OBJECTIVE: To study hesperetin-induced vasorelaxation after depolarizing contraction in human umbilical veins (HUVs) to elucidate the role of L-type Ca²⁺ channel (LTCC) and related signaling pathway. METHODS: Isometric tension recording was performed in HUV rings pre-contracted with K⁺. Hesperetin relaxing mechanism was investigated using a LTCC opener (BayK8644) and blockers of cyclic nucleotides and phosphodiesterases (PDEs). Whole-cell patch-clamping in A7r5 cells, a rat vascular smooth muscle cell line, was performed to study the effect of hesperetin on LTCC current. RESULTS: After depolarizing precontraction, hesperetin induced HUV relaxation concentration-dependently and endothelium-independently; 1 mmol/L hesperetin reduced denuded HUV ring tension by 68.7% ± 4.3% compared to matching vehicle, osmolality, and time controls (P<0.0001). Importantly, hesperetin competitively inhibited BayK8644-induced contraction, shifting the half maximal effective concentration of BayK8644 response from 1.08 nmol/L [95% confidence interval (CI) 0.49-2.40] in vehicle control to 11.30 nmol/L (95% CI 5.45-23.41) in hesperetin (P=0.0001). Moreover, hesperetin elicited further vasorelaxation in denuded HUV rings pretreated with inhibitors of soluble guanylyl cyclase, adenylyl cyclase, PDE3, PDE4, and PDE5 (P<0.01), while rings pretreated with PDE1 inhibitors could not be relaxed by hesperetin (P>0.05). However, simultaneously applying inhibitors of soluble guanylyl cyclase and adenylyl cyclase could not inhibit hesperetin's effect (P>0.05). In whole-cell patch-clamping, hesperetin rapidly decreased LTCC current in A7r5 cells to 66.7% ± 5.8% (P=0.0104). CONCLUSIONS Hesperetin diminishes depolarizing contraction of human vascular smooth muscle through inhibition of LTCC, and not cyclic nucleotides nor PDEs. Our evidence supports direct LTCC interaction and provides additional basis for the use of hesperetin and its precursor hesperidin as vasodilators and may lead to future vasodilator drug development as a treatment alternative for cardiovascular diseases.
Hesperidin/pharmacology* ; Humans ; Calcium Channels, L-Type/metabolism* ; Umbilical Veins/physiology* ; Muscle Contraction/drug effects* ; Animals ; Rats ; Calcium Channel Blockers/pharmacology* ; Vasodilation/drug effects* ; Muscle Relaxation/drug effects*

Spinal α-motoneurons directly innervate skeletal muscles and function as the final common path for movement and behavior. The processes that determine the excitability of motoneurons are critical for the execution of motor behavior. In fact, it has been noted that spinal motoneurons receive various neuromodulatory inputs, especially monoaminergic one. However, the roles of histamine and hypothalamic histaminergic innervation on spinal motoneurons and the underlying ionic mechanisms are still largely unknown. In the present study, by using the method of intracellular recording on rat spinal slices, we found that activation of either H or H receptor potentiated repetitive firing behavior and increased the excitability of spinal α-motoneurons. Both of blockage of K channels and activation of Na-Ca exchangers were involved in the H receptor-mediated excitation on spinal motoneurons, whereas the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were responsible for the H receptor-mediated excitation. The results suggest that, through switching functional status of ion channels and exchangers coupled to histamine receptors, histamine effectively biases the excitability of the spinal α-motoneurons. In this way, the hypothalamospinal histaminergic innervation may directly modulate final motor outputs and actively regulate spinal motor reflexes and motor execution.
Animals ; Histamine ; pharmacology ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; metabolism ; Motor Neurons ; drug effects ; physiology ; Rats ; Receptors, Histamine H2 ; metabolism ; Sodium-Calcium Exchanger ; metabolism

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

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

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

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

OBJECTIVETo explore the modulatory effect of niflumic acid and blocker of calcium channel on the desensitization of gamma aminobutyric acid (GABA)-activated currents in dorsal root ganglion(DRG) neurons from rat.

METHODSThe whole-cell patch-clamp technique was used to observe the modulatory effect of niflumic acid and blocker of calcium channel on the desensitization of GABA-activated currents in neurons freshly dissociated from rat DRG neurons.

RESULTSApplication of GABA (0.1-1 000 micromol/L) could induce concentration-dependent inward currents in some cells (212/223, 95.11%). GABA-(100 micromol/L) activated currents was (1.32 +/- 0.74) nA (n = 84). However, pre-application of niflumic acid (1-100 micromol/L) and nitrendipine (specific blocker of L-calcium channel)(0.1-30 micromol/L) could inhibit the GABA-activated inward current which was identified to be GABAA receptor-mediated current. The inhibitory effects of niflumic acid and nitrendipine were concentration-dependent. The suppression rate of 10 micromol/L niflumic acid and nitrendipine to GABA-activated currents were (31.60% +/- 4.87%) (n = 19) and (43.60% < or = 5.10%) (n = 5), respectively. The desensitization of GABA-activated currents had double exponential characteristic. Tau value was (14.68 +/- 5.11) s (n = 6) and (175.8 +/- 42.67) s (n = 6, r = 0.9647), respectively. Pre-application of niflumic acid (100 micromol/L) and nickel chloride (nonspecific blocker of L-calcium channel) (100 micromol/L) altered tau value of the desensitization of GABA-activated currents, tau value reduced for (4.64 +/- 2.21) s (n = 3), (43.70 +/- 14.34) s ( n = 3, r = 0.9548) and (4.64 +/- 2.21) s (n = 3), (43.70 +/- 14.34) s (n = 3, r = 0.9721).

