Endothelium-denuded Aortic Rings Research Articles

Effects of 3′,4′‐dihydroxyflavonol on vascular contractions of rat aortic rings

1. 3',4'-Dihydroxyflavonol (DiOHF) is an effective vasodilator with anti-oxidant activity. The aim of the present study was to elucidate the effects of DiOHF on vascular contractions. 2. Contractile and relaxation responses were determined in rat endothelium-denuded aortic rings mounted in organ baths. In addition, the phosphorylation of myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI-17) was determined using western blot analysis. Levels of GTP RhoA, as a marker of RhoA activation, were also measured. 3. Cumulative addition of increasing concentrations of NaF (3.0-12.0 mmol/L) or U46619 (1.0-1000 nmol/L) concentration-dependently increased vascular tension. These responses were inhibited by pretreatment of aortic rings with DiOHF (10, 30 or 100 micromol/L), which dose-dependently decreased vascular contractions induced by 8.0 mmol/L NaF, 30 nmol/L U46619, 0.1 micromol/L phenylephrine and 50 mmol/L KCl. 4. The K(+) channel blockers 4-aminopyridine (3 mmol/L), charybdotoxin (10 nmol/L), apamin (500 nmol/L) and glibenclamide (10 micromol/L) had no effect on vascular relaxation induced by DiOHF (1-30 micromol/L). 5. At 30 micromol/L, DiOHF decreased the activation of RhoA and subsequent phosphorylation of MYPT1, CPI-17 and MLC(20) to almost basal levels. 6. In conclusion, DiOHF decreases vascular contraction at least partly by inhibition of the RhoA/Rho-kinase pathway in rat endothelium-denuded aorta. These results suggest that DiOHF may have therapeutic potential in the treatment of cardiovascular diseases.

Vascular Smooth Muscle Dysfunction and Remodeling Induced by Ginsenoside Rg3, a Bioactive Component of Ginseng

Ginseng, one of most well-known herbal medicines, is widely and indiscreetly used among the patients with cardiovascular disorders, raising concern over abuse of this medicine and unwanted effects. In this study, we investigated the effects of ginsenoside Rg3 (Rg3), an active ingredient of ginseng, on vascular contractility and structural integrity to explore its potential vascular toxicity. In isolated rat aorta, Rg3 suppressed the normal agonist-induced contractile response. This suppression persisted even after a rigorous washout. In the endothelium-denuded aortic ring, impairment of vascular contractility by Rg3 was retained, suggesting that vascular smooth muscle was affected. In primary vascular smooth muscle cells, Rg3 abolished agonist-induced Ca(2+) increase, indicating that Ca(2+) regulation was disrupted. Rg3 suppressed the contraction induced by Bay K8644, an L-type Ca(2+) channel activator, whereas store-operated Ca(2+) channel or intracellular Ca(2+) store-mediated contraction was not affected, suggesting that the L-type Ca(2+) channel was selectively impaired by Rg3. These in vitro results were further confirmed in vivo where Rg3 treatment significantly attenuated the agonist-induced pressor response. More importantly, 4-week repeated treatment with Rg3 in normal animals induced eutrophic outward remodeling in the thoracic aorta, that is, it brought about an increased luminal area without changes in the wall area. These results suggest that Rg3 can induce the vascular smooth muscle dysfunction by disturbing Ca(2+) influx from the L-type Ca(2+) channel, ultimately leading to impaired vascular contractility and structural remodeling.

Cochlospermum vitifolium induces vasorelaxant and antihypertensive effects mainly by activation of NO/cGMP signaling pathway

Aim of the study Cochlospermum vitifolium is a medicinal plant used for the treatment of diabetes, hepatobilary and cardiovascular illnesses. The aim of current study was to determine the in vivo antihypertensive and in vitro functional vasorelaxant mechanism of methanol extract of Cochlospermum vitifolium (MECv) and naringenin (NG). Materials and methods Test material was assayed on rat isolated aorta rings test with- and without-endothelium to determine their vasorelaxant mechanism. Also, the in vivo antihypertensive effect was evaluated on spontaneously hypertensive rat (SHR) model. In addition, presence of NG into the extract was confirmed by reverse phase high performance liquid chromatography (RP-HPLC) analysis. Results MECv (120 mg/kg) and NG (50 and 160 mg/kg) showed acute antihypertensive effects on SHR when systolic and diastolic pressure were decreased at 1 h and 24 h after administration, respectively. Vasorelaxant effect of MECv and NG was shifted to the right when endothelium-intact aortic rings were pre-incubated with L-NAME (10 μM) and ODQ (1 μM). Also, NG relaxant curves were displaced to the right in the presence of tetraethylammonium (TEA, 1 mM) and 2-aminopyridine (2-AP, 100 μM) on endothelium-denuded aortic rings. Conclusion Experiments described above showed that MECv play an important role in hypertension regulation through NO synthesis and may be PGI 2 production and potassium channel activation on excessive endothelial dysfunction conditions. Unfortunately, presence of NG into the extract is not significant on bioactivity of the extract; however, this compound could be tested and evaluated as structural scaffold for future drug design for development of antihypertensive agents.

Sex hormones negatively modulate STIM‐1/Orai‐1 activity during hypertension: focus on calcium regulation

We recently showed that augmented vascular activation of STIM1/Orai1 pathway in male hypertensive rats contributes to the sex‐related impairment in intracellular Ca2+handling. We hypothesized that sex hormones contribute to the vascular protection in female hypertensive rats, via a decrease in STIM1/Orai1 activation. Endothelium‐denuded aortic rings from ovariectomized (OVX, for 15 weeks) or intact (SHAM) female stroke prone spontaneously hypertensive (SHRSP) and Wistar‐Kyoto (WKY) rats, were used to functionally evaluate Ca2+ influx‐induced contractions [upon depletion and reloading of intracellular Ca2+ (Ca2+loading period)]. Sarcoplasmic reticulum Ca+2 ATPase was inhibited with thapsigargin (10μM). During the Ca2+loading period, force development was augmented in aortas from SHRSP OVX vs. other groups and increased contraction was normalized with gadolinium (100μM, CRAC channel inhibitor), as well as blockade of STIM1 and Orai1 with neutralizing antibodies. Intracellular Ca2+ store release was increased in aortas from SHRSP vs. WKY, but no differences were observed between SHAM and OVX. Orai1 and STIM1 expression were increased in aortas from SHRSP OVX vs. the other groups. We conclude that sex hormones negatively modulate the expression and activity of STIM1/Orai1, contributing to the sex‐related differences in intracellular Ca2+ handling.Financial support: American Heart Association (AHA 09GRNT2250383)

Pharmacological Characterization of (3-Thienylidene)-3,4-Methylenedioxybenzoylhydrazide: A Novel Muscarinic Agonist With Antihypertensive Profile

Several new bioactive compounds of the N-acylhydrazone class were developed from the safrole, a Brazilian natural product obtained from sassafras oil (Ocotea pretiosa). This work investigated the effects on cardiovascular system of LASSBio-897, a new analogue of the lead compound 3,4-methylenedioxybenzoyl-2-thienylhydrazone named LASSBio-294. Thoracic aorta from Wistar-Kyoto (WKY) rats was prepared for isometric tension recording and for cGMP content determination. Blood pressure (BP) was measured in WKY rats and spontaneously hypertensive rats (SHR) after treatment with 1 mg/kg intravenously of LASSBio-897 and during 14 days' treatment of SHR with 1 mg/kg/day perorally. LASSBio-897 (0.05-1 micromol/l) exhibited a potent vasodilatory activity in phenylephrine (Phe)-contracted aortic rings from WKY rats. This effect was abolished in endothelium-denuded aortic rings and after treatment with the nitric oxide (NO) synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME) or the guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Also, LASSBio 897 (1 micromol/l) increased about 15 times the intracellular content of cGMP. LASSBio-897-induced vasodilation was totally inhibited by the muscarinic antagonist atropine and by the M(3) subtype selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), indicating the involvement of M(3) receptors. Intravenous administration of LASSBio-897 (1 mg/kg) produced significant hypotensive response in both WKY and SHR. The hypotensive effect of LASSBio-897 was also observed during the 14 days of oral administration. The novel N-acylhydrazone derivative LASSBio-897 exhibited a potent vasodilatory activity in aortic rings mediated by the NO/cGMP pathway via activation of endothelial M(3) receptors and was orally effective in reducing BP on SHR.

