ArrayExpress Uploaderarrayexpress_sidhumanthapsigarginhigh potassiumcontrolhigh potassiumthapsigargincontrolcontrolthapsigarginhigh potassium143803Altered Ca2+ handling has both immediate physiological effects and long-term genomic effects on vascular smooth muscle function....16461377E-GEOD-2883/profile/8773/arrayexpressuploader19cellleftmembranemuscleproteinDec.12, 2014Falsetranscription-profilfing-of-human-cerebrovascularE-GEOD-2883_A-AFFY-33Transcription profilfing of human cerebrovascular smooth muscle to identify Ca2+-dependent transcription patternsJun.19, 2014Altered Ca2+ handling has both immediate physiological effects and long-term genomic effects on vascular smooth muscle function. Previously we have shown that elevation of cytoplasmic Ca2+ through voltage-dependent Ca2+ channels (VDCCs) or store-operated Ca2+ channels (SOCCs) results in phosphorylation of the Ca2+/cAMP response element binding protein (CREB) in cerebral arteries. Here we demonstrate that stimulation of these different Ca2+ influx pathways results in transcriptional activation of a distinct, yet overlapping set of genes, and that the induction of selected CRE-regulated genes is prevented by the addition of corresponding Ca2+ channel blockers. Using oligonucleotide array analysis, changes in mRNA levels were quantified following membrane depolarization with K+ or depletion of intracellular Ca2+ stores with thapsigargin in human cerebral vascular smooth muscle cells. Array results for differentially regulated genes containing a CRE were confirmed by quantitative RT-PCR, and corresponding changes in protein expression were shown by Western blot analysis and immunofluorescence. Membrane depolarization induced a transient increase in c-fos mRNA and a sustained increase in early growth response-1 (Egr-1) mRNA and protein that were inhibited by application of the VDCC blocker, nimodipine, and the SOCC inhibitor, 2-aminoethoxydiphenylborate (2-APB). Thapsigargin induced a sustained increase in c-fos mRNA and MAP kinase phosphatase-1 (MKP-1) mRNA and protein, and these effects were decreased by 2-APB but not by nimodipine. Our findings thus indicate that Ca2+ entry through VDCCs and SOCCs can differentially regulate CRE-containing genes in vascular smooth muscle and imply that signals involved in growth modulation are both temporally and spatially regulated by Ca2+. Experiment Overall Design: each of three experiments cell cultures were split three ways; one of the resulting samples was left untreated (C), another was treated with thapsigargin (TG), and the third was treated with elevated K+ (K). The resulting nine samples were used for triplicate estimates of the response of each gene to TG and K treatments. Experiment Overall Design: We tested the prospective hypothesis that genes having a CRE are differentially expressed after TG or K treatment using a permutation test: each of the 22,283 probe sets on the Affymetrix GeneChip was categorized two ways based on 1) whether or not it contains a CRE or not . Independence of CRE and threshold differential expression was rejected by Fisher’s exact test for both TG treatment ( ) and K treatment ( ). Target genes (c-fos, egr-1, and mkp-1 ) were identified based on ranking.http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-2883http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-2883/samples/