EDRF-release and Ca+(+)-channel blockade by magnolol, an antiplatelet agent isolated from Chinese herb Magnolia officinalis, in rat thoracic aorta

Magnolol is an antiplatelet agent isolated from Chinese herb Magnolia officinalis. It inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contractions in rat thoracic aorta. At the plateau of the NE-induced tonic contraction, addition of magnolol caused two phases (fast and slow) of relaxation. These two relaxations were concentration-dependent (10-100 micrograms/ml), and were not inhibited by indomethacin (20 microM). The fast relaxation was completely antagonized by hemoglobin (10 microM) and methylene blue (50 microM), and disappeared in de-endothelialized aorta while the slow relaxation was not affected by the above treatments. Magnolol also inhibited high potassium (60 mM)-induced, calcium-dependent (0.03 to 3 mM) contraction of rat aorta in a concentration-dependent manner. 45Ca(+)+ influx induced by high potassium or NE was markedly inhibited by magnolol. Cyclic GMP, but not PGI2, was increased by magnolol in intact, but not in de-endothelialized aorta. It is concluded that magnolol relaxed vascular smooth muscle by releasing endothelium-derived relaxing factor (EDRF) and by inhibiting calcium influx through voltage-gated calcium channels.     

Antihypertensive and vasorelaxing activities of synthetic xanthone derivatives

A series of xanthones and xanthonoxypropanolamines have been synthesized. The activity of compounds on cardiovascular system was evaluated. All the compounds tested exhibited effective hypotensive activity in anesthetized rats. An oxypropanolamine side chain substituted at the C-3 position of the xanthone nucleus significantly enhanced the hypotensive activity. In rat thoracic aorta, all the compounds tested significantly depressed the contractions induced by Ca(2+) (1.9mM) in high K+(80mM) medium and the phasic and tonic contractions caused by norepinephrine (3 microM). In the rat thoracic aorta, the phenylephrine- and high K+ -induced 45Ca(2+) influx were both inhibited by a selective xanthone derivative, 13. In addition to the previously reported result of 13, evaluated as beta adrenoceptor blocker, the depressor and bradycardia effects of 9 are independent of the parasympathetic passway. These results suggest that 13 showed inhibitory effects on the contractile response caused by high K+ and norepinephrine in rat thoracic aorta are mainly due to inhibition of Ca(2+) influx through both voltage-dependent and receptor-operated Ca(2+) channels. The vasodilating properties of 13 is due to its calcium channel and beta adrenergic blocking effects.     

Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the estrogen-primed rat myometrium

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.     

Mechanism of vascular relaxation by thaligrisine: functional and binding assays

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with alpha-adrenoceptors we examined the effect of the alkaloid on [3H]-(+)-cis diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC50 = 8.9+/-2.9 microM, n=5) and in tail artery (IC50 = 3.04+/-0.3 microM, n=6) or noradrenaline induced contraction in rat aorta (IC50 = 23.0+/-0.39 microM, n=9) and in tail artery (IC50 = 3.8+/-0.9 microM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 = 13.3 microM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC50 = 7.7 microM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [3H]-prazosin than for [3H]-(+)-cis-diltiazem binding sites, with Ki values of 0.048+/-0.007 microM and 1.5+/-1.1 microM respectively. [3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [3H]-prazosin binding site than [3H]-(+)-cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an alpha1-adrenoceptor antagonist and as a Ca2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca2+-stores sensitive to noradrenaline, by blocking Ca2+-entry through voltage-dependent Ca2+-channels.     

Comparison of effects of a potassium channel opener BRL34915, a specific potassium ionophore valinomycin and calcium channel blockers on endothelin-induced vascular contraction

The effects of a potassium (K+) channel opener BRL34915 and a specific K+ ionophore valinomycin on vasoconstriction induced by endothelin (ET) were compared with those of calcium (Ca2+) channel blockers, nicardipine and verapamil, using helical strips from rat thoracic aorta. ET induced potent and persistent contraction in control solution and similar but smaller contraction in Ca2+-free solution. BRL34915 and valinomycin inhibited the ET-induced contraction dose-dependently in control solution, but not in Ca2+-free solution. The ET-induced contraction was also inhibited by nicardipine and verapamil, though less strongly. On the other hand, high K+ (35 mM)-induced vasoconstriction was strongly inhibited by nicardipine and verapamil, but not by BRL34915 or valinomycin. These results support the idea that the extracellular Ca2+-dependent component of the ET-induced contraction may be mediated by Ca2+ influx by a route other than voltage-dependent Ca2+-channels.     

Voltage dependent activation of tonic contraction in cardiac myocytes.

Contractions of isolated single myocytes of guinea pig heart stimulated by rectangular depolarizing pulses consist of a phasic component and a voltage dependent tonic component. In this study we analyzed the mechanism of activation of the graded, sustained contractions elicited by slow ramp depolarization and their relation to the components of contractions elicited by rectangular depolarizing pulses. Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses from the holding potential of -40 to +5 mV or by ramp depolarization shifting voltage within this range within 6 s. [Ca2+]i was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. Myocytes responded to the ramp depolarization between -25 and -6 mV by the slow, sustained increase in [Ca2+]i and shortening, the maximal amplitude of which was in each cell similar to that of the tonic component of Ca2+ transient and contraction. The contractile responses to ramp depolarization were blocked by 200 microM ryanodine and Ca2+-free solution, but were not blocked by 20 microM nifedipine or 100-200 microM Cd2+ and potentiated by 5 mM Ni2+. The responses to ramp depolarization were with this respect similar to the tonic but not to the phasic component of contraction: both components were blocked by 200 microM ryanodine, and were not blocked by Cd2+ or Ni2+ despite complete inhibition of the phasic Ca2+ current. However, the phasic component but not the tonic component of contraction in cells superfused with Ni2+ was inhibited by nifedipine. Both components of contraction were inhibited by Ca2+-free solution superfused 15 s prior to stimulation. CONCLUSIONS: In myocytes of guinea pig heart the contractile response to ramp depolarization is equivalent to the tonic component of contraction. It is activated by Ca2+ released from the sarcoplasmic reticulum by the ryanodine receptors. Their activation and inactivation is voltage dependent and it does not depend on the Ca2+ influx by the Ca2+ channels or reverse mode Na+/Ca2+ exchange, however, it may depend on Ca2+ influx by some other, not yet defined route.     

Calcium mobilization as the endothelium-independent mechanism of action involved in the vasorelaxant response induced by the aqueous fraction of the ethanol extract of Albizia inopinata G. P. Lewis (AFL) in the rat aorta.

In a previous work, we demonstrated that, in normotensive rats, AFL induced a marked hypotension due to a decrease in total peripheral resistances (TPR), partially secondary to the release of NO by the endothelium. NO did not, however, account for the total vasodilation produced by AFL in these rats. The aim of this study was to determine the involvement of the intracellular calcium mobilization in the vasorelaxant action induced by AFL in the rat aorta. In aorta of normotensive rats AFL (10, 20, 40 and 80 microg/ml) inhibited the sustained contractions induced by KCl (80 and 30 mM) and phenylephrine (Phe, 1 microM) with similar IC50 values (54 +/- 6, 52 +/- 4 and 65 +/- 4 microg/ml, respectively). The relaxing response induced by AFL against Phe-induced contractions was modified significantly by the endothelium removal (IC50 = 132 +/- 23 and 65 +/- 4 microg/ml, endothelium removed and intact endothelium aortic rings, respectively). Nevertheless, removal of the endothelium did not significantly change IC50 values when KCl (30 and 80 mM) was used as the contractile agent. The inhibitory effect induced by AFL on high (64.5 mM) K+-induced contraction was potentiated slightly (p < 0.05) by the decrease (from 2.5 to 0.3 mM, Ca2+) and attenuated by the increase (from 2.5 to 7.5 mM Ca2+) in the external [Ca2+]. In addition, in aortas from normotensive rats, AFL antagonized transient contractions induced in Ca2+-free media induced by 1 microM noradrenaline in a concentration-dependent manner, but not those induced by 20 mM caffeine. It is suggested that the remaining vasodilator effect of AFL in normotensive rats is probably due to an inhibition of Ca2+ influx and/or inhibition of intracellular Ca2+ mobilization from the noradrenaline-sensitive stores.     

Hypothyroid state reduces calcium channel function in 18-day pregnant rat uterus

Hypothyroidism significantly reduced the mean amplitude and increased the mean frequency of spontaneous rhythmic contractions in 18 day pregnant rat uterus. Nifedipine (10(-12)-10(-9) M) and diltiazem (10(-10)-10(-6) M) caused concentration related inhibition of the myogenic responses of the uterine strips obtained from both pregnant and hypothyroid state. However, nifedipine was less potent (IC50:2.11 x 10(-11) M) in pregnant hypothyroid state as compared to pregnant control (IC50: 3.1 x 10(-12) M). Similarly, diltiazem was less potent (IC50: 3.72 x 10(-9) M) in inhibiting the uterine spontaneous contractions in hypothyroid than in pregnant rat uterus (IC50:5.37 x 10(-10) M). A similar decrease in the sensitivity to nifedipine and diltiazem for reversal of K+ (100 mM)-induced tonic contraction and K(+)-stimulated 45Ca2+ influx was observed with these calcium channel antagonists in uterus obtained from hypothyroid pregnant rats compared to the controls. Nifedipine-sensitive influx of 45Ca(2+)-stimulated either by K+ (100 mM) or by Bay K8644 (1,4-dihydro-2,6-methyl-5-nitro-4-[2'-(trifluromethyl)phenyl]-3-pyridine carboxylic acid methyl ester) (10(-9) M) was significantly less in uterine strips from hypothyroid rats compared to controls. The results suggest that the inhibition of uterine rhythmic contractions may be attributable to a reduction in rat myometrial Ca2+ channel function in the hypothyroid state.     

Inhibitory actions of eugenol on rat isolated ileum

The effects of eugenol (1-2000 microM) on rat isolated ileum were studied. Eugenol relaxed the basal tonus (IC50 83 microM) and the ileum precontracted with 60 mM KCl (IC50 162 microM), an action unaltered by 0.5 microM tetrodotoxin, 0.2 mM N(G)-nitro-L-arginine methyl ester, 0.5 mM hexamethonium, and 1 microM indomethacin. Eugenol did not alter the resting transmembrane potential (Em) of the longitudinal muscle layer under normal conditions (5.0 mM K+) or in depolarised tissues. Eugenol reversibly inhibited contractions induced by submaximal concentrations of acetylcholine (ACh) and K+ (40 mM) with IC50 values of approximately 228 and 237 microM, respectively. Eugenol blocked the component of ACh-induced contraction obtained in Ca(2+)-free solution (0.2 mM EGTA) or in the presence of nifedipine (1 microM). Our results suggest that eugenol induces relaxation of rat ileum by a direct action on smooth muscle via a mechanism largely independent of alterations of Em and extracellular Ca2+ influx.     

Phorbol ester contracts rabbit thoracic aorta by increasing intracellular calcium and by activating calcium influx

The ability of the phorbol ester tumor promoter, PDB, to activate contraction and stimulate calcium influx was investigated in rabbit thoracic aorta. PDB caused a strong, slowly-developing sustained contraction in physiological salt solution which was concentration-related (0.01 to 10.0 microM). PDB-induced contractions (0.1 microM) in calcium-free medium were attenuated but not prevented. PDB (1.0 microM) maximally stimulated Ca influx above basal control, vehicle = 39.2 +/- 2.2; PDB 1.0 microM = 70.7 +/- 6.7 mumoles Ca/kg tissue; N = 16, p less than 0.01). These data suggest that PDB activates rabbit thoracic aorta by a combination of intracellular and extracellular calcium dependent mechanisms.     

Distribution of the vasoconstrictor and vasorelaxant effects of norbormide along the vascular tree of the rat

Norbormide is a vasoconstrictor of rat peripheral arteries and a relaxant in rat aorta. To characterise norbormide actions within the rat vascular tree we have investigated its effects on the contractile function of rings from several arteries and veins. A maximal norbormide concentration (50 microM) failed to contract thoracic aorta and carotid artery, whereas in pulmonary artery, abdominal aorta, iliac, caudal, and femoral arteries it induced a contractile effect that was respectively 4.8 +/- 0.6, 18.4 +/- 1.5, 39 +/- 5, 144 +/- 7, and 260 +/- 22% of that induced by 90 mM KCl. In pulmonary, carotid, and iliac arteries, and in thoracic and abdominal aorta, 50 microM norbormide inhibited KCl-induced responses. Norbormide (50 microM) contracted all veins investigated. The effect, expressed as % of KCl-induced contraction, was 121 +/- 25, 154 +/- 14.5, 154 +/- 18.2, 203 +/- 19, and 267 +/- 33 for pulmonary vein, thoracic and abdominal vena cava, iliac and jugular veins, respectively. In jugular vein, as previously shown in rat caudal artery, norbormide contraction was abolished in Ca2+-free medium, was unaffected by the Ca2+ channel blocker nifedipine, and was relaxed by SK&F 96365, a blocker of store-operated Ca2+ channels. In conclusion: i) rat veins represent the main target for contractile norbormide action; ii) in both artery and veins norbormide contractions are generally inversely related to the calibre of the vessel; iii) norbormide-induced contraction is mediated by the same mechanism/s in arteries and veins; iiii) in norbormide-contracted arteries the drug activates both contractile and relaxing mechanisms.     