Dual effects of sodium aescinate on vascular tension in rat thoracic aorta

Sodium aescinate (SA) is used as a vasoactive drug in clinical treatment. This study was designed to investigate the effects of SA on rat isolated thoracic aorta and the possible mechanisms. Isometric tension was recorded in response to drugs in organ bath. The effects of SA obeyed an all-or-nothing response. SA in relatively low dose (> or = 50 microg/ml) had an endothelium-independent contractile effect in rat aorta (P<0.01), which depended on extracellular Ca(2+) influx via L-type Ca(2+) channel (P<0.05). SA in relatively high dose (> or = 100 microg/ml) also induced vasoconstriction in Ca(2+)-free medium (P<0.01), which was independent of the activity of inositol-1,4,5-trisphosphate receptor (IP(3)R), ryanodine receptor (RYR), and protein kinase C (PKC). SA in relatively high dose (> or = 100 microg/ml) dilated both endothelium-intact and endothelium-denuded aortic rings precontracted by phenylephrine (PE) or KCl (each P<0.01). SA inhibited extracellular Ca(2+) influx induced by PE or KCl (each P<0.01) and had no activation effect on K(+) channels on vascular smooth muscle. The relaxant effect of SA partly depended on the activity of NO synthase but not on the activity of cyclooxygenase. Taken together, this study indicated that SA had dual effects on vascular tension in rat isolated thoracic aorta.

New Insights in the Contribution of Voltage-Gated Nav Channels to Rat Aorta Contraction

BackgroundDespite increasing evidence for the presence of voltage-gated Na+ channels (Nav) isoforms and measurements of Nav channel currents with the patch-clamp technique in arterial myocytes, no information is available to date as to whether or not Nav channels play a functional role in arteries. The aim of the present work was to look for a physiological role of Nav channels in the control of rat aortic contraction.Methodology/Principal FindingsNav channels were detected in the aortic media by Western blot analysis and double immunofluorescence labeling for Nav channels and smooth muscle α-actin using specific antibodies. In parallel, using real time RT-PCR, we identified three Nav transcripts: Nav1.2, Nav1.3, and Nav1.5. Only the Nav1.2 isoform was found in the intact media and in freshly isolated myocytes excluding contamination by other cell types. Using the specific Nav channel agonist veratridine and antagonist tetrodotoxin (TTX), we unmasked a contribution of these channels in the response to the depolarizing agent KCl on rat aortic isometric tension recorded from endothelium-denuded aortic rings. Experimental conditions excluded a contribution of Nav channels from the perivascular sympathetic nerve terminals. Addition of low concentrations of KCl (2–10 mM), which induced moderate membrane depolarization (e.g., from −55.9±1.4 mV to −45.9±1.2 mV at 10 mmol/L as measured with microelectrodes), triggered a contraction potentiated by veratridine (100 µM) and blocked by TTX (1 µM). KB-R7943, an inhibitor of the reverse mode of the Na+/Ca2+ exchanger, mimicked the effect of TTX and had no additive effect in presence of TTX.Conclusions/SignificanceThese results define a new role for Nav channels in arterial physiology, and suggest that the TTX-sensitive Nav1.2 isoform, together with the Na+/Ca2+ exchanger, contributes to the contractile response of aortic myocytes at physiological range of membrane depolarization.

