Contraction Of Rat Thoracic Aorta Research Articles

Ca2+ entry channels involved in contractions of rat aorta induced by endothelin-1, noradrenaline, and vasopressin.

Endothelin-1 (ET-1) has been shown to activate three types of Ca2+ channel, namely two Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC), and that these channels can be discriminated by Ca2+ channel blockers such as LOE 908 (a blocker of NSCC-1 and NSCC-2) and SK&F 96365 (a blocker of NSCC-2 and SOCC). This study pharmacologically compared Ca2+ entry channels involved in contractions of rat thoracic aorta without endothelium induced by ET-1, noradrenaline (NA), or arginine-vasopressin (AVP). These agonists-induced contractions of aortic rings without endothelium and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of cultured aortic smooth muscle cells were abolished by removal of extracellular Ca2+. A blocker of L-type voltage-operated Ca2+ channel (VOCC), nifedipine had no effect on the responses to ET-1, but it suppressed the responses to NA and AVP to 70% and 65% of control responses, respectively. LOE 908 partially suppressed the nifedipine-resistant responses to ET-1 and AVP, but not those to NA. SK&F 96365 also partially suppressed the nifedipine-resistant responses to ET-1 and AVP, whereas it abolished the responses to NA. LOE 908 in combination with SK&F 96365 abolished the nifedipine-resistant responses to either of the agonists. These results show that the contraction of rat aorta involves different Ca2+ entry channel depending on agonists: (a) NSCC-1, NSCC-2, and SOCC for ET-1; (b) VOCC and SOCC for NA; and (c) VOCC, NSCC-1, NSCC-2, and SOCC for AVP.

Involvement of p44/42 mitogen-activated protein kinases in regulating angiotensin II- and endothelin-1-induced contraction of rat thoracic aorta

In order to elucidate the signal transduction pathway of vascular smooth muscle contraction induced by the activation of receptors for angiotensin II and endothelin-1, we examined whether tyrosine kinases and mitogen-activated protein (MAP) kinases are involved in the development of force of contraction in the rat aorta. Isolated aortic smooth muscles without endothelium were incubated in a modified Krebs–Henseleit solution and stimulated with angiotensin II (100 nM) or endothelin-1 (10 nM). A tyrosine kinase inhibitor genistein (10 μM) reduced the angiotensin II- and endothelin-1-induced aortic contraction, while 10 μM of daidzein (an inactive analogue of genistein) did not. The K + depolarization-induced contraction was not attenuated by 10 μM of genistein. Selective inhibitors of MAP kinase/extracellular signal-regulated kinase (Erk) kinase (MEK) such as PD98059 [2-(2-amino-3-methoxyphenyl)-4 H-1-benzopyran-4-one] and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] inhibited the angiotensin II- and endothelin-1-induced vasocontraction. The p44/42 MAP kinases were phosphorylated in cultured aortic smooth muscle cells and in physiologically contracted aortic vessels stimulated with angiotensin II and endothelin-1 for 5 min. The angiotensin II- and endothelin-1-induced phosphorylations of p44/42 MAP kinases were inhibited by PD98059 as well as U0126 in the intact aorta. These results suggest that the activation of genistein-sensitive tyrosine kinases and p44/42 MAP kinases is involved in the angiotensin II- and endothelin-1-induced rat aortic contraction.

(±)-Domesticine, a novel and selective α 1D-adrenoceptor antagonist in animal tissues and human α 1-adrenoceptors

The pharmacological profile of (±)-domesticine, a novel α 1-adrenoceptor antagonist, was examined in animal tissues and Chinese hamster ovary (CHO) cells expressing cloned human α 1-adrenoceptor subtypes and compared with the properties of BMY-7378 ([8-(2-[4-(2-methoxy-phenyl)-1-piperazinyl]ethyl)-8-azaspirol [4.5]decane-7,9-dione dihydrochloride], the prototypical α 1D-adrenoceptor antagonist. Both (±)-domesticine and BMY-7378 were more potent in inhibiting the phenylephrine-induced contraction in rat thoracic aorta than tail artery or spleen. The selectivity of (±)-domesticine to inhibit phenylephrine-induced contraction in rat thoracic aorta was 32- and 17-fold higher than that in tail artery and spleen, respectively, while that of BMY-7378 it was 125- and 11-fold, respectively. The functional affinity profiles of these compounds for the α 1-adrenoceptor subtypes in animal tissues were consistent with the respective binding affinity profiles in cloned human α 1-adrenoceptor subtypes. (±)-Domesticine displayed a 34- and 9-fold higher selectivity for α 1d-adrenoceptor than for α 1a- and α 1b-adrenoceptor, respectively, while BMY-7378 showed a selectivity for α 1d-adrenoceptor of 102-fold higher than that of α 1a-adrenoceptor and 21-fold higher than that of α 1b-adrenoceptor. Interestingly, in [ 3H]8-OH-DPAT (8-hidroxy-2-(di- n-propyl-amino)tetraline hidrobromide) binding to 5-HT 1A receptors of rat cerebral cortex, (±)-domesticine showed a 183-fold higher selectivity for α 1D-adrenoceptor relative to 5-HT 1A receptor, whereas BMY-7378 displayed a similar affinity at this receptor with respect to the α 1D-adrenoceptor (0.89-fold). Both compounds, however, showed a weak affinity for 5-HT 2A/5-HT 2C receptors in rat frontal cortex. These results suggest that (±)-domesticine is more potent for α 1D-adrenoceptor than for α 1A- or α 1B-adrenoceptor subtypes and it is highly selective compared to 5-HT 1A and other receptors.

Effects of Ba2+ on Norepinephrine-Induced Contraction of Rat Thoracic Aorta in vitro

The aim of this study was to examine the effect of exogenously applied BaCl₂ on the norepinephrine-induced contraction of the rat thoracic aorta. Exogenously applied BaCl₂ (0.3–1 mmol/l) slightly elevated the norepinephrine-induced sustained contraction of the rat thoracic aorta in the absence of nicardipine (1 μmol/l). In the aortic preparation pretreated with nicardipine (1 μmol/l), exogenous BaCl₂ (0.1–3 mmol/l) did not elevate the norepinephrine-induced sustained contraction, but the high concentration of BaCl₂ (10 mmol/l) slightly inhibited the norepinephrine-induced tone. In a Ca²⁺-free Krebs bi- carbonate solution (KBS) containing norepinephrine (1 μmol/l) or a Ca²⁺-free K⁺-rich (60 mmol/l) KBS, exogenously applied BaCl₂ (1–30 mmol/l) caused a sustained contraction of the rat thoracic aorta, and this sustained contraction was completely inhibited by nicardipine (1 μmol/l). Exogenous CaCl₂ (0.1–3 mmol/l) also caused a sustained contraction of the aortic preparation in a Ca²⁺- free KBS containing norephinephrine (1 μmol/l), but such a sustained contraction was partly inhibited by nicardipine (3 μmol/l). These results indicate that Ba²⁺ elevates the norepinephrine-induced tone of the rat isolated thoracic aorta by permeating voltage-dependent Ca²⁺ channels in the absence of nicardipine, but that Ba²⁺ has a minor modification on the norepinephrine-induced sustained contraction of the nicardipine-pretreated preparation.

Calcitonin gene-related peptide causes long-term inhibition of contraction in rat thoracic aorta through a nitric oxide-dependent pathway

Calcitonin gene-related peptide (CGRP) is released into the circulation during pathogenesis of endotoxin and septic shock and appears to partly mediate vascular problems of shock. To explore the function of CGRP during shock, we investigated long-term action of CGRP, alone or in combination with interleukin-1β (IL-1β), another shock mediator, in isolated rings of rat thoracic aorta. CGRP or IL-1β, by themselves, caused significant long-term (3 h) depression of contraction, while the combination of CGRP and IL-1β had no synergistic effects. Dose-response curves to phenylephrine were significantly decreased and shifted to the right when aortic rings were incubated with 1 μM CGRP for 1 h followed by 2 h incubation without CGRP. Inducible nitric oxide synthase (iNOS) inhibitors, S-methylisothiourea sulfate (SMT) and NG-nitro-L-arginine (L-NNA), completely eliminated long-term depressant effect of CGRP. Our results suggest pathology of septic shock may involve long-term inhibition of vascular contraction mediated by CGRP via expression of iNOS.

Ca2+ Entry Channels in Rat Thoracic Aortic Smooth Muscle Cells Activated by Endothelin-1

The contraction of the rat aorta induced by endothelin-1 (ET-1) requires entry of extracellur Ca2+, but involvement of voltage-operated Ca2+ channel is minor. Using whole-cell recordings of patchclamp and monitoring of the intracellular free Ca2+ concentration ([Ca2+]i), we characterized Ca2+ entry channels in A7r5 cells activated by ET-1. ET-1 activates three types of voltage-independent Ca2+ entry channels: two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC). Furthermore, it was found that these channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. NSCC-1 is resistant to SK&F 96365, but sensitive to LOE 908, whereas NSCC-2 is sensitive to both SK&F 96365 and LOE 908. SOCC is sensitive to SK&F 96365, but resistant to LOE 908. Using these channel blockers, we analyzed Ca2+ entry channels involved in the ET-1-induced contractions of rat thoracic aorta and increases in [Ca2+]i of single smooth muscle cells. The responses to lower concentrations of ET-1 (<0.1 nM) were abolished by either SK&F 96365 or LOE 908 alone. In contrast, the responses to higher concentrations of ET-1 (≥1 nM) were suppressed by SK&F 96365 or LOE 908 to about 10% and 35% of controls, respectively, and abolished by combined treatment with SK&F 96365 and LOE 908. These results show that the responses of rat aorta to lower concentrations of ET-1 involve only one Ca2+ channel that is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those to higher concentrations of ET-1 involve NSCC-1, NSCC-2 and SOCC, contributing 10%, 55% and 35%, respectively, to total Ca2+ entry.

P–389 - Inhibitory effects of an extract from Watasenia scintillans on the thromboxane A2 analogue-induced contractions of rat thoracic aorta.

P–389 - Inhibitory effects of an extract from Watasenia scintillans on the thromboxane A2 analogue-induced contractions of rat thoracic aorta.

A new flavone C-glycoside and antiplatelet and vasorelaxing flavones from Gentiana arisanensis.

A new flavone C-glycoside, isovitexin 6"-O-glucoside (1), and three known flavonoids, quercetin, isovitexin, and luteolin-7-O-beta-D-glucoside, have been further isolated from the whole plant of Gentiana arisanensis Hayata. The new compound was characterized by spectral methods and chemical reactions. The antiplatelet effects of isovitexin 6"-O-glucoside (1), isoorientin (2), 2 peracetate (3), isovitexin (4), luteolin 7-O-beta-D-glucoside (5), luteolin (6), isoorientin 6"-O-glucoside (7), and 7 peracetate (8) were studied using washed rabbit platelets. Of the compounds tested, 6 showed potent antiplatelet effects on arachidonic acid (AA)-induced platelet aggregation (IC50 = 43.5 microM). The effect of 2, 5, and 6 on the contraction of rat thoracic aorta was also studied. Compound 6 depressed markedly the contraction induced by Ca2+ (1.9 mM) in high-K+ (80 mM) medium, with an IC50 of about 156 microM and also inhibited noradrenaline (3 microM)-induced phasic and tonic contractions, with an IC50 of about 68 and 72 microM, respectively.

Endothelium-dependent and-independent vasoactive actions of a japanese kampo medicine, saiko-ka-ryukotsu-borei-to

Saiko-ka-ryukotsu-borei-to (TJ-12) is a Japanese kampo medicine used clinically for the treatment of hypertension and atherosclerosis. We investigated the effects of TJ-12 on the contraction of rat thoracic aorta induced by norepinephrine, 5-hydroxytryptamine and high potassium. TJ-12 relaxed endothelium-denuded rings, which had been precontracted with 1 microM norepinephrine, in a dose-dependent manner with an IC50 of 50 micrograms/mL. However, in the presence of TJ-12, endothelium-intact rings initially showed enhanced norepinephrine-induced contraction, followed by relaxation. Interestingly, TJ-12 dose-dependently reversed nitric oxide (2 microM)-induced relaxation of norepinephrine-induced precontraction ofendothelium-denuded rings, with an IC50 of 20 micrograms/mL. In serotonin-contracted rings, TJ-12 caused slight, though statistically significant, relaxation only at high doses (> 200 micrograms/mL). In constrat to these receptor-mediated contractions, TJ-12 failed to affect the tension produced by high potassium 40 mM). These results suggest that the antihypertensive effects of TJ-12 could be related to inhibition of norepinephrine-induced vasoconstriction. In addition, our in vitro experiments revealed an inhibitory effect on nitric oxide-induced relaxation.

Vasodilator Effects of the Extract of the Leaves ofCistus populifolius on Rat Thoracic Aorta

The effects of different extracts of Cistus populifolius L. on smooth muscle were studied. The aqueous extract inhibited in a dose dependent manner the noradrenaline (NA) (10 -9 -1.03 X 10 -6 M) and CaCl 2 (2 x 10 -4 -1.28 x 10 -2 M)-induced contractions in rat thoracic aorta. Furthermore, this extract induced relaxant effects in rat aorta precontracted with NA (10 -6 M) and with K + (80 mM). Two different vasodilator responses were observed on NA-induced contractions: one an endothelium-dependent response abolished by methylene blue but not by indomethacin, and the other an endothelium-independent response unaffected by methylene blue and indomethacin similar to that obtained with a flavone, luteolin (10 -5 M). The search for active constituents included a bioactivity-directed fractionation of the lyophilized aqueous extract (LAE) using vascular reactivity experiments. Four different fractions (Cl 2 CH 2 , Me 2 CO, MeOH, H 2 O) were analysed at two different concentrations (1 and 2 mg (dry plant)/mL). The methanol fraction was the most active on NA precontracted vascular segments with endothelium, whereas the water fraction was the most active on segments without endothelium. These relaxant effects were less on K + (80 mM)-induced contractions. Due to these results we suggest that this plant contains interesting hydrophilic compounds with strong pharmacological action. The endothelium-independent relaxant effect is probably related to flavonoid constituents. However we cannot relate the endothelium-dependent relaxation to known natural compounds in Cistus species. The presence of these constituents with relaxing properties on smooth muscle could account for the use of Cistus plant in traditional medicine.

P-705 - α1-Adrenoceptor subtypes involved in noradrenaline- and oxymetazoline-induced contractions of rat thoracic aorta

P-705 - α1-Adrenoceptor subtypes involved in noradrenaline- and oxymetazoline-induced contractions of rat thoracic aorta

P2-59 - Dual effects of Saiko-ka-ryukotsu-borei-to (TJ-I2) on contraction of rat thoracic aorta elicited by norepinephrine, serotonin and high potassium

P2-59 - Dual effects of Saiko-ka-ryukotsu-borei-to (TJ-I2) on contraction of rat thoracic aorta elicited by norepinephrine, serotonin and high potassium

Cinnamophilin, a novel thromboxane A 2 receptor antagonist, isolated from Cinnamomum philippinense

The pharmacological activity of cinnamophilin ((8 R,8′ S)-4,4′-dihydroxy-3,3′-dimethoxy-7-oxo-8,8′-neolignan), isolated from Cinnamomum philippinense, was studied in isolated rat aorta, guinea-pig trachea and rabbit platelets. Cinnamophilin was found to be a thromboxane A 2 receptor blocking agent in these tissues as revealed by its competitive antagonism of the U-46619 (9,11-dideoxymethanoepoxy-9α,11α-prostaglandin F 2α)-induced contraction of rat aorta and guinea-pig trachea and aggregation of rabbit platelets with pA 2 values of 7.3±0.2, 5.2±0.1 and 6.3±0.3, respectively. Protection against the irreversible vasoconstriction of rat aorta caused by U-46619 (0.05 μM) was obtained by cinnamophilin (10 μM) but not by caffeine (25 mM). Cinnamophilin (1–15 μM) also possessed voltage-dependent Ca 2+ channel blocking action, judging from its antagonism of the high K + (60 mM)- and Bay K 8644 (0.1 μM)-induced contraction in rat thoracic aorta. Cinnamophilin (30 μM) produced a slight relaxation of noradrenaline (3 μM)-induced tonic contractions, and this relaxing effect was abolished in the presence of nifedipine (1 μM). Nifedipine (10 μM) sufficient to inhibit high K +-induced contractions failed to attenuate the contractile response to U-46619. A high concentration of cinnamophilin (100 μM) did not affect the aortic contraction induced by endothelin-1, angiotensin II, carbachol or serotonin. Neither cAMP nor cGMP in rat aorta was increased by cinnamophilin. These results indicate that cinnamophilin is a selective thromboxane A 2 receptor antagonist especially in rat aorta, and also possesses voltage-dependent Ca 2+ channel blocking properties.

ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro.

1. The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)-receptor blocking action of ZD1542 (4(Z)-6-[2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]-4-(3- pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro. 2. ZD1542 caused concentration-dependent inhibition of human platelet microsomal thromboxane B2 (TXB2) production in vitro (IC50 = 0.016 microM); this inhibition was associated with an increase in prostaglandin E2 (PGE2) and PGF2 alpha formation. 3. ZD1542 also inhibited collagen-stimulated TXS in human, rat and dog whole blood giving IC50 values of 0.018, 0.009 and 0.049 microM respectively. The drug did not modify platelet cyclo-oxygenase activity as inhibition of TXB2 formation was associated with a concomitant increase in the levels of PGD2, PGE2 and PGF2 alpha. ZD1542 had little if any effect against cultured human umbilical vein endothelial cell (HUVEC) cyclo-oxygenase (IC50 > 100 microM) and prostacyclin (PGI2) synthase (IC50 = 18.0 +/- 8.6 microM). 4. ZD1542 caused concentration-dependent inhibition of U46619-induced aggregation responses of human, rat and dog platelets yielding apparent pA2 values of 8.3, 8.5 and 9.1 respectively. The drug was selective as, at concentrations up to 100 microM, it did not modify 5-hydroxytryptamine (5-HT) or the primary phases of adenosine diphosphate (ADP) and adrenaline-induced aggregation. Furthermore, ZD1542 (100 microM) modified only weakly the platelet effects of PGD2, PGE1 and PGI2. 5. ZD1542 also caused concentration-dependent inhibition of U46619-mediated contractions of rat thoracic aorta, guinea-pig trachea and lung parenchyma preparations giving apparent pA2 values of 8.6,8.3 and 8.5 respectively. At concentrations approaching three orders of magnitude greater than those required to block U46619-mediated contractions, the drug did not affect the actions of non-prostanoid agonists or exhibit agonist activity in any of the smooth muscle preparations employed; neither did it interact at EP- or FP-receptors.6. In conclusion, the present study demonstrates that ZD1542 is a drug that exhibits both potent,selective TXS inhibition and TXA2 receptor antagonism.

Vasorelaxation of rat thoracic aorta caused by osthole isolated from Angelica pubescens

