The Pharmacogenomics Journal (2010) 10, 134–160 & 2010 Nature Publishing Group All rights reserved 1470-269X/10 $32.00 www.nature.com/tpj ORIGINAL ARTICLE Transcriptome analysis of nicotine-exposed cells from the brainstem of neonate spontaneously hypertensive and Wistar Kyoto rats MFR Ferrari1, EM Reis2, In this study, the effects of nicotine on global gene expression of cultured 3 cells from the brainstem of spontaneously hypertensive rat (SHR) and JPP Matsumoto and normotensive Wistar Kyoto (WKY) rats were evaluated using whole-genome 3 DR Fior-Chadi oligoarrays. We found that nicotine may act differentially on the gene expression profiles of SHR and WKY. The influence of strain was present in 1Departamento de Neurologia, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, 321 genes that were differentially expressed in SHR as compared with WKY SP, Brazil; 2Departamento de Bioquı´mica, brainstem cells independently of the nicotine treatment. A total of 146 genes Instituto de Quimica, Universidade de Sao Paulo, had their expression altered in both strains after nicotine exposure. Sao Paulo, SP, Brazil and 3Departamento de Interaction between nicotine treatment and the strain was observed to Fisiologia, Instituto de Biociencias, Universidade de Sao Paulo, Sao Paulo, SP, Brazil affect the expression of 229 genes that participate in cellular pathways related to neurotransmitter secretion, intracellular trafficking and cell Correspondence: communication, and are possibly involved in the phenotypic differentiation Dr MFR Ferrari, Departamento de Neurologia, between SHR and WKY rats, including hypertension. Further characterization Faculdade de Medicina, Universidade de Sao of their function in hypertension development is warranted. Paulo, Av. Dr Arnaldo, 455, sala 2119, 2 andar, Sao Paulo, SP 01246-903, Brazil. The Pharmacogenomics Journal (2010) 10, 134–160; doi:10.1038/tpj.2009.42; E-mails: [email protected] or [email protected] published online 15 September 2009 Keywords: microarray; hypertension; nicotine; gene expression; primary cell culture; brainstem Introduction The central nervous system may influence the development of hypertension,1 which is a multifactorial disease involving the participation of differentially expressed genes in the brain of hypertensive and normotensive subjects.2 Neurogenic hypertension might be a consequence of the abnormal function of the autonomic nervous system, which may be caused by a number of external and/or intrinsic factors such as the renin–angiotensin system, insulin resistance, dietary salt sensitivity, stress and genetic factors,3 as well as nicotine exposure.4 Spontaneously hypertensive rats (SHR) are a good model to study neurogenic hypertension because they share similarities to human essential hypertension,5 such as the development of hypertension in the early adult life.6 We have earlier reported that nicotine treatment is able to intensify and accelerate the development of hypertension in prehypertensive SHR, without having much effect on normotensive Wistar Kyoto (WKY) rats,7 which may be of relevance to the understanding of the mechanisms involved in the onset of hypertension. Brain nuclei predominantly involved in central cardiovascular control are Received 6 February 2009; revised 31 July 2009; accepted 14 August 2009; published located especially in the brainstem. These areas are also recognized to have a online 15 September 2009 relevant function in the development of hypertension.8,9 Nicotine regulates gene expression of brainstem cells MFR Ferrari et al 135 The nucleus of the solitary tract (NTS), located at the time in fresh medium without nicotine were used as control. dorsal portion of the medulla oblongata, is the major The three plates of each group were polled to obtain one integrative site of cardiovascular information.10,11 Although sample. The experiment was repeated three times using peripheral afferent input is very important for the baro- different pools of animals. At the end of the treatment, cells receptor reflex, the NTS also receives a vast array of from SHR or WKY animals, treated or untreated with projections coming from the spinal cord, the lower nicotine, were lysed for total RNA extraction. brainstem and from the forebrain areas such as hypothala- mus, limbic system and cerebral cortex,12–14 which makes RNA extraction the cardiovascular control an elaborated circuitry. Total RNA was extracted according to the manufacturer’s Blood pressure control is also accomplished by other instructions using the RNAspin Mini Kit (GE Healthcare, brainstem sites such as the rostro- and caudo-ventrolateral UK). The RNA was purified and treated with DNAse before medulla oblongata (RVLM and CVLM), dorsal motor the assessment of its concentration and