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Effects of MaxiK silencing in corporal smooth muscle cells

ABSTRACT: The MaxiK potassium channel is a key modulator of smooth muscle tone. Due to its calcium and voltage sensitivity, MaxiK is activated following depolarization and Ca2+ mobilization, therefore relaxing the muscle. We investigate the effects of silencing MaxiK for 48h in corpus cavernosuml smooth muscle (CCSM) cells to identify possible mechanisms of compensation through molecular crosstalk between pathways regulating smooth muscle tone.

ORGANISM(S): Homo sapiens

PROVIDER: GSE19672 | GEO | 2010/12/25

SECONDARY ACCESSION(S): PRJNA122495

REPOSITORIES: GEO

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Project description:Altered 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 Fisherâs exact test for both TG treatment ( ) and K treatment ( ). Target genes (c-fos, egr-1, and mkp-1 ) were identified based on ranking.

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Effects of MaxiK silencing in corporal smooth muscle cells

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