scholarly journals Quantification of inositol phospholipid breakdown in isolated rat hepatocytes

1993 ◽  
Vol 290 (3) ◽  
pp. 865-872 ◽  
Author(s):  
C J Allan ◽  
J H Exton
Keyword(s):  
Phospholipase C ◽  
Phospholipase D ◽  
Inositol Phosphates ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Phospholipid Hydrolysis ◽  
Inositol Phospholipids ◽  
Inositol Phospholipid ◽  
Inositol Phospholipid Breakdown ◽  
Hydrolysis Of

The hydrolysis of inositol phospholipids induced by vasopressin in hepatocytes during 60 min was quantified chemically. There was a large release of myo-inositol which was abolished by Li+, indicating that it was derived from inositol phosphates and not from phospholipase D action on PtdIns. There was also a large release of inositol phosphates which was increased approx. 2-fold by Li+ at 30 min, but then remained constant, suggesting that inositol phospholipid breakdown declined substantially beyond this time. In cells prelabelled with myo-[3H]inositol and treated with Li+, [3H]PtdIns(4,5)P2 decreased maximally (50%) at 15 s and then recovered to a level at 5 min that was maintained at 25% below control for 40 min. [3H]PtdIns4P and [3H]PtdIns showed slower decreases to approx. 30% below control at 15 min, but with no further changes. Labelled Ins(1,4,5)P3 and Ins(1,3,4)P3 showed 2-4-fold increases within 30 s and then declined to values that were maintained at a constant level above the control, except for [3H]Ins(1,3,4)P3, which showed a second increase. [3H]Ins(1,4)P2 showed a very large increase over 10 min, whereas [3H]Ins4P and [3H]Ins1P showed little change before 6 and 15 min respectively. The total [3H]inositol phosphates showed little further increase after 20 min. These data are consistent with a rapid, but not sustained, hydrolysis of PtdIns-(4,5)P2, but not of PtdIns, by phospholipase C, but do not exclude PtdIns4P as a substrate. Phosphatidate was rapidly increased by vasopressin, whereas diacylglycerol was increased after a 1-2 min lag. Both were maintained at levels 2-3-fold above control for 60 min. The vasopressin-induced increase in inositol phosphates plus myo-inositol (approx. 120 nmol/100 mg) was greater than the increase in diacylglycerol plus phosphatidate (approx. 60 nmol/100 mg) between 10 and 40 min. This indicates that there was substantial further metabolism of these lipids. Addition of 75 mM ethanol resulted in rapid production of phosphatidylethanol in response to vasopressin and a 35% reduction in phosphatidate, but no decrease in diacylglycerol. In summary, the results indicate that inositol phospholipid hydrolysis by phospholipase C can account for most of the diacylglycerol and phosphatidate that accumulate during 60 min of vasopressin action, but that these phospholipids are probably not the major source of the phosphatidate that is formed during the first 2 min by phospholipase D, or of the diacylglycerol and phosphatidate that are formed beyond 30 min.

scholarly journals Epidermal growth factor stimulates distinct diradylglycerol species generation in Swiss 3T3 fibroblasts: evidence for a potential phosphatidylcholine-specific phospholipase C-catalysed pathway

1994 ◽  
Vol 298 (3) ◽  
pp. 655-660 ◽  
Author(s):  
T R Pettitt ◽  
M Zaqqa ◽  
M J Wakelam
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Kinase Inhibitor ◽  
Inositol Phosphates ◽  
Egf Receptors ◽  
3T3 Fibroblasts ◽  
Epidermal Growth ◽  
Hydrolysis Of

Stimulation of 3T3 fibroblasts with epidermal growth factor (EGF) results in an increase in 1,2-diacylglycerol (DAG) mass which is maximal at 25 s, declining at 1 min and returning to basal levels by 30 min. No changes in alkylacylglycerol or alkenylacylglycerol were detected. Three species account for most of this mass increase: 18:0/20:5,n-3, 18:0/20:4,n-6 and 18:0/20:3,n-9. These species are characteristic of the phosphoinositides; however, previous work failed to detect any EGF-stimulated rise in inositol phosphates in these cells [Cook and Wakelam (1992) Biochem. J. 285, 247-253]. This ruled out phosphoinositide hydrolysis by phospholipase C, but raised the possibility of phospholipase D/phosphatidate phosphohydrolase-catalysed hydrolysis of phosphatidylinositol. The inclusion of butanol in the incubation medium failed to block the diacylglycerol changes, indicating that the phospholipase D pathway is not involved and that DAG must be derived from another source, probably via phospholipase C-catalysed hydrolysis of a phosphatidylcholine pool that is particularly rich in these species. The tyrosine kinase inhibitor ST-271 almost abolished the elevation in 18:0/20:5,n-3, 18:0/20:4, n-6 and 18:0/20:3,n-9 at 25 s, but only reduced the rise in total DAG mass by about 50%. The protein kinase C (PKC) inhibitor Ro-31-8220 increased DAG levels at all time points but had no effect on the species profiles. This provides additional evidence for PKC-mediated regulation of cell-surface EGF receptors, since the inhibition of PKC would increase the availability and/or ligand binding affinity of receptors at the plasma membrane and hence increase and prolong the response to EGF.

