Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K+ (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca2+-activated K+, inward rectifier K+ and ATP-sensitive K+ channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca2+ channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, alpha-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway.
4-Aminopyridine/pharmacology ; Action Potentials ; Animals ; Calcium Channel Blockers/pharmacology ; Calcium Channels/metabolism ; Cells, Cultured ; Ketanserin/pharmacology ; Male ; Mesenteric Arteries/drug effects/*metabolism/physiology ; Muscle Contraction ; Muscle, Smooth, Vascular/cytology/drug effects/metabolism/physiology ; Myocytes, Smooth Muscle/drug effects/metabolism/physiology ; Nifedipine/pharmacology ; Potassium Channel Blockers/pharmacology ; Potassium Channels, Voltage-Gated/antagonists & inhibitors/*metabolism ; Protein Kinase Inhibitors/pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptor, Serotonin, 5-HT2A/*metabolism ; Serotonin/*pharmacology ; Serotonin 5-HT2 Receptor Antagonists/pharmacology ; Spiperone/pharmacology ; *Vasoconstriction ; src-Family Kinases/antagonists & inhibitors/*metabolism

Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Membrane ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Humans ; Multiple Myeloma ; drug therapy ; metabolism ; pathology ; Oxides ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels, Voltage-Gated ; drug effects

AIMTo observe the effect of subtypes of Kv channels in rat pulmonary artery smooth muscle cells (PASMCs) on the process of pulmonary vasoconstriction induced by 15-HETE.

METHODSIn the present study, ring of rabbit PA with specific Kv channel blockers were employed to functionally identify certain channel subtypes that took part in the process of 15-HETE induced pulmonary vasoconstriction; RT-PCR and Western blotting analysis were also used to measure the expression of subtypes of Kv in PASMCs exposed to 15-HETE,chronic hypoxia.

RESULTSBlocking of Kv1. 1, Kv1. 2, Kv1. 3 and Kv1. 6 channels did not affect 15-HETE induced vasoconstriction in normoxic rats; 15-HETE did not affect expression of Kv1. 1 and Kv1. 2 channels; 15-HETE significantly downregulated the expression of mRNA and protein of Kv1. 5 and Kv2. 1 in rat PASMCs.

CONCLUSIONThe results suggested that hypoxia may block Kv1. 5 and Kv2. 1 channels via 15-HETE mediated mechanism, leading to decrease numbers of functional Kv1. 5 and Kv2. 1 channels in PASMCs, leading to PA vasoconstriction.

Animals ; Cell Hypoxia ; Cells, Cultured ; Hydroxyeicosatetraenoic Acids ; pharmacology ; Hypoxia ; physiopathology ; Kv1.5 Potassium Channel ; biosynthesis ; genetics ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Potassium Channels, Voltage-Gated ; antagonists & inhibitors ; Pulmonary Artery ; physiopathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Shab Potassium Channels ; biosynthesis ; genetics ; Vasoconstriction ; drug effects

Hepatic stellate cells (HSCs) are known to play a role in the pathogenesis of the increased intrahepatic vascular resistance found in chronic liver diseases. The aim of this study was to evaluate the K+ and Ca2+ currents in cultured HSCs from rat liver, through the patch-clamp technique. Most cells were positive for desmin immunostain after isolation and in alpha-smooth muscle actin immunostain after 10 - 14 days of culturing. Outward and inward rectifying K+ currents were confirmed. Two different types of K+ currents were distinguished: one with the inward rectifying current and the other without. The outward K+ currents consisted of at least four components: tetraethylammonium (TEA) -sensitive current, 4-aminopyridine (4-AP) -sensitive current, pimozide-sensitive current and three blocker-resistant current. The peaks of the outward K+ currents evoked by a depolarizing pulse were decreased to 32.0 +/- 3.0, 62.8 +/- 3.7 and 32.8 +/- 3.5% by 5 mM TEA, 2 mM 4-AP and 15microM pimozide, respectively. Moreover, the combined application of three blockers caused 86.6 +/- 4.8% suppression. The inward currents evoked hyperpolarizing pulses were inwardly rectifying and almost blocked by Ba2+. Elevation of external K+ increased the inward current amplitude and positively shifted its reversal potential. Voltage- dependent Ca2+ currents which were completely abolished by Cd2+ and nimodipine were detected in 14 day cultured HSCs. In this study, the cultured HSCs were found to express outward K+ currents composed of multiple pharmacological components, Ba2+-sensitive inward rectifying K+ current and L-type Ca2+ current.
Animals ; Calcium/*metabolism ; Calcium Channel Blockers/pharmacology ; Calcium Channels, L-Type/*physiology ; Cells, Cultured ; Hepatocytes/cytology/*physiology ; Immunohistochemistry ; Male ; Membrane Potentials/drug effects/physiology ; Patch-Clamp Techniques ; Potassium/*metabolism ; Potassium Channel Blockers/pharmacology ; Potassium Channels, Voltage-Gated/*physiology ; Rats ; Rats, Sprague-Dawley

