Mechanism of vasorelaxation caused by N-Benzylsecoboldine in rat thoracic aorta

The vasorelaxing effect of N-benzylsecoboldine on the rat thoracic aorta was investigated, and we also compare it with nifedipine and cromakalim. In high K⁺ (60 mM) medium, Ca²⁺ (0.03–3 mM)-induced vasoconstriction was inhibited concentration-dependently by N-benzylsecoboldine, whereas this contraction was not altered by cromakalim. Cromakalim relaxed aortic rings precontracted with 15 but not 60 mM of K⁺. N-benzylsecoboldine and nifedipine were more potent and effective in producing relaxation in 60 mM than in 15 mM K⁺-induced contraction. N-benzylsecoboldine was found to be an ₁-adrenoceptor-blocking agent in rat thoracic aorta as revealed by its competitive antagonism of phenylephrine (PE)-induced contraction (pA₂=6.31 ± 0.04, pA₁₀=5.41 ± 0.03). This relaxing effect of N-benzylsecoboldine was not antagonized by indomethacin or methylene blue, and still persisted in endothelium-denuded aorta or in the presence of nifedipine (1 µM). The increase of inositol monophosphate caused by PE in rat aorta was significantly suppressed by N-benzylsecoboldine, but not by nifedipine or cromakalim. High concentration of N-benzylsecoboldine (100 µM) did not affect the contraction induced by B-HT 920, serotonin or PGF₂. Glibenclamide and charybdotoxin did not affect the relaxation of N-benzylsecoboldine in aortic rings precontracted with PE. Neither cGMP nor cAMP levels were changed by N-benzylsecoboldine. We suggest that N-benzyl-secoboldine relaxes rat thoracic aorta by suppressing the Ca²⁺ influx and also has antagonistic effect on ₁-adrenoceptors.     

Formononetin,an isoflavone,relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways

We evaluated the vasorelaxation effects of formononetin, an isoflavone/phytoestrogen found abundantly in Astragalus mongholicus Bunge, on rat isolated aorta and the underlying mechanisms involved. Cumulative administration of formononetin, genistein, daidzein and biochanin A relaxed phenylephrine-preconstricted aorta. Formononetin and biochanin A caused a similar magnitude of relaxation whereas daidzein was least potent. Mechanical removal of endothelium, L-NAME (100 μM) and methylene blue (10 μM) suppressed formononetin-induced relaxation. Formononetin increased endothelial nitric oxide (NO) synthase (eNOS), but not inducible NO synthase, activity with an up-regulation of eNOS mRNA and p-eNOS^Ser1177 protein expression. In endothelium-denuded preparations, formononetin-induced vasorelaxation was significantly reduced by glibenclamide (3 μM) and iberiotoxin (100 nM), and a combination of glibenclamide (3 μM) plus iberiotoxin (100 nM) abolished the relaxation. In contrast, formononetin-elicited endothelium-independent relaxation was not altered by ICI 182,780 (10 μM, an estrogen receptor (ERα/ERβ) antagonist) or mifepristone (10 μM, a progesterone receptor antagonist). In single aortic smooth muscle cells, formononetin caused opening of iberiotoxin-sensitive Ca²⁺-activated K⁺ (BK_Ca) channels and glibenclamide-sensitive adenosine triphosphate (ATP)-dependent K⁺ (K_ATP) channels. Thus, our results suggest that formononetin caused vascular relaxation via endothelium/NO-dependent mechanism and endothelium-independent mechanism which involves the activation of BK_Ca and K_ATP channels.     

Endothelium-independent vasorelaxant effect of sodium ferulate on rat thoracic aorta

AimsThis study was designed to investigate the effects of sodium ferulate (SF) on rat isolated thoracic aortas and the possible mechanisms.Main methodsIsometric tension was recorded in response to drugs in organ bath. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured using Fluo-3 in cultured rat aortic smooth muscle cells (RASMC).Key findingsSF (0.1–30 mM) relaxed the isolated aortic rings precontracted with phenylephrine (PE) and high-K⁺ in a concentration-dependent manner with respective pD₂ of 2.7 ± 0.02 and 2.6 ± 0.06. Mechanical removal of endothelium did not significantly modify the SF-induced relaxation. In Ca²⁺-free solution, SF noticeably inhibited extracellular Ca²⁺-induced contraction in high-K⁺ and PE pre-challenged rings, and suppressed the transient contraction induced by PE and caffeine. The vasorelaxant effect of SF was unaffected by various K⁺ channel blockers such as tetraethylammonium, glibenclamide, 4-aminopyridine, and barium chloride. In addition, SF concentration-dependently reduced the contraction induced by phorbol-12-myristate-13-acetate, an activator of protein kinase C (PKC), in the absence of extracellular Ca²⁺, with the pD₂ of 2.9 ± 0.03. In RASMC, SF had no effect on PE- or KCl-induced [Ca²⁺]_i increase either in the presence or in the absence of external Ca²⁺.SignificanceThese results indicate that SF acts directly as a non-selective relaxant to vascular smooth muscle. The direct inhibition of the common pathway after [Ca²⁺]_i increase may account for the SF-induced relaxation in Ca²⁺-dependent contraction, while the blockage of the PKC-mediated contractile mechanism is likely responsible for the SF-induced relaxation in Ca²⁺-independent contraction.     

Role of Trace Elements,Oxidative Stress and Immune System: a Triad in Premature Ovarian Failure

Cadmium is an environmental pollutant closely linked with cardiovascular diseases that seems to involve endothelium dysfunction and reduced nitric oxide (NO) bioavailability. Knowing that NO causes dilatation through the activation of potassium channels and Na⁺/K⁺-ATPase, we aimed to determine whether acute cadmium administration (10 μM) alters the participation of K⁺ channels, voltage-activated calcium channel, and Na⁺/K⁺-ATPase activity in vascular function of isolated aortic rings of rats. Cadmium did not modify the acetylcholine-induced relaxation. After L-NAME addition, the relaxation induced by acetylcholine was abolished in presence or absence of cadmium, suggesting that acutely, this metal did not change NO release. However, tetraethylammonium (a nonselective K⁺ channels blocker) reduced acetylcholine-induced relaxation but this effect was lower in the preparations with cadmium, suggesting a decrease of K⁺ channels function in acetylcholine response after cadmium incubation. Apamin (a selective blocker of small Ca²⁺-activated K⁺ channels—SK_Ca), iberiotoxin (a selective blocker of large-conductance Ca²⁺-activated K⁺ channels—BK_Ca), and verapamil (a blocker of calcium channel) reduced the endothelium-dependent relaxation only in the absence of cadmium. Finally, cadmium decreases Na⁺/K⁺-ATPase activity. Our results provide evidence that the cadmium acute incubation unaffected the calcium-activated potassium channels (SK_Ca and BK_Ca) and voltage-calcium channels on the acetylcholine vasodilatation. In addition, acute cadmium incubation seems to reduce the Na⁺/K⁺-ATPase activity.     

β-ADRENOCEPTOR-MEDIATED RELAXATION INHIBITED BY TETRAPENTYLAMMONIUM IONS IN RAT MESENTERIC ARTERY

The aim of this study was to examine the contribution of K⁺ channel activation to β-adrenoceptor-mediated relaxation in rat mesenteric arteries. Isoprenaline and fenoterol concentration-dependently relaxed the phenylephrine-preconstricted endothelium-intact arteries of the rat with EC₅₀ values of 0.26 ± 0.03 μM and 0.87 ± 0.12 μM, respectively. β-Adrenoceptor-mediated relaxation was significantly attenuated upon removal of endothelium. Tetrapentylammonium ions (TPA⁺) at low concentrations (1–5 μM) inhibited relaxations induced by β-adrenoceptor agonists in arteries with and without endothelium, while glibenclamide (3 μM) had no effect. TPA⁺ (5 μM) inhibited isoprenaline-induced relaxation in the presence of either iberiotoxin (100 nM) or glibenclamide (3 μM). TPA⁺ did not alter forskolin-induced relaxation. In the presence of 60 mM extracellular K⁺, the relaxations induced by two agonists were reduced in endothelium-intact arteries and abolished in endothelium-denuded arteries. The present results suggest that the activation of TPA⁺-sensitive K⁺ channels contributes toward the relaxations mediated through β- and β₂-adrenoceptor stimulation in rat mesenteric arteries. © 1998 Elsevier Science Inc.     

