Stimulation Of Phosphoinositide Breakdown Research Articles

The Pharmacology of Physostigmine

The Pharmacology of Physostigmine

6beta-Acetoxynortropane: a potent muscarinic agonist with apparent selectivity toward M2-receptors.

A series of tropane derivatives, related in structure to baogongteng A (1), an alkaloid from a Chinese herb, were synthesized. 6beta-Acetoxynortropane (5) had weak affinity (Ki 22 microM) for central (M1) muscarinic receptors in a [3H]quinuclidinyl benzilate binding assay but had extremely high affinity (Ki 2.6 nM) and selectivity for M2-muscarinic receptors expressed in CHO cells. It had 13-fold lower affinity for M4-receptors, 260-fold lower affinity for M3-receptors, and 8200-fold lower affinity for M1-receptors expressed in CHO cells. The 6beta-carbomethoxy analogue (14) of baogongteng A had only weak affinity for M2-muscarinic receptors, as did 6beta-carbomethoxynortropane (13) and 6beta-acetoxytropane (4). In transfected CHO cells, 6beta-acetoxynortropane (5) was an agonist at M2-receptors, based on a GTP-elicited decrease in affinity, and a full agonist with an IC50 of 11 nM at M4-receptors, based on inhibition of cyclic AMP accumulation, while being a full agonist at M1-receptors with an EC50 of 23 nM and a partial agonist at M3-receptors with an EC50 of 3.6 nM, based in both cases on stimulation of phosphoinositide breakdown. All of the 16 tropane derivatives had weak affinities for central alpha4beta2-nicotinic receptors with 6beta-carbomethoxynortropane (13) having the highest affinity, which was still 150-fold less than that of nicotine. 6beta-Acetoxynortropane (5) represents a potent muscarinic agonist with apparent selectivity toward M2-receptors.

Inhibition by heterologously-expressed P2Y2 nucleotide receptors of N-type calcium currents in rat sympathetic neurones.

The P2Y2 nucleotide receptor has previously been shown to stimulate phosphoinositide breakdown. We now show that, when P2Y2 receptors are heterologously expressed by cRNA injection into dissociated rat sympathetic neurones, activation of these receptors by uridine 5'-triphosphate (UTP) or adenosine 5'-triphosphate (ATP) inhibits the N-type voltage-gated calcium current by approximately 65%, with an IC50 of 0.5 microM. Thus, the same molecular species of nucleotide receptor can link to two different effector pathways.

Intra- vs extracellular calcium regulation of neurotransmitter-stimulated phosphoinositide breakdown

The dependence on Ca 2+ of basal, glutamate- and carbachol-stimulated phosphoinositide (PI) turnover was studied on 8-day old rat brain synaptoneurosomes. For that purpose, intracellular and extracellular Ca 2+ concentrations were buffered by bis-(α-aminophenoxy)-ethane- N,N,N′,N′-tetraacetic acid, in its tetra(acetoxymethyl)-ester form (BAPTA-AM) and in its free acid form (BAPTA), respectively. The effects of both forms of the calcium chelator intracellular and extracellular Ca 2+ buffering on intracellular and extracellular Ca 2+ concentration ([Ca 2+] i and [Ca 2+] e) were determined with fluorimetric assay using fura2, either in its acetoxymethyl ester form (fura2-AM) or in its free acid form. Intracellular chelation of Ca 2+ ions with BAPTA-AM induced a dose-dependent reduction of the [Ca 2+] i. Basal inositol phosphate (IP) formation was slightly affected by this [Ca 2+] i buffering, while glutamate and carbachol stimulations of PI hydrolysis were similarly diminished. Chelation of extracellular Ca 2+ ions with BAPTA produced a reduction of both [Ca 2+] e and [Ca 2+] i. Basal IP accumulation was maximally inhibited by 50%. The carbachol-induced PI hydrolysis was completely inhibited in the presence of 200 μM BAPTA, while a substantial residual glutamate-elicited IP response remained (40% of the control response). It is concluded that [Ca 2+] i of synaptoneurosomes is not critical for basal and neurotransmitter-stimulated IP formation, whilst [Ca 2+] e is critical. Glutamate may, in part, stimulate PI breakdown in a Ca 2+-insensitive way.

