ABSTRACT
BACKGROUND: The α2-adrenergic sedative/anesthetic agent dexmedetomidine exerts biphasic effects on isolated arteries, causing endothelium-dependent relaxations at concentrations at or below 30 nM, followed by contractions at higher concentrations. L-arginine is a common substrate of endothelial nitric oxide synthase and arginases. This study was designed to investigate the role of L-arginine in modulating the overall vascular response to dexmedetomidine. METHODS: Isometric tension was measured in isolated aortic rings of Sprague Dawley rats. Cumulative concentrations of dexmedetomidine (10 nM to 10 µM) were added to quiescent rings (with and without endothelium) after previous incubation with vehicle, N-nitro-L-arginine methyl ester hydrochloride (L-NAME; nitric oxide synthase inhibitor), prazosin (α1-adrenergic antagonist), rauwolscine (α2-adrenergic antagonist), L-arginine, (S)-(2-boronethyl)-L-cysteine hydrochloride (arginase inhibitor), N-hydroxy-L-arginine (arginase inhibitor), urea and/or ornithine. In some preparations, immunofluorescent staining, immunoblotting, or measurement of urea content were performed. RESULTS: Dexmedetomidine did not contract control rings with endothelium but evoked concentration-dependent increases in tension in such rings treated with L-NAME (Emax 50 ± 4%) or after endothelium-removal (Emax 74 ± 5%; N = 7 to 12). Exogenous L-arginine augmented the dexmedetomidine-induced contractions in the presence of L-NAME (Emax 75 ± 3%). This potentiation was abolished by (S)-(2-boronethyl)-L-cysteine hydrochloride (Emax 16 ± 4%) and N-hydroxy-L-arginine (Emax 18 ± 4%). Either urea or ornithine, the downstream arginase products, had a similar potentiating effect as L-arginine. Immunoassay measurements demonstrated an upregulation of arginase I by L-arginine treatment in the presence of L-NAME (N = 4). CONCLUSIONS: These results suggest that when vascular nitric oxide homeostasis is impaired, the potentiation of the vasoconstrictor effect of dexmedetomidine by L-arginine depends on arginase activity and the production of urea and ornithine.
Subject(s)
Aorta, Thoracic/drug effects , Arginase/pharmacology , Arginine/pharmacology , Dexmedetomidine/administration & dosage , Hypnotics and Sedatives/administration & dosage , Animals , Fluorescent Antibody Technique , Immunoblotting , Male , Models, Animal , Rats , Rats, Sprague-DawleyABSTRACT
Hypertension is associated with endothelial dysfunction, which favors the release of endothelium-derived contracting factors, including vasoconstrictor prostanoids and reactive oxygen species. Peroxisome proliferator-activated receptor-α (PPAR-α) agonists, clinically used as lipid-lowering drugs, possess antioxidant properties and exert beneficial effects in the vascular system. The present study aimed to identify the mechanism(s) underlying the acute effects of the PPAR-α agonists Wy14643 and fenofibate on endothelium-dependent contractions, in particular those related to oxidative stress, in the aorta of the spontaneously hypertensive rat (SHR). Aortic rings with and without endothelium of male SHRs and normotensive Wistar-Kyoto rats were suspended in organ chambers for isometric tension measurements and homogenized for enzyme activity assays. Contractions to acetylcholine in quiescent SHR aortae with endothelium were reduced by tiron (superoxide anion scavenger), diethyldithiocarbamic acid (superoxide dismutase inhibitor), and acute treatment with either Wy14643 or fenofibrate. Similarly to contractions evoked by acetylcholine, H2O2-induced increases in tension in SHR aortae involved, in succession, phospholipase A2 (PLA2), cyclooxygenase, and thromboxane-prostanoid receptors. Wy14643 or fenofibrate, by decreasing the activity of endothelial Ca2+-independent PLA2, attenuated the contractions to H2O2. In conclusion, the increased oxidative stress in the SHR aorta (mainly increased production of H2O2 and its partially reduced product, hydroxyl radical) contributed to acetylcholine-induced, endothelium-dependent contractions; PPAR-α agonists likely inhibit the H2O2-mediated contractions by inhibiting endothelial Ca2+-independent PLA2. The present study highlights the prospective therapeutic effects of PPAR-α agonists in improving endothelial function in hypertension and other vascular implications due to oxidative stress. NEW & NOTEWORTHY Peroxisome proliferator-activated receptor-α agonists, which are used clinically as lipid-lowering drugs, acutely reduce H2O2-induced contractions in aortae of hypertensive rats by inhibiting the activity of endothelial Ca2+-independent phospholipase A2. These vascular effects of peroxisome proliferator-activated receptor-α agonists suggest that they may help to prevent vascular complications under pathological conditions associated with oxidative stress.
