Kaempferol-induces vasorelaxation via endothelium-independent pathways in rat isolated pulmonary artery.

BACKGROUND: Kaempferol, a flavonoid, is the essential part of human diet. Flavonoids have different pharmacological activities like cardioprotective, anti-inflammatory and anti-oxidant. The aim of current study was to investigate vasorelaxant potential of kaempferol on rat isolated pulmonary artery and to assess the underling mechanisms. METHODS: Tension experiments were conducted on both the branches of main pulmonary artery of rats. Experiments were done using isolated organ bath system by recording tension with the help of data acquisition system, Power Lab. RESULTS: Kaempferol (10-8-10-4.5M) caused concentration-dependent relaxation (Emax 124.33±4.37%; pD2 5.03±0.084) of endothelium-intact pulmonary artery. In endothelium-denuded arterial rings, relaxation produced by kaempferol was not different from intact artery. L-NAME, indomethacin, combination of L-NAME and indomethacin did not show any effect on kaempferol-induced relaxation. Kaempferol-induced relaxation was reduced (Emax 55.53±7.72%) in 60mMK+ pre-contracted pulmonary arterial rings. Iberiotoxin significantly decreased (Emax 71.68±11.84%) the relaxation response. However, glibenclamide, BaCl2, 4-AP (1mM) and ICI182780 did not reduce the kaempferol-induced relaxation. TEA (10mM) and 4-AP (5mM) significantly reduced relaxation. Kaempferol-induced relaxation was significantly attenuated (Emax 94.92±19.60%) in presence of ODQ. H89 significantly decreased (Emax, 98.38±8.55%) the kaempferol-induced relaxation in rat pulmonary arterial rings. HC067047 and apamin did not show any effect on kaempferol-induced relaxation. In endothelium-denuded K+ (80mM)-depolarized arterial rings, kaempferol (10µM) markedly reduced CaCl2-induced contractions (Emax 35.14±6.53% vs. control 69.04±15.19%). CONCLUSION: Kaempferol relaxes rat pulmonary artery in endothelium-independent manner through involvement of BKCa channel, sGC, PKA pathways and inhibition of Ca2+-influx through L-type calcium channels.

Flavonoid galetin 3,6-dimethyl ether attenuates guinea pig ileum contraction through K(+) channel activation and decrease in cytosolic calcium concentration.

Flavonoid galetin 3,6-dimethyl ether (FGAL) has been isolated from the aerial parts of Piptadenia stipulaceae and has shown a spasmolytic effect in guinea pig ileum. Thus, we aimed to characterize its relaxant mechanism of action. FGAL exhibited a higher relaxant effect on ileum pre-contracted by histamine (EC50=1.9±0.4×10(-7) M) than by KCl (EC50=2.6±0.5×10(-6) M) or carbachol (EC50=1.8±0.4×10(-6) M). The flavonoid inhibited the cumulative contractions to histamine, as well as to CaCl2 in depolarizing medium nominally Ca(2+)-free. The flavonoid relaxed the ileum pre-contracted by S-(-)-Bay K8644 (EC50=9.5±1.9×10(-6) M) but less potently pre-contracted by KCl or histamine. CsCl attenuated the relaxant effect of FGAL (EC50=1.1±0.3×10(-6) M), but apamin or tetraethylammonium (1mM) had no effect (EC50=2.6±0.2×10(-7) and 1.6±0.3×10(-7) M, respectively), ruling out the involvement of small and big conductance Ca(2+)-activated K(+) channels (SKCa and BKCa, respectively). Either 4-aminopyridine or glibenclamide attenuated the relaxant effect of FGAL (EC50=1.8±0.2×10(-6) and 1.5±0.5×10(-6) M, respectively), indicating the involvement of voltage- and ATP-sensitive K(+) channels (KV and KATP, respectively). FGAL did not alter the viability of intestinal myocytes in the MTT assay and decreased (88%) Fluo-4 fluorescence, indicating a decrease in cytosolic Ca(2+) concentration. Therefore, the relaxant mechanism of FGAL involves pseudo-irreversible noncompetitive antagonism of histaminergic receptors, KV and KATP activation and blockade of CaV1, thus leading to a reduction in cytosolic Ca(2+) levels.

