Dual effects of guanosine 5'-[gamma-thio]triphosphate on secretion by electroporated human neutrophils.
It is generally believed that G-proteins play stimulatory roles on cell activation. In contrast, we found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a potent inhibitor of Ca(2+)-induced secretion from specific granules (as monitored by vitamin B-12-binding protein). GTP[S] inhibition of specific-granule release occurred in the presence or absence of adenine nucleotides, required Mg2+ (1-3 mM), and was half-maximal at 30 microM-GTP[S]. The dual stimulatory and inhibitory effects of GTP[S] could be readily observed and differentiated when degranulation was monitored over a range of Ca2+ concentrations. Inhibition of specific-granule release by GTP[S] was observed at low Ca2+ concentrations and resulted from shifting the Ca2+ dose-response curves to the right. In contrast, GTP[S] promoted azurophil-granule secretion at relatively high concentrations of Ca2+ and appeared to be due to a general enhancement at all Ca2+ concentrations. A series of hydrolysable and non-hydrolysable nucleotides did not mimic GTP[S] or block its action. Inhibition by GTP[S] occurred in cells which were sensitized with a protein kinase C agonist, suggesting that inhibition of secretion took place distal to this enzyme. However, the inhibitory effects of GTP[S] on specific-granule secretion were reversed by cytochalasin D, which prevents new microfilament formation; this compound also enhanced the stimulation of azurophil-granule release by GTP[S]. We also found that GTP[S] greatly increased the F-actin content of permeabilized neutrophils, whereas Ca2+ (to a lesser extent) decreased F-actin. These data are consistent with the hypothesis that at least two G-proteins are involved in regulating secretion: one which has been previously described as stimulating Ca(2+)-induced secretion (particularly from azurophil granules) and a second, possibly involved in promoting microfilament assembly, which inhibits the discharge of specific granules.
Smolen
J E
JE
Department of Pediatrics, University of Michigan Medical Center, Ann Arbor 48109.
Stoehr
S J
SJ
Kuczynski
B
B
Koh
E K
EK
Omann
G M
GM
D000199:Actins / Q000737:chemistry; D000328:Adult; D002118:Calcium / Q000502:physiology; D002772:Cholera Toxin / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D004558:Electric Stimulation; D019204:GTP-Binding Proteins / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006801:Humans; D008274:Magnesium / Q000502:physiology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000199:Actins; D010566:Virulence Factors, Bordetella; D015638:Cytochalasin D; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium