scholarly journals Relationship between arachidonic acid release and Ca2+-dependent exocytosis in digitonin-permeabilized bovine adrenal chromaffin cells

1990 ◽  
Vol 271 (3) ◽  
pp. 571-574 ◽  
Author(s):  
A Morgan ◽  
R D Burgoyne
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Chromaffin Cells ◽  
Nordihydroguaiaretic Acid ◽  
Inhibitory Effect ◽  
Arachidonic Acid Release ◽  
Adrenal Chromaffin Cells ◽  
Protein Kinase C Inhibitor ◽  
Acid Release ◽  
Adrenal Chromaffin

The relationship between Ca2(+)-dependent arachidonic acid release and exocytosis from digitonin-permeabilized bovine adrenal chromaffin cells was investigated. The phospholipase A2 inhibitors mepacrine, nordihydroguaiaretic acid and indomethacin had no effect on either arachidonic acid release or secretion. The phospholipase A2 activator melittin had no effect on secretion. The specific diacylglycerol lipase inhibitor RG80267 had no effect on secretion, but decreased basal arachidonic acid release to such an extent that the level of arachidonic acid in treated cells in response to 10 microM-Ca2+ was equivalent to that of control cells in the absence of Ca2+. Staurosporine, a protein kinase C inhibitor, was found to abolish Ca2(+)-dependent arachidonic acid release completely, but had only a slight inhibitory effect on Ca2(+)-dependent secretion. It is concluded that arachidonic acid is not essential for Ca2(+)-dependent exocytosis in adrenal chromaffin cells.

scholarly journals Stimulation of Ca2+-independent catecholamine secretion from digitonin-permeabilized bovine adrenal chromaffin cells by guanine nucleotide analogues. Relationship to arachidonate release

1990 ◽  
Vol 269 (2) ◽  
pp. 521-526 ◽  
Author(s):  
A Morgan ◽  
R D Burgoyne
Keyword(s):  
Arachidonic Acid ◽  
Chromaffin Cells ◽  
Binding Protein ◽  
Catecholamine Secretion ◽  
Arachidonic Acid Release ◽  
Adrenal Chromaffin Cells ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Acid Release ◽  
Adrenal Chromaffin

The effect of GTP analogues on catecholamine secretion and [3H]arachidonic acid release from digitonin-permeabilized adrenal chromaffin cells was examined. Several GTP analogues stimulated Ca2(+)-independent exocytosis, with the order of efficacy being XTP greater than ITP greater than guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5′-[gamma-thio]triphosphate (GTP[S]). The stimulatory effect of the GTP analogues appeared to be due to activation of a conventional GTP-binding protein, as it was inhibited by guanosine 5′-[beta-thio]diphosphate (GDP[S]). In contrast, Ca2(+)-dependent exocytosis was only partially inhibited by high doses of GDP[S]. GTP did not stimulate Ca2(+)-independent exocytosis, but instead was found to inhibit secretion caused by micromolar Ca2+. Arachidonic acid (100 microM) also stimulated Ca2(+)-independent catecholamine secretion. Determination of the effect of GTP analogues on release of free [3H]arachidonic acid into the medium showed that it was stimulated by GTP[S] but inhibited by GTP, p[NH]ppG, ITP and XTP. The inhibition of [3H]arachidonic acid release by XTP was not prevented by GDP[S]. These results demonstrate that activation of a GTP-binding protein by certain GTP analogues can induce Ca2(+)-independent secretion in adrenal chromaffin cells and that the effect of GTP analogues on Ca2(+)-independent secretion can be dissociated from generation of arachidonic acid.

scholarly journals Exocytosis from permeabilized bovine adrenal chromaffin cells is differently modulated by guanosine 5′-[γ-thio]triphosphate and guanosine 5′-[β γ-imido]triphosphate. Evidence for the involvement of various guanine nucleotide-binding proteins

1992 ◽  
Vol 284 (2) ◽  
pp. 321-326 ◽  
Author(s):  
G Ahnert-Hilger ◽  
U Wegenhorst ◽  
B Stecher ◽  
K Spicher ◽  
W Rosenthal ◽  
...  
Keyword(s):  
G Protein ◽  
Chromaffin Cells ◽  
Inhibitory Effect ◽  
Adrenal Chromaffin Cells ◽  
Prolonged Incubation ◽  
Permeabilized Cells ◽  
Alpha Subunits ◽  
Cytoplasmic Material ◽  
Streptolysin O ◽  
Adrenal Chromaffin

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.

scholarly journals Role of Phosphorylation Sites and the C2 Domain in Regulation of Cytosolic Phospholipase A2

1999 ◽  
Vol 145 (6) ◽  
pp. 1219-1232 ◽  
Author(s):  
Miguel A. Gijón ◽  
Diane M. Spencer ◽  
Alan L. Kaiser ◽  
Christina C. Leslie
Keyword(s):  
Arachidonic Acid ◽  
Nuclear Envelope ◽  
Phospholipase A2 ◽  
Okadaic Acid ◽  
Cytosolic Phospholipase A2 ◽  
Phosphorylation Sites ◽  
C2 Domain ◽  
Arachidonic Acid Release ◽  
Cytosolic Phospholipase ◽  
Acid Release

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.

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