Inositol Monophosphate Formation Research Articles

Effect of bitter compounds on amylase secretion in murine submandibular glands: Signaling pathway mechanisms

Background Amylase is synthesized in submandibular glands (SMG) and released into the oral cavity to degrade carbohydrates in the mouth. Bitter taste receptors (T2R) belong to the G-protein coupled receptor (GPCR) family and are expressed in the taste cells and also in the digestive tract. Methods The activity of amylase secreted by murine SMG was measured, detecting maltose by Bernfeld's method. Amylase and T2R6 were detected by imunohistochemistry and Western blot. The expression of Ggustducin, Gi, and phospholipase Cβ2 was also studied by Western blot. cAMP levels were measured by radioimmunoassay and inositol monophosphate production was quantified by ELISA. Results Theophylline, denatonium and cycloheximide exerted a dose-dependent inhibition on amylase secretion. This effect was reverted by preincubating SMG with an anti-Gαi antibody. cAMP production was increased by the same compounds, an effect that was also abrogated by an anti-Gαi antibody. Bitter compounds reduced inositol monophosphate formation in SMG and H-89, a protein kinase A inhibitor, reverted this action, revealing that this protein kinase down regulates phospholipase C activity. General significance We demonstrated that theophylline, denatonium and cycloheximide inhibit salivary amylase secretion, activating an intracellular signaling pathway that involves cAMP and phospholipase C, that cross talks via protein kinase A.

(+/-)-Govadine and (+/-)-THP, two tetrahydroprotoberberine alkaloids, as selective alpha 1-adrenoceptor antagonists in vascular smooth muscle cells.

(+/-)-Govadine and (+/-)-THP ((+/-)-2,3,10,11-tetrahydroxytetrahydroprotoberberine HBr) have been shown to inhibit noradrenaline-induced contraction of rat thoracic aortae. The pharmacological activity of the compounds was determined in thoracic aortae and cardiac tissue isolated from the rat and in trachea isolated from the guinea-pig to determine the selectivity of the compounds towards different types of receptor. (+/-)-Govadine and (+/-)-THP were found to be alpha 1-adrenoceptor blocking agents in rat thoracic aorta as revealed by their competitive antagonism of vasoconstriction induced by noradrenaline (pA2 = 6.57 +/- 0.07 and 5.93 +/- 0.06, respectively) or phenylephrine (pA2 = 6.74 +/- 0.08 and 6.06 +/- 0.10, respectively). Removal of endothelium did not affect the antagonistic potencies of (+/-)-govadine (pA2 = 6.83 +/- 0.09) and (+/-)-THP (pA2 = 6.25 +/- 0.06) on phenylephrine-induced vasoconstriction. They were more potent than yohimbine (pA2 = 6.05 +/- 0.05), but less so than phentolamine (pA2 = 7.54 +/- 0.11) and prazosin (pA2 = 9.27 +/- 0.12). (+/-)-Govadine and (+/-)-THP, furthermore, inhibited [3H]inositol monophosphate formation caused by noradrenaline (3 microM) in rat thoracic aorta. (+/-)-Govadine and (+/-)-THP were also alpha 2-adrenoceptor blocking agents with pA2 values 5.50 +/- 0.13 and 5.41 +/- 0.11, respectively. A high concentration of (+/-)-govadine (30 microM) or (+/-)-THP (30 microM) did not, however, affect the contraction induced by the thromboxane receptor agonist U46619, prostaglandin F2 alpha (PGF2 alpha), 5-hydroxytryptamine (5-HT), angiotensin II, endothelin or high K+ in rat aorta denuded of endothelium. Neither the cyclic AMP nor cyclic GMP content of rat thoracic aorta was, furthermore, changed by (+/-)-govadine or (+/-)-THP. Contraction of guinea-pig trachea caused by carbachol, histamine, leukotriene C4 or neurokinin A was not affected by (+/-)-govadine or (+/-)-THP. (+/-)-Govadine or (+/-)-THP also did not block beta 1- or beta 2-adrenoceptor-mediated responses induced by isoprenaline in rat right atria and guinea-pig trachea. It is concluded that (+/-)-govadine and (+/-)-THP are selective alpha 1-adrenoceptor antagonists in vascular smooth muscle.

Inhibition of platelet thromboxane formation and phosphoinositides breakdown by diisoeugenol.

