Sheu-Meei Yu

EDRF-release and Ca+(+)-channel blockade by magnolol, an antiplatelet agent isolated from Chinese herb Magnolia officinalis, in rat thoracic aorta.

Magnolol is an antiplatelet agent isolated from Chinese herb Magnolia officinalis. It inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contractions in rat thoracic aorta. At the plateau of the NE-induced tonic contraction, addition of magnolol caused two phases (fast and slow) of relaxation. These two relaxations were concentration-dependent (10-100 micrograms/ml), and were not inhibited by indomethacin (20 microM). The fast relaxation was completely antagonized by hemoglobin (10 microM) and methylene blue (50 microM), and disappeared in de-endothelialized aorta while the slow relaxation was not affected by the above treatments. Magnolol also inhibited high potassium (60 mM)-induced, calcium-dependent (0.03 to 3 mM) contraction of rat aorta in a concentration-dependent manner. 45Ca(+)+ influx induced by high potassium or NE was markedly inhibited by magnolol. Cyclic GMP, but not PGI2, was increased by magnolol in intact, but not in de-endothelialized aorta. It is concluded that magnolol relaxed vascular smooth muscle by releasing endothelium-derived relaxing factor (EDRF) and by inhibiting calcium influx through voltage-gated calcium channels.

Mechanism of Catecholamine-Induced Proliferation of Vascular Smooth Muscle Cells

BACKGROUND Catecholamines have been shown to aggravate atherosclerosis in animals and humans, and abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the early stage of atherosclerosis. Catecholamines may be involved in such cell growth. Therefore, a series of experiments using cultured VSMC was performed to elucidate their possible mitogenic effect. METHODS AND RESULTS We examined the mitogenic effect of catecholamines using rat aortic smooth muscle cells (VSMC) by measuring [3H]thymidine incorporation, checking with flow cytometry, and counting the cell number directly. Furthermore, the catecholamine-activated signal transduction pathway was assessed by measurement of the formation of inositol 1, 4, 5-triphosphate, intracellular Ca2+ concentration, mitogen-activated protein kinase (MAPK) activity, and mitogenic gene expression. Norepinephrine (NE) and phenylephrine stimulated [3H]thymidine incorporation and cell growth. Clonidine and isoproterenol showed little of such effects. Prazosin was more effective than either yohimbine or propranolol in suppressing the mitogenic effect of NE, indicating that catecholamine-induced VSMC proliferation is mediated by alpha 1-adrenoceptors. The alpha 1-adrenoceptor activation was coupled to pertussis toxin-insensitive Gq-protein and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C and MAPK in VSMC. In response to NE, both 42- and 44-kD MAPK were activated and tyrosine was phosphorylated. alpha 1-Adrenoceptor stimulation with NE also caused accumulation of c-fos, c-jun, and c-myc mRNA. Chloroethylclonidine completely blocked the alpha 1-adrenoceptor-mediated mitogenesis. CONCLUSIONS The effect of catecholamines appears to be mediated via the activation of the chloroethylclonidine-sensitive alpha 1-adrenoceptors that triggers the phosphoinositide hydrolysis and activates the MAPK pathway, leading to DNA synthesis and cell proliferation.

Dicentrine, a natural vascular α1-adrenoceptor antagonist, isolated from Lindera megaphylla

