Brain prostanoid TP receptor-mediated adrenal noradrenaline secretion and EP3 receptor-mediated sympathetic noradrenaline release in rats

Sympathetic nerves release noradrenaline, whereas adrenal medullary chromaffin cells secrete noradrenaline and adrenaline. Therefore, plasma noradrenaline reflects the secretion from adrenal medulla in addition to the release from sympathetic nerves, however the exact mechanisms of adrenal noradrenaline secretion remain to be elucidated. The present study was designated to characterize the source of plasma noradrenaline induced by intracerebroventricularly (i.c.v.) administered bombesin and prostaglandin E2 in urethane-anesthetized rats. Bombesin (1.0 nmol/animal, i.c.v.) elevated plasma noradrenaline and adrenaline, while prostaglandin E2 (0.3 nmol/animal, i.c.v.) elevated only plasma noradrenaline. The bombesin-induced elevations of both catecholamines were attenuated by pretreatments with furegrelate (an inhibitor of thromboxane A2 synthase) [250 and 500 microg (0.9 and 1.8 micromol)/animal, i.c.v.)] and [(+)-S-145] [(+)-(1R,2R,3S,4S)-(5Z)-7-(3-[4-3H]-phenylsulphonyl-aminobicyclo[2.2.1]hept-2-yl)hept-5-enoic acid sodium salt] (an antagonist of prostanoid TP receptors) [100 and 250 microg (250 and 625 nmol)/animal)], and abolished by acute bilateral adrenalectomy. On the other hand, the prostaglandin E2-induced elevation of plasma noradrenaline was not influenced by acute bilateral adrenalectomy. These results suggest that adrenal noradrenaline secretion and sympathetic noradrenaline release are mediated by differential central mechanisms; brain prostanoid TP receptors activated by bombesin are involved in the adrenal noradrenaline secretion, while brain prostanoid EP (probably EP3) receptors activated by prostaglandin E2 are involved in the sympathetic noradrenaline release in rats. Brain prostanoid TP receptors activated by bombesin are also involved in the adrenal adrenaline secretion.     

Oxidative stress and alpha1-adrenoceptor-mediated stimulation of the Cl-/HCO3- exchanger in immortalized SHR proximal tubular epithelial cells

BACKGROUND AND PURPOSE: This study evaluated the signalling coupled to the alpha1-adrenoceptor-induced stimulation of the Cl-/HCO3- exchanger in hypertension. EXPERIMENTAL APPROACH: The Na+ -independent HCO3- transport system activity was assayed as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal) in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rat (SHR) and their normotensive control (Wistar Kyoto rat; WKY). KEY RESULTS: Noradrenaline increased Cl-/HCO3- exchanger activity with EC50 values of 0.6 and 5.3 microM in SHR and WKY cells, respectively. These effects were abolished by prazosin, but not by yohimbine. Phenylephrine increased Cl-/HCO3- exchanger activity in SHR and WKY cells (EC50 of 2.6 and 4.9 microM, respectively). Phenylephrine-mediated increase in Cl-/HCO3- exchanger activity in WKY and SHR cells was inhibited by protein kinase C (PKC), MAPK/ERK kinase (MEK) and p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. The expression of alpha1A- and alpha1B-adrenoceptors was identical in WKY and SHR cells. SHR cells generated more H2O2 than WKY cells. In SHR cells, the NADPH oxidase inhibitor apocynin reduced their increased ability to generate H2O2 and abolished their hypersensitivity to phenylephrine, but failed to affect basal Cl-/HCO3- exchanger activity. H2O2-dependent stimulation of Cl-/HCO3- exchange activity was significantly higher in SHR than in WKY cells. CONCLUSIONS AND IMPLICATIONS: Differences between WKY and SHR cells on their sensitivity to alpha1-adrenoceptor stimulation did not correlate with the abundance of alpha1A- and alpha1B-adrenoceptors and may be related to the increased generation of H2O2, which may amplify the response downstream of alpha1-adrenoceptor activation.     

Blowing off acid: a new tool to study Na+/HCO3- co-transport

Investigation of the physiological functions and possible pathological roles of Na(+)/HCO(3)(-) co-transport in the heart has been hampered by uncertainty over the molecular identity of cardiac Na(+)/HCO(3)(-) co-transporter(s) and the absence of selective pharmacological inhibitors. In their paper published in this issue, Ch'en and colleagues describe the extensive characterization of S0859 as a high-affinity inhibitor of Na(+)/HCO(3)(-) co-transport in cardiac myocytes (Ch'en et al., 2008). The availability of S0859 provides a powerful new tool to investigate the (patho)physiological significance of Na(+)/HCO(3)(-) co-transport in the heart and other tissues.     

Therapeutic administration of 3,4,5-trimethoxy-4'-fluorochalcone,a selective inhibitor of iNOS expression,attenuates the development of adjuvant-induced arthritis in rats

We have previously investigated the effects of a series of dimethoxy- and trimethoxychalcone derivatives, with various patterns of fluorination, on nitric oxide production in LPS-stimulated murine RAW 264.7. The present study was designed to determine if 3,4,5-trimethoxy-4'-fluorochalcone (CH 17) could modulate the production of NO and/or prostaglandins in vivo. On the mouse macrophage cell line RAW 264.7 CH 17 inhibited dose-dependently NO production, with an IC₅₀ value in the nanomolar range, and reduced PGE₂ levels by a 58% at 10 M. This compound had no direct inhibitory effect on iNOS and COX-2 activities. NO reduction was the consequence of inhibition of the expression of iNOS. In vitro experiments indicated that CH 17 is an inhibitor of the nuclear factor-B (NF-B) pathway of cellular activation in macrophages. This compound exhibited in vivo an inhibitory behaviour correlated with its in vitro results on nitrite and PGE₂ accumulation. In the rat adjuvant-induced arthritis, oral administration of CH 17 (25 mg/kg) on days 17–24 after adjuvant injection, significantly inhibited paw oedema, protected from weight loss and reduced the levels of inflammatory mediators (nitrites and PGE₂) in paw homogenates, without affecting PGE₂ levels in stomach homogenates. The profile and potency of this compound, a selective inhibitor of iNOS expression that interferes with NF-B activation, may have relevance for the inhibition of the inflammatory response, representing a new approach to the modulation of different inflammatory pathologies.     

The cyclooxygenase-2/prostaglandin E2 pathway is involved in the somatostatin-induced decrease of epileptiform bursting in the mouse hippocampus

The neuromodulatory peptide somatostatin-14 (SRIF) plays an important inhibitory role in epilepsy, but little is known on the signalling mechanisms coupled to this effect of SRIF. We have previously demonstrated that SRIF induces reduction of epileptiform bursting in a model of interictal-like activity in mouse hippocampal slices. In this same model, we investigated whether the cyclooxygenase 2 (COX-2)/prostaglandin E(2) (PGE(2)) pathway is part of those signalling mechanisms mediating SRIF anti-epileptic actions. Both the expression of COX-2 (mRNA and protein) and the endogenous release of PGE(2) increased in concomitance with epileptiform bursting. In particular, COX-2 protein increased in CA1/CA3 pyramidal layer and in the granular layer of the dentate gyrus. In addition, the selective inhibition of COX-2 by NS-398 markedly decreased endogenous PGE(2) release induced by epileptiform bursting and the epileptiform bursting itself. Similar effects on epileptiform bursting were obtained with another COX-2 inhibitor, i.e., meloxicam. SRIF application counteracted the increase of both COX-2 expression and PGE(2) release which occurred in concomitance with epileptiform bursting. Interestingly, SRIF and NS-398 comparably reduced epileptiform bursting in a non-additive manner and PGE(2) abolished the inhibitory effect of SRIF on epileptiform bursting. These results demonstrate that: i) the COX-2/PGE(2) pathway facilitates epileptiform bursting; and ii) SRIF exerts an anti-epileptic role by coupling to the COX-2/PGE(2) pathway. In conclusion, we have identified a key set of signalling events that underlie anti-convulsant effects of SRIF in a mouse model of hippocampal bursting, thus providing useful data not only to identify alternative intervention points for the modulation of SRIF function, but also to exploit new chemical space for drug-like molecules.     

Induction of UGT1A6 isoform by inflammatory conditions in rat astrocytes

Alteration of drug metabolism under diseased conditions is of clinical importance. We have investigated the effects of inflammatory conditions on phase II drug-metabolizing enzyme activity in rat cultured astrocytes. Lipopolysaccharide (LPS) treatment was used to promote inflammatory conditions. Thus, we reported that LPS initiates an inflammatory response, which is mediated by pro-inflammatory mediators and free radical generation. An increase in astrocyte glucuronidation activity was observed after a 48-h LPS treatment. This increase in glucuronidation activity was associated with an up-regulation of the UGT1A6 isoform mRNA level as shown by RT-PCR and gene reporter assay. Moreover, this endotoxin-induced increase in UGT1A6 expression level was blocked by actinomycin D and cycloheximide, indicating the requirement for RNA and protein synthesis. The UGT1A6 expression enhancement could be prevented by anti-inflammatory drugs (dexamethasone and NS398) or nitric oxide synthase inhibitors (L-NAME and L-NMMA). Moreover, gel shift assay revealed increased activator protein-1 (AP-1) binding activity after LPS treatment. We propose, based on the data presented, that the action of LPS to induce UGT1A6 isoform up-regulation may be mediated by pro-inflammatory mediator accumulation, and AP-1 binding activity increase.     

Stimulatory effect of Coca-Cola<Superscript>TM</Superscript> on gastroduodenal HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> secretion in rats

We examined the effect of various carbonated beverages, especially Coca-Cola^TM, on the HCO₃⁻ secretion in the rat stomach and duodenum. Under urethane anaesthesia, a chambered stomach or a proximal duodenal loop was perfused with saline, and HCO₃⁻ secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The amount of CO₂ contained in these beverages was about 4–7 g/mL. Coca-Cola^TM topically applied to the mucosa for 10 min significantly increased the HCO₃⁻ secretion in both the stomach and the duodenum. The HCO₃⁻ response in the duodenum was totally abolished by indomethacin and also partially inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Likewise, the response in the stomach was also markedly inhibited by either acetazolamide or indomethacin. The mucosal application of Coca-Cola^TM increased the PGE₂ contents in both the stomach and the duodenum. Other carbonated beverages, such as sparkling water, Fanta Grape^TM or cider, also increased the HCO₃⁻ secretion in these tissues. These results suggest that Coca-Cola^TM induces HCO₃⁻ secretion in both the stomach and the duodenum, and these responses may be attributable to both the intracellular supply of HCO₃⁻ generated via carbonic anhydrase, and endogenous PGs, probably related to the acidic pH of the solution. Received 4 August 2006; accepted 10 November 2006     

Prostanoid receptors of the EP3 subtype mediate the inhibitory effect of prostaglandin E2 on noradrenaline release in the mouse brain cortex

Mouse or rat brain cortex slices were preincubated with ³H-noradrenaline and superfused with physiological salt solution containing desipramine. We studied the effects of prostaglandin E₂ (PGE₂), prostaglandin D₂ (PGD₂) and related drugs on the electrically evoked (50 mA, 2 ms, 0.3 Hz) tritium overflow.PGE₂ inhibited the electrically evoked tritium overflow from mouse brain cortex slices; the maximum effect of PGE₂ (79010) was attenuated by the ₂-adrenoceptor agonist talipexole (to 52010) and enhanced by the ₂-adrenoceptor antagonist rauwolscine (to 92%). Rauwolscine was added to the superfusion medium in all subsequent experiments. The effect of PGE₂ was readily reversible upon withdrawal from the medium and remained constant upon prolonged exposure of the tissue to the prostanoid. Studies with EP receptor agonists, mimicking the inhibitory effect of PGE₂, showed the following potencies (pIC₅₀): sulprostone (8.22); misoprostol (8.00); PGE₂ (7.74); PGEZ (7.61); iloprost (5.86). The concentration-response curve of PGE₂ was marginally shifted to the right by the EP₁ receptor antagonist AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid; apparent pA₂ 3.97) and by the TP receptor antagonist vapiprost (4.50). AH 6809, by itself, did not affect the evoked overflow whereas vapiprost increased it. PGD2 inhibited the evoked overflow at high concentrations (pIC₅₀ 4.90); this effect was not altered by the DP receptor antagonist BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2hydroxyethylamino)hydantoin), which, by itself, did not affect the evoked overflow. Indometacin slightly increased the evoked overflow and tended to increase the inhibitory effect of PGE₂. PGE₂ inhibited the electrically evoked tritium overflow also in rat brain cortex slices. The maximum effect (obtained in the presence of rauwolscine) was 61%; the pIC₃₀ value was 7.67.The present study suggests that PGE₂ inhibits noradrenaline release from mouse brain cortex via EP₃ receptors and that its maximum effect is more marked in the mouse than in the rat. The inhibitory effect of PGD₂ (in the mouse brain) does not involve DP receptors and may also be related to EP₃ receptors. The EP₃ receptors interact with a2-adrenoceptors and may be activated by endogenous prostanoids.     

Comparison of the effects of prostaglandins E2 and I2 on testicular nociceptor activities studied in vitro

Summary Effects of exogenous prostaglandin (PG) E₂ and PGI₂ on testicular polymodal receptor activities were compared in in vitro recordings of single- or multi-fiber discharges from canine testis-spermatic nerve preparations. PGI₂ up to 1.4×10^–6 mol/l (cumulative method) or 1.0×10^–5 mol/l (non-cumulative method) excited only weakly some of the receptors, and similar observations were made with PGE2. Both PGs applied cumulatively or non-cumulatively at concentrations above 1.4×10^–8 mol/l augmented the response to bradykinin (9.4×10^–8 mol/l) in more than half of the cases tested. The augmenting effect of PGE₂ lasted longer than that of PGI₂ both with the cumulative and the non-cumulative method. The degree of augmentation tended to increase dependent on concentration, but some cases showed no further increase or rather a decrease in augmentation by PGs at a ten times higher concentration, especially when PGs were applied cumulatively. A second challenge by PG after a short interval (2 min) did not induce augmentation. These phenomena were considered to be tachyphylaxis to PGs. Cross-tachyphylaxis to PGE₂ and PGI₂ was also observed. There was not much difference in excitatory and augmenting potencies between these two PGs, but there was a clear difference in the concentrations of the PGs necessary to induce excitation of polymodal receptors and to facilitate their response to bradykinin. Send offprint requests to T. Kumazawa at the above address     

Thromboxane A2-mediated Cl secretion induced by platelet-activating factor in isolated rat colon

Thromboxane A₂ is a novel endogenous secretagogue of Cl⁻ secretion in the distal colon. Here, we examined if the Cl⁻ secretion caused by platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is mediated by thromboxane A₂ production using isolated mucosae of the rat colon. Furosemide (100 μM) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 300 μM) completely inhibited PAF (10 μM)-induced increase in short-circuit current (Isc) across the mucosa, indicating that PAF caused a Cl⁻ secretion in the rat colon. A selective thromboxane A₂ receptor antagonist (sodium(E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11-dihydrobenz[b,e]oxepine-2-carboxylate monohydrate; KW-3635), and a selective thromboxane synthase inhibitor (sodium 4-[α-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylbenzoate dihydrate; Y-20811) inhibited the PAF-induced Cl⁻ current in a concentration-dependent manner. The IC₅₀ values of KW-3635 and Y-20811 were 2.1 and 0.5 μM, respectively. 30 μM KW-3635 and 1 μM Y-20811 inhibited the PAF response by 92% and 83%, respectively. These inhibitors did not affect the prostaglandin E₂-induced increase in Isc. A 5-lipoxygenase-activating protein inhibitor (3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2,2-dimethyl-propanoic acid sodium; MK-886) (5 μM) did not affect the PAF-induced Cl⁻ current. The present study suggests that the PAF-induced Cl⁻ secretion in the rat colonic mucosa is mainly mediated by a release of thromboxane A₂.     

Effects of nerve stimulation on enzyme secretion from the in vitro rat pancreas and 3H-release after preincubation with catecholamines

Summary In the presence of the cholinergic antagonist atropine, electrical field stimulation (FS) (5–20 Hz) caused a marked, reversible increase in the amylase output from superfused rat pancreatic segments. Adrenaline and noradrenaline evoked dose-dependent increases in amylase output which were similar to those produced by FS. The FS- and catecholamine-evoked amylase secretions were abolished by the -adrenergic antagonist propranolol. The FS-evoked secretion could be abolished by either the removal of external Ca²⁺ or the application of tetrodotoxin (TTX, 2×10^–6 M). FS also resulted in a reversible increase in the fractional efflux of tritium (³H) from rat pancreatic tissues preincubated with either ³H-noradrenaline or ³H-adrenaline. The effects of FS (5–20 Hz) on ³H efflux were abolished by TTX (2×10^–6 M). TTX had no effect on the enhancement of ³H efflux caused by elevation of external potassium concentration (high K⁺, 75 mM). Removal of superfusate Ca²⁺ completely abolished both the FS- and high K⁺-induced increases in ³H efflux. These observations suggest that intrinsic nerve stimulation (i.e. FS) results in the Ca²⁺-dependent release of sympathetic neurotransmitter, noradrenaline, which has a direct secretory action on the rat pancreas. Furthermore, the findings suggest that adrenaline can be taken up by nervous elements. This raises the possibility that uptake and re-release of circulating adrenaline might contribute to the control of rat pancreatic enzyme secretion by the adrenergic nervous system.     

Mechanism of prostaglandin E2-, F2α- and latanoprost acid-induced relaxation of submental veins

The mechanism of prostaglandin E₂-, prostaglandin F_2α- and latanoprost acid (13,14-dihydro-17-phenyl-18,19,20-trinor-prostaglandin F_2α)-induced relaxation of the rabbit submental vein was studied. Prostaglandin E₂ caused maximum relaxation of endothelin-1 precontracted vessels (EC₅₀: 1.8×10⁻⁸ M). Much of the relaxation could be abolished by denuding the endothelium with the nitric oxide synthase inhibitor,

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-NAME (N^G-Nitro-

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-arginine methylester). CGRP-(8–37) (calcitonin gene-related peptide fragment (8–37)), a calcitonin gene-related peptide receptor antagonist, exhibited a partial blocking effect, whereas the tachykinin NK₁ receptor blocker, GR 82334 ([

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-Pro⁹[Spiro-γ-Lactam]Leu¹⁰,Trp¹¹]physalaemin (1–11)), markedly attenuated the response. Both prostaglandin F_2α and the relatively selective FP receptor agonist, latanoprost acid, caused relaxation of the veins to about 50% of the precontracted state in the presence of GR 32191B ([1R-[1α(Z),2β,3β,5α]]-(+)-7-[5-([1,1′-biphenyl]-4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid), a thromboxane receptor antagonist (EC₅₀: for prostaglandin F_2α 7.9×10⁻⁹ M, and for latanoprost acid 4.9×10⁻⁹ M).

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-NAME, as well as denuding the endothelium, completely abolished the effect. In addition, most or at least a large part of the relaxation was also blocked by CGRP-(8–37) as well as GR 82334. These results indicate that the FP receptor-mediated relaxation of veins is based on release of nitric oxide in addition to involvement of calcitonin gene-related peptide and substance P, or some other tachykinin, probably released from perivascular sensory nerves. The more pronounced relaxation induced by prostaglandin E₂ could be due to vasodilator EP receptors in the smooth muscle layer of the veins.     

Caffeine suppresses lipopolysaccharide-stimulated BV2 microglial cells by suppressing Akt-mediated NF-κB activation and ERK phosphorylation

Since the anti-inflammatory effect of caffeine is unclear in microglial cells, we performed whether caffeine attenuates the expression of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Caffeine substantially suppressed the LPS-induced pro-inflammatory mediators nitric oxide (NO), prostaglandin E₂ (PGE₂) and tumor necrosis factor-α (TNF-α) in BV2 microglial cells. These effects resulted from the inhibition of their regulatory genes inducible NO synthase (iNOS), cycloxygenase-2 (COX-2) and TNF-α. In addition, caffeine significantly decreased LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) by suppressing the nuclear translocation of p50 and p65 subunits. A specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), attenuated the LPS-induced expression of iNOS, COX-2 and TNF-α genes. In addition, we elucidated that inhibition of Akt phosphorylation plays a crucial role in caffeine-mediated NF-κB regulation in LPS-stimulated BV2 microglial cells. Caffeine also attenuated the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK) and a specific inhibitor of ERK, PD98059, subsequently downregulated the expression of the pro-inflammatory genes iNOS, COX-2 and TNF-α. Taken together, our data indicate that caffeine suppresses the generation of pro-inflammatory mediators, such as NO, PGE₂ and TNF-α as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting Akt-dependent NF-κB activation and the ERK signaling pathway.     

Induction of integrin β3 in PGE2-stimulated adhesion of mastocytoma P-815 cells to the Arg-Gly-Asp-enriched fragment of fibronectin

We previously demonstrated that prostaglandin (PG) E₂ stimulates adhesion of mastocytoma P-815 cells (P-815 cells) to the Arg-Gly-Asp (RGD)-enriched matrix via the PGE₂ receptor subtype EP4 [Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E₂ receptor subtypes, EP3 and EP4. J Biol Chem 2003;278:17977–81]. Here we investigated the role of various integrin subtypes in the induction of adherent activity in PGE₂-stimulated P-815 cells. FACS analysis showed that P-815 cells express high levels of integrin α4, α5, β1 and β2 subunits and moderate levels of integrin αIIb, αv, β3 and β7 subunits. When treated with PGE₂, the EP4 agonist ONO-AE1-329 or the cell permeable cAMP analogue, 8-Br-cAMP, P-815 cells showed markedly increased cell surface expression of integrin αIIb, αv and β3 subunits, and these expressions were significantly reduced by addition of the protein synthesis inhibitor cycloheximide. Along with increased cell surface expression, mRNA and protein levels of the integrin β3 subunit, but not of integrin αIIb and αv subunits, were simultaneously elevated. On the other hand, adhesion of P-815 cells in response to PGE₂ or 8-Br-cAMP was abolished by antibodies specific for integrin αv and β3 subunits, but not by antibodies for integrin α4, α5, β1, β2 and β7 subunits. Moreover, treatment with tirofiban, an integrin αIIbβ3 antagonist, or eptifibatide, an integrin αvβ3/αIIbβ3 antagonist resulted in a decrease in adhesion of P-815 cells in response to PGE₂ or 8-Br-cAMP. These results suggest that de novo synthesis of the integrin β3 subunit plays a pivotal role in PGE₂-induced adhesion of P-815 cells to the RGD-enriched matrix through EP4-mediated cAMP signaling.     

Role of a pertussis toxin sensitive G-protein in mediating the effects of phorbol esters on receptor activated cyclic AMP accumulation in Jurkat cells

Summary In the human T -cell line, Jurkat, the accumulation of cyclic AMP induced by adenosine is enhanced by tumor-promoting phorbol esters, whereas prostaglandin E₂ receptor-stimulated cAMP accumulation is antagonized (Nordstedt et al. 1989). In the present study we examine the involvement of pertussis toxin sensitive guanine nucleotide binding proteins (G-proteins) in producing the phorbol ester effects.Pertussis toxin pretreatment of the Jurkat cells invariably caused an ADP ribosylation of two G-proteins that inhibit adenylyl cyclase, tentatively identified as G_i2 and G_i3, using Western blots. Pertussis toxin treatment had little effect on basal cAMP accumulation, but sometimes inhibited, sometimes stimulated agonist and cholera toxin induced cAMP accumulation. The latter effect was not mimicked by the B-oligomer. Irrespective of whether pertussis toxin stimulated or inhibited NECA and cholera toxin-induced cAMP accumulation it could not block the effect of phorbol-12,13-dibutyrate (PDBu). The inhibitory effect of PDBu on prostaglandin E2-induced cAMP accumulation was, however, invariably eliminated by pertussis toxin treatment.In conclusion, activation of protein kinase C by phorbol esters reveals a G_i-mediated prostaglandin E receptor-induced inhibition of adenylate cyclase in addition to the prostaglandin E receptor-mediated stimulation of cAMP accumulation in Jurkat cells. The enhancement of adenosine A₂ receptor stimulated CAMP accumulation by PDBu, on the other hand, does not involve a PTX sensitive G_i-protein. Send offprint requests to I. van der Ploeg at the above address     

Inhibitory effects of azelastine on substance P-induced itch-associated response in mice

The anti-pruritic mechanisms of azelastine were studied in mice. Scratching induced by intradermal histamine was inhibited by azelastine (30 mg/kg) and chlorpheniramine (30 mg/kg). Substance P-induced scratching was dose dependently suppressed by azelastine (3-30 mg/kg), but not by chlorpheniramine (10 and 30 mg/kg). Azelastine (30 mg/kg) inhibited the substance P-induced production of leukotriene B4, but not prostaglandin E2, in the skin. Azelastine (3-30 mg/kg) suppressed scratching induced by intradermal injection of leukotriene B4. The results suggest that inhibition of the production and action of leukotriene B4, as well as an anti-histamine action, is involved in the anti-pruritic action of azelastine.     

The effect of acteoside on histamine release and arachidonic acid release in RBL-2H3 mast cells

The effect of acteoside, a phenylpropanoid glycoside isolated from Clerodendron trichotomum Thunberg, on histamine and arachidonic acid release was investigated in RBL 2H3 cells. Histamine was dose-dependently released from RBL 2H3 cells by melittin, arachidonic acid and thapsigargin. In extracellular Ca2+-free solution, basal secretion of histamine increased by two fold. The response of histamine release to melittin and thapsigargin in Ca2+-free solution was significantly decreased, whereas the response to arachidonic acid was significantly increased as compared with those in normal solution. Acteoside inhibited histamine release induced by melittin, arachidonic acid and thapsigargin in a dose-dependent manner in the presence or absence of extracellular Ca2+. However, the inhibitory activity of acteoside was more potent in normal solution than that in Ca2+-free solution. These data suggest that inhibitory mechanism of acteoside on histamine release may be related to extracellular Ca2+. On the other hand, acteoside significantly inhibited arachidonic acid release and prostaglandin E2 production induced by 0.5 microM melittin. It is possible that acteoside may be developed as an anti-inflammatory agent.     

Diphlorethohydroxycarmalol,Isolated from Ishige okamurae,Increases Prostaglandin E2 through the Expression of Cyclooxygenase-1 and -2 in HaCaT Human Keratinocytes

Prostaglandin (PG) E₂, the most abundant prostaglandin in the human body, is synthesized from arachidonic acid via the actions of cyclooxygenase (COX) enzymes. PGE₂ exerts homeostatic, cytoprotective, inflammatory, and in some cases anti-inflammatory effects. Also, it has been reported that PGE₂ is involved in hair growth. Diphlorethohydroxycarmalol (DPHC) is a phlorotannin compound isolated from the brown algae Ishige okamurae, with various biological activities in vitro and in vivo. In this study, the biological effect and mechanism of action of DPHC on prostaglandin synthesis in HaCaT human keratinocytes was examined. The results showed that, in these cells, DPHC significantly and dose-dependently induced PGE₂ synthesis by increasing the protein and mRNA levels of COX-1 and COX-2. Interestingly, DPHC-induced COX-1 expression preceded that of COX-2. Also, while both rofecoxib and indomethacin inhibited PGE₂ production, the latter was seems to be the more potent. From above results, we can expect that DPHC has some beneficial effects via increasing of PGE₂ production.     

Ahcyl2 upregulates NBCe1-B via multiple serine residues of the PEST domain-mediated association

Inositol-1,4,5-triphosphate [IP₃] receptors binding protein released with IP₃ (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiled-coil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B.