The aim of this study was to investigate the inhibitory effect and the underlying mechanism of ethacrynic acid (EA) on the contraction in mice. BL-420S force measuring system was used to measure the tension of mouse tracheal rings. The whole cell patch clamp technique was utilized to record the channel currents of airway smooth muscle (ASM) cells. The calcium imaging system was used to determine the intracellular Ca concentration ([Ca]) in ASM cells. The results showed that EA significantly inhibited the high K (80 mmol/L) and acetylcholine (ACh, 100 µmol/L)-induced contraction of mouse tracheal rings in a dose-dependent manner. The maximal relaxation percentages were (97.02 ± 1.56)% and (85.21 ± 0.03)%, and the median effective concentrations were (40.28 ± 2.20) μmol/L and (56.22 ± 7.62) μmol/L, respectively. EA decreased the K and ACh-induced elevation of [Ca] from 0.40 ± 0.04 to 0.16 ± 0.01 and from 0.50 ± 0.01 to 0.39 ± 0.01, respectively. In addition, EA inhibited L-type voltage-dependent calcium channel (LVDCC) and store-operated calcium channel (SOCC) currents in ASM cells, and Ca influx. Moreover, EA decreased the resistance of the respiratory system (Rrs) in vivo in mice. These results indicated that EA inhibits LVDCC and SOCC, which results in termination of Ca influx and decreases of [Ca], leading to relaxation of ASM. Taken together, EA might be a potential bronchodilator.
Animals ; Calcium ; metabolism ; Calcium Channels, L-Type ; Enzyme Inhibitors ; pharmacology ; Ethacrynic Acid ; pharmacology ; Mice ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; Respiratory System ; cytology ; drug effects

OBJECTIVETo investigate whether the methanol extract of Berberis amurensis Rupr. (BAR) augments penile erection using in vitro and in vivo experiments.

METHODSThe ex vivo study used corpus cavernosum strips prepared from adult male New Zealand White rabbits. In in vivo studies for intracavernous pressure (ICP), blood pressure, mean arterial pressure (MAP), and increase of peak ICP were continuously monitored during electrical stimulation of Sprague-Dawley rats.

RESULTSPreconstricted with phenylephrine (PE) in isolated endotheliumintact rabbit corus cavernosum, BAR relaxed penile smooth muscle in a dose-dependent manner, which was inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one, a soluble guanylyl cclase inhibitor. BAR significantly relaxed penile smooth muscles dose-dependently in ex vivo, and this was inhibited by pretreatment with L-NAME H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one. BAR-induced relaxation was significantly attenuated by pretreatment with tetraethylammonium (TEA, P<0.01), a nonselective K channel blocker, 4-aminopyridine (4-AP, P<0.01), a voltage-dependent K channel blocker, and charybdotoxin (P<0.01), a large and intermediate conductance Ca sensitive-K channel blocker, respectively. BAR induced an increase in peak ICP, ICP/MAP ratio and area under the curve dose dependently.

CONCLUSIONBAR augments penile erection via the nitric oxide/cyclic guanosine monophosphate system and Ca sensitive-K (BK and IK) channels in the corpus cavernosum.

Animals ; Area Under Curve ; Berberis ; chemistry ; Blood Pressure ; drug effects ; Cyclic GMP ; metabolism ; Epoprostenol ; pharmacology ; In Vitro Techniques ; Indomethacin ; pharmacology ; Male ; Models, Biological ; Muscle Relaxation ; drug effects ; Muscle, Smooth ; drug effects ; physiology ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; metabolism ; Penile Erection ; drug effects ; Phenylephrine ; pharmacology ; Plant Extracts ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; metabolism ; Pressure ; Rabbits

OBJECTIVE: To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS). METHODS: VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected. RESULTS: Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01). CONCLUSIONS Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.
Actins ; metabolism ; Animals ; Calcium-Binding Proteins ; metabolism ; Cell Dedifferentiation ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Homocysteine ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Rosuvastatin Calcium ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; X-Box Binding Protein 1 ; metabolism

Calpain activation contributes to hyperglycemia-induced endothelial dysfunction and apoptosis. This study was designed to investigate the role of calpain inhibition in improving diabetic erectile dysfunction (ED) in mice. Thirty-eight-week-old male C57BL/6J mice were divided into three groups: (1) nondiabetic control group, (2) diabetic mice + vehicle group, and (3) diabetic mice + MDL28170 (an inhibitor of calpain) group. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg kg^-1 body weight for 5 consecutive days. Thirteen weeks later, diabetic mice were treated with MDL28170 or vehicle for 4 weeks. The erectile function was assessed by electrical stimulation of the cavernous nerve. Penile tissues were collected for measurement of calpain activity and the endothelial nitric oxide synthase (eNOS)-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining was used to evaluate apoptosis. Caspase-3 expression and activity were also measured to determine apoptosis. Our results showed that erectile function was enhanced by MDL28170 treatment in diabetic mice compared with the vehicle diabetic group. No differences in calpain-1 and calpain-2 expressions were observed among the three groups. However, calpain activity was increased in the diabetic group and reduced by MDL28170. The eNOS-NO-cGMP pathway was upregulated by MDL28170 treatment in diabetic mice. Additionally, MDL28170 could attenuate apoptosis and increase the endothelium and smooth muscle levels in corpus cavernosum. Inhibition of calpain could improve erectile function, probably by upregulating the eNOS-NO-cGMP pathway and reducing apoptosis.
Animals ; Apoptosis/drug effects* ; Calpain/antagonists & inhibitors* ; Cyclic GMP/biosynthesis* ; Diabetes Complications/drug therapy* ; Diabetes Mellitus, Experimental/complications* ; Dipeptides/therapeutic use* ; Endothelium/metabolism* ; Enzyme Inhibitors/therapeutic use* ; Erectile Dysfunction/etiology* ; In Situ Nick-End Labeling ; Male ; Mice ; Mice, Inbred C57BL ; Muscle, Smooth/metabolism* ; Nitric Oxide Synthase Type III/biosynthesis* ; Penis/enzymology* ; Up-Regulation

OBJECTIVETo study the therapeutic mechanism of Zhizhu Pill (ZP) for treating functional dyspepsia (FD) rats.

METHODSTotally 30 ten-day-old male rats were randomly divided into the normal control group (n =10) and the model group (n = 20). The FD rat model was induced using gastric administration of 0.1% iodoacetamide (IA) combined tail clamping. The model was evaluated when rats were 8-week old. Successfully modeled rats were randomly divided into the model group (n = 10) and the ZP group (n = 10). Rats in the normal group and the model group were administered with normal saline by gastrogavage, while those in the ZP group were administered with ZP Decoction (2 mL/100 g) by gastrogavage. All medication lasted for 7 successive days. The contractile activity in in vitro longitudinal gastric muscle was recorded using Power Lab biological signal collecting system. The expression of growth hormone secretagogue receptor (GHSR) in stomach of FD rats was detected using Western blot and immunohistochemistry (IHC).

RESULTSCompared with the normal group, average frequencies of gastric contraction and changing rates of amplitude obviously decreased in the model group (P < 0.05). Results of Western blot and IHC showed that the expression of GHSR decreased in the model group (P < 0.01). Compared with the model group, average frequencies of gastric contraction and changing rates of amplitude obviously increased in the ZP group (P < 0.05). Results of Western blot and IHC showed that the expression of GHSR increased in the ZP group (P < 0.01).

CONCLUSIONZP could promote the gastric motility in FD rats induced by gastric administration of IA combined tail clamping, and its mechanism might be related to up-regulating GHSR protein level.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Dyspepsia ; drug therapy ; Gastrointestinal Motility ; Male ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; metabolism ; Random Allocation ; Rats ; Receptors, Ghrelin ; metabolism

BACKGROUNDClarifying the mechanisms underlying vascular smooth muscle cell (VSMC) proliferation is important for the prevention and treatment of vascular remodeling and the reverse of hyperplastic lesions. Previous research has shown that the gaseous signaling molecule sulfur dioxide (SO2) inhibits VSMC proliferation, but the mechanism for the inhibition of the angiotensin II (AngII)-induced VSMC proliferation by SO2has not been fully elucidated. This study was designed to investigate if SO2inhibited VSMC proliferation in mice with hypertension induced by AngII.