1 The pharmacological activity of dicentrine, isolated from Lindera megaphylla, was determind in rat isolated thoracic aorta, guinea‐pig isolated trachea and human platelet‐rich plasma. 2 Dicentrine was found to be a potent α1‐adrenoceptor blocking agent in rat thoracic aorta as revealed by its competitive antagonism of noradrenaline‐ (pA2 = 8.19 ± 0.09) or phenylephrine (pA2 = 9.01 ± 0.10)‐induced vasoconstriction. These effects still persisted in denuded aorta. It was less potent than prazosin (pA2 = 10.60 ± 0.10), but was more potent than phentolamine (pA2 = 7.53 ±0.10) or yohimbine (pA2 = 6.20 ± 0.05). 3 Inositol monophosphate formation induced by noradrenaline (3 μm) in rat thoracic aorta was suppressed by dicentrine (3–10 μm) and prazosin (3 μm). 4 A high concentration of dicentrine (30 μm) did not affect the aortic contraction induced by the thromboxane receptor agonist U‐46619 (1 μm), angiotensin II (1 μm), high potassium (60 mm) or carbachol (3 μm). 5 Contraction of guinea‐pig trachea caused by histamine or carbachol was slightly inhibited by dicentrine (30 μm), while β‐adrenoceptor relaxation to isoprenaline in trachea was not affected. 6 Aggregation in human platelet‐rich plasma induced by adrenaline (10 μm) was blocked by yohimbine (5 μm). A high concentration of dicentrine (> 30 μm) caused slight inhibition of aggregation, the release reaction and thromboxane formation. Complete blockade was obtained with 150 μm dicentrine. 7 It is concluded that dicentrine is a potent, selective α1‐adrenoceptor antagonist in vascular smooth muscle.

Haemodynamic effects of dicentrine, a novel α1‐adrenoceptor antagonist: comparison with prazosin in spontaneously hypertensive and normotensive Wistar‐Kyoto rats

1 The haemodynamic effects of dicentrine, an aporphine derivative isolated from the plant Lindera megaphylla, were investigated and compared with prazosin in rats. 2 In anaesthetized normotensive Wistar‐Kyoto (WKY) rats, i.v. administration of dicentrine (0.1, 0.5, 1.0 mg kg−1) and prazosin (0.01, 0.05, 0.1 mg kg−1) induced a dose‐related reduction of mean arterial pressure (MAP) which reached a maximal effect 5–10 min after injection and persisted for 2 h. 3 In anaesthetized WKY rats, a higher dose of dicentrine (1.0 mg kg−1, i.v.) did not cause any significant changes in heart rate (HR), cardiac output (CO) and stroke volume (SV) but markedly increased tail blood flow. In contrast, a higher dose of prazosin (0.1 mg kg−1, i.v.) produced a decrease in HR which paralleled the time course of the hypotensive response. 4 The hypotensive activity of dicentrine was completely abolished by α‐adrenoceptor blockade. Both dicentrine and prazosin significantly attenuated pressor responses to noradrenaline but failed, even at maximal hypotensive doses, to impair the pressor effects of angiotensin II or vasopressin. These observations suggest that dicentrine appears to exert its hypotensive action through α1‐adrenoceptor blockade. 5 In conscious normotensive and spontaneously hypertensive (SH) rats, dicentrine (0.5–2.0 mg kg−1, i.v.) and prazosin (0.05–0.2 mg kg−1, i.v.) also evoked dose‐related decreases in MAP which were of greater magnitude in SH rats. Oral administration of dicentrine (5 and 8 mg kg−1) to conscious SH rats caused a hypotensive effect which persisted for over 15 h. 6 These results suggest that dicentrine may have therapeutic potential as an oral antihypertensive drug via α1‐adrenoceptor blockade.

Mechanism of anti-proliferation caused by YC-1, an indazole derivative, in cultured rat A10 vascular smooth-muscle cells.

An indazole derivative, YC-1, was identified in this study to be capable of reversibly and effectively inhibiting proliferation of rat A10 vascular smooth-muscle cells (VSMCs) in vitro. YC-1 (1-100 microM) dose-dependently inhibited [3H]thymidine incorporation into DNA in rat A10 VSMCs that were synchronized by serum depletion and then restimulated by addition of 10% foetal calf serum (FCS), whereas FCS-induced [3H]thymidine incorporation into rat synchronized endothelial cells was unaffected by this agent. The dose of YC-1 required to cause inhibition of FCS-induced proliferation was similar to that necessary for the formation of cellular cyclic GMP (cGMP). Guanylate cyclase activity in soluble fractions of VSMCs was activated by YC-1 (1-100 microM), whereas cGMP-specific phosphodiesterase activity was unaffected by this compound. The anti-proliferative effect of YC-1 was mimicked by 8-bromo-cGMP, a membrane-permeable cGMP analogue, and was antagonized by KT 5823 (0.2 microM), a selective inhibitor of protein kinase G. The anti-proliferative effect of YC-1 was also antagonized by Methylene Blue (50 microM), a guanylate cyclase inhibitor, and was potentiated by 3-isobutyl-1-methylxanthine (500 microM), a phosphodiesterase inhibitor. These results verified that YC-1 is a direct soluble guanylate cyclase activator in A10 VSMCs, and the anti-proliferative effect of YC-1 is mediated by cGMP. YC-1 still inhibited FCS-induced DNA synthesis even when added 10-18 h after restimulation of the serum-deprived A10 VSMCs with 10% FCS. Flow cytometry in synchronized populations revealed an acute blockage of FCS-inducible cell-cycle progression at a point in the G1/S-phase in YC-1 (100 microM)-treated cells. The inhibition of proliferation by YC-1 was demonstrated to be independent of cell damage, as documented by several criteria of cell viability. In conclusion, YC-1 reversibly and effectively inhibited the proliferation of VSMCs, suggesting that it has potential as a therapeutic agent in the prevention of vascular diseases.

