Effect of basic fibroblast growth factor on synthesis of C-type natriuretic peptide in cultured human umbilical vein endothelial cells

AIM: To investigate the effect of basic fibroblast growth factor (bFGF) on C-type natriuretic peptide (CNP) production, release and mRNA expression. METHODS: Human endothelial cell cultured;CNP was mea sured by radioimmunoassay method;CNP mRNA expression was determined by RT-PCR technique.RESULTS: bFGF could augment CNP synthesis in human endothelial cells. Compared with control group,25 ng, 50 ng, 100 ng bFGF increased CNP contents in endothelial cells by 88% (P＜0.05), 95% (P＜0.05), 187% (P＜0.01), respectively.100 ng bFGF also stimulated CNP release from cultured human endothelial cell. In addition, 25 ng, 50 ng and 100 ng bFGF stimulated CNP mRNA expression of cultured human endothelial cells in a dose-dependent manner. CONCLUSION: bFGF might regulate CNP synthesis,release and mRNA expression in cultured umbilical human endothelial cells.     

Effects of adrenomedullin on proliferation of vascular smooth muscle cells induced by urotensin Ⅱ

AIM:To observe the effect of adrenomedullin(ADM)on proliferation of vascular smooth muscle cells(VSMC) induced by urotensin Ⅱ(UⅡ). METHODS:DNA synthesis of cultured rat aortic VSMC was measured by [³H]-TdR incorporation. The activities of mitogen activated protein kinase(MAPK) were determined by isotope tagged with [γ-³²P]-ATP. RESULTS:UⅡ(10^-8mol/L) significantly increased [³H]-TdR incorporation of VSMC and MAPK activities by 38%(P＜0.05) and 260%(P＜0.01) respectively compared with control group. Compared with UⅡ group, 10^-10,10^-9,10^-8mol/L ADM decreased [³H]-TdR incorporation of VSMC by 7%(P＞0.05), 32%(P＜0.05)and 41%(P＜0.01),respectively, and diminished MAPK activities by 24%(P＞0.05), 32%(P＜0.05)and 36%(P＜0.05),respectively. CONCLUSION:ADM inhibits proliferation of VSMC induced by urotensin Ⅱ through inhibiting MAPK activation.     

Inhibition of endothelin on taurine-transportation in rat cardiac myocytes

AIM:To investigate the effect of endothelin(ET) on taurine transportation in rat cardiac myocytes in vitro.METHODS: In cultured cardiac myocytes of neonatal rats, taurine transportation velocity was measured by radio-ligand method. RESULTS: ET(10^-10-10^-8 mol/L) could inhibit taurine transportation in a dose-dependent manner.10^-10,10^-9 and 10^-8mol/L of ET significantly decreased taurine transpotation by 13%, 38% and 71%, respectively (P＜0.01), compared with control group. H₇,BQ123 and Pre-PMA can reverse the inhibition of ET on taurine transportation dramatically(P＜0.01).CONCLUSION:The binding of ET and ET-A receptor might activate protein kinase C,which inhibits taurine transportation in cultured myocytes of rats.     

Metallothionein inhibits homocysteine-induced proliferation of rat vascular smooth muscle cells

AIM:To investigate the effect of metallothionein(MT) on proliferation of rat vascular smooth muscle cells (VSMCs) stimulated by homocysteine and its mechanism. METHODS:VSMCs proliferation was measured by [³-H]-TdR incorporation, mitogen-activated protein kinase(MAPK)activity were determined by immunoprecipitation method, the intracellular contents of MT and malondialdehyde (MDA)were assayed by -hemoglobin saturation method and TBA reaction, respectively, and lactate dehydrogenase (LDH) leakage was measured by NADH oxidation. RESULTS:Hcy(10^-6-10^-4 mmol/L) stimulated [³-H]-TdR incorporation by the VSMCs in a concentration-dependent manner. Compared with control, [³-H]-TdR incorporation in VSMCs treated with 0.1 mmol/L Hcy was increased by 4.2 fold (P＜0.01). Meanwhile, Hcy enhanced MAPK activity, MDA formation and LDH release (P＜0.01)in a concentration-dependent manner. Treatment of VSMCs with MT alone did not change above parameters, compared with control. However, MT (10^-6-10^-4 mol/L)attenuated significantly Hcy-stimulated proliferation of VSMCs (P＜0.01)in a concentration-dependent manner. And MT inhibited obviously Hcy-induced activation of MAPK activity, MDA formation and LDH release. Preincubation of VSMCs with 0.5 mmol/L ZnCl₂ for 6 h induced an increase cellular MT content by 5.7-fold (P＜0.01). The MT-overexpressed VSMCs resisted Hcy-stimulating action on MAPK activity, MDA formation and LDH leakage (P＜0.01). CONCLUSION:These results show that MT has an inhibitory effect on Hcy-induced VSMCs proliferation, and that MT could inhibit Hcy-stimulated MAPK activity and lipid peroxidation.     

Effects of angiotensin II and losartan on collagen synthesis in rat hepatic stellate cells

AIM: To investigate the effects of angiotensin II (Ang II) and AT_1a receptor antagonist (losartan) collagen synthesis in rat hepatic stellate cells (HSCs). METHODS: ① Rat HSCs were isolated, cultured and identified. ② Rat HSCs were incubated in the medium with different concentrations of AngII or losartan, then the quantity of collagen was examined by -proline release assay. RESULTS: ① The yield of HSCs was 2×10⁷-3×10⁷/per rat, their viability and purity was more than 95% and 90%, respectively. ② The yield of collagen in HSCs significantly got a rise in a concentration-dependent manner when HSCs were incubated with AngII (10^－6mol/L-10^－10 mol/L) (P<0.05). While HSCs were influenced by the antagonist of AT_1a (10^－6 mol/L-10^－9 mol/L), the quantity of collagen dropped greatly (P<0.05). CONCLUSIONS: Ang II stimulates HSCs to produce more collagen. Losartan inhibits the cell to synthesize collagen via AT_1a receptor (P<0.05). The results indicate that Ang II may play an important role in the development of hepatic fibrosis, and using AT_1a antagonist may offer a new strategy to prevent hepatic fibrosis.     

Effects of insulin on cardiac fibroblast proliferation and cardiac myocyte hypertrophy

AIM:To study the effect of insulin on proliferation and hypertrophy of cardiac myocytes and its role in the induction of cardiac hypertrophy. METHODS:1. The neonatal rat cardiac myocytes and cardiac fibroblasts were cultured respectively and identified with light microscopy, electron microscopy and immunocytochemistry. 2. Cell proliferation was measured with cell number, metabolic activity and DNA synthesis (with WST-1, BrdU enzyme-linked immunosorbent assay ) and the percentage of S+G₂+M in cell cycle (by flow cytometry ). 3.Cell hypertrophy was evaluated by cell protein content (Coomassie Briliant Blue's method). RESULTS:1. The cultured cells showed the characteristic of cardiac myocytes and cardiac fibroblasts, respectively. 2. After being treated with insulin, the cell number, absorbance of BrdU incorporation and WST-1 cleavage products and the percentage of S+G₂+M of cardiac fibroblasts increased significantly (P＜0.01 orP＜0.05), while the above parameters of cardiac myocytes remained unchanged (P＞0.05). 3. Protein content of cardiac myocytes increased significantly in a dose-dependent manner (P＜0.01 orP＜0.05) in insulin treated groups (10^-10 mol/L-10^-7 mol/L). CONCLUSION:Insulin promoted cardiac fibroblast proliferation and increased myocytes protein content(induced myocyte hypertrophy)in vitroand may play an important role in pathogenesis of cardiac hypertrophyin vivo.     

Role and regulation of calcineurin in angiotensin Ⅱ-stimulated cardiac myocyte hypertrophy of rats

AIM: To study the role and regulation of calcineurin(CaN) in angiotensin II(AngⅡ)-stimulated cardiacmyocyte hypertrophy of rats. METHODS: Using AngⅡ to induce the cultured cardiac myocyte hypertrophy of rats, and investigating the effect of CaN inhibitor on [³H]-leucine incorporation of AngⅡ-stimulated cardiomyocytes and the regulation of various factors on CaN activity in cardiomyocytes.RESULTS: AngⅡ can stimulate the CaN activity in cultured neonatal rat cardiomyocytes in a dose- and time-dependent manner. In cardiac myocytes incubated with 10, 100, 1000 nmol·L^-1 of AngⅡ for 12h, the CaN activities increased respectively by 13%,57%(P＜0.05) and 228%(P＜0.01) compared with that in non-stimulated cardiomyocytes. The CaN activities in AngⅡ-stimulated cardiomyocytes were significantly inhibited by losartan(50 μmol·L^-1), H₇(50 μmol·L^-1)and Fura-2/AM(4 μmol·L^-1),while no effect was observed with PD98059(50 μmol·L^-1).The [³H]-leucine incorporation in AngⅡ-stimulated cardiomyocytes increased by 46%(P＜0.01) compared with that in control group, which was dramatically inhibited by cyclosporin A(0.5~5μg/mL). CONCLUSIONS: Calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, may play an important role in AngⅡ-induced cardiac myocyte hypertrophy. The activation of CaN may dependent on the sustained increases of [Ca²⁺]i and be regulated by some protein kinases (such as PKC,etc.).     

The relations among PAMP,ADM and AngⅡ on vascular tissue

AIM: To elucidate the relations among proadrenomedullin N terminal 20 peptide(PAMP), adrenomedullin(ADM)and angiotensin(Ang).METHODS: Tissue slices of rat aorta were incubated as follows:(I) increasing concentrations of AngⅡ(10^-9, 10^-8, 10^-7 mol/L); increasing concentrations of PAMP(10^-9,10^-8,10^-7mol/L).The tissue and incubation concentrations of PAMP,ADM and AngⅡ were measured by the radioimmunoassay (RIA).RESULTS: The tissue and incubation concentrations of PAMP and ADM were concentration-dependently increased by AngⅡ,but the tissue and incubation concentrations of AngⅡ can not effected by PAMP.CONCLUSION: AngⅡ markedly stimulate the release of ADM and PAMP.It may be one of the factors which regulate the synthesis and release of ADM and PAMP.The regulation may play an important role in homeostasis regulation of cardiovascular system.     

AngiotensinⅡ activates autophagy pathway through elevating ROS level in vascular endothelial cells

AIM: To evaluate the effects of angiotensinⅡ (AngⅡ) on autophagy induction in vascular endothelial cells. METHODS: Human vascular endothelial EA.hy926 cells were used in the study. Intracellular reactive oxygen species (ROS) levels were detected by a microplate reader after the cells were treated with AngⅡ (10^-7 mol/L) or AngⅡ combined with antioxidant N-acetyl-L-cysteine (NAC,50 μmol/L) for 24 h. The protein levels of LC3-Ⅱ was detected by Western blotting after the cells were stimulated by different concentrations (10^-8, 10^-7, 10^-6 mol/L) of AngⅡ for 24 h or by AngⅡ (10^-7mol/L) for different time (0 h, 6 h, 12 h, 24 h, 36 h). The number of autophagosomes was evaluated by fluorescence microscopy after stained with acridine orange. Similarly, the protein level of LC3-Ⅱ and the number of autophagosomes were detected after treated with AngⅡ(10^-7mol/L), AngⅡ combined with autophagy inhibitor 3-methyladenine (3-MA) at concentration of 2 mmol/L or AngⅡ combined with NAC at concentration of 50 μmol/L. RESULTS: Intracellular ROS level and LC3-Ⅱprotein level were significantly increased (P<0.05) after the cells were treated with AngⅡ, accompanied by the significant increase in the number of autophagosomes. AngⅡ-induced autophagy (as showed both in LC3-Ⅱprotein level and autophagosomes) was dramatically down-regulated by the treatment with 3-MA or NAC in EA.hy926 cells (P<0.05). CONCLUSION: AngⅡ induces autophagy through elevating ROS levels in EA.hy926 cells.     

Protective effects of multi-enzyme Ⅱ on vascular endothelial cell injury induced by hyperlipidemia serum

AIM and METHODS: The protective effects of multi-enzyme Ⅱ was studied on cultured endothelial cells which was injuried by hyperlipidemia serum. RESULTS: Hyperlipidemia serum increased ICAM-1 expression on the surface of endothelial cells, and decreased NO₂^- release significantly (P＜0.01). ICAM-1 expression could be reduced and NO₂^- release could be enhanced markedly by multi-enzyme Ⅱ (P＜0.01). CONCLUSION: Multi-enzyme Ⅱ had an obvious protective effect on vascular endothelial cells which was injuried by hyperlipidemia seurm. Multi-enzyme Ⅱ could clean out oxide free radicals effectively because it had the acitive structure of both SOD and CAT.     

The inhibition of nitric oxide synthase for long time up-regulates vascular angiotensin Ⅱ receptor

AIM:To observe the effects of aortal angiotensin Ⅱ(AngⅡ)levels and AngⅡ receptor in the hypertensive rat models. METHODS: Intraperinoneal injection of L-N^ω-nitro-arginine(L-NNA) into rats induced hypertensive model, the binding of aortal AngⅡ receptor and the contents of aortal tissue AngⅡ and plasma NO₂^-+NO₃^-(NOx) were determined.RESULTS:Compared with the control group, the blood pressure of the rats treated with L-NNA was significantly increased by 142%(P＜0.01),the plasma NOx levels were decreased by 48%. However, in the rats treated for 4 weeks the ratio of cardial index was increased by 128%(P＜0.01),the plasma AngⅡ levels weren't significantly changed, the contents of vascular tissue AngⅡ were increased by 612%(P＜0.01) and the Bmax of [¹²⁵I]-AngⅡ was increased by six times (P＜0.01),the affinity was doubled respectively.CONCLUSIONS:Nitric oxide synthase (NOS) inhibition mostly influes the reninangiotensin system in regional tissue. The inhibition for long time up-regulates vascular AngⅡ and AngⅡ receptor, which can support the hypothesis "NOS inhibition can induce AngⅡ-depended hypertension".     

Inhibitory effect of Salidroside on the proliferation of rabbit pulmonary artery smooth muscle cells under hypoxia

AIM: To investigate the effect of Salidroside on the proliferation, DNA synthesis, intracellular Ca²⁺ content of rabbit PASMC (pulmonary artery smooth muscle cells) under hypoxia. METHODS: Techniques of cell culture, MTT test, [³H][³H][³H]-TdR incorporation, fluo-3 and confocal laser scanning microscopy were used. RESULTS: The A value of MTT and [³H][³H]-TdR incorporation of PASMC increased significantly by 62% (P＜0.05) and 138% (P＜0.01) after 24 h hypoxia. Salidroside (32×10^-5 mol/L) inhibited the action of hypoxia on the proliferation of PASMC, the A value of MTT and [³H][³H]-TdR incorporation declined significantly by 29% (P＜0.05) and 37% (P＜0.01) compared with hypoxia group. A calcium channel blocker, verapamil could also inhibit the accelerative effect of hypoxia on the proliferation of PASMC. The intracelluler Ca²⁺ content of PASMC raised markedly under hypoxia, but the effect of hypoxia on the intracelluler Ca²⁺ content could be inhibited by Salidroside. CONCLUSION: Salidroside inhibited the proliferation, DNA synthesis of PASMC induced by hypoxia. The inhibitory action of Salidroside on the increase in intracellular Ca²⁺ concentration under hypoxia might be one of the mechenisms.     

Effects of homocysteine on number and activity of endothelial progenitor cells from peripheral blood

AIM: To investigate whether homocysteine (Hcy) has influences on endothelial progenitor cell (EPCs) number and activity from peripheral blood. METHODS: Total mononuclear cells (MNCs) were plated on fibronectin-coated culture dishes and cultured for 7 days, and then attached cells were stimulated with Hcy or vehicle control for 6 h, 12 h, 24 h and 48 h. The adhesion, proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed, respectively. RESULTS: Incubation of isolated human MNCs with Hcy dose and time-dependently decreased the number of EPCs with maximum at 200 μmol/L for 24 hours (35.7±6.7 vs 62.5±10.6， P＜0.01). In addition, Hcy impaired EPC proliferative (0.531±0.061 vs 0.328±0.055， P＜0.05), migratory (26.3±6.4 vs 6.4±3.7， P＜0.01), adhesive (33.1±8.1 vs 17.4±7.5， P＜0.01) and vasculogenesis capacity (25.4±9.1 vs 10.4±4.7， P＜0.01) in a dose and time-dependent manner. CONCLUSION: It is suggested that Hcy may result in the reduction of EPCs and decrease EPC functional activity.     

Effect of peroxisome proliferator activated receptor δ on mRNA expression of MCP-1 induced by homocysteine in cultured human umbilical vein endothelial cells

AIM: To investigate the effects of peroxisome proliferator activated receptor δ (PPARδ) on the mRNA expression of monocyte chemoattractant protein 1 (MCP-1) induced by homocysteine (Hcy) in human umbilical vein endothelial cells (HUVECs). METHODS: Collagenase was used to isolate endothelial cells from human umbilical vein, and the cells were cultured in vitro . The HUVECs were divided into blank control group, Hcy group, GW0742 (a specific agonist of PPARδ) group and diphenyleneiodonium (DPI,a specific inhibitor of NADPH oxidase) group. RT-PCR was used to examine the mRNA expression of MCP-1 and PPARδ. The protein level of PPARδ was detected by Western blotting.2',7'-Dichlorofluorescin diacetate(DCFH-DA) was added to monitor intracellular production of reactive oxygen species (ROS). RESULTS: Compared with control group, Hcy promoted the mRNA expression of MCP-1 in a concentration-dependent manner, and decreased the mRNA expression of PPARδ in HUVECs. The mRNA expression of MCP-1 was significantly elevated by Hcy at the concentration of 10^-5 mol/L, and the mRNA expression of PPARδ was decreased remarkably (P<0.01). GW0742 decreased the mRNA expression of MCP-1 compared with Hcy group (P<0.01). Hcy remarkably increased the production of ROS compared with control group. Hcy-induced production of ROS was also significantly attenuated by GW0742. CONCLUSION: The activation of PPARδ decreases the Hcy-induced mRNA expression of MCP-1 by suppressing Hcy-stimulated production of ROS.     

ROS mediates regulation of intracellular Ca2+ induced by angiotensin II in primarily cultured medullary neurons

AIM: To investigate the role of reactive oxygen species (ROS) in the regulation of intracellular Ca²⁺ induced by angiotensin II (Ang II) in the primarily cultured medullary neurons. METHODS: Primarily cultured medullary neurons were prepared from 14-day-old embryos of Sprague-Dawley rats in the study. The identification of medullary neurons was assessed by double-labeling immunofluorescence. To explore the role of ROS, mainly the superoxide (O₂^·), the O₂^·generation was measured using the fluorogenic probe dihydroethidium (DHE). To determine intracellular free calcium concentration ([Ca²⁺]_i), the neurons were loaded with the Ca²⁺-specific dye Fura-2/AM. The cell viability after adding Ang II was also examined using CCK-8 assay. RESULTS: Most of the cultured cells were medullary neurons, more than 80% of which were glutamate positive neurons. Ang II (5 μmol/L) increased the level of ROS within 10 min in the medullary neurons. Ang II at 5 μmol/L induced a significant[Ca²⁺]_i increase in the medullary neurons, and the effect of Ang II occurred rapidly and reached a peak within 20 min after administration. The level of[Ca²⁺]_i started to decline after washout. The Ca²⁺ elevation induced by Ang II was significantly decreased by apocynin or TEMPOL. No significant difference in the cell viability between control group and 5 μmol/L Ang II treatment group was observed. CONCLUSION: ROS is involved in the regulation of[Ca²⁺]_i induced by Ang II in the primarily cultured medullary neurons, suggesting a potential intracellular signaling mechanism involved in the Ang II-mediated oxidant regulation of central neural control of blood pressure.     

Effects of leptin on BSEP protein expression and signaling pathway in HepG2 cells

AIM: To investigate the effects of leptin on the expression of bile salt export pump (BSEP) and signaling pathway in human hepatocellular carcinoma cell line HepG2. METHODS: HepG2 cells were cultured in vitro. Leptin at concentrations of 10^-8, 10^-7 and 10^-6 mol/L was used as a stimulating factor. The protein levels of adenosine monophosphate-activated protein kinase alpha subunit (AMPKa), phosphorylated AMPKa (p-AMPKa) and BSEP in the HepG2 cells at 24 h, 48 h and 72 h were detected by Western blotting. The optimal culture time and leptin concentration were selected, and compound C at concentration of 10 μmol/L was added to this group. The protein expression of BSEP was detected by Western blotting. RESULTS: Intervention of HepG2 cells with leptin for 72 h increased the protein expression of AMPKa gradually in a concentration-dependent manner, and leptin at concentration of 10^-6 mol/L induced the strongest AMPKa expression (P<0.01). Intervention of HepG2 cells with leptin for 24 h increased the phosphorylation level of AMPKa gradually in a dose-dependent manner (P<0.01). The effect of leptin on the increase in the protein expression of p-AMPKa was also in a time-dependent manner (P<0.01). After intervention with different concentrations of leptin for 24 h, the protein expression of BSEP in the HepG2 cells was gradually increased by the stimulation of leptin in a concentration- and time-dependent manner (P<0.01). Compared with NC group, the protein expression of BSEP in 10^-6 mol/L leptin group and 10^-6 mol/L leptin+10 μmol/L compound C group was increased at 72 h (P<0.01), and that in 10^-6 mol/L leptin+10 μmol/L compound C group was lower than that in 10^-6 mol/L leptin group at 72 h (P<0.01). CONCLUSION: Leptin promotes the protein expression of BSEP in HepG2 cells by leptin-AMPK-BSEP signaling pathway. Leptin promotes the increases in AMPKa protein and the level of phosphorylation of AMPKa in HepG2 cells.     

