title: Reduction in TRPC4 expression specifically attenuates G-protein coupled receptor-stimulated increases in intracellular calcium in human myometrial cells.
abstract: Canonical transient receptor potential (TRPC) proteins may play a role in regulating changes in intracellular calcium ([Ca(2+)](i)). Human myometrium expresses TRPC4, TRPC1 and TRPC6 mRNAs in greatest relative abundance. Contributions of TRPC4 to increases in [Ca(2+)](i) were assessed in PHM1-41 and primary human uterine smooth muscle (UtSMC) cells using short hairpin RNAs (shRNAs). Based on a reporter assay screen, one shRNA was selected to construct an adenoviral expression vector (TC4sh1). TC4sh1 induced both mRNA and protein TRPC4 knockdown in PHM1-41 cells without affecting expression of other TRPCs. Signal-regulated Ca(2+) entry (SRCE), defined as a stimulus- and extracellular Ca(2+)-dependent increase in [Ca(2+)](i), was measured in PHM1-41 cells treated with oxytocin (G-protein coupled receptor (GPCR)-stimulated), thapsigargin (store depletion-stimulated), and OAG (diacylglycerol-stimulated), using Fura-2. Cells infected with TC4sh1 exhibited attenuated oxytocin-, ATP- and PGF2alpha-mediated SRCE, but no change in thapsigargin- or OAG-stimulated SRCE. Similar results were obtained in primary uterine smooth muscle cells. Additionally, cells expressing TC4sh1 exhibited a significantly smaller increase in channel activity in response to oxytocin administration than did cells infected with empty virus. These data show that, in human myometrial cells, knockdown of endogenous TRPC4 specifically attenuates GPCR-stimulated, but not thapsigargin- or OAG-stimulated extracellular calcium-dependent increases in [Ca(2+)](i). These data imply that, in this cellular context, the mechanisms regulating extracellular Ca(2+)-dependent increases in [Ca(2+)](i) are differentially affected by different signaling pathways.
authors.affiliation: Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Aida
authors.initials: A
authors.lastname: Ulloa
authors.forename: Albert L
authors.initials: AL
authors.lastname: Gonzales
authors.forename: Miao
authors.initials: M
authors.lastname: Zhong
authors.forename: Yoon-Sun
authors.initials: YS
authors.lastname: Kim
authors.forename: Jeremy
authors.initials: J
authors.lastname: Cantlon
authors.forename: Colin
authors.initials: C
authors.lastname: Clay
authors.forename: Chun-Ying
authors.initials: CY
authors.lastname: Ku
authors.forename: Scott
authors.initials: S
authors.lastname: Earley
authors.forename: Barbara M
authors.initials: BM
authors.lastname: Sanborn
mesh_terms: D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D009130:Muscle, Smooth / Q000378:metabolism; D009215:Myometrium / Q000378:metabolism*; D010121:Oxytocin / Q000494:pharmacology; D034622:RNA Interference / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D043562:Receptors, G-Protein-Coupled; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; C450520:seven-transmembrane G-protein-coupled receptor; D010121:Oxytocin; D019284:Thapsigargin; D002118:Calcium