Enhancement of vascular relaxation in rat aorta by phytoestrogens from Curcuma comosa Roxb

The present study aims to examine the effects and mechanisms of Curcuma comosa Roxb., an indigenous medicinal plant containing phytoestrogens, on vascular relaxation. Using an organ bath system, acute exposure of intact or endothelium-denuded aortic rings to the hexane extract of C. comosa or an isolated diarylheptanoid compound, D3, did not induce relaxation. However, pre-incubation of aortic rings for 20 min with hexane extract of C. comosa (10 μg/ml) or the isolated diarylheptanoid compound, D3, (0.1, 1 and 10 μg/ml) markedly enhanced endothelial-dependent relaxation in response to ACh. The hexane extract did not modulate the relaxation of denuded aortic rings in response to SNP, which suggested a predominant effect on endothelial cells rather than on vascular smooth muscle cells. Co-incubation with ICI 182,780 (estrogen receptor antagonist), L-NAME (nitric oxide synthase inhibitor) or ODQ (guanylase cyclase inhibitor) inhibited the enhancing effects of C. comosa on ACh-induced relaxation. These findings suggest that the actions of C. comosa are mediated through estrogen receptor (ER) and NO-cGMP-dependent mechanisms. In addition, C. comosa also increased the phosphorylation of serine 1177 eNOS and serine 473 Akt proteins, and these effects were abolished by ICI 182,780. The results suggest that C. comosa acutely increases endothelium-dependent relaxation of aortic rings through the ER–Akt–eNOS pathway. This is the first evidence indicating non-genomic action of a novel phytoestrogen from C. comosa, on vascular relaxation.

Relaxant Effects of Matrine on Aortic Smooth Muscles of Guinea Pigs

To determine whether matrine, a kind of traditional Chinese medicinal alkaloid, can relax the aortic smooth muscles isolated from guinea pigs and to investigate the mechanism of its relaxant effects. Phenylephrine or potassium chloride concentration-dependent relaxation response of aortic smooth muscles to matrine was studied in the precontracted guinea pigs. Matrine (1 x 10(-4) mol/L -3.3 x 10(3) mol/L) relaxed the endothelium-denuded aortic rings pre-contracted sub-maximally with phenylephrine, in a concentration-dependent manner, and its pre-incubation (3.3 x 10(-3) mol/L) produced a significant rightward shift in the phenylephrine dose-response curve, but had no effects on the potassium chloride-induced contraction. The anti-contractile effect of matrine was not reduced by the highly selective ATP-dependent K+ channel blocker glibenclamide (10(-5) mol/L), either by the non-selective K+ channel blocker tetraethylammonium (10(-3) mol/L), or by the beta-antagonist propranolol (10(-5) mol/L). In either "normal" or "Ca(2+)-free" bathing medium, the phenylephrine-induced contraction was attenuated by matrine (3.3 x 10(-3) mol/L), indicating that the vasorelaxation was due to inhibition of intracellular and extracellular Ca2+ mobilization. Matrine inhibits phenylephrine-induced contractions by inhibiting activation of alpha-adrenoceptor and interfering with the release of intracellular Ca2+ and the influx of extracellular Ca2+.

Altered Endothelium-Dependent Vasoreactivity of Aortic Rings Follows L-Arginine and Potassium Co- Supplementation in Rats

Purpose: The use of L-arginine and potassium chloride separately as supplements has been reported to result in altered vascular reactivity. The concentration of either agent used has varied widely and there has been no report on the outcome of combined supplementation with both agents on vascular reactivity. We therefore designed this study to measure thoracic aortic ring reactivity after five-week co-supplementation with 1% L-arginine and 0.75% potassium chloride.Methods: Endothelium-intact and endothelium-denuded aortic rings obtained from Sprague-Dawley rats which received the combined supplements were subjected to graded concentrations of carbachol or noradrenaline in organ baths. The maximum responses (Emax)and the negative logarithm of the concentration producing 50% of maximum responses (pD2) were computed as indices of vasoreactivity.Results: Co-supplementation significantly enhanced the relaxant effects of carbachol irrespective of the status of the endothelium although relaxation was by far higher in endothelium-intact rings. In concentration-response curves obtained with noradrenaline, Emax and pD2 values were significantly (P < 0.05) reduced in endothelium-intact rings obtainedfrom rats which received the supplements. In endothelium-denuded rings, responses to noradrenaline were not significantly different from those of control.Conclusion: Co-supplementation with the agents enhances the relaxant effect of carbachol independent of the status of the endothelium while attenuating responses to noradrenaline in endothelium-dependent manner. This suggests that oral L-arginine and potassium cosupplementation may possess beneficial vascular effects.

Endothelium-independent vasorelaxation by the selective alpha estrogen receptor agonist propyl pyrazole triol in rat aortic smooth muscle

Abstract Objectives This study investigated the signalling mechanism of the relaxant responses to the estrogen receptor alpha (ERα) agonist PPT (propyl pyrazole triol) in endothelium-denuded rat aortic rings. Methods Several compounds, including protein kinase G (PKG) inhibitors and potassium channel inhibitors, were tested against PPT-dependent rat aortic relaxation. Cyclic GMP and cytosolic calcium responses to PPT in isolated aortic smooth muscle were investigated in parallel. Key findings PPT vasorelaxation was largely reduced by the selective ERα antagonist methyl-piperidinopyrazole (MPP; −91.6 ± 2.5%), by the selective PKG inhibitor Rp-8-Br-cGMP (−78.6 ± 4.9%), by the specific soluble guanylyl cyclase inhibitor ODQ (1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; −85.3 ± 5.2%) and to a lesser extent by the selective BKCa (large-conductance calcium- and voltage-activated potassium channel) inhibitor iberiotoxin (−59.3%), the selective IKCa (intermediate-conductance calcium-activated potassium channel) inhibitor TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; −50.7%) and the voltage-gated potassium channel inhibitor 4-aminopyridine (−40.8%). In isolated aortic smooth muscle, PPT strongly enhanced the cyclic GMP content (+144%) and Rp-8-Br-cGMP largely reduced the PPT-dependent calcium signal (−80.8%). Conclusions ERα receptor stimulation in rat aortic smooth muscle evokes a PKG-signalling pathway, likely triggering relaxation by BKCa and IKCa channel opening.

Endothelium-independent vasorelaxation by the selective alpha estrogen receptor agonist propyl pyrazole triol in rat aortic smooth muscle

This study investigated the signalling mechanism of the relaxant responses to the estrogen receptor (ERalpha) agonist PPT (propyl pyrazole triol) in endothelium-denuded rat aortic rings. Several compounds, including protein kinase G (PKG) inhibitors and potassium channel inhibitors, were tested against PPT-dependent rat aortic relaxation. Cyclic GMP and cytosolic calcium responses to PPT in isolated aortic smooth muscle were investigated in parallel. PPT vasorelaxation was largely reduced by the selective ERalpha antagonist methyl-piperidinopyrazole (MPP; -91.6+/-2.5%), by the selective PKG inhibitor Rp-8-Br-cGMP (-78.6+/-4.9%), by the specific soluble guanylyl cyclase inhibitor ODQ (1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; -85.3+/-5.2%) and to a lesser extent by the selective BKCa (large-conductance calcium- and voltage-activated potassium channel) inhibitor iberiotoxin (-59.3%), the selective IKCa (intermediate-conductance calcium-activated potassium channel) inhibitor TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; -50.7%) and the voltage-gated potassium channel inhibitor 4-aminopyridine (-40.8%). In isolated aortic smooth muscle, PPT strongly enhanced the cyclic GMP content (+144%) and Rp-8-Br-cGMP largely reduced the PPT-dependent calcium signal (-80.8%). ERalpha receptor stimulation in rat aortic smooth muscle evokes a PKG-signalling pathway, likely triggering relaxation by BKCa and IKCa channel opening.

