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 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.

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 Activation of P2z purinoceptors diminishes the muscarinic cholinergic-induced release of inositol 1,4,5-trisphosphate and stored calcium in rat parotid acini. ATP as a co-transmitter in the stimulus-secretion coupling

1995 ◽  
Vol 312 (2) ◽  
pp. 457-464 ◽  
Author(s):  
T D Jørgensen ◽  
J Gromada ◽  
K Tritsaris ◽  
B Nauntofte ◽  
S Dissing
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphate ◽  
Acinar Cells ◽  
Inhibitory Effect ◽  
Coupled Processes ◽  
Parotid Acinar Cells ◽  
Inositol Phosphate Production ◽  
Dependent Inhibition ◽  
Muscarinic Cholinergic ◽  
Rat Parotid

The effect of extracellular ATP on the intracellular free Ca2+ concentration ([Ca2+]i) and inositol phosphate production following stimulation with the muscarinic cholinergic agonist acetylcholine (ACh) was investigated in isolated rat parotid acinar cells. Stimulation of rat parotid acinar cells with ATP4- results in a rise in [Ca2+]i that is due to influx of extracellular Ca2+ and mobilization of Ca2+ from intracellular stores. Stimulation with purinergic agonists revealed that both influx as well as Ca2+ release from intracellular stores was mediated through activation of P2z receptors. The Ca2+ mobilization from intracellular stores was due to production of Ins(1,4,5)P3 and was inhibited by U73122, an inhibitor of phospholipase C-coupled processes. Under Ca(2+)-free conditions ATP4- caused a dose-dependent inhibition (IC50 = 8 microM) of the ACh-evoked Ca2+ release. The inhibitory effect of ATP4- is due to activation of the P2z purinoceptors, which results in a strong reduction in the ACh-induced inositol phosphate production. Prestimulation with 100 microM ATP4- reduced the amount of Ins(1,4,5)P3 formed after maximal ACh stimulation by 91%. In conclusion, the inhibitory effect of ATP4- on the ACh-mediated response is due to interactions of the activated P2z receptor with the phospholipase C-coupled processes underlying the muscarinic cholinergic response.

scholarly journals Differences in the regulation of endothelin-1- and lysophosphatidic-acid-stimulated Ins(1,4,5)P3 formation in rat-1 fibroblasts

1991 ◽  
Vol 280 (3) ◽  
pp. 609-615 ◽  
Author(s):  
R Plevin ◽  
E E MacNulty ◽  
S Palmer ◽  
M J O Wakelam
Keyword(s):  
Phospholipase C ◽  
Lysophosphatidic Acid ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Regulatory Protein ◽  
Permeabilized Cells ◽  
Endothelin 1 ◽  
Kinase C ◽  
Guanine Nucleotide Binding ◽  
Dose Dependent

Endothelin-1 (ET-1)- and lysophosphatidic acid (LPA)-stimulated PtdIns(4,5)P2 hydrolysis has been studied in Rat-1 fibroblasts. Although both agonists caused the dose-dependent accumulation of inositol phosphates, a number of differences were observed. LPA induced a transient increase in Ins(1,4,5)P3 mass which returned to basal levels within 90 s, whereas the response to ET-1 did not desensitize, with levels remaining at 3-4 times basal values for up to 15 min. Stimulated decreases in mass levels of PtdIns(4,5)P2 mirrored Ins(1,4,5)P3 formation for both agonists. Experiments with electropermeabilized cells demonstrated that the effects of both agonists are stimulated by a phospholipase C controlled by a guanine-nucleotide-binding regulatory protein; however, there are differences in the nature of these interactions. The inositol phosphate response to ET-1 is poorly potentiated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and markedly inhibited by guanosine 5′-[beta-thio]diphosphate (GDP[S]), whereas that to LPA is potentiated by GTP[S] but is relatively insensitive to GDP[S]. In addition, LPA decreased the lag time for the onset of GTP[S]-stimulated [3H]InsP3 accumulation, whereas ET-1 was without effect. Phorbol 12-myristate 13-acetate treatment of the cells inhibited LPA-stimulated, but not ET-1-stimulated, inositol phosphate formation in both intact and permeabilized cells, suggesting that the site of protein kinase C-mediated phosphorylation may be blocked in ET-1-stimulated Rat-1 cells. The results indicate that the receptor-G-protein-phospholipase C interaction for the two agonists may not conform to the same model.

scholarly journals Intracellular cross-talk between thyrotropin receptor and A1 adenosine receptor in regulation of phospholipase C and adenylate cyclase in COS-7 cells transfected with their receptor genes

