Inhibitory effects of hesperetin on Nav1.5 channels stably expressed in HEK 293 cells and on the voltage-gated cardiac sodium current in human atrial myocytes.

AIM: Voltage-gated sodium channels composed of a pore-forming α subunit and auxiliary ß subunits are responsible for the upstroke of the action potential in cardiac myocytes. The pore-forming subunit of the cardiac sodium channel Nav1.5, which is encoded by SCN5A, is the main ion channel that conducts the voltage-gated cardiac sodium current (INa) in cardiac cells. The current study sought to investigate the inhibitory effects of hesperetin on human cardiac Nav1.5 channels stably expressed in human embryonic kidney 293 (HEK 293) cells and on the voltage-gated cardiac sodium current (INa) in human atrial myocytes. METHODS: The effects of hesperetin on human cardiac Nav1.5 channels expressed in HEK 293 cells and on cardiac Na+ currents in human atrial myocytes were examined through whole-cell patch-clamp techniques. RESULTS: Nav1.5 currents were potently and reversibly suppressed in a concentration- and voltage-dependent manner by hesperetin, which exhibited an IC50 of 62.99 µmol/L. Hesperetin significantly and negatively shifted the voltage-dependent activation and inactivation curves. Hesperetin also markedly decelerated Nav1.5 current inactivation and slowed the recovery from Nav1.5 channel inactivation. The hesperetin-dependent blockage of Nav1.5 currents was frequency-dependent. Hesperetin also potently and reversibly inhibited Na+ current (INa) in human atrial myocytes, consistently with its effects on Nav1.5 currents in HEK 293 cells. CONCLUSION: Hesperetin is a potent inhibitor of INa in human atrial myocytes and Nav1.5 channels expressed in human embryonic kidney 293 cells. Hesperetin probably functions by blocking the open state and the inactivated state of these channels.

Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts.

AIM: Neferine is an isoquinoline alkaloid isolated from seed embryos of Nelumbo nucifera (Gaertn), which has a variety of biological activities. In this study we examined the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (I(to)) in rabbit heart, and on ex vivo electrophysiology of rabbit hearts. METHODS: Whole-cell Kv4.3 currents were recorded in HEK293 cells expressing human cardiac Kv4.3 channels using patch-clamp technique. Arterially perfused wedges of rabbit left ventricles (LV) were prepared, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG). RESULTS: Neferine (0.1-100 µmol/L) dose-dependently and reversibly inhibited Kv4.3 currents (the IC50 value was 8.437 µmol/L, and the maximal inhibition at 100 µmol/L was 44.12%). Neferine (10 µmol/L) caused a positive shift of the steady-state activation curve of Kv4.3 currents, and a negative shift of the steady-state inactivation curve. Furthermore, neferine (10 µmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation. Neferine-induced blocking of Kv4.3 currents was frequency-dependent. In arterially perfused wedges of rabbit LV, neferine (1, 3, and 10 µmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, and caused dramatic increase of APD10 at Epi sites. CONCLUSION: Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

High glucose enhances angiotensin-II-mediated peroxisome proliferation-activated receptor-gamma inactivation in human coronary artery endothelial cells.

Activation of the renin-angiotensin system plays an important role in the pathogenesis of vascular complications of hyperglycemia. Clinical studies have demonstrated that hypoglycemic effects of peroxisome proliferation-activated receptor-gamma (PPAR-gamma) activation is potentially associated with a significant decrease of cardiovascular disease events in diabetes patients. We assessed the effect of high glucose on the angiotensin II (Ang II), which induced the inactivation of PPAR-gamma and its signal pathways in human coronary artery endothelial cells (HCAECs). The expression of angiotensin II receptor I (AT1R) protein was analyzed by Western blot and knocked down using siRNA. PPAR-gamma activation was examined using a luminometer and a Dual Luciferase Reporter Assay System. Adhesion molecule expressions of HCAECs were measured using ELISA. Both high glucose and Ang II induced a progressive increase in AT1R protein expression on the HCAECs. Troglitazone, a PPAR-gamma activator, significantly increased the transcription activity of PPAR-gamma in HCAECs in vitro. However, activation of PPAR-gamma was significantly inhibited by high glucose and Ang II stimulation. Furthermore, silencing of AT1R expression was able to inhibit the inactivation of PPAR-gamma induced by Ang II and high glucose. Meanwhile, expression of proinflammatory adhesion molecules was increased by high glucose and Ang II in HCAECs, which is blocked by troglitazone and silencing of AT1R expression. These data strongly suggest high glucose enhanced Ang-II-mediated peroxisome proliferation-activated receptor-gamma inactivation and expression of proinflammatory adhesion molecules in human coronary artery endothelial cells.

