scholarly journals A novel tumour promoter, thapsigargin, transiently increases cytoplasmic free Ca2+ without generation of inositol phosphates in NG115-401L neuronal cells

1988 ◽  
Vol 253 (1) ◽  
pp. 81-86 ◽  
Author(s):  
T R Jackson ◽  
S I Patterson ◽  
O Thastrup ◽  
M R Hanley
Keyword(s):  
Inositol Phosphates ◽  
Fluorescent Indicator ◽  
Intracellular Store ◽  
Non Invasive ◽  
Kinase C ◽  
Tumour Promoters ◽  
20 Nm ◽  
Indicator Dye ◽  
Hydrolysis Of ◽  
Stimulation Of

Thapsigargin, a sesquiterpene lactone with potent irritant and tumour-promoting activities, stimulates a rapid (within 15 s) transient increase in intracellular [Ca2+] in the NG115-401L neural cell line, as measured by the fluorescent indicator dye fura-2. This increase in cytoplasmic free [Ca2+] is concentration-dependent (ED50 around 20 nM) and occurs in the absence of extracellular Ca2+. Activation of NG115-401L cells by the inflammatory peptide bradykinin generates inositol phosphates, which parallel increases in intracellular [Ca2+]. However, the rise in cytoplasmic [Ca2+] stimulated by thapsigargin occurs in the absence of detectable production of inositol phosphates. Thapsigargin is unlike phorboid tumour promoters in that it has no action on two non-invasive indicators of phorbol stimulation of these cells, i.e. [3H]choline metabolite production and rise in intracellular pH. These data suggest that thapsigargin releases Ca2+ from an intracellular store by a novel mechanism, independent of the hydrolysis of phosphoinositides and concomitant activation of protein kinase C. Thus thapsigargin may provide a valuable tool for the analysis of intracellular signalling mechanisms.

scholarly journals Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen

1986 ◽  
Vol 235 (3) ◽  
pp. 869-877 ◽  
Author(s):  
W K Pollock ◽  
T J Rink ◽  
R F Irvine
Keyword(s):  
Fold Increase ◽  
Human Platelets ◽  
Free Calcium ◽  
Fura 2 ◽  
Kinase C ◽  
20 Nm ◽  
Indicator Dye ◽  
Arachidonate Release ◽  
Instantaneous Concentration ◽  
Stimulation Of

Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.

scholarly journals Bombesin and platelet-derived growth factor stimulate formation of inositol phosphates and Ca2+ mobilization in Swiss 3T3 cells by different mechanisms

1989 ◽  
Vol 258 (1) ◽  
pp. 177-185 ◽  
Author(s):  
D M Blakeley ◽  
A N Corps ◽  
K D Brown
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Inositol Phosphates ◽  
Platelet Derived Growth Factor ◽  
3T3 Cells ◽  
Kinase C ◽  
Swiss 3T3 ◽  
Stimulation Of

Highly purified platelet-derived growth factor (PDGF) or recombinant PDGF stimulate DNA synthesis in quiescent Swiss 3T3 cells. The dose-response curves for the natural and recombinant factors were similar, with half-maximal responses at 2-3 ng/ml and maximal responses at approx. 10 ng/ml. Over this dose range, both natural and recombinant PDGF stimulated a pronounced accumulation of [3H]inositol phosphates in cells labelled for 72 h with [3H]inositol. In addition, mitogenic concentrations of PDGF stimulated the release of 45Ca2+ from cells prelabelled with the radioisotope. However, in comparison with the response to the peptide mitogens bombesin and vasopressin, a pronounced lag was evident in both the generation of inositol phosphates and the stimulation of 45Ca2+ efflux in response to PDGF. Furthermore, although the bombesin-stimulated efflux of 45Ca2+ was independent of extracellular Ca2+, the PDGF-stimulated efflux was markedly inhibited by chelation of external Ca2+ by using EGTA. Neither the stimulation of formation of inositol phosphates nor the stimulation of 45Ca2+ efflux in response to PDGF were affected by tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast, TPA inhibited phosphoinositide hydrolysis and 45Ca2+ efflux stimulated by either bombesin or vasopressin. Furthermore, whereas formation of inositol phosphates in response to both vasopressin and bombesin was increased in cells in which protein kinase C had been down-modulated by prolonged exposure to phorbol esters, the response to PDGF was decreased in these cells. These results suggest that, in Swiss 3T3 cells, PDGF receptors are coupled to phosphoinositidase activation by a mechanism that does not exhibit protein kinase C-mediated negative-feedback control and which appears to be fundamentally different from the coupling mechanism utilized by the receptors for bombesin and vasopressin.

