scholarly journals Evidence for lithium-sensitive inositol 4,5-bisphosphate accumulation in muscarinic cholinoceptor-stimulated cerebral-cortex slices

1992 ◽  
Vol 287 (2) ◽  
pp. 437-442 ◽  
Author(s):  
S Jenkinson ◽  
R A Challiss ◽  
S R Nahorski
Keyword(s):  
Cerebral Cortex ◽  
Phospholipase D ◽  
Inositol Phosphate ◽  
Intact Cell ◽  
Inositol Phosphate Metabolism ◽  
Kinase C ◽  
Cortex Slices ◽  
A Minor ◽  
Hydrolysis Of ◽  
Stimulation Of

Stimulation of [3H]inositol-prelabelled rat cerebral-cortex slices with carbachol results in the accumulation of four [3H]inositol bisphosphate isomeric species, Ins(1,3)P2, Ins(1,4)P2, Ins(3,4)P2 and Ins(4,5)P2. Although the last isomer ran as a minor peak on h.p.l.c., its accumulation was dramatically enhanced in the presence of Li+ (1 mM), such that at 30 min it represented almost 35% of the total bisphosphate fraction. The accumulation of Ins(4,5)P2 appeared to be very sensitive to Li+ (EC50 = 94 +/- 3 microM), strongly implicating a Li(+)-sensitive metabolism. Evidence for this is provided from the rapid but Li(+)-sensitive decay of Ins(4,5)P2 when muscarinic-receptor stimulation is antagonized by atropine at a time when accumulations have reached a new steady state. Manipulation of phospholipase D by activators and inhibitors of protein kinase C did not suggest a role for phospholipase D hydrolysis of PtdInsP2 in the formation of Ins(4,5)P2. Attempts to reveal Ins(4,5)P2 metabolism, or indeed its synthesis from Ins(1,4,5)P3, were not successful with broken cell preparations and strongly suggest discrete compartmentation of inositol phosphate metabolism in the intact cell.

scholarly journals Accumulation of inositol polyphosphate isomers in agonist-stimulated cerebral-cortex slices. Comparison with metabolic profiles in cell-free preparations

1989 ◽  
Vol 258 (1) ◽  
pp. 23-32 ◽  
Author(s):  
I H Batty ◽  
A J Letcher ◽  
S R Nahorski
Keyword(s):  
Cerebral Cortex ◽  
Inositol Phosphates ◽  
Periodate Oxidation ◽  
Chemical Degradation ◽  
Inositol Polyphosphate ◽  
Tissue Slices ◽  
Inositol 1 ◽  
Agonist Stimulation ◽  
Cortex Slices ◽  
Hydrolysis Of

1. Basal and carbachol-stimulated accumulations of isomeric [3H]inositol mono-, bis-, tris- and tetrakis-phosphates were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. 2. In control samples the major [3H]inositol phosphates detected were co-eluted on h.p.l.c. with Ins(1)P, Ins(4)P (inositol 1- and 4-monophosphate respectively), Ins(1,4)P2 (inositol 1,4-bisphosphate), Ins(1,4,5)P3 (inositol 1,4,5-tris-phosphate) and Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate). 3. After stimulation to steady state with carbachol, accumulation of each of these products was markedly increased. 4. Agonist stimulation, however, also evoked much more dramatic increased accumulations of a second [3H]inositol trisphosphate, which was co-eluted on h.p.l.c. with authentic Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) and of three further [3H]inositol bisphosphates ([3H]InsP2(s]. 5. Examination of the latter by chemical degradation by periodate oxidation and/or h.p.l.c. allowed identification of these as [3H]Ins(1,3)P2, [3H]Ins(3,4)P2 and [3H]Ins(4,5)P2 (inositol 1,3-, 3,4- and 4,5-bisphosphates respectively), which respectively accounted for about 22%, 8% and 3% of total [3H]InsP2 in extracts from stimulated tissue slices. 6. By using a h.p.l.c. method which clearly resolves Ins(1,3,4,5)P4 and Ins(1,3,4,6)P4 (inositol 1,3,4,6-tetrakisphosphate), only the former isomer could be detected in extracts from either control or stimulated tissue slices. Similarly, [3H]inositol pentakis- and hexakis-phosphates were not detectable either in the presence or absence of carbachol under the radiolabelling conditions described. 7. The catabolism of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 by cell-free preparations from cerebral cortex was also studied. 8. In the presence of Mg2+, [3H]Ins(1,4,5)P3 was specifically dephosphorylated via [3H]Ins(1,4)P2 and [3H]Ins(4)P to free [3H]inositol, whereas [3H]Ins(1,3,4)P3 was degraded via [3H]Ins(3,4)P2 and, to a lesser extent, via [3H]Ins(1,3)P2 to D- and/or L-[3H]Ins(1)P and [3H]inositol. 9. In the presence of EDTA, hydrolysis of [3H]Ins(1,4,5)P3 was greater than or equal to 95% inhibited, whereas [3H]Ins(1,3,4)P3 was still degraded, but yielded only a single [3H]InsP2 identified as [3H]Ins(1,3)P2. 10. The significance of these observations with cell-free preparations is discussed in relation to the proportions of the separate isomeric [3H]inositol phosphates measured in stimulated tissue slices.

