scholarly journals Lipopeptides activate Gi-proteins in dibutyryl cyclic AMP-differentiated HL-60 cells

1993 ◽  
Vol 296 (1) ◽  
pp. 245-251 ◽  
Author(s):  
J F Klinker ◽  
A Höer ◽  
I Schwaner ◽  
S Offermanns ◽  
K Wenzel-Seifert ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Phosphatidic Acid ◽  
G Protein ◽  
Acid Formation ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Dibutyryl Cyclic Amp ◽  
Gtp Hydrolysis ◽  
Alpha Subunits ◽  
Gi Proteins

Synthetic lipopeptides activate superoxide-anion (O2-) formation in human neutrophils in a pertussis-toxin (PTX)-sensitive manner, suggesting the involvement of G-proteins of the Gi family in the signal-transduction pathway. We compared G-protein activation by lipopeptides and the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in dibutyryl-cyclic-AMP-differentiated HL-60 cells. The lipopeptide (2S)-2-palmitoylamino-6-palmitoyloxymethyl-7-palmitoyloxy heptanoyl-SK4 (Pam3AhhSK4) and fMLP activated high-affinity GTPase, i.e. the enzymic activity of G-protein alpha-subunits, in HL-60 membranes in a time- and protein-dependent manner, but they had no effect on Mg(2+)-ATPase and Na+/K(+)-ATPase. Pam3AhhSK4 and fMLP increased Vmax. of GTP hydrolysis. Pam3AhhSK4 activated GTP hydrolysis with half-maximal and maximal effects at about 2 microM and 10 microM respectively. Other lipopeptides activated GTP hydrolysis as well. Lipopeptides were less effective than fMLP to activate GTPase. In membranes from PTX-treated cells, the stimulatory effects of lipopeptides and fMLP on GTPase were abolished. In N-ethylmaleimide-treated membranes, the relative stimulatory effect of Pam3AhhSK4 on GTP hydrolysis was enhanced, whereas that of fMLP was diminished. fMLP and Pam3AhhSK4 activated GTPase in an over-additive manner in N-ethylmaleimide-treated membranes. Unlike fMLP, Pam3AhhSK4 did not enhance incorporation of GTP azidoanilide into, and cholera-toxin-catalysed ADP-ribosylation of Gi-protein alpha-subunits in, HL-60 membranes and did not induce rises in cytosolic Ca2+ concentration. Pam3AhhSK4 and fMLP stimulated phosphatidic acid formation in a PTX-sensitive manner. Pam3AhhSK4 itself did not activate O2- formation, but potentiated the stimulatory effects of fMLP. Our data suggest that (i) lipopeptides activate the GTPase of Gi-proteins, (ii) lipopeptides and fMLP activate Gi-proteins differently, (iii) lipopeptides stimulate phospholipase D via Gi-proteins, and (iv) phosphatidic acid formation is not sufficient for activation of O2- formation.

scholarly journals Effect of pertussis toxin and neomycin on G-protein-regulated polyphosphoinositide phosphodiesterase. A comparison between HL60 membranes and permeabilized HL60 cells

1988 ◽  
Vol 256 (2) ◽  
pp. 343-350 ◽  
Author(s):  
S Cockcroft ◽  
J Stutchfield
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Dimethyl Sulphoxide ◽  
Hl60 Cell ◽  
Dependent Manner ◽  
Dibutyryl Cyclic Amp ◽  
Hl60 Cells ◽  
Permeabilized Cells ◽  
Streptolysin O

The promyelocytic HL60 cell can be differentiated with dimethyl sulphoxide or dibutyryl cyclic AMP leading to the appearance of fMetLeuPhe receptors on the cell surface. G-protein-stimulated polyphosphoinositide phosphodiesterase (PPI-pde) activity was assessed in membranes prepared from both differentiated and non-differentiated HL60 cells. Both the extent of the response and the rank order of potency of the GTP analogues to stimulate PPI-pde activation (guanosine 5′-[gamma-thio]triphosphate (GTP[S]) greater than guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5′-[beta gamma-methylene]triphosphate (p[CH2]ppG) remains unchanged after differentiation with dimethyl sulphoxide. In comparison, differentiation by dibutyryl cyclic AMP leads to diminution of PPI-pde activity when stimulated by GTP[S] or fluoride, but not by millimolar concentrations of Ca2+. GTP[S]-stimulated PPI-pde in membranes is sensitive to the presence of Ca2+ (pCa 8-5). Pertussis-toxin pretreatment of intact HL60 cells leads to inhibition of both the secretory response and the formation of inositol phosphates when stimulated by fMetLeuPhe. In contrast, pertussis-toxin pretreatment has no effect on either GTP[S]- or fluoride-stimulated PPI-pde. Neomycin in a concentration-dependent manner inhibits both GTP[S] plus Ca2+ (pCa 5)-stimulated secretion and PPI-pde activation in streptolysin-O-permeabilized cells. The extent of PPI-pde activation in membranes compared with streptolysin-O-permeabilized cells reveals that the membrane preparation does not possess all the components that make up the inositide signalling system.

