae, Araneomorpha) are the second most frequent source of spider Received 22 February 2016 accidents in Southeast Brazil. Severe envenoming from Phoneutria nigriventer produces vision distur- Received in revised form bance, tremor and convulsion, suggesting that the CNS is involved; however, the mechanisms by which 7 April 2016 P. nigriventer venom (PNV) affects the CNS remain poorly understood. The present study aimed to Accepted 14 April 2016 investigate whether PNV directly impairs astrocytes. Cultured astrocytes were exposed to PNV, and Available online 16 April 2016 þ þ þ intracellular Ca2 release and signaling were measured (Fura-2/AM), Na /K -ATPase and Toll-like re- ceptor 4 (TLR4) involvement were investigated, actin filaments were stained (Alexa™ 488-conjugated Keywords: Arthropod venom phalloidin probe), the G-actin/F-actin ratio was determined, and the expression level of connexin 43 2þ 2þ Astrocytes (Cx43) was assessed. Incubation in Ca -free buffer did not change the Ca responses. However, pre- þ Ca2 responses incubation in thapsigargin/caffeine completely abolished these responses, suggesting that PNV-evoked þ þ þ þ Stress fibers Ca2 transients were from intracellular Ca2 stores. Pretreatment with a Na /K -ATPase antagonist þ TLR4 (ouabain) or a TLR4 antagonist (LPS-RS) decreased or increased the Ca2 -evoked transients, respectively. Glutamate Astrocytes showed altered actin filament structure after PNV exposure. PNV treatment increased the þ þ expression levels of Na /K -ATPase and Cx43 but decreased those of TLR4. The present results suggest þ þ that PNV directly affects astrocytes. Na /K -ATPase may thus represent a more specific drug target for controlling the neurotoxicity of PNV. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction In Phoneutria nigriventer (“armed” spider) envenoming, intense local pain is the major symptom reported by victims (96% of Bites from the genus Phoneutria (Ctenidae, Araneomorpha) are envenomed patients). Less than 1% of the accidents cause systemic the second most frequent source of spider accidents in Brazil signs and symptoms, such as arterial hypertension, vomiting, (Gewehr et al., 2013) and are an important public health problem. nausea, agitation, vision disturbance, muscle spasm, tremor, pro- fuse sweating, somnolence, tachycardia, tachypnea, priapism and convulsion (Bucaretchi et al., 2000, 2008). A great number of toxins from P. nigriventer venom (PNV) have Abbreviations: AUC, area under the curve; BBB, blood-brain barrier; Cx43, been isolated and biochemically and pharmacologically character- connexin 43; GFAP, glial fibrillary acidic protein; IL-1b, interleukin-1b;IP, inositol þ þ 3 ized. PNV contains neuroactive toxins that block Ca2 and K 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; LPS, lipopolysac- þ charide; PNV, Phoneutria nigriventer venom; TLR4, Toll-like receptor 4; TNF-a, tu- channels, delay inactivation of Na channels and act on chemical mor necrosis factor-a. receptors resulting in neurotransmission disturbances (Gomez * Corresponding author. et al., 2002; for review see De Lima et al., 2015). The picture of E-mail address: [email protected] (E. Hansson). excitotoxicity which characterizes severe envenoming could be 1 Present address: Department of Animal Physiology and Morphology, Paulista State University (UNESP), 14884-900, Jaboticabal, SP, Brazil; Department of accounted for the effects of these neurotoxins named PhTx-1, PhTx- Morphology, UNESP, 14801-903, Araraquara, SP, Brazil. 2, PhTx-3 and PhTx-4; they exhibit important pharmacological http://dx.doi.org/10.1016/j.neuint.2016.04.005 0197-0186/© 2016 Elsevier Ltd. All rights reserved. 14 C. Raposo^ et al. / Neurochemistry International 96 (2016) 13e23 properties predicting them a potential source for development of numerous adult spiders (male and female) was supplied by Pro- new compounds with biologically-active properties (Escoubas fessor Evanguedes Kalapothakis (Federal University of Minas Ger- et al., 2000; Grishin, 1999; Rash and Hodgson, 2002). ais, Belo Horizonte, MG, Brazil). The quality of the venom was The systemic administration of the whole venom causes blood- evaluated by the observation of experimental envenoming signs in brain barrier (BBB) breakdown in rats and a series of alterations rats: tremors, salivation, flaccid followed by spastic paralysis of legs, related to perivascular astrocytes (For review see Cruz-Ho€fling respiratory anguish, convulsion and sometimes death (Raposo^ et al., 2015). These includes swelling of perivascular end-feet (Le et al., 2007). The lyophilized