This study investigated the role of 15-hydroxyeicosatetraenoic acid (15-HETE) in rabbit pulmonary arterial smooth muscle cells (PASMCs) under hypoxia by using organ bath and whole cell patch-clamp techniques. Neonatal rabbits born into normoxic environment were transferred after first feeding into normal and hypoxic environments with respectively 0.21 and 0.12 fractional inspired oxygen (FiO2). Pulmonary arteries were extracted after 9 d and cut into rings 1.0 approximately 1.5 mm in length for organ bath experiments. Whole cell patch-clamp technique was used to measure the potassium current in the freshly dispersed rabbit PASMCs. The results showed that 15-HETE-induced vasoconstriction was blocked by 4-aminopyridine (5 mmol/L), a Kv channel blocker. The K(ATP) channel blocker glyburide (1 micromol/L) and the BKCa channel blocker tetraethylammonium (10 mmol/L) did not abolish this vasoconstriction. 15-HETE decreased the whole-cell voltage-gated K+ current in the PASMCs. These findings demonstrate that hypoxia blocks Kv channels through a 15-HETE mediated mechanism, leading to PA vasoconstriction.
Animals ; Animals, Newborn ; Cell Hypoxia ; Cells, Cultured ; Female ; Hydroxyeicosatetraenoic Acids ; pharmacology ; Muscle Contraction ; drug effects ; Muscle, Smooth, Vascular ; cytology ; metabolism ; physiology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels, Voltage-Gated ; drug effects ; Pregnancy ; Pulmonary Artery ; cytology ; metabolism ; physiology ; Rabbits ; Vasoconstrictor Agents ; pharmacology

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 relationship between the level of testosterone and the incidence of coronary heart disease is still controversial in the view of the results of clinical and epidemiologic studies. This uncertainty might be partly due to relatively small number of experimental studies undertaken to investigate the cellular mechanism underlying the vascular responses to testosterone. To further investigate the cellular mechanisms of testosterone with respect to vascular response, we investigated the effect of testosterone on contractility and intracellular Ca2+ regulation in a rabbit coronary artery and evaluated the underlying mechanism of testosterone-induced changes of coronary vascular tone by using various pharmacological blockers. Testosterone was found to relax rabbit coronary arteries in a dose-dependent manner, and no significant difference was found in the relaxation response to testosterone with or without endothelium. Similar results were obtained in male and non-pregnant female rabbit coronary arteries. The relaxation response of rabbit coronary arteries to testosterone was greater for PGF2alpha-contracted rings than for KCl contracted rings, which suggest the involvement of K+ channels. Furthermore, the relaxation response to testosterone was significantly reduced by 4-aminopyridine, a sensitive blocker of voltage dependent K+ channels, but not by low doses of tetraethylammonium or iberiotoxin, a Ca2+ activated K+ channel blocker. Testosterone simultaneously reduced the intracellular Ca2+ concentration ([Ca2+]i) and tension, and 4-AP effectively antagonized the testosterone-induced change of [Ca2+]i and tension. Therefore, it may be concluded that the stimulation of voltage dependent K channels is responsible, at least in part, for the testosterone-induced relaxation of rabbit coronary arteries.
Androgens/*pharmacology ; Animals ; Arteries/drug effects ; Calcium/*metabolism ; Coronary Vessels/*drug effects ; Female ; Intracellular Membranes/*metabolism ; Male ; Osmolar Concentration ; Potassium Channels, Voltage-Gated/drug effects/*metabolism ; Rabbits ; Support, Non-U.S. Gov't ; Testosterone/*pharmacology ; *Vasodilation

AIMTo study the effect of magnesium sulfate on transient outward K+ current (IA) and delayed rectifier K+ current (IK) in freshly dissociated hippocampal neurons of rats.

METHODSThe whole-cell patch clamp techniques were used.