Linalool elicits vasorelaxation of mouse aortae through activation of guanylyl cyclase and K(+) channels.

OBJECTIVES: The aim of this study was to investigate the cardiovascular relaxing properties of monoterpene alcohol (-)-linalool (LIN), a principal component of several aromatic plants. METHODS: We assessed the effects of LIN on vascular contractility in mouse aortae and evaluated its underlying mechanisms of action. KEY FINDINGS: We found that LIN dose-dependently relaxed the vascular tonus of mouse thoracic aortae induced by prostaglandin F2 alpha (PGF2α , 3 µm). This effect, however, was reduced by pretreatment with the nitric oxide synthase inhibitor L-NAME (30 µm). Treatment with the inhibitor of soluble guanylyl cyclase ODQ (2 µm) or the K(+) channel blocker TEA (10 mM) partially blocked LIN-induced vasorelaxation. Moreover, addition of TEA after incubation of the rings with L-NAME and ODQ partially blocked LIN-induced vasorelaxation. Furthermore, LIN was able to partially antagonize CaCl2 -induced contractions in high potassium (80 mM) Krebs' solution, whereas LIN did not affect Ca(2+) release from endoplasmic reticulum Ca(2+) stores. CONCLUSION: Our findings indicate that LIN may induce endothelium-dependent vasorelaxation in mouse thoracic aortae by activating soluble guanylyl cyclase and K(+) channels.

Ent-7α-acetoxytrachyloban-18-oic acid and ent-7α-hydroxytrachyloban-18-oic acid from Xylopia langsdorfiana A. St-Hil. & Tul. modulate K(+) and Ca(2+) channels to reduce cytosolic calcium concentration on guinea pig ileum.

In this study we investigated the mechanism underlying the spasmolytic action of ent-7α-acetoxytrachyloban-18-oic acid (trachylobane-360) and ent-7α-hydroxytrachyloban-18-oic acid (trachylobane-318), diterpenes obtained from Xylopia langsdorfiana, on guinea pig ileum. Both compounds inhibited histamine-induced cumulative contractions (slope=3.5±0.9 and 4.4±0.7) that suggests a noncompetitive antagonism to histaminergic receptors. CaCl(2)-induced contractions were nonparallelly and concentration-dependently reduced by both diterpenes, indicating blockade of calcium influx through voltage-dependent calcium channels (Ca(v)). The Ca(v) participation was confirmed since both trachylobanes equipotently relaxed ileum pre-contracted with S-(-)-Bay K8644 (EC(50)=3.5±0.7×10-(5) and 1.1±0.2×10-(5)M) and KCl (EC(50)=5.5±0.3×10-(5) and 1.4±0.2×10-(5)M). K(+) channels participation was confirmed since diterpene-induced relaxation curves were significantly shifted to right in the presence of 5mM tetraethylammonium (TEA(+)) (EC(50)=0.5±0.04×10-(4) and 2.0±0.5×10-(5)M). ATP-sensitive K(+) channel (K(ATP)), voltage activated K(+) channels (K(V)), small conductance calcium-activated K(+) channels (SK(Ca)) or big conductance calcium-activated K(+) channels (BK(Ca)) did not seem to participate of trachylobane-360 spasmolytic action. However trachylobane-318 modulated positively K(ATP), K(V) and SK(Ca) (EC(50)=1.1±0.3×10-(5), 0.7±0.2×10-(5) and 0.7±0.2×10-(5)M), but not BK(Ca). A fluorescence analysis technique confirmed the decrease of cytosolic calcium concentration ([Ca(2+)](c)) induced by both trachylobanes in ileal myocytes. In conclusion, trachylobane-360 and trachylobane-318 induced spasmolytic activity by K(+) channel positive modulation and Ca(2+) channel blockade, which results in [Ca(2+)](c) reduction at cellular level leading to smooth muscle relaxation.

Spasmolytic effect of citral and extracts of Cymbopogon citratus on isolated rabbit ileum.

Cymbopogon citratus, commonly known as lemongrass, has been shown to have antioxidant, antimicrobial and chemo-protective properties. Citral, a monoterpenoid, is the major constituent of C. citratus that gives off a lemony scent and is postulated to be responsible for most of its actions. In addition, C. citratus has been traditionally used to treat gastrointestinal discomforts, however, the scientific evidence for this is still lacking. Thus, the aim of the present study was to investigate the effect of the extracts of various parts of C. citratus (leaves, stems and roots) and citral on the visceral smooth muscle activity of rabbit ileum. The effect of the test substances were tested on the spontaneous contraction, acetylcholine (ACh)- and KCl-induced contractions. Citral at doses between 0.061 mM to 15.6 mM and the extract of leaves at doses between 0.001 mg/mL to 1 mg/mL significantly reduced the spontaneous, ACh- and KCl-induced ileal contractions. When the ileum was incubated in K(+)-rich-Ca(2+)-free Tyrode's solution, it showed only minute contractions. However, the strength of contraction was increased with the addition of increasing concentrations of CaCl(2). The presence of citral almost abolished the effect of adding CaCl(2), while the leaf extract shifted the calcium concentration-response curve to the right, suggesting a calcium antagonistic effect. These results were similar to that elicited by verapamil, a known calcium channel blocker. In addition, the spasmolytic effect of citral was observed to be reduced by the nitric oxide synthase inhibitor, L-NAME. In conclusion, citral and the leaf extract of C. citratus exhibited spasmolytic activity and it appeared that they may act as calcium antagonists. Furthermore, the relaxant effect of citral, but not that of the leaf extract may be mediated by nitric oxide suggesting the presence of other chemical components in the leaf extract other than citral.

Characterization of the plasma and blood anticoagulant potential of structurally and mechanistically novel oligomers of 4-hydroxycinnamic acids.

Recently, we designed sulfated dehydropolymers (DHPs) of 4-hydroxycinnamic acids that displayed interesting anticoagulant properties. Structurally and mechanistically, sulfated DHPs are radically different from all the anticoagulants studied to date. To assess whether their unique mechanism and structure is worth exploiting for further rational design of homogeneous DHP-based molecules, we investigated their anticoagulant potential in human plasma and blood using a range of clotting assays. Sulfated DHPs prolong plasma clotting times, prothrombin and activated partial thromboplastin times at concentrations comparable to the clinically used low-molecular-weight heparin, enoxaparin. Fibrin formation studies on human plasma show that there is a structural dependence of anticoagulant action. Human whole blood studies using thromboelastography and hemostasis analysis system indicate that they are 17-140-fold less potent than enoxaparin. These results demonstrate that sulfated DHPs possess good in-vitro and ex-vivo activity, which will likely be improved through a rational design.

Synthesis and biological activity of thienodiltiazem in isolated heart muscle preparations of guinea pigs.

The new compound thienodiltiazem was synthesized and investigated regarding structure-activity relations and calcium antagonistic properties. Isometric contraction force was measured in guinea-pig papillary muscles and chronotropic activity was studied in right atria of guinea pigs. Compared to the parent drug diltiazem (CAS 42399-41-7) the thieno derivative had a more potent negative chronotropic effect on spontaneously beating right atria and a more potent inotropic effect on papillary muscles. The negative inotropic action was reversed by increasing the extracellular calcium concentration.

Calcium channel blocking activity of calycosin, a major active component of Astragali Radix, on rat aorta.

AIM: To investigate the vasoactivity of calycosin, a major active component of Astragali Radix. METHODS: Experiments were performed on isolated rat thoracic aortic rings pre-contracted with phenylephrine (PHE) or KCl. RESULTS: Calycosin produced a concentration-dependent relaxation on the tissue pre-contracted using PHE with 4.46+/-0.13 of pD(2) and 95.85%+/-2.67% of E(max); or using KCl with 4.27+/-0.05 of pD2 and 99.06%+/-2.15% of Emax, and displaced downwards the concentration-response curves of aortic rings to PHE or KCl. The relaxant effect of calycosin on denuded endothelium aortic rings was the same as on intact endothelium aortic rings, and its vasorelaxant effect was not influenced by L-NAME or indomethacin. In Ca(2+)-free solution, calycosin (30 micromol/L) did not have an effect on PHE (1 micromol/L)-induced aortic ring contraction. The effects of calycosin and nifedipine where somewhat different; calycosin decreased aortic ring contractions induced by the two agonists, but nifedipine displayed a more potent inhibitory effect on KCl-induced contractions than on PHE-induced contractions, and the vascular relaxing effects of calycosin and nifidipine were additive on PHE-induced contraction but not KCl-induced. CONCLUSION: Calycosin is a vasorelaxant. Its action is endothelium-independent and is unrelated to intracellular Ca(2+) release. It is a noncompetitive Ca(2+) channel blocker. The effect of calycosin on Ca(2+) channel blockade may be different from that of dihydropyridines. This study demonstrated a novel pharmacological activity of calycosin, and supplied a theoretic foundation for Astragali Radix application.

[Vasodilation effect of atropine on rat mesenteric artery].

AIM: To study the vasodilation effect of atropine and its mechanism. METHODS: Isometric tension was recorded in isolated rat super mesenteric arteries precontracted by noradrenaline (NE) to study the vasodilation effect of atropine, and to investigate the role of endothelial cell and vascular smooth muscle cell on vasodilation. RESULTS: Atropine was shown to significantly dilate the endothelium-intact and endothelium-denuded arteries precontracted by NE. Nomega-Nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhabitor), indomethacin (cyclooxygenase inhibitor), propranolol (general beta adrenoceptor antagonist) and glibenclamide (ATP sensitive potassium channel inhibitor) showed no effect on vasodilation of atropine. Atropine did not affect the concentration-contraction curve of K+. However, atropine suppressed the contraction induced by NE and CaCl2, but not that by caffeine in the Ca+ -free Krebs solution. CONCLUSION: Atropine showed significant vasodilation effect which may derive, in part, from endothelium. Besides, atropine could inhibit the receptor-mediated Ca2+ -influx and Ca2+ -release, which was inferred to the mechanism of atropine on vasodilation.

Evidence that 17alpha-estradiol is biologically active in the uterine tissue: antiuterotonic and antiuterotrophic action.

