Amplification effect and mechanism of action of ET-1 in U-46619-induced vasoconstriction in pig skin

The aim of this study was to investigate if a low concentration of endothelin-1 (ET-1; 8 x 10(-10) M) may amplify the skin vasoconstrictor effect of other vasoactive substances in the pathogenesis of skin vasospasm. Pig skin flaps (6 x 16 cm) were perfused with Krebs buffer equilibrated with 95% O(2) and 5% CO(2) at 37 degrees C and pH 7.4. Skin perfusion pressure measured by a pressure transducer and skin perfusion assessed by the dermofluorometry technique were used for assessment of skin vasoconstriction. We observed that ET-1 (8 x 10(-10) M) significantly amplified the concentration-dependent (10(-7)-10(-5) M) skin vasoconstrictor effect of norepinephrine. More importantly, we observed for the first time that this low concentration of ET-1 also amplified the concentration-dependent (10(-8)-10(-6) M) skin vasoconstrictor effect of the thromboxane A(2) mimetic U-46619, and this amplification effect of ET-1 was completely blocked by the protein kinase C (PKC) inhibitor chelerythrine (5 x 10(-6) M). Conversely, the PKC activator phorbol 12,13-dibutyrate (10(-7) M) amplified the vasoconstrictor effect of U-46619. Furthermore, the sensitivity of the skin vasculature to the vasoconstrictor effect of extracellular Ca(2+) in U-46619-induced skin vasoconstriction was significantly enhanced in the presence of 8 x 10(-10) M ET-1. Finally, the cyclooxygenase inhibitor indomethacin (5 x 10(-6) M) did not affect the amplification effect of ET-1 on U-46619-induced skin vasoconstriction. We conclude that a low concentration of ET-1 can amplify the skin vasoconstrictor effect of U-46619 independent of endogenous cyclooxygenase products, and the mechanism may involve activation of PKC and increase in sensitivity of the contractile apparatus to Ca(2+) in smooth muscle cells.     

Effect of thromboxane A2 (TXA2) synthase inhibitor and TXA2 receptor antagonist alone and in combination on antigen-induced bronchoconstriction in guinea pigs

Thromboxane A2 (TXA2) causes bronchoconstriction and bronchial hyperresponsiveness. Two types of TXA2 modifiers, one synthase inhibitor and one receptor antagonist, are widely used for the treatment of asthma in Japan. Although the target of TXA2 modifiers is to inhibit bioactivity of TXA2, the pharmacological properties are somewhat different between these drugs. We studied the inhibitory effects of the TXA2 synthase inhibitor CS-518 and the TXA2 receptor antagonist S-1452 alone and in combination on antigen-induced bronchoconstriction in passively sensitized guinea pigs treated with diphenhydramine. Both CS-518 and S-1452 inhibited the antigen-induced bronchoconstriction dose-dependently with the plateau. The combination of these drugs at the maximal inhibitory doses did not have any more effect compared with each single dosing. The combination at the submaximal doses tended to show an additive effect, but the effect was not significant. These findings suggest that other prostanoids such as PGE2, PGI2, PGD2 and PGF2alpha may not take an important role in the antiasthmatic effects of TXA2 modifiers.     

The effect of a novel CCK-antagonist (lorglumide) on human and guinea pig gallbladder strips: a tensiometric study

The effect of a novel CCK-antagonist (lorglumide, CR 1409) was evaluated by "in vitro" tensiometric studies on 16 human (gallstone patients) and 12 guinea pig gallbladder smooth muscle strips. In the animal experiments, increasing doses of lorglumide (0.2-6.5 uM) caused a rightward shift of the dose-response curves of CCK-OP, with an increase of the ED50 from 8.2 nM +/- 1.62 SEM, n = 12; to 100 nM +/- 12, n = 4) without affecting the maximal effect (Emax). Schild plot gave an affinity constant of 7.19. In human gallbladders, the effect of lorglumide was also present (ED50 increased from 47 nM +/- 8 SEM, n = 16; to 300 nM +/- 10 SEM, n = 4) coexisting with a large inter-sample variation for CCK-OP ED50s and maximal contractions, most likely due to the histological changes of the wall in chronic cholecystitis. The affinity constant was similar to that found in animal experiments. We confirm the studies previously reported in animals on the existence of a competitive antagonism of lorglumide on CCK gallbladder receptors. Moreover, our results on gallbladders from gallstone patients show that lorglumide is a highly effective antagonist of CCK-induced contractions despite the presence of chronic cholecystitis. Our study might help for a better comprehension of the role of these new anti-CCK drugs in the treatment of biliary pain.     

Effect of a peptide leukotriene antagonist, ONO-1078 on antigen-induced airway microvascular leakage in actively sensitized guinea pigs.

We examined the effect of ONO-1078, a peptide leukotriene antagonist, on antigen-induced airway microvascular leakage in ovalbumin-sensitized guinea pigs. When guinea pigs were pretreated with mepyramine, ovalbumin challenge increased vascular permeability to Evans blue dye in trachea, main bronchi and intrapulmonary airways. Oral administration of ONO-1078 significantly reduced microvascular leakage in intrapulmonary airways at doses more than 3 mg/kg, but not in trachea. Moreover, oral administration of ONO-1078 significantly reduced SRS-A mediated microvascular leakage into all airway tissues and was more effective in intrapulmonary airways at 3 mg/kg. Simultaneously, ONO-1078 also inhibited SRS-A mediated bronchoconstriction. On the other hand, azelastine (10 mg/kg, p.o.), an anti-asthma agent, failed to inhibit microvascular leakage into the airways. These results suggest that peptide leukotrienes may be important mediators of airway microvascular leakage, and that the inhibitory effect of ONO-1078 on antigen-induced airway microvascular leakage in addition to the blockade of bronchoconstriction may have therapeutic implications for bronchial asthma.     

哮喘豚鼠肺内ADM表达变化及对离体气管条张力的作用

目的：通过观察肾上腺髓质素（ADM）mRNA在豚鼠哮喘模型肺内的表达及对哮喘豚鼠离体气管条张力的影响,研究ADM在支气管哮喘（简称哮喘）发病机制中的作用。方法：用原位杂交方法检测ADM mRNA在豚鼠哮喘模型肺内的表达,用组胺诱导豚鼠离体气管条收缩后,观察不同浓度的ADM对其收缩作用影响。结果：原位杂交结果显示正常及哮喘豚鼠肺内均有ADM mRNA的表达,但哮喘组较正常组明显增多（P＜0．05）,ADM可抑制组胺诱导的哮喘豚鼠离体气管条的收缩,并呈量效关系,当浓度达10^-8mol/L时抑经达到最大,而且即使加大ADM的浓度,抑制率未继续明显增加,并对致敏气管螺旋条的舒张作用明显大于正常气管螺旋条。结论：哮喘时,肺内ADM mRNA的表达明显增多,ADM可抑制组胺诱导的豚鼠离体气管条的收缩,浓度为10^-8mol/L时抑制率达到最大。提示ADM在哮喘发病过程中起重要作用。     

The effect of three novel thromboxane A2 receptor antagonists (S-1452, AA-2414 and ONO-3708) on the increase in pulmonary pressure caused by Forssman anaphylaxis in guinea-pigs.

The effects were studied of three novel thromboxane A2 (TXA2) receptor antagonists (S-1452, AA-2414 and ONO-3708) on the increase in pulmonary pressure caused by Forssman anaphylaxis in guinea-pigs. Three TXA2 antagonists at doses of between 1 and 10 mg/kg administered orally 1 h before the challenge clearly inhibited the pulmonary pressure increase. At a dose of 10 mg/kg, all three antagonists inhibited the pulmonary pressure increase caused by leukotriene D4 (LTD4) and U-46619, but not that caused by histamine. The decrease in peripheral platelet counts caused by Forssman anaphylaxis was also clearly inhibited by the three TXA2 antagonists. However, the decreased peripheral leukocyte counts were unaffected by the three agents. The decrease in serum complement activity (CH50) was inhibited by S-1452 and AA-2414 at a dose of 10 mg/kg. In bronchoalveolar lavage fluid (BALF), significant increases in eosinophils and neutrophils were observed after Forssman anaphylaxis. Three TXA2 antagonists at a dose of 10 mg/kg (except for AA-2414 on eosinophils) did not affect the changes of leukocyte counts in BALF. Moreover, increases in the TXB2 and 6-keto-PGF1 alpha levels of the BALF brought about by Forssman anaphylaxis were unaffected by the three TXA2 receptor antagonists. Histamine and LTD4 were not changed in the BALF after Forssman anaphylaxis. These results indicate the efficacy of TXA2 receptor antagonists on the increase in pulmonary pressure caused by Forssman anaphylaxis in guinea-pigs by direct antagonism to released TXA2.     

Positive inotropic effect of the thromboxane analog U-46619 on guinea pig left atrium: mediation by specific receptors and association with increased phosphoinositide turnover

U-46619, a stable epoxymethano analog of thromboxane A2 elicited a direct positive inotropic effect on guinea pig left atrium paced at a constant rate (EC50 = 2.5 nM). This novel observation contrasts with previous reports of a decrease in myocardial contractility by thromboxane mimetic compounds in coronary-perfused preparations, an action recognized as secondary to vasoconstriction. The positive inotropic effect of U-46619 was competitively antagonized by the specific thromboxane receptor blocker L-655,240 (pA2 = 8.02; identical to that reported in smooth muscle), but was unaffected by blockers of alpha 1-, beta 1-, and H1-receptors and by cyclooxygenase and lipoxygenase inhibitors. Increased tissue levels of inositol phosphates, but not cAMP, were associated with the positive inotropic action of U-46619, in analogy to the actions of alpha 1- and H1-receptor agonists. However, the inotropic effect of U-46619 and the concomitant increase in phosphoinositide breakdown were both selectively antagonized by L-655,240. Thus, U-46619 acts on specific thromboxane receptors in guinea pig left atrium and elicits a positive inotropic effect that probably results from an increase in phosphoinositide metabolism.     

Effect of thromboxane A(2) (TXA(2)) synthase inhibitor and TXA(2) receptor antagonist alone and in combination on antigen-induced bronchoconstriction in guinea pigs

Thromboxane A(2) (TXA(2)) causes bronchoconstriction and bronchial hyperresponsiveness. Two types of TXA(2) modifiers, one synthase inhibitor and one receptor antagonist, are widely used for the treatment of asthma in Japan. Although the target of TXA(2) modifiers is to inhibit bioactivity of TXA(2), the pharmacological properties are somewhat different between these drugs. We studied the inhibitory effects of the TXA(2) synthase inhibitor CS-518 and the TXA(2) receptor antagonist S-1452 alone and in combination on antigen-induced bronchoconstriction in passively sensitized guinea pigs treated with diphenhydramine.Both CS-518 and S-1452 inhibited the antigen-induced bronchoconstriction dose-dependently with the plateau. The combination of these drugs at the maximal inhibitory doses did not have any more effect compared with each single dosing. The combination at the submaximal doses tended to show an additive effect, but the effect was not significant.These findings suggest that other prostanoids such as PGE(2), PGI(2), PGD(2) and PGF(2alpha) may not take an important role in the antiasthmatic effects of TXA(2) modifiers.     

Effects of gestational age on prostaglandin EP receptor expression and functional involvement during in vitro contraction of the guinea pig uterus

Prostaglandin E(2) (PGE(2)) exerts diverse biological effects through four G-protein-coupled cell surface receptor subtypes, EP1-4. This study's objective was to characterize EP1-4 receptor mRNA expression within pregnant guinea pig myometrium during early implantation stage (gestation day [GD] 6) and late stage gestation (GD 50) and evaluate in vitro contractile activity of receptor subtype selective agonists. Using RT-PCR, qualitative gene expression patterns of EP2, EP3, and EP4 mRNA were detected in the myometrium and remained unchanged between the gestational ages. EP1 mRNA remained undetected in pregnant tissue. In vitro contractile activity was evaluated in GD 6 and GD 50 myometrium using vehicle and EP agonists PGE(2), 17-phenyl trinor PGE(2), sulprostone, misoprostol, and CP-533,536. All spasmogens in pregnant myometrium were EP1/EP3 selective agonists, though likely acting via EP3 receptors in this test model. CP-533,536--a highly selective EP2 receptor agonist--and the vehicle failed to induce myometrial contraction at both gestational ages.     

Effects of U-46619 on pulmonary hemodynamics before and after administration of BM-573, a novel thromboxane A2 inhibitor

We studied the effects on pulmonary hemodynamics of U-46619, a thromboxane A2 (TXA2) agonist, before and after administration of a novel TXA2 receptor antagonist and synthase inhibitor (BM-573). Six anesthetized pigs (Ago group) received 6 consecutive injections of U-46619 at 30-min interval and were compared with six anesthetized pigs (Anta group) which received an increasing dosage regimen of BM-573 10 min before each U-46619 injection. Consecutive changes in pulmonary hemodynamics, including characteristic resistance, vascular compliance, and peripheral vascular resistance, were continuously assessed during the experimental protocol using a four-element Windkessel model. At 2 mg/kg, BM-573 completely blocked pulmonary hypertensive effects of U-46619 but pulmonary vascular compliance still decreased. This residual effect can probably be explained by a persistent increase in the tonus of the pulmonary vascular wall smooth muscles sufficient to decrease vascular compliance but not vessel lumen diameter. Such molecule could be a promising therapeutic approach in TXA2 mediated pulmonary hypertension as it is the case in pulmonary embolism, hyperacute lung rejection and endotoxinic shock.     

The effect of calcium antagonists on histamine and leukotriene-induced tracheal microvascular permeability in the guinea pig

The effects of several calcium antagonists, i.e., nifedipine, verapamil and 8-[N,N-diethylamino]-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), were evaluated in situ on agonist-induced increases in permeability of the airway microvasculature in anesthetized guinea pigs. Vascular permeability was measured as tracheal extravascular albumin content by using 125I-bovine serum albumin and the utilization of 51Cr labelled-erythrocytes to correct for blood volume. Intratracheal injections of histamine (1, 10 and 100 micrograms) or leukotriene (LT) D4 (1, 10 and 100 ng) produced dose-dependent increases in extravasated radiolabelled albumin in the trachea. Although histamine produced a greater maximal response than LTD4, the latter provocation was ten times more potent than the former. Nifedipine, a dihydropyridine calcium slow channel blocker, exhibited dose-dependent (30, 100 and 300 micrograms/kg) inhibitory activity against histamine-induced increases in extravascular albumin, while another calcium slow channel blocker, verapamil (100, 300 and 1000 micrograms/kg), exhibited much less activity. TMB-8, a purported intracellular calcium antagonist (1 and 10 mg/kg), was observed to have some inhibitory activity versus histamine. Similar doses of all three calcium antagonists failed to significantly inhibit increases in tracheal microvascular permeability evoked by LTD4. These results suggest that differences in mediator-induced microvascular permeability in the guinea pig trachea are evident depending upon the agonist selected and the pool of calcium utilized.     

In vitro studies of antigen-induced bronchospasm: effect of antihistamine and SRS-A antagonist on response of sensitized guinea pig and human airways to antigen.

Exposure of sensitized guinea pig tracheal rings or human bronchial strips to specific antigen in vitro resulted in a rapidly developing, prolonged contraction that was resistant to washing. Treatment of the tissue with diphenhydramine, a histamine H1 antagonist, before antigen delayed the onset and decreased the amplitude of the initial phase of the contraction but did not reduce the duration. Diphenhydramine treatment after development of the contraction did not relax the airway tissue. Antigen-induced histamine release from guinea pig trachea and from human bronchus was complete within the initial 15% of the duration of the contraction. Treatment of sensitized airway tissue with FPL 55712, a SRS-A antagonist, before antigen selectively inhibited the prolonged phase of the response. FPL 55712 administration after the development of antigen-induced contraction resulted in relaxation. These data suggest that both histamine and SRS-A are involved in the response of sensitized guinea pig and human airway tissue to antigen, with histamine mediating the early phase of the contraction and SRS-A primarily mediating the protracted phase.     

A novel, high-affinity, fluorescent progesterone receptor antagonist. Synthesis and in vitro studies

The present paper describes the chemical synthesis and in vitro characterization of a novel, high-affinity, fluorescent progesterone receptor (PR) antagonist. The three-step synthesis was carried out starting from mifepristone. After demethylation with calcium oxide, the methylamino group was alkylated with 6-bromohexanol, and the resulting compound was reacted with fluorescein 5-isothiocyanate, yielding the fluorescein-mifepristone conjugate. Interaction of the conjugate as well as of its precursors with PR was determined in cell culture (alkaline phosphatase assay and transactivation assay). Antiprogestagenic activity of the intermediates were comparable to that of the parent compound. Even after attachment of the bulky fluorescein moiety, considerable antiprogestagenic activity was maintained. Microscopic studies revealed that fluorescence of the conjugate was almost confined to the nuclei of steroid hormone receptor-positive cells, whereas the nuclei of steroid hormone receptor-negative cells remained unstained. To our knowledge, this is the first report on a fluorescent ligand for PR suitable for studies in living cells. It is proposed that the present fluorescent PR antagonist might serve as a lead compound for the development of contrast agents for PR imaging, e.g., by near-infrared optical imaging.     

In vitro and in vivo pharmacological characterization of the novel NK(1) receptor selective antagonist Netupitant

The novel NK(1) receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK(1) cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK(B) 8.87). In cells expressing NK(2) or NK(3) receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK(B) 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1-10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK(1) agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID(50) 1.5mg/kg) or orally (ID(50) 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK(1) antagonist. Thus this molecule can be useful for investigating the NK(1) receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK(1) receptors has been demonstrated valuable for patients.     

In vitro and in vivo characterization of a novel CCR3 antagonist, YM-344031

Eosinophils play a prominent proinflammatory role in a broad range of diseases, including atopic dermatitis and asthma. Eotaxin-1 and its receptor CCR3 are implicated in the recruitment of eosinophils from blood into inflammatory tissues, therefore inhibition of Eotaxin-1/CCR3 interaction may have therapeutic potential for allergic inflammation with eosinophil infiltration. YM-344031, a novel and selective small molecule CCR3 antagonist, potently inhibited ligand binding (IC(50)=3.0nM), ligand-induced Ca(2+) flux (IC(50)=5.4nM), and the chemotaxis of human CCR3-expressing cells (IC(50)=19.9nM). YM-344031 (1-10mg/kg) orally administered to cynomolgus monkeys significantly inhibited Eotaxin-1-induced eosinophil shape change in whole blood. Additionally, orally administered YM-344031 (100mg/kg) prevented both immediate- and late-phase allergic skin reactions in a mouse allergy model. YM-344031 therefore has potential as a novel and orally available compound for the treatment of allergic inflammation, such as atopic dermatitis and asthma.     

Effects of timolol, indomethacin, and MK-571 on bronchoconstriction to infused leukotriene D4 in guinea pigs

Continuous intravenous infusions of leukotriene D4 produced a prolonged but variable bronchoconstriction (approximately a 200% increase in lung resistance (RL) and a 50% decrease in dynamic compliance (Cdyn] in anesthetized and paralysed guinea pigs that peaked within 1-1.5 min and was followed by a somewhat smaller secondary plateau response. The overall response was delayed (time to peaks) but not significantly reduced by pretreatment with the cyclooxygenase inhibitor indomethacin (1 mg/kg), was markedly potentiated by the beta-adrenoceptor antagonist timolol (5 micrograms/kg), and was partially and completely blocked by pretreatment with 0.1 and 1.0 mg/kg, respectively, of the leukotriene D4 receptor antagonist MK-571. MK-571 prevented the response in indomethacin-treated guinea pigs but was considerably more active at preventing and reversing the potentiated responses (lower dose of leukotriene D4) in animals treated with indomethacin and timolol. Additional studies in indomethacin- and timolol-treated animals demonstrated that MK-571 was active with good duration of action by the aerosol route of administration (30 min and 4 h pretreatment). The technique of infusing leukotrienes into untreated, indomethacin-treated, and indomethacin- and timolol-treated guinea pigs is a useful method to study the action and interaction of leukotriene receptor antagonists.     

The effect of propylbenzilylcholine mustard on contraction and radioligand binding parameters of muscarinic receptors in guinea pig ileum

The receptor occupancy-biological effect relationship for muscarinic receptors in guinea pig ileal smooth muscle has been studied by comparison of radioligand binding and contractile response. Muscarinic receptors in homogenates of ileal smooth muscle were labeled with [3H]-1-Quinuclidinyl benzilate. Treatment with propylbenzilylcholine mustard (PrBCM), to inactivate irreversibly muscarinic receptors, caused a large dose dependent rightward shift of the dose-response curve to three agonistic furtrethonium derivatives with a concomitant decrease in maximal response. Using those data, the fraction of receptors remaining unoccupied (q-values) and "true affinity constants" (-log KA-values) were calculated. Exposure to 20 or 60 nM PrBCM for 15 minutes resulted in a 39% and a 61% reduction in specific [3H]-1-Quinuclidinyl benzilate binding sites respectively to be compared with a 62% and a 85% decrease expected from calculated q-values. KA-values for the methyl and ethyl derivative agreed well with the dissociation constants for the high affinity agonist sites determined from displacement of [3H-]-1-Quinuclidinyl benzilate. The KA-value for the propylfurtrethonium corresponds to the low affinity agonist dissociation constant. The fraction of receptors in the high affinity agonist state differs considerably for the three furtrethonium derivatives investigated. Neither the fraction of receptors in the high affinity agonist state, nor the ratio of dissociation constants for these states is affected by the alkylation of 85% of the functional muscarinic receptors. The inactivation of components of the effector system by PrBCM seems unlikely.     

The effects of thromboxane A2 inhibitors (OKY-046 and ONO-3708) and leukotriene inhibitors (AA-861 and LY-171883) on CCl4-induced chronic liver injury in mice

The effects of OKY-046, a selective thromboxane A2 (TxA2) synthetase inhibitor, ONO-3708, a novel TxA2 receptor antagonist, AA-861, a selective 5-lipoxygenase inhibitor and LY-171883, a peptide leukotrienes (p-LTs) receptor antagonist on the chronic liver injury were investigated in mice. The chronic liver injury was induced by the injection of carbon tetrachloride (CCl4) two times a week for twelve weeks in mice. In chronic liver injury models, significant histopathological changes in the liver and extensive elevation of glutamate transaminase (GOT and GPT) activity were observed. Administration of OKY-046, ONO-3708, AA-861 and LY-171883 for 12 weeks suppressed the elevation of serum GOT and GPT levels and histopathological changes in CCl4-induced chronic liver injury. These results suggest that TxA2 and LTs inhibitors are effective for the onset and development of chronic liver injury in mice.