title: Release of Ca2+ from intracellular organelles by peptide analogues: evidence against involvement of metalloendoproteases in Ca2+ sequestration by the endoplasmic reticulum.
abstract: N-Benzyloxycarbonyl-Gly-Phe-amide (Z-Gly-Phe-NH2), a competitive substrate for metalloendoproteases, mobilizes intracellular Ca2+ and suppresses protein synthesis and processing in a Ca(2+)-dependent, reversible manner. To ascertain whether Z-Gly-Phe-NH2 acts as Ca(2+)-storing organelles, effects of the dipeptide on Ca2+ sequestration by saponin-porated GH3 pituitary cells were examined. Porated preparations sequestered Ca2+ into two compartments with different Ca2+ affinities. Ca2+ accumulation at nM concentrations of free Ca2+ was inhibited by thapsigargin and inositol 1,4,5-triphosphate [Ins(1,4,5)P3], enhanced by oxalate and unaffected by oligomycin. Cation accumulation at microM concentrations of free Ca2+ was sensitive to oligomycin but not to thapsigargin. Z-Gly-Phe-NH2 reduced Ca2+ sequestration by both compartments. The dipeptide mobilized Ca2+ from the high-affinity compartment within 1-2 min without affecting Ca2+ uptake. Ca2+ was mobilized more rapidly by Z-Gly-Phe-NH2 and thapsigargin together than by either agent alone. The presence of a thiol-reducing agent was required for Ca2+ mobilization by Z-Gly-Phe-NH2 but not by thapsigargin or Ins(1,4,5)P3. Ca2+ mobilization by Z-Gly-Phe-NH2 could not be attributed to effects on anion-permeability or to actions at Ins(1,4,5)P3 or ryanodine receptors. Results with assorted peptide analogues did not favour suppression of metalloendoprotease activity in the Ca(2+)-mobilizing action of Z-Gly-Phe-NH2. The more hydrophobic analogue Z-L-Tyr-p-nitrophenyl ester was 60-80-fold more potent in mobilizing Ca2+ from intact and porated cells and perturbed the high-affinity Ca(2+)-sequestering compartment selectively. Z-Gly-Phe-NH2 and Z-L-Tyr-p-nitrophenyl ester are proposed to release Ca2+ from the endoplasmic reticulum through an ion pore with affinity for hydrophobic molecules containing internal peptide bonds.
authors.affiliation: Department of Pharmacology, Robert Wood Johnson Medical School, Piscataway, NJ 08854.
authors.forename: M A
authors.initials: MA
authors.lastname: Brostrom
authors.forename: W L
authors.initials: WL
authors.lastname: Wong Ling
authors.forename: D
authors.initials: D
authors.lastname: Gmitter
authors.forename: C O
authors.initials: CO
authors.lastname: Brostrom
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D004151:Dipeptides / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000378:metabolism*; D015388:Organelles / Q000187:drug effects / Q000378:metabolism*; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D012503:Saponins; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
chemical_list: D004151:Dipeptides; D012503:Saponins; D013729:Terpenes; C066375:carbobenzoxyglycylphenylalanine amide; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008666:Metalloendopeptidases; D002118:Calcium