The pharmacological effects of osthole on isolated rat thoracic aorta were examined. Osthole inhibited norepinephrine (NE, 3 μM)-induced phasic and tonic contractions in rat thoracic aorta in a concentration-dependent manner (40–200 μM). The tonic contraction elicited by NE was also relaxed by the addition of osthole. This relaxing effect of osthole was not affected by indomethacin (20 μM) and was still observed in endothelium-denuded rat aorta. Methylene blue (50 μM) partially antagonized this relaxing effect of osthole. In high-K + medium (80 mM), the Ca 2+ (0.03–3 mM)-induced vasocontraction was inhibited concentration dependently by osthole (20–100 μM). Addition of osthole (100 μM) at the plateau of the K + (80 mM)-induced contraction caused relaxation. Methylene blue (50 μM) did not antagonize this relaxation. In Ca 2+-free medium, the caffeine (10 mM)-induced phasic contraction was also suppressed by osthole in a concentration-dependent manner. Although the cAMP level was not changed by osthole, the cGMP level of rat aorta was increased by osthole in a concentration-dependent manner. The increase in cGMP level caused by osthole was completely blocked by methylene blue. [ 3H]Inositol monophosphate formation caused by NE was not affected by osthole at a concentration of 200 μM. The 45Ca 2+ influx elicited by either NE or high K + was inhibited by osthole in a concentration-dependent manner. It is concluded that osthole relaxes rat thoracic aorta by virtue of its Ca 2+-channel blocking properties and by elevating cGMP levels in vascular smooth muscle.

Interleukin-1 beta alters actin expression and inhibits contraction of rat thoracic aorta.

Previous studies have indicated that interleukin-1 beta (IL-1) inhibits contraction of rat aortas by activating nitric oxide production in vascular smooth muscle cells, with subsequent increases in guanosine 3',5'-cyclic monophosphate (cGMP). This study determined if the effect of IL-1 involves the primary regulatory event in smooth muscle activation, myosin light chain (MLC) phosphorylation. This study also examined whether IL-1 affects contractile protein content. IL-1 (20 ng/ml) significantly decreased stress in response to 0.1 microM phenylephrine with a concomitant decrease in MLC phosphorylation. Incubation with IL-1 for 3 h or longer decreased alpha-smooth muscle actin and increased gamma-actin isoform, with no change in beta-nonmuscle actin or myosin isozyme content. These results suggest that IL-1 inhibition of a vascular smooth muscle contraction may be due to a decrease in activator calcium, which may account for the resultant decrease in MLC phosphorylation. These results also indicate that IL-1 significantly affects contractile protein content, enhancing gamma-actin isoforms and decreasing the vascular smooth muscle specific alpha-isoactin.

Effects of chronic ethanol consumption on α-adrenergic-induced contractions in rat thoracic aorta

1. 1. The effects of chronic oral administration of ethanol (EtOH, 80 mM daily during 15–18 days) on the modulation exerted by the endothelium on the contractions induced by phenylephrine (Ph) and clonidine (C) were studied in rat aortic rings with and without endothelium. 2. 2. The maximal contraction induced by a 70 mM KCl depolarizing solution was similar in control and EtOH treated-rings. 3. 3. EtOH pretreatment significantly enhanced the contractile response to Ph in vessels with intact endothelium, but did not significantly affect the response of endothelium-denuded rings. 4. 4. This effect reflects a reduction of the modulation exerted by the endothelial cells on α 1-adrenergic vasoconstriction. 5. 5. C did not contract control aortic rings with endothelium, but after EtOH treatment, the response reached about 20% of the maximal KCl-induced contraction. 6. 6. EtOH pretreatment significantly enhanced the contractile response to C both in endothelium-denuded and intact aortic rings. 7. 7. The results indicate that chronic EtOH consumption significantly potentiates α-andrenergic-induced contractions in rat aortic rings, probably through interference with the production and/or the release of EDRF.

Synthesis of halogenated trimetoquinol derivatives and evaluation of their beta-agonist and thromboxane A2 (TXA2) antagonist activities.

The 5,8-difluoro (4), 5-iodo (5), 8-iodo (6), and 5-trifluoromethyl (7) derivatives of trimetoquinol (TMQ, 1) have been synthesized and evaluated for their ability to stimulate beta 1 (guinea pig atria) and beta 2 (guinea pig trachea) adrenoceptors as well as for their inhibitory activity against U46619 [a thromboxane A2 (TXA2) mimetic]-mediated contraction of rat thoracic aorta and human platelet aggregation. Both 5 and 6 were considerably less active than TMQ on both beta-adrenergic systems and gave a rank order of stimulatory potency of 1 much greater than 6 greater than or equal to 5. Similarly, iodine substitution at either position also caused a reduction in TXA2 antagonist activity with a rank order potency of 1 greater than 6 much greater than 5. Compared to 1, however, 5-iodo-TMQ (5) showed a marked selectivity for blockade of U46619 responses in rat aorta over human platelets. On beta-systems, 4 had reduced potency compared to TMQ and was similarly nonselective. Introduction of a trifluoromethyl group at the 5-position of TMQ completely abolished both beta 1- and beta 2-adrenergic agonist activities while imparting weak antagonist activity on beta 1 receptors. On TXA2 systems, both 4 and 7 possessed significantly decreased inhibitory activity compared to TMQ. The synthetic approaches to the synthesis of 8-(trifluoromethyl)-TMQ (8) are also described. The enantiomers of the 8-fluoro derivative (3) of TMQ were separated on a preparative Chiralcel OD column and evaluated on beta-adrenergic systems and TXA2 systems. On beta-adrenergic systems, (S)-(+)-8-fluoro-TMQ was at least 10-fold more potent than (R)-(-)-8-fluoro-TMQ. Conversely, (R)-(-)-8-fluoro-TMQ was approximately 14-fold more potent as an antagonist of TXA2-mediated aggregation in human platelets than (S)-(+)-8-fluoro-TMQ. In contrast to platelets, (S)-(+)-8-fluoro-TMQ was an agonist in rat aorta whereas (R)-(-)-8-fluoro-TMQ was an antagonist.

Wortmannin, a microbial product inhibitor of myosin light chain kinase.

We have found that a fungal strain, Talaromyces wortmannin KY12420, produces a potent inhibitor of smooth muscle myosin light chain kinase (MLCK). This active product, designated as MS-54, was isolated and purified from the culture broth of the fungus and identified as wortmannin. The inhibition of MLCK by wortmannin was prevented by a high concentration of ATP. The activity of the catalytic domain, which was disclosed by partial tryptic digestion, was also inhibited by wortmannin. These results suggest that wortmannin acts at or near to the catalytic site of the enzyme. It was shown clearly by kinetic analyses, preincubation studies, and dialysis experiments that the inhibitory action of wortmannin on MLCK was irreversible. Under the condition of preincubation for 3 min, 0.3 microM wortmannin inhibited the activity of MLCK, while 10 microM wortmannin had no effect on the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and calmodulin-dependent protein kinase II, and had little effect on protein kinase C activity. These data expressed clearly the marked selectivity of the compound for MLCK. Furthermore, wortmannin also inhibited both the phosphorylation of myosin light chain and the contraction in rat thoracic aorta stimulated with KCl, which indicates the effectiveness of the compound in the cellular level as an MLCK inhibitor.

Ca2+-Channel Blockade in Rat Thoracic Aorta by Protopine Isolated from Corydalis Tubers

The pharmacological properties and mechanism of the action of protopine on isolated rat thoracic aorta were examined. It inhibited norepinephrine (NE, 3 µM)-induced tonic contraction in rat thoracic aorta in a concentration-dependent manner (25–100 µg/ml). The phasic contraction caused by NE was inhibited only by a high concentration of protopine (100 µg/ml). At the plateau of NE-induced tonic contraction, the addition of protopine also caused relaxation. This relaxing effect of protopine was not antagonized by indomethacin (20 µM) or methylene blue (50 µM), and it still existed in denuded rat aorta or in the presence of nifedipine (2–100 µM). Protopine also inhibited high potassium (60 mM)-induced, calcium-dependent (0.03–3 mM) contraction of rat aorta in a concentration-dependent manner. Neither cAMP nor cGMP level was changed by protopine. Both the formation of inositol monophosphate caused by NE and the phasic contraction induced by caffeine were also not affected by protopine. 45Ca2+ influx caused by either NE or K+ was inhibited by protopine concentration-dependently. It is concluded that protopine relaxed the rat thoracic aorta mainly by suppressing the Ca2+ influx through both voltage- and receptor-operated calcium channels.