Clinical potency of calcium channel blockers in asthma may be related to their inhibition of receptor-mediated phasic responses in vitro.

The calcium channel blockers (CCB) have been clinically effective in exercise-induced asthma. The completeness of protection with the CCB might be related specifically to inhibition of Ca2+ influx or release. To examine this hypothesis, the rank order of potency of inhibition of the CCB, nicardipine, diltiazem and verapamil on the steady-state and kinetic parameters of the phasic and tonic responses to the muscarinic receptor agonist carbachol (10 microM) and KCl (40 mM) in the intact isolated guinea-pig trachea was determined. The Ca2+ channel agonist Bay K 8644 was also examined for its effects on intracellular Ca2+. Nicardipine abolished the KCl response at both 0.1 microM and 1 microM concentrations. The amplitude of the KCl response was inhibited equally by 1 microM diltiazem (61% inhibition) and 1 microM verapamil (68% inhibition). The rate constant of onset of the KCl response was similarly inhibited 60% by diltiazem and 66% by verapamil. Nicardipine abolished the carbachol phasic response at the 1 microM concentration. The amplitude of the phasic response was inhibited equally by 0.1 microM nicardipine (61.3% inhibition), 1 microM diltiazem (64.5% inhibition) and 1 microM verapamil (71% inhibition). The rate constant of decay of the phasic response was inhibited equally by 0.1 microM nicardipine (43% inhibition) and 1 microM diltiazem (29% inhibition). The rate constant of onset of the phasic response was unaffected by nicardipine, diltiazem and verapamil. Only 1 microM nicardipine inhibited the amplitude and rate constant of onset of the tonic response. The only effect of Bay K 8644 (1 microM) was to increase the phasic response amplitude. The CCB demonstrate a similar order of potency for inhibition of the phasic responses and clinical efficacy of the CCB in exercise-induced asthma (nicardipine > verapamil > diltiazem).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of benextramine as an irreversible alpha-adrenergic blocker and as a blocker of potassium-activated calcium channels

An irreversible alpha-adrenergic blocker, benextramine [N,N'-bis(o-methoxybenzylamine-n-hexyl)-cysteamine] was used as a probe to study the possible interrelationship between alpha-adrenoceptors and the K+-activated Ca2+-channels. Benextramine, a tetraamine disulfide, acts irreversibly both on the alpha 1-adrenoceptor (t 1/2 = 3 min) and the alpha 2-adrenoceptors. These studies were carried out on rat brain synaptosomes, [3H]prazosin and [3H]clonidine binding. Benextramine blocked Ca2+ influx in rat brain synaptosomes under both depolarizing (75 mM KCl) and normal conditions (5 mM KCl). Its action at the channel is reversible with IC50 = 10 +/- 5 microM of the net Ca2+ influx. This makes benextramine a most potent Ca2+ blocker compared to verapamil or nicardipine (IC50 = 200 microM and 170 microM, respectively). Pretreatment of rat brain slices with benextramine gave a synaptosomal preparation which was devoid of either alpha 1-adrenergic or alpha 2-adrenergic binding capacity due to the irreversible binding of benextramine, but with an undisturbed Ca2+ influx. Thus, these results suggest that the alpha-adrenoceptors and the Ca2+-channels are independent of each other, and that full occupancy of the alpha-receptors does not affect the net calcium flux.     