Subject(s)
Antihypertensive Agents/pharmacology , Aorta/drug effects , Fenofibrate/pharmacology , Hydrogen Peroxide/toxicity , Hypertension/drug therapy , PPAR alpha/agonists , Phospholipase A2 Inhibitors/pharmacology , Phospholipases A2, Calcium-Independent/antagonists & inhibitors , Pyrimidines/pharmacology , Vasoconstriction/drug effects , Animals , Antioxidants/pharmacology , Aorta/enzymology , Aorta/physiopathology , Disease Models, Animal , Hypertension/enzymology , Hypertension/physiopathology , Male , Oxidative Stress/drug effects , PPAR alpha/metabolism , Phospholipases A2, Calcium-Independent/metabolism , Rats, Inbred SHR , Rats, Inbred WKY , Signal Transduction/drug effectsABSTRACT
Aging is accompanied by endothelial dysfunction due to reduced bioavailability of nitric oxide (NO) and/or reduced endothelium-dependent hyperpolarizations (EDH). This study examines the hypothesis that hypertension aggravates the impairment of EDH-type relaxation due to aging. EDH-type relaxations were studied in superior mesenteric arteries isolated from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats of 12, 36, 60, and 72 weeks of age. EDH-type relaxations in WKY were reduced with aging, and this was associated with an impairment of the function of small-conductance calcium-activated potassium channels (SKC a) and sodium-potassium ATPase (Na-K ATPase). EDH-type relaxation in SHR was smaller than that in WKY arteries, and further reduction occurred with aging. Pharmacological experiments suggested a reduced involvement of SKC a and Na-K ATPase and activation of adenosine monophosphate-activated protein kinase and silent information regulator T1 (sirtuin-1; SIRT1) in mesenteric arteries of 12-week-old SHR. These pharmacological findings suggest that in superior mesenteric arteries of the rat, the reduction in EDH-type relaxation occurs with aging and that such a reduction is exacerbated in hypertension. The latter exacerbation appears to involve proteins associated with the process of cellular senescence and is related to impaired function of SKC a and Na-K ATPase, a phenomenon that is also observed in mesenteric arteries of older normotensive rats.
ABSTRACT
The present study investigated the influence of gender on the changes underlying endothelial dysfunction in hyperlipidemia during aging. Isometric tension in rings (with endothelium) of the aortae and superior mesenteric arteries from apolipoprotein-E deficient mice was determined in wire myographs. Nitric oxide (NO)- and endothelium-dependent hyperpolarization (EDH)-mediated relaxations were smaller in the aortae and mesenteric arteries of 32weeks old males than eight weeks old males. In females, NO- and EDH-mediated relaxations were impaired only at 84weeks of age. The levels of reactive oxygen species were elevated in the blood vessels of 32weeks old males, but not females. Acute in vitro treatment with 17ß-estradiol and apocynin improved NO- and EDH-mediated relaxations in 32weeks old males but not in 84weeks old males. Relaxations to SKA-31, activator of intermediate (IKCa) and small (SKCa) conductance calcium-activated potassium channels, were attenuated in the mesenteric arteries of 32weeks old males. Such impairment was restored by acute treatment with apocynin. These findings suggest that male hyperlipidemic mice develop endothelial dysfunction at an earlier age than females. This endothelial dysfunction is associated with impaired NO bioavailability and reduced IKCa and SKCa activity. Apocynin and 17ß-estradiol restore the endothelial function only in younger male animals but not in older male or female animals.
Subject(s)
Apolipoproteins E/deficiency , Endothelium, Vascular/metabolism , Estradiol/pharmacology , Nitric Oxide/physiology , Sex Characteristics , Vasodilation/physiology , Animals , Dose-Response Relationship, Drug , Endothelium, Vascular/drug effects , Female , Male , Mice , Mice, Knockout , Vasodilation/drug effectsABSTRACT
Heme oxygenase (HO) converts heme to carbon monoxide, bilirubin, and free iron. The present study investigated whether or not HO-1 induction improves vascular relaxations attributable to endothelium-dependent hyperpolarization (EDH). Thirty-six-week-old spontaneously hypertensive rats were treated with the HO-1 inducer hemin, the HO inhibitor zinc protoporphyrin IX (II) (ZnPP), the antioxidant apocynin, or combinations of these compounds. Isolated mesenteric arteries were prepared for measurement of isometric tension, protein presence, and production of reactive oxygen species (ROS). Hemin potentiated acetylcholine-evoked EDH-type relaxations in the presence of N(ω)-nitro-L-arginine methyl ester (l-NAME) and indomethacin, while the combined treatment with ZnPP plus hemin prevented these improvements. The intermediate conductance Ca(2+)-activated K(+) channel (IKCa) blocker TRAM-34 and the Na(+)-K(+)-ATPase blocker ouabain significantly impaired these hemin-potentiated relaxations. NS309-induced TRAM-34- and ouabain-sensitive relaxations were enhanced by hemin. K(+)-induced ouabain-sensitive relaxations and the expression of Na(+)-K(+)-ATPase were increased by hemin. Thus HO-1 induction improves EDH-type relaxations by augmented activation of IKCa and the downstream Na(+)-K(+)-ATPase. Treatment with apocynin showed a similar effect as hemin in impairing ROS production, enhancing K(+)-induced relaxations, and increasing Na(+)-K(+)-ATPase expression, without affecting the expression of HO-1. The effects of hemin and apocynin were not additive. These observations suggest that the effect of HO-1 induction on EDH-type relaxations is possibly due to its antioxidant properties. In vitro treatment with bilirubin, but not carbon monoxide, enhanced EDH-type relaxations and K(+)-induced ouabain-sensitive relaxations, suggesting that the production of bilirubin may be also involved. The present findings reveal that HO-1 may be a potential vascular-specific therapeutic strategy for endothelial dysfunction in hypertension.