Cerebral vasodilator properties of Danshen and Gegen: A study of their combined efficacy and mechanisms of actions

Danshen and Gegen are two commonly used Chinese herbal medicines for treatment of cardiovascular diseases. The aim of the present study was to elucidate the combination effects of these two herbs on cerebral vascular tone and their underlying mechanisms of actions. Basilar artery rings were obtained from rats and precontracted with U46619. Cumulative administrations of aqueous extracts of Danshen, Gegen, or the two herbs combined (DG; ratio 7:3) produced concentration-dependent relaxation of the artery rings. Statistical analysis on these findings produced a combination index (CI) of 1.041 at ED₅₀, which indicates the two herbs produced additive vasodilator effects when used as a combined decoction. Removal of the endothelium had no effect on the vasodilator properties of Danshen, Gegen, and DG. However, their maximum effects (Imax) were significantly blunted by a K_ATP channel inhibitor glibenclamide, a non-selective K⁺ channel inhibitor tetraethylammonium (TEA), and by a combination of K⁺ channel inhibitors (glibenclamide + TEA + iberiotoxin + 4-aminopyridine + barium chloride). In addition, Danshen, Gegen, and DG produced augmentation of K_ATP currents and inhibited Ca²⁺ influx in vascular smooth muscle cells isolated from rat basilar arteries. Furthermore, these agents inhibited CaCl₂-induced contraction in the artery rings. In conclusion, the present study showed that Danshen and Gegen produced additive vasodilator effects on rat cerebral basilar arteries. These effects were independent of endothelium-derived relaxant factors (EDRF), but required the opening of K_ATP channels and inhibition of Ca²⁺ influx in the vascular smooth muscle cells. It is suspected that the cerebral vasodilator effects of Danshen and Gegen produced either on their own or in combination, can help patients with obstructive cerebrovascular diseases.     

Vascular smooth muscle relaxation by endothelium-dependent β1-adrenergic action

1. Norepinephrine (NE) (10⁻⁵M) in rabbit aorta relaxed ring segments with endothelium precontracted with 10⁻⁶M NE, but not segments without endothelium.2. The relaxation was inhibited with metoprolol and methylene blue, but not inhibited with yohimbine and indomethacin.3. NE (10⁻⁵ M) significantly elevated tissue c-GMP levels in segments with endothelium.4. These studies suggest that the vascular relaxation by high doses of NE is mediated by the release of endothelium-derived relaxing factor (EDRF) induced by the stimulation of β₁-adrenoceptor.     

Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1

Plasma membrane Ca²⁺ pumps (PMCA) that expel Ca²⁺ from cells are encoded by four genes (PMCA1–4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca²⁺-Mg²⁺-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K_i value of 46 ± 5 µM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca²⁺-Mg²⁺-ATPase in human embryonic kidney-293 cells with a higher K_i value (105 ± 11 µM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In deendothelialized aortic rings, caloxin 1b1 (200 µM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca²⁺] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium. coronary artery; rat aorta; smooth muscle; endothelium     

Vasorelaxant effect in rat aortic rings through calcium channel blockage: A preliminary in vitro assessment of a 1,3,4-oxadiazole derivative