Effect of beta2-adrenergic agonist clenbuterol on ischemia/reperfusion injury in isolated rat hearts and cardiomyocyte apoptosis induced by hydrogen peroxide.

AIM: To observe the effect of beta2-adrenergic agonist clenbuterol on ischemia/reperfusion (I/R) injury in isolated rat hearts and hydrogen peroxide (H2O2)-induced cardiomyocyte apoptosis. METHODS: Isolated rat hearts were subjected to 30 min global ischemia and 60 min reperfusion on a Langendorff apparatus. Cardiac function was evaluated by heart rate, left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure, maximal rise rate of left ventricular pressure [+dp/dt(max)], and the coronary effluent (CF). Lactate dehydrogenase (LDH) in the coronary effluent, malondialdehyde (MDA), superoxide dismutase (SOD), and Ca2+-ATPase activity in the cardiac tissue were measured using commercial kits. The apoptotic cardiomyocyte was detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay. Bax/Bcl-2 mRNA levels and the expression of caspase-3 were detected by RT-PCR and immunoblotting, respectively. Cultured newborn rat cardiomyocytes were preincubated with clenbuterol, and oxidative stress injury was induced by H2O2. Cell viability and cardiomyocyte apoptosis were evaluated by flow cytometry (FCM). RESULTS: In the isolated rat hearts after I/R injury, clenbuterol significantly improved diastolic function (LVEDP and CF) and Ca2+-ATPase activity. Treatment with clenbuterol increased SOD activity and decreased the MDA level and LDH release compared with the I/R group (P<0.05). Moreover, clenbuterol decreased apoptosis, which was associated with a reduction in TUNEL-positive cells, Bax/Bcl-2 mRNA, and caspase-3 expression. In H2O2-induced cardiomyocyte injury, clenbuterol increased cell viability and attenuated cardiomyocyte apoptosis. Pretreatment with ICI118551 (selective beta2-adrenergic antagonist) decreased these effects compared with the clenbuterol-treated group (P<0.05). CONCLUSION: Clenbuterol ameliorated ventricular diastolic function by enhancing Ca2+-ATPase activity and reduced oxidative stress and cardiac myocyte apoptosis in an experimental rat model of myocardium I/R. It decreased cardiomyocyte apoptosis induced by H2O2 in vitro. It plays a key role in the cardiac protection against myocardium I/R injury.

Vanilloid receptor TRPV1, sensory C-fibers, and activation of adventitial mast cells. A novel mechanism involved in adventitial inflammation.

The immunological mechanisms on adventitial inflammation has received much attention, while the contribution of nerves to adventitial inflammation has largely been ignored. Although the mechanism of initial chemotaxis of the adventitial inflammatory cells remains unknown, vascular nerves were frequently found in the inflammatory lesions of coronary adventitia and adventitial mast cells connect with sensory nerve fibers in atherosclerotic coronary arteries. The sensory nerves in contact with adventitial mast cells contained the neuropeptides SP and CGRP. These neuropeptides play an important role in the amplification of tissue injury by the increase of both vascular permeability and neutrophil recruitment, and the term ''neurogenic inflammation'' has been coined. Activation of adventitial mast cells, with ensuing release of vasoactive compounds, may cause vasoconstriction in atherosclerotic coronary segments. Therefore, we hypothesize that adventitial vanilloid receptor TRPV1 and sensory C-fibers may play a pistol role for adventitial inflammation.

The membrane permeable calcium chelator BAPTA-AM directly blocks human ether a-go-go-related gene potassium channels stably expressed in HEK 293 cells.

