Comparative Study on the Vasorelaxant Effects of Three Harmala Alkaloids in Vitro
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Jpn. J. Pharmacol. 85, 299 – 305 (2001) Comparative Study on the Vasorelaxant Effects of Three Harmala Alkaloids In Vitro Chuen-Chao Shi1, Jyh-Fei Liao1,* and Chieh-Fu Chen1,2 1Department and Institute of Pharmacology, National Yang-Ming University, Taipei 112, Taiwan 2National Research Institute of Chinese Medicine, Taipei 112, Taiwan Received November 6, 2000 Accepted December 19, 2000 ABSTRACT—Three psychological active principles from the seeds of Peganum harmala L., harmine, har- maline and harmalol, showed vasorelaxant activities in isolated rat thoracic aorta preparations precontracted by phenylephrine or KCl with rank order of relaxation potency of harmine > harmaline > harmalol. The vasorelaxant effects of harmine and harmaline (but not harmalol) were attenuated by endothelium removal or pretreatment with a nitric oxide (NO) synthase NM-nitro-L-arginine methyl ester. In cultured rat aortic endothelial cells, harmine and harmaline (but not harmalol) increased NO release, which was dependent on the presence of external Ca2+. In endothelium-denuded preparations, pretreatment of harmine, harmaline or harmalol (3 – 30 mM) inhibited phenylephrine-induced contractions in a non-competitive manner. Receptor binding assays indicated that all 3 compounds interacted with cardiac a 1-adrenoceptors with comparable affinities (Ki value around 31 – 36 mM), but only harmine weakly interacted with the cardiac 1,4-dihydro- 2+ pyridine binding site of L-type Ca channels (Ki value of 408 mM). Therefore, the present results suggested that the vasorelaxant effects of harmine and harmaline are attributed to their actions on the endothelial cells to release NO and on the vascular smooth muscles to inhibit the contractions induced by the activation of receptor-linked and voltage-dependent Ca2+ channels. The vasorelaxant effect of harmalol was not endothe- lium-dependent. Keywords: Harmine, Harmaline, Harmalol, Vasorelaxant, Nitric oxide Harmala alkaloids harmine, harmaline and harmalol frequently followed by a secondary increase; and the effects (Fig. 1) are the psychological active principles from the of harmalol on these two parameters are inconsistent (18). seeds of Peganum harmala L., which are also distributed Recently, we reported the in vivo cardiovascular effect and widely in other medicinal plants and found endogenously in in vitro vasorelaxant effect of harman (Fig. 1), another har- mammalian tissues (1, 2). These harmala alkaloids have a mala alkaloid (19). Our results suggested that the vasore- wide spectrum of pharmacological actions in the central laxant effect of harman may be involved in its hypotensive nervous system such as tremorogenesis (3, 4), hypothermia effect, and the vasorelaxant effect is attributed to its actions (5), hallucinogenesis (6, 7), central monoamine oxidase in- on the endothelial cells to release nitric oxide (NO) and on hibition (8 – 10), convulsive or anticonvulsive actions (11) the vascular smooth muscles to inhibit the contractions and binding to various receptors including 5-HT receptors induced by the activation of receptor-linked and voltage- 2+ and the benzodiazepine binding site of GABAA receptors dependent Ca channels. Although the cardiovascular effects (12 – 14). In addition, these compounds also have antioxi- of these harmala alkaloids may not be of practical impor- dative (15), platelet aggregation inhibitory (16) and immu- tance (or for clinical use), the related information for these nomodulatory effects (17). There are also some reports potential hallucinogens should be important for their toxi- concerning the cardiovascular actions of these harmala cology. Because the chemical structures of these 4 com- alkaloids. For example, it has been reported that harmine pounds are very similar, the present study was carried out reduces systemic arterial blood pressure and total periph- to compare harmine, harmaline and harmalol in terms of eral vascular resistance; harmaline-evoked decreases are vasorelaxant effects on the isolated rat thoracic aorta preparations and stimulating effects on NO release from *Corresponding author. FAX: +886-2-28264372 cultured rat aortic endothelial cells. To compare with har- E-mail: [email protected] man (19), their affinities for cardiac a 1-adrenoceptors and 299 300 C.-C. Shi, J.-F. Liao & C.-F. Chen 37°C. The composition of Krebs’ solution was as follows: 118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.2 mM KH2PO4, 2.5 mM MgSO4, 2.5 mM CaCl2 and 11.1 mM glucose. Isometric tension change was measured with a Grass FT03 force transducer and recorded on a 4-channel polygraph (Gould RS3400 polygraph; Oxnard, CA, USA). Before starting the experiment, all preparations were al- lowed to equilibrate for 60 min, during which time Krebs’ solution was replaced at least twice. Vasorelaxant effects To evaluate the vasorelaxant and endothelium-dependent effects of test compounds, endothelium-intact and -denuded preparations were pretreated with phenylephrine (PE, 0.3 mM) or KCl (60 mM) to produce sustained contractions (19). Lack of functional vascular endothelium was confirmed by the loss of relaxant response to 3 mM acetylcholine before the experiment began. After the contraction had reached a stable plateau, cumulative concentrations of test compound were added. The vasorelaxant effect of test compound was expressed as a percentage of relaxation, and the IC50 (the concentration to produce a 50% maximal relaxation) value was determined from the concentration-response curve by data fitting with computer software GraFit (Erithacus Soft- ware, Staines, Middlessex, UK). The involvement of the mediator for endothelium-related vasorelaxation induced by test compound was examined by pretreatment of preparations with NM-nitro-L-arginine methyl ester (a NO synthase inhibitor), tetraethylammonium (a K+ channel blocker) or indomethacin (a cyclooxygenase inhibitor). A series of experiments was designed to assess the in- volvement of a 1-adrenoceptors in the vasorelaxant effect of test compound in endothelium-denuded aortic preparations. m Fig. 1. Chemical structures of harmala alkaloids. Various concentrations of test compound (3, 10, 30 M) were added 10 min before the construction of cumulative concentration-response curves with PE. The results were expressed as the percentage of the maximum contractile the 1,4-dihydropyridine (DHP) binding site of L-type Ca2+ tension to PE before and after pretreatment with test com- channels were also studied with receptor binding assays. pound. MATERIALS AND METHODS NO measurement The culture of rat aortic endothelial cells and the mea- Isolated thoracic aorta preparations surement of NO in the medium were according to the meth- Sprague-Dawley rats weighing 180 – 280 g were sacri- ods of Wang et al. (20, 21). In brief, endothelial cells were ficed by decapitation. The thoracic aorta was excised and grown in 35-mm2 dishes in 1 ml of Dulbecco’s modified adhesive connective tissues were carefully cleared away. Eagle’s medium supplemented with 10% fetal calf serum The aorta was cut into approximately 3 – 4-mm-long ring and antibiotics. Upon reaching confluence in about 4 days, segments. Denuded aorta ring segments were obtained by the medium was changed to 1 ml of Hanks’ balanced salt gently rubbing with the finger tip. The isolated aorta ring solution (HBSS) with L-arginine (100 mM) and added was suspended under a basal tension of 1 g in a 10 ml organ CaCl2 (to 2.5 mM). The changing over to HBSS was neces- bath containing Krebs’ solution continuously aerated with a sary because it provided the least interference in the assay. 2+ gas mixture of 95% O2 and 5% CO2 and maintained at However, additional Ca was required to make the final Vasorelaxation of Harmala Alkaloids 301 concentration comparable to that in normal Krebs’ solution. (CaCl2), potassium chloride (KCl), EGTA, sodium chloride The cells were then equilibrated for 60 min at 37°C. Ali- (NaCl), magnesium sulfate heptahydrate (MgSO4 · 7H2O), quots (250 ml) of the supernatant were collected for analy- monopotassium phosphate (KH2PO4), sodium bicarbonate sis of nitrite by chemiluminescence, and the total content (NaHCO3), and D-glucose were purchased from Sigma of NO in the medium before test compound treatment was (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medi- calculated and taken as basal 100%. Vehicle (HBSS with- um, fetal calf serum, and HBSS were purchased from out Ca2+) or test compound (1, 10, 100 mM) was then added Gibco Life Technologies (Grand Island, NY, USA). Vana- for 30 min, and then the cell supernatants (100 ml) were dium chloride was purchased from Aldrich (Milwaukee, collected for analysis of nitrite to examine the change of WI, USA). Radioligands [7-methoxy-3H]-prazosin (76.2 Ci NO content. Similar experiments also were carried out in /mmol) and [isopropyl-1,3-3H]-nimodipine (121.1 Ci/mmol) Ca2+-free HBSS to examine the role of external Ca2+. were purchased from NEN, Du Pont (Boston, MA, USA). Samples (100 ml) containing nitrite were measured by add- ing a reducing agent (0.8% VCl3 in HCl) to the purge vessel RESULTS to convert nitrite to NO, which was then carried by a flow of helium to the NO analyzer (Model 280; Sievers Re- As shown in Fig. 2, harmine, harmaline and harmalol search, Boulder, CO, USA). Nitrite concentrations were cal- (0.3 – 100 mM) concentration-dependently relaxed endo- culated by comparison with a standard solution of sodium thelium-intact rings precontracted with PE (0.3 mM), and nitrite. the vasorelaxant responses to harmine and harmaline, but not harmalol, were significantly depressed in endothelium- Receptor binding assays denuded preparations. The IC50 (the concentration to pro- According to previous reports (22, 23), the interaction of duce a 50% maximal relaxation) values of harmine and test compound with a 1-adrenoceptors or the DHP binding harmaline for PE-induced contractions were significantly site of L-type Ca2+ channels was assessed in rat heart mem- increased to 2.1-fold and 1.3-fold, respectively, in endo- brane preparations. In brief, binding assays were initiated thelium-denuded preparations as compared with that in en- by the addition of a receptor membrane preparation in an dothelium-intact preparations (Table 1).