scholarly journals Development of muscarinic-cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a switch in guanine-nucleotide-binding protein coupling

1990 ◽  
Vol 271 (2) ◽  
pp. 443-448 ◽  
Author(s):  
J V Barnett ◽  
S M Shamah ◽  
B Lassegue ◽  
K K Griendling ◽  
J B Galper
Keyword(s):  
Phospholipase C ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Inositol Phosphate ◽  
In Ovo ◽  
Atrial Cells ◽  
Muscarinic Stimulation ◽  
Inositol Phosphate Production ◽  
Guanine Nucleotide Binding ◽  
Stimulation Of

We have demonstrated that muscarinic stimulation of inositol phosphate production in cultured atrial cells from chicks at 14 days in ovo is partially sensitive to inhibition by pertussis toxin. In these cells, muscarinic agonist binding is coupled to phospholipase C activity via at least two guanine-nucleotide-binding proteins (G-proteins), one sensitive to pertussis toxin and the other (Gp) insensitive to pertussis toxin [Barnett, Shamah, Lassegue, Griendling & Galper (1990) Biochem. J. 271, 437-442]. In the current study we demonstrate that during embryonic development of the chick heart, muscarinic stimulation of inositol phosphate production decreases by 50% between days 5 and 14 in ovo in cells cultured from both atrium and ventricle. In atrial cells, however, pertussis toxin-sensitive muscarinic stimulation of inositol phosphate production increased from undetectable levels at day 5 in ovo to 40% of total stimulation at day 12 in ovo. Muscarinic stimulation of inositol phosphate production in the ventricle did not become sensitive to pertussis toxin at any age studied. In permeabilized atrial cells from embryonic chicks at 5 days in ovo, guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated InsP1 levels by 40 +/- 10% (mean +/- S.E.M., n = 3), InsP2 levels by 117 +/- 18% and InsP3 levels by 51 +/- 8%, suggesting that at day 5 in ovo all of the muscarinic-stimulated inositol phosphate production was coupled to phospholipase C via Gp. H.p.l.c. analysis demonstrated that, in spite of these changes in coupling of phospholipase C to different G-proteins, no changes could be demonstrated in the isomers of InsP3 produced in response to carbamylcholine at both days 5 and 14 in ovo. These data demonstrate that embryonic development of the chick atrium is associated with a switch in coupling of muscarinic receptors to phospholipase C from Gp to a pertussis toxin substrate. This developmental switch in coupling of G-proteins may be related to possible developmental switches in levels of muscarinic receptor isoforms or switches in the subtype of phospholipase C.

scholarly journals Muscarinic cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a role of two guanine-nucleotide-binding proteins

1990 ◽  
Vol 271 (2) ◽  
pp. 437-442 ◽  
Author(s):  
J V Barnett ◽  
S M Shamah ◽  
B Lassegue ◽  
K K Griendling ◽  
J B Galper
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Pertussis Toxin ◽  
Inositol Phosphate ◽  
Guanine Nucleotide ◽  
Embryonic Chick ◽  
Atrial Cells ◽  
Inositol Phosphate Production ◽  
Guanine Nucleotide Binding ◽  
Stimulation Of

These studies demonstrate a novel mechanism for the coupling of the muscarinic receptor to phospholipase C activity in embryonic chick atrial cells. In monolayer cultures of atrial cells from hearts of embryonic chicks at 14 days in ovo, carbamylcholine stimulated the sequential appearance of InsP3, InsP2 and InsP1 with an EC50 (concn. causing 50% of maximal stimulation) of 30 microM. In the presence of 15 mM-Li, a 5 min exposure to carbamylcholine (0.1 mM) increased InsP3 levels to a maximum of 47 +/- 12% over basal, InsP2 to 108 +/- 13% over basal and InsP1 to 42 +/- 5% over basal. This effect was blocked by 5 microM-atropine. Incubation of these cells with pertussis toxin (15 h; 0.5 ng/ml) inhibited carbamylcholine-stimulated InsP3, InsP2 and InsP1 formation by 42 +/- 7%, 30 +/- 3% and 48 +/- 7% respectively. The IC50 (concn. causing 50% inhibition) for pertussis toxin inhibition of all three inositol phosphates was 0.01 ng/ml, with a half-time of 6 h at 0.5 ng/ml. This partial sensitivity to pertussis toxin was not due to incomplete ADP-ribosylation of the guanine-nucleotide-binding protein (G-protein), since autoradiography of polyacrylamide gels of cell homogenates incubated with [32P]NAD+ in the presence of pertussis toxin demonstrated that incubation of cells with 0.5 ng of pertussis toxin/ml for 15 h resulted in complete ADP-ribosylation of pertussis toxin substrates by endogenous NAD+. In cells permeabilized with saponin (10 micrograms/ml), 0.1 mM-GTP[S] (guanosine 5′-[gamma-thio]triphosphate) stimulated InsP1 by 102 +/- 15% (mean +/- S.E.M., n = 4), InsP2 by 421 +/- 67% and InsP3 by 124 +/- 33% above basal. Incubation of cells for 15 h with 0.5 ng of pertussis toxin/ml decreased GTP[S]-stimulated InsP1 production in saponin-treated cells by 30 +/- 10% (n = 3), InsP2 production by 45 +/- 7% (n = 4) and InsP3 production by 49 +/- 6% (n = 4). These data demonstrate that in embryonic chick atrial cells at least two independent G-proteins, a pertussis toxin-sensitive G-protein and a pertussis toxin-insensitive G-protein, play a role in coupling muscarinic agonist binding to phospholipase C activation and to inositol phosphate production.

THE ROLE OF GTP-BINDING PROTEINS EXHIBITING GTPase ACTIVITY IN PLATELET ACTIVATION

1987 ◽  
Author(s):  
K H Jakobs ◽  
P Gierschik ◽  
R Grandt
Keyword(s):  
Signal Transduction ◽  
Adenylate Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Inositol Phosphate ◽  
Adenylate Cyclase Inhibition ◽  
Gi Protein ◽  
Stimulation Of

Activation of platelets by agonists acting via cell surface-located receptors apparently involves as an early event in transmembrane signalling an interaction of the agonist-occupied receptor with a guanine nucleotide-binding regulatory protein (G-protein). The activated G-protein, then, transduces the information to the effector molecule, being responsible for the changes in intracellular second messengers. At least two changes in intracellular signal molecules are often found to be associated with platelet activation by agonists, i.e., increases in inositol trisphosphate and diacylglycerol levels caused by activation of a polyphosphoinositide-specific phospholipase C and decrease in cyclic AMP concentration caused by inhibition of adenylate cyclase.Both actions of platelet-activating agents apparently involve G-proteins as transducing elements. Generally, the function of a G-protein in signal transduction can be measured either by its ability to regulate the activity of the effector molecule (phospholipase C or adenylate cyclase) or the binding affinity of an agonist to its specific receptor or by the abitlity of the G-protein to bind and hydrolyze GTP or one of its analogs in response to agonist-activated receptors. Some platelet-activating agonists (e.g. thrombin) can cause both adenylate cyclase inhibition and phospholipase C activation, whereas others induce either inhibition of adenylate cyclase (e.g. α2-adrenoceptor agonists) or activation of phospholipase C (e.g. stable endoperoxide analogs) . It is not yet known whether the simultaneous activation of two signal transduction systems is due to activation of two separate G-proteins by one receptor, to two distinct receptors activating each a distinct G-protein or to activation of two effector molecules by one G-protein.For some of the G-proteins, rather specific compounds are available causing inactivation of their function. In comparison to Gs, the stimulatory G-protein of the adenylate cyclase system, the adenylate cyclase inhibitory Gi-protein is rather specifically inactivated by ADP-ribosylation of its a-subunit by pertussis toxin, “unfortunately” not acting in intact platelets, and by SH-group reactive agents such as N-ethylmaleimide and diamide, apparently also affecting the Giα-subunit. Both of these treatments completely block α2-adrenoceptor-induced GTPase stimulation and adenylate cyclase inhibition and also thrombin-induced inhibition of adenylate cyclase. In order to know whether the G-protein coupling receptors to phospholipase C is similar to or different from the Gi-protein, high affinity GTPase stimulation by agents known to activate phospholipase C was evaluated in platelet membranes. The data obtained indicated that GTPase stimulation by agents causing both adenylate cyclase inhibition and phospholipase C activation is reduced, but only partially, by the above mentioned Gi-inactivating agents, while stimulation of GTPase by agents stimulating only phospholipase C is not affected by these treatments. These data suggested that the G-protein regulating phospholipase C activity in platelet membranes is different from the Gi-protein and may also not be a substrate for pertussis toxin. Measuring thrombin stimulation of inositol phosphate and diacylglycerol formation in saponin-permeabilized platelets, apparently contradictory data were reported after pertussis toxin treatment, being without effect or causing even an increase in thrombin stimulation of inositol phosphate formation (Lapetina: BBA 884, 219, 1986) or being inhibitory to thrombin stimulation of diacylglycerol formation (Brass et al.: JBC 261, 16838, 1986). These data indicate that the nature of the phospholipase C-related G-protein(s) is not yet defined and that their elucidation requires more specific tools as well as purification and reconstitution experiments. Preliminary data suggest that some antibiotics may serve as useful tools to characterize the phospho-lipase-related G-proteins. The possible role of G-protein phosphorylation by intracellular signal molecule-activated protein kinases in attenuation of signal transduction in platelets will be discussed.

scholarly journals G-proteins are not directly involved in the CD3-antigen-mediated production of inositol phosphates in HPB-ALL T-leukaemia cells expressing phospholipase C isoforms γ1 and β3

1993 ◽  
Vol 289 (2) ◽  
pp. 387-394 ◽  
Author(s):  
M Biffen ◽  
M Shiroo ◽  
D R Alexander
Keyword(s):  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
G Protein ◽  
G Proteins ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Cross Linking ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Inositol Phosphate Production

The possible involvement of G-proteins in T cell antigen-receptor complex (TCR)-mediated inositol phosphate production was investigated in HPB-ALL T-cells, which were found to express the phospholipase C gamma 1 and beta 3 isoforms. Cross-linking the CD3 antigen on streptolysin-O-permeabilized cells stimulated a dose-dependent increase in inositol phosphate production, as did addition of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or vanadate, a phosphotyrosine phosphatase inhibitor. It was possible, therefore, that the CD3-antigen-mediated production of inositol phosphates was either via a G-protein-dependent mechanism or by stimulation of protein tyrosine phosphorylation. The CD3-induced inositol phosphate production was potentiated by addition of vanadate, but not by addition of GTP[S]. Guanosine 5′-[beta-thio]diphosphate (GDP[S]) inhibited the rise in inositol phosphates induced by GTP[S], vanadate or cross-linking the CD3 antigen. The increase in protein tyrosine phosphorylation stimulated by vanadate or the OKT3 monoclonal antibody was not observed in the presence of GDP[S], showing that in permeabilized HPB-ALL cells, GDP[S] inhibits the actions of tyrosine kinases as well as G-protein function. Addition of either ADP[S] or phenylarsine oxide inhibited CD3- and vanadate-mediated increases in both tyrosine phosphorylation and inositol phosphate production, but did not inhibit GTP[S]-stimulated inositol phosphate production. On the other hand, pretreatment of cells with phorbol 12,13-dibutyrate inhibited subsequent GTP[S]-stimulated inositol phosphate production but did not inhibit significantly inositol phosphate production stimulated by either OKT3 F(ab')2 fragments or vanadate. Our results are consistent with the CD3 antigen stimulating inositol phosphate production by increasing the level of protein tyrosine phosphorylation, but not by activating a G-protein.

Modulation of phospholipase C pathway and level of Gq alpha/G11 alpha in rat myometrium during gestation

1996 ◽  
Vol 271 (3) ◽  
pp. C895-C904 ◽  
Author(s):  
S. Lajat ◽  
Z. Tanfin ◽  
G. Guillon ◽  
S. Harbon
Keyword(s):  
Phospholipase C ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Receptor Activation ◽  
Similar Increase ◽  
Inositol 1,4,5 Trisphosphate ◽  
M3 Subtype ◽  
Subtype A ◽  
Stimulation Of

The regulation of the receptor-G protein-phospholipase C (PLC) cascade was investigated in rat myometrium at midgestation (day 12) and at term (day 21) comparatively to the estrogen-treated tissue (day 0). Carbachol-mediated generation of [3H]inositol phosphates was insensitive to pertussis toxin and was enhanced at days 12 and 21 two- and threefold, respectively, with no alteration of muscarinic sites (M3 subtype). A similar increase could be detected in the production of inositol 1,4,5-trisphosphate, indicating the stimulation of a PLC degrading phosphatidylinositol 4,5-bisphosphate. AlF4- also enhanced PLC activation during gestation, suggesting pregnancy-related regulations that bypass receptor activation. Immunoreactive G proteins, Gq alpha and G11 alpha, and PLC-beta 3 were detected in all myometrial preparations. The amount of PLC-beta 3 was similar in day 0 and day 21 myometrium, although decreasing by 75% at midgestation. Of significance was the increased amount of Gq alpha in day 12 and day 21 myometrium (3- and 2-fold, respectively) which coincided with the enhanced phosphoinositide breakdown. The upregulation of Gq alpha may contribute to the enhanced PLC activity during pregnancy and at term.

scholarly journals Polyamines inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis. Studies with permeabilized GH3 cells

1988 ◽  
Vol 255 (3) ◽  
pp. 1015-1021 ◽  
Author(s):  
R J H Wojcikiewicz ◽  
J N Fain
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphate ◽  
Pituitary Tumour ◽  
Gh3 Cells ◽  
Inhibitory Effects ◽  
Inositol Phosphate Production ◽  
Ic50 Values ◽  
Marked Selectivity ◽  
Guanine Nucleotide Binding ◽  
Gamma S

[3H]Inositol-labelled GH3 rat anterior pituitary tumour cells were permeabilized with digitonin and were incubated at 37 degrees C in the presence of ATP and Mg2+. [3H]Polyphosphoinositide breakdown and [3H]inositol phosphate production were stimulated by hydrolysis-resistant GTP analogues and by Ca2+. Of the nucleotides tested, guanosine 5′-[gamma-thio]triphosphate (GTP gamma S) was the most effective stimulus. Activation by GTP gamma S appeared to be mediated by a guanine nucleotide-binding (G) protein as GTP gamma S-stimulated [3H]inositol phosphate production was inhibited by other nucleotides with a potency order of GTP = GDP = guanosine 5′-[beta-thio]diphosphate greater than ITP greater than GMP greater than UTP = CTP = adenosine 5′-[gamma-thio]triphosphate. The stimulatory effects of 10 microM-GTP gamma S on [3H]inositol phosphate levels were reversed by spermine and spermidine with IC50 values of approx. 0.25 and 2 mM respectively. Putrescine was inhibitory only at higher concentrations. Similarly, GTP gamma S-induced decreases in [3H]polyphosphoinositide levels were reversed by 2.5 mM-spermine. The inhibitory effects of spermine were not overcome by supramaximal concentrations of GTP gamma S. In contrast, [3H]inositol phosphate production stimulated by addition of 0.3-0.6 mM-Ca2+ to incubation media was only partially inhibited by spermine (5 mM), and spermine was not inhibitory when added Ca2+ was increased to 1 mM. These data show that polyamines, particularly spermine, inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis with a marked selectivity towards the stimulatory effects of GTP gamma S.

scholarly journals Effects of tiazofurin on guanine nucleotide binding regulatory proteins in HL-60 cells

Blood ◽  
1990 ◽  
Vol 75 (3) ◽  
pp. 583-588 ◽  
Author(s):  
SM Kharbanda ◽  
ML Sherman ◽  
DW Kufe
Keyword(s):  
Adenylate Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Binding Sites ◽  
Nucleotide Binding ◽  
Membrane Receptors ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding ◽  
Stimulation Of

Abstract Guanine nucleotide binding proteins (G proteins) are regulatory molecules that couple membrane receptors to effector systems such as adenylate cyclase and phospholipase C. The alpha subunits of G proteins bind to guanosine 5′-diphosphate (GDP) in the unstimulated state and guanosine 5′ triphosphate (GTP) in the active state. Tiazofurin (2-beta- D-ribofuranosylthiazole-4-carboxamide), a specific inhibitor of inosine monophosphate (IMP) dehydrogenase, decreases guanylate synthesis from IMP in HL-60 promyelocytic leukemia cells and depletes intracellular guanine nucleotide pools. This study demonstrates that treatment of HL- 60 cells with tiazofurin is associated with a fourfold increase in membrane binding sites for the nonhydrolyzable analogue GDP beta S. This increase in binding sites was associated with a 3.2-fold decrease in GDP beta S binding affinity. Similar findings were obtained with GTP gamma S. These effects of tiazofurin treatment on guanine nucleotide binding were also associated with decreased adenosine diphosphate- ribosylation of specific G protein substrates by cholera and pertussis toxin. The results further demonstrate that tiazofurin treatment results in inhibition of G protein-mediated transmembrane signaling mechanisms. In this regard, stimulation of adenylate cyclase by prostaglandin E2 was inhibited by over 50% in tiazofurin-treated cells. Furthermore, tiazofurin treatment resulted in inhibition of N- formylmethionylleucylphenylalanine-induced stimulation of phospholipase C. Taken together, these results indicate that tiazofurin acts at least in part by inhibiting the ability of G proteins to function as transducers of intracellular signals.