scholarly journals Activation of V1-receptors by vasopressin stimulates inositol phospholipid hydrolysis and arachidonate metabolism in human platelets

1986 ◽  
Vol 233 (1) ◽  
pp. 83-91 ◽  
Author(s):  
W Siess ◽  
M Stifel ◽  
H Binder ◽  
P C Weber
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Second Messengers ◽  
Inositol Trisphosphate ◽  
Time Lag ◽  
Human Platelets ◽  
Phospholipid Hydrolysis ◽  
Low Concentrations ◽  
Inositol Phospholipid ◽  
High Concentrations

The activation of platelet V1-receptors by vasopressin (0.01-1 microM) induces the rapid formation of inositol phosphates, 1,2-diacylglycerol and phosphatidic acid, indicating inositol phospholipid hydrolysis by phospholipase C. Vasopressin immediately induces the formation of inositol bisphosphate and inositol trisphosphate. Accumulation of inositol 1-monophosphate and inositol 4-monophosphate occurs later after a time lag of 15 s. Low concentrations (10-100 nM) of vasopressin only activate phospholipase C, whereas high concentrations (1 microM) induce activation of phospholipase C and subsequently the production of arachidonate metabolites. Cyclo-oxygenase metabolites are associated with further activation of phospholipase C, release reaction and irreversible platelet aggregation. Vasopressin requires for its action extracellular Mg2+, but not Ca2+. The described platelet changes are not induced by 1-desamino-[8-D-arginine]vasopressin, a V2-receptor agonist, and are blocked by a specific V1-receptor antagonist. The results indicate that platelets possess a V1-receptor that is coupled to polyphosphoinositide hydrolysis by phospholipase C, leading to the formation of 1,2-diacylglycerol and inositol trisphosphate. Those compounds may act as second messengers for platelet responses induced by vasopressin, whereas endoperoxides and thromboxane A2 stimulated by vasopressin may serve as amplifiers for platelet activation.

scholarly journals Muscarinic-agonist and guanine nucleotide activation of polyphosphoinositide phosphodiesterase in isolated islet-cell membranes

1986 ◽  
Vol 240 (3) ◽  
pp. 731-737 ◽  
Author(s):  
M E Dunlop ◽  
R G Larkins
Keyword(s):  
Islet Cell ◽  
Plasma Membranes ◽  
Islet Cells ◽  
Muscarinic Agonist ◽  
Dependent Manner ◽  
Muscarinic Agonists ◽  
Phospholipid Hydrolysis ◽  
Gtp Binding ◽  
Inositol Phospholipids ◽  
Hydrolysis Of

Stimulated hydrolysis of the inositol phospholipids phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was investigated by studying the phosphoinositides produced in a suspended preparation of plasma membranes by transference of 32P from [gamma-32P]ATP. At basal Ca2+ concentration (calculated free Ca2+, 150 nM) phospholipid hydrolysis was stimulated either by the muscarinic agonists carbamoylcholine and bethanecol or by the addition of the non-hydrolysable analogue of GTP, guanosine 5′-[beta gamma-imido]triphosphate [p(NH)ppG]. GTP was without effect on basal hyrolysis. Both GTP and p(NH)ppG enhanced the rapid (within 10 s) hydrolysis of PtdIns4P and PtdIns(4,5)P2 induced by carbamoylcholine in a dose-dependent manner. A rightward shift in the competition curve of carbamoylcholine for bound L-[3H]quinuclidinyl benzilate was seen on addition of GTP or p(NH)ppG (100 microM) under phosphorylating conditions. Pretreatment of intact islet cells with Bordetella pertussis toxin, islet-activating protein (IAP) or treatment of membranes with IAP under conditions which elicited ADP-ribosylation of a protein of Mr 41,000 was without effect on muscarinic binding, phosphoinositide phosphorylation or subsequent hydrolysis by carbamoylcholine. The findings indicate the involvement of a GTP-binding protein in the coupling of the muscarinic receptor to phosphoinositide hydrolysis in the islet cell and suggest that this is distinct from the GTP-binding regulatory component of adenylate cyclase which is covalently modified by IAP.

Conservation of structure in ATP-depleted proximal tubules: role of calcium, polyphosphoinositides, and glycine

1993 ◽  
Vol 265 (5) ◽  
pp. F605-F623 ◽  
Author(s):  
R. Garza-Quintero ◽  
J. M. Weinberg ◽  
J. Ortega-Lopez ◽  
J. A. Davis ◽  
M. A. Venkatachalam
Keyword(s):  
Amino Acids ◽  
Phospholipase C ◽  
Cell Structure ◽  
Proximal Tubules ◽  
Atp Depletion ◽  
The Other ◽  
Antimycin A ◽  
Phospholipid Hydrolysis ◽  
Hydrolysis Of

