Journal Logo

Article

Studies of the Cellular Mechanisms Underlying the Vasorelaxant Effects of Rutaecarpine, a Bioactive Component Extracted from an Herbal Drug

Chiou, Wen-Fei*; Shum, Andrew Yau-Chik; Liao, Jyh-Fei; Chen, Chieh-Fu*†

Author Information
Journal of Cardiovascular Pharmacology: April 1997 - Volume 29 - Issue 4 - p 490-498
  • Free

Abstract

The Chinese herbal drug, Wu-Chu-Yu (an unripe fruit of Evodia rutaecarpa), has been prescribed by Chinese medicinal practitioners for the treatment of gastrointestinal disorders (abdominal pain, dysentery), headache, postpartum hemorrhage, amenorrhea (1), and hypertension (2). In an attempt to characterize its cardiovascular actions, we previously observed that rutaecarpine, an quinazoline alkaloid isolated from Wu-Chu-Yu, dose-dependently relaxed isolated rat mesenteric arteries precontracted by phenylephrine (3). Such vasorelaxant effect was largely endothelium dependent and believed to be coupled to the synthesis or release or both of nitric oxide (NO) as endothelium removal, treatment with the nitric oxide synthase (NOS) inhibitor L-NG-nitroarginine (L-NOARG), and the guanylyl cyclase inhibitor methylene blue (MB) all attenuated such relaxant effect (3). However, the exact mechanisms through which rutaecarpine interacts with the endothelium to stimulate NO production or release or both eventually to induce vasorelaxation have remained unelucidated. Our study was initiated in an attempt to examine the possible underlying mechanisms.

Activation of a pertussis toxin-sensitive G protein (Gi) in the endothelial cell is thought to mediate many of the receptor-operated, endothelium-dependent responses in mammalian arteries (4-6). Involvement of the phospholipase C (PLC)-linked receptor systems, after the activation of pertussis toxin-sensitive G proteins, also has been reported (7-10). These G proteins regulate, for example, receptor-induced phospholipase C activity and may be coupled to receptors controlling the activities of K+- and Ca2+ channels (11-15). Focusing on signal-transduction mechanisms such as G-proteins activation and Ca2+ mobilization and by comparisons with known endothelium-dependent relaxants such as acetylcholine (ACh), histamine, and A23187, we sought to examine the possible involvement and relative roles of these processes.

To define the signal-transduction pathway leading to rutaecarpine-induced nitric oxide-dependent relaxation (NODR), three experimental tools were used in this study. The first tool was pertussis toxin, which has been shown to induce adenosine diphosphate (ADP)-ribosylation of the G protein Gi(16), thereby irreversibly inactivating the inhibitory coupling of some receptors to adenylyl cyclase (17). The second was aluminum fluoride (AlF4), which can stimulate G proteins directly, resulting in continuous activation. The third agent was the PLC antagonist, 1-[6-{[17β-3-methoxyestra-1,2,3(10)-trien-17-yl]amino}hexyl]-1H-pyrrole-2,5-dione (U73122), thought to interfere specifically with PLC at low concentrations (18,19). Additionally, the role of the important messenger Ca2+, including the delineation of the roles of extracellular and intracellular components, on rutaecarpine-induced NODR also was investigated.

METHODS

Preparation of ring segments from rat thoracic aorta

To preserve the functional integrity of the endothelium, ring segments rather than helical strips were used. Adult male Sprague-Dawley rats aged 6-8 weeks (230-280 g) were killed by decapitation and exsanguination. The abdomen and thorax were cut open, and a section of the thoracic aorta was immediately removed and placed in Krebs' solution of the following composition (in mM): NaCl, 118; KCl, 4.7; CaCl2, 1.5; MgCl2, 1.2; KH2PO4, 1.2; NaHCO3, 24; and glucose, 11; pH, 7.3-7.4. The aortae were cleaned of connective tissues and cut into rings ∼4 mm in length. Ring segments were suspended in 5-ml organ baths filled with Krebs' solution, with one end connected to the hooked end of a fixed support post and the other to an isometric transducer (Grass FT03C) through which contractions were amplified and quantified. The arterial rings were routinely left to equilibrate under 1.8-2.0 g resting tension for 60-90 min before being used for experiments. A consistent concentration-response relation to PE was routinely established before the beginning of experiments. Endothelium removal (denudation), when desired, was achieved by inserting into the lumen a fine, Teflon-coated polyethylene spatula and gently rubbing the luminal surface a few times. Functional integrity of the endothelium was evaluated by the preparations' responses to ACh and sodium nitroprusside (Na-NP). Preparations in which the endothelium was intentionally or accidentally damaged would fail to relax in response to a maximal concentration of ACh (3 × 10−6M) but would respond normally to a maximal concentration of Na-NP (3 × 10−8M).

Vasorelaxation evaluation

For the evaluation of relaxation, the arteries were first contracted with PE at concentrations corresponding to the 80% effective concentration (EC80) values (3 × 10−7 and 10−7M, respectively, for endothelium-intact and denuded arteries). After the establishment of consistent relaxant responses to ACh or the calcium ionophore A23187, contraction was again induced in the ring segment by the application of EC80 concentration of PE. Rutaecarpine, its vehicle, or other test drugs were then added cumulatively to the organ bath. Construction of concentration-response curves (CRCs) were based on degrees of relaxation of the PE-induced contractions. A preliminary study had shown no significant differences between results obtained with single or multiple exposures to increasing concentrations of rutaecarpine (i.e., no tachyphylaxis, desensitization, or tissue fatigue was observed). Removal of drugs was achieved by washing the arterial preparation for 1.5 h. No significant loss of vasoconstrictor tone or change in sensitivity to PE was detected after such washouts. However, to avoid the possibly confounding effects of fatigue, no preparation was used for >4 h. Effects of inhibitors were studied by comparing the responses before and after treatments with the inhibitors. A typical protocol would consist of the establishment of the relaxation profile of the test drug (ACh, histamine, A23187, rutaecarpine) on PE-induced contraction. After proper washing, an inhibitor would then be added and, after an appropriate period of incubation, contraction would again be induced by PE, and the relaxation profile of the test drug again observed. However, some of these inhibitors tended to suppress the magnitude of the PE-induced contraction. In such cases, higher concentrations of PE were used to provide uniform starting tensions comparable with those of the controls. In parts of the experiments in which Ca2+-free medium and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) treatment were used, and in which the tension produced by the regular concentration of 3 × 10−7M of PE was severely attenuated, 10−5M PE was used to produce a contraction in which the maximal tension became comparable with, although never quite reached, that of the controls.

Characterization of signal-transduction pathway

Nature of endothelial mediator. To identify the nature of the endothelial mediator in the vasorelaxant actions of rutaecarpine, three likely candidates [NO, prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF)] were assessed by establishing, first, the reference CRC of rutaecarpine's relaxation effects on PE-induced contraction and then incubating the arterial ring with a potential inhibitor of NO biosynthesis, L-Nω-nitro-arginine methyl ester (L-NAME, 3 × 10−4 M) for 10 min (20), the cyclooxygenase inhibitor indomethacin (3 × 10−5 M) for 45 min, or the nonselective K+ channel blocker, tetraethylammonium (TEA; 10−2 M), for 1 h (21) before the construction of post-inhibitor-treatment cumulative CRCs of rutaecarpine and observing for any changes in potencies.

Effects of Ca2+. To delineate the contribution of extracellular or intracellular Ca2+ involved in rutaecarpine-induced endothelium-dependent relaxation (EDR), two different approaches were undertaken. First, effects of rutaecarpine on PE-precontracted artery were established by adding it cumulatively to the Krebs' solution containing the normal Ca2+ concentration of 1.5 mM. The medium was then replaced by Ca2+-free Krebs' solution containing 10−3 M of the Ca2+-chelator EGTA or 10−4 M of TMB-8, an inhibitor of Ca2+ mobilization from intracellular stores (22,23). The vessels were allowed to equilibrate for 10 min in the respective solutions before the addition of PE. In the Ca2+-free conditions, the higher concentration of 10−5 M of PE had to be used to produce a contractile response, the peak tension of which became comparable in magnitude to that of the controls. After attaining steady-state contractions, the CRCs of rutaecarpine were again constructed.

In the second series of experiments, the order of the addition of PE and rutaecarpine was reversed. Aortic rings were preincubated with rutaecarpine (3 × 10−5 M) for 10 min in the respective media (Krebs' with normal Ca2+, Ca2+ free, and with TMB-8) before being contracted by PE. Again, in medium with normal Ca2+, the regular concentration of 3 × 10−7 M PE was used, whereas in Ca2+ free and TMB-8-containing media, the concentration of PE was 10−5 M.

Effects of G-protein inhibition by pertussis toxin. Possible involvement of pertussis toxin-sensitive G protein was examined. Before mounting an experiment, the ring segments were incubated for 2 h at 37°C in either Krebs' solution alone or one that contained 100 ng/ml pertussis toxin. CRCs were then established for ACh, histamine, and rutaecarpine in control and pertussis toxin pretreated preparations precontracted by PE.

Effects of G-proteins activation by aluminum fluoride. Directly to activate G proteins, the ring segments were incubated for 20 min in the presence of a combination of sodium fluoride (NaF; 1, 2, or 3 mM) and aluminum chloride (AlCl3; 10 μM) based on the belief that the resulting AlF4 can interact with the α-subunit of the G proteins, resulting in their activation (24). Contraction was then induced with the regular concentration of 3 × 10−7 M PE. The effects of such treatment on relaxant actions of ACh, Na-NP, A23187, and rutaecarpine were then studied.

Phospholipase C. In comparison with other vasorelaxants, such as ACh and A23187, the role of PLC in the rutaecarpine-induced EDR was studied by using the PLC inhibitor U73122 (25,26). In these experiments, contractile responses to PE were induced in control vessels and vessels that had been pretreated with 10−6 or 10−5 M U73122 for 10 min before the addition of ACh, A23187, or rutaecarpine and observing how their vasorelaxation effects, in the form of CRCs, might be affected.

Chemicals

Phenylephrine HCl, acetylcholine chloride, histamine diphosphate, sodium-nitroprusside, A23187, L-NAME, indomethacin, TEA, pertussis toxin, NaF, and aluminum chloride were obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.), and 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8) and U73122 were purchased from Research Biochemicals International (Natick, MA, U.S.A.). Phenylephrine HCl was dissolved in 0.9% saline containing 0.1% ascorbic acid and stored frozen at −20°C. On the day of the experiments, final dilutions of PE were made with Krebs' solution. Unless stated otherwise, drugs were prepared by using triple-distilled water. Rutaecarpine, A23187, and U73122 were dissolved in dimethylsulfoxide (highest final bath concentration, 0.1%). Pertussis toxin was dissolved in 50% glycerol containing 50 mM sodium phosphate and 0.5 M NaCl (pH 7.2). AlCl3 was dissolved in alcohol; indomethacin was dissolved in Na2CO3. The drugs were kept on ice during the experiments. All concentrations are expressed as final organ bath concentrations (molar).

Rutaecarpine was extracted from the dry fruit of Evodia rutaecarpa in our laboratory, according to the method of Lin et al. (27). Its chemical structure and purity has been identified and determined (27).

Statistics

Results are expressed as the means ± SEM. In rings precontracted with PE, the relaxant responses are normalized as the percentage of relaxation or expressed as the residual tension in grams of the contraction originally induced by PE. N refers to the number of animals from which the aortic rings were prepared. Statistical evaluation of the data was performed by Student's t test for either paired or unpaired observations; a p value of <0.05 was considered significant.

RESULTS

Characteristics of vascular effects of rutaecarpine

Although having few effects on the quiescent preparations at concentrations ranging from 10−7 to 10−4M, rutaecarpine induced a concentration-dependent relaxation in PE-precontracted aortic segments in very much the same manner as ACh or the calcium ionophore A23187, provided the endothelium was intact. The EC50 and the maximal relaxation produced by 10−4M rutaecarpine were 0.9 ± 0.2 μM and 100%, respectively (Fig. 1A, solid circle). Removal of the endothelium by rubbing the lumen of the aorta abolished rutaecarpine-evoked relaxation (Fig. 1A, solidd square), suggesting the dependence on the functional integrity of the endothelium. In contrast, the vasorelaxant action of Na-NP was not significantly affected by endothelium removal (EC50 = 7.6 ± 0.5 nM with endothelium vs. 6.9 ± 0.4 nM without endothelium; n = 8).

FIG. 1
FIG. 1:
Effects of endothelial removal (A, ▪), L-N ω-nitro-arginine methyl ester (B, 3 × 10−4 M; ▪), indomethacin (C, 5 × 10−5 M; ▪), or tetraethylammonium (TEA; D, 10−2 M; ▪) treatments on rutaecarpine (10−7-10−4 M)-induced relaxation in endothelium-intact (•) rat thoracic aorta. Arterial rings were precontracted wit phenylephrine (PE; EC80), and relaxation responses are expressed as residual tension of contraction originally induced by PE. ▴ and ▾ represent the vehicle's (dimethylsulfoxide; DMSO) response in endothelium-intact and -denuded preparations, respectively. Points and vertical bars represent the mean ± SEM (n = 9-13 in each group).

Nature of endothelium mediator in relaxant action of rutaecarpine

The CRCs for cumulative rutaecarpine treatment in endothelium-intact aortic rings, before and after treatment with L-NAME, indomethacin, or TEA, are illustrated in Fig. 1B-D, respectively. None of these treatments affected significantly the basal vascular tone or the developed tension induced by PE. However, L-NAME pretreatment significantly attenuated the relaxant action of rutaecarpine (Fig. 1B). EC50 was increased from 0.9 ± 0.2 to 1.2 ± 0.2 μM, whereas Emax, the contractile response to PE, went from 99.6 ± 1.5% to 12.7 ± 2.3%. In contrast, indomethacin and TEA had practically no effects (Fig. 1C and D).

Effect of CA2+ on rutaecarpine-induced vasodilation

In normal Krebs' solution, both Na-NP and rutaecarpine relaxed PE-precontracted rings in a concentration-dependent fashion. Furthermore, the magnitudes of the relaxant responses remained essentially the same on repeated exposures of the tissues to the relaxing agents (i.e., no desensitization or tachyphylaxis were observed). However, in Ca2+-free solution, the regular PE concentration (3 × 10−7M) produced a much smaller contraction. Therefore a higher concentration of 10−5M (maximal effective concentration) PE was used to produce a comparable starting tension (1.50 ± 0.01 vs. 0.86 ± 0.01 g before and after Ca2+-free medium treatment). In Ca2+-free medium, rutaecarpine at concentrations of ≤10−4M failed significantly to relax PE-precontracted aorta (Fig. 2A). In contrast, Na-NP-induced relaxation was not affected by Ca2+ concentration (maximal relaxation, 100% vs. 100%).

FIG. 2
FIG. 2:
Comparison of relaxation sensitivities to rutaecarpine before (•) and after removal of extracellular Ca2+(▪, 1 mM EGTA-containing Ca2+-free solution) (A), treatment with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8; ▪, 0.1 mM) (B). Arterial rings were contracted with phenylephrine (PE). Relaxation responses in the main figures are expressed as residual tension of contraction originally induced by PE, whereas those in the insets are represented as percentage of PE-induced contraction. Points and vertical bars represent the mean ± SEM (n = 11-14 in each group).

Figure 3 illustrates the effect of TMB-8 (10−4M) on the vasorelaxant responses. Again, because PE-induced contraction (but not the basal tension) was moderately but significantly reduced by TMB-8 treatment, 10−5M instead of the original 3 × 10−7M PE was used to evoke a contractile response of comparable magnitude (1.62 ± 0.02 vs. 0.87 ± 0.01 g before and after TMB-8 treatment). In the control vessel, ACh (10−8 − 10−6M) dose-dependently relaxed PE-precontracted rings from 12.5 ± 2.1, 27.4 ± 4.2, 56.6 ± 5.3, 86.4 ± 3.9, to 100.0% ± 0 (Fig. 3A). Treatment with 10−4M TMB-8 completely abolished the relaxant response to lower concentrations of ACh (10−8−3 × 10−7M), although higher concentrations of ACh (≥10−6M) still retained certain relaxant effects (Fig. 3B). In contrast to ACh, although TMB-8 treatment significantly attenuated the PE-induced contraction in absolute terms (Fig. 2B), it really did not significantly affect the subsequent rutaecarpine-induced NO-dependent relaxation percentage (maximal response, 99.1 ± 2.3% vs. 98.4 ± 1.8%; Fig. 2B, insert).

FIG. 3
FIG. 3:
Typical tracing of relaxation of rat aortic rings induced by cumulative application of acetylcholine (ACh, 10−8−3 × 10−6 M) in control (A) and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8; 10−4 M) pretreated vessels (B). Aortic rings were precontracted by phenylephrine (PE). Vertical and horizontal scales represent 1 g (tension) and 5 min, respectively.

Another approach, consisting of reversing the order of the test drug and PE treatments, was further used to delineate the contributions of extracellular or intracellular Ca2+ to rutaecarpine-induced relaxation. The results are presented as histograms in Fig. 4. In normal Krebs' solution, PE at 3 × 10−7M was able to induce an appreciable contraction. Pretreatment with rutaecarpine (3 × 10−5M) significantly suppressed this PE-evoked maximal contractile force from 1.49 ± 0.13 to 0.92 ± 0.18 g, representing an inhibition of 38.5 ± 2.1%. In EGTA-containing, Ca2+-free solution in which the higher concentration of 10−5M PE was used, rutaecarpine pretreatment failed significantly to attenuate the PE-induced response, with the residual peak contraction remaining at 1.05 ± 0.14 g.

FIG. 4
FIG. 4:
Effect of rutaecarpine (3 × 10−5 M) on phenylephrine-induced maximal contraction. Experiments were conducted in Krebs' solution (normal Ca2+), EGTA containing Ca2+-free solution, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) containing solution, respectively, before (□) and after (▪) rutaecarpine treatment. Relaxant responses are expressed as PE-induced maximal contraction under the various conditions. Points and vertical bars represent the mean ± SEM (*p < 0.01; n = 8-10 in each group).

Effects of Ca2+ release from internal stores was studied in the following manner. Preparations were exposed to TMB-8-containing Krebs' solution for 10 min. Under this condition, PE at 10−5M produced a 0.92 ± 0.16 g contractile tension (peak maximal contraction). Pretreatment with 3 × 10−5M rutaecarpine significantly suppressed the PE-evoked total maximal contraction to 0.57 ± 0.11 g, representing an inhibition of 37.2 ± 1.3%.

Effect of pertussis toxin on vascular response to rutaecarpine

Treatment with pertussis toxin (100 ng/ml, 2 h) did not significantly affect PE-induced contraction. However, relaxation induced by 10−4M histamine, which in our preparation was completely endothelium dependent, was reduced by >50% (from 100% to 38.1 ± 5.3%) by the toxin treatment (Fig. 5A). In contrast, treatment of the rings with the same concentration of toxin did not affect the relaxation potency of rutaecarpine (Fig. 5B).

FIG. 5
FIG. 5:
Effect of pertussis toxin (100 ng/ml, 2 h) on histamine (A) and rutaecarpine-induced (B) concentration-dependent responses. Arterial rings were precontracted with phenylephrine. Relaxant responses are expressed as residual tension of contraction originally induced by PE. Points and vertical bars represent the mean ± SEM (n = 6−8 in each group). •, control group; ▪, pertussis toxin-pretreated group.

Effect of NaF on ACH-, NA-NP-, A23187-, and rutaecarpine-induced responses

The effect of NaF in the continuous presence of AlCl3 (10−5M) was studied. Results demonstrated that, whereas 1 and 2 mM NaF alone had no significant effects on the basal tension or the contractile response induced by PE, it nevertheless affected the ACh, Na-NP, A23187, and rutaecarpine responses, albeit in different manners. As shown in Fig. 6A, 1 mM NaF (solid star) moderately but significantly diminished ACh (10−8-10−5M)-induced relaxant response, whereas 2 mM NaF (solid square) completely inhibited ACh-induced relaxation (Fig. 6A). In contrast, the responsiveness to Na-NP (10−9-10−7M;Fig. 6B), A23187 (10−9-10−6M;Fig. 6C), and rutaecarpine (10−7-10−4M;Fig. 6D) were only minimally affected by 2 mM NaF treatment. EC50 values for Na-NP, A23187, and rutaecarpine in the absence and presence of 2 mM NaF were changed only from 4.5 ± 0.3 to 6.0 ± 1.7 nM, from 11.1 ± 2.9 to 15.2 ± 3.4 nM, and from 1.2 ± 0.2 to 1.5 ± 0.3% μM, respectively, whereas Emax values for Na-NP, A23187, and rutaecarpine, before and after 2 mM NaF treatment, went from 100% to 89.7 ± 4.5%, from 100% to 88.1 ± 2.3%, and from 100% to 87.7 ± 4.4%, respectively. The effects of exposure to an even higher concentration of 3 mM NaF (solid triangle) are shown in Fig. 6B-D. The 3 mM NaF had moderate but nonetheless statistically significant (p < 0.05) effects on Na-NP, A23187, and rutaecarpine. The EC50 and Emax were changed to 15.4 ± 1.8 nM and 69.7 ± 4.0% for Na-NP, to 33.4 ± 2.9 nM and 77.6 ± 3.5% for A23187, and to 2.4 ± 0.8% μM and 76.2 ± 2.9% for rutaecarpine, respectively.

FIG. 6
FIG. 6:
Comparison of relaxant responses of acetylcholine (ACh) (A), sodium-nitroprusside (Na-NP) (B), A23187 (C), and rutaecarpine (D) in the absence (•) or presence of various concentrations of NaF (★:1 mM; ▪, 2 mM; , 3 mM). Arterial rings were precontracted with phenylephrine (PE). Relaxation responses are expressed as residual tension of contraction originally induced by PE. Points and vertical bars represent the mean ± SEM (n = 11-15 in each group).

Effect of U73122 on ACH-, A23187-, and rutaecarpine-induced relaxation

Preliminary studies had shown that pretreatment of rat aorta with 1 or 10 μM U73122 before contraction with PE had no significant effects on either the basal tone or the PE-induced contraction. It, however, concentration-dependently attenuated the vasorelaxant activity of ACh (10−8−3 × 10−6M). Figure 7A shows that 10−6M U73122 greatly reduced responses to ACh (solid squares; p < 0.01), and 10−5M completely abolished the response to ACh (solid triangles; p < 0.001). In contrast, the inhibitory effects of U73122 on A23187 (10−9-10−6M) and rutaecarpine (10−7-10−4M) were insignificant at 10−6M and, although minimally significant at 10−5M(Fig. 7B and C), they fell far short of the pronounced effects on ACh-induced relaxation at the same concentrations.

FIG. 7
FIG. 7:
Comparison of relaxant responses of acetylcholine (ACh) (A), A23187 (B), and rutaecarpine (C) in the absence (•) or presence of various concentrations of 1-[6-{[17β-3-methoxyestra-1,2,3(10)-trien-17-yl]amino}hexyl]-1H-pyrrole-2,5-dione (U73122; ▪, 10−6 M; , 10−5 M). Arterial rings were precontracted with phenylephrine. Relaxant responses are expressed as residual tension of contraction originally induced by PE. Points and vertical bars represent the mean ± SEM (n = 9-12 in each group).

DISCUSSION

Our study clearly demonstrated that rutaecarpine induced endothelium-dependent relaxation in rat thoracic aorta precontracted by PE in very much the same manner as that in rat mesenteric arteries previously reported (3). The endothelium secretes a number of vasoactive substances among which NO, PGl2, and EDHF are three likely candidates as mediators that could lead to relaxation of vascular smooth muscles. Systematic examination with appropriate antagonists in this study revealed that only the NOS inhibitor L-NAME (20) and the guanylyl cyclase inhibitor, methylene blue, abolished the actions of rutaecarpine, whereas the cyclooxygenase inhibitor, indomethacin, or the K+ channel blocker, TEA, had no effects, suggesting that NO and guanylyl cyclase were likely the endothelial mediator and effector responsible for the endothelium-dependent actions of rutacarpine.

For experiments designed to study the possible roles of Ca2+ in the actions of rutaecarpine, both removal of extracellular Ca2+ and treatment with the intracellular antagonist TMB-8 were used. Such treatments affected the PE-induced contraction in both magnitude and pattern. Instead of a steady increase in tension, the contractile response consisted of a fast, but transient, phasic event, followed by a slow but more sustained tonic phase after removal of extracellular Ca2+, consistent with previous observations (28). Of necessity, test drugs were added, often in cumulative fashion for the construction of CRCs, in the sustained tonic phase. The peak tension elicitable by the normally used 3 × 10−7M PE was severely attenuated. To provide an appreciable starting tension, so that any vasorelaxation could be reliably quantitated, a higher concentration of 10−5M PE was used instead. Even then the peak tension generated was still somewhat suppressed. However, in our experience, concentrations of PE >10−5M did not consistently elevate the tension any further. Thus we settled on 10−5M as the standard concentration of PE for these experiments. Interestingly, the rapid phasic component of the PE-induced contraction, believed to be largely the result of mobilization of intracellular Ca2+, was significantly suppressed by TMB-8 treatment, confirming its effectiveness as an antagonist of intracellular Ca2+ mobilization. In our study, the relaxant effect of rutaecarpine appeared to be largely dependent on extracellular Ca2+, as rutaecarpine failed to induce any relaxation in Ca2+-free, EGTA-containing medium, indicating the possible involvement of a transmembrane Ca2+ influx. Because in Ca2+-free medium, a higher concentration of PE had to be used to produce a comparable degree of contraction, it might be argued that the inability of rutaecarpine to relax the artery might have been related to the higher resistance caused by the conceivably heightened contractile response to the high concentration of PE. However, we believe that this was not the case, because, in similarly conducted experiments, the relaxation action of Na-NP was affected neither by the extracellular Ca2+ concentration nor by the higher concentration of PE used (data not shown). On the other hand, treatment with TMB-8 effectively abolished ACh-induced EDR, corroborating the conventional views concerning the importance of these internal Ca2+ stores. In contrast, although TMB-8 treatment attenuated the PE-induced contraction in absolute terms, giving the impression of a suppressed dose-response relation for rutaecarpine-induced vasorelaxation, TMB-8 did not really attenuate the EDR percentage induced by rutaecarpine. This observation suggests that Ca2+ release from intracellular storage sites played a minor role, if any, in the production of EDR induced by rutaecarpine.

Another aim of our study was to characterize the nature of the transduction pathway underlying rutaecarpine-induced EDR. It is well known that G proteins are transduction proteins that couple a large number of membrane-bound receptors to a variety of intracellular effector systems. Certain endothelium-dependent relaxations are mediated by activation of a pertussis toxin-sensitive G protein in the endothelial cells, most likely Gi-protein (4,29). Such inhibitory effect has been reported to occur at a relatively low concentration (100 ng/ml) and after relatively short incubation periods (1-2 h) (4,30). We tested whether the action of rutaecarpine was mediated through G proteins inhibitable by pertussis toxin (PTX). The effects of histamine on the aortic preparation and the signal-transduction pathway also were examined to provide a basis for comparisons. The results indicated that histamine induced a fully endothelium-dependent vasorelaxation in our rat thoracic aortic preparations, as the relaxation was completely abolished with endothelium removal. Previous (3) and preliminary observations in this study (data not shown) had shown that EDRF was released in response to histamine and appeared to be mediated specifically by the histamine H1-receptor because such response can be competitively blocked by the H1-receptor antagonist, triprolidine. In our study, pertussis toxin treatment significantly reduced the histamine-induced EDR by >50%, suggesting that, at least in the rat aorta, a PTX-sensitive G protein, likely Gi, is involved. This is consistent with the findings of others that histamine stimulates increase in inositol triphosphate turnover and EDRF release in bovine coronary artery and is relatively sensitive to PTX (6). Even though a PTX-sensitive G protein may also be involved in the receptor-induced influx of Ca2+, leading to contractions of the smooth-muscle cells (31), the complete dependence on an intact endothelium, as observed in the aorta in our study, would suggest that the Gi-mediated actions of histamine occur mainly in the endothelium. In contrast to that of histamine, the action of rutaecarpine was not affected by pertussis toxin, suggesting the noninvolvement of pertussis toxin-sensitive G proteins. Nevertheless, the apparent lack of involvement of pertussis toxin-sensitive G protein does not in itself preclude the involvement of other species of G proteins.

An additional test of the involvement of G proteins in the action of rutaecarpine was provided by NaF treatment (in the continuous presence of AlCl3). G proteins can be activated indirectly by receptor stimulation, but they can also be activated directly by nonhydrolyzable GTP analogs (e.g., GTP-γS) or F ions (32). It is well known that F is a potent stimulator of Gs, Gi, Gq, and transducin in cell fractions or certain intact cells (33-39). In millimolar concentrations, F is complexed mainly with aluminum, as AlF4, which is then able to interact with the guanosine 5′-diphosphate situated on the α-subunit of the G proteins, resulting in activation by mimicking GTP at its binding site, AlF4 being functionally similar to PO4(24,33). Therefore F activation is often regarded as evidence for involvement of a G protein in a system. In our study, the F donor NaF produced a concentration-dependent inhibition of ACh-induced relaxation, likely through complexing with AlCl3. NaF (1 mM) markedly suppressed and 2 mM NaF completely abolished ACh-induced EDR. In contrast, the same concentrations of NaF had either no or minimal effects on the actions of Na-NP, A23187, or rutaecarpine. Although a higher concentration of 3 mM NaF did produce a significant attenuation of vasorelaxant effects of Na-NP, A23187, and rutaecarpine, it should be borne in mind that NaF can have additional effects on smooth-muscle cells (40,41). Indeed, 3 mM NaF has been observed to enhance the PE-induced contraction in our laboratories and by others (24). Thus the effects observed with 3 mM NaF might not have been the result of simple inhibition, and for that reason, no attempts were made to study the effects of even higher concentrations of NaF. Nevertheless, further antagonizing effects of NaF at higher concentrations cannot excluded. The inhibition of ACh-induced EDR by NaF in our study was, however, unlikely to have been caused by an opposing contractile effect on vascular smooth-muscle cells, because relaxation in response to endothelium-independent vasodilator Na-NP was still elicitable in the presence of 2 mM AlF4. These results suggest that lower concentrations of NaF (≥3 mM) interfered with the release of EDRF evoked only by certain (i.e., ACh) but not all (i.e., A23187 and rutaecarpine) endothelial activators. Taken together, these results suggest that neither the pertussis toxin-sensitive Gi protein nor Gs and Gq were involved in the cellular response to rutaecarpine.

The aminosteroid U73122 has been shown to inhibit the activity of PLC in a variety of cell types (25,26). Thus U73122 appears to be a promising specific inhibitor for PLC-mediated events and may be a valuable tool for the study of pharmacologic events regulated by this second-messenger pathway. In our study, pretreatment with 1 × 10−6M U73122 moderately inhibited, whereas 10−5M U73122 completely abolished, the response of ACh. In contrast, U73122 exerted few or only moderate inhibitory effects on the action of A23187. It is believed that ACh and A23187 represent different modes of activation through which vascular relaxation can be induced. Whereas ACh is believed to be receptor and G protein linked, the calcium ionophore A23187 is thought to bypass both the receptor- and the G proteins-coupled signal transduction by directly translocating Ca2+ ions across the cell membrane (15). Its effects are, therefore, not subject to the same intracellular regulatory mechanisms as are those of receptor-mediated agonists. The relative insensitivity of A23187-induced relaxation to U73122 indicated that U73122 did not interfere with the action of EDRF on vascular smooth muscle. Thus the inhibition of ACh-induced relaxation by U73122 was likely related to an effect at the cellular level of the endothelium. Similar to that of A23187, U73122 treatment had insignificant inhibitory effects on rutaecarpine-induced EDR, implying that the mode of action of rutaecarpine was similar to that of A23187, being independent of receptor activation. These results are consistent with our previous findings that neither endothelial muscarinic receptor, histamine H1-receptor, nor α2-adrenoceptors were involved in the vasorelaxation elicited by rutaecarpine (3).

In platelets, U73122 inhibits agonist-induced inositol 1,4,5-triphosphate (1,4,5-IP3) and [Ca2+]i increases (25,26). From our study, it can also be speculated that IP3 formation and induced Ca2+ mobilization from intracellular stores were likely not involved in rutaecarpine-induced EDR. This possibility was further supported by the fact that treatment with TMB-8, an agent known to interfere with intracellular stores Ca2+ release (42,43), had no effect on rutaecarpine-induced EDR, thus excluding Ca2+ mobilization from intracellular stores by IP3.

In conclusion, rutaecarpine induced a concentration- and endothelium-dependent vasodilatation in rat aorta precontracted by PE. These responses could be inhibited by the removal of extracellular Ca2+ in the medium. EDR induced by rutaecarpine depended primarily on the influx of Ca2+ and not on the mobilization of intracellular Ca2+. Because pertussis toxin did not affect rutaecarpine-induced EDR, whereas NaF and U73122 affected it but not to the same degrees as that induced by ACh, it is speculated that Gi proteins or G proteins-PLC coupling pathway were probably not involved in the action of rutaecarpine on vascular endothelial cells.

Acknowledgment: This work was supported by a grant-in-aid to Professor C. F. Chen from the National Science Council, Taipei, Taiwan, R.O.C. (NSC 85-2331-B-077-001 M04), for which we are deeply grateful.

REFERENCES

1. Li SC, Pen-Tsao Kang Mu (republished by The National Research Institute of Chinese Medicine, 1976, Taipei) 1956, Chapter 32, p. 1064.
2. Kiangsu Institute of Modern Medicine. Encyclopedia of Chinese drugs. Vol 2. Shanghai: Shanghai Scientific and Technical Publications, 1977:1118.
3. Chiou WF, Chou CJ, Liao JF, Shum AYC, Chen CF. The mechanism of the vasodilator effect of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa. Eur J Pharmacol 1994;257:59-66.
4. Flavahan NA, Shimokawa H, Vanhoutte PM. Pertussis toxin inhibits endothelium-dependent relaxations to certain agonists in porcine coronary arteries. J Physiol (Lond) 1989;408:549-60.
5. Shimokawa H, Flavahan N, Vanhoutte PM. Natural course of the impairment of endothelium-dependent relaxations after balloon endothelium removal in porcine coronary arteries. Circ Res 1989;65:740-53.
6. Weinheimer G, Osswald H. Pertussis toxin and N-ethylmaleimide inhibit histamine- but not calcium ionophore-induced endothelium-dependent relaxation. Naunyn-Schmiedebergs Arch Pharmacol 1989;339:14-8.
7. Joseph SK. Receptor-stimulated phosphoinositide metabolism: a role for GTP binding proteins? Trends Biochem Sci 1985;10:297-8.
8. Cockcroft S, Gomperts BD. Role of guanine nucleotide binding protein in the activation of polyphosphoinositide phosphodiesterase. Nature 1985;314:534-6.
9. Smith CD, Uhing RJ, Snyderman R. Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters. J Biol Chem 1987;262:6121-7.
10. Fain JN, Wallace MA, Wojcikiewicz RJH. Evidence for involvement of guanine nucleotide-binding regulatory proteins in the activation of phospholipase by hormones. FASEB J 1988;2:2569-74.
11. Gilman AG. Receptor-regulated G proteins. Trends Neurosci 1986;9:460-3.
12. Taylor CW, Merrit JE. Receptor coupling to polyphosphoinositide turnover: a parallel with the adenylate cyclase system. Trends Pharmacol Sci 1986;7:238-42.
13. Gierschik P, Grandt R, Marquetant R, Jakobs KH. Role of G proteins in signal transduction. J Cardiovasc Pharmacol 1987;10:S6-10.
14. Bimbaumer L, Codina J, Mattera R, et al. Signal transduction by G proteins. Kidney Int 1987;32:14-37.
15. Brown AM, Birnbaumer L. Direct G protein gating of ion channels. Am J Physiol 1988;254:H401-10.
16. Ui M. Pertussis toxin as a probe of receptor coupling to inositol lipid metabolism. In: Phosphoinositides and receptor mechanisms. New York: Alan R. Liss, 1986:163-95.
17. Dolphin AC. Nucleotide binding proteins in signal transduction in health and disease. Trends Neurosci 1987;10:53-7.
18. Smallridge RC, Kiang JG, Gist ID, Fein HG, Galloway RJ. U73122, an aminosteroid phospholipase C antagonist, noncompetitively inhibits thyrotropin-releasing hormone effects in GH3 rat pituitary cells. Endocrinology 1992;131:1883-8.
19. Yule DI, Williams JA. U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells. J Biol Chem 1992;267:13830-5.
20. Gardiner SM, Compton AM, Bennett T, Palmer RMJ, Moncada S. Regional haemodynamic changes during oral ingestion of NG-monomethyl-L-arginine or NG-nitro-L-arginine methyl ester in conscious Brattleboro rats. Br J Pharmacol 1990;101:10-2.
21. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarization of canine coronary smooth muscle. Br J Pharmacol 1988;93:515-24.
22. Chiou CY, Malagodi MH. Studies on the mechanism of action of a new Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride in smooth and skeletal muscle. Br J Pharmacol 1975;53:279-85.
23. Ogawa N, Ono H. Effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release, on autoregulation of renal blood flow in the dog. Naunyn-Schmiedebergs Arch Pharmacol 1988;338:293-6.
24. Zeng YY, Benishin CG, Pang PKT. Guanine nucleotide binding proteins may modulate gating of calcium channels in vascular smooth muscle: I. Studies with fluoride. J Pharmacol Exp Ther 1989;250:343-51.
25. Smith RJ, Sam LM, Justen JM, Bundy GL, Bala GA, Bleasdale JE. Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J Pharmacol Exp Ther 1990;253:688-97.
26. Bleasdale JE, Thakur NR, Gremban RS, et al. Selective inhibition of receptor-coupled phospholipase C-dependent processed in human platelets and polymorphonuclear neutrophils. J Pharmacol Exp Ther 1990;255:756-68.
27. Lin LC, Chou CJ, Chen KT, Chen CF. Flavonoids from Evodia fructus. J Chin Med 1991;2:94-7.
28. Bohr DF. Vascular smooth muscle: dual effect of calcium. Science 1963;137:597-9.
29. Flavahan NA, Vanhoutte PM. G-proteins and endothelial response. Blood Vessels 1990;27:218-23.
30. Lambert TL, Kent RS, Whorton AR. Bradykinin stimulation of inositol polyphosphate production in porcine aortic endothelial cells. J Biol Chem 1986;261:15288-93.
31. Boonen HCM, De Mey JGR. G-proteins are involved in contractile responses of isolated mesenteric resistance arteries to agonists. Naunyn-Schmiedebergs Arch Pharmacol 1990;342:462-8.
32. Bigay J, Deterre P, Pfister C, Chabre M. Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma-phosphate of GTP. EMBO J 1987; 6:2907-13.
33. Sternweis PC, Gilman AG. Aluminium: a requirement for activation of the regulatory component of adenylate cyclase by fluoride. Proc Natl Acad Sci U S A 1982;79:4888-91.
34. Gilman AG. Guanine nucleotide-binding regulatory proteins and dual control of adenylate cyclase. J Clin Invest 1984;37:1-4.
35. Blackmore PF, Bocckino SB, Waynick LE, Exton JH. Role of a guanine nucleotide binding regulatory protein in the hydrolysis of hepatocyte phosphatidyl-inositol 4,5-biphosphate by calcium-mobilizing hormones and the control of cell calcium. J Biol Chem 1985; 260:14477-83.
36. Blackmore PF, Exton JH. Studies on the hepatic calcium-mobilizing activity of aluminium fluoride and glucagon. J Biol Chem 1986;261:11056-63.
37. Kanaho Y, Northup JK, Bokoch GM, Ui M, Gilman AG. The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. J Biol Chem 1985;260:11493-7.
38. Martin TFY, Lucas DO, Bajalien SU, Kowalchyk YA. Thyrotropin-releasing hormone activates a Ca2+-dependent polyphosphoinositide phosphodiesterase in permeable GM3 cells: GTPγS potentiation by a cholera and pertussis toxin-insensitive mechanism. J Biol Chem 1986;261:2918-27.
39. Cockcroft S, Taylor JA. Fluoroaluminates mimic guanosine 5′-[γ-thio]-triphosphate in activating the polyphosphoinositide phosphodiesterase of hepatocyte membrane. Biochem J 1987;241:409-14.
40. Casteels R, Raeymaekers L, Suzuki H, Eldere J. Tension response and Ca release in vascular smooth muscle incubated in Ca-free solution. Pflugers Arch 1981;392:139-45.
41. Nguyen-Duong H. Effects of vasoactive drugs of fluoride-induced contraction of vascular smooth muscle in calcium-free solution. Arzneimittelforschung 1985;358:1246-50.
42. Dougherty RW, Niedel JE. Cytosolic calcium regulates phorbol diester binding affinity in intact phagocytes. J Biol Chem 1986;261:4097-100.
43. Luckhoff A, Pohl U, Mulsch A, Busse R. Differential role of extra- and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Br J Pharmcol 1988;95:189-96.
Keywords:

Rutaecarpine; Nitric oxide; Extracellular Ca2+; Pertussis toxin; Aluminum fluoride; U73122

© Lippincott-Raven Publishers