CONCLUSIONPre-application of niflumic acid exerts a more strong inhibitory effect on the peak value of GABA-activated current, which possibly is through blocking the calcium-activated chloride ion channel to accelerate the desensitization of GABA-activated currents.

Animals ; Animals, Newborn ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; drug effects ; Ganglia, Spinal ; drug effects ; physiology ; Membrane Potentials ; drug effects ; physiology ; Neurons ; drug effects ; physiology ; Niflumic Acid ; pharmacology ; Nitrendipine ; pharmacology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; gamma-Aminobutyric Acid ; pharmacology

Action Potentials ; drug effects ; physiology ; Animals ; Cadmium ; toxicity ; Calcium Channels, L-Type ; physiology ; Myocytes, Cardiac ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley

This article reports the investigation of the effect of carvedilol (Car) on T-type calcium current (I(Ca,T)) of noninfarcted ventricular myocytes in rabbit models of healed myocardial infarction (HMI). Rabbits with left anterior descending artery ligation were prepared and allowed to recover for 8 weeks, as HMI group. Animals undergoing an identical surgical procedure without coronary ligation were served as the sham-operated group (sham group). Whole cell voltage-clamp techniques were used to measure and compare currents in cells from the different groups. Noting that I(Ca,T) density in HMI cells increased markedly to -2.36 +/- 0.12 pA/pF (at -30 mV) compared with cells of sham, where little I(Ca,T) (-0.35 +/- 0.02 pA/pF) was observed. Meanwhile, further analysis revealed a significant hyperpolarizing shift of steady-state activation curve of I(Ca,T) in HMI cells, where the time constants of deactivation were prolonged and the time of recovery from inactivation was shortened. Finally, the amplitude of I(Ca,T) was increased. Carvedilol (1 micromol x L(-1)) was found to decrease the amplitude of I(Ca,T) to -1.38 +/- 0.07 pA/pF through inhibiting process of I(Ca,T) activation. Furthermore, carvedilol delayed recovery from inactivation of I(Ca,T) and shortened the time constants of deactivation in HMI cells. This study suggested that the application of carvedilol in HMI cells contributes to the dynamic changes in I(Ca,T) and may account for reduction of incidence of arrhythmia after myocardial infarction.
Adrenergic beta-Antagonists ; pharmacology ; Animals ; Calcium Channels, T-Type ; drug effects ; Carbazoles ; pharmacology ; Female ; Male ; Myocardial Infarction ; pathology ; physiopathology ; Myocytes, Cardiac ; drug effects ; physiology ; Patch-Clamp Techniques ; Propanolamines ; pharmacology ; Rabbits

OBJECTIVETo study the effect of salvianolic acid A (SAA) on L-type calcium current (I-CaL) in isolated ventricular myocytes of Sprague-Dawley rats.

METHODSSAA powder was dissolved in normal Tyrode's solution to reach the concentrations of 1, 10, 100, and 1000 μmol/L. The traditional whole-cell patch-clamp recording technique was employed to evaluate the effects of SAA on I-CaL in single ventricular myocytes which were prepared by Langendorff perfusion apparatus from Sprague-Dawley rats.

RESULTSSAA (1, 10, 100, and 1000 μmol/L) inhibited I-CaL peak value by 16.23%±1.3% (n=6, P<0.05), 22.9%±3.6% (n=6, P<0.05), 53.4%±3.0% (n=8, P<0.01), and 62.26%±2.9% (n=6, P<0.01), respectively. SAA reversibly inhibited I-CaL in a dose-dependent manner and with a half-blocking concentration (IC(50)) of 38.3 μmol/L. SAA at 100 μmol/L elevated the I-V curve obviously, and shifted the half-active voltage (V(0.5)) from (-15.78±0.86) mV to (-11.24 ±0.77) mV (n=6, P<0.05) and the slope (K) from 5.33±0.74 to 4.35±0.74 (n=6, P>0.05). However, it did not alter the shapes of I-V curve, steady-state inactivation curve, or recovery from inactivation curve.

CONCLUSIONSSAA inhibited I-CaL in a dose-dependent manner. It shifted the steady-state activation curve to a more positive voltage, which indicated that the drug affected the activated state of calcium channels, and suggested that the Ca(2+) antagonistic effect of SAA be beneficial in the treatment of myocardial ischemia reperfusion injury.

Animals ; Caffeic Acids ; chemistry ; pharmacology ; Calcium Channel Blockers ; chemistry ; pharmacology ; Calcium Channels, L-Type ; physiology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Lactates ; chemistry ; pharmacology ; Myocardial Ischemia ; drug therapy ; physiopathology ; Myocytes, Cardiac ; drug effects ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; drug therapy ; physiopathology