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 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 Stimulation of generation of inositol phosphates by carbamoylcholine and its inhibition by phorbol esters and iodide in dog thyroid cells

1989 ◽  
Vol 263 (3) ◽  
pp. 795-801 ◽  
Author(s):  
E Laurent ◽  
J Mockel ◽  
K Takazawa ◽  
C Erneux ◽  
J E Dumont
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Cholera Toxin ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Cholinergic Stimulation ◽  
Kinase C ◽  
Gtp Binding ◽  
Stimulation Of

The action of carbamoylcholine (Cchol), NaF and other agonists on the generation of inositol phosphates (IPs) was studied in dog thyroid slices prelabelled with myo-[2-3H]inositol. The stimulation by Cchol (0.1 microM-0.1 mM) of IPs accumulation through activation of a muscarinic receptor [Graff, Mockel, Laurent, Erneux & Dumont (1987) FEBS Lett. 210, 204-210] was pertussis- and cholera-toxin insensitive. Ins(1,4,5)P3, Ins(1,3,4)P3 and InsP4 were generated. NaF (5-20 mM) also increased IPs generation (Graff et al., 1987); this effect was potentiated by AlCl3 (10 microM) and unaffected by pertussis toxin. Although phorbol dibutyrate (5 microM) abolished the cholinergic stimulation of IPs generation (Graff et al., 1987), it did not affect the fluoride-induced response. Cchol and NaF did not require extracellular Ca2+ to exert their effect, and neither KCl-induced membrane depolarization nor ionophore A23187 (10 microM) had any influence on basal IPs levels, or on cholinergic stimulation. However, more stringent Ca2+ depletion with EGTA (0.1 or 1 mM) decreased basal IPs levels as well as the amplitude of the stimulation by Cchol without abolishing it. Dibutyryl cyclic AMP, forskolin, cholera toxin and prostaglandin E1 had no effect on basal IPs levels and did not decrease the response to Cchol. Iodide (4 or 40 microM) also strongly decreased the cholinergic action on IPs, this inhibition being relieved by methimazole (1 mM). Our data suggest that Cchol activates a phospholipase C hydrolysing PtdIns(4,5)P2 in the dog thyroid cell in a cyclic AMP-independent manner. This activation requires no extracellular Ca2+ and depends on a GTP-binding protein insensitive to both cholera toxin and requires no extracellular Ca2+ and depends on a GTP-binding protein insensitive to both cholera toxin and pertussis toxin. The data are consistent with a rapid metabolism of Ins(1,4,5)P3 to Ins(1,3,4)P3 via the Ins(1,4,5)P3 3-kinase pathway, followed by dephosphorylation by a 5-phosphomonoesterase. Indeed, a Ca2+-sensitive InsP3 3-kinase activity was demonstrated in tissue homogenate. Stimulation of protein kinase C and an organified form of iodine inhibit the Cchol-induced IPs generation. The negative feedback of activated protein kinase C could be exerted at the level of the receptor or of the receptor-G-protein interaction.

Endothelin stimulates phosphatidylcholine hydrolysis through both PLC and PLD pathways in mesangial cells

1994 ◽  
Vol 266 (6) ◽  
pp. F957-F965 ◽  
Author(s):  
E. Baldi ◽  
A. Musial ◽  
M. Kester
Keyword(s):  
Mesangial Cells ◽  
Tetraacetic Acid ◽  
Myristate Acetate ◽  
Inositol Polyphosphates ◽  
Glomerular Function ◽  
Kinase C ◽  
Multiple Signaling ◽  
Dose Dependent ◽  
Hydrolysis Of ◽  
Stimulation Of

Endothelin (ET) is a recently characterized vasoconstrictor hormone that has potent effects on glomerular function. Many vasoconstrictors, like ET, that stimulate phospholipase C (PLC) hydrolysis of polyphosphoinositides also stimulate phosphatidylcholine (PtdCho) hydrolysis via both PLC and phospholipase D (PLD) pathways. We have previously reported that ET stimulates a protein kinase C (PKC)-regulated, intracellular calcium-insensitive PLD activity that forms phosphatidic acid (PA) in rat mesangial cells (MC). We now ask whether ET-induced diglyceride (DG) production is also, in part, a result of either PLC- or PLD-induced hydrolysis of PtdCho. ET induced both a time- and dose-dependent stimulation in DG as measured by radioflux and mass assays. ET-stimulated DG production was still elevated even at time points where inositol polyphosphates had returned to basal levels. In addition, using [3H]choline-labeled cells, ET stimulated [3H]phosphocholine accumulation, suggesting a PLC-mediated hydrolysis of PtdCho. Stimulation of DG was unaffected by the presence of ethanol or propranolol, suggesting that ET-stimulated DG were not a result of a sequential PLD/PA phosphohydrolase activity. We further dissociated PtdCho-dependent PLC and PLD activities because, in contrast to ET-induced stimulation of PLD, the effect of ET on DG formation was mimicked with ionomycin and was inhibited with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid but not ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ET stimulation of DG could not be mimicked by phorbol myristate acetate and was not blocked by PKC inhibition or depletion. Together, these data suggest that ET stimulates multiple signaling pathways in MC that hydrolyze PtdCho via separate PLC and PLD mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Phorbol 12-myristate 13-acetate activates an electrogenic H+-conducting pathway in the membrane of neutrophils

