Invited Symposium: Medicinal Plants and Drug Actions
Discussion and Conclusion
Our initial studies showed that the aqueous extract of A. paniculata possesses a definite hypotensive effect in both the SHR and WKY rats when given by chronic i.p. infusion. The hypotensive responses in these rats suggest that the effects are not related to the level of SBP and non-specific with regard to strain. The results also suggest the likelihood that there may be a variety of mechanisms by which the active compound(s) in A. paniculata exert their hypotensive action.
It is widely accepted that free cytosolic Ca2+ is necessary for the contraction of vascular smooth muscle. KCl has been shown to produce a biphasic contraction in the rat aorta (Sakata & Karaki, 1991) with little separation between the fast and slow components. Both these components depend exclusively upon the influx of extracellular Ca2+ influx through voltage-dependent Ca2+ channel (Karaki & Weiss, 1979, 1984; Marriott, 1988). However contractions induced by high concentrations of norepinephrine show a distinctly biphasis characteristic of a separation between the fast and slow components, the early fast component depending upon the release of intracellular Ca2+, while the slowly developing sustained phase is due to the influx of extracellular Ca2+ (Marriott, 1988) mainly through receptor-operated Ca2+ channels and less through voltage-dependent Ca2+channels (Bolton, 1979; Weiss, 1983). Our experiments showed that DA not only relaxed the phenylephrine and KCl-induced tonic contractions in a concentration-dependent manner but also antagonised the concentration-response curve of phenylephrine in a non-competitive manner. DA also inhibited the responses of KCl-depolarized rings to Ca2+ in a concentration-dependent manner. These results together indicate that DA blocks Ca2+ influx through interference with both voltage- and receptor-operated ion channels.
The contraction of smooth muscle is now believed to be influenced by increases in cytosolic Ca2+ level and Ca2+ sensitivity of contractile elements. Various receptor-agonists are known to increase both these elements. Some vasodilators however selectively inhibit one of these mechanisms, e.g. verapamil, a Ca2+-channel blocker, decreases K+-stimulated Ca2+ influx but not Ca2+ sensitization (Karaki, 1989). This explains the observations of Flaim (1982) and Karaki and Weiss (1988) that Ca2+ channel blockers strongly inhibit the contraction induced by high K+, but are less potent in inhibiting the norepinephrine-induced contraction in vascular smooth muscle. EC50 values showed that DA, like verapamil, was much more potent in relaxing preparations contracted by KCl than by phenylephrine. This finding provides further support for the Ca2+-channel blocking action of DA.
Under Ca2+-free conditions, norepinephrine induced a phasic contraction followed by a tonic contraction, both being due to the intracellular Ca2+ release from internal stores (Flaim, 1982). A similar contraction has been observed after addition of caffeine to Ca2+-free medium, but different mechanisms are cited: caffeine enhances the Ca2+-induced Ca2+ release (CCR) mechanism in vascular smooth muscle (Saida & Van Breemen, 1984; Noguera & D'Ocon, 1992) while norepinephrine enhances the release of intracellular Ca2+ by alpha1-adrenoceptor activation through the action of inositol-1,4,5-trisphosphate (IP3) [Hashimoto et al., 1986; Chiu et al., 1987; Karaki & Weiss, 1988; Daly et al., 1990]. In addition to this difference in the mechanism of intracellular Ca2+-storage pools, caffeine-sensitive Ca2+-storage pools also do not overlap with norepinephrine-sensitive pools (Kanaide et al., 1987). In Ca2+ free medium, DA inhibited or almost abolished contractions induced by norepinephrine and caffeine. Therefore, DA could inhibit the release of intracellular Ca2+ not only from caffeine-sensitive stores, or the enhancement effect on the CCR mechanism, but also from the norepinephrine-sensitive stores or the increase of IP3. However, the possibility that DA depletes the intracellular Ca2+ store by inhibiting the Ca2+ pump cannot be excluded.
The vascular endothelium plays an important role in controlling the vascular tone via secretion of both relaxant and contractile factors (Jaffe, 1985; Vanhoutte et al., 1986). The most potent known are the vasodilators, endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2), and the vasoconstrictors, angiotensin II and endothelin (Luscher, 1994; Persson, 1996). EDRF is now known to be NO (Palmer et al., 1987) or a closely related nitrosothiol (Myers et al., 1990). It is now established that NO is synthesized from the amino acid, L-arginine, by NO synthase, and it stimulates cyclic GMP production by activating soluble guanylate cyclase (Moncada et al., 1991). The relaxing action of DA was attenuated in endothelium-denuded aorta without modifying the maximal response as indicated by the EC50 values. This suggested that the vasorelaxant effect of DA was dependent upon the endothelium. The vasorelaxation caused by DA in intact aorta was shown to persist in the presence of indomethacin (which blocks the formation of PGI2 by inhibiting cyclo-oxygenase) and glibenclamide (which blocks ATP-sensitive K+ channels), implying that this effect was not mediated by PGI2 or ATP-sensitive K+ channel. However, L-NAME (a specific and competitive NO-synthase [NOS] inhibitor) and methylene blue (an inhibitor of NO activation of guanylate cyclase) were able to partially inhibit the relaxant effect of DA on phenylephrine- but not KCl-induced contraction. Taken together, these results suggest that the relaxation of the rat aorta caused by DA may be mediated through the activation of the NOS-guanylate cyclase pathway. The results are consistent with our earlier finding that NO is more effective in inhibiting norepinephrine-induced contraction than high K+-induced contraction, and are also in line with the report of Karaki (1989) that NO (or cGMP) inhibits Ca2+ more strongly than Ca2+ influx.
Endothelium removal will result in the loss of not only NO but also endogenous vasoconstrictors. As these endothelium-derived vasoconstrictors may antagonise the vasorelaxant response of aortic rings to DA, their absence in endothelium-denuded vessels may account for the smaller attenuation of DA-induced vasorelaxation in endothelium-denuded rings, compared to those in endothelium-intact L-NAME- and methylene blue-treated rings. The present level of knowledge suggests that the relationship between K+-induced contraction and endothelium-derived vasoactive substances is not yet clear. It is possible that the absence of endothelium-derived vasorelaxants, other than NO and PGI2, in endothelium-denuded K+-contracted aortae was a factor contributing to the observed decrease in the sensitivity of its response to DA.
We conclude that DA exerts its vasorelaxant activity by the NO-synthase and NO activation of guanylate cyclase pathway as well as the blockade of Ca2+ influx through both voltage- and receptor-operated Ca2+ channels.
DDA was shown to possess a distinct hypotensive effect and to cause bradycardia in anaesthetized SD rats. This initial experimental finding raised the possibility that the cardiovascular activity of DDA may involve a direct action on the heart.
The hypotensive activity of DDA was shown to be unrelated to alpha-adrenoceptors, muscarinic cholinergic or histaminergic receptors, since the application of appropriate specific antagonists failed to affect the hypotensive action of DDA. In the presence of hexamethonium or captopril, the decreases in MAP induced by DDA were significantly attenuated suggesting that the hypotensive action of DDA may involve the autonomic ganglia and the renin-angiotensin system. The data also indicated that the hypotensive effect of DDA may be mediated by beta-adrenoceptors, since the blockade of beta-adrenoceptors by propranolol almost completely abolished this effect of DDA. DDA was found to antagonize beta-adrenoceptor agonist isoproterenol-induced positive chronotropic actions in isolated right atria in a noncompetitive and dose-dependent manner. These findings provide further evidence for a beta-adrenoceptor inhibitory property of DDA and also suggest that DDA may have a direct beta1-adrenoceptor blocking action, since beta-adrenoceptors mediate the positive inotropic and chronotropic effects of the catecholamines.
DDA similarly inhibited vascular smooth muscle contractions induced by phenylephrine and high K+ in a concentration-dependent manner in endothelium-intact aorta. The relaxant effect of DDA seemed to be partially dependent on endothelium since it was significantly attenuated in endothelium-denuded aorta. In Ca2+-free medium, both norepinephrine and caffeine-induced transient contractions were not affected by DDA. Like with DA, the vasorelaxant effect of DDA was partially antagonised by L-NAME and methylene blue but was not affected by indomethacin or glibenclamide. These results suggest that the vasorelaxant activity of DDA may, like DA, be mediated through the NO synthase and activation of guanylate cyclase pathway as well as the blockade of Ca2+ influx through both voltage- and receptor-operated Ca2+ channels. These findings are not surprising, given the similarity in chemical structures of the two compounds.
Additional data for DDA indicate that demonstrate that the hypotensive effect of DDA may also involve beta-adrenoceptors, autonomic ganglion receptor and ACE inhibitory activity. The bradycardia induced by DDA may also contribute to the hypotensive action. Whether DA also has such effects as well has not been established.
Though the vasodilator actions of DA and DDA may not appear to be potent, these compounds could serve as lead molecules for the development of stronger and commercially useful vasodilator compounds.
The authors greatly appreciate Professor Masanori Kuroyanagi, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka-shi 422, Japan for his generosity in supplying DA and DDA for our study. We also wish to thank the NUS for the research scholarship awarded to C.Y. Zhang.
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|Tan, BKH; Zhang, CY; (1998). Pharmacological Mechanisms of Cardiovascular Actions of Diterpenoids of Andrographis Paniculata. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/kwan/tan0674/index.html|
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