ABSTRACT
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.
Subject(s)
Humans , Benzylisoquinolines , Gene Expression Regulation , HEK293 Cells , Patch-Clamp Techniques , QuinidineABSTRACT
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.
Subject(s)
Animals , Male , Rats , Aorta , Cell Biology , Metabolism , Physiology , Calcium Signaling , Cells, Cultured , Large-Conductance Calcium-Activated Potassium Channels , Metabolism , Muscle, Smooth, Vascular , Metabolism , Physiology , Myocytes, Smooth Muscle , Metabolism , Norepinephrine , Pharmacology , Rats, Sprague-Dawley , Ryanodine , Pharmacology , Tacrolimus , Pharmacology , VasoconstrictionABSTRACT
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.
ABSTRACT
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.
Subject(s)
Animals , Male , Rats , Administration, Oral , Alcohol Oxidoreductases , Aryl Hydrocarbon Hydroxylases , Benzylisoquinolines , Metabolism , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP3A , Cytochrome P450 Family 2 , Isoquinolines , Metabolism , Ketoconazole , Pharmacology , Microsomes, Liver , Metabolism , Nelumbo , Chemistry , Phenols , Metabolism , Plants, Medicinal , Chemistry , Quinidine , Pharmacology , Seeds , Chemistry , Spectrometry, Mass, Electrospray IonizationABSTRACT
<p><b>OBJECTIVE</b>To study the relaxant effect of ethanol on the isolated rabbit corpus cavernosum and its possible mechanism.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Ethanol has a relaxant effect on the isolated rabbit corpus cavernosum, which may be associated with the cAMP signaling pathway.</p>
Subject(s)
Animals , Male , Rabbits , Central Nervous System Depressants , Pharmacology , Cyclic AMP , Metabolism , Ethanol , Pharmacology , In Vitro Techniques , Muscle Relaxation , Penile Erection , Physiology , Penis , Physiology , Signal TransductionABSTRACT
<p><b>AIM</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Subject(s)
Animals , Female , Rats , Apoptosis , Fibroblast Growth Factor 1 , Genetics , Pharmacology , Methylnitrosourea , Photoreceptor Cells, Vertebrate , Pathology , Protective Agents , Pharmacology , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Random Allocation , Rats, Sprague-Dawley , Retina , Pathology , Retinitis Pigmentosa , Metabolism , Pathology , bcl-2-Associated X Protein , MetabolismABSTRACT
<p><b>AIM</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Subject(s)
Adult , Female , Humans , Cell Death , Cell Hypoxia , Cells, Cultured , Coronary Vessels , Cell Biology , Metabolism , Endothelial Cells , Cell Biology , Metabolism , Ischemic Preconditioning, Myocardial , L-Lactate Dehydrogenase , Metabolism , Lipopolysaccharides , Pharmacology , Myocardial Reperfusion Injury , Metabolism , Nitric Oxide , Metabolism , Nitric Oxide Synthase Type III , Genetics , RNA, Messenger , Genetics , Staphylococcus aureus , Chemistry , Teichoic Acids , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Subject(s)
Animals , Male , Rabbits , Berberine , Pharmacology , Cyclic AMP , Metabolism , Cyclic GMP , Metabolism , Dose-Response Relationship, Drug , In Vitro Techniques , Muscle, Smooth , Metabolism , Penis , Metabolism , RadioimmunoassayABSTRACT
<p><b>AIM</b>To investigate the effect of capsaicin on IA and IK in cultured rat trigeminal ganglion (TG) neurons.</p><p><b>METHODS</b>Whole-cell patch clamp technique was used to record the IA and IK before and after capsaicin perfusion at different concentrations.</p><p><b>RESULTS</b>In capsaicin-sensitive (CS) neurons, capsaicin was shown to selectively inhibit IA in dose-dependent manner, the IC50 was 0.99 micromol x L(-1). Yet capsaicin showed no inhibitory effect on IK, capsaicin (10 micromol x L(-1)) only slightly inhibited IK by 13.2%. In capsaicin-insensitive (CIS) neurons, capsaicin (1 micromol x L(-1)) showed no significant inhibitory effect on IA and IK, capsaicin (10 micromol x L(-1)) only slightly inhibited IA and IK by 16.8% and 15.3%, respectively. Neither 1 micromol x L(-1) nor 10 micromol x L(-1) capsaicin showed effect on the G-V curve of IA and IK.</p><p><b>CONCLUSION</b>Capsaicin was found to selectively inhibit the IA current in CS neurons, and this effect may contribute to hyperalgesia when capsaicin was first used.</p>