Effects of dicentrine on haemodynamic, plasma lipid, lipoprotein level and vascular reactivity in hyperlipidaemic rats

1 The effects of dicentrine on haemodynamic, plasma lipid, lipoprotein level and vascular reactivity were investigated in Wistar‐Kyoto (WKY) and spontaneously hypertensive (SH) rats, fed a high fat‐high cholesterol diet. 2 In high fat‐high cholesterol (HF‐HC) diet fed WKY and SH rats, oral administration of dicentrine (5 and 10 mg kg−1, twice a day) for 4 weeks caused significant reductions in total plasma cholesterol (CE) by reducing the low density lipoprotein (LDL) fraction, and reductions in total plasma triglyceride (TG) by reducing the very low density lipoprotein (VLDL) fraction. 3 Dicentrine therapy was associated with increased high density lipoprotein (HDL)‐cholesterol levels; thus the ratio of total plasma cholesterol to HDL‐cholesterol was improved. 4 In HF‐HC diet fed conscious WKY and SH rats, oral administration of dicentrine (5 and 10 mg kg−1, twice a day) also evoked dose‐related decreases in mean arterial pressure (MAP) which were of greater magnitude in SH rats. Neither dose of dicentrine caused a significant change in heart rate (HR). 5 The aortic arches from SH rats fed the HF‐HC diet for 8 weeks were significantly more affected by the atherosclerotic lesions than the abdominal aortae and renal arteries of WKY and SH rats. Oral administration of dicentrine (5 and 10 mg kg−1) for 4 weeks did not diminish the atherosclerotic lesion areas in WKY and SH rats. 6 In aortae of the hyperlipidaemic rats, significantly attenuated EC50 values and augmented maximal responses for phenylephrine‐induced contraction were obtained. Endothelium‐dependent relaxation to acetylcholine was abolished, while endothelium‐independent relaxation to nitroprusside was well preserved. Dicentrine therapy caused significantly augmented EC50 values and attenuated maximal responses for phenylephrine‐induced contraction in hyperlipidaemic rats. However, dicentrine neither prevented the impaired relaxation to acetylcholine, nor affected the relaxation to nitroprusside during atherosclerosis progression. 7 It is concluded that dicentrine decreases MAP, plasma CE, LDL‐CE, plasma TG, VLDL‐TG, vascular hyperreactivity to phenylephrine and increases HDL‐CE levels. Dicentrine may thus hold potential for the reduction of two of the major risk factors, hypertension and hyperlipidaemia, for cardiovascular disease.

Effects of dicentrine, a novel α1-adrenoceptor antagonist, on human hyperplastic prostates

The effects of dicentrine, an alpha 1-adrenoceptor antagonist, on human hyperplastic prostates were investigated by radioligand binding and in vitro isometric tension experiments. In human hyperplastic prostates, alpha 1-adrenoceptors were characterized by a binding assay using [3H]prazosin as a radioligand. Specific [3H]prazosin binding was saturable and of high affinity (Kd = 0.2 +/- 0.02 nM) with a maximal number of binding sites (Bmax = 55.2 +/- 3.2 fmol/mg protein). alpha-Adrenoceptor antagonists competed with [3H]prazosin for binding in the order: dicentrine > phentolamine > rauwolscine. Norepinephrine (0.3-100 microM) or phenylephrine (1-300 microM) produced gradual contractions of human hyperplastic prostates. The concentration-response curve of norepinephrine or phenylephrine was shifted in parallel to the right by dicentrine, consistent with a competitive blockade. The pA2 values of dicentrine against norepinephrine and phenylephrine were 8.04 +/- 0.09 and 8.33 +/- 0.11, respectively. These experiments were conducted to confirm that there was no interaction between alpha 1- and alpha 2-adrenoceptors in the tissue. Rauwolscine (1 microM) caused 1.7-fold, while dicentrine (0.1 microM) caused 15.8-fold shift of norepinephrine-induced contraction of human hyperplastic prostates. Combination of rauwolscine with dicentrine caused 17.8-fold shift of norepinephrine-induced prostatic tissue contraction. The contractile response to transmural field stimulation was abolished by pretreatment with tetrodotoxin, and suppressed concentration dependently by dicentrine or prazosin, whereas rauwolscine had little effect. It is concluded that dicentrine inhibits human hyperplastic prostate contractions in response to exogenous and endogenous adrenergic stimulation. Dicentrine may thus hold potential to relieve bladder outlet obstruction caused by benign prostatic hyperplasia via alpha 1-adrenoceptor blockade.

Characterization of the thromboxane (TP-) receptor subtype involved in proliferation in cultured vascular smooth muscle cells of rat.

1 , Jen‐Ai Rd., 1st Section, Taipei, Taiwan 1 The effects of the thromboxane A2 (TxA2)‐mimetic, U‐46619, on the proliferation of vascular smooth muscle cells (VSMCs) were examined in a clonal smooth muscle cell line, A10, which was derived from foetal rat aorta 2 [3H]‐U‐46619 bound to A10 cells of passages 18–20 (pl8‐20) with two classes of sites. The high affinity site showed a Bmax of 3.0 ±1.8 fmol mg−1 protein with a KD value 1.0 ±0.1 nM, while the low affinity site showed a Bmaxx of 43.0 ±6.0 fmol mg protein and KD value of 129.0 ±7.9 nM. However, [3H]‐U‐46619 bound to A10 cells from passages 28–30 (p28‐30) at a single class of site with a Bmax 111.0 ±9.0 fmol mg−1 protein and a Ku value of 175.4 ±22.0 nM 3 Cinnamophilin and SQ29548 inhibited specific [3H]‐U‐46619 binding to pl8‐20 A10 cells in a concentration‐dependent manner with Ki values of 390.0 ±3.2 and 4.6 ±1.0 nM, respectively at a high affinity site, and 2.6 ±0.2 μm and 310.0 ±6.4 nM, respectively at the low affinity site 4 U‐46619 produced isometric contractions of rat aorta in a concentration‐dependent manner with an EC50 7.0 ±1.2 nM. Cinnamophilin and SQ29548 antagonized U‐46619‐induced aortic contractions with pA2 values 6.3 ±0.1 and 8.2 ±0.2, respectively 5 U‐46619 increased [3H]‐thymidine incorporation into DNA of pi8‐20 and p28‐30 A10 cells in a concentration‐dependent manner with EC50 values 362.7 ±27.0 and 302.5 ±20.1 nM, respectively. The U‐46619‐induced increase of [3H]‐thymidine incorporation into DNA of p28‐30 A10 cells was potentiated by PDGF (1 ng ml−1) and FCS (1%) and was inhibited by cinnamophilin (10 μm) and SQ29548 (1 μm) with estimated pKB values 5.4 ±1.2 and 6.3 ±0.9, respectively 6 Cell cycle analysis revealed that U‐46619‐increased cell cycle progression was primarily due to a rapid transition from the DNA synthetic (S) to the G2/mitotic (M) phase. Moreover, U‐46619 also increased protein synthesis and cell numbers in VSMC. All these effects of U‐46619 were inhibited by cinnamophilin and SQ29548 7 U‐46619 caused phosphoinositide breakdown and increased the intracellular Ca2+ concentration in VSMC, effects which were blocked by cinnamophilin and SQ29548 8 These data indicate there are two U‐46619 binding sites in A10 VSMC. The high affinity site is correlated to U‐46619‐induced vasoconstriction while the low affinity site is correlated to U‐46619‐mediated VSMC proliferation. These data also reveal that U‐46619 stimulates the cell cycle progression in VSMC primarily through a rapid transition from S to G2/M. Since cinnamophilin inhibits TP‐receptor‐mediated VSMC proliferation, it may thus hold promising potential for the prevention of atherosclerosis or vascular diseases.

