Mechanisms Involved in the Vasodilator Effect of Curine in Rat Resistance Arteries

• Abstract
• Materials and Methods
• References

Abstract

The vasodilator effect of curine was investigated in the rat small mesenteric arteries. In either endothelium-intact or endothelium-denuded mesenteric arteries, curine induced a concentration-dependent relaxation in rings pre-contracted with noradrenline (10 μM; IC₅₀ = 4.8 ± 1.3 μM and 4.8 ± 1.5 μM, respectively) and KCl (80 mM; IC₅₀ = 6.0 ± 1.3 μM and 13.0 ± 5.6 μM, respectively). Curine also inhibited (IC₅₀ = 4.6 ± 0.9 μM) the concentration-response curves induced by noradrenaline. Contractions dependent on calcium-influx elicited by KCl (80 mM) and noradrenaline (10 μM) were inhibited by curine (10 μM). Finally, contractions induced by noradrenaline (10 μM), in calcium-free medium, were strongly inhibited by curine (10 μM). The above results suggest that the inhibition of influx of calcium ions through voltage-operated calcium channels and non-selective channels, and mobilization of intracellular calcium stores sensitive to noradrenaline are involved in the vasodilator effect of curine.

Chondrodendron platyphyllum A. St. Hil (Miers) is a medicinal plant found in the northwest of Brazil and used for the treatment of fever, malaria and as antispasmodic [1], [2]. Curine (1) is one of the major bisbenzylisoquinoline alkaloids (BBA) isolated from the root barks of C. platyphyllum. Earlier reports had shown that curine presents a non-depolarising neuromuscular blocking action similar to that observed for tubocurarine with lower ganglionary blocking and histamine release actions [3]. However, there is a complete lack of information on the effect of curine in the cardiovascular system, which can be attained after its abortion in the gastrointestinal system.[]

Encouraged by the lack of studies in the vascular system with curine and by the information that some BBA block calcium influx [4], [5], [6], [7], the present work was performed to investigate the effect of curine and its respective mechanism of action in rat small mesenteric arteries.

In the presence of functional endothelium, curine induces a concentration-dependent vasodilatation in rat small mesenteric arteries contracted with 10 μM noradrenaline and 80 mM KCl, with IC₅₀ values of 4.8 ± 1.3 μM and 6.0 ± 1.3 μM, respectively (Fig. [1] A, B). The denudation of the endothelium did not change the vasodilator effect of curine, since the IC₅₀ values observed: 4.8 ± 1.5 μM and 13.0 ± 5.6 μM; were not significantly different for noradrenaline and KCl (Fig. [1] A, B). These results suggest that the relaxant or contractile factors derived from the endothelium did not participate in or modulate the activity of curine in rat small mesenteric arteries.

Recent studies have reported benzylisoquinoline alkaloids as competitive antagonists of alpha₁-adrenergic receptors [8]. In an attempt to verify this effect, we performed concentration-response curves of noradrenaline in the presence and in the absence of various concentrations of curine. We observed that curine induced a concentration-dependent and non-competitive inhibition of curves to noradrenaline with IC₅₀ value of 4.6 ± 0.9 μM in endothelium-intact arteries (Fig. [2]). The value of IC₅₀ observed was similar to those for the vasorelaxant effect of curine in arteries pre-contracted with noradrenaline and KCl. Therefore, our results do not support the competitive antagonism of alpha₁-adrenergic receptors as the mechanism involved in the vasodilator effect of curine in mesenteric arteries.

Curine strongly inhibited the contractile response induced by KCl (80 mM) in mesenteric arteries with functional endothelium (Fig. [3] A). The above results suggest that curine may inhibit the voltage-operated calcium channels in resistance arteries [9]. In an attempt to investigate the effect of curine in other types of channels, such as non-selective channels, we observed the effect of curine in contractions dependent on calcium-influx in arteries stimulated with noradrenaline in calcium-free medium, where the calcium stores were previously depleted. In this experimental condition, curine significantly inhibited contractions dependent on calcium influx (Fig. [3] B) suggesting that inhibition of non-selective channels is involved in the vasorelaxant effect of curine in rat small mesenteric arteries. Together, our results suggest that the inhibition of calcium influx by different types of channel is involved in the vasorelaxant effect of curine.