Increases of intracellular free Ca2+ (Caf) may mediate phospholipid hydrolysis and disintegration in energy-compromised cells; on the other hand, glycine and related amino acids preserve structure. We have examined the effects of increased Caf on phospholipids and structure in ATP-depleted cells, as well as how these actions may be modified by glycine. Incubation of isolated proximal tubules with antimycin A led to ATP depletion, delayed increases of Caf to micromolar levels, polyphosphoinositide (PPI) hydrolysis by phospholipase C, and generalized disintegration of cell structure. Glycine inhibited PPI hydrolysis and preserved cell structure in entirety but did not apparently modify the Caf increases. When overwhelming increases of Caf were induced by the additional presence of a Ca2+ ionophore, glycine did not inhibit either the hydrolysis of PPI or disruption of mitochondria and microvilli. However, the cells remained integrated and unbroken. Incubation in low-Ca2+ medium prevented Caf increases, inhibited PPI hydrolysis, and preserved the structure of mitochondria and microvilli. Nevertheless, there was lethal damage by disintegration of all other membranes. This damage was prevented specifically and completely by glycine. Thus compartments of cells were shown to be differentially susceptible to injury from increased Caf or lack of glycine. Although damage by either factor occurs by distinct mechanisms, glycine also appears to have effects that suppress the deleterious effects of Ca2+ so long as Caf increases are not overwhelming. Our results also suggest that the PPI have a major structural role, which may be compromised by Caf increase during ATP depletion.

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 The zinc chelator 1,10-phenanthroline enhances the stimulatory effects of protein kinase C activators and staurosporine, but not sphingosine and H2O2, on phospholipase D activity in NIH 3T3 fibroblasts

1994 ◽  
Vol 298 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Z Kiss
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Intermediate Step ◽  
Phospholipid Hydrolysis ◽  
3T3 Fibroblasts ◽  
Kinase C ◽  
Zinc Chelator ◽  
Nih 3T3 ◽  
Hydrolysis Of

Protein kinase C (PKC), an enzyme which is believed to mediate the stimulatory effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on phospholipase D (PLD) activity, has a zinc-dependent structure required for phorbol ester binding. Accordingly, zinc or zinc chelators would be expected to promote or inhibit, respectively, the stimulatory effects of PMA on PLD-mediated phospholipid hydrolysis. Instead, treatment of [14C]choline- and [14C]ethanolamine-labelled NIH 3T3 fibroblasts with the high-affinity zinc chelator 1,10-phenanthroline (0.2-1 mM) for 20-30 min was found to enhance the stimulatory effects of PMA on PLD-mediated hydrolysis of phosphatidylcholine and phosphatidylethanolamine. In [14C]palmitic acid-labelled fibroblasts, in the presence of ethanol, phenanthroline also enhanced the stimulatory effect of PMA on the synthesis of phosphatidylethanol, a marker of PLD activity. Addition of zinc (250 microM) to phenanthroline-treated fibroblasts reversed the stimulatory effects of the chelator. The potentiating effects of phenanthroline were also partially reversed by cadmium, whereas iron, lead, copper, magnesium and calcium were without effects. Of the other activators of PLD tested, phenanthroline also enhanced the stimulatory effects of platelet-derived growth factor and staurosporine, but not that of sphingosine and H2O2, on the hydrolysis of both phospholipids. These results suggest that regulation of PLD by PKC activators and staurosporine involves a common intermediate step, which is inhibited by a chelatable cellular pool of zinc.

scholarly journals Relationship between stimulated phosphatidic acid production and inositol lipid hydrolysis in intestinal longitudinal smooth muscle from guinea pig

1987 ◽  
Vol 244 (3) ◽  
pp. 763-768 ◽  
Author(s):  
R S E Mallows ◽  
T B Bolton
Keyword(s):  
Smooth Muscle ◽  
Small Intestine ◽  
Substance P ◽  
Guinea Pig ◽  
Phosphatidic Acid ◽  
Inositol Phosphates ◽  
Muscle Layer ◽  
Phospholipid Hydrolysis ◽  
Longitudinal Smooth Muscle ◽  
Inositol Phospholipid

Accumulation of [32P]phosphatidic acid (PA) and total [3H]inositol phosphates (IPs) was measured in the longitudinal smooth-muscle layer from guinea-pig small intestine. Stimulation with carbachol, histamine and substance P produced increases in accumulation of both [3H]IPs and [32P]PA over the same concentration range. The increase in [32P]PA accumulation in response to carbachol (1 microM-0.1 mM) was inhibited in the presence of atropine (0.5 microM). Buffering the external free [Ca2+] to 10 nM did not prevent the carbachol-stimulated increase in [32P]PA accumulation. Carbachol and Ca2+ appear to act synergistically to increase accumulation of [32P]PA. In contrast, although incubation with noradrenaline also increased accumulation of [3H]IPs, no increase in accumulation of [32P]PA could be detected. These results suggest that an increase in formation of IPs is not necessarily accompanied by an increase in PA formation, and imply the existence of receptor-modulated pathways regulating PA concentrations other than by phospholipase-C-catalysed inositol phospholipid hydrolysis.

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