1995 ◽  
Vol 306 (3) ◽  
pp. 709-715 ◽  
Author(s):  
F Okajima ◽  
H Tomura ◽  
K Sho ◽  
M Akbar ◽  
M A Majid ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Adenylate Cyclase ◽  
Phospholipase C ◽  
Inositol Phosphate ◽  
Term Treatment ◽  
Camp Accumulation ◽  
Thyrotropin Receptor ◽  
A1 Adenosine Receptor ◽  
Kinase C ◽  
Inositol Phosphate Production

COS-7 cells were transiently transfected with human thyrotropin receptor (TSHR) and dog A1 adenosine receptor (A1R) cDNA. TSH stimulated both inositol phosphate production and cyclic AMP (cAMP) accumulation in the cells. An A1 agonist, N6-(L-2-phenylisopropyl)adenosine (PIA), which is ineffective alone, significantly enhanced TSH-induced inositol phosphate production, but insignificantly inhibited TSH-induced cAMP accumulation was revealed by short-term treatment with the protein kinase C inhibitors, staurosporine and K252a, or long-term treatment with 12-myristate 13-acetate, suggesting that endogenous protein kinase C inhibits the A1R-mediated inhibition of the TSHR-adenylate cyclase system. In staurosporine-treated cells, the stimulatory and inhibitory permissive actions of PIA on TSH-induced phospholipase C and adenylate cyclase activation respectively were completely reversed by pretreatment with pertussis toxin whereas intrinsic TSH-induced effects were hardly affected by the toxin. The cross-talk between the signalling pathway for TSHR and that for A1R was not detected in a mixture of cells expressing either TSHR or A1R. We conclude that a single species of A1R, via pertussis-toxin-sensitive GTP-binding proteins, not only inhibits adenylate cyclase but also stimulates phospholipase C in collaboration with an activated TSHR within a single cell expressing both types of receptor.

Ionomycin-stimulated phasic myometrial contractions

1995 ◽  
Vol 269 (4) ◽  
pp. E779-E785
Author(s):  
M. Phillippe ◽  
E. M. Chien ◽  
M. Freij ◽  
T. Saunders
Keyword(s):  
Phospholipase C ◽  
Calcium Release ◽  
Calcium Influx ◽  
Inositol Phosphate ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Calcium Oscillations ◽  
Production Studies ◽  
Inositol Phosphate Production

Ionomycin, a calcium ionophore, facilitates the sustained entry of extracellular calcium; however, in myometrial tissue it stimulates phasic contractions. This study sought to define further this unanticipated effect of ionomycin and to begin to explore the possible mechanism(s) involved. Utilizing rat uterine strips, in vitro isometric contraction studies were performed to determine the effects of ionomycin with and without membrane-permeant inhibitors of cytosolic calcium oscillations. To determine the effects of ionomycin on phospholipase C, qualitative inositol phosphate production studies were performed. The in vitro contraction studies confirmed that ionomycin-stimulated phasic myometrial contractions were potentially dependent on stimulation of phospholipase C, calcium-induced calcium release, and additional calcium influx through dihydropyridine-sensitive membrane calcium channels. The inositol phosphate production studies confirmed that ionomycin stimulated phospholipase C in a dose-related fashion to levels comparable to oxytocin. In summary, these observations have confirmed the ability of ionomycin to generate dose-related phasic myometrial contractions through mechanisms potentially involving the phosphatidylinositol-signaling pathway.

Acute effects of phorbol esters on receptor-mediated IP3, cAMP, and progesterone levels in rat granulosa cells

1989 ◽  
Vol 256 (3) ◽  
pp. E368-E374
Author(s):  
J. S. Davis ◽  
L. L. Weakland ◽  
R. G. Coffey ◽  
L. A. West
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Granulosa Cells ◽  
Phorbol Esters ◽  
Inositol Phosphate ◽  
Inhibitory Effect ◽  
Inhibitory Effects ◽  
Kinase C ◽  
Inositol Phospholipid

Luteinizing hormone (LH) stimulates the formation of adenosine 3',5'-cyclic monophosphate (cAMP) and inositol trisphosphate (IP3) in rat granulosa cells. This report describes the effects of protein kinase C activators on second messenger generation in isolated rat granulosa cells. The protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) completely inhibited LH-stimulated inositol phosphate accumulation. The inhibitory effects of TPA were rapid (5-15 min) and concentration dependent with 50 nM TPA producing maximally inhibitory effects. However 30-min incubations with 10-100 nM TPA had no effect on LH-stimulated cAMP or progesterone levels. The inhibitory effect of TPA could not be overcome by high concentrations of LH. TPA also inhibited gonadotropin-releasing hormone-stimulated phospholipase C activity, although to a much lesser extent. Increased inositol phosphate degradation and reduced inositol phospholipid synthesis were unlikely explanations for the effects of TPA. The results indicate that phorbol esters modulate the inositol phospholipid-phospholipase C transmembrane signaling system in rat granulosa cells. The results suggest that phorbol esters may alter the coupling of the hormone receptor complex to phospholipase C.