Subject(s)
Antihypertensive Agents/pharmacology , Endothelium, Vascular/drug effects , Heme Oxygenase (Decyclizing)/metabolism , Hemin/pharmacology , Hypertension/drug therapy , Mesenteric Arteries/drug effects , Vasodilation/drug effects , Vasodilator Agents/pharmacology , Animals , Antioxidants/pharmacology , Bilirubin/metabolism , Blood Pressure/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Endothelium, Vascular/enzymology , Endothelium, Vascular/physiopathology , Enzyme Inhibitors/pharmacology , Heme Oxygenase (Decyclizing)/antagonists & inhibitors , Hypertension/enzymology , Hypertension/physiopathology , Male , Mesenteric Arteries/enzymology , Mesenteric Arteries/physiopathology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Potassium Channel Blockers/pharmacology , Potassium Channels, Calcium-Activated/antagonists & inhibitors , Potassium Channels, Calcium-Activated/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Reactive Oxygen Species/metabolism , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/metabolism , Up-RegulationABSTRACT
Rapid non-genomic effects of 17ß-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17ß-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17ß-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17ß-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17ß-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.
Subject(s)
Estrogen Receptor alpha/metabolism , Binding Sites , Cell Line , Cell Membrane , Estradiol/metabolism , Estrogen Receptor alpha/agonists , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/genetics , Gene Expression , Humans , Ligands , Lipoylation , Phytoestrogens/metabolism , Protein Binding , Protein Isoforms , Protein Processing, Post-TranslationalABSTRACT
Experiments were designed to determine if Wy14643 ([[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thio]-acetic acid), a preferential agonist at peroxisome proliferator-activated receptors (PPAR) α, improves vascular function in hypertension, and if so, the mechanism(s) involved. Isometric tension was measured in isolated thoracic aorta of spontaneously hypertensive rats (SHR). Wy14643-induced relaxations in SHR aortic rings were greater than those induced by fenofibrate or rosiglitazone (PPARα or PPARγ agonists, respectively) and were larger in rings with endothelium than those without. Both MK886 [(1-[(4-chlorophenyl)methyl]-3-1,1-dimethylethyl)thio]-(α,α-dimethyl-5-1-methylethyl)-1H-indole-2-propanoic acid (PPARα antagonist) and GW9662 (2-chloro-5-nitrobenzanilide) (PPARγ antagonists) inhibited Wy14643-induced relaxations. The inhibitory effect of MK886 was more pronounced in rings with endothelium than those without. In SHR aortic rings with endothelium, L-NAME (N(ω)-nitro-L-arginine methyl ester, nitric oxide synthase inhibitor), ODQ (1H-1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one, soluble guanylyl cyclase inhibitor) and compound C [adenosine monophosphate-activated protein kinase (AMPK) inhibitor] reduced Wy14643-induced relaxations. Western blotting studies indicated that Wy14643 and fenofibrate, but not rosiglitazone, increased the phosphorylation of endothelial nitric oxide synthase and AMPK; these effects were abolished by compound C but not L-NAME. Endothelium-dependent contractions evoked by acetylcholine in quiescent SHR aorta in the presence of L-NAME were reduced by Wy14643 and fenofibrate but not by rosiglitazone. MK886, but not GW9662, prevented this effect. Wy14643 and fenofibrate inhibited acetylcholine-induced prostanoid release to the same extent. These findings suggest that PPARα agonists induce nitric oxide-mediated relaxation through activation of AMPK and reduce the release of endothelium-dependent contracting factors. Because also of the ability to activate smooth muscle PPARγ to induce relaxation, Wy14643 offers additional protection against vascular dysfunction of spontaneous hypertension.