Antagonistic effect of captopril on activation and injury of vascular endothelial cells induced by lipopolysaccharide

AIM: To investigate the antagonistic action of Captopril (Cap) on the activation and injury of human umbilical vascular endothelial cells (HUVECs) induced by lipopolysaccharide(LPS) and the possible mechanisms. METHODS: After 18 h exposure of the cultured HUVECs to LPS (1 mg/L), or LPS (1 mg/L) plus Cap at the concentration of 10^-7mol/L, 10^-5mol/L and 10^-3mol/L, the expression of vWF protein in the conditioned media was tested by enzyme-linked immunosorbent assay (ELISA), the expression of ICAM-1 protein in HUVECs was determined by indirect immunofluorescence technique with flow cytometry as well. In addition, the expression of TNFα mRNA was determined by in situ hybridization. RESULTS: The results of ELISA and indirect immunofluorescence technique showed that exposure to LPS at a concentration of 1 mg/L led to a significant increase in the vWF and ICAM-1 expression in HUVECs as compared to the control (P＜0. 05), whereas they were somewhat decreased when exposed to Cap at three increasing concentrations mentioned above, especially in the Cap (10^-3mol/L) plus LPS group (P＜0.05). Cap inhibited vWF secretion and ICAM-1 expression of HUVECs caused by LPS in a concentration-dependent manner. In situ hybridization revealed that the expression of TNFα mRNA was inhibited by Cap both in a concentration of 10^-3mol/L, and in a lower concentration of 10^-5mol/L. CONCLUSION: Cap antagonizes the activation and injury of HUVECs induced by LPS, which may be related to the decrease in TNFα mRNA expression.     

Effect of urotensin II on cultured pulmonary arterial smooth muscle cells of rabbits

AIM: The effect of urotensin II (U-II) on proliferation of cultured pulmonary arterial smooth muscle cells (PASMCs) of rabbits and its mechanism are investigated. METHODS: PASMCs were isolated using explant technique. RPASMCs were incubated in serum-free medium with different concentrations of nicardipine, calcimodulin antagonist W₇, PKC inhibitor H₇ or MAPK inhibitor (PD₉₈₀₅₉), with or without U-II. RPASMC proliferation was examined by MTT assay and by the increase in [³H]-thymidine incorporation into DNA. RESULTS: U-II (10^-9 mol/L-10^-7 mol/L) increased A value of PASMCs by MTT assay and [³H]-thymidine incorporation in PASMCs in a dose-dependent manner. U-II induced a maximal effect at a concentration of 10^-7 mol/L. A value and [³H]-thymidine incorporation rose 42.9% and 68.5% (P<0.05), respectively. U-II had no effect at a concentration of 10^-10 mol/L. Nicardipine, W₇, H₇, PD₉₈₀₅₉ (10^-7 mol/L-10^-5 mol/L) inhibited the effect of U-II in inducing increase of A value and -thymidine incorporation in a dose-dependent manner, with the maximal inhibitory rate of 42.3%, 19.6%, 23.2%, 10.5% (P<0.05) in A value and 46.6%, 9.8%, 21.7%, 14.7% (P<0.05) in [³H]-thymidine incorporation at concentration of 10^-5 mol/L, respectively. CONCLUSION: Our results suggest that U-II may induce proliferation of PASMCs of rabbits by Ca²⁺, CaM, PKC and MAPK signal transduction pathway.