title: Mechanisms of luteinizing-hormone exocytosis in Staphylococcus aureus-alpha-toxin-permeabilized sheep gonadotropes.
abstract: We have used primary gonadotropes permeabilized with the pore-forming protein Staphylococcus aureus alpha-toxin to investigate luteinizing hormone (lutropin, LH) exocytosis. The diameter of the alpha-toxin pores (2-3 nm) allows the exchange of small molecules, whereas larger cytosolic proteins are retained. Because of the slow exchange of small molecules through the pores, we have developed a protocol which combines prolonged pre-equilibration of the permeabilized cells at 0 degrees C before stimulation with strong Ca2+ buffering. Under these conditions, increasing the free Ca2+ concentration from 0.1 microM to 10 microM [EC50 (concentration effecting half-maximal response) 2-3 microM] resulted in a 15-20-fold increase in LH exocytosis. LH exocytosis was maximal in the first 3 min and completed by 12 min. When permeabilized cells were equilibrated for prolonged periods in the absence of MgATP, Ca2(+)-stimulated LH secretion gradually declined (greater than 90% decrease by 60 min). Addition of MgATP (5 mM) rapidly restored full Ca2(+)-stimulated LH secretion. MgATP supported Ca2(+)-stimulated LH secretion at a half-maximal concentration of 1.5 mM. UTP and adenosine 5'-[gamma-thio]triphosphate were 40 and 31% as effective as MgATP, whereas other nucleotide triphosphates were ineffective. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; 50 nM) stimulated LH exocytosis at free Ca2+ concentrations as low as 1 nM and was additive with Ca2+ at higher free Ca2+ concentrations. PMA-stimulated exocytosis required MgATP at concentrations similar to those required for Ca2(+)-stimulated LH exocytosis. These results demonstrate that LH exocytosis can be triggered both by micromolar Ca2+ concentrations or, in the virtual absence of Ca2+, by PKC activation. Both mechanisms of stimulated exocytosis have an absolute requirement for millimolar ATP. Because they retain cytosolic proteins, alpha-toxin-permeabilized cells may have advantages over alternative permeabilization methods provided that conditions are used that compensate for slow diffusion through alpha-toxin pores.
authors.affiliation: Department of Chemical Pathology, University of Cape Town Medical School, Observatory, Republic of South Africa.
authors.forename: P A
authors.initials: PA
authors.lastname: van der Merwe
authors.forename: R P
authors.initials: RP
authors.lastname: Millar
authors.forename: I K
authors.initials: IK
authors.lastname: Wakefield
authors.forename: J S
authors.initials: JS
authors.lastname: Davidson
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D005089:Exocytosis; D006460:Hemolysin Proteins; D007700:Kinetics; D007986:Luteinizing Hormone / Q000378:metabolism*; D008297:Male; D009919:Orchiectomy; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology; D012756:Sheep; D013211:Staphylococcus aureus; D013755:Tetradecanoylphorbol Acetate
chemical_list: D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D012265:Ribonucleotides; C021091:staphylococcal alpha-toxin; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D007986:Luteinizing Hormone; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium