scholarly journals Phosphoinositide hydrolysis by guanosine 5′-[γ-thio]triphosphate-activated phospholipase C of turkey erythrocyte membranes

1988 ◽  
Vol 252 (2) ◽  
pp. 583-593 ◽  
Author(s):  
T K Harden ◽  
P T Hawkins ◽  
L Stephens ◽  
J L Boyer ◽  
C P Downes
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Specific Radioactivity ◽  
Major Product ◽  
Erythrocyte Membranes ◽  
Low Concentrations ◽  
Phosphate Response ◽  
Phosphatidylinositol 4 ◽  
Rate Of Accumulation

Phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] of turkey erythrocytes were labelled by using either [32P]Pi or [3H]inositol. Although there was little basal release of inositol phosphates from membranes purified from labelled cells, in the presence of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) the rate of accumulation of inositol bis-, tris- and tetrakis-phosphate (InsP2, InsP3 and InsP4) was increased 20-50-fold. The enhanced rate of accumulation of 3H-labelled inositol phosphates was linear for up to 20 min; owing to decreases in 32P specific radioactivity of phosphoinositides during incubation of membranes with unlabelled ATP, the accumulation of 32P-labelled inositol phosphates was linear for only 5 min. In the absence of ATP and a nucleotide-regenerating system, no InsP4 was formed, and the overall inositol phosphate response to GTP[S] was decreased. Analyses of phosphoinositides during incubation with ATP indicated that interconversions of PtdIns to PtdIns4P and PtdIns4P to PtdIns(4,5)P2 occurred to maintain PtdIns(4,5)P2 concentrations; GTP[S]-induced inositol phosphate formation was accompanied by a corresponding decrease in 32P- and 3H-labelled PtdIns, PtdIns4P and PtdIns(4,5)P2. In the absence of ATP, only GTP[S]-induced decreases in PtdIns(4,5)P2 occurred. Since inositol monophosphate was not formed under any condition, PtdIns is not a substrate for the phospholipase C. The production of InsP2 was decreased markedly, but not blocked, under conditions where Ins(1,4,5)P3 5-phosphomonoesterase activity in the preparation was inhibited. Thus the predominant substrate of the GTP[S]-activated phospholipase C of turkey erythrocyte membranes is PtdIns(4,5)P2. Ins(1,4,5)P3 was the major product of this reaction; only a small amount of Ins(1:2-cyclic, 4,5)P3 was released. The effects of ATP on inositol phosphate formation apparently involve the contributions of two phenomena. First, the P2-receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) greatly increased inositol phosphate formation and decreased [3H]PtdIns4P and [3H]PtdIns(4,5)P2 in the presence of a low (0.1 microM) concentration of GTP[S]. ATP over the concentration range 0-100 microM produced effects in the presence of 0.1 microM-GTP[S] essentially identical with those observed with 2MeSATP, suggesting that the effects of low concentrations of ATP are also explained by a stimulation of P2-receptors. Higher concentrations of ATP also increase inositol phosphate formation, apparently by supporting the synthesis of substrate phospholipids.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Convulxin-induced platelet aggregation is accompanied by a powerful activation of the phospholipase C pathway

1994 ◽  
Vol 298 (1) ◽  
pp. 87-91 ◽  
Author(s):  
A Faili ◽  
J Randon ◽  
I M Francischetti ◽  
B B Vargaftig ◽  
M Hatmi
Keyword(s):  
Platelet Aggregation ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Platelet Activating Factor ◽  
Phosphatidyl Inositol ◽  
High Concentration ◽  
Adp Release ◽  
Pathway Formation ◽  
Phosphatidylinositol 4

Platelet aggregation and stimulation of phosphoinositide-specific phospholipase C (PLC) by thrombin and by convulxin (Cvx), a non-enzymic snake venom glycoprotein, were compared. Cvx-stimulated production of inositol phosphates by washed platelets was independent of the cyclo-oxygenase pathway, formation of platelet-activating factor and ADP release, but prostacyclin (prostaglandin I2), a stimulator of cyclic AMP formation, suppressed its effects on platelet and PLC activation. Kinetic analysis showed that inositol 1,4,5-trisphosphate formation reached its maximal value 15 s after platelet stimulation with Cvx and persisted for at least 5 min. Neomycin sulphate (10 mM), which complexes phosphatidylinositol 4-phosphate and phosphatidyl-inositol 4,5-bisphosphate, decreased the production of inositol phosphates, partially prevented platelet aggregation induced by a high concentration of Cvx (10 nM) and abolished both platelet aggregation and inositol phosphate formation induced by thrombin (2 units/ml) and by a stable prostaglandin H2 analogue, U46619 (1 microM). In contrast with neomycin sulphate, Na2SO4 had no significant effect against all agonists tested. It is concluded that platelet activation by Cvx is partially mediated by PLC and involves other mechanisms as well.

scholarly journals The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+

1985 ◽  
Vol 231 (2) ◽  
pp. 269-278 ◽  
Author(s):  
R V Farese ◽  
J S Davis ◽  
D E Barnes ◽  
M L Standaert ◽  
J S Babischkin ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
De Novo ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Specific Radioactivity ◽  
Phospholipid Content ◽  
Phospholipid Synthesis ◽  
Rat Adipose Tissue ◽  
Phosphatidylinositol 4 ◽  
Protein Kinase C Activation

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated ‘second messenger’ which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular ‘messenger’ for controlling metabolic processes during insulin action.

scholarly journals Guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate in HL-60 granulocytes. Evidence that the guanine nucleotide acts by relieving phospholipase C from an inhibitory constraint

1990 ◽  
Vol 271 (3) ◽  
pp. 743-748 ◽  
Author(s):  
M Camps ◽  
C F Hou ◽  
K H Jakobs ◽  
P Gierschik
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Anion Exchange Chromatography ◽  
Major Product ◽  
Exchange Chromatography ◽  
High Protein ◽  
High Protein Concentration ◽  
Inositol Phosphate Accumulation ◽  
Stimulation Of

Myeloid differentiated human leukaemia (HL-60) cells contain a soluble phospholipase C that hydrolysed phosphatidylinositol 4.5-bisphosphate and was markedly stimulated by the metabolically stable GTP analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] occurred at 1.5 microM and 30 microM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5′-[beta-thio]diphosphate. ATP, adenosine 5′-[gamma-thio]triphosphate, UTP) did not affect phospholipase C activity, GTP[S] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTP[S] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. approximately 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTP[S]-stimulated phospholipase C activity. In the absence of GTP[S], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated inositol phosphate formation were linear with time whether studied at low or high protein concentration, these results suggest that (a) phospholipase C is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating a soluble GTP-binding protein.

scholarly journals Interfacial hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate by turkey erythrocyte phospholipase C

1994 ◽  
Vol 298 (2) ◽  
pp. 499-506 ◽  
Author(s):  
S R James ◽  
R A Demel ◽  
C P Downes
Keyword(s):  
Phospholipase C ◽  
Surface Pressure ◽  
Inositol Phosphates ◽  
Specific Radioactivity ◽  
Initial Pressure ◽  
Maximum Activity ◽  
First Order Kinetics ◽  
High Specific Radioactivity ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of

The activity of a beta-isoform of phospholipase C (PLC) partially purified from turkey erythrocyte cytosol was assayed using phospholipid monolayers formed at an air-water interface. PLC was rapidly purified at least 8000-fold by a sequence of ion-exchange, hydrophobic and heparin chromatographies. 33P-labelled substrates were prepared using partially purified PtdIns kinase and PtdIns4P 5-kinases, respectively, and purified by h.p.l.c. using an amino-cyano analytical column. Using such 33P-labelled phosphoinositides of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphates and their subsequent dissolution and quenching in the subphase. Under these conditions, PtdIns4P hydrolysis obeyed approximately first-order kinetics whereas PtdIns(4,5)P2 hydrolysis was zero-order at least until 80% of the substrate had been degraded. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure and with the most permissive pressures in the middle of the range investigated. The optimum surface pressure for hydrolysis of PtdIns4P was approx. 25 mN/m, but for PtdIns(4,5)P2 the maximum activity occurred at the markedly higher surface pressure of 30 mN/m. These data are discussed in terms of the substrate specificity and likely regulation of PLC beta isoforms engaged in degrading their substrate in biological membranes.

scholarly journals The decrease in phosphatidylinositol 4,5-bisphosphate in ADP-stimulated washed rabbit platelets is not primarily due to phospholipase C activation

1986 ◽  
Vol 237 (2) ◽  
pp. 327-332 ◽  
Author(s):  
J D Vickers ◽  
R L Kinlough-Rathbone ◽  
J F Mustard
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Shape Change ◽  
Specific Radioactivity ◽  
Amine Storage ◽  
Rabbit Platelets ◽  
Storage Granules ◽  
Amine Storage Granules ◽  
Platelet Shape

Addition of 10 micron-ADP to washed rabbit platelets caused platelet shape change and aggregation without release of the contents of the amine-storage granules, and caused a transient decrease (8.8% at 10 s) in the amount of phosphatidylinositol 4,5-bisphosphate (PIP2). By 20 s the decrease in PIP2 was no longer apparent, but by 60 s the amount of PIP2 was again decreased. Addition of thrombin (1 unit/ml), which causes platelet shape change, aggregation and the release of the contents of the amine-storage granules, caused a decrease in the amount of PIP2 (8.0% at 10 s); at 60 s the amount of PIP2 was not significantly different from that in controls. In platelets prelabelled with [3H]glycerol, the specific radioactivity of PIP2 was increased at 10 s in ADP-stimulated platelets, and unchanged in thrombin-stimulated platelets. In platelets prelabelled with [3H]inositol and incubated with 20 mM-Li+ to inhibit the degradation of the inositol phosphates to inositol, there was no increase in the labelling of inositol trisphosphate (IP3) upon stimulation with ADP. In contrast, stimulation with thrombin caused a significant increase in the labelling of IP3 at 10 s. These differences in the changes in polyphosphoinositide metabolism in ADP- and thrombin-stimulated platelets are consistent with the hypothesis that the decrease in PIP2 in ADP-stimulated platelets may be due not to degradation of PIP2 by phospholipase C, but rather to a shift in the equilibrium between PIP2 and phosphatidylinositol 4-phosphate (PIP). Increases in the labelling of phosphatidic acid at 10 s and of inositol bisphosphate and inositol phosphate after 20 s are consistent with phospholipase C being stimulated through some other mechanism that leads to the degradation of PIP and phosphatidylinositol; one possibility is that ADP causes an increase in cytoplasmic Ca2+.

Phosphoinositide breakdown in blowfly salivary glands

1984 ◽  
Vol 246 (1) ◽  
pp. C141-C147 ◽  
Author(s):  
I. Litosch ◽  
H. S. Lee ◽  
J. N. Fain
Keyword(s):  
Salivary Glands ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Ionophore A23187 ◽  
Phosphoinositide Breakdown ◽  
Transient Loss ◽  
Phosphatidylinositol 4

In blowfly salivary glands, 5-hydroxytryptamine stimulated a rapid and sustained loss of [3H]inositol, [32P]phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. There was a corresponding increase in labeled inositol phosphates. In the absence of Ca2+, 5-hydroxytryptamine stimulated a rapid but transient loss of labeled phosphatidylinositol 4,5-bisphosphate. By 5 min, the amount of labeled phosphatidylinositol 4,5-bisphosphate recovered to control values. The divalent ionophore A23187 stimulated loss of labeled phosphatidylinositol 4,5-bisphosphate and increased the amount of labeled phosphatidylinositol. In homogenates, Ca2+ stimulated phosphatidylinositol 4,5-bisphosphate breakdown but not phosphatidylinositol breakdown. These results suggest that hormone-stimulated breakdown of labeled phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate occurs through a phospholipase C and is relatively independent of extracellular Ca2+. There is also a Ca2+-activated conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol.

The interaction of arginine vasopressin and oxytocin with bovine adrenal medulla cells

1989 ◽  
Vol 121 (1) ◽  
pp. 133-139 ◽  
Author(s):  
A. H. Taylor ◽  
G. St J. Whitley ◽  
S. S. Nussey
Keyword(s):  
Arginine Vasopressin ◽  
Chromaffin Cells ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Binding Capacity ◽  
Camp Production ◽  
Low Concentrations ◽  
Inositol Phosphate Accumulation ◽  
Adrenal Chromaffin ◽  
Induced Changes

ABSTRACT Binding of [3H]arginine vasopressin (AVP) and [3H]oxytocin to primary monolayer cultures of bovine adrenal chromaffin cells was time-dependent, and the binding sites for each peptide were specific and saturable. Studies with the V1 AVP antagonist d(CH2)5Tyr(Me)2-AVP, the V2 agonist 1-deamino-8-d-AVP and the V2 antagonist d(CH2)5d-Leu2,Val4-AVP indicated that the AVP receptor was V1 in specificity. Scatchard plots showed that each ligand interacted with a single high-affinity, low-capacity binding site: oxytocin dissociation constant (Kd) 0·29 ± 0·02 nmol/l, maximum binding capacity (Bmax) 7·6 ± 0·2 fmol/106 cells (or 4500 ± 102 sites/cell) (n = 3); AVP Kd 0·09±0·02 nmol/l, Bmax 5·1±0·63 fmol/106 cells (or 3050 ± 318 sites/cell) (n = 3). Although forskolin in concentrations from 1 nmol/l to 1 mmol/l stimulated cyclic AMP (cAMP) production in isolated chromaffin cells, this did not result in detectable catecholamine release. Neither AVP nor oxytocin in concentrations between 10 pmol/l and 10 μmol/l stimulated cAMP production in these cells. Vasopressin in concentrations as low as 10 pmol/l stimulated a sixfold increase in total inositol phosphates; the dose–response curve was triphasic. Oxytocin had little effect on total inositol phosphate accumulation at low concentrations, but concentrations above micromolar stimulated total inositol phosphate production approximately fourfold. There was no measurable release of catecholamines in response to either peptide. The physiological consequences of these AVP-induced changes in inositol phosphate concentrations remain to be elucidated. Journal of Endocrinology (1989) 121, 133–139

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.

scholarly journals Relationships between the degree of cross-linking of surface immunoglobulin and the associated inositol 1,4,5-trisphosphate and Ca2+ signals in human B cells

1992 ◽  
Vol 284 (2) ◽  
pp. 447-455 ◽  
Author(s):  
F M McConnell ◽  
S B Shears ◽  
P J L Lane ◽  
M S Scheibel ◽  
E A Clark
Keyword(s):  
B Cells ◽  
Tyrosine Kinase ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Cross Linking ◽  
Immunoglobulin Receptor ◽  
Surface Immunoglobulin ◽  
Human B Cells ◽  
Inositol 1,4,5 Trisphosphate

Cross-linking of surface immunoglobulin (Ig) receptors on human B cells leads to the activation of a tyrosine kinase. The activated tyrosine kinase subsequently phosphorylates a number of substrates, including phospholipase C-gamma. This enzyme breaks down phosphoinositol bisphosphate to form two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate, leading to the activation of protein kinase C and the release of intracellular Ca2+ respectively. We have used h.p.l.c. and flow cytometry to measure accurately the inositol phosphate turnover and Ca2+ release in anti-Ig-stimulated human B cells. In particular, we have examined the effect of dose of the cross-linking antibody on the two responses. The identity of putative messenger inositol phosphates has been verified by structural analysis, and the amounts of both inositol phosphates and Ca2+ present have been quantified. In the Ramos Burkitt lymphoma, which is very sensitive to stimulus through its Ig receptors, both inositol phosphate production and Ca2+ release were found to be related to the dose of anti-Ig antibody applied. This suggests that phospholipase C-mediated signal transduction in human B cells converts the degree of cross-linking of the immunoglobulin receptor quantitatively into intracellular signals.

scholarly journals Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands

1983 ◽  
Vol 216 (3) ◽  
pp. 633-640 ◽  
Author(s):  
C P Downes ◽  
M M Wusteman
Keyword(s):  
Molecular Mechanisms ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Muscarinic Agonist ◽  
Parotid Glands ◽  
Muscarinic Agonists ◽  
Inositol 1 ◽  
Inositol 1,4,5 Trisphosphate ◽  
Rat Parotid ◽  
Phosphatidylinositol 4

The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.

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