RELEASE OF CA2+ FROM INTRACELLULAR ORGANELLES BY PEPTIDE ANALOGS - EVIDENCE AGAINST INVOLVEMENT OF METALLOENDOPROTEASES IN CA2+ SEQUESTRATION BY THE ENDOPLASMIC-RETICULUM

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 Ca2+-dependent, reversible manner. To ascertain whether Z-Gly-Phe-NH2 acts at Ca2+-storing organelles, effects of the dipeptide on Ca2+ sequestration by saponin-porated GH(3) 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)P-3], enhanced by oxalate and unaffected by oligomycin. Cation accumulation at mu M 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)P-3. Ca2+ mobilization by Z-Gly-Phe-NH2 could not be attributed to effects on anion-permeability or to actions at Ins(1,4,5)P-3 or ryanodine receptors. Results with assorted peptide analogues did not favour suppression of metalloendoprotease activity in the Ca2+-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 Ca2+-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.