BACKGROUND: 17alpha-Estradiol has been considered as the hormonally inactive isomer of 17beta-estradiol. Recently, nongenomic (smooth muscle relaxation) and genomic (light estrogenic activity) effects of 17alpha-estradiol have been reported, but no reports have yet determined its possible antiestrogenic activity. Therefore, this study investigated: the nongenomic action of 17alpha-estradiol on uterine contractile activity and its potential agonist-antagonist activity on uterine growth. METHODS: Uterine rings from rats were isometrically recorded. Different concentrations (0.2-200 microM) of 17alpha-estradiol were tested on spontaneous contraction and equimolarly compared with 17beta-estradiol. To examine the mechanism of 17alpha-estradiol action, its effect was studied in presence of beta2-antagonist (propranolol), antiestrogens (tamoxifen and ICI 182,780) or inhibitors of protein synthesis (cycloheximide) and transcription (actinomycin D). Moreover, contractions induced by high potassium (KCl) solution or calcium in depolarized tissues by KCl-calcium free solution were exposed to 17alpha-estradiol. Collaterally, we performed an uterotrophic assay in adult ovariectomized rats measuring the uterine wet weight. The administration for three days of 0.3 microM/day/Kg 17beta-estradiol was equimolarly compared with the response produced by 17alpha-estradiol. Antiuterotrophic activity was assayed by administration of 0.3 microM/day/Kg 17beta-estradiol and various doses ratios (1:1, 1:3, 1:5, and 1:100) of 17alpha-estradiol. RESULTS: The estradiol isomers elicited an immediate relaxation, concentration-dependent and reversible on spontaneous contraction. 17alpha-Estradiol presented lower potency than 17beta-estradiol although it did not antagonize 17beta-estradiol-induced relaxation. Relaxation to 17alpha-estradiol was not inhibited by propranolol, tamoxifen, ICI 182,780, cycloheximide or actinomycin D. The KCl contractions were also sensitive to 17alpha-estradiol-induced relaxation and calcium contractions in depolarized tissues were markedly prevented by 17alpha-estradiol, implying a reduction of extracellular calcium influx through voltage-operated calcium channels (VOCCs). Uterotrophic assay detected significant increase in uterine weight using 17alpha-estradiol, which was significantly minor as compared with 17beta-estradiol. 17alpha-Estradiol, at all doses ratios, significantly antagonized the hypertrophic response of 17beta-estradiol. CONCLUSION: 17alpha-Estradiol induces a relaxing effect, which may be independent of the classical estrogen receptor, nongenomic action, apparently mediated by inactivation of VOCCs. 17alpha-Estradiol is also a weak estrogen agonist (uterotrophic response); likewise, 17alpha-estradiol may act as an antiestrogen (antiuterotrophic response). The overall data document a nongenomic relaxing action and a novel antiestrogenic action of 17alpha-estradiol, which are relevant in estrogen-mediated uterine physiology.

Induces vasodilatation of rat mesenteric artery in vitro mainly by inhibiting receptor-mediated Ca(2+)-influx and Ca(2+)-release.

The purpose of this study was to investigate the effect of atropine on peripheral vasodilation and the mechanisms involved. The isometric tension of rat mesenteric artery rings was recorded in vitro on a myograph. The results showed that atropine, at concentrations greater than 1 microM, relaxed the noradrenalin (NA)-precontracted rat mesenteric artery in a concentration-dependent manner. Atropine-induced vasodilatation was mediated, in part, by an endothelium-dependent mechanism, to which endothelium-derived hyperpolarizing factor may contribute. Atropine was able to shift the NA-induced concentration-response curve to the right, in a non-parallel manner, suggesting the mechanism of atropine was not mediated via the (alpha1-adrenoreceptor. The beta-adrenoreceptor and ATP sensitive potassium channel, a voltage dependent calcium channel, were not involved in the vasodilatation. However, atropine inhibited the contraction derived from NA and CaCI2 in Ca(2+)-free medium, in a concentration dependent manner, indicating the vasodilatation was related to the inhibition of extracellular Ca2+ influx through the receptor-operated calcium channels and intracellular Ca2+ release from the Ca2+ store. Atropine had no effect on the caffeine-induced contraction in the artery segments, indicating the inhibition of intracellular Ca2+ release as a result of atropine most likely occurs via the IP3 pathway rather than the ryanodine receptors. Our results suggest that atropine-induced vasodilatation is mainly from artery smooth muscle cells due to inhibition of the receptor-mediated Ca(2+)-influx and Ca(2+)-release, and partly from the endothelium mediated by EDHF.

Antispasmodic effect of Achillea nobilis L. subsp. sipylea (O. Schwarz) Bässler on the rat isolated duodenum.

The aim of the present study was to investigate the antispasmodic effect of the total extract of Achillea nobilis L. subsp. sipylea (Schwarz) Bässler (Asteraceae) on rat duodenum. In the first part of experiments, cumulative dose-response curves for acetylcholine (Ach) were obtained and then dose-response curves are repeated after addition of atropine, papaverine and different doses of the extract. In the second part, cumulative dose-response curves to CaCl(2) were obtained in the absence and presence of verapamil and different doses of the extract. In the third part, papaverine and extract were applied to the tissues after contraction with K(+). The extract has exhibited an inhibitory effect on the dose-response curves induced by Ach and CaCl(2) on rat duodenum and significantly reduced the maximal response in a concentration-dependent manner. A similar effect was observed with papaverine but not with atropine on the dose-response curves obtained by ACh. Verapamil also reduced the maximal response in curves induced by CaCl(2). The present results demonstrate that total extract of A. nobilis subsp. sipylea exerts antispasmodic activity on rat duodenum.

Acute relaxant effects of 17-beta-estradiol through non-genomic mechanisms in rabbit carotid artery.

Estrogens could play a cardiovascular protective role not only by means of systemic effects but also by means of direct effects on vascular structure and function. We have studied the acute effects and mechanisms of action of 17-beta-estradiol on vascular tone of rabbit isolated carotid artery. 17-Beta-estradiol (10, 30, and 100 microM) elicited concentration-dependent relaxation of 50 mM KCl-induced active tone in male and female rabbit carotid artery. The stereoisomer 17-alpha-estradiol showed lesser relaxant effects in male rabbits. Endothelium removal did not modify relaxation induced by 17-beta-estradiol. The NO synthase inhibitor L-NAME (100 microM) only reduced significantly relaxation produced by 30 microM 17-beta-estradiol. Relaxation was not modified by the estrogen receptor antagonist ICI 182,780 (1 microM), the protein synthesis inhibitor cycloheximide (1 microM), and the selective K(+) channel blockers charybdotoxin (0.1 microM) and glibenclamide (1 microM). CaCl(2) (30 microM -10 mM) induced concentration-dependent contraction in rabbit carotid artery depolarized by 50 mM KCl in Ca(2+) free medium. Preincubation with 17-beta-estradiol (3, 10, 30, or 100 microM) or the L-type Ca(2+) channel blocker nicardipine (0.01, 0.1, 1, or 10 nM) produced concentration-dependent inhibition of CaCl(2)-induced contraction. In conclusion, 17-beta-estradiol induces endothelium-independent relaxation of rabbit carotid artery, which is not mediated by classic estrogen receptor and protein synthesis activation. The relaxant effect is due to inhibition of extracellular Ca(2+) influx to vascular smooth muscle, but activation of K(+) efflux is not involved. Relatively high pharmacological concentrations of estrogen causing relaxation preclude acute vasoactive effects of plasma levels in the carotid circulation.

Inhibition of the mitochondrial permeability transition by aldehydes.

Fructose has been shown to protect hepatocyte viability during hypoxia or exposure to mitochondrial electron transport inhibitors. We report here that the fructose metabolite D-glyceraldehyde (D-GA) is a good inhibitor of the mitochondrial permeability transition pore (PTP) in isolated rat liver mitochondria. We propose that a substantial portion of the protective effect of fructose on hepatocytes is due to D-GA inhibition of the permeability transition. Aldehydes which are substrates of the mitochondrial aldehyde dehydrogenase (mALDH) afford protection, while poor substrates do not. Protection is prevented by the ALDH inhibitor chloral hydrate. We propose that the NADH/NAD(+) ratio is the key to protection. The aldehydes phenylglyoxal (PGO) and 4-hydroxynonenal (4-HNE), which have previously been shown to inhibit the PTP, apparently function by a different mechanism independent of mALDH activity. Both PGO or 4-HNE are themselves potent inhibitors of ALDH, and their protective effect cannot be blocked by an ALDH inhibitor.

Involvement of calcium ion in the stimulated shoot elongation of arrowhead tubers under anaerobic conditions.

Shoot elongation of arrowhead (Sagittaria pygmaea Miq.) tubers was stimulated in anaerobic conditions. The anaerobic elongation was attributed to stimulation of cell elongation in the middle of the shoots. The anaerobic elongation of the shoots was severely inhibited by ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The EGTA inhibition was completely nullified by exogenous CaCl2, which acts as an enhancer of anaerobic elongation. Moreover, calcium channel blockers, verapamil, diltiazem and LaCl3, inhibited the anaerobic elongation enhanced by CaCl2. These results showed that calcium plays an important role in stimulating the elongation in anaerobic conditions. Incorporation of 45Ca into the shoot tissues was measured to determine the involvement of calcium uptake in anaerobic elongation. Incorporation of 45Ca into the cell sap, which was collected from frozen and thawed shoots after thorough washing with LaCl3, was significantly stimulated in anaerobic conditions. Verapamil and diltiazem prevented the stimulation of 45Ca incorporation in anaerobic conditions. These results suggest that calcium uptake from the medium serves to enhance shoot elongation of arrowhead tubers under anaerobic conditions.

[The effect of kudingcha (Ilex latifolia Thunb.) on the contraction of isolated guinea-pig tracheal smooth muscle in vitro].

OBJECTIVE: To study the effects of Kudingcha (Ilex latifolia Thunb., IL) on the isolated guinea pig tracheal smooth muscle in vitro. METHOD: By obtaining CaCl2 and Histamine accumulative dose-response curves, to observe the influences of IL on the contraction of tracheal strips induced by calcium and some asthmogenic mediators. RESULT: CaCl2 and histamine caused significant contraction of tracheal smooth muscle and pD2 was 3.55 and 5.34 respectively. After incubated with IL, the dose-response curves of CaCl2 and histamine were significantly shifted to the right, and the maximal contractile force was reduced. IL could also inhibit isolated tracheal strip contraction induced by acetylcholine 3 x 10(-6) mol.L-1 and histamine 3 x 10(-6) mol.L-1, and IC50 was 0.16 mg.ml-1 and 0.21 mg.ml-1. CONCLUSION: Kudingcha has significant dilated effects on tracheal smooth muscle.

Calcium antagonistic activity of Bacopa monniera on vascular and intestinal smooth muscles of rabbit and guinea-pig.

The present study demonstrates the calcium antagonistic activity in ethanol extract of Bacopa monniera. The plant extract inhibited the spontaneous movements of both guinea-pig ileum (IC50 = 24+/-4 microg/ml) and rabbit jejunum (IC50 = 136+/-9 microg/ml). A marked reduction in acetylcholine- and histamine-induced responses (0.0001-10 microM) in the ileum was evident in the presence of extract (260 microg/ml). The acetylcholine (1 microM)-induced contraction in the ileum was also inhibited by the extract (100-700 microg/ml) in a concentration dependent way (IC50 = 285+/-56 microg/ml). All these results indicate a direct action of the extract on smooth muscles. Calcium chloride-induced responses in the rabbit blood vessels and jejunum were attenuated in the presence of plant extract (10-700 microg/ml) implying a direct interference of plant extract with influx of calcium ions in the cells. However, the lack of modification of either noradrenaline- or caffeine-induced contractions in the presence of extract suggests that extract has no detectable effect on mobilization of intracellular calcium. These results indicate that spasmolytic effect of the B. monniera extract in smooth muscles is predominantly due to inhibition of calcium influx via both voltage and receptor operated calcium channels of the cell membrane.

Nonspecific inhibition of myogenic tone by PD98059, a MEK1 inhibitor, in rat middle cerebral arteries.