Activation of adenosine A2 receptors stimulates phosphoinositide metabolism in rat peripheral nerve.

The effects of adenosine analogues on phosphoinositide metabolism in rat sciatic nerve were examined. Sciatic nerve segments were prelabeled with [3H]-cytidine and incubated in the presence of LiCl and varying concentrations of adenosine analogues. The formation of [3H]cytidine monophosphate phosphatidic acid [3H]-CMP-PA) was determined as an index of phosphoinositide breakdown. Liponucleotide accumulation was elevated significantly in the presence of 5'-N-ethylcarbox-amidoadenosine (NECA), a nonselective analogue, and two different A2-selective analogues, N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine and 2-p-(2-carboxyethyl)phenethylamino-NECA (CGS 21680), but not by N6-cyclopentyladenosine, an A1-selective analogue. The stimulatory action of CGS 21680 was blocked by the A2-selective adenosine receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 1,3-dipropyl-7-methylxanthine. Inositol phosphate formation was also stimulated to a comparable degree by CGS 21680 and this response was antagonized by DMPX. Carbamylcholine, which was previously shown to stimulate phosphoinositide breakdown, also enhanced the accumulation of CMP-PA. When adenosine analogues and carbamylcholine were simultaneously present, their effects were additive. Taken together, these data suggest that an adenosine receptor, possibly of the A2 subtype, is coupled to enhanced phosphoinositide hydrolysis in peripheral nerve. However, adenosine-receptor activation does not appear to modulate phosphoinositide hydrolysis stimulated via muscarinic receptors.

The morphologic change of endothelial cells by ancrod-generated fibrin is triggered by αvβ3 integrin binding and the subsequent activation of a G-protein coupled phospholipase C

The mechanism of morphologic change of human cultured umbilical vein endothelial cells (HUVECs) caused by fibrin was investigated. Ancrod, a thrombin-like enzyme, did not cause morphologic alteration of HUVEC by itself at concentrations ranging from 0.01 to 10 U/ml. However, when 0.02 U/ml of ancrod was added to cultured HUVEC monolayers in the presence of citrated plasma, it caused pronounced morphologic change of HUVEC after 6–10 h incubation period. Gly-Pro-Arg-Pro (4 mg/ml), an inhibitor of fibrin polymerization, prevented the morphologic alteration, indicating that the morphologic alteration was caused by the polymerized fibrin. The morphologic change of HUVEC caused by ancrod-generated fibrin was not observed in the presence of an intracellular calcium mobilization inhibitor TMB-8 (50 μM), and the morphologic alteration was also less pronounced with BAPTA(15 μM)-loaded HUVECs and HUVECs pretreated with EGTA (1.2 mM). Ancrod (in Medium 199) itself did not stimulate phosphoinositide breakdown of HUVEC. However, when ancrod was present in plasma, it caused an increase of [ 3H]IP 1 of HUVECs preloaded with [ 3H]myoinositol. This IP 1 increment was inhibited by Gly-Pro-Arg-Pro. The increase of IP 1 was significantly inhibited by the pretreatment of monoclonal antibodies 23C6 and 7E3 directed against α v β 3 integrin. Neomycin (1 mM) and pertussis toxin (100 ng/ml), but not aspirin or mepacrine, blocked this enhanced phosphoinositide breakdown. The morphologic change was also prevented by the monoclonal antibodies, 23C6 and 7E3. These results suggest that both intra- and extra-cellular calcium participate in the event of morphologic change of HUVEC caused by ancrod-generated fibrin, and the morphologic change is mediated, at least in part, by fibrin binding to integrin α v β 3 on HUVECs causing the subsequent activation of the endogenous G-protein coupled phospholipase C.

