OBJECTIVETo explore the acute effects of testosterone at the physiological level on PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells (VSMCs).

METHODSVSMCs from the thoracic aorta of male Sprague-Dawley rats were cultured using the explant method. The subconfluent VSMCs were incubated with serum-free medium for 24 hours to obtain quiescent non-dividing cells and then treated with the indicated agents. For the measurement of [Ca2+]i, the VSMCs were loaded with fura-2. Changes of [Ca2+]i were determined ratiometrically with a Nikon TE-2000E system.

RESULTSThe resting level of [Ca2+]i was around 100 nmol/L in the VSMCs. Exposing cells to perfusate containing 10 micromol/L PGF2alpha triggered an immediate and transient peak in [Ca2+]i, which gradually decreased afterwards. Interference at the peak with the physiological concentration (40 nmol/L) of testosterone significantly decreased the peak-to-baseline time of [Ca2+]i, compared with ethanol vehicle (104.9 +/- 27.0 s vs 153.5 +/- 40.4 s, P < 0.01). Pretreatment with testosterone at 40 nmol/L for 2 minutes also reduced the peak-to-baseline time of [Ca2+]i significantly in comparison with the ethanol control (120.6 +/- 32.0 s vs 151.4 +/- 27.4 s, P < 0.01), but it had no significant effect on the peak level of PGF2alpha-induced intracellular Ca2+ (390.0 +/- 126.0 nmol/L vs 403.4 +/- 160.7 nmol/L, P > 0.05).

CONCLUSIONTestosterone at physiological concentration inhibits PGF2alpha-induced Ca2+ fluxes, probably via receptor-operated calcium channels by a non-genomic mechanism in VSMCs, which may be involved in the vasodilatory effect of testosterone.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Dinoprost ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Myocytes, Smooth Muscle ; metabolism ; Rats ; Rats, Sprague-Dawley ; Testosterone ; metabolism ; physiology

AIMTo explore the effects of sinomenine(Sin) on intracellular free calcium ([Ca2+]i) and the activity of PKC (protein kinase C) of the cultured aortic vascular smooth muscle cells (VSMC) during ischemia and hypoxia.

METHODSThe effect of Sin on changes in [Ca2+]i were determined in cultured rabbit VSMC after exposure to high K+, norepinephrine (NE) and caffeine (Caf). Fluorescent Ca2+ -indicater fura-2/AM was used. The effects of Sin were compared with that of verapamil (Ver). The hypoxia model was made, then the activity of PKC was measured by y scintillation counting instrument.

RESULTSSin (10 x 10(-6) mol x L(-1), 3 x 10(-5) mol x L(-1) 10(-4) mol x L(-1)) inhibited the elevation of [Ca2+]i induced by high K+ -depolarization in a concentration dependent manner. In addition, Sin inhibited the elevation of [Ca2+]i induced by NE in the presence of extracellular Ca2+. In the absence of extracellular Ca2+, Sin (3 x 10(-5) mol.L(-1)) also had no blocking effect on the NE-induced [Ca2+]i increase. It was found that the activity of PKC treated with Sin in VSMC cytoplasm and cell membrane in normal condition increased, the activity of PKC in cytoplasm in ischemia and hypoxia situation increased, but the activity of PKC in cell membrane decreased. When VSMC was treated with Sin, the activity of PKC in cytoplasm decreased and that of cell membrane increased.

CONCLUSIONThe results suggest that Sin might decrease the[Ca2+] i of VSMC by blocking both VDC and ROC, could regulate the PKC activities induced by ischemia and hypoxia.

Animals ; Aorta ; cytology ; drug effects ; Calcium ; metabolism ; Cell Hypoxia ; Cells, Cultured ; Cytoplasm ; metabolism ; Morphinans ; pharmacology ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; physiology ; Protein Kinase C ; metabolism ; Rabbits

Proliferation of vascular smooth muscle cells (VSMCs) is often accompanied by changes in intracellular actin distribution. The changes are controlled by the signal transduction pathways of protein kinase C/mitogenic activated protein kinase (PKC-MAPK), but the mechanism is unclear. In order to study the effect of insulin on the intracellular signal transduction (PKC-MAPK) probably involved in the modulation of proliferation and redistribution of actins in the VSMCs, the DNA synthesis, MAPK activities and its gene expression, and the redistribution of intracellular actins were investigated in the isolated VSMCs of SHR pretreated with PKC inhibitor and/or insulin, respectively. We found that insulin treatment resulted in proliferation of the VSMCs and an increase in [(3)H] TdR incorporation. Meanwhile, the activities and expression of MAPK increased significantly compared to the control group. These effects of insulin were blocked by PKC inhibitor. In addition, insulin caused a redistribution of the intracellular actins in VSMCs, which was also inhibited by PKC inhibitor. It is, therefore, suggested that these effects of insulin on VSMCs proliferation and distribution of the intracellular actins may be mediated by the MAPK signal transduction pathway.
Actins ; metabolism ; Animals ; Cell Division ; drug effects ; In Vitro Techniques ; Insulin ; pharmacology ; Mitogen-Activated Protein Kinases ; physiology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; enzymology ; metabolism ; Protein Kinase C ; physiology ; Rats ; Rats, Inbred SHR ; Tissue Distribution

The present study was designed to investigate the electrophysiological characteristics of rat conduit pulmonary artery smooth muscle cells (PASMCs) and the response to acute hypoxia. PASMCs of the 1st to 2nd order branches in the conduit pulmonary arteries were obtained by enzymatic isolation. The PASMCs were divided into acute hypoxia preconditioned group and normoxia group. Hypoxia solutions were achieved by bubbling with 5% CO2 plus 95% N2 for at least 30 min before cell perfusion. Potassium currents were compared between these two groups using whole-cell patch clamp technique. The total outward current of PASMCs was measured under normoxia condition when iBTX [specific blocking agent of large conductance Ca-activated K(+) (BK(Ca)) channel] and 4-AP [specific blocking agent of delayed rectifier K(+) (K(DR)) channel] were added consequently into bath solution. PASMCs were classified into three types according to their size, shape and electrophysiological characteristics. Type I cells are the smallest with spindle shape, smooth surface and discrete perinuclear bulge. Type II cells show the biggest size with banana-like appearance. Type III cells have the similar size with type I, and present intermediary shape between type I and type II. iBTX had little effect on the total outward current in type I cells, while 4-AP almost completely blocked it. Most of the total outward current in type II cells was inhibited by iBTX, and the remaining was sensitive to 4-AP. In type III cells, the total outward current was sensitive to both iBTX and 4-AP. Acute hypoxia reduced the current in all three types of cells: (1614.8+/-62.5) pA to (892.4+/-33.6) pA for type I cells (P<0.01); (438.3+/-42.8) pA to (277.5+/-44.7) pA for type II cells (P<0.01); (1 042.0+/-37.2) pA to (613.6+/-23.8) pA for type III (P<0.01), and raised the resting membrane potentials (E(m)) in all these three types of cells: (-41.6+/-1.6) mV to (-18.6+/-1.5) mV (P<0.01), (-42.3+/-3.8) mV to (-30.6+/-3.0) mV (P<0.01), (-43.3+/-1.6) mV to (-28.4+/-1.4) mV (P<0.01), for type I, II, III cells, respectively. These results suggest that acute hypoxia suppresses the potassium current and improves the E(m) in PASMCs. These effects may be involved in the modulation of constriction/relaxation of conduit artery under acute hypoxia. Different distribution of K(DR) and BK(Ca) channels in these three types of PASMCs might account for their different constriction/relaxation response to acute hypoxia.
4-Aminopyridine ; pharmacology ; Animals ; Calcium ; metabolism ; Cell Hypoxia ; Male ; Membrane Potentials ; drug effects ; Muscle, Smooth, Vascular ; cytology ; physiology ; Myocytes, Smooth Muscle ; physiology ; Peptides ; pharmacology ; Potassium Channels ; physiology ; Pulmonary Artery ; cytology ; physiology ; Rats ; Rats, Sprague-Dawley

AIMTo study whether store-operated Ca2+ channel (SOC) is present in rat colonic smooth muscle cells.

METHODSIntracellular Ca2+ ([Ca2+]i) changes induced by thapsigargin- or caffeine-activated SOC channel were measured in enzymatically dissociated rat colonic smooth muscle cells with the fluorescent indicator Fura-2/AM.

RESULTSIn the absence of external Ca2+ , the sarco-endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (1 micromol/L) and ryanodine receptor (RyR) activator caffeine both transiently elevated [Ca2+]i from (68.32 +/- 3.43) nmol/L to (240.85 +/- 12.65 ) nmol/L, (481.25 +/- 34.77) nmol/L. A subsequent reintroduction of Ca2+ into the extracellular solution resulted in [Ca2+]i further elevated to (457.55 +/- 19.80) nmol/L, (1005.93 +/- 54.62) nmol/L; (643.88 +/- 34.65) nmol/L, (920.16 +/- 43.25) nmol/L, respectively. And the elevated response was blocked by lanthanum (1 mmol/L), but was insensitive to L-type voltage calcium channels blocker verapamil and membrane depolarization.

CONCLUSIONSOC activated by store depletion are present in rat colonic smooth muscle cells. And we further prove the existence of such Ca2+ channels in excitable cells.

Animals ; Caffeine ; pharmacology ; Calcium ; metabolism ; Calcium Channels ; physiology ; Colon ; cytology ; Fura-2 ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Rats ; Rats, Wistar ; Thapsigargin ; pharmacology

OBJECTIVETo investigate the effect of salidroside on hypoxia-induced apoptosis of corpus cavernosum smooth muscle cells (CCSMCs) in rats.

METHODSRat CCSMCs were cultured in vitro by the enzyme digestion method and identified by immunofluorescent staining of anti-alpha-SMA and anti-Desmin. The non-toxic dose of salidroside was determined by MTT assay. Low-oxygen mixed gas (1% O2, 5% CO2, and 94% N2) was piped into a modular incubator chamber to induce hypoxia. The CCSMCs were divided into a normal, a hypoxia, and a 32 microg/mL salidroside intervention group. The apoptosis of the CCSMCs was detected by flow cytometry and the expression of the caspase-3 protein determined by Western blot.

RESULTSThe majority of the CCSMCs were positive for alpha-SMA and Desmin at immunofluorescent staining. Salidroside at < 32 microg/ml produced no obvious toxicity to CCSMCs. Compared with the normal control group, the rates of early and late apoptosis of CCSMCs were both increased significantly in the hypoxia group ([12.77 +/-1.41]% vs [18.69 +/- 1.29]%, P < 0.01 and [14.63 +/- 2.00]% vs [21.03 +/- 1.530]% , P < 0.05). Western blot showed a markedly increased expression of cleaved caspase-3 (P < 0.01). Intervention with 32 microg/ml salidroside significantly reduced hypoxia-induced early apoptosis of CCSMCs ([13.46% +/- 1.87]%, P < 0.01) and decreased the expression of cleaved caspase-3 (P < 0.01).

CONCLUSIONSalidroside can reduce the expression of cleaved caspase-3 and inhibit hypoxia-induced apoptosis of CCSMCs in rats.

Animals ; Apoptosis ; drug effects ; physiology ; Caspase 3 ; metabolism ; Cell Hypoxia ; physiology ; Cells, Cultured ; Glucosides ; pharmacology ; Humans ; Male ; Myocytes, Smooth Muscle ; cytology ; drug effects ; enzymology ; Penis ; cytology ; drug effects ; Phenols ; pharmacology ; Rats

To discuss the effect of puerarin (Pue) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) and discuss whether its mechanism is achieved by regulating reactive oxygen. PASMCs of primarily cultured rats (2-5 generations) were selected in the experiment. MTT, Western blot, FCM and DCFH-DA were used to observe Pue's effect the proliferation of PASMCs. The Western blot was adopted to detect whether ROS participated in Pue's effect in inhibiting PASMC proliferation. The PASMCs were divided into five groups: the normoxia group, the hypoxia group, the hypoxia + Pue group, the hypoxia + Pue + Rotenone group and the hypoxia + Rotenone group, with Rotenone as the ROS blocker. According to the results, under the conditions of normoxia, Pue had no effect on the PASMC proliferation; But, under the conditions of hypoxia, it could inhibit the PASMC proliferation; Under the conditions of normoxia and hypoxia, Pue had no effect on the expression of the tumor necrosis factor-α (TNF-α) among PASMCs, could down-regulate the expression of hypoxia-induced cell cycle protein Cyclin A and proliferative nuclear antigen (PCNA). DCFH-DA proved Pue could reverse ROS rise caused by hypoxia. Both Rotenone and Pue could inhibit the up-regulated expressions of HIF-1α, Cyclin A, PCNA caused by anoxia, with a synergistic effect. The results suggested that Pue could inhibit the hypoxia-induced PASMC proliferation. Its mechanism may be achieved by regulating ROS.
Animals ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hypoxia ; pathology ; Isoflavones ; pharmacology ; Male ; Myocytes, Smooth Muscle ; drug effects ; physiology ; Proliferating Cell Nuclear Antigen ; analysis ; Pulmonary Artery ; cytology ; drug effects ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism

OBJECTIVETo observe the effect of emodin on motility signal transduction and calcium ion in colonic smooth muscle cells (SMC) in rats with bacterial peritonitis caused multiple organ dysfunction syndrome (MODS).

METHODSObservation was conducted in colon of MODS model rats on (1) effects of emodin on the contraction of muscular strip and cells of colonic smooth muscle, and influences of specific myoglobulin light chain kinase inhibitor (ML-7) and selective proteinkinase C inhibitor (Calphostin C) on these effects; and (2) effect of emodin on calcium ion in SMC.

RESULTSEmodin could directly contract the muscular strip and cells of smooth muscle; ML-7 and Calphostine could inhibit these contractile action to some extent. Under MODS condition, emodin could still increase the intracellular calcium ion concentration; this effect could be inhibited by heparin (inosamine triphosphate receptor inhibitor IP3 and ryanodine receptor inhibitor in MODS model but the calcium chelator EGTA and nifedipine (the specific cell membrane voltage dependent calcium channel blocker) showed no influence on it.

CONCLUSIONEmodin could directly contract the colonic smooth muscle in MODS model rats, which is mediated by raise the signal path MLCK of calcium ion and the PKCa path for increase calcium sensibility. The mechanism of increasing calcium ion is mainly through IP3 and RyR the two calcium ion channel receptor in the sarcoplasm.

Animals ; Calcium ; metabolism ; Colon ; drug effects ; Emodin ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Gastrointestinal Motility ; drug effects ; In Vitro Techniques ; Male ; Multiple Organ Failure ; physiopathology ; Muscle Contraction ; drug effects ; Muscle, Smooth ; cytology ; drug effects ; Myocytes, Smooth Muscle ; drug effects ; physiology ; Rats ; Rats, Wistar ; Signal Transduction

PURPOSE: This study was designed to investigate the change of peroxisome proliferator-activated receptor gamma (PPARgamma) after the infection of the human coronary artery smooth muscle cells (HCSMCs) with Chlamydia pneumoniae (C. pneumoniae) and the effect of PPARgamma agonist on the expression of PPARgamma of C. pneumoniae-infected HCSMCs. MATERIALS AND METHODS: To determine the effect of PPARgamma agonist on the proliferation of C. pneumoniae-infected HCSMCs, rosiglitazone at various concentrations was applied 1 hour before inoculation of HCSMCs. RESULTS: The expression of PPARgamma mRNA in HCSMCs increased from 3 hours after C. pneumoniae infection and reached that of noninfected HCSMCs at 24 hours (p < 0.05). The expression of PPARgamma protein in HCSMCs also increased from 3 hours after C. pneumoniae and persisted until 24 hours as compared with that of noninfected HCSMCs (p < 0.05). The pretreatment of HCSMCs with rosiglitazone followed by the infection with C. pneumoniae augmented the expression of PPARgamma mRNA and protein (p < 0.05) and decreased cell proliferation. CONCLUSION: Our results showed that the expression of PPARgamma increases in response to C. pneumoniae infection and rosiglitazone further augmented the expression of PPARgamma. It is suggested that rosiglitazone could ameliorate the chronic inflammation in the vessel wall induced by C. pneumoniae by augmenting PPARgamma expression.
Blotting, Western ; Cell Line ; Cell Proliferation/drug effects ; Chlamydophila pneumoniae/growth & development/*physiology ; Gene Expression Regulation/drug effects ; Humans ; Muscle, Smooth, Vascular/cytology/drug effects/metabolism ; Myocytes, Smooth Muscle/drug effects/*metabolism/microbiology ; PPAR gamma/genetics/*metabolism ; RNA, Messenger/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Thiazolidinediones/pharmacology

Airway remodeling is an important pathological feature of asthma and the basis of severe asthma. Proliferation of airway smooth muscle cells (ASMCs) is a major contributor to airway remodeling. As an important Ca(2+) channel, transient receptor potential vanilloid 1 (TRPV1) plays the key role in the cell pathological and physiological processes. This study investigated the expression and activity of TRPV1 channel, and further clarified the effect of TRPV1 channel on the ASMCs proliferation and apoptosis in order to provide the scientific basis to treat asthmatic airway remodeling in clinical practice. Immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of TRPV1 in rat ASMCs. Intracellular Ca(2+) was detected using the single cell confocal fluorescence microscopy measurement loaded with Fluo-4/AM. The cell cycles were observed by flow cytometry. MTT assay and Hoechst 33258 staining were used to detect the proliferation and apoptosis of ASMCs in rats respectively. The data showed that: (1) TRPV1 channel was present in rat ASMCs. (2) TRPV1 channel agonist, capsaicin, increased the Ca(2+) influx in a concentration-dependent manner (EC50=284.3±58 nmol/L). TRPV1 channel antagonist, capsazepine, inhibited Ca(2+) influx in rat ASMCs. (3) Capsaicin significantly increased the percentage of S+G2M ASMCs and the absorbance of MTT assay. Capsazepine had the opposite effect. (4) Capsaicin significantly inhibited the apoptosis, whereas capsazepine had the opposite effect. These results suggest that TRPV1 is present and mediates Ca(2+) influx in rat ASMCs. TRPV1 activity stimulates proliferation of ASMCs in rats.
Animals ; Antipruritics ; pharmacology ; Apoptosis ; physiology ; Bronchi ; cytology ; metabolism ; Calcium Signaling ; drug effects ; physiology ; Capsaicin ; analogs & derivatives ; pharmacology ; Cell Proliferation ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; TRPV Cation Channels ; antagonists & inhibitors ; metabolism