Subject(s)
Aorta, Thoracic/drug effects , Hypertension/physiopathology , PPAR alpha/physiology , Peroxisome Proliferators/pharmacology , Pyrimidines/pharmacology , Animals , Aorta, Thoracic/physiology , Fenofibrate/pharmacology , In Vitro Techniques , Male , PPAR alpha/agonists , PPAR gamma/agonists , PPAR gamma/physiology , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Rosiglitazone , Thiazolidinediones/pharmacology , Vasoconstriction/drug effectsABSTRACT
The present study investigated the mechanism underlying the transient potentiation of vasoconstriction by hypoxia in isolated porcine coronary arteries. Isometric tension was measured in rings with or without endothelium. Hypoxia (Po(2) <30 mmHg) caused a transient further increase in tension (hypoxic augmentation) in contracted (with U46619) preparations. The hypoxic response was endothelium dependent and abolished by inhibitors of nitric oxide synthase [N(ω)-nitro-L-arginine methyl ester (L-NAME)] or soluble guanylyl cyclase (ODQ and NS2028). The addition of DETA NONOate (nitric oxide donor) in the presence of L-NAME restored the hypoxic augmentation, suggesting the involvement of the nitric oxide pathway. However, the same was not observed after incubation with 8-bromo-cyclic GMP, atrial natriuretic peptide, or isoproterenol. Assay of the cyclic GMP content showed no change upon exposure to hypoxia in preparations with and without endothelium. Incubation with protein kinase G and protein kinase A inhibitors did not inhibit the hypoxic augmentation. Thus the hypoxic augmentation is dependent on nitric oxide and soluble guanylyl cyclase but independent of cyclic GMP. The hypoxic augmentation persisted in calcium-free buffer and in the presence of nifedipine, ruling out a role for extracellular calcium influx. Hypoxia did not alter the intracellular calcium concentration, as measured by confocal fluorescence microscopy. This observation and the findings that hypoxic augmentation is enhanced by thapsigargin (sarco/endoplasmic reticulum calcium ATPase inhibitor) and inhibited by HA1077 or Y27632 (Rho kinase inhibitors) demonstrate the involvement of calcium sensitization in the phenomenon.
Subject(s)
Coronary Vessels/metabolism , Cyclic GMP/metabolism , Endothelium, Vascular/metabolism , Hypoxia/metabolism , Nitric Oxide/metabolism , Vasoconstriction , Animals , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Calcium Channels/metabolism , Coronary Vessels/drug effects , Coronary Vessels/physiopathology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic GMP-Dependent Protein Kinases/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiopathology , Enzyme Inhibitors/pharmacology , Guanylate Cyclase/antagonists & inhibitors , Guanylate Cyclase/metabolism , Hypoxia/physiopathology , Microscopy, Confocal , Microscopy, Fluorescence , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase Type III/antagonists & inhibitors , Nitric Oxide Synthase Type III/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Soluble Guanylyl Cyclase , Swine , Vasoconstriction/drug effects , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/metabolismABSTRACT
AIMS: We investigated how modification of levels of the sex hormones 17ß-estradiol and testosterone affects vascular contraction and nongenomic vascular effects of 17ß-estradiol. METHODS: Male and female rats were treated with vehicle, 17ß-estradiol (25 µg/kg/day) or testosterone (1 mg/kg/day) for 14 consecutive days after sham gonadectomy or gonadectomy was performed. Isometric tensions were then measured from mesenteric arteries of each group of rats. RESULTS: Contraction to phenylephrine was increased in mesenteric arteries from rats with or without gonadectomy treated with testosterone for 14 days compared to their intact controls. Contraction to phenylephrine was reduced in mesenteric arteries of rats with or without gonadectomy treated with 17ß-estradiol for 14 days compared to their intact controls. Incubation of mesenteric arteries with 17ß-estradiol (1 nmol/l) for 30 min reduced contraction to phenylephrine in mesenteric arteries of rats that were treated with testosterone for 14 days. This acute incubation of 17ß-estradiol had no effect on arteries from rats that were treated with 17ß-estradiol for 14 days. The acute effect of 17ß-estradiol (1 nmol/l) is preserved in arteries without endothelium. CONCLUSION: Our results suggest that 14 days' testosterone treatment enhances while 14 days' 17ß-estradiol treatment suppresses contraction as well as the nongenomic effects of 17ß-estradiol in the vascular smooth muscles.
Subject(s)
Estradiol/pharmacology , Estrogens/pharmacology , Gonadal Steroid Hormones/blood , Mesenteric Arteries/physiology , Adrenergic alpha-Agonists/metabolism , Adrenergic alpha-Agonists/pharmacology , Animals , Body Weight , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Evaluation, Preclinical , Endothelium, Vascular/physiology , Estradiol/blood , Estrogens/blood , Female , Gonadal Steroid Hormones/pharmacology , Humans , Male , Muscle, Smooth, Vascular/physiology , Phenylephrine/metabolism , Phenylephrine/pharmacology , Random Allocation , Rats , Rats, Sprague-Dawley , Sex Factors , Testosterone/blood , Testosterone/pharmacology , Vasoconstriction/drug effects , Vasoconstriction/physiology , Vasodilation/drug effects , Vasodilation/physiologyABSTRACT
The aim of the present study was to investigate the signaling mechanisms underlying the non-genomic effects of estrogen in rat superior mesenteric arteries. Isometric tension was recorded in rings with or without endothelium. Changes in cyclic nucleotide levels and protein kinase (PK) activities were measured. Localization of estrogen receptors (ER) and caveolin-1 were visualized by confocal microscopy. 17ß-Estradiol elicited a concentration-dependent relaxation. The relaxation was reduced by SQ 22536 (adenylyl cyclase inhibitor) and KT 5823 (PKG inhibitor) while ODQ (guanylyl cyclase inhibitor) and KT 5720 (PKA inhibitor) had no effect. At the physiological concentration of 1 nM, 17ß-estradiol had no significant effect on relaxation but enhanced the relaxation to sodium nitroprusside. The enhancement of relaxation by 17ß-estradiol was blocked by SQ 22536 and KT 5823. Although 1 nM 17ß-estradiol or 10 nM sodium nitroprusside given alone had minimal effects on PKG activity, in their combined presence, a significant increase in PKG activity was observed. Confocal microscopy demonstrated that ERα and ERß colocalized with caveolin-1 and PKG in vascular smooth muscle cells. The present findings suggest that 17ß-estradiol enhances relaxation of vascular smooth muscle of the rat superior mesenteric artery by activating adenylyl cyclase, leading to an increase in cAMP which cross activates PKG in the caveolae. No detectable increase in total cAMP level was detected as these changes occurred in the caveolae. These results are consistent with the notion that 17ß-estradiol mediates its effect in the distinct microdomains of the caveolae of the plasma membrane with colocalization of adenylyl cyclase and PKG.