scholarly journals Effects of tiazofurin on guanine nucleotide binding regulatory proteins in HL-60 cells

Blood ◽  
1990 ◽  
Vol 75 (3) ◽  
pp. 583-588
Author(s):  
SM Kharbanda ◽  
ML Sherman ◽  
DW Kufe
Keyword(s):  
Adenylate Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Binding Sites ◽  
Nucleotide Binding ◽  
Membrane Receptors ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding ◽  
Stimulation Of

Guanine nucleotide binding proteins (G proteins) are regulatory molecules that couple membrane receptors to effector systems such as adenylate cyclase and phospholipase C. The alpha subunits of G proteins bind to guanosine 5′-diphosphate (GDP) in the unstimulated state and guanosine 5′ triphosphate (GTP) in the active state. Tiazofurin (2-beta- D-ribofuranosylthiazole-4-carboxamide), a specific inhibitor of inosine monophosphate (IMP) dehydrogenase, decreases guanylate synthesis from IMP in HL-60 promyelocytic leukemia cells and depletes intracellular guanine nucleotide pools. This study demonstrates that treatment of HL- 60 cells with tiazofurin is associated with a fourfold increase in membrane binding sites for the nonhydrolyzable analogue GDP beta S. This increase in binding sites was associated with a 3.2-fold decrease in GDP beta S binding affinity. Similar findings were obtained with GTP gamma S. These effects of tiazofurin treatment on guanine nucleotide binding were also associated with decreased adenosine diphosphate- ribosylation of specific G protein substrates by cholera and pertussis toxin. The results further demonstrate that tiazofurin treatment results in inhibition of G protein-mediated transmembrane signaling mechanisms. In this regard, stimulation of adenylate cyclase by prostaglandin E2 was inhibited by over 50% in tiazofurin-treated cells. Furthermore, tiazofurin treatment resulted in inhibition of N- formylmethionylleucylphenylalanine-induced stimulation of phospholipase C. Taken together, these results indicate that tiazofurin acts at least in part by inhibiting the ability of G proteins to function as transducers of intracellular signals.

Immunological characterization of the guanine-nucleotide binding proteins Gi and Go in rat islets of Langerhans

1992 ◽  
Vol 8 (2) ◽  
pp. 103-108 ◽  
Author(s):  
N. S. Berrow ◽  
G. Milligan ◽  
N. G. Morgan
Keyword(s):  
G Proteins ◽  
Islets Of Langerhans ◽  
Pertussis Toxin ◽  
Nucleotide Binding ◽  
Molecular Forms ◽  
Antigenic Determinants ◽  
Guanine Nucleotide ◽  
Rat Islets ◽  
Guanine Nucleotide Binding ◽  
Rat Islets Of Langerhans

ABSTRACT Inhibition of insulin secretion from rat islets of Langerhans is known to involve at least one pertussis toxin-sensitive guanine-nucleotide binding (G) protein. We have used antisera raised against unique antigenic determinants of different members of the family of pertussis toxin-sensitive G proteins to identify these proteins in rat islets. Antiserum SG1, which recognizes both Gi1 and Gi2, reacted with an islet protein having an approximate Mr of 40 000. Antiserum IlC (Gi1 specific) failed to recognize any islet proteins, suggesting that Gi2 is present in much greater amounts than Gi1. Indeed, Gi1 levels were below the detection limit of a sensitive immunogold/silver-staining method, indicating that it may be absent from the cells of rat islets. Two different antisera were used to identify Go-like G proteins in rat islet homogenates. Both antisera reacted with a protein band which, under appropriate conditions, could be resolved to reveal two separate proteins of Mr 39–40 000. Thus, at least two molecular forms of Go are present in rat islets. Subcellular fractionation indicated that all three G proteins identified in this study (Gi2 and two forms of Go) are localized to islet membranes. No immunoreactivity could be detected in the cytosolic fraction.

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