scholarly journals Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase

1984 ◽  
Vol 220 (3) ◽  
pp. 803-809 ◽  
Author(s):  
C J Dix ◽  
A D Habberfield ◽  
B A Cooke
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
G Protein ◽  
Tumour Cell ◽  
Tumour Cells ◽  
Dependent Manner ◽  
Dibutyryl Cyclic Amp ◽  
Heterologous Desensitization ◽  
Homologous Desensitization

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.

scholarly journals Inhibition by somatostatin of amylase secretion induced by calcium and cyclic AMP in rat pancreatic acini

1994 ◽  
Vol 304 (2) ◽  
pp. 531-536 ◽  
Author(s):  
H Ohnishi ◽  
T Mine ◽  
I Kojima
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Pertussis Toxin ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Amylase Secretion ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Pancreatic Acini

It has recently been shown that somatostatin inhibits amylase secretion from isolated pancreatic acini by reducing cyclic AMP (cAMP) production [Matsushita, Okabayashi, Hasegawa, Koide, Kido, Okutani, Sugimoto and Kasuga (1993) Gastroenterology 104, 1146-1152]. To date, however, little is known as to the other mechanism(s) by which somatostatin inhibits amylase secretion in exocrine pancreas. To investigate the action of somatostatin independent of cAMP generation, we examined the effect of somatostatin in isolated rat pancreatic acini stimulated by 1 microM calcium ionophore A23187 and 1 mM 8-bromo-cyclic AMP (8Br-cAMP). Somatostatin inhibited amylase secretion evoked by a combination of A23187 and 8Br-cAMP in a dose-dependent manner. The maximum inhibition was obtained by 10(-7) M somatostatin, and at this concentration somatostatin inhibited the effect of A23187 and 8Br-cAMP by approximately 30%. In electrically permeabilized acini, an elevation of free calcium concentration resulted in an increase in amylase secretion and cAMP enhanced the secretion evoked by calcium. cAMP shifted the dose-response curve for calcium-induced secretion leftwards and elevated the peak value of secretion. Somatostatin inhibited the effect of cAMP on calcium-induced amylase secretion by shifting the dose-response curve to the right. To determine the involvement of a G-protein(s), we examined the effect of somatostatin in acini pretreated with pertussis toxin. Pretreatment of acini with pertussis toxin completely blocked somatostatin-inhibition of amylase-secretion evoked by A23187 and 8Br-cAMP. These results indicate that somatostatin decreases amylase secretion induced by cAMP and calcium by reducing the calcium sensitivity of exocytosis. A pertussis toxin-sensitive G-protein is also involved in this step.

Abscisic acid induction of GTP hydrolysis in maize coleoptile plasma membranes

1998 ◽  
Vol 25 (5) ◽  
pp. 539 ◽  
Author(s):  
Helen R. Irving
Keyword(s):  
Abscisic Acid ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Plasma Membranes ◽  
Dependent Manner ◽  
Gtpase Activity ◽  
Heterotrimeric G Proteins ◽  
Gtp Hydrolysis ◽  
Maize Coleoptile

Since receptor-coupled G proteins increase GTP hydrolysis (GTPase) activity upon ligands binding to the receptor, a study was undertaken to determine if abscisic acid (ABA) induced such an effect. Plasma membranes isolated from etiolated maize (Zea mays L.) coleoptiles were enriched in GTPase activity relative to microsomal fractions. Vanadate was included in the assay to inhibit the high levels of vanadate sensitive low affinity GTPases present. Under these conditions, GTPase activity was enhanced by Mg2+, stimulated by mastoparan, and inhibited by GTPγS indicating the presence of either monomeric or heterotrimeric G proteins. The combination of NaF and AlCl3 is expected to inhibit heterotrimeric G protein activity but had little effect on GTPase activity in maize coleoptile membranes. Cholera toxin enhanced basal GTPase activity, confirming the presence of heterotrimeric G proteins in maize plasma membranes. Pertussis toxin also slightly enhanced basal GTPase activity in maize membranes. Abscisic acid enhanced GTPase activity optimally at 5 mmol/L Mg2+ in a concentration dependent manner by 1.5-fold at 10 µmol/L and up to three-fold at 100 µmol/L ABA. Abscisic acid induced GTPase activity was inhibited by GTPγS, the combination of NaF and AlCl3, and pertussis toxin. Overall, these results are typical of a receptor-coupled G protein responding to its ligand.

scholarly journals Mechanism of GTP hydrolysis by G-protein alpha subunits.