BAPTA-AM is a well-known membrane permeable Ca(2+) chelator. The present study found that BAPTA-AM rapidly and reversibly suppressed human ether a-go-go-related gene (hERG or Kv11.1) K(+) current, human Kv1.3 and human Kv1.5 channel currents stably expressed in HEK 293 cells, and the effects were not related to Ca(2+) chelation. The externally applied BAPTA-AM inhibited hERG channels in a concentration-dependent manner (IC(50): 1.3 microM). Blockade of hERG channels was dependent on channel opening, and tonic block was minimal. Steady-state activation V(0.5) of hERG channels was negatively shifted by 8.5 mV (from -3.7+/-2.8 of control to -12.2+/-3.1 mV, P<0.01), while inactivation V(0.5) was negatively shifted by 6.1 mV (from -37.9+/-2.0 mV of control to -44.0+/-1.6 mV, P<0.05) with application of 3 microM BAPTA-AM. The S6 mutant Y652A and the pore helix mutant S631A significantly attenuated blockade by BAPTA-AM at 10 microM causing profound blockade of wild-type hERG channels. In addition, BAPTA-AM inhibited hKv1.3 and hKv1.5 channels in a concentration-dependent manner (IC(50): 1.45 and 1.23 microM, respectively), and the blockade of these two types of channels was also dependent on channel opening. Moreover, EGTA-AM was found to be an open channel blocker of hERG, hKv1.3, hKv1.5 channels, though its efficacy is weaker than that of BAPTA-AM. These results indicate that the membrane permeable Ca(2+) chelator BAPTA-AM (also EGTA-AM) exerts an open channel blocking effect on hERG, hKv1.3 and hKv1.5 channels.

Raloxifene inhibits transient outward and ultra-rapid delayed rectifier potassium currents in human atrial myocytes.

The selective estrogen receptor modulator raloxifene is widely used in the treatment of postmenopausal osteoporosis, and has cardioprotective properties. However, effects of raloxifene on cardiac ion channels are unclear. The present study was designed to investigate the effects of raloxifene and beta-estradiol on transient outward and ultra-rapid delayed rectifier potassium currents (Ito1 and IKur) in human atrial myocytes with a whole cell patch-clamp technique. Ito1 was inhibited by raloxifene in a concentration-dependent manner with an IC50 of 0.9 microM. Raloxifene at 1 microM decreased Ito1 by 40.2+/-1.9% (at +50 mV, n=14, P<0.01 vs control). Time-dependent recovery from inactivation was slowed, and time to peak and time-dependent inactivation of Ito1 were significantly accelerated, while steady-state voltage dependent activation and inactivation of Ito1 were not affected by raloxifene. In addition, raloxifene remarkably suppressed IKur (IC50=0.7 microM). Raloxifene at 1 microM decreased IKur by 57.3+/-3.3% (at +50 mV, n=10, P<0.01 vs control). However, beta-estradiol inhibited Ito1 (IC50=10.3 microM) without affecting IKur. The inhibitory effects of raloxifene and beta-estradiol on Ito1 and/or IKur were unaffected by the estrogen receptor antagonist ICI 182,780. Our results indicate that raloxifene directly inhibits the human atrial repolarization potassium currents Ito1 and IKur. Whether raloxifene is beneficial for supraventricular arrhythmias remains to be studied.

Protective effect of non-mitogenic human acidic fibroblast growth factor on hepatocyte injury.

