Coagulation factor Xa stimulates platelet-derived growth factor release and mitogenesis in cultured vascular smooth muscle cells of rat.

The mitogenic effect of activated coagulation factor X (factor Xa) was examined in cultured aortic smooth muscle cells (VSMC) from Wistar-Kyoto rats (WKY). Factor Xa stimulated DNA synthesis and cell growth in VSMC, not through the phospholipase C-protein kinase C pathway because increase of inositol monophosphate (IP) accumulation and intracellular Ca2+ concentration was not observed, but probably via the PDGF receptor tyrosine kinase pathway since the pathway's components, Ras, Raf-1, MAPK (both 42 and 44 kD), and the transcription factors, c-Fos and c-Jun, were activated. These appeared to be effected by the serine protease activity of factor Xa, since in the presence of serine protease inhibitors such as PMSF, leupeptin, benzamidine, TAP anticoagulant, and TLCK, the latter three being specific inhibitors of the factor Xa, active site, the effects were completely blocked. Anti-factor Xa mAb, 5224, which specifically negated the activity of factor Xa, also inhibited completely the mitogenic effect of factor Xa, but not that of thrombin. Addition of PDGF did not affect the effect of factor Xa, which, however, was inhibited by anti-PDGF-AB antibody. This observation and the activation of PDGF receptor tyrosine kinase pathway suggested that the factor Xa might exert its effect via PDGF-like function. Direct measurement confirmed that factor Xa stimulated the release of PDGF from VSMC. Factor Xa, therefore, exerts serine protease activity on VSMC, causing somehow the release of PDGF, that in turn acts on the PDGF receptor tyrosine kinase; the pathway is then turned on, leading eventually to DNA synthesis and cell proliferation.

Antiplatelet effects of clausine-D isolated from Clausena excavata.

Clausine-D inhibited concentration-dependently the aggregation and release reaction of washed rabbit platelets induced by arachidonic acid and collagen, without affecting those induced by U46619, PAF and thrombin. The IC50 values of clausine-D on arachidonic acid- and collagen-induced platelet aggregation were calculated to be 9.0 +/- 1.1 and 58.9 +/- 0.9 microM, respectively. Thromboxane B2 and prostaglandin D2 formation in platelets caused by arachidonic acid were also suppressed. Clausine-D inhibited increased intracellular concentration of calcium in platelets caused by arachidonic acid and collagen, and also abolished the generation of inositol monophosphate caused by arachidonic acid, but not that by collagen, U46619, PAF and thrombin. In human citrated platelet-rich plasma, clausine-D inhibited the secondary phase, but not the primary phase, of aggregation induced by epinephrine and ADP. These results indicate that the antiplatelet effect of clausine-D is due to inhibition of the formation of thromboxane A2.

Antioxidant properties of demethyldiisoeugenol.

The antioxidant properties of demethyldiisoeugenol were investigated in this study using various models. Demethyldiisoeugenol inhibited Fe(2+)-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC50 1.8 +/- 0.1 microM. Demethyldiisoeugenol was more effective than alpha-tocopherol and BHT in reducing the stable free radical diphenyl-2-picrylhydrazyl (DPPH). It also scavenged superoxide anion generated by xanthine/xanthine oxidase and peroxyl radical (ROO.) derived from 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) in aqueous system with a stoichiometric factor of 1.3 +/- 0.2. Furthermore, it prevented conjugated-diene formation and apolipoprotein B (apo B) oxidation of LDL. However, demethyldiisoeugenol did not scavenge 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN)-derived peroxyl radical in hexane. It also did not chelate Fe2+, did not inhibit xanthine oxidase activity or possessed hydroxyl radical (.OH) scavenging activity. Experimental results indicate that demethyldiisoeugenol is a potentially effective antioxidant and can protect rat brain homogenate and LDL against oxidation.

Antioxidant properties of isotorachrysone isolated from Rhamnus nakaharai.

Isotorachrysone inhibited iron-induced lipid peroxidation with an IC50 value of 1.64 +/- 0.08 microM in rat brain homogenates, and was comparable in potency to butylated hydroxytoluene and was more potent than alpha-tocopherol or desferrioxamine. The mechanism of antioxidant properties were then examined. Isotorachrysone could scavenge the stable free radical diphenyl-p-picrylhydrazyl. And it was an efficient direct scavenger of water-soluble peroxyl radicals with stoichiometry factor of 0.53 +/- 0.05 in the aqueous phase and also toward lipid-soluble peroxyl radicals in tissue homogenates. The oxygen consumption during peroxidation induced by radicals on human erythrocyte ghosts was suppressed by isotorachrysone. Furthermore, it was reactive towards superoxide anion with a second-order rate constant of 5.06 +/- 0.65 x 10(5) M-1 S-1. But it did not react with hydrogen peroxide detected within the sensitivity limit of our assay. Using ascorbate/iron ion/H2O2 as a hydroxyl radical generating system and deoxyribose as a probe, isotorachrysone was effective with hydroxyl radicals with a second-order rate constant of 3.88 +/- 0.54 x 10(11) M-1 S-1 under stimulation by iron-EDTA. On the other hand, isotorachrysone retarded the peroxidation of human low density lipoprotein (LDL) initiated by both aqueous and lipophilic peroxyl radicals. And it also suppressed copper-catalyzed human LDL oxidation, as measured by fluorescence intensity, electrophoretic mobility, and thiobarbituric acid-reactive substances formation in a concentration-dependent manner. Our results show that isotorachrysone is potentially an effective and versatile antioxidant, and can help protecting LDL against oxidation.

Vasodilatory action mechanisms of apigenin isolated from Apium graveolens in rat thoracic aorta.

The effect of apigenin, isolated from Apium graveolens, on the contraction of rat thoracic aorta was studied. Apigenin inhibited the contraction of aortic rings caused by cumulative concentrations of calcium (0.03-3 mM) in high potassium (60 mM) medium, with an IC50 of about 48 microM. After pretreatment it also inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contraction in a concentration (35-140 microM)-dependent manner with an IC50 of 63 microM. At the plateau of NE-induced tonic contraction, addition of apigenin caused relaxation. This relaxing effect of apigenin was not antagonized by indomethacin (20 microM) or methylene blue (50 microM), and still existed in endothelial denuded rat aorta or in the presence of nifedipine (2-100 microM). Neither cAMP nor cGMP levels were changed by apigenin. Both the formation of inositol monophosphate caused by NE and the phasic contraction induced by caffeine in the Ca(2+)-free solution were unaffected by apigenin. 45Ca2+ influx caused by either NE or K+ was inhibited by apigenin concentration-dependently. It is concluded that apigenin relaxes rat thoracic aorta mainly by suppressing the Ca2+ influx through both voltage- and receptor-operated calcium channels.

Inhibition on platelet activation by shikonin derivatives isolated from Arnebia euchroma.

Acetylshikonin, teracrylshikonin, beta,beta-dimethylacrylshikonin and shikonin, isolated from Arnebia euchroma, inhibited collagen (10 micrograms/ml)-induced aggregation of washed rabbit platelets in a concentration-dependent manner with IC50 values of 2.1 +/- 0.2, 2.8 +/- 0.3, 4.2 +/- 0.5 and 10.7 +/- 0.7 microM, respectively. Acetylshikonin also inhibited the aggregation and ATP release of washed rabbit platelets induced by arachidonic acid (AA, 100 microM), U46619 (1 microM), platelet-activating factor (PAF, 3.6 nM) and thrombin (0.1 U/ml) in a concentration-dependent manner. The IC50 values of acetylshikonin on the inhibition of these four agonists-induced platelet aggregation were 3.1 +/- 0.4, 2.2 +/- 0.2, 8.0 +/- 0.6 and 12.7 +/- 1.0 microM, respectively. The thromboxane B2 formation caused by collagen, PAF and thrombin was inhibited by acetylshikonin, while formations of thromboxane B2 and prostaglandin D2 caused by AA were not inhibited. Acetylshikonin did not inhibit cyclooxygenase activity since it did not attenuate prostaglandin E2 formation after incubation of sheep vesicular gland microsomes with AA. Acetylshikonin suppressed both the rise of intracellular Ca2+ concentration and the generation of [3H]inositol monophosphate caused by these five aggregation inducers. Platelet cyclic AMP level was unaffected by acetylshikonin. These data indicate that acetylshikonin inhibits platelet activation by suppression of phosphoinositide breakdown.

Isoorientin-6"-O-glucoside, a water-soluble antioxidant isolated from Gentiana arisanensis.

The antioxidant activities of isoorientin-6"-O-glucoside were studied using various models. Isoorientin-6"-O-glucoside was more potent than Trolox, probucol and butylated hydroxytoluene (BHT) in reducing the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). It also scavenged superoxide anion, peroxyl and hydroxyl radicals that were generated by xanthine/xanthine oxidase, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and Fe3+-ascorbate-EDTA-H2O2 system, respectively. The IC50 value, stoichiometry factor and second-order rate constant were 9.0+/-0.8 microM, 1.8+/-0.1 and 2.6 X 10(10) M(-1) s(-1) for superoxide generation, peroxyl and hydroxyl radicals. However, isoorientin-6"-O-glucoside did not inhibit xanthine oxidase activity or scavenge hydrogen peroxide (H2O2), carbon radical or 2,2'-azobis(2,4-dimethyl-valeronitrile) (AMVN)-derived peroxyl radical in hexane. Isoorientin-6"-O-glucoside inhibited Cu2+-induced oxidation of human low-density lipoprotein (LDL) as measured by fluorescence intensity, thiobarbituric acid-reactive substance formation and electrophoretic mobility. Since isoorientin-6"-O-glucoside did not possess pro-oxidant activity, it may be an effective water-soluble antioxidant that can prevent LDL against oxidation.

Antioxidant properties of butein isolated from Dalbergia odorifera.

The antioxidant properties of butein, isolated from Dalbergia odorifera T. Chen, were investigated in this study. Butein inhibited iron-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC50, 3.3+/-0.4 microM. It was as potent as alpha-tocopherol in reducing the stable free radical diphenyl-2-picrylhydrazyl (DPPH) with an IC0.200, 9.2+/-1.8 microM. It also inhibited the activity of xanthine oxidase with an IC50, 5.9+/-0.3 microM. Besides, butein scavenged the peroxyl radical derived from 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) in aqueous phase, but not that from 2,2-azobis(2, 4-dimethylvaleronitrile) (AMVN) in hexane. Furthermore, butein inhibited copper-catalyzed oxidation of human low-density lipoprotein (LDL), as measured by conjugated dienes and thiobarbituric acid-reactive substance (TBARS) formations, and electrophoretic mobility in a concentration-dependent manner. Spectral analysis revealed that butein was a chelator of ferrous and copper ions. It is proposed that butein serves as a powerful antioxidant against lipid and LDL peroxidation by its versatile free radical scavenging actions and metal ion chelation.

CIS-19, a novel platelet activating factor receptor antagonist: in vitro and in vivo studies.

The effects of CIS-19 (cis-2-(3,4-dimethoxyphenyl)-6-isopropoxy-7-methoxyl-1-(N-methylforma mido)-1,2, 3,4-tetrahydronaphthalene) was determined in vitro in rabbit platelets and in vivo in rats and guinea-pigs. CIS-19 inhibited in a selective and concentration-dependent manner the aggregation and ATP release reaction of rabbit platelets induced by PAF (4 nM). The IC50 values of CIS-19 on PAF-induced aggregation of washed platelets and platelet-rich plasma were 11.3 +/- 2.7 and 16.8 +/- 3.0 microM respectively. BN52021 also inhibited PAF-induced aggregation of washed platelets with an IC50 value of 11.7 +/- 2.8 microM. CIS-19 inhibited [3H]PAF (4 nM) binding to washed rabbit platelets with an IC50 value of 1.5 +/- 0.2 microM. The concentration-response curve of PAF-induced aggregation of washed platelets was shifted rightwards by CIS-19 with pA2 and pA10 values of 7.1 (6.8-7.3 for 95% confidence limit) and 6.1 (5.8-6.2) respectively. The thromboxane B2 formation of washed platelets caused by AA, collagen or thrombin was not affected by CIS-19 of concentrations below 400 microM. CIS-19 (25 microM) completely blocked PAF-induced, but not collagen- or thrombin-induced [3H]inositol monophosphate formation of washed platelets. When CIS-19 (2.5 and 5 mg/kg) was injected i.v. into the femoral vein, it did not affect the blood pressure of rats, but antagonized PAF (2.5 micrograms/kg, i.v.)-induced hypotensive shock either preventively or curatively. CIS-19 (2.5 and 5 mg/kg) also blocked PAF (50 ng/kg)-induced, but not AA (50 micrograms/kg)-induced, bronchoconstriction in guinea-pigs. It is concluded that CIS-19 is an effective PAF receptor antagonist not only in vitro, but also in vivo.

Antiplatelet effects of chelerythrine chloride isolated from Zanthoxylum simulans.

Chelerythrine chloride is an antiplatelet agent isolated from Zanthoxylum simulans. Aggregation and ATP release of washed rabbit platelets caused by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin were inhibited by chelerythrine chloride. Less inhibition was observed in platelet-rich plasma. The thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, ionophore A23187 and thrombin was decreased by chelerythrine chloride. Phosphoinositides breakdown caused by collagen and PAF was completely inhibited by chelerythrine chloride, while that of thrombin was only partially suppressed. Chelerythrine chloride inhibited the intracellular calcium increase caused by arachidonic acid, PAF, collagen and thrombin in quin-2/AM-loaded platelets. The cyclic AMP level of washed platelets did not elevated by chelerythrine chloride. The antiplatelet effect of chelerythrine chloride was not dependent on the incubation time and the aggregability of platelets inhibited by chelerythrine chloride was easily recovered after sedimenting the platelets by centrifugation and then the platelet pellets were resuspended. Chelerythrine chloride did not cause any platelet lysis, since lactate dehydrogenase activity was not found in the supernatant. These data indicate that the inhibitory effect of chelerythrine chloride on rabbit platelet aggregation and release reaction is due to the inhibition on thromboxane formation and phosphoinositides breakdown.

Antiplatelet actions of panaxynol and ginsenosides isolated from ginseng.

The antiplatelet effect of panaxynol isolated from the diethyl ether layer was compared with those of ginsenosides from the butanol layer of Panax ginseng. Panaxynol (0.1 mg/ml) inhibited markedly the aggregation of washed platelets induced by collagen, arachidonic acid, ADP, ionophore A23187, PAF and thrombin while ginsenosides had no significant effect on the aggregation but ginsenoside Ro (1 mg/ml) inhibited the ATP release of platelets. Less inhibitory effect of panaxynol was observed in the aggregation of platelet-rich plasma. Thromboxane B2 formation of platelets was inhibited by panaxynol but not by ginsenosides. The antiplatelet effect of panaxynol was dependent on the incubation time and the aggregability of platelets inhibited by panaxynol could not easily be recovered after washing the platelets. In human platelet-rich plasma, panaxynol prevented secondary aggregation and completely blocked ATP release from platelets induced by epinephrine and ADP. Both panaxynol and ginsenoside Rg2 inhibited the rise of intracellular calcium caused by collagen. It is concluded that panaxynol is the most potent antiplatelet agent in ginseng and its mechanism of action is chiefly due to the inhibition of thromboxane formation.

Protection of oxidative hemolysis by demethyldiisoeugenol in normal and beta-thalassemic red blood cells.

The purpose of this study was to evaluate the ability of demethyldiisoeugenol to protect normal and beta-thalassemic human red blood cells (RBCs) against oxidative damage in vitro. Oxidative hemolysis and lipid peroxidation of normal and beta-thalassemic human RBCs induced by aqueous peroxyl radical were suppressed by demethyldiisoeugenol in a concentration-dependent manner. The formation of proteins with high molecular weight and concomitant decrease of the low-molecular-weight proteins of RBCs challenge with aqueous peroxyl radical were inhibited by demethyldiisoeugenol. It also prevented the shortening of the Russell's viper venom (RVV)-clotting time mediated by prelytic radical-treated RBCs. In contrast, demethyldiisoeugenol inhibited oxidative hemolysis but not those metHb and ferrylHb formations caused by hydrogen peroxide (H2O2) in normal RBCs. Furthermore, demethyldiisoeugenol did not prevent the consumption of the cytosolic antioxidant, glutathione (GSH), in radical-treated RBCs. It also did not cause of a loss of sulfhydryl group during incubation with GSH. However, the diphenyl-2-picrylhydrazyl (DPPH) scavenging activity of demethyldiisoeugenol was dramatically increased in the presence of GSH. These results imply that demethyldiisoeugenol can regenerate from its oxidized form to its active reduced form in the presence of GSH. It may be useful in diminishing oxidative damage to pathological RBCs.

Scavenger and antioxidant properties of prenylflavones isolated from Artocarpus heterophyllus.

The antioxidant properties of prenylflavones, isolated from Artocarpus heterophyllus Lam., was evaluated in this study. Among them, artocarpine, artocarpetin, artocarpetin A, and cycloheterophyllin diacetate and peracetate had no effect on iron-induced lipid peroxidation in rat brain homogenate. They also did not scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl. In contrast, cycloheterophyllin and artonins A and B inhibited iron-induced lipid peroxidation in rat brain homogenate and scavenged 1,1-diphenyl-2-picrylhydrazyl. They also scavenged peroxyl radicals and hydroxyl radicals that were generated by 2,2'-azobis(2-amidinopropane) dihydrochloride and the Fe3+-ascorbate-EDTA-H2O2 system, respectively. However, they did not inhibit xanthine oxidase activity or scavenge superoxide anion, hydrogen peroxide, carbon radical, or peroxyl radicals derived from 2,2'-azobis(2,4-dimethylvaleronitrile) in hexane. Moreover, cycloheterophyllin and artonins A and B inhibited copper-catalyzed oxidation of human low-density lipoprotein, as measured by fluorescence intensity, thiobarbituric acid-reactive substance and conjugated-diene formations and electrophoretic mobility. It is concluded that cycloheterophyllin and artonins A and B serve as powerful antioxidants against lipid peroxidation when biomembranes are exposed to oxygen radicals.

Comparison of the platelet aggregation induced by three thrombin-like enzymes of snake venoms and thrombin.

Platelet aggregation induced by three thrombin-like enzymes of snake venoms was compared with that by thrombin. Acutin was isolated from Agkistrodon acutus venom and thrombocytin and batroxobin were from Bothrops atrox venom. The fibrinogen-clotting activities were 700, 170 and 7 U/mg for batroxobin, acutin and thrombocytin, respectively. They induced aggregation and ATP release of washed rabbit platelets. The aggregating activity of thrombin was 10(2), 10(4) and 10(5) times more potent than those of thrombocytin, acutin and batroxobin, respectively. Platelet-activating potency of the thrombin-like enzymes was correlated with their effectiveness on the retractility and elasticity of the clots. Platelet aggregation induced by thrombin or thrombocytin could be inhibited by heparin with antithrombin III while that by acutin or batroxobin could not. Indomethacin showed weak inhibition on the aggregation while the ADP-scavenging system, creatine phosphate/creatine phosphokinase, inhibited the aggregation induced by the three thrombin-like enzymes but not that by thrombin. Platelet aggregation induced by the thrombin-like enzymes could not be inhibited by PAF antagonists-BN 52021, kadsurenone or L-652,731. In the presence of EGTA, only thrombin could induce ATP release from platelets. Thrombin-like enzymes and low concentration of thrombin did not form thromboxane B2. Nitroprusside and prostaglandin E1 completely inhibited the aggregation, mepacrine and imipramine showed marked inhibition while verapamil had only weak inhibition. It is concluded that the aggregation induced by the thrombin-like enzymes is different from that of thrombin and mainly due to ADP released from platelets.

Trimucytin: a collagen-like aggregating inducer isolated from Trimeresurus mucrosquamatus snake venom.

Trimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytin- and collagen-induced platelet aggregation and ATP release.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of platelet thromboxane formation and phosphoinositides breakdown by osthole from Angelica pubescens.

Osthole, isolated from Chinese herb Angelica pubescens, inhibited platelet aggregation and ATP release induced by ADP, arachidonic acid, PAF, collagen, ionophore A23187 and thrombin in washed rabbit platelets. It showed a weak activity in platelet-rich plasma. Osthole inhibited the thromboxane B2 formation caused by arachidonic acid, collagen, ionophore A23187 and thrombin in washed platelets, and also the thromboxane B2 formation caused by the incubation of lysed platelet homogenate with arachidonic acid. The generation of inositol phosphates in washed platelets caused by collagen, PAF and thrombin was suppressed by osthole. These data indicate that the inhibitory effect of osthole on platelet aggregation and release reaction was due to the inhibition of thromboxane formation and phosphoinositides breakdown.

ADP-mimicking platelet aggregation caused by rugosin E, an ellagitannin isolated from Rosa rugosa Thunb.

Among the nine ellagitannins, rugosin E was the most potent platelet aggregating agent with an EC50 of 1.5 +/- 0.1 microM in rabbit platelets and 3.2 +/- 0.1 microM in human platelets. The aggregations caused by rugosin E and ADP were inhibited by EGTA, PGE1, mepacrine, sodium nitroprusside and neomycin, but not by indomethacin, verapamil, TMB-8, BN52021 and GR32191B. Rugosin E-induced thromboxane formation was suppressed by indomethacin, EGTA, PGE1, verapamil, mepacrine, TMB-8 and neomycin. ADP-scavenging agents, such as CP/CPK and apyrase inhibited concentration-dependently ADP (20 microM)-, but not rugosin E (5 microM)-induced platelet aggregation. In thrombin (0.1 U/ml)-treated and degranulated platelets, rugosin E and ADP still caused 63.5 +/- 3.0% and 61.2 +/- 3.5% of platelet aggregation, respectively. Selective ADP receptor antagonists, ATP and FSBA inhibited rugosin E- and ADP-induced platelet aggregations in a concentration-dependent manner. Both rugosin E and ADP did not induce platelet aggregation in ADP (1 mM)-desensitized platelets. In contrast to ADP, rugosin E did not decrease cAMP formation in washed rabbit platelets. Both rugosin E and ADP did not cause phosphoinositide breakdown in [3H]myo-inositol-labeled rabbit platelets. In fura-2/AM-load platelets, both rugosin E and ADP induced increase in intracellular calcium concentration and these responses were inhibited by ATP and PGE1. All these data suggest that rugosin E may be an ADP receptor agonist in rabbit platelets.

Activation by high potassium of a novel voltage-operated Ca2+ channel in rat spleen.

1. High potassium produced a concentration-dependent contraction in rat isolated spleen. 2. The high potassium-induced contraction of rat spleen was abolished in Ca(2+)-free Krebs solution containing 1 mM EGTA, and the subsequent addition of 3 mM Ca2+ restored the high potassium-induced contraction to the control level. 3. Nifedipine, verapamil, diltiazem, Cd2+, Ni2+, Co2+, R-(+)-Bay K 8644 and pimozide inhibited and relaxed high potassium-induced contraction of rat spleen with IC50 and EC50 values much higher than those values in rat aorta. 4. In addition, high potassium-stimulated contraction of rat spleen was insensitive to omega-conotoxin GVIA, omega-conotoxin MVIIC and omega-agatoxin IVA. 5. The high potassium-induced contraction of rat spleen was also unaffected by tetrodotoxin (TTX), prazosin, chloroethylclonidine (CEC), yohimbine, propranolol, atropine, diphenhydramine, cimetidine, ketanserin, 3-tropanyl-indole-3-carboxylate, saralasin, indomethacin, nordihydroguaiaretic acid, GR32191B, domperidone, naloxone, chlorpromazine, suramin, (+/-)-2-amino-5-phosphonopentanoic acid, 6,7-dinitroquinoxaline-2,3-dione (DNQX), L-659,877, L-703,606, lorglumide, PD 135,158 N-methyl-D-glucamine, benextramine, amiloride, dantrolene, TMB-8, econazole, staurosporine and neomycin. 6. Forskolin and sodium nitroprusside relaxed high potassium-induced contraction of rat spleen with EC50 values of 0.55 +/- 0.04 and 20.0 +/- 2.7 microM, respectively. 7. It is concluded that high potassium may activate a novel, pharmacologically uncharacterized voltage-operated Ca2+ channel in rat spleen.

YC-1 inhibited human platelet aggregation through NO-independent activation of soluble guanylate cyclase.

1. Our previous study demonstrated that YC-1, a derivative of benzylindazole, is a novel activator of soluble guanylate cyclase (sGC) in rabbit platelets. This work investigated whether the antiplatelet effect of YC-1 was mediated by a nitric oxide (NO)/sGC/cyclic GMP pathway in human platelets. 2. In human washed platelets, YC-1 inhibited platelet aggregation and ATP released induced by U46619 (2 microM), collagen (10 micro ml(-1)) and thrombin (0.1 u ml(-1)) in a concentration-dependent manner with IC50 values of (microM) 2.1 +/- 0.03, 11.7 +/- 2.1 and 59.3 +/- 7.1, respectively. 3. In a 30,000 g supernatant fraction from human platelet homogenate, YC-1 (5-100 microM) increased sGC activity in a concentration-dependent manner. At the same concentration-range, YC-1 elevated cyclic GMP levels markedly, but only slightly elevated cyclic AMP levels in the intact platelets. 4. MY-5445, a selective inhibitor of cyclic GMP phosphodiesterase, potentiated the increases in cyclic GMP caused by YC-1, and shifted the concentration-anti-aggregation curve of YC-1 to the left. In contrast, HL-725, a selective inhibitor of cyclic AMP phosphodiesterase, did not affect either the increases in cyclic nucleotides or the anti-aggregatory effect caused by YC-1. 5. Methylene blue, an inhibitor of sGC, blocked the increases of cyclic GMP caused by YC-1, and attenuated markedly the anti-aggregatory effect of YC-1. The adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA) did not affect YC-1-induced inhibition of platelet aggregation. 6. Haemoglobin, which binds NO, prevented the activation of sGC and anti-aggregatory effect caused by sodium nitroprusside, but did not affect YC-1 response. 7. These results would suggest that YC-1 activates sGC of human platelets by a NO-dependent mechanism, and exerts its antiplatelet effects through the sGC/cyclic GMP pathway.

Characterization of the thromboxane (TP-) receptor subtype involved in proliferation in cultured vascular smooth muscle cells of rat.

1. The effects of the thromboxane A2 (TxA2)-mimetic, U-46619, on the proliferation of vascular smooth muscle cells (VSMCs) were examined in a clonal smooth muscle cell line, A10, which was derived from foetal rat aorta. 2. [3H]-U-46619 bound to A10 cells of passages 18-20 (p18-20) with two classes of sites. The high affinity site showed a Bmax of 3.0 +/- 1.8 fmol mg-1 protein with a KD value 1.0 +/- 0.1 nM, while the low affinity site showed a Bmax of 43.0 +/- 6.0 fmol mg-1 protein and KD value of 129.0 +/- 7.9 nM. However, [3H]-U-46619 bound to A10 cells from passages 28-30 (p28-30) at a single class of site with a Bmax 111.0 +/- 9.0 fmol mg-1 protein and a KD value of 175.4 +/- 22.0 nM. 3. Cinnamophilin and SQ29548 inhibited specific [3H]-U-46619 binding to p18-20 A10 cells in a concentration-dependent manner with Ki values of 390.0 +/- 3.2 and 4.6 +/- 1.0 nM, respectively at a high affinity site, and 2.6 +/- 0.2 microM and 310.0 +/- 6.4 nM, respectively at the low affinity site. 4. U-46619 produced isometric contractions of rat aorta in a concentration-dependent manner with an EC50 7.0 +/- 1.2 nM. Cinnamophilin and SQ29548 antagonized U-46619-induced aortic contractions with pA2 values 6.3 +/- 0.1 and 8.2 +/- 0.2, respectively. 5. U-46619 increased [3H]-thymidine incorporation into DNA of p18-20 and p28-30 A10 cells in aconcentration-dependent manner with EC50 values 362.7 +/- 27.0 and 302.5 +/- 20.1 nm, respectively. The U-46619-induced increase of [3H]-thymidine incorporation into DNA of p28 -30 AO0 cells was potentiatedby PDGF (1 ng ml-1) and FCS (1%) and was inhibited by cinnamophilin (10 microM) and SQ29548 (1 microM)with estimated pKB values 5.4 +/- 1.2 and 6.3 +/- 0.9, respectively.6. Cell cycle analysis revealed that U-46619-increased cell cycle progression was primarily due to a rapidtransition from the DNA synthetic (S) to the G2/mitotic (M) phase. Moreover, U-46619 also increasedprotein synthesis and cell numbers in VSMC. All these effects of U-46619 were inhibited bycinnamophilin and SQ29548.7. U-46619 caused phosphoinositide breakdown and increased the intracellular Ca2+ concentration inVSMC, effects which were blocked by cinnamophilin and SQ29548.8 These data indicate there are two U-46619 binding sites in AlO VSMC. The high affinity site is correlated to U-46619-induced vasoconstriction while the low affinity site is correlated to U-46619-mediated VSMC proliferation. These data also reveal that U-46619 stimulates the cell cycle progression in VSMC primarily through a rapid transition from S to G2/M. Since cinnamophilin inhibits TPreceptor-mediated VSMC proliferation, it may thus hold promising potential for the prevention of atherosclerosis or vascular diseases.