The study was undertaken on the basis of several reports in the literature that relaxation of vascular smooth muscles is a good treatment strategy in hypertension, angina and other cardiovascular disorders. Oxadiazoles have been reported to have effect on vascular smooth muscles and calcium influx. The goals of our current in vitro study were to investigate the effect of a 1,3,4-oxadiazole derivative on vascular smooth muscles in rat aorta, and to elucidate the associated signaling pathway. NOX-1 induced a relaxation of vascular smooth muscles in both endothelium intact and denuded rat aortic rings precontracted with norepinephrine or phenylephrine or KCl. NOX-1 also significantly antagonized cumulative dose-response effect of norepinephrine, phenylephrine, KCl or calcium with reduction in submaximal contractions. Verapamil, an L-type of calcium channel blocker, effectively attenuated phenylephrine and calcium induced contractions in aortic rings. Incubation with NOX-1 and verapamil did not significantly alter the dose-response curve of phenylephrine or calcium compared to verapamil treatment alone indicating L-type Ca²⁺ channel blockage leads to loss of NOX-1 activity. Hence it can be concluded NOX-1 exhibited vasorelaxant action by inhibiting calcium influx from extracellular space to intracellular space through L-type of calcium channels.     

Dietary ascorbic acid deficiency in guinea pigs: No effect on ethanol preference,spiroperidol binding,or monoamine oxidase activity

Ascorbic acid levels are commonly reported to be decreased in alcoholics. Although this deficiency could be due to dietary factors, there is evidence that ascorbic acid may be involved in the metabolism and acute effects of ethanol, possibly related to the pathogenesis of alcoholism. Therefore, we examined ethanol preference in guinea pigs receiving an ascorbate deficient $\text{vs}$ a normal diet. Brain and spleen ascorbic acid levels were dramatically decreased, but ethanol preference was not altered by the acute dietary deficiency of this vitamin. In addition, an acute stressor (cold water swim), alone or in combination with ascorbate deficiency, had no effect on ethanol preference. At termination of the experiment, two measures of brain aminergic function (MAO activity and ³H-spiroperidol binding), purportedly altered by ethanol or ascorbic acid or both, were not associated with tissue ascorbate levels.     

Endothelium-dependent and -independent relaxation of rat aorta induced by extract of Schizophyllum commune

Schizophyllum commune (SC) is widely consumed by Chinese, especially in southern part of China. The aim of the present study was to assess the extract of SC on vascular tone and the mechanisms involved. Experiments were performed on aorta of 18-week-old male Sprague-Dawley rats. Dried SC was extracted with 50% ethanol, 90% ethanol and deionized water, respectively. The effects of SC on the isometric tension of rat aortic rings were measured. Protein expression for the endothelial nitric oxide synthase (eNOS) was also determined in the primarily cultured rat aortic arterial endothelial cells (RAECs). The results showed that the water extract of SC induced a marked relaxation in aortic rings with or without endothelium. After the pretreatments of N^ω-nitro-l-arginine methyl ester, indomethacin, RP-cAMP, and methylene blue, the SC-induced relaxation was significantly decreased. In addition, the contraction due to Ca²⁺ influx and intracellular Ca²⁺ release was also inhibited by SC. Furthermore, expression of the eNOS protein was significantly elevated in RAECs after treatment of SC. In conclusion, the water extract of SC induces an endothelium-dependent and -independent relaxation in rat aorta. The relaxing effect of SC involves the modulation of NO-cGMP-dependent pathways, PGI₂-cAMP-depedent pathways, Ca²⁺ influx though calcium channels and intracellular Ca²⁺ release.     

A novel mechanism of vascular relaxation induced by sodium nitroprusside in the isolated rat aorta

Sodium nitroprusside (SNP) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle (VSM). Endothelium modulates the vascular tone through the release of vasoactive agents, such as NO. The aim of this study was to investigate the contribution of the endothelium on SNP vasorelaxation, NO release and Ca²⁺ mobilization. Vascular reactivity experiments showed that endothelium potentiates the SNP-relaxation in rat aortic rings and this effect was abolished by l-NAME. SNP-relaxation in intact endothelium aorta was inhibited by NOS inhibitors for the constitutive isoforms (cNOS). Furthermore, endogenous NO is involved on the SNP-effect and this endogenous NO is released by cNOS. Moreover, Ca²⁺ mobilization study shows that l-NAME inhibited the reduction of Ca²⁺-concentration in VSM cells and reduced the increase in Ca²⁺-concentration in endothelial cells induced by SNP. This enhancement in Ca²⁺-concentration in the endothelial cells is due to a voltage-dependent Ca²⁺ channels activation. The present findings indicate that the relaxation and [Ca²⁺]_i decrease induced by SNP in VSM cells is potentiated by endothelial production of NO by cNOS-activation in rat aorta.     

Restricted ability of human mast cell tryptase to activate proteinase-activated receptor-2 in rat aorta

We investigated the potential of human mast cell tryptase to induce relaxation of rat aorta. Trypsin and the selective PAR2-activating peptide (PAR2-AP) SLIGRL-NH2 stimulated robust relaxation of phenylephrine-precontracted rat aortic rings. However, human lung tryptase (1-100 nM) either in the presence or absence of heparin failed to induce any significant relaxation. Notwithstanding, incubation of the aorta with tryptase (100 nM), following the addition of a peptide corresponding to the cleavage/activation sequence of rat PAR2 (rPAR2), resulted in relaxation of precontracted tissue due to the proteolytic release of the PAR2-AP SLIGRL/ from the parent peptide. Thus, tryptase was enzymatically active in the bioassay system. Preincubation of aorta with neuraminidase to remove cell-surface sialic acid unmasked the ability of tryptase to induce relaxation of the aorta, but had no effect on relaxation induced by trypsin, SLIGRL-NH2, or acetylcholine (Ach). Like trypsin and SLIGRL-NH2, the tryptase-induced relaxation was inhibited by either removal of the endothelium or pretreatment of the tissue with NG-nitro-L-arginine methyl ester (L-NAME), suggesting an endothelium-derived nitric oxide mechanism. Interestingly, tryptase in the presence of heparin failed to induce relaxation of precontracted neuraminidase-treated rat aorta. We conclude that tryptase-induced relaxation of rat aorta, most likely via PAR2, is tightly regulated by heparin and cell-surface sialic acid.     

Vasorelaxant responses to endomorphins, nociceptin, albuterol, and adrenomedullin in isolated rat aorta

The endogenous peptides endomorphins 1 and 2 are newly discovered, potent, selective mu-opioid receptor agonists. In the present study, the effects of endomorphins 1 and 2 on vascular smooth muscle tone were investigated on isolated rings from rat aorta with and without endothelium. In rings precontracted with phenylephrine, endomorphins 1 and 2 at concentrations of 0.1 and 1.0 microM, nociceptin at concentrations of 1-100 microM, and adrenomedullin at concentrations of 0.01-1.0 microM induced concentration dependent relaxant responses. The endomorphins and nociceptin were less potent than adrenomedullin. No relaxation was induced by endomorphins 1 and 2 in aortic rings denuded of endothelium and precontracted with phenylephrine. The results of the present studies demonstrate that the endomorphins relax aortic vascular smooth muscle from the rat aorta by an endothelium-dependant mechanism.     

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta

AimTo investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta.MethodsThe relaxation response to NaOH-induced extracellular alkalinization (7.4–8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10^?6 M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10^?5 M), NG-nitro-l-arginine methyl ester (L-NAME, 10^?4 M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10^?7 M), 2,5-dimethylbenzimidazole (DMB, 2 × 10^?5 M) and methyl-β-cyclodextrin (10^?2 M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 μM), in the presence and absence of DMB (2 × 10^?5 M).ResultsThe extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca²⁺/calmodulin and Na⁺/Ca²⁺ exchanger (NCX), and partially blunted by the caveolae disassembly.ConclusionsThese results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca²⁺ concentration and activating the Ca²⁺/calmodulin-dependent NOS. In turn, NO is released promoting relaxation.     

(±)-Praeruptorin A enantiomers exert distinct relaxant effects on isolated rat aorta rings dependent on endothelium and nitric oxide synthesis

Praeruptorin A is a coumarin compound naturally occurring in the roots of Peucedanum praeruptorum Dunn., a commonly used traditional Chinese medicine for the treatment of certain respiratory diseases and hypertension. Although previous studies indicated the relaxant effects of (±)-praeruptorin A on tracheal and arterial preparations, little is known about the functional characteristics of the enantiomers. In the present study, the two enantiomers were successfully isolated and identified by using a preparative Daicel Chiralpak AD-H column, and their relaxant effects on aorta rings were observed and compared. (+)-Praeruptorin A showed more potent relaxation than (?)-praeruptorin A against KCl- and phenylephrine-induced contraction of rat isolated aortic rings with intact endothelium. Removal of the endothelium remarkably reduced the relaxant effect of (+)-praeruptorin A but not that of (?)-praeruptorin A. Pretreatment of aortic rings with N^ω-nitro-l-arginine methyl ester (l-NAME, an inhibitor of nitric oxide synthase) or methylene blue (MB, a soluble guanylyl cyclase inhibitor) resulted in similar changes of the relaxant effects of the two enantiomers to endothelium removal. Molecular docking studies also demonstrated that (+)-praeruptorin A was in more agreement to nitric oxide synthase pharmacophores than (?)-praeruptorin A. On the other hand, the two enantiomers of praeruptorin A could slightly attenuate the contraction of rat aortic rings induced by internal Ca²⁺ release from sarcoplasmic reticulum (SR). These findings indicated that (+)-praeruptorin A and (?)-praeruptorin A exerted distinct relaxant effects on isolated rat aorta rings, which might be mainly attributed to nitric oxide synthesis catalyzed by endothelial nitric oxide synthase.     

Na+K+-ATPase Activity and K+ Channels Differently Contribute to Vascular Relaxation in Male and Female Rats

Gender associated differences in vascular reactivity regulation might contribute to the low incidence of cardiovascular disease in women. Cardiovascular protection is suggested to depend on female sex hormones’ effects on endothelial function and vascular tone regulation. We tested the hypothesis that potassium (K⁺) channels and Na⁺K⁺-ATPase may be involved in the gender-based vascular reactivity differences. Aortic rings from female and male rats were used to examine the involvement of K⁺ channels and Na⁺K⁺-ATPase in vascular reactivity. Acetylcholine (ACh)-induced relaxation was analyzed in the presence of L-NAME (100 µM) and the following K⁺ channels blockers: tetraethylammonium (TEA, 2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 µM) and charybdotoxin (ChTX, 0.1 µM). The ACh-induced relaxation sensitivity was greater in the female group. After incubation with 4-AP the ACh-dependent relaxation was reduced in both groups. However, the dAUC was greater in males, suggesting that the voltage-dependent K⁺ channel (K_v) participates more in males. Inhibition of the three types of Ca²⁺-activated K⁺ channels induced a greater reduction in R_max in females than in males. The functional activity of the Na⁺K⁺-ATPase was evaluated by KCl-induced relaxation after L-NAME and OUAincubation. OUA reduced K⁺-induced relaxation in female and male groups, however, it was greater in males, suggesting a greater Na⁺K⁺-ATPase functional activity. L-NAME reduced K⁺-induced relaxation only in the female group, suggesting that nitric oxide (NO) participates more in their functional Na⁺K⁺-ATPase activity. These results suggest that the K⁺ channels involved in the gender-based vascular relaxation differences are the large conductance Ca²⁺-activated K⁺ channels (BK_Ca) in females and K_v in males and in the K⁺-induced relaxation and the Na⁺K⁺-ATPase vascular functional activity is greater in males.     

High-affinity sodium-dependent uptake of ascorbic acid by rat osteoblasts

Summary Ascorbic acid is essential for the formation of bone by osteoblasts, but the mechanism by which osteoblasts transport ascorbate has not been investigated previously. We examined the uptake ofl-[¹⁴C]ascorbate by a rat osteoblast-like cell line (ROS 17/2.8) and by primary cultures of rat calvaria cells. In both systems, cells accumulatedl-[¹⁴C]ascorbate during incubations of 1–30 min at 37°C. Unlike propionic acid, which diffuses across membranes in protonated form, ascorbic acid did not markedly alter cytosolic pH. Initial ascorbate uptake rate saturated with increasing substrate concentration, reflecting a high-affinity interaction that could be described by Michaelis-Menten kinetics (apparentK _m =30±2 m andV _max=1460±140 nmol ascorbate/g protein/min in ROS 17/2.8 cells incubated with 138mm extracellular Na⁺). Consistent with a stereoselective carrier-mediated mechanism, unlabeledl-ascorbate was a more potent inhibitor (IC₅₀=30±5 m) ofl-[¹⁴C]ascorbate transport than wasd-isoascorbate (IC₅₀=380±55 m). Uptake was dependent on both temperature and Na⁺, since it was inhibited by cooling to 4°C and by substitution of K⁺, Li⁺ or N-methyl-d-glucamine for extracellular Na⁺. Decreasing the external Na⁺ concentration lowered both the affinity of the transporter for ascorbate and the apparent maximum velocity of transport. We conclude that osteoblasts possess a stereoselective, high-affinity, Na⁺-dependent transport system for ascorbate. This system may play a role in the regulation of bone formation.     

Evidence for electrogenic sodium-dependent ascorbate transport in rat astroglia

The dependence of ascorbate uptake on external cations was studied in primary cultures of rat cerebral astrocytes. Initial rates of ascorbate uptake were diminished by lowering the external concentrations of either Ca²⁺ or Na⁺. The Na⁺-dependence of astroglial ascorbate uptake gave Hill coefficients of approximately 2, consistent with a Na⁺-ascorbate cotransport system having stoichiometry of 2 Na⁺1 ascorbate anion. Raising external K⁺ concentration incrementally from 5.4 to 100 mM, so as to depolarize the plasma membrane, decreased the initial rate of ascorbate uptake, with the degree of inhibition depending on the level of K⁺. The depolarizing ionophores gramicidin and nystatin slowed ascorbate uptake by astrocytes incubated in 5.4 mM K⁺; whereas, the nondepolarizing ionophore valinomycin did not. Qualitatively similar results were obtained whether or not astrocytes were pretreated with dibutyryl cyclic AMP (0.25 mM for 2 weeks) to induce stellation. These data are consistent with the existence of an electrogenic Na⁺-ascorbate cotransport system through which the rate of ascorbate uptake is modulated by endogenous agents, such as K⁺, that alter astroglial membrane potential.     

N-acetylcysteine-induced vasodilation involves voltage-gated potassium channels in rat aorta

AimsN-acetylcysteine (NAC) has a protective effect against vascular dysfunction by decreasing the level of reactive oxygen species (ROS) in experimental and human hypertension. This study was designed to examine whether NAC would relax vascular rings in vitro via nitric oxide–cyclic guanosine monophosphate (NO–cGMP) pathway, extracellular Ca²⁺ and/or K⁺ channels.Main methodsRat aortic arteries were mounted in an organ bath, contracted with 0.1, 0.5 or 1 µmol/L phenylephrine to plateau, and the vasodilatory effect of NAC was examined in the absence or presence of ROS scavengers, inhibitors of NO–cGMP pathway or K⁺ channels. Vascular smooth muscle cells (VSMCs) were loaded with a calcium sensitive fluorescent dye fluo-3 AM, and [Ca²⁺]_i was determined with laser-scanning confocal microscopy.Key findingsNAC (0.1–4 mmol/L) dose-dependently relaxed rat aorta pre-contracted with phenylephrine. Endothelium removal, endothelial nitric oxide synthase inhibitor N^ω-Nitro-l-arginine (L-NNA) (100 µmol/L) or soluble guanylyl cyclase (sGC) inhibitor (ODQ) (10 µmol/L) did not affect NAC-induced vasodilation. In contrast, NAC-induced vasodilation was blunted after extracellular calcium was removed and calcium imaging showed that 4 mmol/L NAC quickly decreased [Ca²⁺]_i in fluo-3 AM loaded VSMCs. NAC-induced vasodilation was significantly reduced in the presence of voltage-gated K⁺ channels (Kv) inhibitor 4-aminopyridine (4-AP).SignificanceThe vasodilatory effect of NAC may be explained at least partly by activation of voltage-gated K⁺ channels.