The objective of the study was to investigate the mechanism of the relaxant activity of the
The use of alternative therapies, herbs, and supplements occurs at a very high rate among patients with cardiovascular disease including hypertension [
Several medicinal plants have been largely studied and their therapeutic potential has been demonstrated in animals, aiming to provide a scientific basis for the therapeutic applications. In this context, we highlight the impact and importance of medicinal plants containing diterpenoids and their action antihypertensive. For example,
Terpenoids constitute the largest family of natural products [
Biological assays have shown that the diterpenes exert hypotensive and antihypertensive action. The kaurenoic acid relaxes isolated rat aorta by blocking extracellular Ca2+ influx blocked, activation of NO-cGMP pathway, and the opening of K+ channels [
Based on this premise, it is evident that the importance of studies characterizes the cardiovascular activities of kaurane-type diterpene, because these compounds can be a promising source for the discovery of numerous bioactive molecules and the development of novel antihypertensive agents [
In a broad pharmacological screening performed in our laboratory, we observed in isolated mesenteric superior rings of rats that KA-acetoxy produced a relaxant action on smooth muscle. The present study aimed to elucidate the mechanism of this vasorelaxant effect induced by KA-acetoxy in rat isolated mesenteric rings.
The leaves of
The dried and powdered leaves (5.230 g) were extracted in a Soxhlet apparatus. The solvents used were first hexane, then chloroform, and finally EtOH. The hexane extract yielded after cooling a white precipitate. This precipitate, after filtration and washing with hot hexane, yielded KA-acetoxy (5.38 g). KA-acetoxy was identified by means of spectroscopic methods, mainly 1D and 2D NMR as
Chemical structure of KA-acetoxy.
The following drugs were used: atropine sulfate, acetylcholine hydrochloride (ACh), indomethacin,
Male Wistar rats (200–300 g) were used in all experiments. Experimental protocols and procedures were approved by the Laboratório de Tecnologia Farmacêutica Animal Care and Use Committee. Animals were housed under conditions of controlled temperature (
Rats were killed by stunning and exsanguination. The superior mesenteric artery was removed, cleaned from connective tissue and fat and sectioned in rings (1-2 mm), which were suspended by cotton threads in organ baths containing 10 mL of Tyrode’s solution (composition in mM: NaCl: 158.3: KCl: 4.0; CaCl2: 2.0; MgCl2: 1.05; NaH2PO4: 0.42; NaHCO3: 10.0; and glucose: 5.6.), gassed with carbogenic mixture (95% O2 and 5% CO2), and maintained at 37°C for isometric
In the first set of experiments, the ability of KA-acetoxy to cause vascular relaxation was evaluated in both endothelium-intact and endothelium-denuded mesenteric artery rings previously contracted by Phe (10
In the second set of experiments, after the stabilization period, rings without endothelium were precontracted with KCl 80 mM on the tonic phase and different concentrations of KA-acetoxy (10−6–1 mM) were added cumulatively to organ bath. The extent of relaxation was expressed as the percentage of phenylephrine- or KCl-induced contraction.
To investigate the possible mechanism(s) responsible for KA-acetoxy induced relaxation, the preparations with endothelium were precontracted with Phe for 30 min after being early incubated with one of the following inhibitors: atropine (1
Phe-induced sustained contractions were obtained in endothelium-intact and endothelium-denuded mesenteric artery rings incubated with tetraethylammonium, a nonselective inhibitor of K+ channels (TEA, 5 mM), and then concentration-response curves to KA-acetoxy were obtained. The TEA was added 30 minutes before the contractions with Phe.
In order to access the effects of KA-acetoxy on voltage-gated Ca2+ channels, superior mesenteric artery rings were bathed for 15 min in Ca2+-free Tyrode’s solution, prepared by omitting only CaCl2 and then exposed for an additional 15 min to a high K+ (60 mM) Ca2+-free solution. Under this new experimental condition, cumulative concentration response curves to CaCl2 (ranging from 1
The effect of KA-acetoxy on phenylephrine- or caffeine-sensitive calcium intracellular stores was assessed by using a protocol described by Sakata and Karaki [
In order to study the effect of KA-acetoxy on inducing relaxation, two pharmacological parameters were analysed: the
Table
Comparison of
Phenylephrine 10 ( |
|
pD2 |
---|---|---|
(condition) | (percentage of relaxation) | (value) |
Endothelium intact | 92.8 ± 3.7 | 6.0 ± 0.3 |
Endothelium denuded | 95.2 ± 2.8 | 4.6 ± 0.2*** |
L-NAME (100 |
67.3 ± 4.3*** | 4.1 ± 0.1*** |
Indomethacin (10 |
61.2 ± 7.4*** | 4.5 ± 0.3* |
L-NAME + indomethacin | 66.9 ± 3.6*** | 4.7 ± 0.3* |
Atropine (1 |
73.6 ± 6.6* | 4.4 ± 0.2*** |
ODQ (10 |
77.7 ± 3.6* | 4.6 ± 0.5* |
Hydroxycobalamine (10 |
68.1 ± 6.7** | 4.6 ± 0.3* |
L-arginine (1000 |
91.4 ± 2.9 | 5.4 ± 0.4 |
L-NAME + L-arginine | 100 ± 0 | 5.3 ± 0.4 |
Values are expressed as means ± SEM of six experiments. These experiments were performed in mesenteric rings with functional endothelium. *
The magnitude of contraction induced by Phe in rings with and without endothelium functional was 0.42 and 0.44 g, respectively. Similarly, the magnitude of contraction induced by KCl in rings without endothelium was 0.40 g. There were no significant differences between the magnitudes.
The incubation with L-NAME (100
In endothelium-intact and denuded rings precontracted with Phe (10
Effect of TEA in the relaxant effect of KA-acetoxy.
Phenylephrine 10 ( |
|
pD2 |
---|---|---|
(condition) | (%) relaxation | (value) |
Endothelium intact | 92.8 ± 3.7 | 6.0 ± 0.3 |
Endothelium denuded | 95.2 ± 2.8 | 4.6 ± 0.2 |
Endothelium intact + TEA (5 mM) | 90.7 ± 3.1a | 5.4 ± 0.4a |
Endothelium denuded + TEA (5 mM) | 100 ± 0b | 4.4 ± 0.2b |
Values are expressed as means ± SEM of six experiments. The data were analysed by one-way Anova followed by the Bonferroni post-test.
aCompared to endothelium intact.
bCompared to endothelium denuded.
Under this experimental condition, KA-acetoxy produced a nonparallel and concentration-dependent rightward shift of the CaCl2 concentration-response curve significantly reducing the maximal response as illustrated in Figure
Concentration-response curves for CaCl2 before (• control,
In mesenteric rings under a Ca2+-free solution, KA-acetoxy not inhibited transient contractions induced by 10
Effects of KA-acetoxy (100
The present work was performed in order to investigate possible vasodilator effects of the KA-acetoxy in the isolated rat superior mesenteric artery. It was observed that diterpene induced concentration-dependent vasorelaxation in the isolated rat superior mesenteric artery. The results also suggest that there are two components of the vasodilatory effect: one endothelium dependent and the other endothelium independent.
The vascular endothelium plays an important role in homeostasis by modulating vascular smooth muscle tone and acts as a main target site in hypertension and atherosclerosis. Regulation of vasodilatation by the endothelium is determined by three main components; NO, prostacyclin and endothelium derived hyperpolarizing factor (EDHF). These endothelium-derived relaxing factors diffuse to adjacent smooth muscle cells and cause them relaxation [
L-arginine, a NO precursor, antagonized the effect of L-NAME, but when added alone it did not affect the relaxations induced by KA-acetoxy. The inability of this NO synthase substrate to increase the relaxation induced by KA-acetoxy may be explained in terms of there being sufficient amounts of L-arginine in the vascular endothelium. The
In most vascular beds, the stimulation of muscarinic receptors (M3 subtype) produces an intense dilation, despite the lack of vascular cholinergic innervation [
It is well known that NO induces vascular smooth muscle relaxation through activation of guanylyl cyclase, leading to the accumulation of cyclic GMP. Relaxation of vascular smooth-muscle by NO-cGMP signaling involves a sequence of steps. NO released activates soluble guanylyl cyclase. This enzyme catalyzes the conversion of GPT to cGMP. cGMP-activaed protein kinase G inhibits Ca2+ influx, augments Ca2+ sequestration, and decreases the sensitivity of contractile elements to Ca2+ [
EDHF plays little role in vasoactive responses of conduit vessels, it mediates a major component of the response to endothelium-dependent vasodilators in resistance arteries [
The vasodilation mediated by membrane hyperpolarization is attributed to a rise in K+ permeability. Direct activation of K+ channels on arterial smooth muscle cells normally hyperpolarizes the cell membrane and thus inhibits Ca2+ influx through voltage sensitive Ca2+ channels [
The maintenance of smooth muscle contraction depends on Ca2+ influx from the extracellular space through voltage- and/or receptor-operated calcium channels (VOCCs and/or receptor operated calcium channels, resp.) [
Thus, it is proposed that the residual vasorelaxant effect of the diterpene is due to a mechanism independent of endothelium, possibly a blocking activity on the Ca2+ channels. Based on this assumption, we evaluated the effect of KA-acetoxy on endothelium-denuded rings precontracted with K+-depolarizing solutions (KCl 80 mM). KA-acetoxy was capable to inhibit contractility induced by KCl (80 mM) in endothelium-denuded mesenteric rings. This result suggests that KA-acetoxy could inhibit Ca2+ influx through VOCCs.
In order to strengthen the above hypothesis, KA-acetoxy was tested in the presence of CaCl2-induced contractions in a depolarizing medium without calcium. This protocol was based on the fact that CaCl2-induced contractions are elicited, almost exclusively, through Ca2+ influx, since the depolarization promoted by high concentrations of extracellular K+ induces the opening of voltage-dependent Ca2+ channels [
The release of intracellular stored Ca2+ is mainly regulated by IP3 receptor system (IP3Rs) and ryanodine receptor system (RyRs). The former induces Ca2+ release directly when the receptors are bound to IP3. The later may function through a Ca2+ induced Ca2+ release (CICR) mechanism when the receptors are activated by caffeine [
KA-acetoxy is a closely related derivate with kaurenoic cid (
Recently, we observed that KA-acetoxy induced hypotension activity in animals with essential hypertension (Lyon hypertensive rats) (unpublished data). It is well known that small arteries, as the superior mesenteric artery, play an important role in the determination of the peripheral resistance and in the regulation of blood pressure [
In addition, only substances that inhibit contractions in KCl test model are worth further examination in a search for naturally occurring calcium-antagonists. In this context, the KA-acetoxy was able to inhibit contractility induced by KCl. In this context, it is possible to suggest that the KA-acetoxy could exert antihypertensive action in vivo as other diterpenes [
In summary, the present study demonstrated that the KA-acetoxy produced a concentration and endothelium-dependent and -independent vasorelaxation in superior mesenteric artery rings. Endothelium-dependent relaxation appears to be due to endothelial muscarinic receptors activation, NO and PGI2 release, as well as probably the participation of EDHF. Endothelium-independent relaxation KA-acetoxy acts through inhibition of the Ca2+ influx.
Financial support from CNPq-Brazil is acknowledged gratefully. The authors report no conflict of interests. The authors alone are responsible for the content and writing of this paper.