Activation of MAP kinase kinase, also called ERK kinase (MEK), may lead to desinhibition of thin filament regulatory proteins and we therefore investigated the acute effects of the potent MEK inhibitor, PD98059 on the contractile properties of pressurized rat middle cerebral arteries. Cerebral arteries (diameter 100-150 microm) were mounted on a pressure myograph and PD98059 (10 microM, 40 microM) significantly inhibited (15% and 64%) myogenic tone (P < 0.001). At these concentrations, PD98059 also significantly reduced the vasopressin (0.1 microM)- and KCl (60 mM)-induced tone. Cumulative addition of exogenous Ca2+ (0.4-1.6 mM) increased myogenic tone to approximately 50% of constriction at 80 mmHg. This effect was inhibited by PD98059 (P < 0.001). These results demonstrate that pressure-induced myogenic tone is inhibited by PD98059 at the concentrations that have been reported to be selective for inhibition of MEK and the MAP kinase cascade. However, our results also demonstrate that PD98059 may have nonspecific effects on voltage-sensitive Ca2+ entry in vascular smooth muscle.

Effects of the protein tyrosine kinase inhibitor, herbimycin A, on prolactin gene expression in GH3 and 235-1 pituitary tumor cells.

The high basal level of prolactin (PRL) gene expression in rat pituitary GH3 cells is maintained through the spontaneous activity of voltage-sensitive calcium channels (VSCCs). This can be observed experimentally by addition of 0.5 mM CaCl2 to GH3 cells cultured in a low calcium, serum-free medium. CaCl2 specifically induces PRL gene expression and this induction is inhibited by VSCC blockers. PRL gene expression is also stimulated by several hormones and growth factors. In the present study, we examined the effects of tyrosine kinase inhibitors on the ability of CaCl2, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and thryrotropin-releasing hormone (TRH) to increase PRL mRNA levels. Of several PTK inhibitors used, one PTK inhibitor, herbimycin A, specifically inhibited the CaCl2-induced increase in cytoplasmic and nuclear prolactin (PRL) mRNA without affecting cell viability, cell-cell and cell-matrix adhesion, or the expression of several other genes. The effects of herbimycin A were reversible. In cells pretreated with herbimycin A, PRL mRNA levels were reduced by 69 +/- 12% (P < 0.001; n = 4). Western blot analysis using anti-phosphotyrosine antibody revealed a decrease of 91 +/- 1% (P < 0.001; n = 4) in the phosphotyrosine content of proteins in the molecular weight range of 130-160 kDa. After changing the medium back to SFM plus 0.5 mM CaCl2, levels of PRL mRNA increased over a period of several hours, and this increase was accompanied by the tyrosine phosphorylation of two or more proteins in the approximate size range of 130-160 kDa. Herbimycin A also inhibited PRL gene expression in the independently-derived 235-1 lactotrope cell line and lowered the tyrosine specific phosphorylation of protein(s) in a similar size range. Herbimycin A inhibited the ability of bFGF, EGF and TRH to stimulate PRL gene expression in GH3 cells. Again, in cells pretreated with herbimycin A, bFGF induced a reappearance of tyrosine-specific phosphorylation, followed by a reappearance of PRL mRNA. These findings provide evidence for a role for at least one PTK which is necessary for basal and stimulated PRL gene expression.

Spasmolytic effects of tetrazepam on rat duodenum and guinea-pig ileum.

This study was designed to examine the inhibitory effects exerted by tetrazepam isolated rat duodenum and guinea pig ileum contractive responses and to further clarity the mechanisms involved. Tetrazepam produced concentration-dependent and complete relaxation of muscle contractions induced by KCl (80 mM) in guinea-pig ileum and this relaxant action was not antagonized by pretreatment with hexamethonium (0.1 mM), antagonist for nicotinic receptors, or atropine (1 microM), antagonist for muscarinic receptors, or PK 11195 (1 microM) antagonist for peripheral-type benzodiazepines receptors. Tetrazepam also modified the concentration-response curves of CaCl2 in calcium-free and high K/ depolarizing medium as soon as concentration-response curves of acetylcholine in Tyrode solution. The results suggested that tetrazepam inhibits the contractile responses to guinea-pig ileum and rat duodenum, probably through a reduction of calcium influx by way of calcium channels and these events are not related to high-affinity peripheral benzodiazepine binding sites.