METHODSThirty-six male C57 mice were randomly divided into control, AngII, and AngII + SO2groups. Mice in AngII group and AngII + SO2group received a capsule-type AngII pump implanted under the skin of the back at a slow-release dose of 1000 ng·kg-1·min-1. In addition, mice in AngII + SO2received intraperitoneal injections of SO2donor. Arterial blood pressure of tail artery was determined. The thickness of the aorta was measured by elastic fiber staining, and proliferating cell nuclear antigen (PCNA) and phosphorylated-extracellular signal-regulated kinase (P-ERK) were detected in aortic tissues. The concentration of SO2 in serum and aortic tissue homogenate supernatant was measured using high-performance liquid chromatography with fluorescence determination. In the in vitro study, VSMC of A7R5 cell lines was divided into six groups: control, AngII, AngII + SO2, PD98059 (an inhibitor of ERK phosphorylation), AngII + PD98059, and AngII + SO2 + PD98059. Expression of PCNA, ERK, and P-ERK was determined by Western blotting.

RESULTSIn animal experiment, compared with the control group, AngII markedly increased blood pressure (P < 0.01) and thickened the aortic wall in mice (P < 0.05) with an increase in the expression of PCNA (P < 0.05). SO2, however, reduced the systemic hypertension and the wall thickness induced by AngII (P < 0.05). It inhibited the increased expression of PCNA and P-ERK induced by AngII (P < 0.05). In cell experiment, PD98059, an ERK phosphorylation inhibitor, blocked the inhibitory effect of SO2on VSMC proliferation (P < 0.05).

CONCLUSIONSERK signaling is involved in the mechanisms by which SO2inhibits VSMC proliferation in AngII-induced hypertensive mice via ERK signaling.

Angiotensin II ; pharmacology ; Animals ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hypertension ; chemically induced ; drug therapy ; Male ; Mice ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Signal Transduction ; drug effects ; Sulfur Dioxide ; therapeutic use

OBJECTIVETo evaluate the effect of ulinastatin on hypoxia-induced phenotype modulation of pulmonary artery smooth muscle cells (PASMCs) and explore the underlying mechanism.

METHODSCultured PASMCs from SD rats were exposed to normoxic condition, normoxia with ulinastatin treatment, hypoxia, or hypoxia with ulinastatin treatment. After 24 h of exposures, the cells were examined for SM-α-actin and caplonin expressions with immunofluorescence assay and for cell migration with CCK-8 andH-TdR assays. Western blotting was used for detecting the expressions of PPAR-γ in the cells, and PPAR-γ-responsive firefly luciferase reporter was employed for measuring the transcriptional activity of PPAR-γ. The PPAR-γ inhibitor GW9662 was used to explore the mechanism of the inhibitory effect of ulinastatin on hypoxia induced-phenotype modulation of PASMCs by measuring the changes in cell proliferation and migration.

RESULTSUlinastatin obviously enhanced the expressions of SM-α-actin and calponin (P<0.05), inhibited the proliferation and migration (P<0.05), and up-regulated the expression of PPAR-γ in PASMCs exposed to hypoxia (P<0.05). Pretreatment of the cells with GW9662 abolished the effect of ulinastatin on hypoxia-induced phenotype modulation of PASMCs and enhanced the cell proliferation and migration (P<0.05).

CONCLUSIONUlinastatin inhibits hypoxia-induced phenotype modulation of PASMCs from rats possibly by up-regulating the expression of PPAR-γ.

Actins ; metabolism ; Animals ; Calcium-Binding Proteins ; metabolism ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Glycoproteins ; pharmacology ; Microfilament Proteins ; metabolism ; Myocytes, Smooth Muscle ; cytology ; drug effects ; PPAR gamma ; metabolism ; Phenotype ; Pulmonary Artery ; cytology ; Rats ; Rats, Sprague-Dawley ; Up-Regulation

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.
Animals ; Aorta ; cytology ; metabolism ; physiology ; Calcium Signaling ; Cells, Cultured ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Male ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; physiology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Norepinephrine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Ryanodine ; pharmacology ; Tacrolimus ; pharmacology ; Vasoconstriction

