Involvement of cAMP and β-adrenoceptors in the relaxing effect elicited by flavonoids on rat uterine smooth muscle

1 The effect of the flavonoids genistein (3–100 μM), kaempferol (3–60 μM) and quercetin (1–100 μM) on KCI (60 mM)-induced tonic contraction in rat smooth muscle was assayed. In the same way, the modification of these effects in the presence of an inhibitor of protein kinase (PKA) (Rp-cAMPS), an inhibitor of phosphodiesterase (papaverine) and β-adrenoreceptor blocking agents (propranolol and atenolol) was studied. 2 The flavonoids totally relaxed the KCI-induced tonic contraction (IC₅₀: genistein 20.2 ± 2.0 μM, n = 11; kaempferol 10.1 ± 1.6 μM, n = 8; quercetin 13.2 ± 1.2 μM, n = 8). 3 The incubation with Rp-cAMPS (10 and 100 μM) 30 min prior to KCI shifted the dose-response curve of the flavonoids to the right, increasing their IC₅₀ up to 27.8 ± 3.8 and 31.9 ± 7.3 μM, respectively, for genistein; 24.7 ± 0.2 and 19.6 ± 4.9 μM, respectively, for kaempferol; 18.8 ± 2.2 and 18.4 ± 1.5 μM, respectively, for quercetin. 4 Papaverine (3–100 μM) also relaxed the contraction induced by KCI and this effect was significantly displaced to the right with Rp-cAMPS (10 μM) (IC₅₀ 12.1 ± 2.2 vs. 16.5 ± 3.1 μM). Papaverine (3 μM) added to the organ bath 15 min before the contractile agent increased the relaxing effect of the flavonoids and significantly decreased their IC₅₀ (genistein 20.2 ± 2.0 vs. 9.8 ± 1.4 μM; kaempferol 10.1 ± 1.6 vs. 6.6 ± 0.7 μM; quercetin 13.2 ± 1.2 vs. 7.8 ± 1.4 μM). 5 The incubation with atenolol (10 μM) did not alter the relaxing effect of the flavonoids. In the same experimental conditions, propranolol (10 μM) did not modify the effect of genistein and kaempferol, but shifted the response curve of quercetin significantly to the right (13.2 ± 1.2 vs. 17.7 ± 3.4 μM). 6 The results suggest that genistein, kaempferol and quercetin produced the relaxation of uterine smooth muscle by increasing intracellular cAMP. β-Adrenoceptors could also be involved in the effect of quercetin.     

Contribution of cAMP to the inhibitory effect of non-steroidal anti-inflammatory drugs in rat uterine smooth muscle

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Progesterone and pregnanolone derivatives relaxing effect on smooth muscle

• 1.1. The effect of gestagens, 5α-hydroxyprogesterone (10⁻⁶−6 × 10⁻⁵ M), 5β-hydroxyprogesterone (10⁻⁶−3 × 10⁻⁵ M), progesterone (6 × 10⁻⁶−10⁻⁴ M), pregnanolone (10⁻⁶−10⁻⁵ M), allopregnanolone (10⁻⁶−10⁻⁴ M) and epipregnanolone (10⁻⁶−6 × 10⁻⁵ M) on rat uterine contractions induced by KCl (60 mM), has been assayed.
• 2.2. All drugs assayed relaxed the tonic-contraction induced by KCl in a concentration-dependent way. The respectives IC₅₀ were 31.3 ± 4.1 × 10⁻⁶ M (progesterone), 8.9 ± 0.8 × 10⁻⁶ M (5α-hydroxyprogesterone 3.8 ± 0.3 × 10⁻⁶ M (5β-hydroxyprogesterone), 3.1 ± 0.1 × 10⁻⁶ M (pregnanolone), 21.2 ± 3.1 × 10⁻⁶ M (allopregnanolone) and 6.3 ± 1.3 × 10⁻⁶ M (epipregnanolone). This relaxing effect was partially or totally counteracted by CaCl₂ (1–10 mM)
• 3.3. Cycloheximide (10 μg/ml) significantly shifted to the right the effect of allopregnanolone but not the effect of the other drugs. Actinomycin D (5 μg/ml) did not modify the effect of allopregnanolone.
• 4.4. Our results suggest that the relaxing effect of gestagens in the rat uterus could be related to inhibition on calcium influx and mainly occur through non-genomic mechanisms.

     

The relaxing effect of aluminum and lanthanum on rat and human gastric smooth muscle in vitro

M. HAVA and A. HURWITZ, The relaxing effect of aluminum and lanthanum on rat and human gastric smooth muscle in vitro, European J. Pharmacol. 22 (1973) 156–161.Aluminum and lanthanum relaxed and partly blocked the acetylcholine-evoked contractions of rat and human stomach smooth muscle strips. In the perfused isolated rat stomach, intraluminal administration of 3 ml of 5 mM AlCl₃ or LaCl₃ caused a reversible reduction of contractions evoked by serosal administration of acctylcholine in the organ bath after a 90-min delay. The concentration of aluminum in solution in rat stomachs after administration of aluminum hydroxide in vivo was 0.015 M. This concentration was 30 times that which blocked contraction of isolated muscie strips and 3 times the concentration effective in perfused rat stomachs in vitro.     

Relaxant effect induced by wogonin from Scutellaria baicalensis on rat isolated uterine smooth muscle

Context: Wogonin is a flavone derivative isolated from Scutellaria baicalensis Georgi (Labiatae) root, which is a traditional Chinese drug used as an anti-inflammatory and for management of dysmenorrhea.

Objective: The effect of wogonin on the uterus has not yet been examined. We investigated the relaxant effects of wogonin on contractile activity of isolated uterine strips of rats.

Materials and methods: The effect of wogonin on spontaneous uterine contraction, and uterine contraction induced by agonists, K⁺-depolarization and oxytocin in Ca²⁺-free solution was observed. To clarify the type of potassium channel, we tested the effects of 4-aminopyridine, tetraethylammonium and glibenclamide.

Results: Wogonin reduced the contractile amplitude of uterine strip smooth muscle of rats in a dose-dependent manner. The concentration of wogonin for reducing the contraction amplitude by 50% (IC₅₀) on spontaneous contractions was 60.5 μM. Wogonin also inhibited the contraction induced by three agonists (oxytocin, prostaglandin F_2α and acetylcholine). For the uterine strips pretreated with oxytocin in Ca²⁺-free solution or K⁺-depolarization, wogonin showed relaxant effect on the induced uterine contractions. In addition, whereas the inhibitive effect of wogonin on the contraction of uterine smooth muscle in rats could be partly blocked by 4-aminopyridine and tetraethylammonium, it was not influenced by glibenclamide.

Discussion and conclusion: Wogonin significantly inhibited the contraction of rat uterine smooth muscle probably through the inhibition of the inflow of extracellular calcium into cells via cell membrane, and intracellular release of calcium ions. In addition, the relaxant effect induced by wogonin might be due in part to the opening of voltage-dependent and large conductance Ca²⁺-activated K⁺ channels.     

Involvement of cyclic AMP-dependent and -independent mechanisms in the relaxation of rat detrusor muscle via beta-adrenoceptors

We investigated the cAMP-dependent and -independent mechanisms of relaxation via beta-adrenoceptor in rat detrusor muscle with and without pre-contraction. A microdialysis technique was used to measure detrusor tension and cAMP level on the same detrusor tissue. In non-contracted tissue, isoproterenol, clenbuterol (beta2-adrenoceptor agonist) and FR165101, ((8S)-8-{[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino}-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yl)oxy]acetic acid hydrochloride (beta3-adrenoceptor agonist) relaxed detrusor muscle and cAMP levels also increased in a concentration dependent manner. SQ22536 (adenylyl cyclase inhibitor) markedly suppressed relaxation, suggesting that beta-adrenoceptor-mediated relaxation may be attributed mainly to cAMP-dependent mechanism. In high K+ pre-contracted tissue, although relaxation advanced in a concentration dependent manner, cAMP production reached a plateau at concentrations of more than 10(-7) M. SQ22536 had only a small inhibitory effect. However, large-conductance, Ca2+-activated K+ (BK(Ca)) channel inhibitors, charybdotoxin and iberiotoxin markedly suppressed relaxation. These results suggest that in addition to cAMP-dependent pathway, BK(Ca) channels are involved in the beta-adrenoceptor agonists-induced relaxation in pre-contracted detrusor muscle.     

The relaxing effect of SKF 38393 on the catch contraction of Mytilus smooth muscle

The relaxing effect of SKF 38393 on the catch contraction in the anterior byssus retractor muscle (ABRM) of Mytilus and the effect of SKF 38393 on the cyclic AMP (cAMP) levels in the ABRM were investigated. The catch contraction was relaxed by SKF 38393 in a dose-dependent manner. The dose-response curves of SKF 38393 were shifted in parallel to the right by butaclamol (3 X 10(-6) and 6 X 10(-6) M) and haloperidol (3 X 10(-6) and 3 X 10(-5) M). Their pA2 values were 5.69 +/- 0.05 and 5.80 +/- 0.07. The cAMP levels in the ABRM were not altered by SKF 38393 at a concentration (10(-5) M) sufficient to relax. These findings indicate that the dopamine receptors of the ABRM are D-1 like receptors but somewhat different from those of the vertebrates.     

Effects of silica nanoparticles on isolated rat uterine smooth muscle

In spite of their widespread use, toxicity of silica nanoparticles (SiO₂ NPs) to mammalian has not been extensively investigated. In the present study, it is aimed to investigate the effects and the mechanism of action of 20?nm sized SiO₂ NPs on isolated uterine smooth muscle. A total number of 84 preparations of uterine strips were used in the experiments. Study was designed as four groups: group I (control), group II (0.2?mM SiO₂ NPs), group III (0.4?mM SiO₂ NPs) and group IV (0.8?mM SiO₂ NPs). Spontaneous contractions were recorded using mechanical activity recording system. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) levels were measured using the spectrophotometric methods. Apoptosis of the cells was detected using immunofluorescence staining assay. SiO₂ NP distribution and ultrastructural changes were determined by transmission electron microscopy. In groups II–IV, the frequency of contraction was significantly lower than that of the group I, whereas the contraction energy significantly decreased only in group IV. SOD and GSH-Px activities were significantly lower in experimental groups compared to the control group. MDA level and apoptotic cells were significantly higher in all SiO₂ groups compared to the control group. Numerous SiO₂ NPs in cytoplasm and connective tissue were observed in all dose groups. These findings showed that 20?nm sized SiO₂ NPs enter the connective tissue and cytoplasm of uterine muscle cells and cause oxidative stress and apoptosis leading to impaired uterine contractile activity.     

The beta-adrenoceptors in the smooth muscle of pig coronary arteries

An attempt has been made to classify the coronary vascular β-adrenoceptors as ‘β₁’ or ‘β₂’ by studying the effect of 3 different β-blocking agents on the isoprenaline-induced relaxation of isolated strips of pig coronary arteries. The study includes propranolol as a potent blocker of both cardiac and peripheral vascular β-adrenoceptors, H 93/26, 1-isopropylamino-3-[4-(2-methoxyethyl)-phenoxyl]-2-propanol, as a ‘cardioselective’ β-blocker, and H 35/25, 2-isopropylamino-1-(4-methylphenyl)- 1-propanol, as a blocker with predominant action on the peripheral adrenoceptors. The differential influence of the latter 2 substances is illustrated by experiments on isolated rat atria and rat portal veins. The coronary artery strips resembled the atrial effectors in their sensitivity to all 3 blockers but they differed from the venous smooth muscle. It is suggested that the β-adrenoceptors of the coronary arteries should be placed in the β₁ category.     

Immunohistochemical and functional studies on calcium-sensing receptors in rat uterine smooth muscle

1. Activation of calcium‐sensing receptors (CaS) leads to relaxation of vascular smooth muscle. However, the role of CaS in uterine smooth muscle is unknown. Therefore the aim of the present study was to investigate the expression and function of CaS in the uterus. 2. The expression of CaS in the oestrogen‐dominated rat uterus was investigated using immunohistochemistry. The effects of putative CaS ligands on oxytocin‐induced contractions of longitudinally orientated uterine strips from oestrogen‐dominated rats were determined at reduced extracellular Ca²⁺concentrations using conventional organ bath techniques. 3. Immunohistochemical evidence showed the presence of CaS in the endometrium and smooth muscle layers of the rat uterus. Oxytocin‐induced contractions were inhibited by cations (Gd³⁺ > Ca²⁺ = Mg²⁺), polyamines (spermine > spermidine) and the positive allosteric modulators cinacalcet and calindol. However (R)‐ and (S)‐cinacalcet were equipotent, indicating a lack of stereoselectivity, and the negative allosteric modulator calhex‐231 also caused dose‐dependent relaxation. In addition, although intermediate‐conductance calcium‐activated potassium channels and cytochrome P450‐dependent signal transduction have been implicated in CaS‐induced relaxation of vascular smooth muscle, neither Tram‐34 nor miconazole (1 μmol/L), which block these pathways, respectively, had any effect on the ability of cinacalcet to inhibit oxytocin‐induced contractions. 4. Calcium‐sensing receptors are expressed in smooth muscle layers of the rat uterus and their ligands produce potent relaxation of longitudinally orientated uterine strips. However, the pharmacological profile of inhibition of contractility by CaS ligands is not consistent with a role for CaS in the regulation of uterine contractility in the rat.     

Nitroprusside enhances isoprenaline-induced increases in cAMP in rat aortic smooth muscle

Low concentrations of sodium nitroprusside (SNP) and of isoprenaline acted synergistically to inhibit the phenylephrine-induced contraction of rat aortic smooth muscle. In experiments with these concentrations, SNP enhanced the increases in smooth muscle cAMP caused by isoprenaline by 4- to 5-fold, whereas the SNP-induced increases in tissue cGMP were unaffected by isoprenaline. We conclude that cAMP is likely to mediate the synergistic inhibition of the contraction of rat aortic smooth muscle by these compounds.     

Calcium-dependent smooth muscle excitatory effect elicited by the venom of the hydrocoral Millepora complanata.

In the present paper, we describe the results obtained from a preliminary pharmacological and biochemical study of the fire coral Millepora complanata, a regular component of coral reefs in the Mexican Caribbean. The protein-containing crude extract obtained from M. complanata (tested from 0.001 to 1000 microg protein/ml) caused a concentration-dependent stimulation of spontaneous contractions of the guinea pig ileum. The extract (EC(50)=11.55+/-2.36 microg/ml) was approximately 12-fold less potent than ionomycin (EC(50)=0.876+/-0.25 microg/ml) and its maximum induced contraction (1mg protein/ml) was equivalent to 68% of the response to 60mM KCl. FPLC size exclusion chromatography of the M. complanta extract afforded 12 primary fractions, of which only FV (containing proteins with molecular weights ranging from 17 to 44 kDa) and FVIII (consisting of peptides with molecular weights lesser than 1.8k Da) elicited an excitatory effect when tested at the EC(50) of the original extract. After incubation in Ca(2+)-free medium, the ileal response to FV and FVIII was significantly reduced. Blockage of L-type Ca(2+) channels with nifedipine (1 microM) inhibited FV and FVIII-evoked contractions. Cd(2+) (10 microM), an unspecific blocker of voltage-activated calcium channels, also antagonized FV and FVIII-induced effects, whereas the Na(+) channel blocker tetrodotoxin (10nM) did not significantly affect FV and FVIII responses. These results suggest that the contractions induced by the bioactive fractions obtained from the crude extract of M. complanata are caused mainly by a direct action on smooth muscle cells, via an increase in Ca(2+) permeability that occurs, at least partly, through L-type voltage-dependent Ca(2+) channels found in the cell membrane of smooth muscle.     

Calcium and depolarization-dependent effect of pregnenolone derivatives on uterine smooth muscle

• 1.1. The effects of several gestagens (pregnenolone [1 to 30 μm], 20α-hydroxypregnenolone [1 to 30 μM]), and 20β-hydroxypregnenolone [1 to 30 μm]) on rat uterine contraction induced by KC1 (60 mM) and CaCl₂ (30 μM to 6 mM) have been assayed.
• 2.2. The three drugs relaxed the tonic contraction induced by KCI in a concentration-dependent way. The respective EC₅₀ values were: 27.6±1.58 μM (pregnenolone), 4.1±0.12 μM (20α-hydroxypregnenolone), and 11.2±1.04 μM (20β-hydroxypregnenolone). CaCl₂ (1 to 10 mM) totally counteracted the relaxing effect of pregnenolone but only partially compared to that of 20α- or 20β-hydroxy-pregnenolone.
• 3.3. CaC1₂ (30 μM to 6 mM) produced concentration-dependent contraction of rat uterus in medium lacking calcium plus 30, 60, or 90 mM of KC1. The EC₅₀ values of CaCl₂ were: 0.38±0.072, 0.183±0.015, and 0.183±0.015 μM in a medium with 30, 60, or 90 mM of KCI, respectively.
• 4.4. Pregnenolone (10 μM) did not significantly modify the EC₅₀ of CaC1₂ in a medium with 30, 60, or 90 mM of KCI. However, 20β-hydroxypregnenolone (10 μM) antagonized, in a noncompetitive manner, the concentration-response curve to CaC1₂.
• 5.5. 20α-Hydroxypregnenolone (4 μM) antagonized the concentration-response curve to CaCl₂ in a competitive manner. This antagonism was directly related to the concentration of KCI in the medium.
• 6.6. Our results suggest a different calcium antagonist effect of the three gestagens assayed.

     

Prostaglandin synthesis inhibitors: effect on angiotensin II- and oxytocin-induced contractions in rat uterine smooth muscle.

1 Eicosatetraynoic acid, the acetylene analogue of arachidonic acid, which inhibits both the cyclo-oxygenase and lipoxygenase pathways, reduced the contractile response of rat uterine smooth muscle to either angiotensin II or oxytocin. 2 Indomethacin, an inhibitor of cyclo-oxygenase, did not reduce the response to angiotensin II but did abolish the contractile response to low doses of oxytocin. 3 Nordihydroguaiaretic acid, a lipoxygenase inhibitor, totally abolished the uterine response to either oxytocin or angiotensin II. 4 The contractile response to carbachol, a cholinoceptor agonist, was unaffected by pretreatment with any of the cyclo-oxygenase or lipoxygenase inhibitors. 5 From these findings, it can be implied that some product of the arachidonate lipoxygenase pathway augments peptide-induced contractions of the rat uterus.     

Mechanism of rat uterine smooth muscle contraction induced by endothelin-1.

1. Endothelin (ET)-1 has been demonstrated to cause contraction of uterine smooth muscle. We investigated the role of ET receptor subtypes (ETA and ETB receptors) in ET-1-induced contraction of rat uterine smooth muscle by using the ETA receptor antagonist BQ-123 and the ETB receptor agonist BQ-3020. 2. ET-1 caused a contraction with superimposed oscillations of the rat isolated uterus suspended in Krebs-Ringer solution; both the amplitude of contraction as well as the oscillation frequency increased in a dose-dependent manner (10(-11)-10(-7)M). 3. BQ-123 (10(-6)M) markedly shifted the dose-response curve of ET-1 for both contractile effects and oscillation frequency to the right. 4. BQ-3020 (10(-11)-3 x 10(-7) M) did not cause uterine contraction; neither did it affect the dose-response curve of ET-1 for either the contractile effect or the increase in oscillation frequency. Thus, stimulation of ETB receptors is not involved in these responses. 5. The present findings suggest that ET-1-induced contractile responses and the increase in oscillation frequency in rat uterine smooth muscle is mediated through ETA receptors, and that ETB receptors play no role in these responses.     

The effect of metyrapone on uterine prostaglandin output and smooth muscle activity

Metyrapone, at low doses (0.5–1.0 mM), stimulated the output of both PGE and PGF from the isolated uterus of the pregnant rat determiend following extraction of bath fluid, chromatographic separation and bioassay of the prostaglandin. At higher doses (2–4 mM), metyrapone inhibited PGF output, but had no effect on PGE output. Uterine activity was rapidly inhibited by metyrapone in a dose-related manner. This inhibition was not related to PG output as, at 1 mM metyrapone, activity was inhibited and PG output stimulated. Both metyrapone and papaverine produced dose-dependent inhibition of the activity of the isolated rabbit ileum, papaverine being 10 times more potent than metyrapone.

Propranolol antagonised the response of the ileum to isoprenaline, but had no effect on the response to metyrapone. These observations confirm earlier data, suggesting that metyrapone exerts a differential effect on uterine PGE and PGF production and indicate that metyrapone has a direct inhibitory effect on smooth muscle activity.