AIM: To study whether non-mitogenic human acidic fibroblast growth factor (nm-haFGF) has protective effects on H(2)O(2)-induced hepatocyte injury in vitro and CCl(4)-induced hepatocyte injury in vivo. METHODS: (i) HL-7702 hepatocytes were incubated with different concentrations of nm-haFGF for 12 h, and then the activity of lactate dehydrogenase (LDH) in culture medium was detected, and genomic DNA electrophoresis analysis was observed after being exposed to H(2)O(2) (8 mmol/L) for 4 h. Proximately, apoptotic rates and protein expressions of Bcl-2 and Bax of HL-7702 cell were detected after being exposed to H(2)O(2) (0.2 mmol/L) for 20 h. (ii) Being injected intraperitoneally with nm-haFGF, mice were treated with CCl(4) intraperitoneally to induce hepatic injury. Twenty-four hours later, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured and histopathologic changes were evaluated. RESULTS: (i) In vitro tests: LDH activities and apoptotic rates decreased, the protein expression of Bcl-2 increased and Baxdecreased in nm-haFGF-treated groups at the concentrations of 100 150 and 200 ng/mL, compared with that in the model control group, which was treated with H(2)O(2) alone. The genomic DNA remained nearly intact at the concentrations of 150 and 200 ng/mL. (ii) In vivo tests: serum ALT and AST in nm-haFGF-treated groups (10 mug/kg and 20 mug/kg) were much lower as compared to the model control group, which was treated with CCl(4) alone. Histological examination showed that nm-haFGF markedly ameliorated hepatocytes vacuolation, cloudy swelling and inflammatory cells infiltration induced by CCl(4). CONCLUSION: nm-haFGF had protective effects against H(2)O(2)-induced hepatocyte injury in vitro and CCl(4)-induced acute liver injury in vivo.

Adventitial inflammation: a possible pathogenic link to the instability of atherosclerotic plaque.

A variety of cells, including fibroblasts, mast cells, macrophages, and ganglionic cells, are present in coronary artery adventitia. In the infarct-related coronary arteries of myocardial infarction patients, the majority of mast cells are found in the outer layer of the adventitia. Neurogenic stimulation of mast cells in the adventitia of coronary arteries may release vasoactive compounds, such as histamine and leukotrienes, which can contribute to the complex neurohormonal response that leads to abnormal coronary vasoconstriction. Lymphocytes and bacteria are also present mainly in the adventitial layer. Chlamydia pneumoniae is directly involved in the development of adventitial and plaque inflammation (pan-arteritis), leading to plaque rupture. Adventitial O(2)(-) may also play an extensive role in the control of vascular tone. Therefore, adventitial inflammation may play a pivotal role for atherosclerotic lesion development and atheroma instability.

[Metabolic products and pathway of neferine in rat liver].

The present study utilized LC-MS and HPLC approaches to characterize the metabolites of neferine in rat liver after an oral administration of 20 mg x kg(-1), and investigated the involvement of CYP450 isoforms in the metabolism of neferine by their selective inhibitors in vitro, separately. In positive ionization mode, besides neferine, four metabolites (M1-M4) were detected. M2 (the major metabolite) and M4 were identified as liensinine and isoliensinine by comparison with reference substances. Moreover, according to the analysis of metabolic rule of parent drug (neferine), M1 and M3 may be desmethylliensinine and desmethyl-isoliensinine, respectively. Furthermore, the metabolism of neferine in rat liver microsomes showed that the percentage inhibition of the major metabolism (liensinine) formation was 80.5% by quinidine (10 micromol x L(-1), selective CYP2D1 inhibitor) and 25.7% by ketoconazole (1 micromol x L(-1), selective CYP3A1 inhibitor). Neferine was mainly metabolized by CYP2D1 or CYP3A1 to liensinine, isoliensinine, desmethyl-liensinine and desmethyl-isoliensinine.

[Relaxant effect of ethanol on isolated rabbit corpus cavernosum and its mechanism].

OBJECTIVE: To study the relaxant effect of ethanol on the isolated rabbit corpus cavernosum and its possible mechanism. METHODS: The tension of isolated smooth muscle strips was recorded by the platform physiological graphed, and the concentrations of cAMP and cGMP in the rabbit corpus cavernosum were measured by 125I radioimmunoassay. RESULTS: The 1.25% (V/V) ethanol significantly augmented the corporal relaxation induced by isoprenaline (10(-9) - 10(-5) mol/L). Ethanol-induced relaxation was inhibited by 100 micromol/L and 300 micromol/L SQ22536 (an adenylate cyclase inhibitor). Emax was depressed from (105.12 +/- 3.39) % to (97.00 +/- 2.57) % in the presence of 100 micromol/L SQ22536 or (91.09 +/- 2.42) % in the presence of 300 micromol/L SQ22536. EC50 was increased from (1.18 +/- 0.09)% (V/V) to (1.36 +/- 0.10) % in the presence of 100 micromol/L SQ22536 (P < 0.05) or (1.68 +/- 0.13) % (in the presence of 300 micromol/L SQ22536) (P < 0.05) respectively. Ethanol significantly elevated the level of cAMP but not that of cGMP in the isolated rabbit corpus cavernosum, and it also significantly enhanced the activity of the adenylate cyclase (AC) extracted from the rabbit corpus cavernosum in a dose-dependent manner. CONCLUSION: Ethanol has a relaxant effect on the isolated rabbit corpus cavernosum, which may be associated with the cAMP signaling pathway.

Platelet derived TGF-ß promotes cervical carcinoma cell growth by suppressing KLF6 expression.

Platelets in the primary tumor microenvironment play crucial roles in regulating tumor growth, metastasis, and angiogenesis, but the underlying mechanisms are unclear. Here, we show that platelet releasates exhibited a proliferative effect on HeLa cells, and this effect correlated with a reduction of KLF6 expression. After incubation with either washed human platelets or collagen-related peptide (CRP) activated platelet releasates, expression of KLF6 in the HeLa cervical tumor cell line was markedly reduced. However, no significant difference was observed between control HeLa cells and HeLa cells incubated with resuspended activated platelet pellet. Moreover, the platelets' promoting effect on HeLa cell growth was significantly abolished in KLF6 silenced HeLa cells. In addition, blocking TGF-ß signaling with SB431542, a TGF-ß receptor inhibitor, also counteracted the effect of platelets on proliferation and KLF6 expression in HeLa cells. From these findings, we conclude that platelet derived TGF-ß promotes proliferation of HeLa cells by decreasing the expression of KLF6. The discovery that KLF6 is a key target of platelet-derived TGF-ß signaling in HeLa cells identifies a potential new therapeutic target for the prevention and treatment of cervical carcinoma.

Platelet releasates promote the proliferation of hepatocellular carcinoma cells by suppressing the expression of KLF6.

Platelets in the primary tumor microenvironment play crucial roles in the regulation of tumor progression, but the mechanisms underlying are poorly understood. Here, we report that platelet releasates exerted a proliferative effect on hepatocellular carcinoma (HCC) cells both in vitro and in vivo. This effect depended on a reduction of KLF6 expression in HCC cells. After incubation with either platelets or platelet granule contents, SMMC.7721 and HepG2 cells exhibited significant increases in proliferation and decreases in apoptosis. However, no effect was observed when incubating cancer cells with resuspended activated platelet pellet which exhausted of releasates. Platelet releasates also increased the population of HCC cells in the S and G2/M phases of the cell cycle and reduced the cell population in the G0/G1 phase. Moreover, knocking down KLF6 expression significantly diminished the platelet-mediated enhancement of HCC growth. In addition, blocking TGF-ß signaling with the TGF-ß receptor inhibitor SB431542 counteracted the effect of platelets on KLF6 expression and proliferation of HCC cells. Based on these findings, we conclude that platelet releasates, especially TGF-ß, promote the proliferation of SMMC.7721 and HepG2 cells by decreasing expression of KLF6. This discovery identifies a potential new therapeutic target for the prevention and treatment of hepatocellular carcinoma.

Inhibitory effects of neferine on Nav1.5 channels expressed in HEK293 cells.

Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition (IC50) being 26.15 µmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 µmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.

Antiplatelet activity of chrysin via inhibiting platelet αIIbß3-mediated signaling pathway.

SCOPE: Propolis is thought to help prevent thrombotic and related cardiovascular diseases in humans. Chrysin, a bioflavonoids compound found in high levels in propolis and in honey, has been reported to possess antiplatelet activity. However, the mechanism by which it inhibits platelet function is unclear. METHODS AND RESULTS: The effects of chrysin on agonist-activated platelet-aggregation, granule-secretion, and integrin αIIbß3 activation were examined. Its effects on the phosphorylation of Akt, GSK3ß, MAPKs, and several proteins of the glycoprotein VI (GPVI) signaling pathway were also studied on collaged-activated platelets. In addition, human platelet spreading on immobilized fibrinogen was also tested. We found that chrysin dose dependently inhibited platelet aggregation and granule secretion induced by collagen, as well as platelet aggregation induced by ADP, thrombin, and U46619. Chrysin also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen. Biochemical analysis revealed that chrysin inhibited collagen-induced activation of Syk, PLCγ2, PKC, as well as the phosphorylation of Akt and ERK1/2. Additionally, chrysin attenuated phosphorylation of molecules such as FcγRIIa, FAK, Akt, and GSK3ß in platelet spreading on immobilized fibrinogen. CONCLUSIONS: Our findings indicate that chrysin suppresses not only integrin αIIbß3-mediated "inside-out" signaling, but also the "outside-in" signal transmission. This implies that chrysin may represent a potential candidate for an antiplatelet agent.

Inhibitory effects of hesperetin on Kv1.5 potassium channels stably expressed in HEK 293 cells and ultra-rapid delayed rectifier K(+) current in human atrial myocytes.

In the present study, the inhibitory effects of hesperetin (HSP) on human cardiac Kv1.5 channels expressed in HEK 293 cells and the ultra-rapid delayed rectifier K(+) current (Ikur) in human atrial myocytes were examined by using the whole-cell configuration of the patch-clamp techniques. We found that hesperetin rapidly and reversibly suppressed human Kv1.5 current in a concentration dependent manner with a half-maximal inhibition (IC50) of 23.15 µΜ with a Hill coefficient of 0.89. The current was maximally diminished about 71.36% at a concentration of 300µM hesperetin. Hesperetin significantly positive shifted the steady-state activation curve of Kv1.5, while negative shifted the steady-state inactivation curve. Hesperetin also accelerated the inactivation and markedly slowed the recovery from the inactivation of Kv1.5 currents. Block of Kv1.5 currents by hesperetin was in a frequency dependent manner. However, inclusion of 30µM hesperetin in pipette solution produced no effect on Kv1.5 channel current, while the current were remarkable and reversibly inhibited by extracellular application of 30µM hesperetin. We also found that hesperetin potently and reversibly inhibited the ultra-repaid delayed K(+) current (Ikur) in human atrial myocytes, which is in consistent with the effects of hesperetin on Kv1.5 currents in HEK 293 cells. In conclusion, hesperetin is a potent inhibitor of Ikur (which is encoded by Kv1.5), with blockade probably due to blocking of both open state and inactivated state channels from outside of the cell.

Tacrolimus inhibits vasoconstriction by increasing Ca(2+) sparks in rat aorta.

The present study attempted to test a novel hypothesis that Ca(2+) sparks play an important role in arterial relaxation induced by tacrolimus. Recorded with confocal laser scanning microscopy, tacrolimus (10 µmol/L) increased the frequency of Ca(2+) sparks, which could be reversed by ryanodine (10 µmol/L). Electrophysiological experiments revealed that tacrolimus (10 µmol/L) increased the large-conductance Ca(2+)-activated K(+) currents (BKCa) in rat aortic vascular smooth muscle cells (AVSMCs), which could be blocked by ryanodine (10 µmol/L). Furthermore, tacrolimus (10 and 50 µmol/L) reduced the contractile force induced by norepinephrine (NE) or KCl in aortic vascular smooth muscle in a concentration-dependent manner, which could be also significantly attenuated by iberiotoxin (100 nmol/L) and ryanodine (10 µmol/L) respectively. In conclusion, tacrolimus could indirectly activate BKCa currents by increasing Ca(2+) sparks released from ryanodine receptors, which inhibited the NE- or KCl-induced contraction in rat aorta.

Endogenous nitric oxide mediates lipoteichoic acid induced preconditioning on reoxygenation injury of cultured human coronary artery endothelial cells.