Subject(s)
Adenylyl Cyclases/metabolism , Cyclic GMP-Dependent Protein Kinases/metabolism , Estradiol/pharmacology , Mesenteric Artery, Superior/drug effects , Vasodilator Agents/pharmacology , Animals , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Enzyme Activation/drug effects , Male , Mesenteric Artery, Superior/enzymology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/enzymology , Rats , Rats, Sprague-DawleyABSTRACT
AIMS: Soluble guanylyl cyclase (sGC) is a heterodimer. The dimerization of the enzyme is obligatory for its function in mediating actions caused by agents that elevate cyclic guanosine monophosphate (cGMP). The present study aimed to determine whether sGC dimerization is modulated by thiol-reducing agents and whether its dimerization influences relaxations in response to nitric oxide (NO). METHODS AND RESULTS: The dimers and monomers of sGC and cGMP-dependent protein kinase (PKG) were analysed by western blotting. The intracellular cGMP content was measured by enzyme-linked immunosorbent assay. Changes in isometric tension were determined in organ chambers. In isolated porcine coronary arteries, the protein levels of sGC dimer were decreased by the thiol reductants dithiothreitol, l-cysteine, reduced l-glutathione and tris(2-carboxyethyl) phosphine. The effect was associated with reduced cGMP elevation and attenuated relaxations in response to nitric oxide donors. The dimerization of sGC and activation of the enzyme were also decreased by dihydrolipoic acid, an endogenous thiol antioxidant. Dithiothreitol at concentrations markedly affecting the dimerization of sGC had no significant effect on the dimerization of PKG or relaxation in response to 8-Br-cGMP. Relaxation of the coronary artery in response to a NO donor was potentiated by hypoxia when sGC was partly inhibited, coincident with an increase in sGC dimer and enhanced cGMP production. These effects were prevented by dithiothreitol and tris(2-carboxyethyl) phosphine. CONCLUSION: These results demonstrate that the dimerization of sGC is exquisitely sensitive to thiol reductants compared with that of PKG, which may provide a novel mechanism for thiol-dependent modulation of NO-mediated vasodilatation in conditions such as hypoxia.
Subject(s)
Coronary Vessels/enzymology , Coronary Vessels/physiology , Guanylate Cyclase/chemistry , Guanylate Cyclase/physiology , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/physiology , Vasodilation/physiology , Animals , Coronary Vessels/drug effects , Cyclic GMP/metabolism , Cyclic GMP-Dependent Protein Kinases/chemistry , Cyclic GMP-Dependent Protein Kinases/physiology , Dimerization , Dithiothreitol/pharmacology , Female , Hypoxia/enzymology , Hypoxia/physiopathology , In Vitro Techniques , Male , Nitric Oxide/pharmacology , Nitroso Compounds/pharmacology , Protein Structure, Quaternary/drug effects , Soluble Guanylyl Cyclase , Sulfhydryl Reagents/pharmacology , Sus scrofa , Vasodilation/drug effectsABSTRACT
Genistein, a phytoestrogen present in soybeans, has well established vasodilator properties. The present study examined the mechanisms involved in the rapid vascular effects of genistein. Endothelium-dependent relaxations and contractions, induced by acetylcholine and the calcium ionophore A23187, were obtained in isolated aortic rings from male spontaneously hypertensive rats (SHR). Acute exposure to genistein potentiated relaxations and reduced contractions induced by the two agonists. Both effects of genistein were not affected by transcription- and translation-inhibitors or by tyrosine kinase inhibition. The potentiation of acetylcholine and A23187-induced relaxation by genistein was inhibited by NF023 and GP antagonist-2A, selective G(i) and G(q) α-subunit antagonists, respectively, but not by NF449, a selective G(s) α-subunit antagonist. These G protein antagonists did not alter the inhibitory effect of genistein on acetylcholine and A23187-induced contractions. The potentiation of A23187-induced relaxations by genistein was not inhibited by the conventional estrogen receptor (ER) antagonist, ICI 182,780, but inhibited by the specific ER-α antagonist, MPP, and by the epidermal growth factor receptor (EGFR) inhibitor, AG1478. It was mimicked by heparin-binding epidermal growth factor (HB-EGF). Activation of EGFR and endothelial nitric oxide synthase (eNOS) was detected in genistein-treated rings using Western blotting. These data suggest that the rapid vascular actions of genistein are mediated by non-genomic pathways and are unrelated to its tyrosine kinase inhibitory properties. Furthermore, genistein transactivates EGFR through membrane ERα via G protein-coupled pathways. This in turn enhances eNOS phosphorylation and hence endothelial function in the aorta of the SHR.
