AIM To provide toxicokinetics data for toxicity studies of repeated doses of sodium 9-[2-(phosphonomethoxy) ethyl] adenine (PMEA-Na). METHODS The concentrations of PMEA-Na in plasma and urine were determined by HPLC/MS/MS method after single and multiple iv administrations in dogs. Data were executed by the statistical moment method to acquire the toxicokinetics parameters. Serum biochemical tests and histopathological examination were performed. RESULTS The system exposure of PMEA-Na in dogs was dose-dependent over the dose range of 1.0-6.0 mg·kg-1. The areas under the plasma concentration-time curve of PMEA-Na after single and multiple iv administrations at 1.0, 3.0 and 6.0 mg·kg-1 dosage were (2.3±0.5), (8.4±1.6), (17.5±3.7) and (5.0±0.4), (15.9±3.2), (30.3±4.7)mg·L-1·h, respectively. The urinary excretion of PMEA-Na in 72 h after iv administration was (87.0±4.8)% at the dose of 3.0 mg·kg-1. In 6.0 mg·kg-1 dose group, liver enzyme activity of glutamic-pyruvic transaminase and serum levels of total bilirubin, blood urea nitrogen, creatinine and triglycerides were all significantly elevated; glucose level significantly decreased comparing with the control group. Histopathological observation showed distinct pathological changes in liver and kidney tissues of 6.0 mg·kg-1 dose group. CONCLUSION There was evidence of toxicity after repeated-dose (14 d) of PMEA-Na in dogs and the major toxicity target organs were the kidney and liver.

AIM To investigate the effects of honokiol on ischemic neurological deficiency and on the scavenging ability of ischemia reperfusion (I-R) brain tissue for reactive oxygen species (ROS). METHODSCerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in rats. I-R in mice were induced by blood stream pause in bilateral common carotid arteries for 30 min and reperfusion for 30 min. The activities of ROS scavenging enzymes were determined with colorimetric methods. RESULTS Intravenous honokiol in 5-50 μg·kg-1 significantly decreased the neurological deficiency score, and diminished cerebral infarction volume in rats. In I-R brain tissue of mice, intravenous honokiol in 7-70 μg·kg-1 evidently enhanced the activities of superoxide dismutase, catalase, glutathione peroxidase and peroxidase, and markedly lowered lipid peroxidative product malondialdehyde content. Moreover honokiol significantly increased Na+-K+-ATPase activity in I-R brain tissue. CONCLUSION Honokiol ameliorates the neurological deficiency behavior and diminishes infarction volume in MCAO rats; and enhances cerebral scavenging ability for ROS and Na+-K+-ATPase activity in cerebral I-R mice. It is indicated that honokiol is a protective agent for cerebral ischemia and I-R.

AIM To investigate whether dipfluzine (Dip) possesses antiarrhythmic effect on experimental arrhythmias and effect on cytosolic calcium in ventricular myocytes of guinea-pig. METHODS Experimental arrhythmias were induced by strophanthin G infusion through jugular vein in guinea-pigs and by myocardial ischemia-reperfusion (I-R) in rats respectively. Cytosolic calcium concentration ([Ca2+]i) of isolated guinea-pig ventricular myocytes was examined with laser confocal scanning microscope. RESULTSIn guinea-pigs pretreatment with Dip 20 mg·kg-1 increased the dosages of strophanthin G required to induce ventricular premature contraction (VP), ventricular tachycardia (VT), ventricular fibrillation (VF) and cardiac arrest (CA), pretreatment with Dip 10 mg·kg-1 increased the dosages of strophanthin G required to induce VP. In the I-R-induced arrhythmic model of rats, Dip 20 mg·kg-1 decreased the number of rats exhibiting VT, VF and CA, and the number of rats exhibiting VF and CA was decreased by Dip 10 mg·kg-1. Both Dip and verapamil (Ver) decreased [Ca2+]i of the ventricular myocytes in normal Tyrode′s solution. The Ca2+ overload evoked by high extracellular Ca2+ levels was inhibited by Dip and Ver, and the prophylactic effect of Dip was less than that of Ver, while the curative effect of Dip was more obvious than that of Ver. CONCLUSION Dip has antiarrhythmic effect, which is likely related to the modulation on the intracellular calcium homeostasis.

AIM To study the effect of genistein (GST), a protein tyrosine kinases inhibitor, on L-type calcium channel currents (Iba,L or Ica,L, dependent on permeating ion used) in freshly dispersed colon smooth muscle cells from guinea-pig. METHODS Single colon smooth muscle cells were enzymatically dissociated from guinea-pig. L-type calcium currents were measured by conventional whole-cell patch-clamp techniques. RESULTS The peak amplitudes of Iba,L elicited to 10 mV test potential from a holding potential of -80 mV, were reversibly and dose-dependently reduced by GST (10-100 μmol·L-1) with an IC50 value of (39.9±3.6)μmol·L-1. Bath application of GST shifted the steady-state inactivation curves of Iba,L in a hyperpolarized direction (about 10 mV, P＜0.01) without altering their slopes. The peak amplitudes of Iba,L were also inhibited but to a less extent by daidzein, an inactive analogue of GST. Sodium orthovanadate 1 mmol·L-1, a potent inhibitor of protein tyrosine phosphatases, blocked GST-induced inhibition of Ica,L. CONCLUSION GST can block L-type calcium channel activity in guinea-pig colon smooth muscle cells via tyrosine kinase pathway.