venom was stored at À20 C and Sueur et al., 2003, 2004, 2005; Raposo^ et al., 2007, 2012, 2014), was dissolved immediately before use. upregulations of the glial fibrillary acidic protein (GFAP) and cal- cium metabolism-associated protein S-100, tumor necrotic factor- 2.2. Primary astrocyte cultures and treatments alpha (TNF-a) and interferon-gamma (IFN-g)(Cruz-Ho€fling et al., 2009), aquaporin-4 (Stavale et al., 2013) and acute, but transient Rat primary cortical astrocytes, SpragueeDawley rats from increases in the major gap junction protein connexin 43 (Cx43) embryonic day 19, were purchased from Invitrogen (Thermo Fisher (Raposo^ et al., 2014). In vitro, PNV promotes the activation of the Scientific, Waltham, MA, USA) and prepared according to the multi-drug resistance protein (MRP-1), an efflux protein of astro- manufacturer's instructions with some modifications. Briefly, one cytes that impedes the access of xenobiotics into the brain (Raposo^ vial containing 1  106 viable cells was rapidly thawed and gently et al., 2014). However, the mechanisms involved following the rinsed with astrocyte growth medium consisting of Dulbecco's direct impairment of astrocytes by PNV have yet to be described. modified Eagle medium (DMEM) (high glucose) supplemented Astrocytes are key cellular elements in both tripartite synapses with 15% fetal bovine serum (both from Invitrogen). After centri- (Santello et al., 2012) and neurovascular units (Bechmann et al., fugation, the supernatant was removed, and the cells were resus- þ 2007). They exhibit excitability based on the movement of Ca2 pended in astrocyte growth medium. The cells were plated at a ions through their intracellular compartments and on plasma- seeding density of 1  104 cells per cm2 on an uncoated glass þ lemmal Ca2 influx (Blomstrand et al., 1999). Upon activation by coverslip (nr 1, 20 mm in diameter) (Bergman Labora, Stockholm, physiological or pathological stimulation, multiple receptors Sweden) placed in a 12-well plate. The cells were incubated at trigger the production of inositol 1,4,5-trisphosphate (IP3) and 37 C, 5% CO2 and 90% humidity, and the medium was replaced 2þ subsequent IP3-induced Ca release from the endoplasmic retic- twice a week. The astrocytes were used after 16e17 days in culture. ulum (Finkbeiner, 1993). þ þ Na /K -ATPase is an energy-transducing ion pump that indi- 2.3. Calcium imaging 2þ þ þ rectly modulates Ca signaling. Na /K -ATPase and the IP3 re- 2þ 2þ ceptor (IP3R) can form signaling microdomains that influence Ca Astrocytes were incubated at room temperature in the Ca - homeostasis in astrocyte networks in the presence of specific li- sensitive fluorophore Fura-2/AM (Invitrogen Molecular Probes, þ gands or inflammatory stimuli. Those changes in Ca2 homeostasis Eugene, OR, USA) for 30 min (8 ml in 990 ml Hank's HEPES buffered þ can develop into Ca2 oscillations (Miyakawa-Naito et al., 2003; saline solution [HHBSS], consisting of 137 mM NaCl, 5.4 mM KCl, Forshammar et al., 2011). Furthermore, an intact cytoskeleton is 0.4 mM MgSO4, 0.4 mM MgCl2, 1.26 mM CaCl2, 0.64 mM KH2PO4, 2þ required for the physiologic propagation of Ca responses (Liu 3.0 mM NaHCO3, 5.5 mM glucose, and 20 mM HEPES dissolved in et al., 2007). Ankyrin B, a protein associated with the cytoskel- distilled water, pH 7.4). All substances used during the experiment þ þ eton, interacts with Na /K -ATPase and with IP3R, linking the were diluted in the same solution. The fluorophore was dissolved in þ pump to the Ca2 responses from internal cell stores and the 40 ml of dimethyl sulfoxide (DMSO) and 10 ml of pluronic acid integrity of the cytoskeleton. In addition, astrocytes form physically (Molecular Probes, Leiden, The Netherlands). After incubation, the coupled networks mediated by gap junctions, which facilitate the cells were rinsed three times with HHBSS before exposure to PNV. þ intercellular transmission of Ca2 signaling (Cotrina et al., 1998). Three different sets of experiments were conducted, with the Evidence supports an important role for TLRs in the regulation following aims. 1) To investigate whether PNV directly affects as- of inflammation and tissue repair. In astrocytes, the stimulation of trocytes and whether the observed effects are dose-dependent, the TLR4 leads to the activation of NF-kB, which regulates the expres- cells were exposed to three different concentrations of PNV (1.4, sion of genes involved in immune responses, including cytokines 1400 or 14000 ng/ml). At 1 min preceding the start of the experi- such as TNF-a and interleukin-1b (IL-1b)(Kielian, 2006).