We investigated the effect of curine in contractions induced by noradrenaline in arteries maintained in calcium-free medium, which are believed to occur due to the release of calcium from InsP₃ stores [10], and to be inhibited by alkaloids [6], [11]. We observed that curine dramatically inhibited these contractions (Fig. [3] C), suggesting that this alkaloid may inhibit the mechanisms involved in the release of calcium from stores sensitive to InsP₃.

Our results allow us to conclude that curine is a vasodilator alkaloid that acts through inhibition of voltage-operated and non-selective calcium channels and by inhibition of contractile mechanisms dependent on the release of calcium from stores sensitive to InsP₃. These results also suggest that the absorption of curine present in C. platyphyllum preparations used in the folk medicine may induce undesirable alterations in the peripheral vascular resistance and consequently in the blood pressure.

Materials and Methods

Animal experiments were performed according to the recommendations of the Brazilian Council for Animal Care and the Ethics Committee of the Universidade Federal de Minas Gerais. Male Wistar rats were killed by cervical dislocation and exsanguinated. Branch II or III of mesenteric arteries were mounted as described by Côrtes et al. [12]. The presence of functional endothelium was assessed by the ability of acetylcholine (ACh; 1 μM) to induce more than 50 % relaxation of vessels pre-contracted with noradrenaline (3 μM). Curine was added cumulatively during the tonic contractions induced by noradrenaline (10 μM), and KCl (80 mM) in tissues suspended in normal Krebs-Henseleit. The vasodilator effect of curine was expressed as percentage decrease in maximal contraction induced by the contractile agents, considering 100 % relaxation as the point when the basal line was reached. Alternatively, concentration-response curves to noradrenaline were performed in the absence and in the presence of different concentrations (1, 3 and 6 μM) of curine and its inhibitory effect was calculated as the percentage of reduction of the maximal response to noradrenaline. Values of inhibitory concentration 50 % (IC₅₀) were calculated graphically from concentration-response curves of curine or by linear regression of the inhibitory effect of different concentrations of curine over concentration-response curves of noradrenaline. A single concentration of curine (10 μM) was added to the organ bath 15 min prior to the addition of KCl (80 mM) and noradrenaline (10 μM) in vessels maintained in Ca²⁺-free (without CaCl₂ plus 0.2 mM EGTA) solution. For experiments performed with noradrenaline, the intracellular calcium stores were depleted after addition of noradrenaline (10 mM) twice, and contractions for both agonists were observed only after addition of CaCl₂ (2.5 mM). The effect of curine was also investigated over the contractile responses of noradrenaline (10 μM) performed in Ca²⁺-free solution [6]. Noradrenaline induced phasic contractile responses, which were considered as control in the absence of curine and used to calculate the inhibitory effect observed in the presence of curine. Results are expressed as the mean ± s. e.m of five experiments. Student’s t-test was used to analyze the results, and was considered significant when p < 0.05. Acetylcholine chloride, noradrenaline bitartrate, and Chaps were purchased from Sigma (USA). Spectroscopically pure curine was isolated as described elsewhere [13], [14]. Curine was solubilized in a mixture of distilled water/chremophor as a 10^-1 M solution and diluted to the desired concentration with distilled water just before use. The final concentration of chremophor never exceeded 0.1 %, which was without effect when exposed to control preparations. The other compounds were freely dissolved in water.

References

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Steyner F. Côrtes

Laboratório de Farmacologia Cardiovascular

Departamento de Farmacologia, UFMG

Av. Antonio Carlos, 6627

Pampulha. 31270-901, Belo Horizonte - MG

Brazil

Phone: +55-31-3499-2726

Fax: +55 31-3499-2695

Email: sfcortes@icb.ufmg.br

References

• 1 Corrêa M P. Dicionário de Plantas Úteis do Brasil e das Exóticas Cultivadas. Vols. 1 - 6 J. Di Giorgio, Rio de Janeiro; 1974
  Search in Google Scholar
• 2 Brandão M, Botelho M, Krettli E. Antimalarial experimental chemotherapy using natural products.  Ciência e Cultura. 1985;  37 1152-63
  PubMedSearch in Google Scholar
• 3 Tang X -C, Feng J, Wang Y E, Liu M -Z. Neuromuscular blocking activity of alkaloids of Cyclea hainanensis .  Acta Pharmacologica Sinica. 1980;  1 17-22
  PubMedSearch in Google Scholar
• 4 Felix J P, King V F, Shevell J L, Garcia M L, Kaczorowski G J, Bick I R, Slaughter R S. Bis(benzylisoquinoline) analogs of tetandrine block L-type calcium channels: evidence for interaction at the diltiazem-binding site.  Biochemistry. 1992;  31 11 793-800
  PubMedSearch in Google Scholar
• 5 Low A M, Berdik M, Sormaz L, Gataiance S, Buchanan M R, Kwan C Y, Daniel E E. Plant alkaloids, tetrandrine and hernandezine, inhibit calcium-depletion stimulated calcium entry in human and bovine endothelial cells.  Life Sciences. 1996;  58 2327-35
  CrossrefPubMedSearch in Google Scholar
• 6 Freitas M R, Côrtes S F, Thomas G, Barbosa-Filho J M. Modification of Ca²⁺ metabolism in the rabbit aorta as a mechanism of spasmolytic action of warifteine, a bisbenzylisoquinoline alkaloid isolated from the leaves of Cissampelos sympodialys Eichl. (Menispermaceae).  Journal of Pharmacy and Pharmacology. 1995;  48 332-6
  PubMedSearch in Google Scholar
• 7 Liu Q Y, Li B, Gang J M, Karpinski E, Pang P KT. Tetrandrine, a Ca⁺⁺ antagonist: effects and mechanisms of action in vascular smooth muscle cells.  Journal of Pharmacology and Experimental Therapeutics. 1995;  273 32-9
  PubMedSearch in Google Scholar
• 8 Chulia S, Ivorra M D, Martinez S, Elorriaga M, Valiente M, Noguera M A, Lugnier C, Advenier C, D’Ocon P. Relationships between structure and vascular activity in a series of benzylisoquinolines.  British Journal of Pharmacology. 1997;  122 409-16
  CrossrefPubMedSearch in Google Scholar
• 9 Karaki H, Ozaki H, Hori M, Mitsui-Saito M, Amano K I, Harada K I, Shigeki M, Nakazawa H, Won K J, Sato K. Calcium movements, distribution, and functions in smooth muscle.  Pharmacological Reviews. 1997;  49 157-230
  PubMedSearch in Google Scholar
• 10 Lagaud G JL, Randriamboavonjy V, Roul G, Stoclet J C, Andriantsitohaina R. Mechanism of Ca²⁺ release and entry during contraction elicited by norepinephrine in rat resistance arteries.  American Journal of Physiology. 1999;  276 H300-308
  PubMedSearch in Google Scholar
• 11 Leung Y M, Kwan C Y, Loh T T. Dual effects of tetrandrine on cytosolic calcium in human leukaemic HL-60 cells: intracellular calcium release and calcium entry blockade.  British Journal of Pharmacology. 1994;  113 767-74
  PubMedSearch in Google Scholar
• 12 Côrtes S F, Rezende B A, Corriu C, Medeiros I A, Teixeira M M, Lopez M J, Lemos V S. Pharmacological evidence for the activation of potassium channels as the mechanism involved in the hypotensive and vasorelaxant effect of dioclein in rat small resistance arteries.  British Journal of Pharmacology. 2001;  133 849-58
  CrossrefPubMedSearch in Google Scholar
• 13 Mambu L, Martin M T, Razafimahela D, Ramanitrahasimbola D, Rasoanaivo P, Frappier F. Spectral characterization and antiplasmodial activity of bisbenzylisoquinolines from Isolona ghesquiereina .  Planta Medica. 2000;  66 537-40
  Thieme ConnectPubMedSearch in Google Scholar
• 14 Barbosa-Filho J M, Da-Cunha E VL, Cornélio M L, Dias C S, Gray A I. Cissaglaberrimine, an aporphine alkaloid from Cissampelos glaberrima .  Phytochemistry. 1997;  44 59-61
  CrossrefPubMedSearch in Google Scholar

Steyner F. Côrtes

Laboratório de Farmacologia Cardiovascular

Departamento de Farmacologia, UFMG

Av. Antonio Carlos, 6627

Pampulha. 31270-901, Belo Horizonte - MG

Brazil

Phone: +55-31-3499-2726

Fax: +55 31-3499-2695

Email: sfcortes@icb.ufmg.br