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.
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Research Article|
December 15 1989
Mechanisms of luteinizing-hormone exocytosis in Staphylococcus aureus-α-toxin-permeabilized sheep gonadotropes
P A van der Merwe;
P A van der Merwe
1Medical Research Council Regulatory Peptides Research Unit, Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, Republic of South Africa
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R P Millar;
R P Millar
1Medical Research Council Regulatory Peptides Research Unit, Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, Republic of South Africa
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I K Wakefield;
I K Wakefield
1Medical Research Council Regulatory Peptides Research Unit, Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, Republic of South Africa
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J S Davidson
J S Davidson
1Medical Research Council Regulatory Peptides Research Unit, Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, Republic of South Africa
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1989 London: The Biochemical Society
1989
Biochem J (1989) 264 (3): 901–908.
Citation
P A van der Merwe, R P Millar, I K Wakefield, J S Davidson; Mechanisms of luteinizing-hormone exocytosis in Staphylococcus aureus-α-toxin-permeabilized sheep gonadotropes. Biochem J 15 December 1989; 264 (3): 901–908. doi: https://doi.org/10.1042/bj2640901
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