Thrombin-induced calcium movements in platelet activation

The thrombin-induced Ca2+ fluxes and their coupling to platelet aggregation of the human platelet were studied using quin2 as a measure of the cytoplasmic Ca2+ concentration [( Ca2+]cyt) and chlorotetracycline (CTC) as a measure of internally sequestered Ca2+. Evidence is given that the CTC fluorescence change is proportional to the free internal Ca2+ concentration in the dense tubular lumen. The intracellular quin2 concentration was 1 mM and analysis showed that it did not perturb the processes reported herein. The value of [Ca2+]cyt at rest and during thrombin activation was analyzed in terms of Ca2+ influx, Ca2+ release, Ca2+ sequestration, and Ca2+ extrusion. Influx was distinguished from internal release by removing extracellular Ca2+ 1 min before thrombin activation. In the presence of 2 mM external Ca2+, the thrombin-induced Ca2+ influx accounts for most of the increase in [Ca2+]cyt (over 80%). Thrombin-induced Ca2+ influx and release have somewhat different EC50 values (0.17 U/ml vs. 0.35 U/ml). The contribution of influx can be inhibited by verapamil, bepridil and Cd2+ (IC50 values of 19 microM, 2 microM and 50 microM). The influx results were analyzed in terms of a thrombin-activated channel. Indomethacin pretreatment experiments suggest that activation of the arachidonic pathway accounts for approx. 50% of the influx-related [Ca2+]cyt elevation. Elevation of [Ca2+]cyt by intracellular release is not inhibited by verapamil or Cd2+ but is inhibited by bepridil with a high IC50 (25 microM). It is only 15-20% inhibited by indomethacin and is thus not dependent on thromboxane A2 formation. The release reaction does not require Ca2+ influx. The rate of thrombin-activated platelet aggregation is shown to have an approximately fourth-power dependence on [Ca2+]cyt with an apparent Km of 0.4 microM. Comparisons of aggregation rates of the partially thrombin-activated vs. fully thrombin-activated, partially verapamil-inhibited conditions suggest that this dependence on [Ca2+]cyt is the major determinant of the aggregation behavior. Analysis shows that calcium influx is the major pathway for elevating [Ca2+]cyt by thrombin when physiological concentrations of external Ca2+ are present.     

Influence of ryanodine receptor agonist and antagonist on development of potassium contracture in phasic (twitch) and tonic fibers of frog skeletal muscle

We compared the influence of external calcium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of ryanodine-sensitive Ca channels of the sarcoplasmic reticulum on the characteristics of potassium contracture in phasic and tonic frog skeletal muscle fibers. The duration of contracture in tonic fibers, as contrasted to the phasic ones, is not limited by the presence of Ca²⁺. The tonic contractile response is virtually indifferent to dantrolene and is much less sensitive to chlorocresol than the phasic one (1 mM vs. 0.25 mM). In phasic fibers, the K⁺ contracture on the chlorocresol background is quite similar in amplitude and dynamics to that in control, whereas tonic fibers exhibit response summation without relaxation upon removal of excessive K⁺. One can suggest that in phasic fibers the Ca²⁺ influx can directly create a level sufficient to sustain contraction, while in tonic fibers its effect is mediated by Ca-dependent activation of the beta isoform of the ryanodine-sensitive channel.     

Tricyclic antidepressants inhibit voltage-dependent calcium channels and Na(+)-Ca2+ exchange in rat brain cortex synaptosomes

We have used a resting (5 mM K+) or depolarizing (60 mM K+) choline-based medium, and a nondepolarizing sodium-based or choline-based medium, to characterize the inhibitory potential of tricyclic antidepressants against the voltage-dependent calcium channels or the Na(+)-Ca2+ exchange process, respectively, in synaptosomes from rat brain cortex. Imipramine, desipramine, amitriptyline, and clomipramine inhibited net K(+)-induced 45Ca uptake with similar IC50 values (26-31 microM), and this uptake was also inhibited by diltiazem with an IC50 of 36 microM; these results indicate an inhibition of voltage-dependent calcium channels by tricyclic antidepressants. The net uptake of 45Ca induced by Na(+)-Ca2+ exchange was also inhibited by the four tricyclic antidepressants tested, but not by diltiazem; imipramine (IC50 = 94 microM) was a more potent inhibitor of this process than desipramine (IC50 = 151 microM), and the IC50 values of amitriptyline (107 microM) and clomipramine (97 microM) were similar to that of imipramine. Some degree (approximately 25%) of brain calcium channel blockade could be present at the steady-state concentrations of tricyclic antidepressants expected to occur therapeutic use of these compounds to treat depression or panic disorder.     