Maitotoxin-elicited calcium influx in cultured cells: Effect of calcium-channel blockers

Maitotoxin elicited a marked influx of 45Ca 2+ into NIH 3T3 fibroblast cells. The influx was blocked by imidazoles (econazole, miconazole, SKF 96365, clotrimazole, calmidazolium) with ic 50 values from 0.56 to 3 μM. Phenylalkylamines (verapamil, methoxyverapamil) and nitrendipine were less potent, and diltiazem was very weak. Among other calcium blockers, the diphenylbutylpiperidines fluspirilene and penfluridol, the diphenylpropylpiperidine loperamide, and the local anesthetic proadifen were quite active with ic 50 values of 2–4 μM. The pattern of inhibition of maitotoxin-elicited calcium influx did not correspond to the ability of the agents to block elevation of calcium that ensues through calcium-release activated calcium (CRAC) channels after activation of phosphoinositide breakdown by ATP in HL-60 cells. The imidazoles did block CRAC channels, but fluspirilene, penfluridol, loperamide and proadifen were ineffective. Loperamide actually appeared to enhance influx of calcium via the activated CRAC channels. The imidazoles, in particular calmidazolium, caused an apparent influx of calcium and caused a stimulation of phosphoinositide breakdown in HL-60 cells.

Carbachol, but not norepinephrine, NMDA, ionomycin, ouabain, or phorbol myristate acetate, increases inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices.

Ionomycin, a Ca2+ ionophore, stimulated phosphoinositide breakdown in rat brain cortical slices incubated in the presence of 1.2 mM Ca2+, but, unlike muscarinic cholinergic stimulation, it had little effect on inositol 1,3,4,5-tetrakisphosphate accumulation. However, at 2 min, the increase in inositol 1,4,5-trisphosphate due to 10 microM ionomycin was equivalent to that seen with 1 mM carbachol. Phorbol 12-myristate 13-acetate or high K+ (30 mM) increased inositol 1,4,5-trisphosphate, but not inositol 1,3,4,5-tetrakisphosphate accumulation. The stimulation of inositol 1,4,5-trisphosphate accumulation due to ionomycin, unlike that seen with carbachol, was abolished in buffer containing 0.2 mM Ca2+. The increase in inositol 1,3,4,5-tetrakisphosphate accumulation in brain slices due to 1 mM carbachol ranged from 55 to 68% of that for inositol 1,4,5-trisphosphate. Norepinephrine, NMDA, veratridine, and ouabain also increased inositol 1,4,5-trisphosphate, but had minimal effects on inositol 1,3,4,5-tetrakisphosphate accumulation. These results suggest that there is something unique about the stimulation of inositol 1,3,4,5-tetrakisphosphate accumulation by carbachol, which is also the only one of these agents that is able to activate phosphoinositidase C beta 1 in isolated rat brain membranes.

Effects of ATP and UTP in pheochromocytoma PC12 cells: evidence for the presence of three P2 receptors, only one of which subserves stimulation of norepinephrine release.

1. In pheochromocytoma PC12 cells ATP and, to a lesser extent, 2-methylthioATP stimulate phosphoinositide breakdown, release of intracellular calcium, and influx of external calcium, leading to stimulation of norepinephrine release. In contrast, although UTP also stimulates phosphoinositide breakdown, release of intracellular calcium, and influx of external calcium, there is no stimulation of norepinephrine release. 2. 2-MethylthioATP, presumably acting at P2y receptors, and UTP, presumably acting at P2u receptors, in combination elicit a phosphoinositide breakdown greater than that elicited by either alone. Intracellular levels of calcium measured with Fura-2 increase to greater levels with ATP than with UTP and are sustained, while the UTP intracellular levels of calcium rapidly return to basal values. Both ATP and UTP cause a similar influx of 45 Ca2+ presumably by stimulation of a P2 receptor directly linked to a cation channel. 3. It is proposed that PC12 cells contain two distinct G protein-coupled P2 receptors that activate phospholipase C and a P2 receptor linked to a cation channel. The P2y receptor sensitive to ATP (and to 2-methylthioATP) causes the depletion of a pool of intracellular calcium, sufficient to activate so-called "receptor-operated calcium entry". The sustained elevation of intracellular calcium after ATP treatment is proposed to result in stimulation of norepinephrine release and activation of calcium-dependent potassium channels and sodium-calcium exchange pathways. 4. The P2u receptor sensitive to UTP (and to ATP) causes only a transient elevation in levels of intracellular calcium, perhaps from a different pool, insufficient to activate so-called receptor-operated calcium entry. Further sequelae do not ensue, and the functional role of the UTP-sensitive P2u receptor is unknown.