Endothelin activation of phospholipase D: dual modulation by protein kinase C and Ca2+

1993 ◽  
Vol 264 (5) ◽  
pp. F845-F853
Author(s):  
M. M. Friedlaender ◽  
D. Jain ◽  
Z. Ahmed ◽  
D. Hart ◽  
R. L. Barnett ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Intracellular Signaling ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Eta Receptor ◽  
Ca2 Channels ◽  
Stimulation Of

Previous work from this laboratory has identified an endothelin (ET) type A (ETA) receptor on cultured rat renal medullary interstitial cells (RMIC), coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), dihydropyridine-insensitive receptor-operated Ca2+ channels, and phospholipase A2. The current studies explored a role for ET stimulation of phosphatidylcholine-specific phospholipase D (PC-PLD) in intracellular signaling of this cell type. ET stimulated PLD activation, as measured by phosphatidic acid (PA) or phosphatidylethanol (PEt) accumulation, in a time- and concentration-dependent manner. Inhibition of diacylglycerol (DAG) kinase by ethylene glycol dioctanoate or 6-(2)4-[(4-fluorophenyl)-phenylmethylene]-1-piperadinyl]ethy l-7-methyl-5H - thiaxolo-[3,2-alpyrimidin]-5-one (R 59022) failed to blunt PA accumulation, indicating that PLD, and not DAG, was the source of PA. Inhibition of PA phosphohydrolase (PAP) by propranolol increased late accumulation of PA, suggesting that the prevailing metabolic flow was in the direction of PA to DAG. Phorbol 12-myristate 13-acetate (PMA) augmented ET-evoked PEt accumulation, whereas downregulation of protein kinase C (PKC) obviated agonist-induced PEt production. PMA augmentation of PLD activity proceeded independent of cytosolic free Ca2+ concentration. Ca2+ derived from either intracellular or extracellular sources enhanced ET-related PEt accumulation but was without effect in PKC-downregulated cells. Collectively, these observations indicate that ET stimulates PLD production in RMIC. PKC is the major regulator of this process, with Ca2+ playing a secondary, modulatory role. In addition, these data suggest that PC-PLD is coupled to the ETA receptor.

scholarly journals Rapid accumulation and sustained turnover of inositol phosphates in cerebral-cortex slices after muscarinic-receptor stimulation

1989 ◽  
Vol 260 (1) ◽  
pp. 237-241 ◽  
Author(s):  
I H Batty ◽  
S R Nahorski
Keyword(s):  
Cerebral Cortex ◽  
Muscarinic Receptor ◽  
Metabolic Flux ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Receptor Activation ◽  
Inositol Polyphosphate ◽  
Receptor Stimulation ◽  
Cortex Slices ◽  
Rapid Kinetics

The rapid kinetics of [3H]inositol phosphate accumulation and turnover were examined in rat cerebral-cortex slices after muscarinic-receptor stimulation. Markedly increased [3H]inositol polyphosphate concentrations were observed to precede significant stimulated accumulation of [3H]inositol monophosphate. New steady-state accumulations of several 3H-labelled products were achieved after 5-10 min of continued agonist stimulation, but were rapidly and effectively reversed by subsequent receptor blockade. The results show that muscarinic-receptor activation involves phosphoinositidase C-catalysed hydrolysis initially of polyphosphoinositides rather than of phosphatidylinositol. Furthermore, prolonged carbachol stimulation is shown not to cause receptor desensitization, but to allow persistent hydrolysis of [3H]phosphatidylinositol bisphosphate and permit sustained metabolic flux through the inositol tris-/tetrakis-phosphate pathway.

scholarly journals Stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine by endothelin, a complete mitogen for Rat-1 fibroblasts

1990 ◽  
Vol 272 (3) ◽  
pp. 761-766 ◽  
Author(s):  
E E MacNulty ◽  
R Plevin ◽  
M J O Wakelam
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Endothelin 1 ◽  
Kinase C ◽  
Choline Phosphate ◽  
Rapid Generation ◽  
Endothelin 3 ◽  
Hydrolysis Of ◽  
Phosphatidylcholine Synthesis ◽  
Stimulation Of

The mitogenic activity of endothelin and its ability to stimulate PtdIns(4,5)P2 and phosphatidylcholine turnover in Rat-1 fibroblasts was studied. Stimulated incorporation of [3H]thymidine occurred in the absence of any other added growth factors. The endothelins stimulated rapid generation of both Ins(1,4,5)P3 and choline. Endothelin-1 and endothelin-2 were equipotent in stimulating both responses, but endothelin-3 was less potent. Endothelin-1-stimulated Ins(1,4,5)P3 generation reached a maximum at 5 s and then declined; however, the response was long-lived, with a 4.5-fold elevation over basal still observed after 15 min. Endothelin-stimulated choline generation was observed with no increase in choline phosphate; indeed, the apparent level of this metabolite fell after 30 min of stimulation, presumably due to the observed stimulation of phosphatidylcholine synthesis. The endothelin-stimulated increase in choline generation was abolished in cells where protein kinase C was down-regulated. However, endothelin-stimulated choline generation was greater than that observed in response to a protein kinase C-activating phorbol ester, raising the possibility that the peptide activates phospholipase D by both protein kinase C-dependent and -independent mechanisms.

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 Phorbol ester and bryostatin differentially regulate the hydrolysis of phosphatidylethanolamine in Ha-ras- and raf-oncogene-transformed NIH 3T3 cells

1991 ◽  
Vol 276 (2) ◽  
pp. 505-509 ◽  
Author(s):  
Z Kiss ◽  
U R Rapp ◽  
G R Pettit ◽  
W B Anderson
Keyword(s):  
Phospholipase D ◽  
Second Messengers ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Intact Cells ◽  
Pkc Inhibitor ◽  
Kinase C ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

Previously it was reported that transformation of NIH 3T3 fibroblast by the Ha-ras, v-src, v-fms, and A-raf oncogenes decreased the stimulatory effects of phorbol 12-myristate 13-acetate (PMA; ‘TPA’), an activator of protein kinase C (PKC), on the phosphorylation of an endogenous 80 kDa substrate and on 86Rb uptake [Wolfman, Wingrove, Blackshear & Macara (1987) J. Biol. Chem. 262, 16546-16552], as well as on sphingomyelin synthesis [Kiss, Rapp & Anderson (1988) FEBS Lett. 240, 221-226]. Here, we investigated how transformation affects the PMA-stimulated hydrolysis of phosphatidylethanolamine (PtdEtn), a recently characterized mechanism which may contribute to the generation of the second messengers phosphatidic acid and 1,2-diacylglycerol. The effects of PMA were compared with those of bryostatin, a non-tumour-promoter activator of PKC. Transformation of NIH 3T3 cells with Ha-ras, v-raf, or A-raf enhanced the stimulatory effect of PMA on the phospholipase D-mediated hydrolysis of PtdEtn. On the other hand, the effects of bryostatin on PtdEtn hydrolysis were only slightly increased, if at all, in cells transformed with these oncogenes. In crude membrane preparations isolated from these transformed cells, PMA, but not bryostatin, enhanced the combined stimulatory effects of ATP and the GTP analogue guanosine 5′-[gamma-thio]triphosphate on phospholipase D-mediated PtdEtn hydrolysis. The PKC inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine inhibited the stimulatory effect of PMA only in intact cells. These results indicate that transformation of cells by certain oncogenes differentially affects phospholipase D-mediated hydrolysis of PtdEtn induced by PMA and bryostatin, suggesting that the action of PMA might involve two different mechanisms.

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