AIMTo study the effects of oxyphenamone (Oxy) on activation of Ca(2+)-activated K+ channels in rabbit mesenteric vascular smooth muscle cells.

METHODSTo measure the effect of Oxy on the Ca(2+)-activated K+ channel (BK (Ca) channel) activity in rabbit mesenteric vascular smooth muscle cells by using whole cell patch clamp techniques.

RESULTSOxy reversibly increase BK (Ca) channel activity in rabbit mesenteric artery smooth muscle cells. Application of Oxy (0.1 mumol.L-1) to the perfusion solution caused significant increase in outward currents and its effect was completely abolished by washout; The outward currents K+ was inhibited by TEA (7.5 mmol.L-1); Oxy activated the BK (Ca) channel in a dose-dependent manner (0.01-10 mumol.L-1).

CONCLUSIONOxy directly increase the activity of BK (Ca) channel activity in rabbit mesenteric vascular smooth muscle cells in dose-dependent manner.

Animals ; Cardiotonic Agents ; pharmacology ; Mesenteric Arteries ; cytology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Organic Chemicals ; Patch-Clamp Techniques ; Potassium Channels, Calcium-Activated ; drug effects ; Rabbits

The present study was aimed to investigate the role of cyclin D1 in human pulmonary artery smooth muscle cells (HPASMCs) proliferation and migration induced by cigarette smoke extract (CSE). The eukaryotic expression vector of antisense cyclin D1 gene (pIRES2-EGFP-ascyclin D1) was recombinated. The recombinant and empty vector were separately transfected into normal HPASMCs using liposome. Then the cells were treated with or without 5% CSE. The cells were randomly divided into six groups: control group, vector group, antisense cyclin D1 group, 5% CSE group, vector+5% CSE group and antisense cyclin D1+5% CSE group. The expressions of cyclin D1 mRNA and protein were detected by real-time fluorescence RT-PCR and Western blot, respectively. The proliferation of HPASMCs was examined by cell cycle analysis, MTT assay and proliferation cell nuclear antigen (PCNA) immunocytochemical staining. The migration of HPASMCs was measured by Transwell cell test. The results showed that the eukaryotic expression vector of antisense cyclin D1 gene was constructed and transfected into HPASMCs successfully. The cyclin D1 mRNA and protein levels in antisense cyclin D1 group were significantly lower than those in control group (P<0.05). In 5% CSE group, the cyclin D1 mRNA and protein levels were elevated significantly compared with those in control group (P<0.05), and the indicators of cell and migration in antisense cyclin D1+5% CSE group were remarkably lower than those in 5% CSE group (P<0.05). These results suggest that CSE could promote HPASMCs proliferation and migration through up-regulation of cyclin D1 expression. PIRES2-EGFP-ascyclin D1 could attenuate CSE-induced proliferation and migration of HPASMCs by suppressing the expression of cyclin D1, which implicates that cyclin D1 might be involved in the process of HPASMCs proliferation and migration stimulated by CSE.
Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin D1 ; physiology ; Humans ; Muscle, Smooth, Vascular ; cytology ; pathology ; Myocytes, Smooth Muscle ; cytology ; pathology ; Pulmonary Artery ; cytology ; pathology ; Smoke ; adverse effects ; Tobacco ; adverse effects

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

The large conductance Ca2+ activated K+ channel (BK channel) has been considered to play an important role in the excitability and contractility of vascular smooth muscle cells. Activation of the BK channel causes the hyperpolarization and relaxation of vascular smooth muscle cells. It has been reported that fatty acids can affect the BK channel activity and its concentration is increased significantly during myocardial ischemia. These reports suggest that fatty acids may contribute to the ischemic coronary vasodilation by increasing the BK channel activity. However, the underlying mechanism of fatty acid-induced activation of the BK channel is still uncertain. In the present study, we measured the effect of fatty acids on the BK channel activity in rabbit coronary smooth muscle cells by using patch clamp method and also examined its underlying mechanism. Arachidonic acid (AA) dissolved in DMSO activated the BK channel in a dose-dependent manner (from 0.5 to 10 microM), and DMSO (0.1%) alone had no effect on the activity of the BK channel. Arachidonic acid activated BK channels in both cell-attached and inside-out patches, but the onset and recovery of this effect were slower in the cell-attached patch configuration. The BK channel activity was also increased by other fatty acids, including myristic acid, linoleic acid, palmitoleic acid and palmitic acid. Long chain fatty acids were more effective than short chain fatty acids (myristic acid), and there was no statistical difference between the effect of saturated (palmitic acid) and unsaturated fatty acids (palmitoleic acid) on the BK channel activity. The concentration of Ca2+ and Mg2+ in the bathing solution had no appreciable effects on the AA-induced increase of BK channel activity. From the above results, it may be concluded that fatty acids directly increase the BK channel activity and may contribute to the ischemic coronary vasodilatation in rabbit coronary smooth muscle cells.
Animal ; Calcium/physiology ; Cells, Cultured ; Coronary Vessels/cytology/drug effects/*physiology ; Fatty Acids/*pharmacology/physiology ; Female ; Male ; Membrane Potentials/drug effects ; Muscle, Smooth, Vascular/cytology/drug effects/*physiology ; Potassium Channels/*drug effects ; Rabbits ; Support, Non-U.S. Gov't

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

Hypoxia-induced 15-hydroxyeicosatetraenoic acid (15-HETE) is an essential mediator to constrict pulmonary arteries (PA). The signaling pathway involved in 15-HETE-induced PA vasoconstriction remains obscure. The aim of the present study was to test the hypothesis that hypoxic PA constriction induced by 15-HETE was possibly regulated by the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway. PA ring tension measurement, Western blot and immunocytochemistry were used in the study to determine the possible role of ERK1/2 in 15-HETE-induced PA vasoconstriction. The organ bath for PA rings tension study was employed. Adult male Wistar rats were raised in hypoxic environment with fractional inspired oxygen (FIO2, 0.12) for 9 d. PA 1~1.5 mm in diameter were dissected and cut into 3 mm long rings for tension study. ERK1/2 up-stream kinase (MEK) inhibitor PD98059, which blocks the activation of ERK1/2, was used. The results showed that pretreatment of PD98059 significantly blunted 15-HETE-induced PA vasoconstrictions in the rings from hypoxic rat. Moreover, in endothelium-denuded rings, PD98059 also significantly attenuated 15-HETE-induced vasoconstriction. Phosphorylation of ERK1/2 in pulmonary arterial smooth muscle cells (PASMCs) of rat was enhanced evidently when stimulated by 15-HETE. Thus, the data suggest that ERK1/2 signaling pathway is involved in 15-HETE-induced hypoxic pulmonary vasoconstriction.
Animals ; Flavonoids ; pharmacology ; Hydroxyeicosatetraenoic Acids ; antagonists & inhibitors ; pharmacology ; Hypoxia ; physiopathology ; MAP Kinase Signaling System ; physiology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; Pulmonary Artery ; cytology ; drug effects ; physiopathology ; Rats ; Rats, Wistar ; Vasoconstriction ; drug effects

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

Chloride channels have been identified in vascular smooth muscle cells (SMCs). It has been shown that these channels are involved in myogenic tone regulation and neuromuscular transmission in various vascular beds. However, whether the chloride channels are responsible for the formation of excitatory junction potentials (EJPs) of SMCs in the spiral modiolar artery (SMA) remains unelucidated. In the present study, the effects of chloride channel blockers (niflumic acid, NFA; indanyloxyacetic acid 94, IAA-94; disodium 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonate, DIDS) on EJP were explored in guinea pigs, using intracellular recording techniques on acutely isolated SMA. It was found that EJP was evoked in the majority of the SMCs (75%, n=49) with an adequate electronic stimulation. The amplitude of the EJP was partially blocked (30% approximately 80%) by combined application of alpha(1) receptor antagonist (prazosin) and alpha(2) receptor antagonist (idazoxan) at concentration of up to 1 micromol/L, and P(2x) receptor antagonist (PPADS, 10 approximately 100 micromol/L). NFA (100 micromol/L) could further inhibit the residual EJP in the presence of alpha(1), alpha(2)-adrenergic and P(2x) receptor antagonists. IAA-94 or DIDS not only inhibited the amplitude but also shortened the duration of EJP. Decrease of extracellular chloride concentration from 135.6 mmol/L to 60 mmol/L would enhance EJP. Moreover, IAA-94 (100 micromol/L) and DIDS (200 mumol/L) could reverse the enhancement of EJP by low extracellular Cl(-). NFA (100 micromol/L) could also block the residual depolarizations evoked by norepinephrine (NE, 1 approximately 50 micromol/L). Based on these results, it is inferred that NE could activate a novel adrenoceptor to open the chloride channel on the membrane of the SMCs, leading to a transmembrane Cl(-) current. This current is involved, at least partially, in the formation of EJP.
Adrenergic alpha-Antagonists ; pharmacology ; Animals ; Arteries ; physiology ; Chloride Channels ; antagonists & inhibitors ; Cochlea ; blood supply ; Excitatory Postsynaptic Potentials ; drug effects ; Female ; Guinea Pigs ; Male ; Muscle, Smooth, Vascular ; cytology ; physiology ; Myocytes, Smooth Muscle ; drug effects ; physiology ; Norepinephrine ; pharmacology

The purpose of the present study is to investigate the effect of isoliquiritigenin (ISL) on the cerebral basilar artery in spontaneously hypertensive rats (SHR). The change of SHR systolic pressure was measured by tail artery pressure measurement instrument before and after ISL intervention. After perfusion with 1 × 10(-5) mol/L phenylephrine (PE), 1 × 10(-5) mol/L PE + 1 × 10(-4) mol/L ISL and 1 × 10(-5) mol/L PE, the diameter of the cerebral basilar artery separated from SHR was measured by pressure myograph. The current of large-conductance calcium-activated potassium (BKCa) channel of SHR single vascular smooth muscle cell (VSMC) was recorded by whole-cell patch-clamp technique and the cGMP levels of basilar artery was evaluated by ELISA. The results showed that 1) after intervention with ISL for 14 days, the systolic pressure of SHR was decreased from (218.3 ± 1.6) mmHg to (119.2 ± 1.9) mmHg (P < 0.01), but there was no difference in systolic pressure between ISL-treated SHR and Wistar-Kyoto (WKY) rat; 2) 1 × 10(-4) mol/L ISL relaxed the SHR cerebral basilar artery (P < 0.01); 3) ISL significantly increased the outward current density of VSMC from SHR cerebral basilar artery (P < 0.01, n = 6), and the effect could be reversed by 1 × 10(-3) mol/L TEA (a BKCa channel inhibitor), but 3 × 10(-4) mol/L 4-AP (a Kv channel inhibitor) had no effect on the enhanced current density induced by ISL in VSMC; 4) 1 × 10(-5) mol/L Methylene blue (a sGC inhibitor) significantly inhibited the ISL-enhanced current density in VSMC (P < 0.05, n = 6); 5) ISL significantly increased the cGMP level of SHR basilar artery (P < 0.05, n = 6). The results suggest that the role of the ISL in relaxing the SHR cerebral basilar artery may be related to its effect in enhancing BKCa current by increasing the levels of cGMP in the VSMC.
Animals ; Basilar Artery ; drug effects ; Blood Pressure ; Cerebral Arteries ; drug effects ; Chalcones ; pharmacology ; Cyclic GMP ; physiology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Patch-Clamp Techniques ; Potassium Channels, Calcium-Activated ; physiology ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Systole

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