Vasorelaxing effect in rat thoracic aorta caused by denudatin B, isolated from the Chinese herb, Magnolia fargesii

Denudatin B is an antiplatelet agent isolated from the flower buds of Magnolia fargesii. We studied the effects of denudatin B on the vasoconstriction of rat thoracic aorta induced by high potassium (K+) solution, norepinephrine (NE) and caffeine, and to elucidate its mode of action. The contraction of rat aorta caused by high K+ (60 mM) and cumulative concentrations of CaCl2 (0.03-3 mM) was inhibited concentration dependently by denudatin B with an IC50 of 21.2 micrograms/ml. NE (3 microM)-induced phasic and tonic contractions of rat aorta were inhibited by pretreatment with denudatin B (10-100 micrograms/ml). The relaxing action of denudatin B persisted in denuded aorta, in Ca2(+)-free and EGTA (2 mM)-containing medium. The vasorelaxing effects were not affected by indomethacin (20 microM), hemoglobin (10 microM) or methylene blue (50 microM) and were not accompanied by PGI2 formation. In quin-2/AM-loaded cultured rat vascular smooth muscle cells, denudatin B (100 micrograms/ml) inhibited the increase of intracellular calcium caused by NE (3 microM) in the presence or absence of extracellular calcium. Denudatin B did not affect the caffeine (10 mM)-induced contraction and the increase in intracellular calcium. Denudatin B (100 micrograms/ml) increased the cGMP, but not the cAMP level in intact and denuded aorta. The 45Ca2+ influx induced in rat aorta by high K+ (60 mM) or NE (3 microM) was markedly inhibited by denudatin B in a concentration-dependent manner. These results indicate that denudatin B relaxed vascular smooth muscle by inhibiting the Ca2+ influx through voltage-gated and receptor-operated Ca2+ channels; its effect to increase cGMP may enhance the vasorelaxation.

Vasoconstricting effect in rat aorta caused by thaliporphine isolated from the plant Neolitsea konishii K

Thaliporphine (0.1-100 microM) produced sustained, concentration-dependent contraction in isolated rings of rat aorta. Thaliporphine (ED50 = 1.5 +/- 0.5 microM) was less potent than endothelin (ED50 = 3.9 +/- 0.4 nM), but was more potent than Bay K 8644 (ED50 = 5.5 +/- 0.6 microM). Thaliporphine also contracted guinea-pig trachea and ileum preparations, and increased the force of beating of rat left atria, but was less potent on these tissues than on rat aorta. Thaliporphine-induced contraction of rat aorta was not affected by prazosin (0.3 microM), atropine (1 microM), saralasin (10 microM) or ketanserin (10 microM). However, the contraction was slightly potentiated by the removal of endothelium. Preincubation of the rat aorta in Ca(2+)-free Krebs solution (containing 1 mM EGTA) for 15 min completely abolished thaliporphine-induced contractions, and the subsequent addition of 3 mM CaCl2 restored thaliporphine-induced contractions to the control level. In rat aorta, thaliporphine-induced contractions were significantly reduced by nifedipine (1-30 nM) or verapamil (0.01-0.3 microM), and significantly increased by Bay K 8644 (0.1 microM) or KCl (20 mM). The pA2 values for nifedipine and verapamil against thaliporphine-induced contractions were 9.4 +/- 0.1 and 8.4 +/- 0.2, respectively. The results indicate that thaliporphine exerts its vasoconstrictor effect on rat aorta mainly by promoting Ca2+ entry.