AIMTo investigate the effects of blockers of the three kinds of potassium channels: voltage-dependent K+ channel(KV), calcium-activated K+ channel(KCa) and ATP-sensitive K+ channel(KATP), on the proliferation of rat bronchial smooth muscle cells (BSMCs).

METHODSThe effects of three kinds of potassium channel blockers on the proliferation of BSMCs were detected by MTT method, immunocytochemistry staining and flow-cytometry. Their effects on the dynamic changes of Ca2+ concentration in BSMCs were investigated by the fluorophotometry of Fura-2/AM.

RESULTSThe KV blocker 4-aminopyridine (4-AP) was shown to significantly increase the expression of proliferating cell nucleus antigen in cultured rat BSMCs (P < 0.01), but the KCa blocker tetraethylammonium (TEA) and KATP blocker glibenclamide(Glib) did not show such effect (P > 0.05). 4-AP was found to significantly increase the optical density value of the cultured BSMCs (P < 0.01) by MTT method and the numbers of S + G2M BSMCs(P < 0.05) by flow-cytometry. TEA and Glib did not show such effects. 4-AP significantly increased the Ca2+ concentration in cultured BSMCs(P < 0.01). TEA and Glib did not show such effects.

CONCLUSIONThis result suggests that inhibition of KV activity can increase intracellular Ca2+ and proliferation of rat BSMCs, but inhibition of KCa and KATP showed no effect.

4-Aminopyridine ; pharmacology ; Animals ; Bronchi ; cytology ; Calcium ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; Glyburide ; pharmacology ; Muscle, Smooth ; drug effects ; metabolism ; Potassium Channel Blockers ; pharmacology ; Potassium Channels, Calcium-Activated ; antagonists & inhibitors ; Potassium Channels, Voltage-Gated ; antagonists & inhibitors ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tetraethylammonium ; pharmacology

The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.
Cell Separation ; methods ; Culture Media ; Pinellia ; physiology ; Protoplasts ; physiology ; Regeneration

Amino Acid Sequence ; Base Sequence ; Genes, Bacterial ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Staphylococcus epidermidis ; enzymology ; genetics ; beta-Lactamases ; chemistry ; genetics

BACKGROUNDIt has been demonstrated that only 10%-20% cigarette smokers finally suffer chronic obstructive pulmonary disease (COPD). The underlying mechanism of development remains uncertain so far. Nitric oxide (NO) has been found to be closely associated with the pathogenesis of COPD, the alteration of NO synthase (NOS) expression need to be revealed. The study aimed to investigate the alterations of NOS isoforms expressions between smokers with and without COPD, which might be helpful for identifying the susceptibility of smokers developing into COPD.

METHODSPeripheral lung tissues were obtained from 10 nonsmoker control subjects, 15 non-COPD smokers, and 15 smokers with COPD. Neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) mRNA and protein levels were measured in each sample by using real-time polymerase chain reaction and Western blotting.

RESULTSINOS mRNA was significantly increased in patients with COPD compared with nonsmokers and smokers with normal lung function (P < 0.001, P = 0.001, respectively). iNOS protein was also higher in COPD patients than nonsmokers and smokers with normal lung function (P < 0.01 and P = 0.01, respectively). However, expressions of nNOS and eNOS did not differ among nonsmokers, smokers with and without COPD. Furthermore, there was a negative correlation between iNOS protein level and lung function parameters forced expiratory volume in 1 s (FEV₁) (% predicted) (r = -0.549, P = 0.001) and FEV₁/forced vital capacity (%, r = -0.535, P = 0.001).

CONCLUSIONSThe expression of iNOS significantly increased in smokers with COPD compared with that in nonsmokers or smokers without COPD. The results suggest that iNOS might be involved in the pathogenesis of COPD, and may be a potential marker to identify the smokers who have more liability to suffer COPD.

Adult ; Aged ; Blotting, Western ; Female ; Humans ; Isoenzymes ; genetics ; metabolism ; Lung ; enzymology ; pathology ; Male ; Middle Aged ; Nitric Oxide Synthase ; genetics ; metabolism ; Pulmonary Disease, Chronic Obstructive ; enzymology ; pathology ; Real-Time Polymerase Chain Reaction

The purpose of this study was to explore the effect of proteasome inhibitor, bortezomib (Bzb), on osteoblast in pathologic status of myeloma bone disease. The myeloma bone disease was modeled by co-culture of mouse myeloma cell RPMI8226 with osteoblast line MC-3T3E1 from mouse calvaria, and intervenient culture of supernatant. The inhibitory effect of Bzb on proliferation of MC-3T3E1 assayed by modified MTT method, the apoptosis of MC-3T3E1 cells was determined by flow cytometry with Annexin V/PI staining, the expressions of osteoblast markers, Runx2/cbfa1, osteocalcin (OCN) and osterix (OSX) in MC-3T3E1 treated with Bzb were detected by RT-PCR and Western blot respectively. Experiments were divided into 3 group: single cultured, co-cultured and supernatant-interveniently cultured groups. The results showed the Bzb in higher concentration inhibited proliferation of MC-3T3E1 cells in a dose-dependent manner, with the IC(50) of 38.1 nmol/L for 48 hours, the Bzb in low concentration (5 nmol/L) did not show the inhibitory effect on proliferation of MC-3T3E1 in single cultured group (p>0.10), but could decrease apoptotic rate of MC-3T3E1 by 32.5% and 24.6% respectively in cocultured and supernatant-interveniently cultured groups, moreover increased the expression of osteoblast-related gene OSX, OCN mRNA and protein (p<0.05), while no obvious change of Runx2/cbfa1 expression was observed (p>0.05). It is concluded that the proteasome inhibitor, Bzb, in low concentration promotes the activity of osteoblast internal mechanisms, and prevents the apoptosis of osteoblasts induced by myeloma cells. In addition, it can up-regulate transcription and expression of osteoblast markers related to Runx2/cbfa1 path way, thus may protect osteoblasts in myeloma bone disease.
3T3 Cells ; Animals ; Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Mice ; Multiple Myeloma ; pathology ; Pyrazines ; pharmacology

Animals ; Antineoplastic Agents ; administration & dosage ; Chemoembolization, Therapeutic ; methods ; Female ; Hepatic Artery ; Liver Neoplasms, Experimental ; therapy ; Oligopeptides ; administration & dosage ; Rats ; Rats, Wistar

BACKGROUNDAlthough it is recognized that bronchial smooth muscle cells (BSMCs) play a key role in airway remodeling during chronic asthma, it is not well understood how BSMCs exert their inflammatory functions. The extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is an important signaling pathway in chronic asthma, but its influence on secretion by BSMCs has not been well-studied. We investigated the impact of ERK1/2 signaling pathway on secretion by BSMCs in a rat model of chronic asthma in this study.

METHODSTo create a rat model of chronic asthma, Wistar rats underwent ovalbumim (OVA) injection and eight weeks of inhalation. BSMCs were isolated and cultured in vitro. Epidermal growth factor, PD98059 and ERK1/2 antisense oligonucleotide were used to explore the role of ERK1/2 signaling pathway. The expression of P-ERK1/2 (phospho-ERK1/2) in BSMCs was analyzed by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Secretion of BSMCs was detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSPhospho-ERK1/2 expression was increased in BSMCs of chronic asthmatic rats compared with the controls. PD98059 inhibited expression of phospho-ERK1/2 protein, while treatment with an antisense oligonucleotide inhibited the expression of P-ERK1/2 mRNA and protein. BSMCs obtained from the chronic asthma group secreted significantly greater quantities of growth factors (transforming growth factor (TGF)-beta(1), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF)), cytokines (regulated upon activation, normal T cell-expressed and secreted (RANTES) and eotaxin), and extracellular matrix (fibronectin and collagen I) compared with normal controls. Epidermal growth factor stimulated secretion in both groups, but the response of the chronic asthma group was more intense. Both PD98059 and antisense oligonucleotide suppressed secretion by BSMCs in chronic ashmatic rats. Antisense oligonucleotide reduced the level of RANTES nearly to that of normal controls, while PD98059 could not.

CONCLUSIONThese results suggest that ERK1/2 signaling pathway may play an important role in the augmented secretion of BSMCs in chronic asthmatic rats, and ERK1/2 antisense oligonucleotide effectively inhibits the process.

Animals ; Asthma ; metabolism ; Bronchi ; secretion ; Chemokine CCL5 ; secretion ; Chronic Disease ; Disease Models, Animal ; MAP Kinase Signaling System ; physiology ; Male ; Mitogen-Activated Protein Kinase 1 ; physiology ; Mitogen-Activated Protein Kinase 3 ; physiology ; Myocytes, Smooth Muscle ; secretion ; Rats ; Rats, Wistar ; Transforming Growth Factor beta1 ; secretion ; Vascular Endothelial Growth Factor A ; secretion

To investigate the regulatory effect of extracellular signal-regulated kinase (ERK) signaling pathway on airway smooth muscle cell (ASMC) proliferation in chronic asthmatic rats, the rat model of chronic asthma was established, and ERK agonist epidermal growth factor (EGF) and inhibitor PD98059 were used in the cell culture. ASMC proliferation was examined by flow cytometry analysis, methyl thiazolyl tetrazolium (MTT) colorimetric assay, [(3)H]-thymidine (TdR) incorporation and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of ERK mRNA, ERK protein, phosphorylated ERK1/2 (p-ERK1/2) protein were observed by RT-PCR and Western blot. The results showed that in chronic asthmatic group, compared with that in the control group, the percentage of cells at G(0)/G(1) phase was significantly decreased and the percentage of cells at S+G(2)/M phase was significantly increased. Absorbance (A(490)), DNA synthesis and the expression of PCNA protein in ASMCs in chronic asthmatic group were significantly increased. The expressions of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMCs in chronic asthmatic group were significantly increased compared with those in the control group. After treatment with PD98059, the percentage of cells at S+G(2)/M phase, A(490), DNA synthesis and the expression of PCNA protein in ASMCs in chronic asthmatic group were significantly decreased; the expressions of ERK mRNA, ERK1/2 protein, p-ERK1/2 protein and the activation ratio of ERK in ASMCs in chronic asthmatic group were significantly decreased compared with those in the control group. After treatment with EGF, the percentage of cells at S+G(2)/M phase, A(490), DNA synthesis and the expression of PCNA protein in ASMCs in chronic asthmatic group were significantly increased compared with those before treatment; and PD98059 markedly inhibited the effect of EGF. These results suggest that the endogenous proliferation activity of ASMCs in chronic asthmatic rats significantly increases compared with that in the control rats, and ERK1/2 participates in this process. The ERK signaling pathway might play an important role in regulating ASMC proliferation, leading to asthmatic airway remodeling.
Animals ; Asthma ; enzymology ; pathology ; Bronchi ; pathology ; Cell Proliferation ; Cells, Cultured ; Chronic Disease ; Colorimetry ; Enzyme Activation ; Extracellular Signal-Regulated MAP Kinases ; analysis ; genetics ; physiology ; Flow Cytometry ; Myocytes, Smooth Muscle ; pathology ; Rats

This work was designed to explore the role of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in migration of bronchial smooth muscle cells (BSMCs) of chronic asthmatic rats. To make chronic asthma model, Wistar rats underwent ovabumin (OVA) injection and eight-week inhalation. BSMCs were cultured in vitro. The expression of ERK1/2 in BSMCs was analyzed by immunocytochemistry, Western blot and RT-PCR. Migration of BSMCs was detected by both plate test and Boyden cell test. Results showed: (1) With Western blot technique, the ratio of p-ERK1/2 to total ERK1/2 in chronic asthmatic group was obviously higher than that in the control group (0.55 +/- 0.05 vs 0.48 +/- 0.04, n=10, P<0.01). (2) With RT-PCR, the relative A values of ERK1 and ERK2 mRNA in airways of chronic asthmatic rats were 1.83 +/- 0.24 and 1.07 +/- 0.11, respectively, which were significantly increased compared with that in the control group (0.58 +/- 0.14 and 0.51 +/- 0.12, n=10, P<0.01). (3) In plate test, the migration of BSMCs of chronic asthmatic rats was 2.9 times of that in the control group and reached 5.0 times by epidermal growth factor (EGF) stimulation, but decreased to 1.7 times by 30 mumol/L PD98059. (4) In Boyden cell test, the migration of BSMCs of chronic asthmatic rats was 1.9 times of that in the control group, and reached 3.1 times by EGF stimulation, but decreased to 1.45 times by 30 mumol/L PD98059. Our results indicate that the migration ability of BSMCs of chronic asthmatic rats increases, and ERK1/2 signaling pathway may play an important role in this process.
Animals ; Asthma ; chemically induced ; physiopathology ; Bronchi ; pathology ; Cell Movement ; physiology ; Cells, Cultured ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; physiology ; Mitogen-Activated Protein Kinase 3 ; metabolism ; physiology ; Myocytes, Smooth Muscle ; pathology ; Ovalbumin ; Rats ; Rats, Wistar ; Signal Transduction ; physiology