Vasorelaxant properties of acid and neutral fractions of Dimerocostus strobilaceus Kuntze used by Kuna Indians of Panama

Ethnopharmacological relevanceDimerocostus strobilaceus is used by the Kuna Indians of Panama for the treatment of hypertension and other cardiovascular diseases. Aim of the studyWe investigated the vascular effects of acid and neutral fractions obtained from methanol and dichloromethane extracts of Dimerocostus strobilaceus. Materials and methodsThe acid and neutral methanol fractions (A-MeOH and N-MeOH) or acid and neutral dichlorometanic fractions (A-DCM and N-DCM) were tested using isolated rat aortic rings with or without endothelium pre-contracted by phenylephrine. We examined the ability of these different fractions at different concentrations to modify vascular responses induced by angiotensin II using endothelium-denuded aortic rings from Spontaneously Hypertensive Rats (SHR). ResultsIn aortic rings with intact endothelium A-MeOH, N-MeOH and A-DCM fractions produced a concentration-dependent vasorelaxation (62.4±5.2, 64.5±5.0 and 63.7±5.0%, respectively), whereas the N-DCM fraction did not produce any vasorelaxant effect. Maximal relaxation evocated by vasoactive fractions was substantially inhibited on aortic rings without endothelium.Our study demonstrates that A-MeOH, N-MeOH, A-DCM and N-DCM significantly reduce contractile responses induced by angiotensin-II on aortic rings. ConclusionsOur findings may contribute to a better understanding of the potential link between vascular properties observed with Dimerocostus strobilaceus and their ethnobotanical use.

Increased Activation of Stromal Interaction Molecule-1/Orai-1 in Aorta From Hypertensive Rats

Disturbances in the regulation of cytosolic calcium (Ca 2+ ) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca 2+ entry. Although STIMs act as Ca 2+ sensors for the intracellular Ca 2+ stores, Orai is the putative pore-forming component of Ca 2+ release–activated Ca 2+ channels at the plasma membrane. We hypothesized that augmented activation of Ca 2+ release–activated Ca 2+ /Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca 2+ influx. Depletion of intracellular Ca 2+ stores, which induces Ca 2+ release–activated Ca 2+ activation, was performed by placing arteries in Ca 2+ free-EGTA buffer. The addition of the Ca 2+ regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 μmol/L), an inhibitor of the sarcoplasmic reticulum Ca 2+ ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca 2+ release–activated Ca 2+ channel inhibitors 2-aminoethoxydiphenyl borate (100 μmol/L) or gadolinium (100 μmol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca 2+ levels in hypertension.

Endogenous methyl palmitate modulates nicotinic receptor-mediated transmission in the superior cervical ganglion

Nitric oxide (NO) is identified as the endothelium-derived relaxing factor and a neurotransmitter with a superfusion bioassay cascade technique. By using a similar technique with rat superior cervical ganglion (SCG) as donor tissue and rabbit endothelium-denuded aortic ring as detector tissue, we report here that a vasodilator, which is more potent than NO, is released in the SCG upon field electrical stimulation (FES) or addition of nicotine. Release of this vasodilator was enhanced by arginine analogs, including N(omega)-nitro-l-arginine (a NO synthase inhibitor), suggesting that it is not NO. Analysis by gas chromatography/mass spectrometry identified 2 saturated fatty acids, palmitic acid methyl ester (PAME) and stearic acid methyl ester (SAME), being released from the SCG upon FES in the presence of arginine analogs. Exogenous PAME but not SAME induced significant aortic dilation (EC(50) = 0.19 nM), indicating that PAME is the potent vasodilator. Release of PAME and SAME was significantly diminished in chronically decentralized SCG but not denervated SCG, suggesting the preganglionic origin. Furthermore, release of both fatty acids was calcium- and myosin light chain kinase-dependent, suggesting that both were released from axoplasmic vesicular stores. Electrophysiological studies further demonstrated that PAME but not SAME inhibited nicotine-induced inward currents in cultured SCG and the alpha7-nicotinic acetylcholine receptor-expressing Xenopus oocytes. Endogenous PAME appears to play a role in modulation of the autonomic ganglionic transmission and to complement the vasodilator effect of NO.

The vasodilator mechanism of sulfur dioxide on isolated aortic rings of rats: Involvement of the K + and Ca 2+ channels

The present study was designed to investigate vasodilator effect of endogenous gaseous sulfur dioxide (SO 2) and roles of Ca 2+ channels and K + channels in relaxations of SO 2 in isolated rat aortic rings. Isolated rat aortic rings were perfused in organ baths and changes in isometric tension were recorded. The results showed that: (1) SO 2 could relax isolated aortic rings contracted by norepinephrine in a dose-dependent manner (EC 50, 1247.38 ± 98.32 µM). The vasorelaxant effect of SO 2 at basal (110.34 ± 35.22 µM) and low concentrations (< 450 µM) was endothelium-dependent, while it was endothelium-independent at high concentrations (> 500 µM). (2) The vasorelaxation of 1500 µM SO 2 on both endothelium-intact and endothelium-denuded aortic rings was partially inhibited by nifedipine, an L-type calcium-channel blocker. (3) The vasoconstriction responses induced by CaCl 2 were inhibited by 1500 µM SO 2 on both endothelium-intact and endothelium-denuded aortic rings. (4) The initial fast vasoconstriction induced by intracellular Ca 2+ release was enhanced by 1500 µM SO 2, but the sustained vasoconstriction evoked by extracellular Ca 2+ influx was inhibited by 1500 µM SO 2. (5) Pretreated by 1500 µM SO 2, the vasoconstriction responses induced by norepinephrine or KCl were enhanced at low concentrations and inhibited at high concentrations. (6) The SO 2-induced vasorelaxation was partially inhibited by tetraethylammonium (TEA) and glibenclamide for both endothelium-intact and endothelium-denuded rings. For the endothelium-intact rings, the vasorelaxant effects induced by 30 and 300 µM SO 2 were partially inhibited by iberiotoxin. These results led to the conclusions that endogenous gaseous SO 2 could cause vasorelaxation on rat aortic rings in a dose-dependent manner. The vasorelaxant effects of SO 2 at basal and low concentrations were endothelium-dependent, which might be partly related to big-conductance Ca 2+-activated K + (BK Ca) channel. The mechanism of SO 2-induced vasorelaxation at high concentrations was shown to be endothelium-independent, which might be related to ATP-sensitive K + (K ATP) channel and L-type calcium-channel as well as possible alterations in Ca-influx and release pathways.

Relaxation evoked by extracellular Ca 2+ in rat aorta is nerve-independent and involves sarcoplasmic reticulum and L-type Ca 2+ channel

The perivascular nerve network expresses a Ca 2+ receptor that is activated by high extracellular Ca 2+ concentrations and causes vasorelaxation in resistance arteries. We have verified the influence of perivascular nerve fibers on the Ca 2+-induced relaxation in aortic rings. To test our hypothesis, either pre-contracted aortas isolated from rats after sensory denervation with capsaicin or aortic rings acutely denervated with phenol were stimulated to relax with increasing extracellular Ca 2+ concentration. We also studied the role of the endothelium on the Ca 2+-induced relaxation, and we verified the participation of endothelial/nonendothelial nitric oxide and cyclooxygenase–arachidonic acid metabolites. Additionally, the role of the sarcoplasmic reticulum, K + channels and L-type Ca 2+ channels on the Ca 2+-induced relaxation were evaluated. We have observed that the Ca 2+-induced relaxation is completely nerve independent, and it is potentiated by endothelial nitric oxide (NO). In endothelium-denuded aortic rings, indomethacin and AH6809 (PGF 2α receptor antagonist) enhance the relaxing response to Ca 2+. This relaxation is inhibited by thapsigargin and verapamil, while was not altered by tetraethylammonium. In conclusion, we have shown that perivascular nervous fibers do not participate in the Ca 2+-induced relaxation, which is potentiated by endothelial NO. In endothelium-denuded preparations, indomethacin and AH6809 enhance the relaxation induced by Ca 2+. The relaxing response to Ca 2+ was impaired by verapamil and thapsigargin, revealing the importance of L-type Ca 2+ channels and sarcoplasmic reticulum in this response.

Scutellarin‐induced endothelium‐independent relaxation in rat aorta

Scutellarin is a flavonoid extracted from the traditional Chinese herb, Erigeron breviscapus Hand Mazz. In the present study, the vasorelaxant effects of scutellarin and the underlying mechanism were investigated in isolated rat aorta. Scutellarin (3, 10, 30, 100 microm) caused a dose-dependent relaxation in both endothelium-intact and endothelium-denuded rat aortic rings precontracted with noradrenaline bitartrate (IC(50) = 7.7 +/- 0.6 microm), but not with potassium chloride. Tetraethylammonium, glibenclamide, atropine, propranolol, indomethacin and N(G)-nitro-l-arginine methyl ester had no influence on the vasorelaxant effect of scutellarin, which further excluded the involvement of potassium channels, muscarinic receptor, nitric oxide pathway and prostaglandin in this effect. Pretreatment with scutellarin decreased the tonic phase, but not the phasic phase of the noradrenaline bitartrate induced tension increment. Scutellarin also alleviated Ca(2+)-induced vasoconstriction in Ca(2+)-depleted/noradrenaline bitartrate pretreated rings in the presence of voltage-dependent calcium channel blocker verapamil. The noradrenaline bitartrate evoked intracellular calcium increase was inhibited by scutellarin. Scutellarin had no effect on phorbol-12,13-diacetate induced contraction in a calcium-free bath solution. These results showed that scutellarin could relax thoracic artery rings in an endothelium-independent manner. The mechanism seems to be the inhibition of extracellular calcium influx independent of the voltage-dependent calcium channel.

Vasoconstrictive effect of hydrogen sulfide involves downregulation of cAMP in vascular smooth muscle cells

Hydrogen sulfide (H(2)S), a new endogenous mediator, produces both vasorelaxation and vasoconstriction. This study was designed to examine whether cAMP mediates the vasoconstrictive effect of H(2)S. We found that NaHS at a concentration range of 10-100 microM (yields approximately 3-30 microM H(2)S) concentration-dependently reversed the vasodilation caused by isoprenaline and salbutamol, two beta-adrenoceptor agonists, and forskolin, a selective adenylyl cyclase activator, in phenylephrine-precontracted rat aortic rings. Pretreatment with NaHS (10-100 microM) for 5 min also significantly attenuated the vasorelaxant effect of salbutamol and forskolin. More importantly, NaHS (5-100 microM) significantly reversed forskolin-induced cAMP accumulation in vascular smooth muscle cells. However, NaHS produced significant, but weaker, vasoconstriction in the presence of N(G)-nitro-l-arginine methyl ester (100 microM), a nitric oxide synthase inhibitor, or in endothelium-denuded aortic rings. Blockade of ATP-sensitive potassium channels with glibenclamide (10 microM) failed to attenuate the vasoconstriction induced by H(2)S. Taken together, we demonstrated for the first time that the vasoconstrictive effect of H(2)S involves the adenyly cyclase/cAMP pathway.

Isometric contraction increases endothelial nitric oxide synthase activity via a calmodulin antagonist-sensitive pathway in rat aorta

In this work, the possibility that isometric contraction activates endothelial nitric oxide synthase (eNOS) in a calcium/calmodulin (Ca 2+/CaM)-dependent manner was examined in rat thoracic aorta. Step-wise stable contractile responses (precontractions) to phenylephrine were obtained in endothelium-intact and endothelium-denuded aortic rings. The subsequent addition of the NO synthase inhibitor, N Gnitro- l-arginine methyl ester ( l-NAME), or the soluble guanylyl cyclase inhibitor, ODQ, further augmented precontractions in a concentration-dependent manner. The amplitude of l-NAME- and ODQ-induced increases in tone were dependent on the level of precontraction; the maximal increments for l-NAME and ODQ were observed in arteries precontracted with phenylephrine at 67% of its maximal effect. Likewise, in endothelium-intact non-contracted arteries, l-NAME and ODQ induced small but significant increases in tone. Neither l-NAME nor ODQ had any effect in endothelium-denuded preparations. In endothelium-intact aortic rings precontracted with high K + solutions, l-NAME also elicited supplementary contractions dependent on precontraction level. The CaM antagonist, calmidazolium, inhibited in a concentration-dependent, noncompetitive, manner the effects of l-NAME on the tone of endothelium-intact phenylephrine-precontracted aortic rings. These results suggest that isometric contraction increases the activity of eNOS by means of the Ca 2+/CaM complex in rat aorta.