To investigate 1alpha,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1alpha,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1alpha,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1alpha,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.
Acid Phosphatase/metabolism ; Animals ; Blotting, Western ; Calcitriol/*pharmacology ; Calcium Channel Agonists/pharmacology ; *Cell Differentiation ; Cell Line ; Cell Proliferation ; Gene Expression Regulation, Enzymologic/*drug effects ; Isoenzymes/metabolism ; Matrix Metalloproteinase 9/*genetics/metabolism ; Mice ; Osteoclasts/*cytology/*enzymology ; Tetrazolium Salts ; Thiazoles

Animals ; Bone Density Conservation Agents ; pharmacology ; Calcitriol ; pharmacology ; Calcium Channel Agonists ; pharmacology ; Humans ; Pulmonary Fibrosis ; metabolism ; Signal Transduction ; drug effects ; Transforming Growth Factor beta ; metabolism ; Wnt Proteins ; metabolism

BACKGROUNDIt has been reported that endogenous or exogenous hydrogen sulfide (H(2)S) exerts physiological effects in the vertebrate cardiovascular system. We have also demonstrated that H(2)S acts as an important regulator of electrophysiological properties in guinea pig papillary muscles and on pacemaker cells in sinoatrial nodes of rabbits. This study was to observe the electrophysiological effects of H(2)S on human atrial fibers.

METHODSHuman atrial samples were collected during cardiac surgery. Parameters of action potential in human atrial specialized fibers were recorded using a standard intracellular microelectrode technique.

RESULTSNaHS (H(2)S donor) (50, 100 and 200 µmol/L) decreased the amplitude of action potential (APA), maximal rate of depolarization (V(max)), velocity of diastolic (phase 4) depolarization (VDD) and rate of pacemaker firing (RPF), and shortened the duration of 90% repolarization (APD(90)) in a concentration-dependent manner. ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide (Gli, 20 µmol/L) partially blocked the effects of NaHS (100 µmol/L) on human atrial fiber cells. The L-type Ca(2+) channel agonist Bay K8644 (0.5 µmol/L) also partially blocked the effects of NaHS (100 µmol/L). An inhibitor of cystathionine γ-lyase (CSE), DL-propargylglycine (PPG, 200 µmol/L), increased APA, V(max), VDD and RPF, and prolonged APD(90).

CONCLUSIONSH(2)S exerts a negative chronotropic action and accelerates the repolarization of human atrial specialized fibers, possibly as a result of increases in potassium efflux through the opening of K(ATP) channels and a concomitant decrease in calcium influx. Endogenous H(2)S may be generated by CSE and act as an important regulator of electrophysiological properties in human atrial fibers.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; pharmacology ; Action Potentials ; drug effects ; Calcium Channel Agonists ; pharmacology ; Calcium Channels, L-Type ; metabolism ; Cystathionine gamma-Lyase ; metabolism ; Electrophysiology ; methods ; Glyburide ; pharmacology ; Heart Atria ; drug effects ; metabolism ; Humans ; Hydrogen Sulfide ; metabolism ; In Vitro Techniques ; KATP Channels ; antagonists & inhibitors ; metabolism ; Sulfides ; pharmacology

