AIMTo investigate the role and mechanism of the bradykinin(BK) B1 receptor in the antiproliferative effects of the angiotensin-converting enzyme inhibitor (ACEI) captopril on rat cardiac fibroblasts (CFs) treated with angiotensin II (Ang II).

METHODSNeonatal rat cardiac fibroblasts were randomly treated with Ang II, captopril, B2 receptor antagonist (icatibant) or B1 receptor antagonist (des-Arg10, Leu9-kallidin). Thiazolyl blue (MTT) and flow cytometry (FCM) were used to evaluate cell number and cell cycle, respectively. Nitric oxide (NO) and intracellular cGMP level were measured by colorimetry and radioimmunoassay.

RESULTSAfter incubating the fibroblasts with 10(-7) mol/L Ang II for 48 hours, the percentage of CFs in the S stage and the value of MTT A490 nm were significantly increased (P < 0.01 vs control), and this increase was inhibited by 10(-5) mol/L captopril; however, NO and cGMP level were significantly higher than with Ang II alone (P < 0.01). 10(-5) mol/L icatibant attenuated the effects of captopril, which were blunted further by dual blockade of both B1 and B12.

CONCLUSIONActing via the B2 receptor, BK contributes to the antiproliferative effects of ACEI on CFs. In the absence of the B2 receptor, the B1 receptor may assume some of the functions of the B2 receptor and contribute to inhibition of CFs proliferation by ACEI.

Animals ; Bradykinin B1 Receptor Antagonists ; pharmacology ; Captopril ; pharmacology ; Cell Proliferation ; drug effects ; Heart Ventricles ; Myocytes, Cardiac ; cytology ; Rats ; Rats, Sprague-Dawley ; Receptor, Bradykinin B1 ; metabolism

OBJECTIVETo investigate the underlying mechanism for the selective modulation of the permeability of blood-tumor barrier (BTB) by small dose of bradykinin (BK).

METHODSC6 glioma cells were treated with BK, and changes of intracellular nitric oxide (NO) and intracellular calcium level were measured with fluorescent spectrophotometer.

RESULTSThe initial application of BK easily triggered extracellular calcium influx, which resulted in intracellular calcium store release in C6 glioma cells. The above mechanism was also named ryanodine mediated calcium induced calcium release (CICR). We also detected a long-lasting intracellular NO elevation in C6 glioma cells upon BK treatment. Further study showed that ryanodine mediated CICR contributed greatly to the secondary NO elevation induced by BK treatment.

CONCLUSIONThese results suggested that BK triggered CICR in C6 glioma cells and the associated NO generation might be the underlying mechanism for the selective modulation of BTB permeability by BK.

Animals ; Bradykinin ; pharmacology ; Calcium ; metabolism ; Cell Line, Tumor ; Glioma ; pathology ; Intracellular Fluid ; drug effects ; Nitric Oxide ; metabolism ; Rats ; Ryanodine ; pharmacology ; Spectrometry, Fluorescence ; methods ; Time Factors

OBJECTIVETo evaluate the inhibitory effect and related mechanism of bradykinin on mechanical stress induced myocardial hypertrophy.

METHODSNeonatal rat cardiomyocytes were isolated and cultured in silicon plates. All cardiomyocytes were randomly divided into three groups: control group, mechanical stretch group (mechanical stretch of silicon plates to 120% for 30 min) and mechanical stretch plus bradykinin group (1×10(-8) mol/L for 24 h before stretch). The protein synthesis and surface area of cardiomyocytes were detected by [(3)H] leucine incorporation and immunofluorescence of α-MHC, respectively. mRNA expression of atrial natriuretic peptide (ANP) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) was detected by real time-PCR, the phosphorylation of calcineurin (CaN), the expression of Angiotensin II receptor 1 (AT1R) and angiotensin converting enzyme (ACE)by Western blot.

RESULTSThe surface area of cardiomyocytes of mechanical stretch group [(973 ± 103) µm(2)] was significantly enlarged than in control group [(312 ± 29) µm(2)] and this effect could be partly attenuated by bradykinin [(603 ± 74) µm(2), all P < 0.05]. Mechanical stretch also significantly increased the protein synthesis, up-regulated the expression of ANP and decreased the expression of SERCA2, and these effects could be partly reversed by pretreatment with bradykinin. Moreover, bradykinin partly abolished the mechanical stretch-induced increases in CaN phosphorylation, up-regulation of AT1R but preserved the expression of ACE.

CONCLUSIONSBradykinin significantly attenuates mechanical stretch-induced myocardial hypertrophy through inhibition of Ca(2+)/CaN pathway.

Animals ; Bradykinin ; pharmacology ; Calcineurin ; metabolism ; Calcium ; metabolism ; Cell Enlargement ; drug effects ; Cells, Cultured ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Rats ; Stress, Mechanical

OBJECTIVETo investigate the effects of bradykinin (BK) on the proliferation, apoptosis and differentiation of human keratinocyte (HKC) and the underlying mechanisms.

METHODSHKCs were cultured together with 1 x 10(-4) - 1 x 10(-9) mol/L of BK. With methyl thiotetrazole (MTT) and trypan blue staining it was shown that the BK in dose of 1 x 10(-4) mol/L possessed most powerful inhibitory effect, and the survival rate of HKC was 69.3%. Therefore, BK was employed in the dose of 1 x 10(-4) mol/L in the following studies. When the growth of HKCs reached the logarithmic phase, BK in the concentration of 1 x 10(-4) mol/L was added, and it was categorized as the test group (E). HKCs without BK served as the control group (C). The cell cycle and apoptosis were detected by flow cytometry after being cultured for 24 and 48 hours. The change in intracellular calcium [Ca(2+)](i) was determined by means of laser scanning confocal microscopy with calcium fluorescence probe Fluo-3/AM technique. The expression of HKC differentiation labeling protein keratin10 (K10) and involucrin were detected with Strept Avidin-Biotin Complex (SABC) immunocytochemical assay.

RESULTSThe cell ratio in G0/G1 phase in E group increased by 34.57% while in S phase decreased by 58.91% in reference to that in C group. The G1/S phase switching of HKCs was obviously inhibited by BK, and apoptosis was stimulated (apoptotic rate of 15.34% in E group vs 5.60% in C group, P < 0.05). The [Ca(2+)](i) increased transiently in HKCs by 163.0% in E group after 3 minutes of BK activation and decreased thereafter in reference to that in C group. The K10 expression in HKC was down-regulated in E group with positive cell rate of 2.20%, which was lower than that of C group (6.89%, P < 0.05).

CONCLUSIONThe cell cycle process of HKC could be inhibited by high concentration of BK with increased apoptosis and an increase in [Ca(2+)](i), which might be the mechanism of inhibition of growth of HKC in vitro. Furthermore, the epithelial regeneration and HKC differentiation can also be inhibited by BK.

Apoptosis ; drug effects ; Bradykinin ; pharmacology ; Cell Cycle ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Humans ; Keratinocytes ; cytology ; metabolism ; Keratins ; metabolism

OBJECTIVETo investigate whether cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1) are involved in the bradykinin-induced delayed protection.

METHODSCardiac contractility, lactate dehydrogenase (LDH) and infarct area were analyzed in isolated rat hearts undergoing ischemia-reperfusion injury induced by Langendorff method.

RESULTConscious rats received bradykinin (40 microg/kg), and the isolated hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion 24 h later. Bradykinin pretreatment would improve post-ischemic performance, and reduced the release of LDH and infarct size. COX-2 inhibitor celecoxib (3 mg/kg) abolished bradykinin-induced protection, leading to poorer myocardial performance, release of more LDH and larger infarct sizes. Administration of HO-1 inhibitor ZnPP IX(20 microg/kg) before bradykinin partially abrogated the delayed protection. Pretreatment with the mitochondrial ATP sensitive potassium channel(mitoK(ATP) antagonist 5-HD before or 24 h after bradykinin administration also abolished the effect of protection.

CONCLUSIONThe results indicate that activation of HO-1 and COX-2 might be involved in the delayed cardioprotection evoked by bradykinin, and mitoK(ATP) channel may serve as both a trigger and a mediator in the cardioprotection.

Animals ; Bradykinin ; pharmacology ; Celecoxib ; Cyclooxygenase 2 ; metabolism ; Cyclooxygenase Inhibitors ; pharmacology ; Heme Oxygenase-1 ; metabolism ; In Vitro Techniques ; Ischemic Preconditioning, Myocardial ; methods ; Male ; Myocardial Reperfusion Injury ; enzymology ; prevention & control ; Potassium Channels ; physiology ; Pyrazoles ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sulfonamides ; pharmacology

AIMTo establish a new amino acid structure descriptor that can be applied to polypeptide QSAR studies.

METHODSThe new amino acid structure descriptor c-scales were derived from a principal components analysis of 167 amino acid structure descriptor indexes by theoretic calculation. The c1,c2,c3-scales were related to 3D structural features of amino acid such as steric, electronic and conformation properties etc. G/PLS regression method was used to find out the relationship between the c-scales and the biological activity and developed QSAR models of the polypeptides.

RESULTSUsing the established method, we developed accordingly QSAR models of Bitter tasting dipeptide, ACE inhibitors and bradykinin-potentiating pentapeptides and their r2 and XV-r2 were more than 0.70.

CONCLUSIONThe c-scales can quantitatively describe the 3D structural features of any coded and non-coded amino acid and can be used to establish a QSAR model of good predictability.

Amino Acid Sequence ; Amino Acids ; chemistry ; Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Bradykinin ; pharmacology ; Least-Squares Analysis ; Peptides ; chemistry ; pharmacology ; Principal Component Analysis ; Protein Conformation ; Quantitative Structure-Activity Relationship ; Structure-Activity Relationship

AIMTo investigate the effects of bradykinin on voltage-dependent sodium channel currents in rat dorsal root ganglion neurons (DRG).

METHODSWhole-cell patch clamp technique was used to determine sodium channel current.

RESULTSBradykinin at 0.01 - 10.0 micromol/L dose dependently increased the frequency of repetitive firing of DRG. Bradykinin at 0.01 - 10.0 micromol/L dose dependently enhanced the TTX-R sodium current, and had no effect on TTX-S sodium current.

CONCLUSIONMechanism underlying the inflammation induced by bradykinin is related to the TTX-R sodium channel.

Action Potentials ; drug effects ; Animals ; Bradykinin ; pharmacology ; Ganglia, Spinal ; drug effects ; physiology ; Membrane Potentials ; drug effects ; Neurons ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Sodium Channels ; drug effects ; physiology

AIMTo study the action of RMP-7 and its derivative on transporting liposome across the blood brain barrier (BBB) into the brain.

METHODSRMP-7 and DSPE-PEG-NHS [[1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly (ethylene-glycol)]-hydroxy succinamide]] were conjugated together in mild condition and MALDI-TOF-MS (Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry) was used to determine their molecular ratio. An in vitro BBB model was established and used to determine in vitro bioactivity of RMP-7 and its derivative. The fluorescence of brain slices and the Evens Blue (EB) concentration in the brain, liver, spleen, lung and kidney of each group were used to evaluate the in vivo bioactivity of RMP-7 and its derivative on transporting liposome across the BBB.

RESULTSThe average molecular weight (MW) of the reaction product was 4,900, while those of DSPE-PEG-NHS and RMP-7 were 3,224 and 1,098. The results demonstrated that RMP-7 was conjugated to DSPE-PEG-NHS at the molecular ratio of 1:1, so the product was DSPE-PEG-RMP-7. RMP-7 and DSPE-PEG-RMP-7 was shown to improve the transporting of peralcohol enzyme across the in vitro BBB model 2-3 times higher than the peralcohol enzyme only. DSPE-PEG-RMP-7 could facilitate the transporting of EB into brain more easily than RMP-7.

CONCLUSIONBoth RMP-7 and DSPE-PEG-RMP-7 could facilitate the transporting of liposome across the BBB, especially DSPE-PEG-RMP-7.

Animals ; Biological Transport ; Blood-Brain Barrier ; drug effects ; Bradykinin ; analogs & derivatives ; pharmacology ; Brain ; metabolism ; Drug Carriers ; Drug Delivery Systems ; Evans Blue ; pharmacokinetics ; Liposomes ; pharmacokinetics ; Phosphatidylethanolamines ; Polyethylene Glycols ; Rats ; Rats, Sprague-Dawley ; Tissue Distribution

AIMTo study the permeability of nerve growth factor (NGF) liposomes (NGF-L, NGF-SSL, NGF-SSL-T) on the blood-brain barrier (BBB) model and the distribution in vivo, and analyze the correlation between the results in vitro and in vivo.

METHODSThe BBB model in vitro was established by using mouse brain microvascullar endothelial cell, and the model was applied to study the permeability of NGF liposomes. The distribution of NGF of each group was studied by 125I labeled and SDS-PAGE method.

RESULTSThe highest encapsulation proportion was 34%, and the mean size of NGF liposomes was below 100 nm. The permeability of NGF liposomes on in vitro BBB model showed that the liposome could promote NGF to transport across the BBB, the permeability of NGF-SSL-T was the highest. The distribution in the brain showed in an order of NGF concentration NGF-SSL-T > NGF-SSL + RMP-7 > NGF-SSL > NGF-L. There was a close relationship between P(e) (permeability coefficient on in vitro BBB model) and BUI (brain uptake constant in vivo).

CONCLUSIONLiposomes can promote NGF to transport across the BBB, and the transporting ability BBB of NGF-SSL-T which RMP-7 incorporated into the surface of NGF liposomes is the best.

Animals ; Biological Transport ; drug effects ; Blood-Brain Barrier ; Bradykinin ; analogs & derivatives ; pharmacology ; Brain ; metabolism ; Cell Membrane Permeability ; Drug Delivery Systems ; Endothelial Cells ; cytology ; Liposomes ; Male ; Mice ; Nerve Growth Factor ; administration & dosage ; pharmacokinetics ; Particle Size ; Rats ; Tissue Distribution

Diverse signaling pathways have been proposed to regulate store-operated calcium entry (SOCE) in a wide variety of cell types. However, it still needs to be determined if all of these known pathways operate in a single cell type. In this study, we examined involvement of various signaling molecules in SOCE using human fibroblast cells (HSWP). Bradykinin (BK)-stimulated Ca2+ entry, previously shown to be via SOCE, is enhanced by the addition of vanadate, an inhibitor of tyrosine phosphatases. Furthermore, SOCE is regulated by cytochrome P-450, as demonstrated by the fact that the products of cytochrome P-450 activity (14,15 EET) stimulated SOCE while econazole, an inhibitor of cytochrome P450, suppressed BK-stimulated Ca2+ entry. In contrast, Ca2+ entry was unaffected by the guanylate cyclase inhibitor LY83583, or the membrane permeant cyclic GMP analog 8-bromo-cyclic GMP (8-Br-cGMP). Neither nitric oxide donors nor phorbol esters affected BK-stimulated Ca2+ entry. SOCE in HSWP cells is primarily regulated by tyrosine phosphorylation and the cytochrome P-450 pathway, but not by cyclic GMP, nitric oxide, or protein kinase C. Thus, multiple pathways do operate in a single cell type leading to the activation of Ca2+ entry and some of these signaling pathways are more prominently involved in regulating calcium entry in different cell types.
Vanadates/pharmacology ; Tetradecanoylphorbol Acetate/pharmacology ; Protein-Tyrosine-Phosphatase/*metabolism ; Phosphotyrosine/metabolism ; Phosphorylation/drug effects ; Nitric Oxide/metabolism ; Humans ; Fibroblasts ; Epidermal Growth Factor/pharmacology ; Enzyme Inhibitors/pharmacology ; Econazole/pharmacology ; Cytochrome P-450 Enzyme System/antagonists & inhibitors/*metabolism ; Cyclic GMP/analogs & derivatives/metabolism ; Cells, Cultured ; Calcium Channels/*metabolism ; Calcium/metabolism ; Bradykinin/pharmacology