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*

OBJECTIVETo study the effect of gastrodin on isolated thoracic aorta rings of rats and to investigate the potential mechanism.

METHODSA perfusion model of isolated thoracic aorta rings of rats was applied. The effect of cumulative gastrodin (5, 50, 100,150, 200, and 250 μmol/L) on endothelium-intact aorta rings was investigated. The same procedure was applied to observe the effect of gastrodin on endothelium-intact/denuded aorta rings pre-contracted with 10(-6) mol/L phenylephrine hydrochloride (PE). The aorta rings incubated by 200 mmol/L gastrodin in the Ca(2+)-free (K-H) solution was contracted by using PE. The effect of 200 mmol/L gastrodin on endothelium-denuded aorta rings pre-contracted with 60 mmol/L KCl was also observed.

RESULTSCompared with the denuded gastrodin group, the intact gastrodin group could significantly relax the PE-contracted aorta rings (P<0.01). In Ca(2+)-free (K-H) solution KHS, the PE-induced contraction rate of aorta rings pre-incubated by gastrodin was 6.5%±0.7%, which was significantly less than the control group (11.8%±0.9%,P<0.01). However, after 3 mmol/L CaCl2 was added, the Ca(2+)-induced contraction in the gastrodin group (51.7%±2.4%) was similar to that in the control group (49.8%±2.8%). The contractile rate of rings in the KCl-contracted gastrodin group (96.3%±0.6%) was not significantly different from that in the control group (96.8%±1.2%).

CONCLUSIONSGastrodin has the effect of vasorelaxation on isolated thoracic aorta rings of rats. The mechanism of the vasorelaxation of gastrodin may mainly work through the inhibition of inositol 1, 4, 5-trisphosphosphate receptor on the sarcoplasmic reticulum of the arterial smooth muscle, which leads to the reduction of the Ca(2+) released from the sarcoplasmic reticulum.

Animals ; Aorta, Thoracic ; drug effects ; physiology ; Benzyl Alcohols ; pharmacology ; Calcium ; metabolism ; Endothelium, Vascular ; physiology ; Female ; Glucosides ; pharmacology ; In Vitro Techniques ; Male ; Phenylephrine ; pharmacology ; Rats ; Rats, Wistar ; Vasodilation ; drug effects

Scutellarin (SCU), a flavonoid from a traditional Chinese medicinal plant. Our previous study has demonstrated that SCU relaxes mouse aortic arteries mainly in an endothelium-depend-ent manner. In the present study, we investigated the vasoprotective effects of SCU against HR-induced endothelial dysfunction (ED) in isolated rat CA and the possible mechanisms involving cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG). The isolated endothelium-intact and endothelium-denuded rat CA rings were treated with HR injury. Evaluation of endothelium-dependent and -independent vasodilation relaxation of the CA rings were performed using wire myography and the protein expressions were assayed by Western blotting. SCU (10-1 000 μmol·L(-1)) could relax the endothelium-intact CA rings but not endothelium-denuded ones. In the intact CA rings, the PKG inhibitor, Rp-8-Br-cGMPS (PKGI-rp, 4 μmol·L(-1)), significantly blocked SCU (10-1 000 μmol·L(-1))-induced relaxation. The NO synthase (NOS) inhibitor, NO-nitro-L-arginine methylester (L-NAME, 100 μmol·L(-1)), did not significantly change the effects of SCU (10-1 000 μmol·L(-1)). HR treatment significantly impaired ACh-induced relaxation, which was reversed by pre-incubation with SCU (500 μmol·L(-1)), while HR treatment did not altered NTG-induced vasodilation. PKGI-rp (4 μmol·L(-1)) blocked the protective effects of SCU in HR-treated CA rings. Additionally, HR treatment reduced phosphorylated vasodilator-stimulated phosphoprotein (p-VASP, phosphorylated product of PKG), which was reversed by SCU pre-incubation, suggesting that SCU activated PKG phosphorylation against HR injury. SCU induces CA vasodilation in an endothelium-dependent manner to and repairs HR-induced impairment via activation of PKG signaling pathway.
Animals ; Apigenin ; pharmacology ; Cell Adhesion Molecules ; drug effects ; Cell Hypoxia ; Coronary Vessels ; drug effects ; Cyclic GMP ; analogs & derivatives ; metabolism ; pharmacology ; Cyclic GMP-Dependent Protein Kinases ; Glucuronates ; pharmacology ; Microfilament Proteins ; drug effects ; NG-Nitroarginine Methyl Ester ; metabolism ; pharmacology ; Phosphoproteins ; drug effects ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; complications ; physiopathology ; Signal Transduction ; drug effects ; Thionucleotides ; metabolism ; pharmacology ; Vasodilation ; drug effects ; physiology

OBJECTIVEIn our study, the function of the third-order branches of the mesentenc artery was measured by microvascular ring technique, which can be used to detect microvascular function in some disease related to microvascular dysfunction.