Subject(s)
Aorta, Thoracic/drug effects , ErbB Receptors/metabolism , Estrogen Receptor alpha/metabolism , GTP-Binding Proteins/metabolism , Genistein/metabolism , Nitric Oxide Synthase Type III/physiology , Signal Transduction/drug effects , Vasodilation/physiology , Animals , Aorta, Thoracic/enzymology , Aorta, Thoracic/metabolism , Enzyme Activation/drug effects , Enzyme Activation/physiology , ErbB Receptors/genetics , Genistein/pharmacology , Genistein/therapeutic use , Hypertension/enzymology , Hypertension/metabolism , Hypertension/physiopathology , Male , Organ Culture Techniques , Protein Binding/drug effects , Protein Binding/physiology , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Signal Transduction/physiology , Vasodilation/drug effectsABSTRACT
Dexmedetomidine is an α(2)-adrenoceptor agonist and anesthetic. The present study was designed to characterize the receptor subtypes and the downstream mechanisms of the vascular effects of dexmedetomidine in small (mesenteric artery) and large (aorta) arteries ex vivo. Isometric tension was measured in Sprague-Dawley rat mesenteric and aortic rings (with or without endothelium). To study relaxations, cumulative concentrations of dexmedetomidine, 5-bromo-N-(2-imidazolin-2-yl)-6-quinoxalinamine, (UK14304), or clonidine were added to rings contracted with 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F(2α) (U46619) in the presence or absence of indomethacin; N(ω)-nitro-l-arginine methyl ester hydrochloride (l-NAME); 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole maleate (BRL44408); 2-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione dihydrochloride (ARC239); l-657,743, (2S-trans)-1,3,4,5',6,6',7,12b-octahydro-1',3'-dimethyl-spiro[2H-benzofuro[2,3-a]quinolizine-2,4'(1'H)-pyrimidin]-2'(3'H)-one hydrochloride hydrate (MK912); rauwolscine; prazosin; or pertussis toxin. To study contractions, dexmedetomidine was added to quiescent rings without endothelium or after incubation with l-NAME, rauwolscine, prazosin, indomethacin, or 3-[(6-amino-(4-chlorobenzensulfonyl)-2-methyl-5,6,7,8-tetrahydronaphth)-1-yl]propionic acid (S18886). Dexmedetomidine evoked relaxation at low concentrations (10 pM-30 nM) followed by contraction at higher concentrations (>30 nM) in the mesenteric artery. In the aorta, the relaxation was significantly smaller. The relaxation to dexmedetomidine depended on nitric oxide, endothelium, and G(i) protein, and it was mediated by α(2A/D)-adrenoceptors and possibly α(2B)-adrenoceptors. The contraction was mediated mainly by α(2B)- and α(1)-adrenoceptors and involved the action of prostanoids. UK14304 and clonidine induced greater and smaller relaxations, respectively, than dexmedetomidine. In conclusion, depending on the concentration used and the presence of functional endothelium, dexmedetomidine may evoke both relaxation and contraction in isolated arteries. The vascular effects also vary depending on the blood vessel studied. Its vascular effect is different from that of clonidine and UK14304.
