Effects Of Da And Dda On Isolated Rat Thoracic Aorta

Further studies were done with DA and DDA to investigate their effects on phenylephrine- and high-K + -induced contractions of the rat thoracic aorta.

The results showed that both DA and DDA had a vasorelaxant property as they inhibited contractions induced by phenylephrine and high K+ in a concentration-dependent manner in endothelium-intact aorta. They also antagonized the concentration-response curve of phenylephrine in a non-competitive manner. The effect was attenuated in endothelium-denuded aorta without modifying the maximal response. This suggested that the vasorelax-ant effect of DA and DDA was partly dependent on the endothelium.

The vascular endothelium plays an important role in controlling the vascular tone via secretion of both relaxant and contractile factors (21). The most potent known are the vasodilators NO and PGI2 and the vasoconstrictors angiotensin II and endothelin. NO is synthesized from the amino acid L-arginine, a family of nitric oxide synthetase (NOS) isoenzymes, including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS

(iNOS). NO stimulates cyclic GMP production by activating soluble guanyl-ate cyclase (21) and thus causes vasodilatation.

Like verapamil, both DA and DDA produced a much greater vasorelaxant effect in aorta precontracted by KCl than by phenylephrine. In Ca2+-free medium, these diterpenoids antagonized Ca2 + -induced vasocon-traction in a concentration-dependent manner and almost abolished both caffeine- and norepinephrine-induced transient contractions. Their vasorelaxant effects were partly antagonized by the competitive nitric oxide (NO)-synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), and also by methylene blue, a soluble guanylate cyclase inhibitor, but were unaffected by both indomethacin, a cyclo-oxygenase inhibitor and glibenclamide, an ATP-sensitive K +-channel blocker. These results suggest that the vasorelaxant activity of DA and DDA may be mediated via the activation of nitric oxide synthase and guanylate cyclase, as well as the blockade of Ca2 + influx through both voltage- and receptor-operated Ca2+ channels. Compared to DA, DDA had a stronger vasorelaxant activity.

Figure 7 The effects of ganglionic, a- and h-adrenergic, muscarinic cholinergic and histaminergic receptor blocking agents, and captopril on the hypotensive acatin of DDA. Columns represent the mean percent change in MAP of six animals; bars indicate the SEM. *Denotes that hypotensive responses of DDA were significantly reduced from those of control (p < 0.05, t-test).

Figure 7 The effects of ganglionic, a- and h-adrenergic, muscarinic cholinergic and histaminergic receptor blocking agents, and captopril on the hypotensive acatin of DDA. Columns represent the mean percent change in MAP of six animals; bars indicate the SEM. *Denotes that hypotensive responses of DDA were significantly reduced from those of control (p < 0.05, t-test).

VI. EFFECTS OF DDA IN ANESTHETIZED SD RATS

AND ISOLATED RAT RIGHT ATRIA

We found that DDA produced significant decreases in both MAP and heart rate in a dose-dependent manner (Fig. 6). The ED50 value for MAP was 3.4 mmol/kg. Pharmacological antagonist studies were subsequently done with this dose. We found that the hypotensive action of DDA was not mediated through effects on the a-adrenoceptor, muscarinic cholinergic, or histamin-ergic receptors, for it was not affected by phentolamine, atropine, pyrilamine, or cimetidine (Fig. 7). However, in the presence of propranolol, hexametho-nium, and captopril, the hypotensive effect was negated or attenuated, suggesting the involvement of h-adrenoceptors, autonomic ganglia receptor, and ACE.

In spontaneously beating isolated rat right atria, DDA caused a negative chronotropic effect (Fig. 8), indicating that it may have direct h1-adrenoceptor blocking action on the heart in addition to its a-adrenergic receptor inhibitory activity. The bradycardic effect of DDA may also contribute to the hypotensive action. This discordant finding, compared to

Figure 8 Effect of DDA (•, n = 10) on the beating rate of isolated right atria from normotensive rats. Vehicle-treated control group (DMSO, o, n = 5). Points represent the mean F SEM of values. All points on the DDA curve were significantly different from the corresponding points on the control (p < 0.05, t-test).

Figure 8 Effect of DDA (•, n = 10) on the beating rate of isolated right atria from normotensive rats. Vehicle-treated control group (DMSO, o, n = 5). Points represent the mean F SEM of values. All points on the DDA curve were significantly different from the corresponding points on the control (p < 0.05, t-test).

the earlier finding of a lack of effect of FB on the heart of the anesthetized SD rat, may be explained by the fact that DDA is either absent or else present in FB in such small amounts as to have no significant effect on the heart rate.

VII. EFFECTS OF DA AND DDA ON ENDOTHELIAL CELL

PRODUCTION OF NITRIC OXIDE

As our earlier studies suggested that the relaxation of the isolated rat aorta caused by DA and DDA may be mediated through the L-arginine-NO synthase pathway and NO activation of guanylate cyclase, it was decided to study the effects of DDA on NO production by human endothelial cell cultures. NO was quantified spectrometrically by the accumulation of nitrite produced by endothelial cells cultured in F12 medium for up to 48 hr. This method has been documented by previous workers (22,23).

We found that DA and DDA significantly stimulated NO production by endothelial cells in a concentration-dependent manner. This suggests another possible mechanism for their hypotensive effect.

Figure 9 Comparison of DA, DDA, and FB by TLC.
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