Dataset Information

Relationship between arachidonate release and exocytosis in permeabilized human neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMetLeuPhe), guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and Ca2+.

ABSTRACT: The role of two G-proteins, Gp and Ge, in the stimulus-secretion pathway has been proposed on the basis of studies where GTP analogues have been introduced into permeabilized cell preparations. In this study, evidence is provided that two G-proteins are also involved when a receptor-directed agonist is used. Intact human neutrophils were made refractory to formylmethionyl-leucyl-phenylalanine (fMetLeuPhe) stimulation by metabolic inhibition and then permeabilized with streptolysin O to compare the intracellular requirements for exocytosis from specific and azurophilic granules and arachidonate release. In the presence of 1 microM-Ca2+ and 1 mM-MgATP, fMetLeuPhe or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induce secretion from both granule types as well as arachidonate release. Secretion and arachidonate release owing to fMetLeuPhe can occur in the absence of ATP, conditions under which G-protein-mediated activation of phospholipase C is suppressed. GTP[S]-induced secretion can also occur in the absence of MgATP, but GTP[S]-induced arachidonate release cannot. It is concluded that fMetLeuPhe, like GTP[S], stimulates secretion by interacting with another G-protein-mediated reaction apart from Gp. Evidence is provided that a possible target for the second G-protein-mediated reaction involved in fMetLeuPhe-induced secretion (but not GTP[S]-induced secretion) is phospholipase A2.

SUBMITTER: Cockcroft S

PROVIDER: S-EPMC1150022 | biostudies-other | 1991 Apr

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O.

Dataset Information

Relationship between arachidonate release and exocytosis in permeabilized human neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMetLeuPhe), guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and Ca2+.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure