title: [The role of protein kinase C-mediated phosphorylation of type III inositol 1, 4, 5-triphosphate receptor in cholecystokinin octapeptide induced calcium mobilization in gastric antral smooth muscle cells].
abstract: OBJECTIVE
  To study the effects of sulfated cholecystokinin octapeptide (sCCK-8S) on intracellular calcium release and extracellular calcium influx in gastric antral smooth muscle cells (SMC) and the mechanism thereof.
  
  
  METHODS
  (1) Longitudinal muscle (LM) and circular muscle (CM) strips of gastric antrum and pylorus were isolated from SD rats and suspended in a tissue chamber to record the contractile responses by polyphysiography. (2) Immunoprecipitation, electrophoresis, and immunoblotting were used to detect the phosphorylation of type III inositol 1, 4, 5-triphosphate receptor (InsP(3)R(3)) in the SMCs. (3) The responsiveness of gastric SMC to CCK-8S was examined by using fura-2-loaded microfluorimetric measurement of intracellular calcium concentration ([Ca(2+)]i). (4) The current of L-type calcium channels (ICaL) was recorded by using patch-clamp techniques.
  
  
  RESULTS
  (1) Significant changes to CCK-8S were found in the mean contractile amplitude of the CM and frequency of LM of gastric antrum and could be suppressed by CCK-A receptor (CCK-AR) antagonist and ATPase inhibitors. (2) CCK-8S stimulation of SMC resulted in PKC-dependent phosphorylation of the InsP(3)R(3). (3) CCK-8S-evoked significant increase in [Ca(2+)]i [from (69 +/- 7) mol/L to (472 +/- 36) nmol/L, P < 0.01] could be suppressed by CCK-AR antagonist, ATPase inhibitors and protein kinase C (PKC) activator; whereas on condition that extracellular calcium was removed or L-type calcium inhibitor nifedipine was added a small but significant increase of [Ca(2+)]i could be still elicited by CCK-8S. (4) CCK-8S-intensified calcium current [from (-56 +/- 7) pA to (-89 +/- 6) pA, P < 0.01] could be apparently inhibited by respective administration of nifedipine, ATPase inhibitors, and calcium dependent chloride channel (I(Cl-Ca)) blocker (all P < 0.01).
  
  
  CONCLUSIONS
  CCK-8S-evoked [Ca(2+)]i increase in gastric antral SMCs depends on the release of intracellular calcium stores, which is regulated by PKC mediated phosphorylation of InsP(3)R(3). The released intracellular calcium in turn activates the L-type voltage-dependent calcium channels (VDCC) through the activation of calcium dependent chloride channels, and ultimately results in the occurrence of contraction response of smooth muscles.
authors.affiliation: Department of Digestive Medicine, People's Hospital of Wuhan University, Wuhan 430060, China.
authors.forename: Xin-Min
authors.initials: XM
authors.lastname: Si
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.forename: He-Sheng
authors.initials: HS
authors.lastname: Luo
authors.forename: Shelley Chireyath
authors.initials: SC
authors.lastname: Paul
authors.forename: Peng
authors.initials: P
authors.lastname: Lü
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000502:physiology; D066298:In Vitro Techniques; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D009543:Nifedipine / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D011706:Pyloric Antrum / Q000166:cytology / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D012844:Sincalide / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: C090019:8-sulfocholecystokinin octapeptide; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011493:Protein Kinase C; D009543:Nifedipine; D012844:Sincalide; D002118:Calcium