Subject(s)
Aorta/drug effects , Dexmedetomidine/pharmacology , Mesenteric Arteries/drug effects , Muscle Contraction/drug effects , Muscle Relaxation/drug effects , Muscle, Smooth, Vascular/drug effects , Receptors, Adrenergic, alpha-2/metabolism , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Adrenergic alpha-1 Receptor Antagonists/pharmacology , Adrenergic alpha-2 Receptor Agonists/pharmacology , Adrenergic alpha-2 Receptor Antagonists/pharmacology , Animals , Brimonidine Tartrate , Clonidine/pharmacology , Endothelium, Vascular/drug effects , In Vitro Techniques , Indomethacin/pharmacology , Male , Models, Biological , NG-Nitroarginine Methyl Ester/pharmacology , Naphthalenes/pharmacology , Pertussis Toxin/pharmacology , Prazosin/pharmacology , Propionates/pharmacology , Quinoxalines/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Thromboxane/antagonists & inhibitorsABSTRACT
Vitamin D has cardiovascular protective effects besides regulating calcium homeostasis. To examine the chronic in vivo effect of a physiological dose of 1,25-dihydroxyvitamin D(3) on the occurrence of endothelium-dependent contractions, spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were treated with the vitamin D derivative for 6 wk. The serum 1,25-dihydroxyvitamin D(3) level of both treated WKY and SHR was significantly higher than in untreated rats while the mean arterial blood pressure of the treated SHR was significantly lower than that of control SHR. Aortic rings with or without endothelium were studied in conventional organ chambers for isometric force measurement. Confocal microscopy was used to measure the cytosolic free calcium concentration (with the fluorescent dye fluo 4) and reactive oxygen species (ROS; with dichlorodihydrofluorescein diacetate). Reverse transcription PCR and Western blotting were used to determine the mRNA and protein expression level of cyclooxygenase-1 (COX-1), prostacyclin synthase, and thromboxane synthase. The endothelium-dependent concentration-contraction curves to both acetylcholine- and A-23187-induced contractions were shifted to the right in aortas from treated SHR but not from treated WKY. The chronic treatment normalized the relaxations of contracted preparations to acetylcholine. There were no significant differences in the increases in cytosolic free calcium concentration evoked by acetylcholine and A-23187 between control and treated groups. The endothelial ROS level was higher in SHR than WKY aortas and reduced by the chronic treatment. The gene and protein expression studies indicated that the overexpression of COX-1 observed in SHR aorta was reduced by the chronic treatment. These results demonstrate that chronic treatment with 1,25-dihydroxyvitamin D(3) modulates vascular tone and this modulation is accompanied by a lowered blood pressure, reduced expression of COX-1 mRNA and protein, and reduced ROS level in SHR. The reduction in endothelium-dependent contractions does not involve the surge in endothelial cytosolic calcium concentration that initiates the contractions.
Subject(s)
Aorta/drug effects , Blood Pressure/drug effects , Endothelium, Vascular/drug effects , Hypertension/physiopathology , Vasoconstriction/drug effects , Vitamin D/pharmacology , Animals , Aorta/metabolism , Aorta/physiopathology , Blood Pressure/physiology , Calcitriol/blood , Calcium/metabolism , Cyclooxygenase 1/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Endothelium, Vascular/metabolism , Endothelium, Vascular/physiopathology , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Reactive Oxygen Species/metabolism , Vasoconstriction/physiologyABSTRACT
Acute inhibition of nitric-oxide synthase (NOS) unmasks the release of endothelium-derived contracting factors (EDCFs). The present study investigated whether chronic inhibition of NOS modulates endothelium-dependent contractions. Eighteen-week-old male Sprague-Dawley rats were treated by daily gavage for 6 weeks with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (60 mg/kg) or vehicle (distilled water; 1 ml/kg). Chronic treatment with L-NAME increased arterial blood pressure. Isometric tension was measured in aortic rings with or without endothelium. Endothelium-dependent relaxations to acetylcholine and the calcium ionophore 5-(methylamino)-2-[(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)-ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid (A23187) were reduced in preparations from L-NAME-treated rats. The reduction in relaxation to A23187 was partially reversed by L-arginine (1 mM). In quiescent aortic rings, A23187 caused contractions in the presence of L-NAME and intact endothelium. The A23187-induced contractions were greater in rings from the L-NAME-treated rats than in those from the control group. These contractions were abolished by the cyclooxygenase (COX)-2 inhibitor N-[2-cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS-398) and the thromboxane-prostanoid (TP) receptor antagonist 3-((6R)-6-{[(4-chlorophenyl)sulfonyl]amido}-2-methyl-5,6,7,8-tetrahydronaphthalen-1-yl)propanoate (S18886), but not by the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560). Chronic L-NAME treatment reduced the level of nitric oxide in the plasma but increased COX activity in the aortic rings. Western blotting and immunohistochemical staining showed that endothelial NOS expression was reduced in the aortae of the chronic L-NAME-treated group. COX-1 expression was augmented slightly, whereas COX-2 expression was up-regulated markedly. The TP receptor expression was comparable with control. These experiments demonstrate that chronic NOS inhibition increases endothelium-dependent contractions of the rat aorta by inducing COX-2 expression and augmenting the production of EDCF.