To study the effect of genistein on the proliferation of cardiac fibroblasts(CF), CFs were cultured from neonatal rat hearts, DNA synthesis of the cells was determined by incorporation of ［3H］TdR into DNA, the cell cycle was measured by flow cytometric analysis. Genistein(0.5－50 μmol*L-1) attenuated 2.5% fetal calf serum-induced proliferation of CF in concentration-dependent manner. Genistein(50 μmol*L-1) arrested CF cell progression at G2/M phase. The results suggest that genistein be a potential substance for treatment of cardiac fibrosis.

To study the effect and mechanisms of 18 α-glycyrrhizic acid (18 α-GA) on cytochrome P450 (CYP) enzymes, the expression of CYP1A1, CYP2E1 and CYP3A was determined in rat hepatocyte sandwich cultures by using enzyme assay and semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR). The results showed that the activities of CYP1A1 (7-ethoxyresorufin O-deethylase, EROD), CYP2E1(aniline hydroxylase, ANH) and CYP3A (erythromycin N-demethylase, ERD) were decreased in concentration-dependent manner after treatment with 18 α-GA(50－400 mg*L-1), and at the concentration of 200 mg*L-1 inhibitory rate reached the maximum (the maximum inhibitory rate was 59.6%, 69.7% and 44.7%, respectively). The time course revealed that the inhibition reached plateau level at d 4 of culure. 18 α-GA Decreased CYP1A1, CYP2E1 and CYP3A1 mRNA expression in dose-dependent manner, the maximum inhibitory rate was 44.5% , 58.1% and 37.1%, respectively. The results suggest that 18 α-GA down-regulate CYP expression at the transcriptive levels.

1-(2,6-Dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino) propane hydrochloride(DDPH) caused parallel rightward shifts of the phenylephrine(Phe) concentration-contractile response curves and did not suppress the maximal contractile response to Phe (pA2=7.24) in isolated rabbit urinary bladder smooth muscle. DDPH decreased the parameters of cystometry in urethane-anesthetized rats. Thirty minutes after DDPH (25 and 50 mg*kg-1 ig) administration, bladder capacity, voiding pressure, voiding threshold pressure were significantly decreased. With the observation of light-microscope and electron-microscope technique, DDPH (25 and 50 mg*kg-1*d-1 ig for 4 weeks) also inhibited the development of testosterone propionate-induced benign prostatic hyperplasia in rats. The results indicate that DDPH may inhibit benign prostatic hyperplasia and improve the urinary flow.

To analyse the effect of tetrandrine(Tet) on proliferation of aortic vascular smooth muscle cells ( AVSMC), AVSMC were isolated and cultured from sham-operated rats(Sham), renovascular hypertensive rats〔RHR, 18 weeks after two kidney one clip(2K1C) operation〕, and Tet (50 mg*kg-1*d-1 po for 9 weeks from week 9 after 2K1C operation)treated RHR. The proliferation of AVSMC was detected by MTT method, and the DNA synthesis was evaluated by ［3H］-thymidine incorporation. The results showed that ①The ultrastructure of aorta suggested that AVSMC in RHR had transferred from contractile phenotype to synthetic phenotype; ②Compared to Sham, AVSMC from RHR showed a higher proliferative property with a higher cell number and an increased growth rate stimulated by norepinephrine(NE) or angiotensinⅡ(AngⅡ); ③Compared to untreated RHR, AVSMC from Tet treated RHR showed a reduced reactivity to NE- or AngⅡ-stimulated proliferation and growth rate; ④Tet(0.1－10 μmol*L-1) treated in vitro induced a concentration-dependent depression in ［3H］ thymidine-incorporation stimulated by NE or AngⅡ in AVSMC from either RHR or Sham. This study provides an evidence of increased reactivity to NE or AngⅡ in AVSMC of RHR. Tet inhibits the proliferation and DNA synthesis in AVSMC, depresses the susceptibility of AVSMC to AngⅡ and NE, both contribute to the regression effect on hypertensive vascular remodeling.

Effects of preconditioning(PC) and praeruptorine C(Pra-C) on intracellular free calcium level(［Ca2+］i) and NO content in hypertrophied vascular smooth muscle cells (VSMC) with hypoxia were studied. ①The aorta VSMC of rats with renovascular hypertension induced by two-kidney-one-clip(2K1C) were isolated and cultured. Fura-2/AM was as a calcium fluorescent indicator. It was seen that Pra-C(20 mg*kg-1*d-1，ig, for 9 weeks from week 9 after 2K1C) and PC (3 cycles of 5 min pure N2 and 5 min 95%O2+5%CO2) antagonisted the elevated activities(higher ［Ca2+］i) induced by KCl and norepinephrine in VSMC with hypo- xia (30 min pure N2). ② In hypertrophied VSMC stimulated by angiotensinⅡ, combination of Pra-C(10 μmol*L-1 for 24 h) and PC enhanced NO content to the normal level in hypertrophied VSMC with hypo- xia. The results suggest that combination of Pra-C and PC have cooperative protection for hypertrophied VSMC with hypoxia injury.

By using decapitating, intravenous injection of saturated MgCl2 and legation of bilateral carotid arteries with vagi, the effects of 1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethyl- amino) propane hydrochloride(DDPH) on survival time in mice were studied. With the model of cerebral ischemia for 20 min and reperfusion for 10 min, effects of DDPH on the superoxide dismutases(SOD) activity and malondialdehyde (MDA) content in brain tissue and pathological changes were studied. The results indicated that DDPH at dosages of 3,6,12,24 mg*kg-1 ip 30 min before ischemia prolonged the survival time significantly. Meanwhile, DDPH was found to increase the activity of SOD and reduce the content of MDA, as well as mitigate pathological damage of neuron after cerebral ischemia and reperfusion in mice. The results suggest that DDPH has protective effects on brain ischemia.