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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.

Summary

In a previous work, we demonstrated that, in normotensive rats, AFL induced a marked hypotension due to a decrease in total peripheral resistances (TPR), partialy 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 [micro]g/ml) inhibited the sustained contractions induced by KCl (80 and 30 mM) and phenylephrine (Phe, 1 [micro]gM) with similar I[C.sub.50] values (54 [+ or -] 6, 52 [+ or -] 4 and 65 [+ or -] 4 [micro]g/ml, respectively). The relaxing response induced by AFL against Phe--induced contractions was modified significantly by the endothelium removal (I[C.sub.50] = 132 [+ or -] 23 and 65 [+ or -] 4 [micro]g/ml, endothelium removed and intact endothelium aortic rings, respectively). Nevertheless, removal of the endothelium did not significantly change I[C.sub.50] 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.sup.+]-induced contraction was potentiated slightly (p < 0.05) by the decrease (from 2.5 to 0.3 mM, [Ca.sup.2+]) and attenuated by the increase (from 2.5 to 7.5 mM [Ca.sup.2+]) in the external [[Ca.sup.2+]]. In addition, in aortas from normotensive rats, AFL antagonized transient contractions induced in [Ca.sup.2+]-free media induced by 1 [micro]gM 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 [Ca.sup.2+] influx and/or inhibition of intracellular [Ca.sup.2+] mobilization from the noradrenaline-sensitive stores.

Key words: Albizia inopinata, calcium mobilization, vasodilation, rat aorta

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Introduction

In a previous study on conscious, unrestrained normotensive rats, we demonstrated that the aqueous fraction of the ethanolic extract of the leaves (AFL) of Albizia inopinata (Harms) G.P. Lewis, produced a significant and dose-dependent hypotension associated with increases in heart rate and cardiac output, and with a strong reduction in TPR, at least partialy secondary to the release of NO by the vascular endothelium (Pires et al. 2000).

The aim of this study was to investigate the involvement of the intracellular calcium mobilization in the vasorelaxant action induced by the aqueous fraction of the ethanol extract of the leaves (AFL) of Albizia inopinata G.P. Lewis, in the aorta of normotensive rats.

Material and Methods

Animals

Male Wistar normotensive rats (200-350 g each) were used for all experiments. The animals were housed under conditions of controlled temperature (21 [+ or -] 1 [degrees]C) and exposed to a 12-hour, light-dark cycle with free access to food and tap water.

* Phytochemical screening of the aqueous fraction of the leaves of Albizia inopinata (Harms) G.P. Lewis (AFL). AFL was prepared as reported previously by Pires et al. (2000). A phytochemical screening of AFL demonstrated the presence of saponins and alkaloids only. When required, AFL was dissolved in water and filtered, and known volumes were dried to determine the concentration, as only 70% of the crude extract was soluble. This factor was used to calculate the final concentration of AFL.

Drugs

The following drugs were used: L(-) phenylephrine hydrochloride (Pfizer), noradrenaline hydrochloride (Pfizer); sodium pentobarbital (Halocarbon); heparin sodium salt (Roche); caffeine, ([+ or -]) sodium nitroprusside, acetylcoline hydrochloride, [N.sup.[omega][epsilon]]-Nitro-L-arginine methyl ester (L-NAME) hydrochloride; ethyleneglycol bis ([beta]-aminoethylether)-N,N,N'N'-tetraacetic acid (EGTA) (all from Sigma); ethylenediaminetetraacetic acid dihydrate dissodium salt (EDTA) (Merck). The stock solutions were dissolved on distilled water and kept at 0[degrees]C. All the drugs were dissolved on distilled water immediately before the experiments, except EGTA.