Effects of tachykinins on phosphoinositide metaboism in the hypothalamus: is the NK1 receptor involved?

Substance P (SP) has been shown to stimulate the hydrolysis of inositol phospholipids in peripheral tissues and in the brain. In mammalian peripheral tissues, three tachykinin receptor subclasses, neurokinin 1 (NK1), neurokinin 2 (NK2) and neurokinin 3 (NK3), have been identified. The purpose of our study was to pharmacologically characterize the SP receptors in the hypothalamus using phosphoinositide breakdown as a functional response. SP, previously described as a NK1 agonist, and Neurokinin A (NKA), previously described as a NK2 agonist, stimulated phosphoinositide breakdown in the hypothalamus in a dose-dependent fashion, with SP being more potent than NKA. The NK2-selective antagonist L-659,877, at a dose of 10 −6 M, abolished the effect of SP (10 −8 M) without affecting basal phosphoinositide breakdown. However, this NK2-selective antagonist did not inhibit the NKA-induced stimulation on phosphoinositide metabolism. The NK-1-selective antagonist L-668,169 stimulated phosphoinositide metabolism at a concentration of 10 −6 M, but not at 10 −8 M. This NK1-receptor antagonist did not significantly inhibit the effect of SP on phosphoinositide metabolism. Spantide II, another NK1-selective antagonist, also stimulated phosphoinositide metabolism at a dose of 10 −6 M. Like L-668,169, spantide II failed to inhibit the SP-induced stimulation of phosphoinositide metabolism, and even potentiated the response to SP. The blockade of the SP-induced phosphoinositide breakdown by the NK2-receptor antagonist (L-659,877), the absence of blockade of the NKA-induced phosphoinositide metabolism by the NK2 receptor antagonist and the absence of inhibition by the NK1 receptor antagonists (L-688,169 and spantide II) suggest that the SP-induced phosphoinositide metabolism in the hypothalamus is mediated by a receptor subclass different from those mediating the peripheral actions of SP.

A new selective bioassay for tachykinin NK 3 receptors based on inositol monophosphate accumulation in the guinea pig ileum

The selective agonists of tachykinin NK 1, NK 2 and NK 3 receptors, respectively [Pro 9]substance P, [Lys 5,MeLeu 9,Nle 10]neurokinin A-(4–10) and senktide, stimulated phosphoinositide breakdown in slices of the guinea pig ileum. This was also the case with septide which has recently been found to act on a new type tachykinin receptors in this issue. The NK 1, NK 2 and septide-evoked responses were completely antagonized in the combined presence of (±)-CP-96,345 and MEN 10,376 which are potent and selective antagonists of tachykinin NK 1 and NK 2 receptors respectively in the guinea pig ileum. Like senktide, other available NK 3 receptor agonists, such as [MePhe 7]neurokinin B, [MeVal 7]neurokinin B, [Pro 7]neurokinin B and and DiMe-C7, stimulated phosphoinositide hydrolysis in either the absence or combined presence of (±)-CP-96,345 and MEN 10,376, although senktide was the most potent. Therefore, following the blockade of tachykinin NK 1, NK 2 and septide-sensitive receptors, the accumulation of inositol monophosphate appears to be a valuable, rapid and sensitive bioassay for determining the activity of NK 3 receptor agonists and putative NK 3 receptor antagonists.

Characterization of prostaglandin F2 alpha receptor of mouse 3T3 fibroblasts and its functional expression in Xenopus laevis oocytes.

Prostaglandin (PG) F2 alpha increased [3H]thymidine incorporation into quiescent NIH 3T3 cells, stimulated phosphoinositide breakdown, and raised intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner with ED50 values of 2.0 x 10(-8) M, 4.6 x 10(-8) M, and 7.5 x 10(-8) M, respectively. The increase in [3H]thymidine incorporation with PGF2 alpha was additive with that seen with epidermal growth factor (EGF) or insulin. The peak [Ca2+]i increase with PGF2 alpha was still obvious in the absence of extracellular Ca2+ and was insensitive to islet activating protein (IAP) pretreatment. Membranes prepared from NIH 3T3 cells exhibited a specific binding for PGF2 alpha, which was sensitive to GTP gamma S but not sensitive to IAP pretreatment. Xenopus laevis oocytes injected with NIH 3T3 cell mRNA between 18S and 28S rRNA fractionated by sucrose gradient, expressed a PGF2 alpha-specific Cl- current when examined by voltage clamp. This Cl- current was also insensitive to IAP pretreatment and not affected by extracellular Ca2+ concentration ([Ca2+]o). These results indicate 1) that the NIH 3T3 cells expressed a specific PGF2 alpha receptor which is linked to phosphoinositide-specific phospholipase C (PLC) activation and to mobilization of Ca2+ via an IAP-insensitive G-protein(s), 2) that this PGF2 alpha receptor may play an active role in the proliferation of NIH 3T3 cells, and 3) that this PGF2 alpha receptor can be expressed in the oocyte system.

Effect of thiol reagents on phosphoinositide hydrolysis in rat brain synaptoneurosomes.

Some divalent ions, such as Cd2+ and Zn2+, are able to stimulate phosphoinositide (PI) breakdown and to inhibit receptor-mediated PI metabolism. These ions are also known to react with the free -SH groups of proteins. This prompted us to investigate the effects of more potent sulphydryl reagents, Hg2+ and p-chloromercuric benzosulphonic acid (PCMBS), on the inositol phosphate (IP) accumulation triggered by the neuroactive substances: glutamate, carbachol and K+, using synaptoneurosomes from 8-day-old rat forebrains. Hg2+ and PCMBS, depending on their concentration, had two distinct effects on IP accumulation: at low doses, Hg2+ (from 1 to 10 microM) and PCMBS (0.1 mM) by themselves stimulated PI breakdown, inhibited glutamate-elicited IP accumulation and had additive effects with respect to carbachol-induced IP stimulation. At higher doses, Hg2+ (from 0.01 to 1 mM) inhibited both basal and neuroactive substance-stimulated IP accumulation. PCMBS (1 mM), provoked only an inhibition of the agonist-stimulated IP formation. Monitoring membrane potential and intracellular Ca2+ with the fluorescent dyes diSC2(5) and fura2, respectively, indicated that these mercurials could strongly depolarize the synaptoneurosomal membrane and produce a Ca2+ influx dependent on extracellular Ca2+. The stimulatory effects of low concentrations of mercurials on PI turnover could be linked to the depolarization they provoke and the subsequent Ca2+ rise, which in turn is known to stimulate some phospholipase C enzymes. The inhibitory effects observed at high concentrations might be due to a loss of activity of proteins involved in PI breakdown, as all receptor-mediated IP accumulations were inhibited.

A pertussis toxin-sensitive mechanism of endothelin action in porcine coronary artery smooth muscle.

1. Endothelin-1 (ET-1)-induced contraction of porcine coronary artery strips may be mediated via at least two intracellular signalling mechanisms, the activation of dihydropyridine-sensitive voltage-dependent Ca2+ channels and the stimulation of phosphoinositide breakdown. Here we have investigated the possible involvement of pertussis toxin (PT)-sensitive guanosine-5'-triphosphate (GTP)-binding proteins (G-proteins) in ET-1-induced activation of these two signalling pathways in porcine coronary artery smooth muscle. 2. Increase in extracellular K+ concentration (10, 15 mM) shifted the dose-response relationship for the ET-1-induced contraction to the left. 3. The dihydropyridine Ca2+ channel blocker, nifedipine (10(-8) M), induced a rightward shift in the dose-response curve for ET-1. Pretreatment of the arterial strips with PT (0.1 microgram ml-1) induced a similar rightward shift of the ET-1 dose-response curve but not of the KCl response. Nifedipine (10(-8) M) did not further attenuate the ET-1-induced contraction in the PT-pretreated strips. 4. The pretreatment with PT significantly reduced 45Ca2+ uptake of the arterial strips stimulated by ET-1, but had no effect on ET-1-induced production of inositol phosphates. 5. The contractile response of the arterial strips to phorbol dibutyrate, an active phorbol ester, was not significantly affected by 10(-8) M nifedipine. 6. We confirmed that the pretreatment of the tissue with PT induced ADP-ribosylation of a 41 kDa membrane protein. 7. These findings indicate that activation of dihydropyridine-sensitive voltage-dependent Ca2+ channels by ET-1 in this tissue is mediated via a PT-sensitive G-protein in a manner apparently independent of the ET-1-induced activation of protein kinase C. It is concluded that the action of ET-1 in porcine coronary artery is mediated via two distinct signal transduction pathways, which are coupled to PT-sensitive and PT-insensitive GTP-binding proteins, respectively.

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation.

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.

Effects of mastoparan and related peptides on phosphoinositide breakdown in HL-60 cells and cell-free preparations

In differentiated HL-60 cells the amphiphilic peptide mastoparan induces a dose-dependent stimulation of phosphoinositide breakdown with an EC 50 value of 9μM. Such stimulation can be markedly reduced by pretreatment of the cells with pertussis toxin (100 ng/ml, 2h). In membranes obtained from differentiated HL-60 cells, guanine nucleotides stimulate the formation of IP2 and IP3. Calcium ions also induce phosphoinositide breakdown in this preparation independent of the presence of guanine nucleotides. In HL-60 cell membranes, mastoparan inhibited GTPγS-stimulation of phosphoinositide breakdown with an IC 50 value of 3 μM. Such inhibitory activity of mastoparan also was present in membranes from cells pretreated with pertussis toxin. Calcium-induced stimulation of phosphoinositide breakdown was not significantly inhibited by mastoparan. The analogs mastoparan-X and polistes mastoparan had similar inhibitory activity, whereas the analog des-Ile 1-Asn 2-mastoparan was inactive. In permeabilized HL-60 cells mastoparan also inhibited phosphoinositide breakdown. Another amphiphilic peptide, melittin, was inactive in HL-60 intact cells, but similar to mastoparan, inhibited guanine nucleotide-induced phosphoinositide breakdown in HL-60 cell membranes and permeabilized cells. Thus, mastoparan peptides can stimulate phosphoinositide breakdown in intact HL-60 cells, probably through the interaction with a guanine nucleotide binding protein. In permeabilized cells an in cell membranes, mastoparan induces inhibition of guanine nucleotide-mediated phosphoinositide breakdown presumably through an interaction with an intracellular site. The inhibitory action of mastoparan and melittin is probably related to the amphiphilic character of these peptides.

Phosphoinositide breakdown is associated with Fc-gamma RII-mediated activation of 5'-lipoxygenase in murine eosinophils.

In this report we present data on the ability of murine eosinophils to generate inositol phosphate derivatives, and their relationship with the activation of 5'-lipoxygenase by a Fc-gamma R-dependent mechanism. The addition of anti-IgG F(ab')2 to mouse eosinophils, previously sensitized with IgG, induces inositol phosphate generation after 2 min and after 10 min of stimulation. Maximal generation of inositol tris and inositol tetrakis phosphate has been detected after 15 min of stimulation, and the optimal concentration of anti-IgG F(ab')2 was found to be 25 micrograms. Inositol tris phosphate formation is also observed at 5 min after the addition of the calcium ionophore A23187 (5 microM). We also report that neomycin, an inhibitor of phosphoinositide-phospholipase C, inhibits Fc-gamma R-mediated phosphoinositide breakdown in a dose-dependent manner (88% inhibition at 150 microM of neomycin). The possible involvement of phosphoinositide breakdown in the activation of 5'-lipoxygenase has been investigated. Using streptolysin-O permeabilized cells and different doses of neomycin that inhibit phosphoinositide breakdown, we have demonstrated a parallel decrease in LTC4 released by these cells, using either A23187 (86% inhibition at 200 microM of neomycin) or anti-IgG F(ab')2 (82.4% inhibition at 100 microM of neomycin). [Ca2+]i elevation has been observed by loading the cells with the fluorescent calcium indicator Fura-2 penta-acetoxy methyl ester and after stimulating with the anti-Fc-gamma RII mAb (2.4G2). It is likely that the activation of murine eosinophils by a Fc-gamma R mechanism stimulates phosphoinositide breakdown as a primary step that leads to the activation of murine 5'-lipoxygenase, producing the formation of leukotriene C4.

Alpha - 1 - stimulated phosphoinositide breakdown in cultured cardiomyocytes: Diacylglycerol production and composition in docosahexaenoic acid supplemented cells

The fatty acid pattern of phosphatidylinositol and other inositol phospholipids is reported to be predominantly 1-stearoyl, 2-arachidonyl. However, literature does not report data about the effect of a modification of this fatty acid composition on the production and acidic pattern of the diacylglycerol (DAG) formed during phosphoinositide hydrolysis. Culturing cardiomyocytes in a docosahexaenoic acid supplemented medium, we obtained an homogeneous cell population whose phospholipid fatty acid pattern was strongly different from control cells, and which produced, after α1-adrenergic stimulation with phenylephrine, an higher amount of DAG. This DAG was different from control DAG in fatty acid composition, too. This structurally different DAG could be responsible for a different activation pattern of protein kinase C.

Primary cultures of corticostriatal cells from newborn rats: A model to study muscarinic receptor subtypes regulation and function

In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small population of choline acetyltransferase (ChAT) immunoreactive neurons, corresponding to approximately 3% of the total cell number, and synthesizes increasing amounts of acetylcholine (ACh) from the third day in vitro (DIV), which reaches a plateau around the 10 day of culture. Muscarinic cholinergic receptors (mAChR), measured by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB), are detectable from the fifth DIV and increase linearly during the time of culture. At the twelfth DIV, the density of mAChRs (approximately 600 fmol/mg protein) is comparable to the density of mAChR in adult rat cortex. These receptors are coupled to second messenger systems, since muscarinic agonists inhibit adenylate cyclase activity and stimulate phosphoinositide breakdown with efficacies and potencies similar to those found in adult rat cortex. Moreover, by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, we were able to demonstrate the presence of the m1, m3, and m4 mAChR subtype mRNAs in this neuronal culture at 12 DIV. Our data suggest that corticostriatal neuronal cultures develop in vitro ACh-synthesizing neurons and functionally active cholinergic receptors. This therefore makes them ideally suited to study the development and properties of brain mAChR subtypes.

Agonist-induced desensitization of cholinergically stimulated phosphoinositide breakdown is independent of endogenously activated protein kinase C in HT-29 human colon carcinoma cells

Activation of M3 muscarinic receptors in HT-29 cells by carbachol rapidly increases polyphosphoinositide breakdown. Pretreatment of these cells with carbachol (0.1 mM) for 5 h completely inhibits the subsequent ability of carbachol to increase [3H]inositol monophosphate ([3H]InsP) accumulation, paralleled by a total loss of muscarinic binding sites. In contrast, protein kinase C (PK-C)-mediated desensitization by incubation with phorbol esters [PMA (phorbol 12-myristate 13-acetate)], leading to a time- and dose-dependent inhibition of cholinergically stimulated InsP release (95% inhibition after 4 h with 0.1 microM-PMA), is accompanied by only a 40% decrease in muscarinic receptor binding, which suggests an additional mechanism of negative-feedback control. Neither carbachol nor PMA pretreatment had any effect on receptor affinity. Incubation with carbachol for 15 min caused a small increase of membrane-associated PK-C activity (15% increase, P less than 0.05) as compared with the potency of phorbol esters (PMA) (3-4-fold increase, P less than 0.01). Long-term incubation (4-24 h) with PMA resulted in a complete down-regulation of cytosolic and particulate PK-C activity. Stimulation of InsP release by NaF (20 mM) was not affected after a pretreatment with phorbol esters or carbachol, demonstrating an intact function of G-protein and phospholipase-C (PL-C) at the effector side. Determination of PL-C activity in a liposomal system with [3H]PtdInsP2 as substrate, showed no change in PL-C activity after carbachol (13 h) and short-term PMA (2.5 h) pretreatment, whereas long-term preincubation with phorbol esters (13 h) caused a small but significant decrease in PL-C activity (19%, P less than 0.05). Our results indicate that agonist-induced desensitization of phosphoinositide turnover occurs predominantly at the receptor level, with a rapid loss of muscarinic receptors. Exogenous activation of PK-C by phorbol esters seems to dissociate the interaction between receptor and G-protein/PL-C, without major effects on total cellular PL-C activity.