Sildenafil increases the cyclic guanosine monophosphate (cGMP) by inhibition of a phosphodiesterase 5, thereby leading to an antinociceptive effect. The increased cGMP may exert the effect on an L-type calcium channel through the activation of protein kinase G (PKG). The purpose of this study was to examine the possible involvement of a PKG-L-type calcium channel on the effect of sildenafil at the spinal level. Catheters were inserted into the intrathecal space of male SD rats. Pain was induced by applying 50 microliter of a 5% formalin solution to the hindpaw. The sildenafil-induced effect was examined after an intrathecal pretreatment of a PKG inhibitor (KT 5823), or a L-type calcium channel activator (FPL 64176). Intrathecal sildenafil produced an antinociceptive effect during phase 1 (0~10 min interval) and phase 2 (10~60 min interval) in the formalin test. Intrathecal KT 5823 and FPL 64176 attenuated the antinociceptive effect of sildenafil during both phases. Sildenafil is effective against both acute pain and the facilitated pain state at the spinal level. In addition, the inhibition of an L-type calcium channel by activation of the PKG may contribute to the antinocieptive mechanism of sildenafil in the spinal cord.
Animals ; Calcium Channel Agonists/pharmacology ; Calcium Channels, L-Type/*physiology ; Carbazoles/pharmacology ; Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors/*physiology ; Dose-Response Relationship, Drug ; Male ; Pain/drug therapy/*physiopathology ; Pain Measurement ; Piperazines/*pharmacology/therapeutic use ; Protein Kinase Inhibitors/pharmacology ; Purines/pharmacology/therapeutic use ; Pyrroles/pharmacology ; Rats ; Rats, Sprague-Dawley ; Sulfones/*pharmacology/therapeutic use

OBJECTIVE: To explore the protective effect of calcium channel agonist BayK8644 preconditioning on the lungs against ischemia-reperfusion injury (I/R) and its mechanism in rabbits. METHODS: Forty rabbits were randomly divided into 4 groups (10 in each group): a sham-operated group (Sham), an I/R group (I/R), an ischemic preconditioning (IP) group (IP), and a BayK8644 preconditioning group(BayK8644).The wet to dry weight (W/D) ratio, superoxide dismutase (SOD) activity, myleoperoxidase (MPO), and malondialdehyde (MDA) contents of the lung tissues were measured after the operation. Morphological and ultrastructural changes of the lung tissue were observed by light and electron microscope. The expression of pulmonary surfactant-associated protein-A (SP-A) was examined with immunohistochemistry. RESULTS: The W/D ratio, MPO and MDA contents of the lung tissue in the BayK8644 group and IP group were significantly lower than those in the I/R group (P<0.05), while the SOD activity and expression of SP-A of the lung tissue in the BayK8644 group and IP group were significantly higher than those in the I/R group(P<0.05), The morphological changes and ultrastructural changes were less notable in the BayK8644 group and IP group, compared with the I/R group. There was no significant difference in SOD activity, MPO and MDA contents between the BayK8644 group and IP group (P>0.05). CONCLUSION Appropriate BayK8644 preconditioning can induce transient Ca²+ influx, and elicit strong protection against the lung ischemia-reperfusion injury, which can simulate the endogenous protective effect of ischemic preconditioning.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; pharmacology ; Animals ; Calcium Channel Agonists ; pharmacology ; Female ; Ischemic Preconditioning ; methods ; Lung ; blood supply ; Male ; Rabbits ; Random Allocation ; Reperfusion Injury ; prevention & control

OBJECTIVETo study the central role of ginkgolide B (BN52021) in regulating cardiovascular function of nerve center by examining the effects of ginkgolide B on the electrical activity of rat paraventricular nucleus (PVN) neurons in hypothalamic slice preparation and to elucidate the mechanism involved.

METHODSExtracellular single-unit discharge recording technique.

RESULTS(1) In response to the application of ginkgolide B (0.1, 1, 10 micromol/L; n = 27) into the perfusate for 2 min, the spontaneous discharge rates (SDR) of 26 (26/27, 96.30%) neurons were significantly decreased in a dose-dependent manner. (2) Pretreatment with L-glutamate (L-Glu, 0.2 mmol/L) led to a marked increase in the SDR of all 8 (100%) neurons in an epileptiform pattern. The increased discharges were suppressed significantly after ginkgolide B (1 micromol/L) was applied into the perfusate for 2 min. (3) In 8 neurons, perfusion of the selective L-type calcium channel agonist, Bay K 8644 (0.1 micromol/L), induced a significant increase in the discharge rates of 8 (8/8, 100%) neurons, while ginkgolide B (1 micromol/L) applied into the perfusate, could inhibit the discharges of 8 (100%) neurons. (4) In 8 neurons, the broad potassium channels blocker, tetraethylammonium (TEA, 1 mmol/L) completely blocked the inhibitory effect of ginkgolide B (1 micromol/L).

CONCLUSIONThese results suggest that ginkgolide B can inhibit the electrical activity of paraventricular neurons. The inhibitory effect may be related to the blockade of L-type voltage-activated calcium channel and potentially concerned with delayed rectifier potassium channel (K(DR)).

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; pharmacology ; Action Potentials ; drug effects ; Analysis of Variance ; Animals ; Animals, Newborn ; Calcium Channel Agonists ; pharmacology ; Dose-Response Relationship, Drug ; Drug Interactions ; Fibrinolytic Agents ; pharmacology ; Ginkgolides ; pharmacology ; Glutamic Acid ; pharmacology ; In Vitro Techniques ; Lactones ; pharmacology ; Neural Inhibition ; drug effects ; Neurons ; drug effects ; Paraventricular Hypothalamic Nucleus ; cytology ; Potassium Channel Blockers ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Tetraethylammonium ; pharmacology

To study the role of resveratrol in the discharges of neurons in paraventricular nucleus (PVN) in hypothalamic slices, extracellular single-unit discharge recording technique was used. The effects of resveratrol were examined with glass microelectrodes in the rat PVN neurons at resting potential level. The results were as follows: (1) In response to the application of resveratrol (0.05, 0.5, 5.0 μmol/L, n=29) to the superfusate for 2 min, the spontaneous discharge rate (SDR) of neurons in 28/29 (96.6%) hypothalamic slices significantly decreased in a dose-dependent manner; (2) Pretreatment with L-glutamate (0.2 mmol/L) led to a marked increase in the SDR in all 8/8 (100%) slices in an epileptiform pattern. The increased discharges were suppressed by the application of resveratrol (5.0 mmol/L) in all 8 slices; (3) In 8 slices, perfusion of the selective L-type calcium channel agonist, Bay K8644 (0.1 μmol/L), induced a significant increase in the discharge rate in 8/8 (100%) slices. Resveratrol (5.0 μmol/L) significantly attenuated the increased SDR in all 8 slices; (4) Pretreatment with the nitric oxide synthase (NOS) inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME, 50 μmol/L) increased SDR in 7/8 (87.5%) slices, but did not affect the inhibitory effect of resveratrol (5.0 μmol/L). These results suggest that resveratrol inhibits the electrical activity of PVN neurons and exerts neuroprotective actions on central neurons. The inhibitory effect of resveratrol is possibly related to the blockade of L-type calcium channel, but not due to NO release.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; pharmacology ; Action Potentials ; Animals ; Calcium Channel Agonists ; pharmacology ; Calcium Channels, L-Type ; metabolism ; Glutamic Acid ; pharmacology ; In Vitro Techniques ; Microelectrodes ; NG-Nitroarginine Methyl Ester ; pharmacology ; Neurons ; drug effects ; Paraventricular Hypothalamic Nucleus ; cytology ; Rats ; Rats, Sprague-Dawley ; Stilbenes ; pharmacology

The effects of ginkgolide B on the carotid sinus baroreflex (CSB) were studied in the perfused isolated carotid sinus of 30 anesthetized Sprague-Dawley male rats. The results were as follows. (1) By perfusing with ginkgolide B (0.1, 1, 10 μmol/L), the functional curve of the baroreflex was shifted to the right and upward. There was a marked decrease in peak slope (PS) and reflex decrease (RD) in mean arterial pressure (P<0.01), while the threshold pressure (TP), equilibrium pressure (EP) and saturation pressure (SP) were significantly increased (P<0.05, P<0.01). Among the functional parameters of CSB, the changes in PS, RD, TP, EP and SP were dose-dependent. (2) Pretreatment with Bay K8644 (500 nmol/L), an agonist of L-type calcium channel, completely eliminated the effects of ginkgolide B (1 μmol/L) on the CSB. (3) Pretreatment with tetraethylammonium (TEA, 1 mmol/L), an inhibitor of potassium channel, completely abolished the above effects of ginkgolide B (1 μmol/L) on the CSB. These results suggest that ginkgolide B inhibits the CSB in anesthetized rats, which is mediated by decreased calcium influx and increased potassium efflux in baroreceptor nerve endings.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; pharmacology ; Animals ; Baroreflex ; drug effects ; Calcium Channel Agonists ; pharmacology ; Calcium Channels, L-Type ; Carotid Sinus ; physiopathology ; Ginkgolides ; pharmacology ; Lactones ; pharmacology ; Male ; Potassium Channel Blockers ; pharmacology ; Pressoreceptors ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tetraethylammonium ; pharmacology