AIM: To explore the effects of lipoteichoic acid (LTA) induced delayed preconditioning (PC) on hypoxia-reoxygenation (H/R) injury of cultured human coronary artery endothelial cells (HCAECs), and to investigate the potential role of endogenous nitric oxide (NO) participated in the protective mechanism. METHODS: HCAECs were incubated for 2 h in a hypoxic atmosphere and reoxygenated for 4 h in a normoxic atmosphere. The delayed PC was induced by pretreatment with LTA (30 or 300 microg x L(-1)) for 4 h before 24 h recovery. The extent of cellular injury after reoxygenation was assessed by the percentage of cellular injury with Trypan blue exclusion and by the amount of lactate dehydrogenase (LDH) in culture media. The NO level of the culture media was measured spectrophotometrically. Furthermore, HCAECs were exposed to 300 microg x L(-1) of LTA for 4 h, and to detect the expression of eNOS mRNA by RT-PCR method after cells were recovered from different points. RESULTS: LTA pretreatment significantly decreased the percentage of the killed cell and the concentration of LDH in media. Also, LTA pretreatment obviously raised the concentrations of NO in culture media. The protective effects of LTA were abrogated by pretreatment with N-monomethyl-L-arginine (L-NMMA). Moreover, the expression of eNOS mRNA was significantly upregulated after HCAECs exposure to LTA for 4 h following 2 h or 4 h recovery. CONCLUSION: LTA could induce the delayed protection against H/R induced endothelial injury and dysfunction of cultured HCAECs. NO produced by eNOS acts initially as a trigger and subsequently as a mediator of delayed PC.

[Protective effect of non-mitogenic haFGF on retinal injury induced by N-methyl-N-nitrosourea in Sprague-Dawley rats].

AIM: To study the effect of non-mitogenic human acidic fibroblast growth factor (nm-haFGF) on retinal injury induced by N-methyl-N-nitrosourea (MNU) in Sprague-Dawley rats and its mechanism. METHODS: Female rats of 50-days-old were injected with MNU (60 mg x kg(-1)) intraperitoneally, and three doses of nm-haFGF (1.25 microg, 2.5 microg and 5 microg in one eye of each rat) were injected, separately, into vitreous body of one eye of each rat twice a day at 0 and 12 h after MNU treatment. 24 h later, apoptotic index of photoreceptor cells was detected by TUNEL labeling and the expressions of Bcl-2 and Bax were analyzed by Western blotting. At the 7th day, retinal injury was evaluated based on retinal thickness. RESULTS: Compared with model group, apoptotic index of photoreceptor cells was significantly reduced in nm-haFGF groups at the dose of 1.25 microg and 2.5 microg in one eye of each rat at 24 h, and the total retinal thickness as well as the outer retinal thickness markedly increased 7 days after MNU, respectively. The expressions of Bcl-2 increased and that of Bax decreased adversely after being injected with different doses of nm-haFGF. CONCLUSION: nm-haFGF partially suppressed retinal injury induced by MNU in Sprague-Dawley rats. The mechanism could be related to up-regulation of Bcl-2 and down-regulation of Bax.

[Effects of berberine on cyclic GMP and cyclic AMP levels in rabbit corpus cavernosum in vitro].

OBJECTIVE: To further investigate the action mechanisms of berberine (Ber), and assess the effects of Ber on the in vitro formation of cGMP and cAMP in the isolated rabbit corpus cavernosum. METHODS: Isolated segments of the rabbit corpus cavernosum were exposed to different concentrations of Ber, and, the dosage-dependent accumulations of cGMP and cAMP were determined in the tissue samples by means of 125I radioimmunoassay. Responses of the isolated tissue preparations to Ber were compared with those obtained with the reference compound sildenafil (Sil). RESULTS: Ber increased cGMP concentrations directly (P < 0.05). In the presence of sodium nitroprusside (SNP), a stimulatory agent of cGMP, both Ber and Sil increased cGMP with increasing dosage (P < 0.01), the EC, values being 1.32 and 0.67 micromol/L respectively. With the same concentration, neither Ber nor Sil influenced the cAMP level significantly (P > 0.05). In the presence of PGE1, a stimulator of cAMP, Ber and Sil also raised the cAMP level concentration (P < 0.01 ), the EC, values being 4.90 (Ber) and 6.53 (Sil) micromol/L respectively. CONCLUSION: Ber can increase cGMP and cAMP concentrations in the corpus cavernosum smooth muscles, which may contribute to its action of relaxing corpus cavernosum smooth muscles.