METHODSIsolated, fixed, standardized and then activated the third-order branches of rat mesenteric artery. Microvascular tone was measured by systolic and diastolic drags respectively, with the help of DMT tension apparatus and PowerLab data acquisition system.

RESULTSThe third-order branches of rat mesenteric artery showed excellent response to vasoactive drugs. The contraction effect of norepinephrine (NE) reached 19 in mN. When acetylcholine (Ach) or sodium nitroprusside (SNP) of 10(9)-10(5)mol/L was added, vascular tones showed gradient drop: 80% of maximal relaxation when adding ACh, while 95% of maximal relaxation when adding SNP.

CONCLUSIONThe third-order branches of the mesenteric artery function was successfully detected by using microvascular ring technique.

Acetylcholine ; pharmacology ; Animals ; In Vitro Techniques ; Male ; Mesenteric Arteries ; drug effects ; physiology ; Nitroprusside ; pharmacology ; Norepinephrine ; pharmacology ; Rats ; Vasoconstrictor Agents ; pharmacology ; Vasodilation ; physiology ; Vasodilator Agents ; pharmacology

OBJECTIVETo investigate the changes of vasoconstriction and vasodilatation under different temperature conditions and the protective effects of Vitamin E (Vit E) against endothelial injury induced by hypothermia.

METHODSThe tail arterial rings were prepared for isometric tension recording using multi wire myograph system. The effect of temperature on relaxation and construction was evaluated. Incubate the arterial rings with different concentration of Vit E when they were exposed to hypothermia, then acetylcholine (ACh)-induced endothelium-dependent relaxation was investigated to evaluate the activity of endothelial.

RESULTS(1) The hypothermia could enhanced the dose-dependent construction induced by PE in mice tail artery. (2) Exposure to hypothermia also resulted in increase of sodium nitroprusside (SNP)-induced re-After incubation with Vit E, the vascular relaxation responses to ACh increased in an endothelium-dependent manner, when compared with the hypothermia-treated group.

CONCLUSIONThe vascular function of constriction was attenuated by hypothermia, while the relaxation was increased. Vit E could prevent the hypothermia-induced decrease in vascular endothelial cells.

Animals ; Arteries ; drug effects ; physiology ; Cold Temperature ; Hypothermia ; In Vitro Techniques ; Male ; Mice ; Prazosin ; pharmacology ; Solanaceous Alkaloids ; pharmacology ; Vasoconstriction ; drug effects ; Vasodilation ; drug effects ; Vasodilator Agents ; pharmacology ; Vitamin E ; pharmacology

OBJECTIVETo investigate the vasodilative effect of paeonol in rat mesenteric artery and the mechanisms responsible for it.

METHODSRats were anaesthetized and sacrificed. The superior mesenteric artery was removed, dissected free of adherent tissue and cut into 2.0 mm long cylindrical segments. Isometric tension of artery rings was recorded by a myograph system in vitro. Concentration-relaxation curves of paeonol (17.8 μ mol/L to 3.16 mmol/L) were recorded on artery rings precontracted by potassium chloride (KCl) and concentration-contraction curves of KCl, 5-hydroxytryptamine (5-HT), noradrenaline (NA) or calcium chloride (CaCl2) were recorded in the presence of paeonol (10(-4.5), 10(-3.8), 10(-3.5) mol/L) respectively. And also, concentration-relaxation curves of paeonol were recorded in the presence of different potassium channel inhibitors and propranolol on rings precontracted with KCl respectively. To investigate the role of intracellular Ca(2+) release from Ca(2+) store, the contraction induced by NA (100 μ mol/L) and CaCl2 (2 mmol/L) in Ca(2+) free medium was observed in the presence of paeonol respectively.

RESULTSPaeonol relaxed artery rings precontracted by KCl in a concentration-dependent manner and the vasodilatation effect was not affected by endothelium denudation. Paeonol significant decreased the maximum contractions (Emax) induced by KCl, CaCl2, NA and 5-HT, as well as Emax induced by NA and CaCl2 in Ca(2+) -free medium, suggesting that paeonol dilated the artery via inhibiting the extracellular Ca(2+) influx mediated by voltage-dependent calcium channel, and receptor-mediated Ca(2+)-influx and release. Moreover, none of glibenclamide, tetraethylammonium, barium chlorded and propranolol affected the paeonol-induced vasodilatation, indicating that the vasodilatation was not contributed to ATP sensitive potassium channel, calcium-activated potassium channel, inwardly rectifying potassium channel, and β-adrenoceptor.

CONCLUSIONPaeonol induces non-endothelium dependent-vasodilatation in rat mesenteric artery via inhibiting voltage-dependent calcium channel-mediated extracellular Ca(2+) influx and receptor-mediated Ca(2+) influx and release.

Acetophenones ; pharmacology ; Adrenergic beta-Antagonists ; pharmacology ; Animals ; Calcium ; metabolism ; Calcium Chloride ; pharmacology ; Endothelium, Vascular ; drug effects ; physiology ; Extracellular Space ; drug effects ; metabolism ; Female ; In Vitro Techniques ; Intracellular Space ; drug effects ; metabolism ; Male ; Mesenteric Arteries ; drug effects ; physiology ; Norepinephrine ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Potassium Chloride ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Serotonin ; pharmacology ; Vasoconstriction ; drug effects ; Vasodilation ; drug effects

This study is to investigate the impairment and possible mechanism of endothelium-dependent relaxation of mice mesenteric arteries induced by mmLDL. Wire myography was employed to examine endothelial function of mesenteric arteries. Ultramicrostructure of mesenteric vascular beds were detected by transmission electron microscope. The results showed that endothelium cell edema and peeling, vascular elastic membrane fracture traces in mmLDL group. Endothelium-dependent relaxation was decreased in a time-dependent and dose-dependent manner by using mmLDL, compared with normal arteries. In endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation, the Rmax and pIC50 were decreased from (63 +/- 5) % and 6.42 +/- 0.09 of normal saline control to (31 +/- 3) % and 5.67 +/- 0.07 in mmLDL group (P < 0.001, P < 0.001), respectively. In nitric oxide (NO)-mediated relaxation, the Rmax and pIC50 were decreased from (45 +/- 4) % and 5.93 +/- 0.08 in normal saline control to (32 +/- 4) % and 5.43 +/- 0.11 in mmLDL group (P < 0.05, P < 0.01), respectively. There is no significant alteration of prostacyclin I2 (PGI2) pathway between these two groups. In conclusion, mmLDL induced the impairment of the ultramicrostructure of mesenteric vascular endothelium cell as well as the endothelium-dependent relaxation. The latter includes the dysfunction of NO- and EDHF pathway mediated endothelium-dependent relaxation.
Animals ; Biological Factors ; antagonists & inhibitors ; physiology ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; ultrastructure ; Epoprostenol ; antagonists & inhibitors ; physiology ; Female ; Lipoproteins, LDL ; administration & dosage ; pharmacology ; Male ; Mesenteric Arteries ; cytology ; physiology ; ultrastructure ; Mice ; Mice, Inbred ICR ; Microscopy, Electron, Transmission ; Nitric Oxide ; antagonists & inhibitors ; physiology ; Vasodilation ; drug effects

OBJECTIVETo investigate the vasodilating effect of capsaicin (CAP) on rat mesenteric artery and its mechanism.

METHODSThe third branch of the superior mesenteric artery in male Sprague-Dawley rat (250-350 g) was excised, the periadventitial fat and connective tissue were removed and the mesenteric artery was dissected into 2 mm rings. Each ring was placed in a 5 ml organ bath of DMT 610M system and the tension was recorded.

RESULTSCAP (10(-9)-10(-5) mol/L) relaxed endothelium-intact and endothelium-denuded mesenteric artery pre-constricted by phenylephrine (10(-5) mol/L), and the vasodilation in endothelium-intact mesenteric artery was stronger than that in endothelium-denuded one. Pretreatment with either L-NAME (3 X10(-4) mol/L), an inhibitor of nitric oxide synthase(NOS), or CGRP8-37 (2 X 10(-6) mol/L), an antagonist of calcitonin gene-related peptide (CGRP), for 30 min significantly attenuated the relaxation of endothelium-intact mesenteric artery induced by CAP. CGRP (10(-10)-3 X10(-8) mol/L) relaxed endothelium-intact and endothelium-denuded mesenteric artery pre-constricted by phenylephrine, and the vasodilation in endothelium-intact mesenteric artery was stronger than that in endothelium-denuded one. Substance P did not relax the mesenteric artery pre-constricted by phenylephrine.

CONCLUSIONCAP has partial endothelium-dependent relaxation effect on rat mesenteric artery, which may be mediated by activating the endothelial NOS-NO pathway. The endothelium-independent relaxation in rat mesenteric artery induced by CAP may be mediated by CGRP.

Animals ; Calcitonin Gene-Related Peptide ; metabolism ; Capsaicin ; pharmacology ; In Vitro Techniques ; Male ; Mesenteric Arteries ; drug effects ; physiology ; Peptide Fragments ; metabolism ; Rats ; Rats, Sprague-Dawley ; Vasodilation ; drug effects

Cinnamyl alcohol (CAL) is known as an antipyretic, and a recent study showed its vasodilatory activity without explaining the mechanism. Here we demonstrate the vasodilatory effect and the mechanism of action of CAL in rat thoracic aorta. The change of tension in aortic strips treated with CAL was measured in an organ bath system. In addition, vascular strips or human umbilical vein endothelial cells (HUVECs) were used for biochemical experiments such as Western blot and nitrite and cyclic guanosine monophosphate (cGMP) measurements. CAL attenuated the vasoconstriction of phenylephrine (PE, 1 microM)-precontracted aortic strips in an endothelium-dependent manner. CAL-induced vasorelaxation was inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 10(-4) M), methylene blue (MB; 10(-5) M) and 1 H-[1,2,4]-oxadiazolole-[4,3-a] quinoxalin-10one, (ODQ; 10(-6) or 10(-7) M) in the endothelium-intact aortic strips. Atrial natriuretic peptide (ANP; 10(-8) or 10(-9) M) did not affect the vasodilatory effect of CAL. The phosphorylation of endothelial nitric oxide synthase (eNOS) and generation of nitric oxide (NO) were stimulated by CAL treatment in HUVECs and inhibited by treatment with L-NAME. In addition, cGMP and PKG1 activation in aortic strips treated with CAL were also significantly inhibited by L-NAME. Furthermore, CAL relaxed Rho-kinase activator calpeptin-precontracted aortic strips, and the vasodilatory effect of CAL was inhibited by the ATP-sensitive K+ channel inhibitor glibenclamide (Gli; 10(-5) M) and the voltage-dependent K+ channel inhibitor 4-aminopyridine (4-AP; 2 x 10(-4) M). These results suggest that CAL induces vasorelaxation by activating K+ channels via the NO-cGMP-PKG pathway and the inhibition of Rho-kinase.
Animals ; Aorta/drug effects/metabolism/physiology ; Atrial Natriuretic Factor/pharmacology ; Cyclic GMP/*metabolism ; Cyclic GMP-Dependent Protein Kinases/*metabolism ; Dipeptides/pharmacology ; Human Umbilical Vein Endothelial Cells/drug effects/metabolism ; Humans ; Male ; Methylene Blue/pharmacology ; NG-Nitroarginine Methyl Ester/pharmacology ; Nitric Oxide/*metabolism ; Nitric Oxide Synthase/metabolism ; Oxadiazoles/pharmacology ; Phenylephrine/pharmacology ; Phosphorylation ; Potassium Channel Blockers/pharmacology ; Potassium Channels/*agonists ; Propanols/*pharmacology ; Quinoxalines/pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Vasoconstriction/*drug effects ; Vasodilation/drug effects ; rho-Associated Kinases/antagonists & inhibitors/*metabolism

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS-/- mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor NG-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca2+ levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca2+ levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol-treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca2+ chelator, calmodulin antagonist, and CaMKKbeta siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca2+-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca2+/CaMKKbeta-dependent eNOS phosphorylation and Ca2+-dependent eNOS dimerization.
Animals ; Aorta/*drug effects/physiology ; Enzyme Activation/drug effects ; Furans/*pharmacology ; Gene Deletion ; Human Umbilical Vein Endothelial Cells/drug effects/metabolism ; Humans ; Lignans/*pharmacology ; Mice ; Mice, Inbred C57BL ; Nitric Oxide/metabolism ; Nitric Oxide Synthase Type III/genetics/*metabolism ; Phosphatidylinositol 3-Kinases/metabolism ; Phosphoinositide Phospholipase C/metabolism ; Phosphorylation/drug effects ; Protein Multimerization/*drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; Vasodilation/*drug effects