Xanthorrhizol induces endothelium-independent relaxation of rat thoracic aorta

Xanthorrhizol, a bisabolene isolated from the medicinal plant Iostephane heterophylla, was assayed on rat thoracic aorta rings to elucidate its effect and likely mechanism of action, by measuring changes of isometric tension. Xanthorrhizol (1, 3, 10, 30 and 100 microg/mL) significantly inhibited precontractions induced by KCI-; (60mM), noradrenaline (10(-6) M) or CaCl2 (1.0 mM). Increasing concentrations of external calcium antagonized the inhibitory effect on KCl-induced contractions. The vasorelaxing effect of xanthorrhizol was not affected by indomethacin (10 microM) or L-NAME (100 microM) in intact rat thoracic aorta rings precontracted by noradrenaline, which suggested that the effect was not mediated through either endothelium-derived prostacyclin (PGI2) or nitric oxide release from endothelial cells. Endothelium removal did not affect the relaxation induced by xanthorrhizol on rat thoracic aorta rings, discarding the participation of any substance released by the endothelium. Xanthorrhizol inhibitory effect was greater on KCI- and CaCl2-induced contractions than on those induced by noradrenaline. Xanthorrhizol inhibitory effect in rat thoracic aorta is likely explained for interference with calcium availability by inhibiting calcium influx through both voltage- and receptor-operated channels.     

The monoterpene alkaloid cantleyine from Strychnos trinervis root and its spasmolytic properties.

Cantleyine, a monoterpene alkaloid isolated from the root bark of Strychnos trinervis, was submitted to a broad spectrum pharmacological screening, in which the principal effect observed was a nonspecific relaxation of isolated smooth muscles. Cantleyine relaxed (IC50 2.1 x 10(-4) M) the guinea-pig trachea, pre-contracted by carbachol and antagonized in a nonspecific manner; carbachol (IC50 2.1 x 10(-4) M) and histamine (IC50 1.4 x 10(-4) M) induced contractions in the guinea-pig ileum; and phenylephrine (IC50 3.8 x 10(-4) M) responses in the rat aorta. Cantleyine antagonized (pD'2, 3.82) cumulative concentration response curves to histamine in the ileum in a noncompetitive, reversible (slope, 4.84) and concentration dependent manner. The tonic contractions induced by histamine and KCl were also inhibited in a concentration-dependent and reversible manner (IC50 7.2 x 10(-5) and 1.8 x 10(-4) M, respectively), suggesting that cantleyine should be acting on voltage-dependent Ca2+ channels. This hypothesis was confirmed by the observation that cantleyine inhibited (pD'2, 3.35), in a concentration dependent manner, the CaCl2 induced contraction in depolarizing medium. These results suggest that cantleyine produces nonspecific spasmolytic effects in smooth muscle and that in guinea-pig ileum this effect is, in part, due to the inhibition of Ca+2 influx through voltage-dependent Ca2+ channels.     

Phorbol esters inhibit alpha 1-adrenergic receptor-stimulated phosphoinositide hydrolysis and contraction in rat aorta: evidence for a link between vascular contraction and phosphoinositide turnover

We investigated the actions of two biologically active phorbol esters, phorbol dibutyrate (PDB) and phorbol myristate acetate (PMA), on receptor-stimulated phosphoinositide hydrolysis in rat aorta. We found both PDB and PMA potently inhibited norepinephrine (NE) stimulated PI hydrolysis in rat aortic rings. The biologically inactive phorbol, 4-alpha-phorbol was ineffective. In the presence of the calcium channel antagonist nitrendipine, PDB potently inhibited both the phasic and tonic components of NE-induced contraction. These results suggest a functional coupling between receptor-stimulated PI turnover and vascular contraction. They also suggest a mode of feed-back regulation in vascular tissue involving phorbol esters in receptor-stimulated PI hydrolysis.     

Endothelium-independent vasorelaxation by leonurine, a plant alkaloid purified from Chinese motherwort

Leonurine, a plant alkaloid present in Chinese motherwort, induced concentration- dependent and endothelium-independent relaxation of phenylephrine (PE)- pretreated rat aortic arterial rings. The IC50 values for leonurine were 86.4+/-10.4 and 85.9+/-17.2 microM in the presence and absence of endothelium respectively. It inhibited the responses of aortic smooth muscle to PE in Ca2+ free medium containing 100 microM EGTA, suggesting a possible action on the release of intracellular Ca2+. Leonurine is not a specific alpha-adrenoceptor blocker, since it also caused a concentration-dependent inhibition of vascular contractile responses to KCl with an IC50 value of 96.4+/-13.4 microM, suggesting that leonurine also blocks the L-type Ca2+-channel. In addition, leonurine relaxed the aortic contraction induced by prostaglandin F2alpha (PGF2alpha). These inhibitory effects of leonurine were reversible and did not affect the resting tension. In conclusion, these findings suggest that leonurine is an effective inhibitor of vascular smooth tone, probably acting by inhibiting the Ca2+ influx and the release of intracellular Ca2+.