The regulation of luteinizing hormone exocytosis in α-toxin permeabilized sheep anterior pituitary cells
type: CreativeWork
description: Although exocytosis is the major mechanism by which cells secrete products into their environment, little is known about the mechanism of this fundamental process. Previous studies on the regulation of luteinizing hormone (LH) exocytosis have used intact cells exclusively. It is not possible, however, to determine the precise requirements for exocytosis in intact cells since the cytosol is not directly accessible. Permeabilization of the plasma membrane allows experimental manipulation of the intracellular milieu while preserving the exocytic apparatus. The diameter of the atoxin pores (2-3 nm) allowed the exchange of small molecules such as ATP while larger cytosolic proteins such as lactate dehydrogenase were retained. Because of the slow exchange of small molecules through a-toxin pores a protocol was developed which combines prolonged pre-equilibration of the permeabilized cells at 0°C before stimulation with strong Ca²⁺ buffering. Under these conditions an increase in the [Ca²⁺]free stimulated a 15-20 fold increase in LH exocytosis (EC₅₀ pCa 5.5). After 12-15 minutes the rate of exocytosis declined and the cells became refractory to Ca²⁺. At resting [Ca²⁺]free (pea 7), cAMP stimulated a rapid, 2 - 3 fold, increase in LH exocytosis. cAMP caused a modest enhancement of Ca²⁺-stimulated LH exocytosis by causing a left shift in the EC₅₀ for Ca²⁺ from pCa 5.6 to pCa 5.9. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) synergistically enhanced cAMP-stimulated LH exocytosis, an effect which was further augmented by increasing the [Ca²⁺]free· Gonadotrophin-releasing hormone (GnRH) was found to stimulate cAMP production in intact pituitary cells. Since previous studies have shown that GnRH activates PKC and stimulates a rise in cytosolic [Ca²⁺]free, these results suggest that a synergistic interaction of the cAMP, PKC and Ca²⁺ second messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis. When permeabilized ce…
abstract: Although exocytosis is the major mechanism by which cells secrete products into their environment, little is known about the mechanism of this fundamental process. Previous studies on the regulation of luteinizing hormone (LH) exocytosis have used intact cells exclusively. It is not possible, however, to determine the precise requirements for exocytosis in intact cells since the cytosol is not directly accessible. Permeabilization of the plasma membrane allows experimental manipulation of the intracellular milieu while preserving the exocytic apparatus. The diameter of the atoxin pores (2-3 nm) allowed the exchange of small molecules such as ATP while larger cytosolic proteins such as lactate dehydrogenase were retained. Because of the slow exchange of small molecules through a-toxin pores a protocol was developed which combines prolonged pre-equilibration of the permeabilized cells at 0°C before stimulation with strong Ca²⁺ buffering. Under these conditions an increase in the [Ca²⁺]free stimulated a 15-20 fold increase in LH exocytosis (EC₅₀ pCa 5.5). After 12-15 minutes the rate of exocytosis declined and the cells became refractory to Ca²⁺. At resting [Ca²⁺]free (pea 7), cAMP stimulated a rapid, 2 - 3 fold, increase in LH exocytosis. cAMP caused a modest enhancement of Ca²⁺-stimulated LH exocytosis by causing a left shift in the EC₅₀ for Ca²⁺ from pCa 5.6 to pCa 5.9. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) synergistically enhanced cAMP-stimulated LH exocytosis, an effect which was further augmented by increasing the [Ca²⁺]free· Gonadotrophin-releasing hormone (GnRH) was found to stimulate cAMP production in intact pituitary cells. Since previous studies have shown that GnRH activates PKC and stimulates a rise in cytosolic [Ca²⁺]free, these results suggest that a synergistic interaction of the cAMP, PKC and Ca²⁺ second messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis. When permeabilized ce…
author: Van der Merwe, Philip Anton
language: eng
date published: 1990
date modified: 2026-07-22T22:22:52Z
provider: University of Cape Town
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Division of Chemical Pathology

PropertyValue
property ID: Handle
value: http://hdl.handle.net/11427/26556