Diisoeugenol inhibited the platelet aggregation and ATP release of rabbit platelets caused by ADP, arachidonic acid, platelet-activating factor (PAF), collagen and thrombin. Prolongation of the incubation time of platelets with diisoeugenol did not cause further inhibition and the aggregability of platelets could not be restored after washing. In human platelet-rich plasma, diisoeugenol inhibited the biphasic aggregation and ATP release induced by adrenaline and ADP in a concentration-dependent manner. Thromboxane B2 formation caused by arachidonic acid, collagen and thrombin was markedly inhibited by diisoeugenol in a concentration-dependent manner. Diisoeugenol also inhibited the formation of inositol monophosphate caused by collagen, PAF and thrombin. The cAMP level of washed platelets was not changed by diisoeugenol. It is concluded that the antiplatelet effect of diisoeugenol is due to the inhibition of thromboxane formation and phosphoinositides breakdown.

Pharmacological activity of DC-015, a novel potent and selective alpha 1-adrenoceptor antagonist.

The pharmacological activity of 3-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)-2,3-dihydroimidaz o(1,2 -c)quinazolin-5(6H)-one (DC-015), a newly synthesized quinazoline derivative, was determined in rat isolated thoracic aorta and pressor responses were determined in spontaneously hypertensive rats (SHR). Experimental results indicated that DC-015 is an alpha 1-adrenoceptor-blocking agent in rat thoracic aorta as revealed by its competitive antagonism of phenylephrine-induced vasocontraction (pA2 = 10.54 +/- 0.55). These effects still persisted in denuded aorta. It was as potent as prazosin (pA2 = 10.04 +/- 0.63). At higher concentration (1.0 microM), DC-015 also expressed 5-hydroxytryptamine (5-HT) receptor competitive antagonism, but this 5-HT blocking effect was not found in the prazosin-administration group. [3H]Inositol monophosphate formation stimulated by phenylephrine (30 microM) in rat thoracic aorta was diminished by DC-015 (3 and 10 nM) and prazosin (10nM); whereas the cAMP content of rat thoracic aorta was not altered by DC-015 and prazosin. Furthermore, intravenous administration of DC-015 and prazosin (both at 0.01, 0.05 and 0.1 mg/kg-1) induced a dose-dependent reduction of mean arterial pressure which reached a maximal effect at 5 mm after injection and persisted over 2 h in SHR. A higher dose of DC-015 (0.1 mg/kg-1, i.v.) did not cause any significant changes in heart rate, whereas, the same dose of prazosin (0.1 mg/kg-1, i.v.) produced a decrease which seems to parallel the time course of the hypotensive response. We can conclude that the DC-015 is a potent, highly selective alpha 1-adrenoceptor antagonist in vascular smooth muscle.

CSH72—A Specific Antagonist of Platelet‐activating Factor in Rabbit Platelets

CSH72 (N-methyl-1-benzyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline HCl) inhibited the aggregation and ATP-release reaction induced by platelet-activating factor (PAF) in a concentration-dependent manner in rabbit washed platelets. The IC50 values of CSH72, kadsurenone and BN52021 on PAF (3·6 nM)-induced platelet aggregation were 61·5 ± 7·2, 18·0 ± 2·8 and 10·6 ± 2·3 μM, respectively. CSH72 also concentration-dependently inhibited [3H]PAF (3·6 nM) binding to washed platelets with an IC50 value of 46·1 ± 1·9 μM. The concentration-response curve of PAF-induced platelet aggregation was shifted to the right by CSH72 with pA2 and pA10 values of 5·47 and 4·68, respectively. CSH72 (300 μM) significantly inhibited PAF (3·6 nM)-induced thromboxane B2 formation but exhibited no effects on those induced by arachidonic acid (100 μM) and thrombin (0·1 units mL−1). CSH72 (300 μM) also completely abolished [3H]inositol monophosphate formation induced by PAF but not that caused by collagen or thrombin in the presence of indomethacin (20 μM). It is suggested that CSH72 is a specific PAF-receptor antagonist as an antiplatelet aggregatory agent.

Antiplatelet effect of marchantinquinone, isolated from Reboulia hemisphaerica, in rabbit washed platelets.

Platelet activation is involved in serious pathological situations, including atherosclerosis and restenosis. It is important to find efficient antiplatelet medicines to prevent fatal thrombous formation during the course of these diseases. Marchantinquinone, a natural compound isolated from Reboulia hemisphaerica, inhibited platelet aggregation and ATP release stimulated by thrombin (0.1 units mL(-1)), platelet-activating factor (PAF; 2 ng mL(-1)), collagen (10 microg mL(-1)), arachidonic acid (100 microM), or U46619 (1 microM) in rabbit washed platelets. The IC50 values of marchantinquinone on the inhibition of platelet aggregation induced by these five agonists were 62.0 +/- 9.0, 86.0 +/- 7.8, 13.6 +/- 4.7, 20.9 +/- 3.1 and 13.4 +/- 5.3 microM, respectively. Marchantinquinone inhibited thromboxane B2 (TxB2) formation induced by thrombin, PAF or collagen. However, marchantinquinone did not inhibit TxB2 formation induced by arachidonic acid, indicating that marchantinquinone did not affect the activity of cyclooxygenase and thromboxane synthase. Marchantinquinone did inhibit the rising intracellular Ca2+ concentration stimulated by the five platelet-aggregation inducers. The formation of inositol monophosphate induced by thrombin was inhibited by marchantinquinone. Platelet cAMP and cGMP levels were unchanged by marchantinquinone. The results indicate that marchantinquinone exerts antiplatelet effects by inhibiting phosphoinositide turnover.

SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride], a new nonpeptide antagonist of the bradykinin B1 receptor: biochemical and pharmacological characterization.

The biochemical and pharmacological properties of a novel non-peptide antagonist of the bradykinin (BK) B(1) receptor, SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride] were evaluated. SSR240612 inhibited the binding of [(3)H]Lys(0)-des-Arg(9)-BK to the B(1) receptor in human fibroblast MRC5 and to recombinant human B(1) receptor expressed in human embryonic kidney cells with inhibition constants (K(i)) of 0.48 and 0.73 nM, respectively. The compound selectivity for B(1) versus B(2) receptors was in the range of 500- to 1000-fold. SSR240612 inhibited Lys(0)-desAr(9)-BK (10 nM)-induced inositol monophosphate formation in human fibroblast MRC5, with an IC(50) of 1.9 nM. It also antagonized des-Arg(9)-BK-induced contractions of isolated rabbit aorta and mesenteric plexus of rat ileum with a pA(2) of 8.9 and 9.4, respectively. Antagonistic properties of SSR240612 were also demonstrated in vivo. SSR240612 inhibited des-Arg(9)-BK-induced paw edema in mice (3 and 10 mg/kg p.o. and 0.3 and 1 mg/kg i.p.). Moreover, SSR240612 reduced capsaicin-induced ear edema in mice (0.3, 3 and 30 mg/kg p.o.) and tissue destruction and neutrophil accumulation in the rat intestine following splanchnic artery occlusion/reperfusion (0.3 mg/kg i.v.). The compound also inhibited thermal hyperalgesia induced by UV irradiation (1 and 3 mg/kg p.o.) and the late phase of nociceptive response to formalin in rats (10 and 30 mg/kg p.o.). Finally, SSR240612 (20 and 30 mg/kg p.o.) prevented neuropathic thermal pain induced by sciatic nerve constriction in the rat. In conclusion, SSR240612 is a new, potent, and orally active specific non-peptide bradykinin B(1) receptor antagonist.

Role of protein kinase C in BSA-AGE-mediated inducible nitric oxide synthase expression in RAW 264.7 macrophages

In the present study, the roles of protein kinase C (PKC) in BSA-derived advanced glycosylation end products (BSA-AGEs)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression were investigated. Treatment of RAW 264.7 cells with BSA-AGEs caused dose- and time-dependent increases in NO release and iNOS expression in RAW 264.7 cells, whereas BSA alone had no effect on iNOS induction. The tyrosine kinase inhibitor (genistein), the phosphatidylinositol-specific phospholipase C inhibitor (U-73122), the phosphatidylcholine-specific phospholipase C inhibitor (D-609), and the PKC inhibitors (staurosporine, Ro 31-8220, and Go 6976) all inhibited BSA-AGE-induced NO release and iNOS expression in RAW 264.7 cells. Stimulation of RAW 264.7 cells with BSA-AGEs resulted in the formation of inositol monophosphate; the response was attenuated by U-73122 and genistein. BSA-AGEs stimulated PKC-α, -βI, -δ, and -η but not -ζ translocation from the cytosol to the membrane. However, incubation of RAW 264.7 cells with BSA-AGEs increased phosphorylation of PKC-ζ at threonine-410, which reflects activation of PKC-ζ, indicating the possible involvement of these PKC isoforms in AGE-mediated effects. Pretreatment of RAW 264.7 cells with U-73122, D-609, and genistein reduced the AGE-stimulated translocation of PKC-α, -βI, -δ, and -η and activation of PKC-ζ. Taken together, these data suggest that BSA-AGEs might activate PKC and subsequently induce iNOS expression and NO release.

The antiplatelet activity of Geiji-Bokryung-Hwan, Korean traditional formulation, is mediated through inhibition of phospholipase C and inhibition of TxB 2 synthetase activity

Geiji-Bokryung-Hwan (GBH), consisting of herbes of Cinnamomi ramulus (Geiji), Poria cocos (Bokryun), Mountan cortex radicis (Mokdanpi), Paeoniae radix (Jakyak), and Persicae semen (Doin), on antiplatelet activity in human platelet suspensions was studied. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes and GBH (15 and 30 μg/ml) significantly inhibited [ 3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. GBH did not significantly affect nitrate production in collagen (10 μg/ml)-induced human platelet aggregation. Various concentrations of GBH (0, 5, 10, 15, and 30 μg/ml) dose-dependently inhibited [ 3H]inositol monophosphate formation stimulated by collagen (10 μg/ml) in [ 3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 min). These results indicated that the antiplatelet activity of GBH may possibly be due to the inhibition of phospholipase C (PLC) activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A 2 formation, and then inhibition of [Ca 2+] i mobilization of platelet aggregation stimulated by agonists. In conclusion, GBH suppressed PLC in a dose-dependent manner, and may have pharmaceutical applications. These data suggest that GBH extracts merit investigation as a potential anti-atherosclerogenic agent in humans.

The Antiplatelet Activity of Danggijakyaksan by Inhibition of Phospholipase C

We investigated the effects of the traditional Korean prescription, Danggijakyaksan (DJS) on antiplatelet activity in human platelet suspensions. The effect of oriental medicinal prescriptions, Danggijakyaksan consisting of 6 herbes of Paeoniae Radix (2 g), Poria Cocos (1.33 g), Angelicae Sinensis Radix (1 g), Cnidii Rhizoma (1 g), Atractylodis Macrocephalae Rhizoma (1.33 g) and Alismatis Rhizoma (1.66 g), was studied. In this study, the mechanism involved in the antiplatelet activity of DJS in human platelet suspensions was investigated. Danggijakyaksan did not significantly affect the thromboxane synthetase activity of aspirin‐treated platelet microsomes and DJS (20 and 40 µg/mL) significantly inhibited [3H]arachidonic acid (AA) released in collagen‐activated platelets but not in unactivated‐platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. Danggijakyaksan did not significantly affect nitrate production in collagen (10 µg/mL)‐induced human platelet aggregation. On the other hand, various concentrations of DJS (10, 20, and 40 µg/mL) dose‐dependently inhibited [3H]inositol monophosphate (IP) formation stimulated by collagen (10 µg/mL) in [3H]myoinositol‐loaded platelets at different incubation times (1, 2, 3, and 5 min). It is concluded that the antiplatelet activity of DJS may possibly be due to the inhibition of phospholipase C activity, leading to reduced phosphoinositide breakdown, followed by the inhibition of thromboxane A2 formation, and then inhibition of [Ca2+]i mobilization of platelet aggregation stimulated by agonists.

SSR240600 [(R)-2-(1-[2-[4-[2-[3,5-bis(trifluoromethyl)phenyl]acetyl]-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl]- 4-piperidinyl)-2-methylpropanamide], a centrally active nonpeptide antagonist of the tachykinin neurokinin-1 receptor: I. biochemical and pharmacological characterization.

The biochemical and pharmacological properties of a novel antagonist of the tachykinin neurokinin 1 (NK1) receptor, SSR240600 [(R)-2-(1-[2-[4-[2-[3,5-bis(trifluoromethyl)phenyl]acetyl]-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl]-4-piperidinyl)-2-methylpropanamide], were evaluated. SSR240600 inhibited the binding of radioactive substance P to tachykinin NK1 receptors in human lymphoblastic IM9 cells (K(i) = 0.0061 nM), human astrocytoma U373MG cells (K(i) = 0.10 nM), and human brain cortex (IC50 = 0.017 nM). It also showed subnanomolar affinity for guinea pig NK1 receptors but was less potent on rat and gerbil NK1 receptors. SSR240600 inhibited [Sar(9),Met(O2)(11)]substance P-induced inositol monophosphate formation in human astrocytoma U373MG cells with an IC50 value of 0.66 nM (agonist concentration of 100 nM). It also antagonized substance P-induced contractions of isolated human small bronchi with a pIC50 value of 8.6 (agonist concentration of 100 nM). The compound was >100- to 1000-fold more selective for tachykinin NK1 receptors versus tachykinin NK2 or NK3 receptors as evaluated in binding and in vitro functional assays. In vivo antagonistic activity of SSR240600 was demonstrated on tachykinin NK1 receptor-mediated hypotension in dogs (3 and 10 microg/kg i.v.), microvascular leakage (1 and 3 mg/kg i.p.), and bronchoconstriction (50 and 100 microg/kg i.v.) in guinea pigs. It also prevented citric acid-induced cough in guinea pigs (1-10 mg/kg i.p.), an animal model in which central endogenous tachykinins are suspected to play a major role. In conclusion, SSR240600 is a new, potent, and centrally active antagonist of the tachykinin NK1 receptor, able to antagonize various NK1 receptor-mediated pharmacological effects in the periphery and in the central nervous system.

Biochemical and pharmacological activities of SSR 146977, a new potent nonpeptide tachykinin NK3 receptor antagonist.

SSR 146977 is a potent and selective antagonist of the tachykinin NK3 receptor. In Chinese hamster ovary cells expressing the human tachykinin NK3 receptor, SSR 146977 inhibited the binding of radioactive neurokinin B to NK3 receptors (Ki = 0.26 nM), senktide (10 nM) induced inositol monophosphate formation (IC50 = 7.8-13 nM), and intracellular calcium mobilization (IC50 = 10 nM). It antagonized [MePhe7]neurokinin B induced contractions of guinea pig ileum (pA2 = 9.07). Senktide (30 nM) induced firing rate increase of noradrenergic neurons in the guinea pig locus coeruleus and dopaminergic neurons in the guinea pig substantia nigra was also blocked by SSR 146977 (50 and 100 nM, respectively). In vivo, in the respiratory system, SSR 146977 inhibited bronchial hyperresponsiveness to acetylcholine, bronchial microvascular permeability hypersensitivity to histamine (doses of 0.1-1 mg/kg i.p.), and cough (doses of 0.03-1 mg/kg i.p.) provoked by citric acid in guinea pigs. In the central nervous system, SSR 146977 inhibited turning behaviour (ID50 = 0.2 mg/kg i.p. and 0.4 mg/kg p.o.) and prevented the decrease of locomotor activity (10 and 30 mg/kg i.p) mediated by the stimulation of NK3 receptors in gerbils. In guinea pigs, SSR 146977 antagonized senktide-induced acetylcholine release in the hippocampus (0.3 and 1 mg/kg i.p) and norepinephrine release in the prefrontal cortex (0.3 mg/kg i.p.). It also prevented haloperidol-induced increase of the number of spontaneously active dopamine A10 neurons (1 and 3 mg/kg i.p.).

Circular muscle contraction, messenger signalling and localization of binding sites for neurokinin A in human sigmoid colon.

1. Neurokinin (NK)A is the endogenous ligand for the tachykinin NK2 receptor. In the present study, tachykinins and selective receptor agonists were tested as contractile agonists in human colon circular muscle and [125I]-NKA was used to localize binding sites in human colon. 2. In strips of circular muscle, removal of mucosa and submucosa significantly (P < 0.05) increased the potency and the maximum response achieved by NKA. 3. The rank order of potency of tachykinin and selective receptor agonists in contracting circular muscle strips was NKA > or = [Lys5,MeLeu9,Nle10]NKA(4-10) > or = neuropeptide (NP)gamma > or = [betaAla8]NKA(4-10) >> NKB > substance P (SP) >> senktide approximate to [Pro9]SP. 4. Specific binding sites for [125I]-NKA were densely localized over circular muscle and muscularis mucosae. Weak specific binding was seen on longitudinal muscle and taenia coli, whereas no binding sites were seen on mucosa, ganglia or blood vessels. 5. In circular muscle, the selective NK2 receptor agonist [LysS,MeLeu9,Nle10]NKA(4-10) produced weak increases (maximum 37%) in inositol monophosphate formation with a pD2 of 6.8+/-0.51 (n = 3). Carbachol (100 micromol/L) was also a weak stimulant (maximum 45%). These agonists were over 10-fold more efficacious in stimulation of inositol monophosphate in rat urinary bladder. 6. In conclusion, [125I]-NKA binding sites localized on human colon circular muscle were characterized as NK2 receptors. Functionally, the tachykinin NK2 receptor is mediating circular smooth muscle contraction. Although the human NK2 receptor is coupled to the phosphatidylinositol pathway, other second messenger mechanisms may also operate in this tissue.

Pharmacological and biochemical investigation of receptors for the toad gut tachykinin peptide, bufokinin, in its species of origin.

This is the first report of the development of a new radioligand [125I]Bolton-Hunter bufokinin ([125I]BH-bufokinin) and its use in the characterisation of tachykinin receptors in the small intestine of the cane toad, Bufo matrinus. The binding of [125I]BH-bufokinin to toad intestinal membranes was rapid, saturable, of high affinity and to a single population of binding sites with KD 0.57 nM and Bmax 3.1 fmol mg wet weight tissue(-1). The rank order of affinity of tachykinins to compete for [125I]-BH bufokinin binding revealed similarities with that of the mammalian NK1 receptor, being bufokinin (IC50, 1.7 nM)>physalaemin (6.7 nM)>substance P (SP, 10.7 nM)> or =neuropeptide gamma (NPgamma, 12.4 nM)> or =kassinin (17.8 nM)>scyliorhinin I (35.3 nM)> or =eledoisin (40.6 nM)> or =carassin (43.2 nM)> or =neurokinin A (NKA, 57.8 nM)> or =neurokinin B (NKB, 77.5 nM)>scyliorhinin II (338 nM). The mammalian NK3-selective agonist senktide was a very weak competitor. The radioligand [125I]neurokinin A showed no specific binding to toad intestinal membranes. In the toad isolated small intestine, the maximum contractile response to bufokinin was over 150% greater than that to acetylcholine in longitudinal muscle, whereas responses to bufokinin and acetylcholine were similar in circular muscle. Bufokinin was the most potent agonist (EC501 0.34 nM) and produced a long-lasting contraction. Other tachykinins such as physalaemin, SP and kassinin were also potent contractile agents. The potency values of mammalian and amphibian tachykinins derived from functional studies (pD2) correlated significantly with those from binding assays (pKi). The data for fish and molluscan tachykinins, however, showed poor correlation. Contractions to bufokinin and SP were unaffected by atropine, indomethacin and tetrodotoxin. The highly selective NK1 receptor antagonists CP 99994, GR 82334 and RP 67580 were ineffective in both binding and functional studies. Bufokinin increased inositol monophosphate formation in a concentration-dependent manner with an EC50 value of 10.7 nM, suggesting that the tachykinin receptor may be coupled to phosphoinositol hydrolysis. In summary, this study provides evidence for a high-affinity, bufokinin-preferring, NK1-like tachykinin receptor in the toad small intestine. This is probably not the receptor which mediates contraction to carassin, scyliorhinin II and eledoisin. The study also provides evidence that bufokinin and its receptor play an important physiological role in regulating intestinal motility.

The Antiplatelet Activity of Rutaecarpine, an Alkaloid Isolated from Evodia rutaecarpa, Is Mediated through Inhibition of Phospholipase C

In this study, the mechanism involved in the antiplatelet activity of rutaecarpine in human platelet suspensions was investigated. In platelet suspensions (4.5×10 8/ml), rutaecarpine (100 and 200 μM) did not influence the binding of FITC-triflavin to platelet glycoprotein IIb/IIIa complex. Additionally, rutaecarpine (200 μM) did not significantly change the fluorescence of platelet membrane labeled with diphenylhexatriene (DPH). On the other hand, rutaecarpine (50 and 100 μM) dose-dependently inhibited the increase in intracellular free Ca 2+ of Fura 2-AM loaded platelets stimulated by collagen. Moreover, rutaecarpine (100 and 200 μM) did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. Furthermore, retaecarpine (100 and 200 μM) significantly inhibited [ 3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. Rutaecarpine (100 and 200 μM) did not significantly affect nitrate production in collagen (10 μg/ml)-induced human platelet aggregation. On the other hand, various concentrations of rutaecarpine (50, 100, and 200 μM) dose-dependently inhibited [ 3H]inositol monophosphate formation stimulated by collagen (10 μg/ml) in [ 3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 minutes). It is concluded that the antiplatelet activity of rutaecarpine may possibly be due to the inhibition of phospholipase C activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A 2 formation, and then inhibition of [Ca 2+]i mobilization of platelet aggregation stimulated by agonists.

Inhibitory mechanisms of naloxone on human platelets.

1. In the present study, naloxone was tested for its antiplatelet activities in human platelet-rich plasma (PRP). In human PRP, naloxone (0.1-0.5 mmol/L) inhibited aggregation stimulated by a variety of agonists (i.e. collagen, adenosine diphosphate (ADP), U46619 and adrenaline). 2. Naloxone (0.1-0.5 mmol/L) did not significantly affect cyclic adenosine monophosphate and cGMP levels in human washed platelets, whereas naloxone (0.5 mmol/L) significantly inhibited thromboxane B2 formation stimulated by collagen (5 micrograms/mL) in human washed platelets. 3. Naloxone (0.5 mmol/L) significantly inhibited [3H]-inositol monophosphate formation of [3H]-myoinositol-loaded platelets stimulated by collagen and U46619. Moreover, naloxone did not influence the binding of 125I-triflavin to platelet membranes. Triflavin is an Arg-Gly-Asp-containing specific fibrinogen receptor antagonist. 4. Addition of naloxone (0.5 mmol/L) to platelet preparations tagged with diphenylhexatriene (DPH) resulted in a considerable decrease in relative fluorescence intensity. 5. It is suggested that the anti-platelet effects of naloxone may be caused, at least partly, by the induction of conformational changes in the platelet membrane initially, followed by the inhibition of thromboxane A2 formation and phosphoinositide breakdown of platelets stimulated by agonists.

Metabotropic glutamate receptor agonists stimulate polyphosphoinositide hydrolysis in primary cultures of rat hepatocytes

The metabotropic glutamate (mGlu) receptor agonists, quisqualate and (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), but not ( RS)-3,5-dihydroxyphenylglycine or (2 S,3 S,4 S)- α-(carboxycyclopropyl)glycine, stimulated [ 3 H ]inositolmonophosphate ([ 3 H ]InsP) formation in primary cultures of rat hepatocytes. 1 S,3 R-ACPD-stimulated [ 3 H ]InsP formation was inhibited by α-methyl-4-carboxyphenylglycine, indicating that cultured hepatocytes express functional mGlu receptors coupled to polyphosphoinositide hydrolysis. The identity of these receptors is not similar to that of any of the known mGlu receptor subtypes characterized in heterologous expression systems.

Mechanisms Involved in the Antiplatelet Activity of Tetramethylpyrazine in Human Platelets

Tetramethylpyrazine is the active ingredient of a Chinese herbal medicine. In this study, tetramethylpyrazine was tested for its antiplatelet activities in human platelet suspensions. In human platelets, tetramethylpyrazine (0.5–1.5 mM) dose-dependently inhibited both platelet aggregation and ATP-release reaction induced by a variety of agonists (i.e., ADP, collagen, and U46619). Tetramethylpyrazine (0.5 mM) did not significantly change the fluorescence of platelet membranes labeled with diphenylhexatriene, even at the high concentration (1.5 mM). Furthermore, tetramethylpyrazine (0.5–1.5 mM) dose-dependently inhibited [ 3H]inositol monophosphate formation stimulated by collagen (5 μg/ml) in [ 3H]myoinositol loaded platelets. Tetramethylpyrazine (0.5–1.5 mM) also dose-dependently inhibited the intracellular free Ca +2 rise of Fura 2-AM loaded platelets stimulated by collagen (5 μg/ml). Moreover, tetramethylpyrazine (0.5–1.5 mM) inhibited thromboxane B 2 formation stimulated by collagen. At a higher concentration (1.0 mM), tetramethylpyrazine has also been shown to influence the binding of FITC-triflavin to platelet glycoprotein IIb/IIIa complex. Triflavin, a specific glycoprotein IIb/IIIa complex antagonist purified from Trimeresurus flavoviridis venom. It is concluded that the antiplatelet activity of tetramethylpyrazine may possibly involve two pathways: 1) at a lower concentration (0.5 mM), tetramethylpyrazine is shown to inhibit phosphoinositide breakdown and thromboxane A 2 formation; and 2) at a higher concentration (1.0 mM), it leads to the inhibition of platelet aggregation through binding to the glycoprotein IIb/IIIa complex.

HISTAMINE‐STIMULATED PHOSPHOLIPASE C SIGNALLING IN THE ADRENAL CHROMAFFIN CELL: EFFECTS ON INOSITOL PHOSPHOLIPID METABOLISM AND TYROSINE HYDROXYLASE PHOSPHORYLATION

1. The present report gives a detailed account of histamine-stimulated phospholipase C (PLC) activity in bovine adrenal chromaffin cells. 2. Histamine activation of H1 receptors stimulates PLC with a biphasic sensitivity to extracellular Ca2+. The initial response (the first 15 s stimulation) was not reduced by the removal of extracellular Ca2+, whereas the maintenance of PLC activity beyond this time required Ca2+ influx. 3. Phospholipase C activity in response to a 10 min incubation with histamine was inhibited by La3+ (3 mmol/L) or SKF96365 (10 mumol/L). Nifedipine (10 mumol/L), but not omega-agatoxin IVA (100 nmol/L) or omega-conotoxin GVIA (300 nmol/L), produced a partial inhibition of PLC activity. The response was also partially inhibited by a reduction in the extracellular Cl- concentration (40 mmol/L) or by the inclusion of the Cl- channel blocker N-phenylanthranilic acid (300 mumol/L). 4. Kinetic analysis of the rate of turnover of the various inositol phosphate isomers in response to histamine suggested that the inositol monophosphates were being produced from a source in addition to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) metabolism. This conclusion was supported by the differential action of pertussis toxin and neomycin on Ins(1,4,5)P3 formation compared with inositol monophosphate formation. 5. We have attempted to identify a defined role for the intracellular Ca2+ mobilized in these cells in response to histamine. After short incubations (up to 3 min), histamine was able to regulate the site-specific phosphorylation of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. This observation has important implications for a possible role for the PLC signalling pathway in controlling the rate of catecholamine biosynthesis.

Low-affinity thromboxane receptor mediates proliferation in cultured vascular smooth muscle cells of rats.

We examined the binding properties and mitogenic effects of U46619, using cultured vascular smooth muscle cells (VSMCs), by ligand-binding assay, measuring [3H]thymidine and [3H]leucine incorporation, checking with flow cytometry, and counting the cell number. The U46619-activated mitogenic signal-transduction pathway was assessed by measuring formation of inositol monophosphate (IP); [Ca2+]i; mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK), and p74raf-1 activities; and GTP-bound Ras. [3H]U46619 bound to cultured VSMCs from Wistar-Kyoto (WKY) rats at a single class of site (Kd: 15.5 +/- 2.6 nmol/L). However, it bound to VSMCs from spontaneously hypertensive rats (SHRs) at two classes of sites (Kd: 2.3 +/- 0.6 nmol/L and 1.4 +/- 0.5 mumol/L). U46619 increased DNA and protein synthesis, cell number, IP formation, [Ca2+]i, and MAPK and MAPKK activities, with EC50 values close to its Kd value for the low-affinity binding site in VSMCs from SHR. Prostaglandin (PG) E2 and PGF2 alpha showed little of such mitogenic effects. All these effects of U46619 were inhibited by SQ29548, staurosporine, or pretreatment of VSMCs with phorbol 12-myristate 13-acetate for 24 hours. However, U46619 stimulation did not lead to a significant increase in the Ras-GTP complex or p74raf-1 activity. In conclusion, the mitogenic effect of U46619 appears to be mediated via the activation of low-affinity thromboxane binding sites that trigger phosphoinositide hydrolysis and activate the MAPK pathway, leading to DNA synthesis and cell proliferation.