2Aiii), than in the group that received 8-Br-cGMP and alcohol (Fig

2Aiii), than in the group that received 8-Br-cGMP and alcohol (Fig. cGK confers alcohol resistance by preventing [Ca2+]i disruptions. Alcohol resistance, determined by neuronal survival after 24 hours of alcohol exposure, was examined in main cerebellar granule neuron (CGN) cultures derived from 5 to 7 day-old neonatal mice with an activator, 8-Br-cGMP, and/or an inhibitor, Rp-8-pCPT-cGMPS, of cGK signaling. Intracellular Ca2+ responses to alcohol were measured by ratiometric Ca2+ imaging in Fura-2-loaded CGN cultures after 8-Br-cGMP treatment. Our results indicate that activating cGK with 8-Br-cGMP before alcohol administration provided neuroprotection, Tafamidis (Fx1006A) which the cGK inhibitor, Rp-8-pCPT-cGMPS, blocked. Alcohol exposure elevated [Ca2+]i, whereas 8-Br-cGMP pretreatment reduced both the level of the alcohol-induced rise in [Ca2+]i as well as the number of cells that responded to alcohol by increasing [Ca2+]i. These findings associate alcohol resistance, mediated by cGK signaling, to reduction of the prolonged and harmful increase in [Ca2+]i from alcohol exposure. [16-19] and models of FASD [20, 21] to trigger neuronal death by disrupting intracellular Ca2+ ([Ca2+]i). Conversely, cGK has been demonstrated to regulate and inhibit [Ca2+]i release in multiple contexts [22, 23], including cerebellar tissue [24]. This evidence has led us to examine the relationship between cGK modulation of [Ca2+]i and alcohol-induced neuronal death. In this study, we tested the hypothesis that cGMP activation of cGK can prevent the alcohol-induced [Ca2+]i elevations and producing neuronal death. Experiments using alcohol-vulnerable CGN cultures examined 1) neuronal survival and 2) [Ca2+]i following alcohol exposure in the presence of activators and/or inhibitors of cGK signaling. Our findings suggest that cGK mediates alcohol resistance by moderating alcohols disruption of [Ca2+]i. Materials and methods Animals A breeding colony of C57BL/6 mice (in the beginning obtained from Jackson Laboratories, Bar Harbor, ME) was housed in The University or college of Iowa Animal Care Facility. All animal experiments were approved by The University or college of Iowa Institutional Animal Care and Use Committee and performed according to the guidelines of the National Institutes of Health. Cell culture Main CGN cultures offer a well-defined and manageable biological system widely used to investigate the direct neuronal effects of alcohol [25]. These cultures do not proliferate and any cell number reduction is due to decreased neuronal survival [6]. CGN cultures were established from neonatal mice (5C7 days aged) as previously Rabbit Polyclonal to Cortactin (phospho-Tyr466) explained [18]. For all those Tafamidis (Fx1006A) treatment conditions in a single experiment, cultures derived from each mouse litter were established in duplicate or triplicate and constituted one replicate. CGN were plated into poly-D-lysine (PDL, 50 mg/ml)-coated 96-well culture trays (1.5 106 cells/ml; 300 l/well) for cell survival experiments, and into six-well culture trays (1.0 106 cells/ml; 2.0 ml/well) which contained PDL-coated (100 mg/ml) glass coverslips (25 mm diameter) for Ca2+ imaging experiments. CGN cultures were incubated overnight at 37C in humidified air flow (5% CO2) prior to use. Pharmacological treatments and alcohol exposure Alcohol exposure at 400 mg/dl (87 mM ethanol) was chosen for most experiments which began 24 hours after plating. Although chronic alcoholics can achieve similar blood alcohol concentrations [26], it is a high physiological dose. We previously exhibited that alcohol elicits Ca2+-dependent cell death in a concentration-dependent manner in CGN cultures and that lower alcohol concentrations (100 and 200 mg/dl) produced cell death to a smaller degree [6, 18]. Since this indicates that higher alcohol concentrations are eliciting the same effect with greater magnitude, 400 mg/dl was used to more readily accomplish statistical significance. This alcohol concentration also matches blood alcohol levels often achieved in rodent studies of alcohol neuroteratology [10, 12, 27-29] and our previous studies [8, 9, 18, 30]. Cultures were also treated with one or more of the following: PBS (vehicle control); 8-Br-cGMP, a cGMP analogue that activates cGK, or Rp-8-pCPT-cGMPS, a specific inhibitor of cGK. Exposure occurred 30 minutes before adding alcohol as described. Tafamidis (Fx1006A) Culture trays in cell survival experiments were placed in sealed containers containing alcohol baths of equivalent concentration (0 or 400 mg/dl) to that of.