1992 ◽  
Vol 281 (3) ◽  
pp. 697-701 ◽  
Author(s):  
A Kapus ◽  
K Szászi ◽  
E Ligeti
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nadph Oxidase ◽  
Phorbol Esters ◽  
Charge Compensation ◽  
Kinase C ◽  
Low Concentrations ◽  
Conducting Pathway ◽  
20 Nm ◽  
Stimulation Of

The mode of activation of an H(+)-conducting pathway present in the membrane of neutrophils was investigated. (1) Resting neutrophils released protons through an electrogenic Cd(2+)-inhibitable (K0.5 approximately 20 microM) route when a pH gradient and appropriate charge compensation was provided. (2) The rate of H+ efflux was stimulated over 2.5-fold by 4 beta-phorbol 12-myristate 13-acetate (PMA; K0.5 approximately 0.7 nM) or by 4 beta-phorbol 12,13-dibutyrate (K0.5 approximately 20 nM) even when the NADPH oxidase was blocked by p-chloromercuribenzoate. (3) Staurosporine inhibited the effect of PMA. (4) The H+ egress was not enhanced by 4 alpha-phorbol 12,13-didecanoate. (5) Low concentrations of Cd2+ (less than 40 microM) inhibited the H+ flux without influencing the oxidase. The results raise the possibility that protein kinase C could be involved in the activation of an electrogenic H(+)-conducting pathway in the membrane of neutrophils. The activation of this route by phorbol esters seems to be independent of the stimulation of NADPH oxidase.

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 Cellular mechanisms of adrenaline-induced hyperpolarization in renal epitheloid MDCK cells

1991 ◽  
Vol 274 (1) ◽  
pp. 243-248 ◽  
Author(s):  
J Pfeilschifter ◽  
M Paulmichl ◽  
E Wöll ◽  
R Paulmichl ◽  
F Lang
Keyword(s):  
Cell Membrane ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Mdck Cells ◽  
Extracellular Fluid ◽  
Potential Difference ◽  
Cellular Mechanisms ◽  
Kinase C ◽  
Stimulation Of ◽  
In Cells

The effects of adrenaline on the potential difference across the cell membrane, on formation of inositol phosphates and on intracellular Ca2+ ([Ca2+]i) were analysed in cells without or with pretreatment with pertussis toxin or phorbol 12-myristate 13-acetate (PMA). In untreated cells, adrenaline leads to a sustained hyperpolarization, a stimulation of Ins(1,4,5)P3 and Ins(1,3,4,5,)P4 formation and a transient increase in [Ca2+]i from 78 +/- 7 to 555 +/- 43 nM, followed by a plateau of 260 +/- 23 microM. In the absence of extracellular Ca2+ the effect of adrenaline on both potential difference and [Ca2+]i is transient. In cells pretreated with pertussis toxin, the effects of adrenaline on InsP3 and [Ca2+]i are still preserved, but the effect on potential difference is transient. In cells pretreated with PMA, the effect of adrenaline on InsP3 formation is severely decreased and that on [Ca2+]i abolished, whereas a transient hyperpolarizing effect is still present. This transient hyperpolarization is abolished by additional pretreatment with pertussis toxin. The observations suggest that adrenaline hyperpolarizes the cell membrane of MDCK cells by several distinct mechanisms. First, adrenaline stimulates the formation of InsP3 and InsP4, which at least in part accounts for the release of intracellular Ca2+ and the entry of Ca2+ from the extracellular fluid. Stimulation of phospholipase C is not mediated by pertussis-toxin-sensitive G-proteins, but apparently is inhibited by activation of protein kinase C. Second, adrenaline hyperpolarizes the cell membrane by a mechanism independent from increase in [Ca2+]i which is sensitive to pertussis toxin but is, at least in part, insensitive to PMA.

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