scholarly journals Formation of inositol phosphate isomers in GH3 pituitary tumour cells stimulated with thyrotropin-releasing hormone. Acute effects of lithium ions

1987 ◽  
Vol 248 (2) ◽  
pp. 463-470 ◽  
Author(s):  
P J Hughes ◽  
A H Drummond
Keyword(s):  
Inositol Phosphate ◽  
Cell Types ◽  
Pituitary Tumour ◽  
Thyrotropin Releasing Hormone ◽  
Gh3 Cells ◽  
Acute Effects ◽  
Releasing Hormone ◽  
Inositol Phosphate Metabolism ◽  
Trh Stimulation ◽  
Gh3 Cell

With a h.p.l.c. system, the inositol mono-, bis- and tris-phosphate isomers found in [3H]inositol-labelled GH3 cells were resolved and identified. These cells possess at least ten distinct [3H]inositol-containing substances when acid-soluble extracts are analysed by anion-exchange h.p.l.c. These substances were identified by their co-elution with known inositol phosphate standards and, to a limited extent, by examining their chemical structure. Two major inositol monophosphate (InsP) isomers were identified, namely Ins1P and Ins4P, both of which accumulate after stimulation with the hypothalamic releasing factor (TRH) (thyrotropin-releasing hormone). Three inositol bisphosphate (InsP2) isomers were resolved, of which two were positively identified, i.e. Ins(1,4)P2 and Ins(3,4)P2. TRH treatment increases both of these isomers, with Ins(1,4)P2 being produced at a faster rate than Ins(3,4)P2. The third InsP2 isomer has yet to be fully identified, although it is co-eluted with an Ins(4,5)P2 standard. This third InsP2 is also increased after TRH stimulation. In common with other cell types, the GH3 cell contains two inositol trisphosphate (InsP3) isomers: Ins(1,4,5)P3, which accumulates rapidly, and Ins(1,3,4)P3, which is formed more slowly. The latter substance appears simultaneously with its precursor, inositol 1,3,4,5-tetrakisphosphate. We also examined the effects of acute Li+ treatment on the rates of accumulation of these isomers, and demonstrated that Li+ augments TRH-mediated accumulation of Ins1P, Ins4P, Ins(1,4)P2, the presumed Ins(4,5)P2 and Ins(1,3,4)P3. These results suggest that the effects of Li+ on inositol phosphate metabolism are more complex than was originally envisaged, and support work carried out by less sophisticated chromatographic analysis.

Potassium chloride effects on the hormonal signal transduction mechanisms underlying phasic myometrial contractions

1995 ◽  
Vol 146 (3) ◽  
pp. 485-493 ◽  
Author(s):  
M Phillippe ◽  
E K Chien
Keyword(s):  
Signal Transduction ◽  
Potassium Chloride ◽  
Phospholipase C ◽  
Inositol Phosphate ◽  
Cytosolic Calcium ◽  
Calcium Oscillation ◽  
Uterine Tissue ◽  
Production Studies ◽  
Inositol Phosphate Production ◽  
Transduction Mechanisms

Abstract These studies sought to test the hypothesis that potassium-stimulated phasic myometrial contractions utilize cytosolic calcium oscillation-like mechanisms comparable to those activated in response to oxytocin. Uterine tissue was obtained from pro-oestrus/oestrus Sprague-Dawley rats. In vitro isometric contraction studies were performed using longitudinal myometrial strips; computer digitalized contraction data were analyzed for contraction area, and normalized for tissue cross-section area. Dose–response studies were performed using potassium chloride with and without inhibitors of cytosolic calcium oscillation mechanisms. Qualitative inositol-phosphate production studies were performed after preloading uterine tissue with [3H]inositol; subsequently, the individual inositol-phosphates produced in response to stimulation were isolated by anion exchange chromatography. Potassium chloride over a concentration of 10 to 30 mm produced a dose-related increase in phasic contractile activity. The potassium-stimulated phasic contractions were significantly suppressed in response to inhibition of phospholipase C, stimulation of protein kinase C, inhibition of calcium-induced calcium release, and prevention of extracellular calcium influx. The qualitative inositol-phosphate production studies confirmed activation of phospholipase C in response to 20 mm potassium. These studies have provided support for the hypothesis that potassium-stimulated phasic myometrial contractions activate intracellular signal transduction mechanisms comparable to those activated in response to hormonal uterotonic agonists. Journal of Endocrinology (1995) 146, 485–493

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