PURPOSE: Proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. Rutin is a major representative of the flavonol subclass of flavonoids and has various pharmacological activities. Currently, data are lacking regarding its effects on VSMC proliferation induced by intermittent hyperglycemia. Here, we demonstrate the effects of rutin on VSMC proliferation and migration according to fluctuating glucose levels. MATERIALS AND METHODS: Primary cultures of male Otsuka Long-Evans Tokushima Fatty (OLETF) rat VSMCs were obtained from enzymatically dissociated rat thoracic aortas. VSMCs were incubated for 72 h with alternating normal (5.5 mmol/L) and high (25.0 mmol/L) glucose media every 12 h. Proliferation and migration of VSMCs, the proliferative molecular pathway [including p44/42 mitogen-activated protein kinases (MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2), p38 MAPK, phosphoinositide 3-kinase (PI3K), c-Jun N-terminal protein kinase (JNK), nuclear factor kappa B (NF-kappaB), and Akt], the migratory pathway (big MAPK 1, BMK1), reactive oxygen species (ROS), and apoptotic pathway were analyzed. RESULTS: We found enhanced proliferation and migration of VSMCs when cells were incubated in intermittent high glucose conditions, compared to normal glucose. These effects were lowered upon rutin treatment. Intermittent treatment with high glucose for 72 h increased the expression of phospho-p44/42 MAPK (extracellular signal regulated kinase 1/2, ERK1/2), phospho-MEK1/2, phospho-PI3K, phospho-NF-kappaB, phospho-BMK1, and ROS, compared to treatment with normal glucose. These effects were suppressed by rutin. Phospho-p38 MAPK, phospho-Akt, JNK, and apoptotic pathways [B-cell lymphoma (Bcl)-xL, Bcl-2, phospho-Bad, and caspase-3] were not affected by fluctuations in glucose levels. CONCLUSION: Fluctuating glucose levels increased proliferation and migration of OLETF rat VSMCs via MAPK (ERK1/2), BMK1, PI3K, and NF-kappaB pathways. These effects were inhibited by the antioxidant rutin.
Animals ; Caspase 3/metabolism ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Flavonoids/*pharmacology ; Glucose/*metabolism/pharmacology ; JNK Mitogen-Activated Protein Kinases ; MAP Kinase Kinase 1 ; Male ; Mitogen-Activated Protein Kinase 3 ; Muscle, Smooth, Vascular/cytology/*drug effects/enzymology ; Myocytes, Smooth Muscle/metabolism ; NF-kappa B/metabolism ; Phosphatidylinositol 3-Kinases ; Protein Kinase Inhibitors/*pharmacology ; Rats ; Rats, Inbred OLETF ; Rats, Long-Evans ; Reactive Oxygen Species/metabolism ; Rutin/*pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism

OBJECTIVETo observe the effect of Pinggan Qianyang Recipe (PQR) on inhibiting angiotensin II (Ang II) induced proliferation and migration of vascular smooth muscle cells (VSMCs) and changes of DNA methylation.

METHODSVSMCs were cultured using tissue explant method, and PQR containing serum was prepared. Primarily cultured VSMCs were divided into four groups, the normal group, the model group, the folate group (folic acid intervention) , and the PQR group. The proliferation and migration of VSMCs was duplicated by Ang II. After 24-h Ang II induced culture, 40 microg/mL folic acid was added to the folate group for 48 h, while 5% PQR containing serum was added to the PQR group for 48 h. The cell growth curve of VSMCs was drawn by using Cell Counting Kit (CCK-8). The proliferative activity of VSMC was determined by MTT assay. The migration of VSMCs was measured by Millicell chamber. The general level of cytosine methylation in cell nucleus was detected via 5-mC antibodies immunofluorescence, and mRNA expression levels of DNA methyltransferase 1 (DNMT1) were measured by Real-time q-polymerase chain reaction (q-PCR).

RESULTSVSMCs were promoted by Ang II at 10(-6) mol/L for 24 h. Compared with the normal group, the proliferative activity and migration quantity of VSMCs obviously increased, and DNA methylation level obviously decreased (P < 0.05, P < 0.01). Compared with the model group, the cell growth, proliferative activity and migration quantity of VSMCs obviously decreased and the general DNA methylation level increased in the folate group and the PQR group (P < 0.05, P < 0.01). Compared with the normal group, the mRNA expression of DNMT1 decreased in the model group (P < 0.01). Compared with the model group, mRNA expression of DNMT1 in Ang II induced VSMCs was obviously enhanced in the folate group and the PQR group (P < 0.01).

CONCLUSIONSPQR could inhibit Ang II induced proliferation and migration of VSMCs, and cause high genomic DNA methylation level. Changes of DNA methylation might be associated with DNMT1 expression.

Angiotensin II ; pharmacology ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects