The M2 muscarinic receptor antagonist methoctramine activates mast cells via pertussis toxin-sensitive G proteins

Methoctramine, a selective M₂ muscarinic cholinergic receptor antagonist, has been reported to activate phosphoinositide breakdown at high concentrations. Its polyamine structure suggests a putative activation of guanine nucleotide-binding proteins (G proteins). Incubation of methoctramine with rat peritoneal mast cells resulted in a dose-dependent noncytotoxic histamine release, with an EC₅₀ of 20μM and a maximum effect at 1mM. Atropine, pirenzepine and HHSiD neither inhibited methoctramine-induced histamine release nor stimulated histamine release. Histamine release and inositol phosphates generation induced by methoctramine were both inhibited by pertussis toxin pretreatment. Benzalkonium chloride, a selective inhibitor of histamine secretion induced by basic secretagogues, inhibited the secretory response to methoctramine. [p-Glu⁵, d-Trp^7,9,10]-SP_5–11 (GPAnt-2), a well-characterized antagonist of G proteins, blocked the methoctramine-induced histamine release when the antagonist was allowed to reach its intracellular target by streptolysin O-permeabilization. The response to methoctramine was prevented by the hydrolysis of sialic acid residues of the cell surface by neuraminidase. The response of mast cells was restored by permeabilization of the plasma membrane. These results demonstrate that methoctramine, following its entry into the cell and the involvement of pertussis toxin-sensitive G proteins, activates phosphoinositide hydrolysis leading to mast cell exocytosis. Received: 26 September 1997 / Accepted: 1 December 1997     

Iloprost: Intracellular Ca2+-dependent Contractile Effect on Isolated Smooth Muscle Cells from Guinea-pig Ileum

Prostaglandin E₂ (PGE₂) and iloprost induced a concentration-dependent contraction of smooth muscle cells isolated from the circular layer of guinea-pig ileum. PGE₂- and iloprost-induced contractions were inhibited by the selective EP₁-receptor antagonist, SCI9220 (1-acetyl-2-(8-chloro-10, 11-dihydrodibenz (b,f) (1,4) oxazepine-10-carbonyl)-hydrazine), indicating the involvement of the EP₁ subtype of the PGE₂ receptor. When cells were incubated for 10 min in the presence of strontium (4 mm L^?1), an inhibitor of the release of Ca²⁺ from intracellular store, the contractile effect of PGE₂ and iloprost was inhibited. In contrast, incubation of cells in Ca²⁺-free medium, Ca²⁺-free medium plus EGTA, or in the presence of nifedipine, an organic Ca²⁺-channel blocker, did not alter the PGE₂- and iloprost-induced contraction. These observations suggest that the myogenic effect of PGE₂ and iloprost on intestinal smooth muscle is dependent on the release of intracellular calcium.     

Intermolecular cross-talk between the prostaglandin E2 receptor (EP)3 subtype and thromboxane A2 receptor signalling in human erythroleukaemic cells

Background and purpose:

In previous studies investigating cross-talk of signalling between prostaglandin (PG)E₂ receptor (EP) and the TPα and TPβ isoforms of the human thromboxane (TX)A₂ receptor (TP), 17-phenyl trinor PGE₂-induced desensitization of TP receptor signalling through activation of the AH6809 and SC19220-sensitive EP₁ subtype of the EP receptor family, in a cell-specific manner. Here, we sought to further investigate that cross-talk in human erythroleukaemic (HEL) 92.1.7 cells.

Experimental approach:

Specificity of 17-phenyl trinor PGE₂ signalling and its possible cross-talk with signalling by TPα/TPβ receptors endogenously expressed in HEL cells was examined through assessment of agonist-induced inositol 1,4,5-trisphosphate (IP)₃ generation and intracellular calcium ([Ca²⁺]_i) mobilization.

Key results:

While 17-Phenyl trinor PGE₂ led to activation of phospholipase (PL)Cβ to yield increases in IP₃ generation and [Ca²⁺]_i, it did not desensitize but rather augmented that signalling in response to subsequent stimulation with the TXA₂ mimetic U46619. Furthermore, the augmentation was reciprocal. Signalling by 17-phenyl trinor PGE₂ was found to occur through AH6809- and SC19920-insensitive, Pertussis toxin-sensitive, G_i/G_βγ-dependent activation of PLCβ. Further pharmacological investigation using selective EP receptor subtype agonists and antagonists confirmed that 17-phenyl trinor PGE₂-mediated signalling and reciprocal cross-talk with the TP receptors occurred through the EP₃, rather than the EP₁, EP₂ or EP₄ receptor subtype in HEL cells.

Conclusions and Implications:

The EP₁ and EP₃ subtypes of the EP receptor family mediated intermolecular cross-talk to differentially regulate TP receptor-mediated signalling whereby activation of EP₁ receptors impaired or desensitized, while that of EP₃ receptors augmented signalling through TPα/TPβ receptors, in a cell type-specific manner.     

Prostaglandins inhibit secretion of histamine and pancreastatin from isolated rat stomach ECL cells

1. The present study examines the effect of naturally occurring prostanoids and prostaglandin (PG) congeners on gastrin- and pituitary adenylate cyclase-activating peptide (PACAP)-evoked histamine and pancreastatin secretion from isolated rat stomach ECL cells.
2. ECL cells (75–85% purity) were isolated from rat stomach using pronase digestion followed by repeated counter-flow elutriation and cultured for 48 h before secretion experiments. The release of histamine and pancreastatin was determined by radioimmunoassay.
3. None of the PGs tested stimulated the release of either histamine or pancreastatin.
4. PGE₁ and PGE₂ inhibited both gastrin- and PACAP-evoked histamine and pancreastatin secretion (IC₅₀=1–2×10⁻¹⁰ M). Most other naturally occuring prostanoids and PG congeners had no or little inhibitory effect. The PGE analogues misoprostol and sulprostone were more potent (IC₅₀=0.9×10⁻¹¹ M and 2×10⁻¹¹ M respectively) than PGE₁ and PGE₂. The rank order of potency was misoprostol>sulprostone>PGE₁=PGE₂, suggesting the involvement of the so-called EP₃ receptor.
5. The effects of PGs on the stomach ECL cells may be direct or indirect, for instance through the stimulated release of somatostatin from contaminating D cells (2–3%). However, the amount of somatostatin in the cell culture after 48 h was below the limit of detection, and somatostatin immunoneutralization did not prevent misoprostol from inhibiting secretion from the ECL cells.
6. The misoprostol-induced inhibition was reversed by pertussis toxin suggesting the involvement of G-protein subunits Gα₀ and/or Gα_i.
7. In view of the potency by which PGE₁, PGE₂, misoprostol and sulprostone inhibited the stimulated release of histamine and pancreastatin, we suggest that the ECL cells represent a primary target for prostaglandins acting via an EP₃ receptor in the oxyntic mucosa.
8. The results suggest that the clinically useful effect of misoprostol as an anti-ulcer drug reflects its ability to inhibit stomach ECL-cell histamine secretion.

     

Prostaglandins of the E series inhibit monoamine release via EP3 receptors: proof with the competitive EP3 receptor antagonist L-826,266

Prostaglandin E₂ (PGE₂) and its analogue sulprostone inhibit noradrenaline and serotonin release in rodent tissues. We examined whether the receptor involved is blocked by the EP₃ antagonist L-826,266, whether such receptors also occur on central cholinergic neurones and retinal dopaminergic cells, whether PGE₂ is produced by the degradation of the endocannabinoid virodhamine and whether EP₃ receptor activation stimulates ³⁵S-GTPγS binding. Transmitter release was studied as electrically evoked tritium overflow in superfused tissues preincubated with ³H-noradrenaline (which in the guinea pig retina labels dopaminergic cells), ³H-serotonin or ³H-choline. ³⁵S-GTPγS binding, a measure of G protein activation, was studied in mouse and guinea pig hippocampal membranes. L-826,266 antagonised the effect of sulprostone on noradrenaline release in the rat cortex, yielding a Schild plot-based pA₂ value of 7.56. Apparent pA₂ values in mouse cortex and rat vas deferens (noradrenaline release) and rat cortex (serotonin release) were 7.55, 7.87 and 7.67, respectively. PGE₂ did not affect acetylcholine release in rat brain and dopamine release in guinea pig retina. In seven mice tissues, noradrenaline release was inhibited by sulprostone but not affected by virodhamine. ³⁵S-GTPγS binding was not altered by sulprostone but stimulated by the cannabinoid agonist WIN 55,212-2. Prostaglandins of the E series inhibit monoamine release via EP₃ receptors at which L-826,266 is a competitive antagonist. EP₃ receptors that inhibit transmitter release are not present on central cholinergic neurones and retinal dopaminergic cells. Virodhamine is not converted to PGE₂. An EP₃ receptor model based on ³⁵S-GTPγS binding could not be identified.     

Effects of ATP on Phosphoinositide Hydrolysis and Prostaglandin E2 Generation in Rabbit Astrocytes

Extracellular ATP secreted from stimulated nerves plays a role in neurotransmission. This study examined the effects of extracellular ATP on phospholipase A₂ and C signalling pathways in rabbit astrocytes. ATP caused prostaglandin E₂ (PGE₂) generation and phosphoinositide hydrolysis in a time- and concentration-dependent manner. A P_2y purinoceptor-selective agonist, 2-methylthio-ATP also caused phosphoinositide hydrolysis, but not PGE₂ generation. A P_2x purinoceptor-selective agonist, α,β-methylene-ATP did not cause either phosphoinositide hydrolysis or PGE₂ generation. Although pertussis toxin had no effect on 2-methylthio-ATP-induced phosphoinositide hydrolysis, it markedly decreased ATP-induced PGE₂ generation, with significant inhibition of phosphoinositide hydrolysis. Dexamethasone and indomethacin which potently inhibited ATP-induced PGE₂ generation, caused partial inhibition of phosphoinositide hydrolysis, suggesting that pertussis toxin-sensitive component of ATP-induced phospholipase C activation is mediated by cyclo-oxygenase metabolites of arachidonic acid. These results suggest that a stimulation of P_2y receptor results in phospholipase C activation in a pertussis toxin-insensitive manner, and that a P₂ receptor other than the P_2y or P_2x subtypes is involved in ATP-induced phospholipase A₂ activation via a pertussis toxin-sensitive G protein.     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

Prostaglandin E2 suppression of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by interacting with prostanoid receptors of the EP3-subtype

1. We have demonstrated recently that exogenous prostaglandin E₂ (PGE₂) inhibits electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. In the present study, we have attempted to characterize the pre-junctional prostanoid receptor(s) responsible for the inhibitory action of PGE₂ and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea-pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS-evoked [³H]-ACh release.
2. In epithelium-denuded tracheal strips pretreated with indomethacin (10 μM), PGE₂ (0.1 nM–1 μM) inhibited EFS-evoked [³H]-ACh release in a concentration-dependent manner with an EC₅₀ and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP-receptor agonist, PGF_2α and the stable thromboxane mimetic, U46619 (each at 1 μM), also inhibited [³H]-ACh release by 48%, 41% and 35%, respectively. PGD₂ (1 μM) had no significant effect on [³H]-ACh release.
3. The selective TP-receptor antagonist, ICI 192,605 (0.1 μM), completely reversed the inhibition of cholinergic neurotransmission induced by U-46619, but had no significant effect on similar responses effected by PGE₂ and PGF_2α.
4. A number of EP-receptor agonists mimicked the ability of PGE₂ to inhibit [³H]-ACh release with a rank order of potency: GR63799X (EP₃-selective)>PGE₂>M&B 28,767 (EP₃ selective)>17-phenyl-ω-trinor PGE₂ (EP₁-selective). The EP₂-selective agonist, AH 13205 (1 μM), did not affect EFS-induced [³H]-ACh release.
5. AH6809 (10 μM), at a concentration 10 to 100 times greater than its pA₂ at DP-, EP₁- and EP₂-receptors, failed to reverse the inhibitory effect of PGE₂ or 17-phenyl-ω-trinor PGE₂ on [³H]-ACh release.
6. These results suggest that PGE₂ inhibits [³H]-ACh release from parasympathetic nerves supplying guinea-pig trachea via an interaction with prejunctional prostanoid receptors of the EP₃-receptor subtype. Evidence for inhibitory prejunctional TP- and, possibly, IP-receptors was also obtained although these receptors may play only a minor role in suppressing [³H]-ACh release when compared to receptors of the EP₃-subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea-pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.

     

Prostaglandin E2 couples through EP4 prostanoid receptors to induce IL-8 production in human colonic epithelial cell lines

Background and purpose:

Prostaglandin (PG) E₂ and interleukin (IL)-8 are simultaneously increased during the inflammation that characterizes numerous pathologies such as inflammatory bowel disease. IL-8 is a potent neutrophil chemo-attractant and activator, and can initiate and/or exacerbate tissue injury. PGE₂ signals principally through prostanoid receptors of the EP₂ and/or EP₄ subtypes to promote cAMP-dependent cellular functions. The aim of this study was to identify the role of the EP₂ and EP₄ receptor subtype(s) on two human colonic epithelial cell lines (Caco-2 and T84), in regulating PGE₂-induced IL-8 production.

Experimental approach:

To identify the causative receptor, we knocked-down and over-expressed EP₂ and EP₄ receptor subtypes in colonic epithelial cells and studied the effect of several selective EP₂/EP₄ receptor agonists and antagonists. The inductions of IL-8 and EP receptor mRNA and protein expression were determined by real-time PCR and western blot analysis. The affinity of PGE₂ and Bmax values for the EP₂ and EP₄ receptor on colonic epithelial cells were determined by radioligand-binding assays with [³H]PGE₂.

Key results:

PGE₂ had the highest affinity for the EP₄ receptor subtype and promoted a robust stimulation of cAMP-dependent IL-8 synthesis. This effect was mimicked by a selective EP₄ receptor agonist, ONO-AE1-329, and abolished by silencing the EP₄ receptor gene by using siRNA techniques, a selective EP₄ receptor antagonist (ONO-AE3-208) and a selective inhibitor (Rp-cAMP) of cAMP-dependent protein kinase.

Conclusions and implications:

These findings suggest that initiation and progression of colonic inflammation induced by IL-8 could be mediated, at least in part, by PGE₂ acting via the EP₄ receptor subtype.     

Membrane depolarization by activation of prostaglandin E receptor EP3 subtype of putative serotonergic neurons in the dorsal raphe nucleus of the rat

A whole-cell current-clamp study using a thin slice preparation of the rat brain was carried out to elucidate the function of prostaglandin E (PGE) receptor EP₃, subtype in the dorsal raphe nucleus (DR), where mRNA of this subtype is highly expressed. Bath application of PGE₂ or M&B 28767, an EP₃ agonist, depolarized the membrane of the large DR neurons in a concentration-dependent manner between 10^–9 and 10^–6 M. These neurons showed hyperpolarization of membrane potential to 10 or 50 M serotonin. Neither an EP₂ receptor agonist, butaprost, an EP₂/EP₄ receptor agonist, 11-deoxy-PGE₁, nor an EP₁ receptor agonist, 17-phenyl-PGE₂, had any effect on large DR neurons between 10^–9 and 10^–6 M. The M&B 28767-induced depolarization was observed in a Ca²⁺-free, high Mg²⁺ (5 mM) solution containing 0.3 M tetrodotoxin, and occurred equally well when intracellular Cl^– was replaced by gluconate. These results suggest that direct agonist-activation of EP₃ receptor depolarizes the membrane by a cationic conductance, leading to excitation of DR neurons, and indicate a physiological implication that EP₃ receptor may modulate the serotonergic inhibition of neuronal activities.     

Muscarinic receptors in canine colonic circular smooth muscle. II. Signal transduction pathways.

We have, in the accompanying work, demonstrated the coexistence of M2 and M3 muscarinic receptors in the circular smooth muscle of canine colon. In the present study, the effects of muscarinic receptor stimulation on phosphoinositide turnover and adenylate cyclase activity were examined. In myo-[3H]inositol-labeled circular smooth muscle strips, carbachol caused a concentration-dependent (EC50 = 5 microM) increase in [3H]inositol phosphate production. The more M3 receptor-selective muscarinic antagonist pirenzepine (KB = 53 nM) was approximately 60 times more potent than the more M2-selective agent AF-DX 116 (KB = 3 microM) in blocking carbachol-elicited accumulation of [3H]inositol phosphates. The carbachol-stimulated increase in [3H]inositol phosphate accumulation was not affected by pretreatment of the tissue with pertussis toxin (200 ng/ml, 3 hr). Within the first minute, carbachol (100 microM) caused a rapid and transient increase of [3H]inositol 1,4,5-trisphosphate production that oscillated continuously in the presence of agonist (120 min). The accumulation of [3H]inositol 1,3,4-trisphosphate was also extremely rapid, reaching a peak at 15 sec. The accumulation of [3H]inositol monophosphate was delayed and progressively increased over 30 min. [3H]inositol 1,3,4,5-tetrakisphosphate, although not detectable in the first minute, accumulated to significant levels over 30 min in the presence of agonist. Addition of carbachol in the adenylate cyclase assay caused inhibition of forskolin-stimulated [32P]cAMP production and blocked forskolin-stimulated cAMP accumulation in the intact tissue. The inhibitory effects of carbachol on adenylate cyclase were blocked by atropine, AF-DX 116, and 4-diphenylacetoxy-N-methylpiperidine methobromide but were unaffected by the more M3-selective agent pirenzepine (1 microM). Pretreatment of tissues with pertussis toxin completely eliminated M2 receptor-mediated inhibition of adenylate cyclase activity, without altering inositol 1,4,5-trisphosphate accumulation. We conclude that muscarinic receptor stimulation of inositol trisphosphate production is mediated by the M3 receptor coupled to a pertussis toxin-insensitive GTP-binding protein and results in the rapid formation of inositol tetrakisphosphate, whereas inhibition of adenylate cyclase activity is mediated by the M2 subtype of muscarinic receptor coupled to the pertussis toxin-sensitive GTP-binding protein Gi.     

Effect of NMDA receptor ligands on mast cell histamine release, a reappraisal

Natural polyamines have been proposed to induce histamine release from mast cells through a direct interaction with G proteins. Alternatively, the polyamine binding site of ionotropic N-methyl-d-aspartate (NMDA) receptors has been suggested as a target for spermine on mast cells. We reexamined both hypotheses. Incubation of rat peritoneal mast cells with spermine resulted in a concentration-dependent histamine release (EC₅₀ 270 μM). Incubation with NMDA receptor agonists, glutamate or NMDA, associated to the co-agonist glycine, did not induce secretion. Western blot experiments did not reveal NMDA R1, R2a, R2b or R2c subunit expression in rat peritoneal mast cell membranes. The NMDA receptor antagonist at the glycine site, L-689,560, did not modify, at relevant concentrations, the spermine-induced secretion. The NMDA receptor antagonists, ifenprodil and LY 235959, and the NMDA channel blocker, MK801, slightly inhibited, at high concentrations, the secretory effect of spermine. The polyamine arcaine, an antagonist of the NMDA receptor polyamine binding site, induced histamine secretion (EC₅₀ 350 μM). Both spermine- and arcaine-induced effects were independent upon extracellular calcium and were largely inhibited by treatment of mast cells with pertussis toxin or benzalkonium chloride. The response to spermine and arcaine was prevented by the hydrolysis of sialic acid residues of the cell surface by neuraminidase, and was restored by permeabilization of the plasma membrane with streptolysine-O, indicating that polyamines act intracellularly. These results confirm the involvement of pertussis toxin-sensitive G proteins in the secretory effect of polyamines and demonstrate the absence of NMDA receptors on rat peritoneal mast cells. Nonselective effects of some NMDA receptor ligands on mast cells cannot be excluded. Received: 28 December 1998 / Accepted: 19 March 1999     

Effect of the tridecamer of compound 48/80, a Ca2+-dependent histamine releaser,on phospholipid metabolism during the early stage of histamine release from rat mast cells

When the tridecamer component of compound 48/80 (Fraction D, Fr. D), a Ca²⁺-dependent histamine releaser, was incubated with rat mast cells that had been prelabeled with [³²P]phosphate, [³H]inositol or [³H]glycerol, it induced a rapid decrease in [³²P]phosphatidylinositol-4,5-bisphosphate (PIP₂) followed by increases of [³H]inositol-1,4,5-triphosphate (Ins P₃) and [³H]diacylglycerol during the 10 sec prior to detectable histamine release. Fr.D-induced changes of the metabolism of these compounds occurred even in the absence of Ca²⁺, but to a lesser extent than in the presence of Ca²⁺. In contrast, the accumulation of [³H]arachidonic acid into phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidic acid (PA) in [³H]arachidonic acid-prelabeled mast cells was Ca²⁺-dependently stimulated by Fr.D with a concomitant decrease in [³H]phophatidylethanolamine (PE). These Ca²⁺-dependent changes in PC and PE were not observed in mast cells preloaded with [³²P]phosphate, while [³²P]PI and [³²P]PA increased Ca²⁺ independently. Fr.D also increased ⁴⁵Ca²⁺ uptake by mast cells within 5 sec after the stimulation. These results indicate that Fr.D binding to mast cell Ca²⁺ independently induces rapid changes of PI cycle-related metabolism of plasma membrane components, while it also induces Ca²⁺-dependent accumulation of arachidonic acid into PC, PI and PA in association with the decrease of PE, which may be important during the latent period prior to the Ca²⁺-dependent release of histamine from Fr.D-stimulated mast cells.     

Characterization of the prostanoid receptors mediating inhibition of PAF-induced aggregation of guinea-pig eosinophils

1. Prostanoids induce a wide range of biological actions which are mediated by specific membrane-bound receptors. We have recently shown that the E-type prostaglandins, PGE₁ and PGE₂, effectively inhibit eosinophil aggregation induced by platelet-activating factor (PAF). In an attempt to determine which prostanoid receptor(s) were involved, we investigated the effects of a range of selective prostanoid agonists and antagonists on eosinophil homotypic aggregation induced by PAF.
2. Both PGE₁ and PGE₂ (10⁻¹⁰ to 10⁻⁶ M) induced a concentration-related inhibition of the aggregation response induced by PAF. PGE₁ was more effective than PGE₂ but PGE₂ was slightly more potent than PGE₁ (approximate IC₅₀ values for PGE₁ and PGE₂ of 1.5×10⁻⁸ M and 5×10⁻⁹ M, respectively).
3. The EP₂-selective agonists, 11-deoxy-PGE₁, butaprost and AH13205, and the EP₂/EP₃-selective agonist, misoprostol, also inhibited PAF-induced aggregation. The rank order of potency for EP₂-selective agonists was 11-deoxy-PGE₁ > misoprostol > butaprost = AH13205. The protein kinase A inhibitor, KT5720 (10⁻⁶ M), reversed the inhibitory effects of 11-deoxy-PGE₁ (10⁻⁶ M) and AH13205 (10⁻⁵ M).
4. The EP₁/EP₃-selective agonist, sulprostone, and the EP₁-selective agonist, 17-phenyl-ω-trinor PGE₂, had no significant inhibitory activity when tested at concentrations up to 10⁻⁶ M. The EP₄-receptor antagonist, AH23848B, had no effect on PAF-induced aggregation and did affect the inhibitory activity of PGE₁.
5. The IP-selective agonist, cicaprost (up to 10⁻⁶ M), and the IP/EP₁-receptor agonist, iloprost (up to 10⁻⁵ M), had no significant effect on PAF-induced eosinophil aggregation. However, iloprost significantly augmented the inhibitory effects of a maximally inhibitory concentration of PGE₂.
6. PGD₂ (10⁻⁵ M) had no effect on eosinophil aggregation and the inhibitory activity of PGE₁ on PAF-induced eosinophil aggregation was not altered by the DP-selective receptor antagonist, BWA868C.
7. The results presented here suggest that the inhibition of PAF-induced eosinophil aggregation by prostanoids is mediated by the occupation of EP₂-receptors. It is important to note that the effects of naturally occuring prostanoids, such as PGE₂, on eosinophil aggregation occur at low concentrations highlighting a potential role for EP₂ receptors in regulating eosinophil function in vivo.

     

Effect of okadaic acid on histamine release from rat peritoneal mast cells activated by anti-IgE

Abstract— The effect of okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A, on histamine release from mast cells has been investigated. Okadaic acid strongly and dose-dependently inhibited histamine release from mast cells induced by anti-IgE. The IC50 value of okadaic acid on histamine release induced by anti-IgE was 3·2 Nm. However, okadaic acid failed to inhibit histamine release induced by A23187 and compound 48/80. Moreover, okadaic acid showed no effect on the initial rise in intracellular Ca²⁺, Ca²⁺-mobilization from intracellular Ca²⁺-stores and the generation of inositol trisphosphate. These results suggest a possible involvement of protein phosphatase 2A in the histamine release from mast cells induced by anti-IgE.     

Effect of a new potent H2-receptor antagonist 3[[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl]thio]-N2-sulfamoylpropionamidine (YM-11170) on gastric mucosal histamine-sensitive adenylate cyclase from guinea pig

The effect of 3[[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl]thio]-N²-sulfamoylpro-pionamidine (YM-11170), a new thiazole H₂-receptor antagonist bearing propionamidine at the terminus of a side chain, on histamine-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] of gastric mucosa from the guinea pig was studied and compared with that of cimetidine. YM-11170 displaced the concentration-stimulation curve of histamine-sensitive adenylate cyclase to the right with a pA₂ of 7.65 (K_i, = 2.25 × 10^?8M). Stimulation of gastric adenylate cyclase by 0.1 mM histamine was competitively inhibited by YM-11170 and cimetidine in a dose-dependent manner, with ic₅₀ values of 5.9 × 10^?7M and 1.4 × 10^?5M respectively. Hippocampal histamine-sensitive adenylate cyclase in the presence of 0.1 mM histamine was also competitively inhibited by YM-11170 with an ic₅₀ of 1.1 × 10^?7 M. YM-11170 did not affect Gpp(NH)p-, NaF-, PGE₂-stimulated or basal activity of the gastric adenylate cyclase. These data, together with other results, indicate that YM-11170 is a highly selective and potent H₂-receptor antagonist which competes with histamine at the receptor site on the histamine-sensitive adenylate cyclase.     

An MCASE approach to the search of a cure for Parkinson's Disease.

Background

Prostaglandin E₂ (PGE₂) has multiple physiologic roles mediated by G protein coupled receptors designated E-prostanoid, or "EP" receptors. Evidence supports an important role for the EP₂ receptor in regulating fertility, vascular tone and renal function.

Results

The full-length rabbit EP₂ receptor cDNA was cloned. The encoded polypeptide contains 361 amino acid residues with seven hydrophobic domains. COS-1 cells expressing the cloned rabbit EP₂ exhibited specific [³H]PGE₂ binding with a K_d of 19.1± 1.7 nM. [³H]PGE₂ was displaced by unlabeled ligands in the following order: PGE₂>>PGD₂=PGF_2α=iloprost. Binding of [³H]PGE₂ was also displaced by EP receptor subtype selective agonists with a rank order of affinity consistent with the EP2 receptor (butaprost>AH13205>misoprostol>sulprostone). Butaprost free acid produced a concentration-dependent increase in cAMP accumulation in rabbit EP₂ transfected COS-1 cells with a half-maximal effective concentration of 480 nM. RNase protection assay revealed high expression in the ileum, spleen, and liver with lower expression in the kidney, lung, heart, uterus, adrenal gland and skeletal muscle. In situ hybridization localized EP₂ mRNA to the uterine endometrium, but showed no distinct localization in the kidney. EP2 mRNA expression along the nephron was determined by RT-PCR and its expression was present in glomeruli, MCD, tDL and CCD. In cultured cells EP2 receptor was not detected in collecting ducts but was detected in renal interstitial cells and vascular smooth muscle cells. EP2 mRNA was also detected in arteries, veins, and preglomerular vessels of the kidney.

Conclusion

EP2 expression pattern is consistent with the known functional roles for cAMP coupled PGE₂ effects in reproductive and vascular tissues and renal interstitial cells. It remains uncertain whether it is also expressed in renal tubules.     

A pertussis toxin-sensitive G protein is required to induce histamine release from rat peritoneal mast cells by bradykinin.

Bradykinin, kallidin (Lys-bradykinin) and [Thi 5,8, D-Phe7]-bradykinin, a functional B2 antagonist, induce histamine release from rat peritoneal mast cells. The histamine release is dependent upon added calcium when mast cells are placed in calcium-free medium 30 min before being triggered with the kinins. Histamine release was dose-dependently inhibited by pertussis toxin (1-100 ng/ml) and by benzalkonium chloride (0.1-3 micrograms/ml). The efficiency of ionophore A23187 on histamine release was affected neither by pertussis toxin nor by benzalkonium chloride. The parallel response of rat peritoneal mast cells to kinins and to substance P suggest that these peptides have the same mechanisms of action i.e. activation of a pertussis toxin-sensitive G protein and of phospholipase C defining a peptidergic triggering pathway of mast cells.     

Inhibitory effect of prostaglandins on dopamine release from the retina

1. Prostaglandins have been shown to modulate transmitter release from both central and peripheral neuroeffector junctions. In the present study, we examined the effect of prostaglandins on [³H]-dopamine release from isolated, superfused rabbit retina.2. Both naturally occurring and synthetic prostaglandins produced concentration-dependent reduction of electrically evoked [³H]-dopamine overflow without affecting basal tracer efflux. The rank order of potencies of the agonists was: sulprostone > 16, 16-dimethyl PGE₂>PGE₂> > 11-deoxy-PGE₁>PGF_2α.3. The PGE₂-mediated inhibition of field stimulated [³H]-dopamine release was not blocked by the selective EP₁-receptor antagonist, AH6809 (5–30 μM).4. The cyclooxygenase inhibitor, flurbiprofen (3 μM) had no effect on basal or evoked [³H]-dopamine overflow nor did it affect the inhibition caused by PGE₂ suggesting that endogenous prostaglandins are not involved in the regulation of dopamine release in the retina.5. The inhibition of [³H]-dopamine release produced by submaximal concentrations of PGE₂, apomorphine and melatonin were not additive indicating that presynaptic PGE₂, D₂- and melatonin receptors coexist at sites for neurotransmitter release and may share a common mechanism for regulation of dopamine release.6. We conclude that prostaglandin-induced inhibition of electrically evoked [³H]-dopamine release from the rabbit retina may be mediated by specific prostaglandin receptors of the EP₃ subtype.     

Hydrogen peroxide stimulation of CFTR reveals an Epac-mediated,soluble AC-dependent cAMP amplification pathway common to GPCR signalling

BACKGROUND AND PURPOSE

H₂O₂ is widely understood to regulate intracellular signalling. In airway epithelia, H₂O₂ stimulates anion secretion primarily by activating an autocrine PGE₂ signalling pathway via EP₄ and EP₁ receptors to initiate cytic fibrosis transmembrane regulator (CFTR)-mediated Cl⁻ secretion. This study investigated signalling downstream of the receptors activated by H₂O₂.

EXPERIMENTAL APPROACH

Anion secretion by differentiated bronchial epithelial cells was measured in Ussing chambers during stimulation with H₂O₂, an EP₄ receptor agonist or β₂-adrenoceptor agonist in the presence and absence of inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca²⁺]_I) changes were followed by microscopy using fura–2-loaded cells and PKA activation followed by FRET microscopy.

KEY RESULTS

Transmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were both necessary for H₂O₂ and EP₄ receptor-mediated CFTR activation in bronchial epithelia. H₂O₂ and EP₄ receptor agonist stimulated tmAC to increase exchange protein activated by cAMP (Epac) activity that drives PLC activation to raise [Ca²⁺]_i via Ca²⁺ store release (and not entry). Increased [Ca²⁺]_i led to sAC activation and further increases in CFTR activity. Stimulation of sAC did not depend on changes in [HCO₃⁻]. Ca²⁺-activated apical K_Ca1.1 channels and cAMP-activated basolateral K_V7.1 channels contributed to H₂O₂-stimulated anion currents. A similar Epac-mediated pathway was seen following β₂-adrenoceptor or forskolin stimulation.

CONCLUSIONS AND IMPLICATIONS

H₂O₂ initiated a complex signalling cascade that used direct stimulation of tmACs by Gαs followed by Epac-mediated Ca²⁺ crosstalk to activate sAC. The Epac-mediated Ca²⁺ signal constituted a positive feedback loop that amplified CFTR anion secretion following stimulation of tmAC by a variety of stimuli.