scholarly journals Effect of deoxycholate on guanine-nucleotide-dependent carbachol stimulation of phosphoinositidase C in mouse brain cortical membranes

1995 ◽  
Vol 312 (2) ◽  
pp. 445-449 ◽  
Author(s):  
N Bas ◽  
A Garcia
Keyword(s):  
G Protein ◽  
Mouse Brain ◽  
Bile Salts ◽  
Response Curve ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Guanine Nucleotide ◽  
Phosphoinositide Cycle ◽  
Cortical Membranes ◽  
Stimulation Of

Demonstration of guanine-nucleotide-dependent neurotransmitter stimulation of phosphoinositide breakdown in brain membranes has generally required the presence of the detergent, deoxycholate (DOC), in the assay medium. In the present study, by using mouse brain cortical membranes labelled with [3H]inositol in the presence of CMP through the reverse PtdIns synthase reaction, we have been able to show guanosine 5′-[gamma-thio]triphosphate (GTP[S])-dependent carbachol (CCh) stimulation of the formation of [3H]inositol phosphates in the absence of DOC and have studied how the detergent affects the response. The results of our study indicate that DOC affects the muscarinic receptor-G-protein-phosphoinositidase C (PIC) transduction system in several ways. First, it enhances agonist-induced PIC activity towards [3H]PtdInsP and [3H]PtdInsP2 and, secondly, it decreases the potency for GTP[S] stimulation of PIC, thus enhancing the agonist-induced leftward shift of the dose-response curve for GTP[S]. Additionally, DOC appears to increase the activity of the enzymes of the phosphoinositide cycle, PtdIns 4-kinase, Ins(1,4,5)P3 5-phosphatase and Ins(1,4)P2 1-phosphatase, thus altering the proportion of phosphoinositide substrates and inositol phosphate products. These observations advise caution in drawing conclusions about PIC substrate specificity and the potency of both guanine nucleotides and agonists from experiments performed in membranes in the presence of DOC or related bile salts.

scholarly journals Guanine nucleotides stimulate production of inositol trisphosphate in rat cortical membranes

1985 ◽  
Vol 232 (3) ◽  
pp. 799-804 ◽  
Author(s):  
R A Gonzales ◽  
F T Crews
Keyword(s):  
Inositol Phosphates ◽  
Adenine Nucleotides ◽  
Inositol Trisphosphate ◽  
Guanine Nucleotides ◽  
Guanine Nucleotide ◽  
Inositol Phospholipids ◽  
Cortical Membranes ◽  
Gamma S ◽  
Hydrolysis Of ◽  
Stimulation Of

The guanine nucleotides guanosine 5′[beta, gamma-imido]triphosphate (Gpp[NH]p), guanosine 5′-[γ-thio]-triphosphate (GTP gamma S), GMP, GDP and GTP stimulated the hydrolysis of inositol phospholipids by a phosphodiesterase in rat cerebral cortical membranes. Addition of 100 microM-Gpp[NH]p to prelabelled membranes caused a rapid accumulation of [3H)inositol phosphates (less than 30 s) for up to 2 min. GTP gamma S and Gpp [NH]p caused a concentration-dependent stimulation of phosphoinositide phosphodiesterase with a maximal stimulation of 2.5-3-fold over control at concentrations of 100 microM. GMP was as effective as the nonhydrolysable analogues, but much less potent (EC50 380 microM). GTP and GDP caused a 50% stimulation of the phospholipase C at 100 microM and at higher concentrations were inhibitory. The adenine nucleotides App[NH]p and ATP also caused small stimulatory effects (64% and 29%). The guanine nucleotide stimulation of inositide hydrolysis in cortical membranes was selective for inositol phospholipids over choline-containing phospholipids. Gpp[NH]p stimulated the production of inositol trisphosphate and inositol bisphosphate as well as inositol monophosphate, indicating that phosphoinositides are substrates for the phosphodiesterase. EGTA (33 microM) did not prevent the guanine nucleotide stimulation of inositide hydrolysis. Calcium addition by itself caused inositide phosphodiesterase activation from 3 to 100 microM which was additive with the Gpp[NH]p stimulation. These data suggest that guanine nucleotides may play a regulatory role in the modulation of the activity of phosphoinositide phosphodiesterase in rat cortical membranes.