OBJECTIVETo investigate the effect of Ca(2+) influx through L-type Ca(2+) channels on normal and hyperpolarized membrane potential of arteriole smooth muscle cells (ASMCs) in rats.

METHODSThe ASMCs isolated from normal rats and those with severe hemorrhagic shock were labeled with DiBAC4 (3) for membrane potential detection.

RESULTSCa(2+) influx caused hyperpolarization of the membrane potential in the normal ASMCs but depolarization in the cells from rats with hemorrhagic shock, and this effect could be inhibited by TEA.

CONCLUSIONCa(2+)-activated potassium channels activated by Ca(2+) influx through L-type Ca(2+) channels in normal ASMCs to cause hyperpolarization but leads directly to membrane potential depolarization in ASMCs from rats with severe hemorrhagic shock. This finding can be meaningful for treatment of vascular hyporeactivity in advanced stage of severe shock.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; pharmacology ; Animals ; Arterioles ; physiopathology ; Calcium Channel Agonists ; pharmacology ; Calcium Channels, L-Type ; metabolism ; Female ; Male ; Membrane Potentials ; drug effects ; Muscle, Smooth, Vascular ; physiopathology ; Potassium Channels, Calcium-Activated ; metabolism ; Rats ; Rats, Wistar ; Shock, Hemorrhagic ; physiopathology

The effects of resveratrol on the discharges of neurons in CA1 area of rat hippocampal slices were examined by using extracellular recording technique. The results are as follows: (1) In response to the application of resveratrol (0.05, 0.5, 5.0 micromol/L, n=52) into the superfusate for 2 min, the spontaneous discharge rate of 46/52 (88.5%) neurons was significantly decreased in a dose-dependent manner; (2) Application of L-glutamate (0.2 mmol/L) into the superfusate led to a marked increase in discharge rate of all 8 (100%) slices in an epileptiform pattern. The increased discharges were suppressed by application of resveratrol (5.0 micromol/L); (3) In 7 slices, perfusion of the selective L-type calcium channel agonist, Bay K8644 (0.1 micromol/L), induced a significant increase in the discharge rate of 6/7 (85.7%) slices. The increased discharges were suppressed by application of resveratrol (5.0 micromol/L); (4) In 9 slices, perfusion of nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 micromol/L) into the superfusate significantly augmented the discharge rate of 7/9 (77.8%) slices. Resveratrol (5.0 micromol/L) applied into the superfusate reduced the increased discharges of all 7/7 (100%) neurons; (5) In 10 units, the large-conductance Ca(2+)-activated K(+) channel blocker (tetraethylammonium chloride, TEA, 1 mmol/L) significantly increased the discharge rate of 9/10 (90%) slices. Resveratrol (5.0 micromol/L) applied into the superfusate inhibited the discharges of 8/9 (88.9%) slices. These results suggest that resveratrol inhibits the electrical activity of CA1 neurons. This effect may be related to the blockade of L-type calcium channel and a subsequent reduction of calcium influx, and probably has no association with large-conductance Ca(2+)-activated K(+) channel.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; pharmacology ; Animals ; Calcium Channel Agonists ; pharmacology ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; Electrophysiology ; Glutamic Acid ; pharmacology ; Hippocampus ; cytology ; physiology ; Male ; Neurons ; physiology ; Rats ; Rats, Sprague-Dawley ; Stilbenes ; pharmacology