RESULTSMagnesium sulfate reversibly reduced the amplitudes of IA and IK in a concentration-dependent and voltage-dependent, but not frequency-dependent manner. Half-blocking concentration (IC50) on IA and IK were 6.30 mmol.L-1 and 7.60 mmol.L-1, respectively. Magnesium sulfate (6 mmol.L-1) affected the activation process of IA and IK. Before and after application of the drug, the half-activation voltages of IA were (7 +/- 6) mV and (-7 +/- 11) mV (n = 10, P < 0.01), and the half-activation voltages of IK were (20 +/- 6) mV and (28 +/- 4) mV (n = 10, P < 0.01), but the slope factors were not changed. In addition, magnesium sulfate (6 mmol.L-1) also affected the inactivation process of IA. Before and after application of the drug, the half-inactivation voltages of IA were (-65 +/- 5) mV and (-89 +/- 6) mV (n = 10, P < 0.01).

CONCLUSIONMagnesium sulfate inhibited IA and IK in freshly dissociated hippocampal neurons of rats, which might contribute to protect the central neuronal system (CNS) against damages induced by ischemia and oxygen deprivation.

Animals ; Cell Separation ; Delayed Rectifier Potassium Channels ; Female ; Hippocampus ; cytology ; Magnesium Sulfate ; pharmacology ; Male ; Neurons ; drug effects ; physiology ; Neuroprotective Agents ; pharmacology ; Patch-Clamp Techniques ; Potassium Channels ; drug effects ; metabolism ; Potassium Channels, Voltage-Gated ; Rats ; Rats, Wistar

AIMTo study mRNA expression difference of voltage-dependent potassium channels in the brain of scopolamine-induced memory impaired rats.

METHODSMemory impairments induced in rats by scopolamine (1 mg.kg-1) were assessed in the Morris water maze test. After rats were injected intraperitoneally with scopolamine for 6 days, the mRNA expression level of five voltage-dependent potassium channels, Kv1.4, Kv1.5, Kv2.1, Kv4.2 and Kv4.3 were detected in the rat cortex and hippocampus by RT-PCR.

RESULTSScopolamine (1 mg.kg-1) was shown to significantly induce memory impairment in rats. The mRNA levels of Kv4.2 were decreased by 28.8% and 33.9% in the cortex and hippocampus, respectively. The mRNA levels of Kv1.4 and Kv2.1 were increased in the hippocampus by 111.7% and 64.3%, respectively. There were no differences in the brain mRNA levels of other voltage-dependent potassium channels in scopolamine-induced memory impaired rat.

CONCLUSIONThe mRNA expression levels of voltage-dependent potassium channels changed significantly in the brain of scopolamine-induced memory impaired rats.

Adjuvants, Anesthesia ; Animals ; Cerebral Cortex ; drug effects ; metabolism ; Hippocampus ; drug effects ; metabolism ; Male ; Maze Learning ; Memory Disorders ; chemically induced ; metabolism ; Potassium Channels, Voltage-Gated ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; Random Allocation ; Rats ; Rats, Wistar ; Scopolamine Hydrobromide

AIMTo investigat the effects of donepezil on delayed rectifier-like potassium currents (IK) in rat hippocampus and neocortex.

METHODSWhole cell configuration of the patch-clamp techniques were used to characterize IK in acutely isolated rat hippocampal and neocortical pyramidal neurons.

RESULTSThe slowly inactivating outward currents (IK) were recorded in all cells under investigation. Donepezil in micromolar concentrations were shown to supress the IK of all cells in a dose-dependent and voltage-dependent manner. The steady-state activation curves of IK were characterized by half-activation potentials of -15.5 mV in hippocampal and -4.1 mV in neocortical pyramidal neurons and were changed to -26.2 mV and -18.6 mV, respectively, after perfusion with donepezil (10 mumol.L-1).

CONCLUSIONAt concentrations as low as 1 mumol.L-1, donepezil was found to block the IK in a voltage-dependent manner in hippocampus and neocortex. This effect may be synergistic with the anticholinesterase activity of donepezil to increase its therapeutic effectiveness.

Animals ; Cholinesterase Inhibitors ; pharmacology ; Delayed Rectifier Potassium Channels ; Hippocampus ; cytology ; In Vitro Techniques ; Indans ; pharmacology ; Male ; Neocortex ; cytology ; Neurons ; drug effects ; metabolism ; Patch-Clamp Techniques ; Piperidines ; pharmacology ; Potassium Channels ; drug effects ; physiology ; Potassium Channels, Voltage-Gated ; Pyramidal Cells ; drug effects ; metabolism ; Rats ; Rats, Wistar