Effects of insulin, pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: Evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism

1987 ◽  
Vol 7 (7) ◽  
pp. 533-541 ◽  
Author(s):  
John E. Hesketh ◽  
Gillian P. Campbell
Keyword(s):  
Protein Synthesis ◽  
Phospholipase C ◽  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Cell Protein ◽  
3T3 Cells ◽  
3T3 Fibroblasts ◽  
Kinase C ◽  
Ribosomal Activity

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 μM) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.

Rapid response of protein synthesis to insulin in 3T3 cells: Effects of protein kinase C depletion and differences from the response to serum repletion

1986 ◽  
Vol 6 (9) ◽  
pp. 797-804 ◽  
Author(s):  
John E. Hesketh ◽  
Gillian P. Campbell ◽  
Peter J. Reeds
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Actinomycin D ◽  
Calf Serum ◽  
Cell Protein ◽  
Foetal Calf Serum ◽  
3T3 Cells ◽  
Kinase C

Quiescent 3T3 cells grown in media containing 4% foetal calf serum showed different responses to insulin and to serum repletion (to 12%). Insulin stimulated protein synthesis within 1 h and this early response was insensitive to actinomycin D. The later insulin response showed progressive sensitivity to actinomycin D. The serum response was slower, not occurring until 1 h, and was inhibited by actinomycin D. Depletion of cell protein kinase C by pre-treatment with phorbol ester caused a total block of the immediate response to insulin but had little effect on the response to serum or the later response to insulin. Acute phorbol ester treatment stimulated protein synthesis.

Evidence that v-Src-induced phospholipase D activity is mediated by a G protein

1994 ◽  
Vol 14 (6) ◽  
pp. 3676-3682
Author(s):  
H Jiang ◽  
K Alexandropoulos ◽  
J Song ◽  
D A Foster
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Type D ◽  
Kinase C ◽  
Intracellular Signals ◽  
Phosphatidic Acid Phosphatase ◽  
Phosphorylation Event ◽  
Gamma S

v-Src-induced increases in diglyceride are derived from phosphatidylcholine via a type D phospholipase (PLD) and a phosphatidic acid phosphatase. v-Src-induced PLD activity, as measured by PLD-catalyzed transphosphatidylation of phosphatidylcholine to phosphatidylethanol, is inhibited by GDP beta S, which inhibits G-protein-mediated intracellular signals. Similarly, v-Src-induced increases in diglyceride are also blocked by GDP beta S. In contrast to the PLD activity induced by v-Src, PLD activity induced by the protein kinase C agonist, 12-O-tetradecanoylphorbol-13-acetate (TPA), was insensitive to GDP beta S. Consistent with the involvement of a G protein in the activation of PLD activity by v-Src, GTP gamma S, a nonhydrolyzable analog of GTP that potentiates G-protein-mediated signals, strongly enhanced PLD activity in v-Src-transformed cells relative to that in parental BALB/c 3T3 cells. The effect of GTP gamma S on PLD activity in v-Src-transformed cells was observed only when cells were prelabeled with [3H]myristate, which is incorporated exclusively into phosphatidylcholine, the substrate for the v-Src-induced PLD. There was no difference in the effect of GTP gamma S-induced PLD activity on v-Src-transformed and BALB/c 3T3 cells when the cells were prelabeled with [3H]arachidonate, which is not incorporated into phospholipids that are substrates for the v-Src-induced PLD. Similarly, GDP beta S inhibited PLD activity in v-Src-transformed cells much more strongly than in BALB/c 3T3 cells when [3H]myristate was used to prelabel the cells. The GTP-dependent activation of PLD by v-Src was dependent upon the presence of ATP but was unaffected by either cholera or pertussis toxin. These data suggest that v-Src induces PLD activity through a phosphorylation event and is mediated by a cholera and pertussis toxin-insensitive G protein.

scholarly journals Evidence that v-Src-induced phospholipase D activity is mediated by a G protein.

1994 ◽  
Vol 14 (6) ◽  
pp. 3676-3682 ◽  
Author(s):  
H Jiang ◽  
K Alexandropoulos ◽  
J Song ◽  
D A Foster
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Type D ◽  
Kinase C ◽  
Intracellular Signals ◽  
Phosphatidic Acid Phosphatase ◽  
Phosphorylation Event ◽  
Gamma S

v-Src-induced increases in diglyceride are derived from phosphatidylcholine via a type D phospholipase (PLD) and a phosphatidic acid phosphatase. v-Src-induced PLD activity, as measured by PLD-catalyzed transphosphatidylation of phosphatidylcholine to phosphatidylethanol, is inhibited by GDP beta S, which inhibits G-protein-mediated intracellular signals. Similarly, v-Src-induced increases in diglyceride are also blocked by GDP beta S. In contrast to the PLD activity induced by v-Src, PLD activity induced by the protein kinase C agonist, 12-O-tetradecanoylphorbol-13-acetate (TPA), was insensitive to GDP beta S. Consistent with the involvement of a G protein in the activation of PLD activity by v-Src, GTP gamma S, a nonhydrolyzable analog of GTP that potentiates G-protein-mediated signals, strongly enhanced PLD activity in v-Src-transformed cells relative to that in parental BALB/c 3T3 cells. The effect of GTP gamma S on PLD activity in v-Src-transformed cells was observed only when cells were prelabeled with [3H]myristate, which is incorporated exclusively into phosphatidylcholine, the substrate for the v-Src-induced PLD. There was no difference in the effect of GTP gamma S-induced PLD activity on v-Src-transformed and BALB/c 3T3 cells when the cells were prelabeled with [3H]arachidonate, which is not incorporated into phospholipids that are substrates for the v-Src-induced PLD. Similarly, GDP beta S inhibited PLD activity in v-Src-transformed cells much more strongly than in BALB/c 3T3 cells when [3H]myristate was used to prelabel the cells. The GTP-dependent activation of PLD by v-Src was dependent upon the presence of ATP but was unaffected by either cholera or pertussis toxin. These data suggest that v-Src induces PLD activity through a phosphorylation event and is mediated by a cholera and pertussis toxin-insensitive G protein.