Solutions

The normal physiological salt solution contained (mM), NaCl 118.0; KCl 4.7; NaHC[O.sub.3] 25.00; Ca[Cl.sub.2].2[H.sub.2]O 2.5; glucose 11.1; K[H.sub.2]P[O.sub.4] 1.2 and MgS[O.sub.4].7[H.sub.2]O 1.2. In some experiments, the concentration of Ca[Cl.sub.2] was changed to 0.3 mM or 7.5 mM. Isosmotic 65.4 mM [K.sup.+] solution was made by substituting 60 mM NaCl in the normal solution with equimolar KCl. [Ca.sup.2+]-free solution was made by omitting Ca[Cl.sub.2] and adding 1 mM ethyleneglycol bis ([beta]-aminoethyllether)-N,N,N',N'-Tetraacetic acid (EGTA). These solutions were saturated with a mixture of 95% [O.sub.2] and 5% C[O.sub.2] at 37[degrees]C (pH 7.4).

Muscle preparations using isolated rat aortic rings

Male Wistar rats were killed by a blow on the neck and exsanguination. Rat thoracic aortic rings (2-4 mm) were dissected free of connective tissue and fat. Endothelium was removed by gently rubbing the intimal surface with a finger moistened with normal physiological salt solution, and suspended by platinum hooks for isometric tension recordings in a Kreb's-Henseleit solution maintained at 37[degrees]C and gassed with a mixture of 95% [O.sub.2] and 5% C[O.sub.2]. The rings were allowed to equilibrate for 1 h under a resting tension of 1 g.

Contractile tension

Muscle tension was recorded isometrically with a force-displacement transducer (7003, Ugo basile, Comerio, VA, Italy) coupled to a physiograph (Gemini 2, Ugo Basile, Comerio, VA, Italy). Passive tension of 1 g was applied initially and tissues were allowed to equilibrate in a 10 ml bath for 60 min before the beginning of the experimental period. AFL was applied cumulatively when the contractile tension induced by a stimulant reached a steady level or applied before the addition of a stimulant. The I[C.sub.50] values were calculated by linear regression of individual concentration-response curves.

Experimental protocols

* Effect of AFL on isolated rat aortic rings. In this experiment, two successive contractions of similar magnitude to each agonist were induced. Following stabilization of the preparations, during the tonic phase of the second response, increasing concentrations of AFL (10, 20, 40 and 80 [micro]g * m[l.sup.-1]) were added cumulatively to isolated aortic preparations pre-contracted with Phe 1 [micro]gM or KCl 80 mM and 30 mM. The presence of endothelium was verified by the relaxation of the aortic rings after addition of 1 [micro]gM acetylcholine. The percentage of inhibition was measured by comparing the response before and after addition of AFL. I[C.sub.50] values were calculated by linear regression of the individual curves.

Influence of endothelium removal on the relaxing response of AFL against phenylephrine and KCl-induced contractions

In this experiment, concentration-response curves (CCR) to AFL (10, 20, 40 and 80 [micro]g * m[l.sup.-1]) were obtained after Phe (1 [micro]gM)- and KCl (80 and 30 mM)-induced contractions in endothelium-free rings. The effectiveness of endothelium removal was verified by the loss of the relaxing response of the preparations (pre-contracted with Phe) to 1 [micro]gM acetylcholine.

Influence of extracellular calcium on the vasorelaxant response induced by AFL

This protocol was conducted according to Sakata and Karaki (1991). In endothelium-denuded aortic rings, the [[Ca.sup.2+]] of the K-H solution was changed (to 0.3, 2.5 and 7.5 mM) 10 min before the addition of a stimulant (1 [micro]gM noradrenaline or 65.4 mM [K.sup.+]). After two CCR of magnitude similar to a stimulant were obtained, in the tonic phase of the second one, AFL (10, 20, 40 and 80 [micro]g * m[l.sup.-1]) was then added (cumulatively) to the preparations. I[C.sub.50] values were calculated as described previously.

Effect of AFL on noradrenaline and caffeine-sensitive intracellular calcium stores

The effect of AFL (20 and 80 [micro]g * m[l.sup.-1]) on intracellular calcium stores sensitive to noradrenaline or caffeine was investigated by using the method described by Sakata and Karaki, 1991. After the equilibration period, the tissues were exposed to 65.4 mM [K.sup.+] solution for 3 min. The tissues were then washed with [Ca.sup.2+]-free solution (CFS) followed by the addition of 1 [micro]gM noradrenaline or 20 mM caffeine. After washing the tissues with normal krebs solution, high potassium was added for 3 min ([Ca.sup.2+] loading). The preparations were washed with CFS followed again by the application of noradrenaline or caffeine. This procedure was repeated until two similar transient contractions to the agonist had been obtained. The experiment was then repeated with AFL being added 2 min before the application of noradrenaline or caffeine.

Data analysis

Data are expressed as mean [+ or -] s.e.mean. Statistical analyses were performed by means of paired and unpaired Student's t-tests. Values were considered to be significantly different when p < 0.05. Linear regressions were performed using the least square method.

Results

Vasorelaxant effects of AFL on isolated normotensive rat aortic rings on sustained contractions induced by Phe and KCl (Table 1)

AFL (10, 20, 40 and 80 [micro]g/ml) antagonized the contractions induced by all the agents tested in a concentration dependent manner. Table 1 also shows that in isolated intact endothelium aortic rings of normotensive rats, AFL (10, 20, 40 and 80 [micro]g/ml) inhibited the sustained contractions induced by 80 mM and 30 mM KCl and 1 [micro]M Phe with similar I[C.sub.50] values (54 [+ or -] 6, 52 [+ or -] 4, 65 [+ or -] 4 [micro]g/ml, respectively).

Influence of endothelium removal on the relaxing response of AFL against Phe- and KCl-induced contractions

As shown in Table 1, the relaxing response induced by AFL (10, 20, 40 and 80 [micro]g/ml) against Phe-induced contractions was modified significantly by endothelium removal (I[C.sub.50] value = 132 [+ or -] 23 and 65 [+ or -] 4 [micro]g/ml, endothelium removed vs. intact endothelium aortic rings). Nevertheless, removal of the endothelium did not change significantly I[C.sub.50] values when KCl (30 and 80 mM) was used as the contractile agent (Table 1).

[FIGURE 1 OMITTED]

Influence of extracellular calcium on the vasorelaxant response induced by AFL

As shown in Table 2, in normotensive rats, the inhibitory effect on high (65.4 mM) [K.sup.+]-induced contraction was potentiated slightly (p < 0.05) by the decrease (from 2.5 to 0.3 mM [Ca.sup.2+]) in external [Ca.sup.2+] concentration ([[Ca.sup.2+]][.sub.ext]) and was antagonized by the increase (from 2.5 to 7.5 mM [Ca.sup.2+]) in the [[Ca.sup.2+]][.sub.ext]. Although changes in the [[Ca.sup.2+]][.sub.ext] modified the inhibitory effect on noradrenaline-induced contraction, a statistically significant difference was not obtained (Table 2).

Effect of AFL on noradrenaline and caffeine-sensitive intracellular calcium stores

Fig. 1 shows that in aortic rings from normotensive rats, AFL (20 and 80 [micro]g/ml) antagonized transient contractions induced in [Ca.sup.2+]-free media by 1 [micro]gM noradrenaline in a concentration-dependent manner. Therefore, even at a high concentration (80 [micro]g/ml) AFL was not capable to antagonize transient contrations induced by caffeine -20 mM (Fig. 1).

Discussion

The major finding of this study was that AFL can exert its non-specific spasmolytic actions in rat isolated aortic rings by inhibiting calcium influx and some of the calcium signaling elements involved in the smooth-muscle-contraction event.

It is well-established that the release of endothelium-derived relaxing factors (EDRFs) from endothelial cells leads to relaxation of vascular smooth muscle cells and plays a critical role in the maintenance of vascular tone (Moncada and Higgs, 1993). In order to determine whether part of the relaxant effect produced by AFL in isolated aortic rings could be due to the release of EDRFs, we performed experiments in aortic preparations in which the endothelium was removed mechanically. In these preparations, the relaxant effect induced by the aqueous fraction of Albizia inopinata was attenuated significantly. These results indicated clearly that relaxing factors released by the endothelium play an important role in the vasorelaxant response induced by AFL. In a previous work (Pires et al. 2000), we demonstrated that the inhibition of nitric-oxide synthase (L-NAME) as well as the inhibition of cyclooxygenase-1 (indomethacin) attenuated the relaxing action induced by AFL 80 [micro]g/ml. Taken together, these results indicated clearly that relaxing factors released by the endothelium, mostly NO and prostacyclin, may play an important role in the vasorelaxant response induced by AFL.

In intact aortic preparations, we found that AFL was able to antagonize, in a concentration-dependent manner, KCl (30 and 80 mM)- and Phe-induced contractions. Its is well known that KCl induces smooth-muscle contraction through activation of voltage-dependent calcium channels and subsequent release of calcium from the sarcoplasmic reticulum (Nasa et al. 1992; Gurney, 1994), whereas phenylephrine-induced vasoconstriction is mediated by the stimulation of G-protein coupled to [alpha]-adrenoceptors (Perez-Viscaino et al. 1988). In both cases, the major effect is an increasing in intracellular calcium concentration through calcium entry. We suggest therefore that the residual vasorelaxant effect observed after endothelium removal is due to an endothelium-independent mechanism, possibly linked to a calcium influx blocking activity.

The inhibitory effect of AFL on high potassium (65.4 mM [K.sup.+])-induced contraction was antagonized by the increase in the external [Ca.sup.2+] concentration. These results are similar to those obtained with verapamil and other calcium channel blockers (Karaki and Weiss, 1984; Sakata and Karaki, 1991).

In vascular smooth muscle, it has been shown that [Ca.sup.2+] channel blockers inhibit strongly the high [K.sup.+]-induced contraction, with less inhibitory effect on the noradrenaline-induced contraction (Karaki and Weiss, 1984; Sakata and Karaki, 1991). By contrast, AFL inhibited the contractions induced by high [K.sup.+] and noradrenaline at similar concentrations. This result indicates that the inhibitory effect of AFL is not attributable solely to a calcium channel blocker-like action.

This led us to investigate if AFL could exert its non-specific vasorelaxant actions by interfering with the calcium release induced by phosphoinositide production following receptor activation. We investigated the effect of AFL on noradrenaline- and caffeine-induced transient contractions in [Ca.sup.2+]-free media. AFL inhibited transient contractions induced by noradrenaline but not those induced by caffeine. It has been suggested that the noradrenaline-induced release of calcium is attributable to receptor-mediated formation of inositol 1, 4, 5-trisphosphate, whereas the caffeine-induced calcium release is due to [Ca.sup.2+]-induced [Ca.sup.2+]-release mechanism (for review see Karaki and Weiss, 1988). Since caffeine-induced transient contractions in [Ca.sup.2+]-free media were not inhibited by the aqueous fraction, we suggest that the spasmolytic effect of AFL is not due to an inhibition of the smooth-muscle-contractile elements. Thus, AFL may relatively selectively inhibit the [Ca.sup.2+] release due to inositol 1,4,5-trisphosphate in the rat aorta.

In summary, it is suggested that the remaining endothelium-independent vasodilatador effect of AFL in the aortic rings is due to an inhibition of [Ca.sup.2+] influx. Furthemore, the non-specific spasmolitic action of AFL can be attributed to its ability to inhibit the intracelular calcium mobilization from the noradrenaline-sensitive stores.
Table 1. Vasorelaxant effect of increasing concentrations of AFL (10,
20, 40 and 80 [micro]g/m[l.sup.-1]) on isolated rat aortic rings (n =
7-10) intact and after endothelium removal, pre-contracted with Phe (1
[micro]M) and KCl (80 and 30 mM). Results are means [+ or -] s.e.m. *p <
0.05 vs endothelium intact.

Contractile agent I[C.sub.50] values ([micro]g/ml)
 Endothelium Endothelium
 Intact Denuded

Phe (1 [micro]M) 65 [+ or -] 4 132 [+ or -] 23 *
KCl (80 mM) 54 [+ or -] 6 69 [+ or -] 4
KCl (30 mM) 52 [+ or -] 4 48 [+ or -] 3

Table 2. Concentration of AFL required to induce a 50% inhibition of
contractions in isolated rat aortic rings (n = 7) pre-contracted with
isosmotic solution [K.sup.+] (65.4 mM) or noradrenaline (1 [micro]M)
under different extracellular concentrations of calcium. Results are
means [+ or -] s.e.m., * p < 0.05 versus [Ca.sup.2+] 2.5 mM.

Condition [K.sup.+] (65.4 mM) Noradrenaline (1 [micro]M)
 I[C.sub.50] values I[C.sub.50] values
 ([micro]g/ml) ([micro]g/ml)

[Ca.sup.2+] 0.3 mM 30.4 [+ or -] 3.9 * 79.6 [+ or -] 6.6
[Ca.sup.2+] 2.5 mM 44.8 [+ or -] 1.4 77.1 [+ or -] 5.1
[Ca.sup.2+] 7.5 mM 58.1 [+ or -] 6.3 * 66.1 [+ or -] 4.3


Acknowledgements

The authors wish to express their sincere thanks to Jose Crispim Duarte for technical assistance. Financial support from CAPES and CNPq-Brazil is also gratefully acknowledged.

References

Gurney AM (1994) Mechanisms of drugs-induced vasodilatation. J Pharm Pharmacol 46: 242-251

Karaki H, Weiss GB (1988) Calcium release in smooth muscle. Life Sciences 42: 111-122

Karaki H, Weiss GB (1984) Calcium channels in smooth muscle. Gastroenterology 87: 960-970

Moncada S, Higgs EA (1993) The L-arginine-nitric oxide pathway. N Engl J Med 29: 2002-2012

Nasa Y, Ichihara K, Yoshida R, Abiko Y (1992) Positive inotropic and negative chronotropic effect of (-)-cis-diltiazem in rat isolated atria. Br J Pharmacol 105: 696-702

Perez-Viscaino E, Cogolludo A L, Villamor E, Tamargo J (1988) Role of [K.sup.+] channel opening and stimulation of cyclic GMP in the vasorelaxant effects of nicorandil in isolated piglet pulmonary and mesenteric arteries: relative efficacy and interactions between both pathways. Br J Pharmacol 123: 781-1022

Pires S L S, Assis T S, Nogueira E A R, Almeida R N, Barbosa-Filho J M, Medeiros I A (2000) Endothelium-derived nitric oxide is involved in the hypotensive and vasorelaxant responses induced by the aqueous fraction of the ethanolic extract of the leaves of Albizia inopinata (Harms) G. P. Lewis in rats. Phytomedicine 7: 91-98

Sakata K, Karaki H (1991) Effects of a novel smooth muscle relaxant, KT -362, on contraction on cytosolic [Ca.sup.2+] level in the rat aorta. Br J Pharmacol 102: 174-178

S. S. Maciel, K. L. G. Dias, and I. A. Medeiros

Laboratorio de Tecnologia Farmaceutica, Universidade Federal da Paraiba, Joao Pessoa, Brazil

Address

Simone dos Santos Maciel, Laboratorio de Tecnologia Farmaceutica, Universidade Federal da Paraiba, Caixa Postal 5009, 58.051-970-Joao Pessoa, PB, Brazil

e-mail: isacmed@uol.com.br
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Author:Maciel, S.S.; Dias, K.L.G.; Medeiros, I.A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:3BRAZ
Date:Feb 1, 2004
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