1994 ◽  
Vol 91 (21) ◽  
pp. 9828-9831 ◽  
Author(s):  
C. Kleuss ◽  
A. S. Raw ◽  
E. Lee ◽  
S. R. Sprang ◽  
A. G. Gilman
Keyword(s):  
G Protein ◽  
Gtp Hydrolysis ◽  
Alpha Subunits

scholarly journals MEDIATION OF IMMUNOLOGIC DISCHARGE OF LYSOSOMAL ENZYMES FROM HUMAN NEUTROPHILS BY GUANOSINE 3',5'-MONOPHOSPHATE

1974 ◽  
Vol 140 (1) ◽  
pp. 225-238 ◽  
Author(s):  
L. J. Ignarro ◽  
W. J. George
Keyword(s):  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Lysosomal Enzymes ◽  
Extracellular Calcium ◽  
Human Neutrophils ◽  
Enzyme Release ◽  
Dibutyryl Cyclic Amp ◽  
Relationship Of ◽  
The Relationship ◽  
Stimulation Of

The purpose of this investigation was to elucidate the relationship of cyclic GMP and calcium to the immunologic discharge of lysosomal enzymes from purified human neutrophils. Contact of neutrophils with a variety of immunologic stimuli, including zymosan particles treated with either normal or rheumatoid arthritic (RA) serum, heat-aggregated (agg) IgG, particulate and immobilized agg IgG each treated with RA serum, and zymosan-treated serum, provoked the discharge of ß-glucuronidase, but not cytoplasmic lactate dehydrogenase, and stimulated the accumulation of cyclic GMP. Both enzyme release and elevation of cyclic GMP levels required the presence of extracellular calcium as neither cellular event proceeded in its absence. Cholinergic enhancement of the immunologic secretion of ß-glucuronidase from neutrophils by acetylcholine was associated with a concomitant accumulation of cyclic GMP. These actions of acetylcholine on neutrophils did not proceed in the absence of extracellular calcium. Whereas the concentrations of cyclic GMP in neutrophils were elevated by both immune reactants and a combination of the latter and acetylcholine, cyclic AMP levels remained unaltered. Thus, cyclic GMP, but not cyclic AMP, was associated with the immunologic and pharmacologic discharge of lysosomal enzymes from neutrophils. Contrariwise, cyclic AMP, but not cyclic GMP, was associated with inhibition of lysosomal enzyme release. For example, dibutyryl cyclic AMP and epinephrine inhibited the release of ß-glucuronidase from neutrophils that was elicited by each of the immune reactants tested. Moreover, cyclic AMP levels in the cells were elevated markedly in every instance that enzyme discharge was inhibited by epinephrine. Epinephrine did not alter the neutrophil concentrations of cyclic GMP at times when those of cyclic AMP were elevated. The data in this report constitute partial evidence that the immunologic discharge of lysosomal enzymes from human neutrophils is mediated or signaled by intracellular cyclic GMP and that calcium is linked to this stimulation of enzyme secretion.

scholarly journals Effects of verapamil on lipolysis due to dibutyryl cyclic AMP

1984 ◽  
Vol 220 (1) ◽  
pp. 321-324 ◽  
Author(s):  
H Goko ◽  
S Takashima ◽  
S Shimizu ◽  
S Kagawa ◽  
A Matsuoka
Keyword(s):  
Cyclic Amp ◽  
Adrenergic Receptors ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Dibutyryl Cyclic Amp ◽  
Alpha Adrenergic Receptors ◽  
Biphasic Effect ◽  
Adrenergic Antagonists ◽  
Dose Dependent ◽  
Isolated Rat

The effects of verapamil, a calcium antagonist, on lipolysis in isolated rat adipocytes were studied. Verapamil (100 microM) potentiated lipolysis due to dibutyryl cyclic AMP (Bt2cAMP) at submaximal concentrations, with or without extracellular Ca2+. Lipolysis due to 0.5 mM-Bt2cAMP was potentiated by verapamil in a dose-dependent manner up to 200 microM, whereas at concentrations higher than 100 microM the stimulatory effect of verapamil was progressively diminished with or without extracellular Ca2+. Verapamil showed only an inhibitory effect on lipolysis due to adrenaline (0.1-10 microM) or 3-isobutyl-1-methylxanthine (IBMX; 25-200 microM). The stimulatory effect of verapamil on lipolysis due to Bt2cAMP was not blocked by alpha-adrenergic antagonists. These results suggest (i) that verapamil has a biphasic effect on lipolysis due to Bt2cAMP and only an inhibitory effect on that due to adrenaline or IBMX, and (ii) that extracellular Ca2+ or alpha-adrenergic receptors are not involved in the action of verapamil.

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