quality by spectro- nucleus of the vagus, area postrema and locus coeruleus photometry and agarose gel electrophoresis, respectively. (in pons).8,15–17 We have recently shown that nicotine is able to interfere Target labeling and microarray hybridizations with the glutamatergic and angiotensinergic neurotransmis- Global measurements of gene expression in cells from sion system preferentially in SHR as compared with WKY normotensive and hypertensive rats after the exposition to rats,7,18 indicating a possible function of nicotine in nicotine and their controls were obtained using the interacting with neurotransmitter systems, which might CodeLink Expression Bioarray System (GE Healthcare), contribute to the development of hypertension. which interrogates 34 000 annotated genes and expressed Glutamatergic and angiotensinergic are not the only sequence tags expressed in the rat genome. Three different neurotransmitter systems altered in SHR, but other systems samples of the each group were used in microarray are also possibly involved in strain differences between SHR hybridization constituting three biological replicates. and WKY rats, related or not related to the development of Target labeling and hybridization was performed strictly hypertension.19–21 as recommended by the array manufacturer (GE Health- High-density oligoarrays allow the simultaneous measure- care). In brief, the purified and high-quality total RNA was ment of global gene expression levels and is a valuable tool used in reverse transcription reactions to convert messenger to study pathologies associated to the central nervous RNA (mRNA) in double-stranded cDNA before biotin system.22–24 labeling. cRNA was synthesized by in vitro transcription of In this study, we aimed to evaluate the effects of nicotine cDNA and simultaneously labeled with biotin-NTP mix. The exposure on gene expression of cultured neurons and glia samples were filtered to recover biotinylated cRNA. Assess- from pons and medulla oblongata of newborn SHR and ment of cRNA concentration, purity and quality was carried WKY rats using whole-genome oligoarrays. out using the Agilent 2100 Bioanalyzer (Agilent Technolo- gies, CA, USA). All the samples presented high quality and purity (260/280 ratio of 2.0). Materials and methods Labeled cRNA targets were fragmented at 94 1C for 20 min before hybridization with arrays at 37 1C during 18 h. After Cell cultures and nicotine treatment the hybridization, the bioarrays were washed and bound All the procedures were approved by the Institutional targets were detected after incubation with Cy5-Streptavidin Committee for animal care of the Institute of Biosciences, (GE Healthcare). The bioarrays were washed, dried and University of Sao Paulo. Cell culture methodology was protected from light. All samples were processed in parallel described in details elsewhere.25 Briefly, pons and medulla and the arrays were incubated simultaneously using oblongata of 1-day old SHR and WKY rats (n ¼ 20 for each the same working solution to limit technical variation strain) from the animal facilities of the Department of across the experiments. After the hybridization, bioarrays Physiology, Institute of Biosciences, University of Sao Paulo were scanned immediately in a GenePix 4000B scanner were dissected out and dissociated in cold isotonic salt (Molecular Dynamics, USA). solution, pH 7.4. Cells were suspended in Neurobasal A media (Invitrogen) supplemented with L-glutamine Analysis of microarray data (250 mmol l–1, Sigma), glutamax (250 mmol l–1, Gibco), B27 CodeLink Expression Analysis software (GE Healthcare) was (2%, Gibco) and gentamicin (40 mg l–1, Gibco). Viable cells used to extract background-subtracted spot intensities from were counted and plated on poly-D-lysine-coated culture microarray images. Measurements that were below the dishes (35 mm, Nunclon, USA) at the concentration of intensity of negative controls plus 2 s.d. were excluded 1800 cells mm–2. Cultures were kept in a humidified incu- and 23 814 genes with valid measurements in all three bator with 5% CO2/95% air, at 37 1C, for 9 days before biological replicates of brainstem cells from normotensive or nicotine exposure. hypertensive animals cultured in the presence or absence of Cultured cells (n ¼ 3 from each strain) were treated with nicotine were further analyzed. To make experiments 10 mM of nicotine (Sigma), diluted in fresh culture medium, comparable, intensity data from different hybridizations over 24 h. Cell cultures (n ¼ 3) kept for the same period of were normalized by the quantile method.26 A two-way The Pharmacogenomics Journal Nicotine regulates gene expression of brainstem cells MFR Ferrari et al 136 analysis of variance (ANOVA) was applied (PowerArray 6.25 ml of DEPC water