title: Expression of functional Stim1-mKO1 in rat submandibular acinar cells by retrograde ductal injection of an adenoviral vector.
abstract: OBJECTIVE
  Adenoviruses are used for gene transfer to salivary glands cells in vivo. We constructed an adenovirus vector that expressed a fusion protein of human Stim1 and the fluorescent protein mKO1 (Ad-Stim1-mKO1), and used it to investigate the molecular dynamics and functions of exogenously expressed proteins in living salivary acinar cells.
  
  
  METHODS
  Ad-Stim1-mKO1 was transferred to rat submandibular glands by retrograde ductal injection. Expression and distribution of Stim1-mKO1 were examined by confocal microscopy. In addition, the effects of Stim1-mKO1 on store-operated Ca(2+) entries were examined in fura-2-loaded cells.
  
  
  RESULTS
  The expression of Stim1-mKO1 was detected in approximately 40% of rat submandibular acini, whereas the expression in HSY-EA1 cells was ∼80%. Confocal microscopy revealed Stim1-mKO1 fluorescence along the endoplasmic reticulum-like network in the cytoplasm of both HSY-EA1 and dispersed acinar cells. The depletion of intracellular Ca(2+) stores with thapsigargin (ThG), a sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) inhibitor, led to the translocation of Stim1-mKO1 to the peripheral region in these cells. In addition, expression of Stim1-mKO1 enhanced store-operated Ca(2+) entry in these cells.
  
  
  CONCLUSIONS
  We succeeded in expressing Stim1-mKO1 fluorescent protein in the salivary glands of live animals by retrograde ductal injection of an adenoviral vector. This method allowed us to investigate the functions and molecular dynamics of these expressed molecules in living salivary acinar cells.
authors: Department of Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan. moritat@hoku-iryo-u.ac.jp
authors: Takao
authors: T
authors: Morita
authors: Akihiko
authors: A
authors: Tanimura
authors: Akiko
authors: A
authors: Shitara
authors: Yuko
authors: Y
authors: Suzuki
authors: Akihiro
authors: A
authors: Nezu
authors: Taishin
authors: T
authors: Takuma
authors: Yosuke
authors: Y
authors: Tojyo
mesh_terms: D061354:Acinar Cells / Q000378:metabolism*; D000256:Adenoviridae; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D016257:Fura-2 / Q000494:pharmacology; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000502:physiology; D018613:Microscopy, Confocal; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D000071737:Stromal Interaction Molecule 1; D013363:Submandibular Gland / Q000166:cytology* / Q000378:metabolism; D019284:Thapsigargin / Q000378:metabolism
chemical_list: D002614:Chelating Agents; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D019284:Thapsigargin; D002118:Calcium; D016257:Fura-2