scholarly journals Carbachol and histamine stimulation of guanine-nucleotide-dependent phosphoinositide hydrolysis in rat brain cortical membranes

1989 ◽  
Vol 261 (1) ◽  
pp. 29-35 ◽  
Author(s):  
E Claro ◽  
A Garcia ◽  
F Picatoste
Keyword(s):  
Rat Brain ◽  
Inositol Phosphates ◽  
Phosphoinositide Hydrolysis ◽  
Maximal Effect ◽  
Guanine Nucleotide ◽  
Effect Curve ◽  
Guanine Nucleotide Binding ◽  
Cortical Membranes ◽  
Nucleotide Binding Proteins ◽  
Stimulation Of

Guanine nucleotides have been shown to stimulate phosphoinositide breakdown in brain membranes, but no potentiation of such an effect by agonist was demonstrated. We have studied the effect of carbachol and histamine on guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulation of inositol phosphates formation in [3H]inositol-labelled rat brain cortical membranes. In this preparation, GTP[S] enhancement of phosphoinositide hydrolysis required the presence of MgATP and low Ca2+ concentration (100 nM). Carbachol potentiation of the GTP[S] effect was only observed when 1 mM-deoxycholate was also added. Under these conditions, stimulated production of [3H]inositol phosphates was linear for at least 15 min, and [3H]inositol bisphosphate [(3H]IP2) accounted for approx. 80%, whereas the amount of [3H]inositol trisphosphate [(3H]IP3) was very low. Stimulation by GTP[S] was concentration-dependent (half-maximal effect at 0.86 microM), and its maximal effect (815% over basal) was increased by 1 mM-carbachol (1.9-fold) and -histamine (1.7-fold). Both agonists decreased the slope index of the GTP[S] concentration/effect curve to values lower than unity, suggesting the appearance of some heterogeneity in the population of guanine-nucleotide-binding proteins (G-proteins) involved. The carbachol and histamine effects were also concentration-dependent, and were inhibited by atropine and mepyramine respectively. Fluoroaluminate stimulated phosphoinositide hydrolysis to a higher extent than GTP[S] plus carbachol, and these stimulations were not additive, indicating that the same polyphosphoinositide phospholipase C-coupled G-protein mediates both effects.

G protein in stimulation of PI hydrolysis by CCK in isolated rat pancreatic acinar cells

1988 ◽  
Vol 255 (5) ◽  
pp. E652-E659 ◽  
Author(s):  
T. Matozaki ◽  
C. Sakamoto ◽  
M. Nagao ◽  
H. Nishizaki ◽  
S. Baba
Keyword(s):  
G Protein ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Secretory Rate ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Phosphate Accumulation ◽  
Maximal Stimulation ◽  
Inositol Phosphate Accumulation ◽  
Stimulation Of

To clarify the possible role of a guanine nucleotide-binding protein (G protein) in the signal transducing system activated by cholecystokinin (CCK), actions of CCK on rat pancreatic acini were compared with those of fluoride, a well-known activator of stimulatory (Gs) or inhibitory (Gi) G protein. When acini were incubated with increasing concentrations of either CCK-octapeptide (CCK8) or NaF, a maximal stimulation of amylase release from acini occurred at 100 pM CCK8 or 10 mM NaF, respectively; this secretory rate decreased as CCK8 or NaF concentration was increased. NaF caused an increased in cytoplasmic Ca2+ concentration from the internal Ca2+ store and stimulated accumulation of inositol phosphates in acini, as observed with CCK. However, NaF-stimulated Ca2+ mobilization had a lag period before detectable stimulation and was potentiated by AlCl3. These stimulatory effects of NaF appeared to be independent of cellular adenosine 3',5'-cyclic monophosphate (cAMP). Pretreatment with cholera toxin or pertussis toxin did not affect CCK8- or NaF-induced inositol phosphate accumulation or Ca2+ mobilization. 5'-Guanylimidodiphosphate activated the generation of inositol phosphates in the [3H]inositol-labeled pancreatic acinar cell membrane preparation, with half-maximal and maximal stimulation at 1 and 10 microM, respectively. Furthermore, the effects of submaximal CCK concentrations on inositol phosphate accumulation in membranes were markedly potentiated in the presence of 100 microM GTP, which alone was ineffective. Combined findings of the present study strongly suggest that pancreatic CCK receptors are probably coupled to the activation of polyphosphoinositide (PI) breakdown by a G protein, which appears to be fluoride sensitive but is other than Gs- or Gi-like protein.

scholarly journals Guanine nucleotide regulation of phospholipase C activity in permeabilized rabbit neutrophils. Inhibition by pertussis toxin and sensitization to submicromolar calcium concentrations

1986 ◽  
Vol 239 (1) ◽  
pp. 97-102 ◽  
Author(s):  
P G Bradford ◽  
R P Rubin
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Pertussis Toxin ◽  
Response Curve ◽  
Regulatory Protein ◽  
Guanine Nucleotide ◽  
Nucleotide Regulation ◽  
Guanine Nucleotide Regulatory Protein ◽  
Stimulation Of

Rabbit neutrophils labelled with [3H]inositol and permeabilized with saponin produced [3H]inositol trisphosphate (InsP3) when incubated with stable analogues of GTP or millimolar concentrations of Ca2+. [3H]InsP3 production elicited by guanosine 5′-[gamma-thio]triphosphate was enhanced by the chemoattractant formylmethionyl-leucyl-phenylalanine and inhibited by pertussis-toxin pretreatment. A pertussis-toxin-sensitive stimulation of [3H]InsP3 concentration was also observed with guanosine 5′-[beta gamma-imido]triphosphate, but not with guanosine 5′-[beta-thio]diphosphate or GTP. Millimolar Ca2+ alone was sufficient to stimulate [3H]InsP3 production; however, in the presence of guanosine 5′-[gamma-thio]triphosphate, the Ca2+ dose-response curve was shifted to submicromolar concentrations. These findings directly confirm the role of a pertussis-toxin-sensitive guanine nucleotide regulatory protein (G protein) in chemoattractant-stimulated phospholipase C activity in rabbit neutrophils. Moreover, the ability of guanine nucleotides to sensitize phospholipase C to physiologically relevant Ca2+ concentrations suggests that the role of the activated G protein may be to enhance the apparent affinity of phospholipase C for Ca2+ and thus to activate the enzyme without an increase in the Ca2+ concentration.

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.

Eicosapentaenoic Acid Interferes with U46619-Stimulated Formation of Inositol Phosphates in Washed Rabbit Platelets

1989 ◽  
Vol 62 (04) ◽  
pp. 1116-1120 ◽  
Author(s):  
N Chetty ◽  
J D Vickers ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
J F Mustard
Keyword(s):  
Signal Transduction ◽  
Fatty Acid Composition ◽  
Eicosapentaenoic Acid ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Dense Granule ◽  
Detectable Change ◽  
Membrane Phospholipids ◽  
Rabbit Platelets ◽  
Stimulation Of

SummaryEicosapentaenoic acid (EPA) inhibits platelet responsiveness to aggregating agents. To investigate the reactions that are affected by EPA, we examined the effect of preincubating aspirintreated rabbit platelets with EPA on stimulation of inositol phosphate formation in response to the TXA2 analogue U46619. Stimulation of platelets with U46619 (0.5 μM) caused aggregation and slight release of dense granule contents; aggregation and release were inhibited by preincubation of the platelets with EPA (50 μM) for 1 h followed by washing to remove unincorporated EPA. Incubation with EPA (50 μM) for 1 h did not cause a detectable increase in the amount of EPA in the platelet phospholipids. When platelets were prelabelled with [3H]inositol stimulation with U46619 of control platelets that had not been incubated with EPA significantly increased the labelling of mos1tol phosphates. The increases in inositol phosphate labelling due to U46619 at 10 and 60 s were partially inhibited by premcubat10n of the platelets with 50 μM EPA. Since the activity of cyclo-oxygenase was blocked with aspirin, inhibition of inositol phosphate labelling in response to U46619 indicates either that there may be inhibition of signal transduction without a detectable change in the amount of EPA in platelet phospholipids, that changes in signal transduction require only minute changes in the fatty acid composition of membrane phospholipids, or that after a 1 h incubation with EPA, activation of phospholipase C is affected by a mechanism that is not directly related to incorporation of EPA.

Substance P stimulation of polyphosphoinositide hydrolysis in rat anterior pituitary membranes involves a GTP-dependent mechanism

1991 ◽  
Vol 130 (1) ◽  
pp. 63-70 ◽  
Author(s):  
S. E. Mau ◽  
T. Saermark
Keyword(s):  
Substance P ◽  
Pertussis Toxin ◽  
Anterior Pituitary ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Sodium Deoxycholate ◽  
Pituitary Cells ◽  
Guanine Nucleotide ◽  
Nk1 Receptor ◽  
Gtp Binding

ABSTRACT Substance P (SP) stimulates polyphosphoinositide breakdown in the rat anterior pituitary through an NK-1 receptor. In the present study we present evidence that the coupling between the SP–NK1 receptor complex and polyphosphoinositide-specific phospholipase C (PI-PLC) in rat anterior pituitary membranes may involve a mechanism consistent with a GTP-binding protein. The formation of inositol phosphates from [3H]myo-inositol-labelled anterior pituitary membranes induced by SP was potentiated by GTP and non-hydrolysable guanine nucleotides. The stimulatory effects of SP alone and SP plus GTP could be blocked by addition of GDP-β-S (guanosine 5-O-(thiodiphosphate)) in excess. Basal and SP plus guanine nucleotide-induced inositol phosphate formation were stimulated by fluoride, whereas the effect of SP alone was inhibited. Pretreatment of anterior pituitary membranes with sodium deoxycholate attenuated the inositol phosphate response elicited by GTP and GTP-γ-S, whereas basal and SP-stimulated inositol phosphate production showed a peak at 1 mg sodium deoxycholate/ml. SP, fluoride and guanine nucleotide stimulatory effects on hydrolysis of polyphosphoinositide (PPI) were unaffected by pretreatment of anterior pituitary cells with cholera or pertussis toxin for 12 h. Treatment of anterior pituitary membranes with cholera and pertussis toxin yielded [32P]ADP-ribosylation of two proteins with molecular masses of 45 and 41 kDa respectively. We conclude that SP coupling to PI-PLC through the NK1 receptor in the rat anterior pituitary involves a GTP-binding mechanism distinct from the G-proteins associated with adenylate cyclase, Gs and Gi. Journal of Endocrinology (1991) 130, 63–70

Adrenergic stimulation of inositol phosphate accumulation in tracheal epithelium

1989 ◽  
Vol 66 (1) ◽  
pp. 504-508 ◽  
Author(s):  
T. Bainbridge ◽  
R. D. Feldman ◽  
M. J. Welsh
Keyword(s):  
Adrenergic Receptor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Adrenergic Stimulation ◽  
Cl Secretion ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation ◽  
Stimulation Of

To determine whether inositol phosphates are important second messengers in the regulation of Cl- secretion by airway epithelia, we examined the relationship between inositol phosphate accumulation and Cl- secretion in response to adrenergic agonists. We found that epinephrine stimulated Cl- secretion and inositol phosphate accumulation with similar concentration dependence. Although isoproterenol stimulated Cl- secretion, there was no effect of beta-adrenergic receptor activation on inositol phosphate accumulation. In contrast, alpha 1-adrenergic receptor activation stimulated inositol phosphate accumulation but failed to induce Cl- secretion. Another Cl- secretagogue, prostaglandin E1, also failed to stimulate inositol phosphate accumulation. These data suggest that inositol phosphate accumulation is neither sufficient nor required for stimulation of Cl- secretion in cultured canine tracheal epithelial cells.

scholarly journals Effects of pertussis toxin on growth factor-stimulated inositol phosphate formation and DNA synthesis in Swiss 3T3 cells

1988 ◽  
Vol 249 (3) ◽  
pp. 917-920 ◽  
Author(s):  
C W Taylor ◽  
D M Blakeley ◽  
A N Corps ◽  
M J Berridge ◽  
K D Brown
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Phosphoinositide Hydrolysis ◽  
Polypeptide Growth Factors ◽  
Swiss 3T3 ◽  
Swiss 3T3 Cell ◽  
Stimulation Of

We have compared the effects of pretreatment of Swiss 3T3 cell with pertussis toxin on the stimulation of DNA synthesis and phosphoinositide hydrolysis in response to a wide variety of mitogens. The toxin substantially inhibited the stimulation of DNA synthesis in response to a phorbol ester or various peptide and polypeptide growth factors irrespective of their ability to activate phosphoinositidase C. Production of inositol phosphates in response to platelet-derived growth factor, fibroblast growth factor and prostaglandin F2 alpha were unaffected by the toxin while bombesin- and vasopressin-stimulated formation of inositol phosphates were inhibited by only 27 and 23% respectively. These results argue against a major role for a pertussis toxin-sensitive G protein in coupling any of these mitogen receptors to activation of a phosphoinositidase C. Furthermore, the results suggest that the widespread inhibitory effects of pertussis toxin on mitogen-stimulated DNA synthesis may be unrelated to the toxin's limited actions on phosphoinositide hydrolysis.

Rapid stimulation of Ins (1,4,5)P3 production in rat aorta by NE: correlation with contractile state

1993 ◽  
Vol 264 (1) ◽  
pp. H126-H132
Author(s):  
V. Pijuan ◽  
I. Sukholutskaya ◽  
W. G. Kerrick ◽  
M. Lam ◽  
C. van Breemen ◽  
...  
Keyword(s):  
Time Course ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Rat Aorta ◽  
Release Mechanism ◽  
Maximal Increase ◽  
Contractile State ◽  
Rapid Stimulation ◽  
Relationship Of ◽  
Stimulation Of

Rapid stimulation of Ins(1,4,5)P3 production in rat aorta by NE: correlation with contractile state. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H126-H132, 1993.--The isomeric composition of inositol phosphates generated in response to norepinephrine (NE) stimulation and the relationship of inositol phosphate production to release of intracellular Ca2+ as measured by contraction were characterized in rat aorta prelabeled with [3H]inositol. NE stimulated a rapid and transient increase in labeled D-myo-inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] levels. A maximal increase in labeled Ins(1,4,5)P3 occurred within 15 s of stimulation followed by a decline to control levels at 5 min. D-Myo-inositol 1,3,4-trisphosphate [Ins-(1,3,4)P3] and D-myo-inositol 1-monophosphate [Ins(1)P] levels also increased rapidly in response to NE. In contrast to the transient production of Ins(1,4,5)P3, Ins(1,3,4)P3 and Ins(1)P production was maintained in the presence of NE. Half-maximal stimulation of Ins(1,4,5)P3 production and Ca2+ release occurred at 0.3 microM NE, and maximal effects were obtained with 10 microM NE. The concentration-response curve and time course for production of Ins(1,4,5)P3 correlated with the neurotransmitter-induced Ca2+ release from intracellular stores, indicating that the level of Ins(1,4,5)P3 regulated the Ca(2+)-release mechanism. In the continued presence of NE, the intracellular pools did not completely refill with Ca2+ despite the return of Ins-(1,4,5)P3 levels to basal at 5 min. These results demonstrate that NE stimulates a rapid increase in Ins(1,4,5)P3 that correlates with contraction in Ca(2+)-free buffer. The reuptake of Ca2+ into intracellular stores is regulated by a mechanism that may not involve Ins(1,4,5)P3.

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