Pregnancy enhances G protein activation and nitric oxide release from uterine arteries

2001 ◽  
Vol 280 (5) ◽  
pp. H2069-H2075 ◽  
Author(s):  
L. P. Thompson ◽  
C. P. Weiner
Keyword(s):  
Nitric Oxide ◽  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Nitric Oxide Synthase Inhibitor ◽  
Protein Activation ◽  
Nitric Oxide Release ◽  
G Protein Activation ◽  
Hormonal Modulation ◽  
Synthase Inhibitor

We hypothesized that pregnancy modulates receptor-mediated responses of the uterine artery (UA) by altering G protein activation or coupling. Relaxation and contraction to NaF (0.5–11.5 mM), acetylcholine (10−9–10−5 M), and bradykinin (10−12–3 × 10−5 M) were measured in isolated UA of pregnant and nonpregnant guinea pigs. Responses were measured in the presence and absence of either cholera toxin (2 μg/ml) or pertussis toxin (Gαs and Gαiinhibitors, respectively). NaF relaxation was endothelium dependent and nitro-l-arginine sensitive (a nitric oxide synthase inhibitor). Relaxation to NaF, acetylcholine, and bradykinin were potentiated by pregnancy. Cholera but not pertussis toxin increased relaxation to acetylcholine and bradykinin in UA from nonpregnant animals, had no effect in UA from pregnant animals, and abolished the pregnancy-induced differences in acetylcholine relaxation. Cholera toxin potentiated the bradykinin-induced contraction of UA of both pregnant and nonpregnant animals, whereas pertussis toxin inhibited contraction of UA from pregnant animals only. Therefore, pregnancy may enhance agonist-stimulated endothelium-dependent relaxation and bradykinin-induced contraction of UA by inhibiting GTPase activity or enhancing Gαs but not Gαi activation in pregnant animals. Thus the diverse effects of pregnancy on UA responsiveness may result from hormonal modulation of G proteins coupled to their specific receptors.

scholarly journals Insulin and vasopressin elicit inhibition of cholera-toxin-stimulated adenylate cyclase activity in both hepatocytes and the P9 immortalized hepatocyte cell line through an action involving protein kinase C

1995 ◽  
Vol 312 (3) ◽  
pp. 769-774 ◽  
Author(s):  
L Zeng ◽  
M D Houslay
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Cell Line ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
3T3 Cells ◽  
Kinase C ◽  
Nih 3T3

Incubation of hepatocytes or the SV40-DNA-immortalized hepatocyte P9 cell line with cholera toxin led to a time-dependent activation of adenylate cyclase activity, which occurred after a defined lag period. When added together with cholera toxin, each of the hormones insulin and vasopressin was capable of attenuating the maximum stimulatory effect achieved by cholera toxin over a period of 60 min through a process which could be blocked by the compounds staurosporine and chelerythrine. Attenuating effects on cholera-toxin-stimulated adenylate cyclase activity could also be elicited by using either the protein kinase C (PKC)-stimulating phorbol ester PMA (phorbol 12-myristate 13-acetate) or the protein phosphatase inhibitor okadaic acid. Alkaline phosphatase treatment of membranes reversed the inhibitory effect of PMA. Cholera toxin also stimulated the adenylate cyclase activity of intact CHO (Chinese-hamster ovary) and NIH-3T3 cells, but this activity was insensitive to the addition of PMA. Overexpression of various PKC isoforms in CHO cell lines did not confer sensitivity to inhibition by PMA upon cholera-toxin-stimulated adenylate cyclase activity. Rather, overexpression of the gamma isoform of PKC allowed PMA to stimulate adenylate cyclase activity in CHO cells. It is suggested that the PKC-mediated phosphorylation of a membrane protein attenuates cholera-toxin-stimulated adenylate cyclase activity in hepatocytes and P9 cells. The cellular selectivity of such an action may be due to the target for this inhibitory action of PKC being a particular isoform of adenylate cyclase which provides the major activity in hepatocytes and P9 cells, but not in either CHO or NIH-3T3 cells.

Pertussis toxin sensitive G-protein coupling of HDL receptor to phospholipase C in human platelets

1992 ◽  
Vol 67 (5) ◽  
pp. 559-567 ◽  
Author(s):  
H. Nazih ◽  
D. Devred ◽  
F. Martin-Nizard ◽  
V. Clavey ◽  
J.C. Fruchart ◽  
...  
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Pertussis Toxin ◽  
Human Platelets ◽  
G Protein Coupling ◽  
Protein Coupling

Dual Regulation of Phospolipase C Activity by G Proteins

Physiology ◽  
1993 ◽  
Vol 8 (2) ◽  
pp. 61-63
Author(s):  
H Deckmyn ◽  
C Van Geet ◽  
J Vermylen
Keyword(s):  
Adenylate Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Inhibitory Pathway ◽  
Close Parallelism

Some subtypes of phosphatidylinositide-specific phospholipase C (PLC) are activated via pertussis toxin-sensitive or -insensitive G proteins. However, a G protein-dependent PLC inhibitory pathway also may exist. The resultant picture is of dual regulation of PLC, showing a close parallelism with the dual regulation of adenylate cyclase.

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