Subject(s)
Aorta, Thoracic/drug effects , Cyclooxygenase 2/biosynthesis , Endothelium, Vascular/physiology , Muscle, Smooth, Vascular/drug effects , Nitric Oxide Synthase/antagonists & inhibitors , 6-Ketoprostaglandin F1 alpha/blood , Animals , Aorta, Thoracic/physiology , Cyclooxygenase 1/biosynthesis , Cyclooxygenase Inhibitors/pharmacology , Dinoprost/blood , In Vitro Techniques , Isometric Contraction , Male , Muscle Relaxation , Muscle, Smooth, Vascular/physiology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase Type III/biosynthesis , Rats , Rats, Sprague-Dawley , Thromboxane B2/blood , Up-RegulationABSTRACT
Phospholipase A(2) (PLA(2)), a regulatory enzyme found in most mammalian cells, catalyzes the breakdown of membrane phospholipids to arachidonic acid. There are two major cytosolic types of the enzyme, calcium-dependent (cPLA(2)) and calcium-independent (iPLA(2)) PLA(2). The present study investigated whether or not iPLA(2) plays a role in the endothelium-dependent contractions of the aorta of the spontaneously hypertensive rat and its normotensive counterpart, the Wistar-Kyoto rat. The presence of iPLA(2) in the endothelial cells was identified by using immunochemistry and immunoblotting. Aortic rings with and without the endothelium were suspended in organ chambers for isometric tension recording. The production of prostanoids was measured by using enzyme immunoassay kits. iPLA(2) was densely distributed in endothelial cells of the aorta of both strains. At 3 x 10(-6) M, the selective iPLA(2) inhibitor, bromoenol lactone (BEL), abrogated endothelium-dependent contractions induced by acetylcholine but not those evoked by the calcium ionophore A-23187. The effects of BEL were similar in the aortae of Wistar-Kyoto and spontaneously hypertensive rats. The nonselective PLA(2) inhibitor quinacrine abolished the contractions triggered by both acetylcholine and A-23187, whereas the store-operated calcium channel inhibitor SKF-96365 prevented only the acetylcholine-induced contraction. The acetylcholine- but not the A-23187-induced release of 6-keto prostaglandin F(1alpha) was inhibited by BEL. The release of thromboxane B(2) by either acetylcholine or A-23187 was not affected by BEL. In conclusion, iPLA(2) plays a substantial role in the generation of endothelium-derived contracting factor evoked by acetylcholine.
Subject(s)
Acetylcholine/pharmacology , Aorta/enzymology , Endothelium, Vascular/enzymology , Hypertension/enzymology , Muscle Contraction/drug effects , Muscle, Smooth, Vascular/enzymology , Phospholipases A2, Calcium-Independent/metabolism , Animals , Calcimycin/pharmacology , Disease Models, Animal , Hypertension/physiopathology , Imidazoles/pharmacology , Ionophores/pharmacology , Male , Muscle Contraction/physiology , Naphthalenes/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Pyrones/pharmacology , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Vasodilator Agents/pharmacologyABSTRACT
BACKGROUND: Epigallocatechin-3-gallate (EGCG), which has been shown to have potent antioxidant effect, comprises 80% of catechins in Chinese green tea. This study was to investigate whether cigarette smoke (CS) exposure would induce lung morphological changes and oxidative stress in the CS-exposed rat model, and whether Chinese green tea (Lung Chen tea with EGCG as its main active ingredient) consumption would alter oxidative stress in sera and lung leading to protection of CS-induced lung damage. METHODS: Sprague-Dawley rats were randomly divided into four groups, i.e. sham air (SA), 4% CS, 2% Lung Chen tea plus SA or 4% CS. Exposure to SA or 4% CS was performed for 1h/day for 56 days in ventilated smoking chambers. Sera and lung tissues were collected 24h after last CS exposure for histology and all biochemical assays. RESULTS: Airspace enlargement and goblet cell hyperplasia were observed after 56-day CS exposure alone, which were abolished in the presence of green tea consumption. Serum 8-isoprostane level was significantly elevated (p<0.01) as well as lung superoxide dismutase (SOD) and catalase activities in CS-exposed rats compared to SA-exposed rats (p<0.05), which returned to the levels of SA-exposed rats after Chinese green tea consumption. CONCLUSION: These results indicate that increased levels of systemic oxidative stress after CS exposure play an important role in the induction of lung damage. Chinese green tea may have the ability to suppress CS-induced oxidative stress that leads to protection of lung injury.
Subject(s)
Antioxidants/pharmacology , Catechin/analogs & derivatives , Goblet Cells/drug effects , Lung Injury/drug therapy , Oxidative Stress/drug effects , Tea/chemistry , Animals , Catechin/pharmacology , Goblet Cells/pathology , Hyperplasia/pathology , Inhalation Exposure , Random Allocation , Rats , Rats, Sprague-Dawley , Smoking , Tobacco Smoke Pollution/adverse effectsABSTRACT
Rho kinase is involved in the pathogenesis of hypertension, which favors the occurrence of endothelium-dependent contractions. The present study was designed to determine the effects of two Rho kinase inhibitors, HA1077 [1-(5-isoquinolinesulfonyl)-homopiperazine (fasudil)] and Y27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride], on endothelium-dependent and -independent contractions. Isometric tension of 1-year-old spontaneously hypertensive rat and Wistar Kyoto aortae were measured. In the presence of N(omega)-nitro-L-arginine methyl ester, HA1077, and Y27632 reduced endothelium-dependent contractions caused by acetylcholine and the calcium ionophore 5-(methylamino)-2-[[2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)-ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid (A23187). The Rho kinase inhibitors did not significantly affect prostacyclin production measured as 6-keto prostaglandin F(1alpha). They nearly abolished endothelium-independent contractions to (5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2-oxabicyclo-[2.2.1]hept-5-yl]-5-heptenoic acid (U46619), prostaglandin F(2alpha), and phenylephrine. Western blotting revealed a comparable expression of Rho kinase in the aortae of the two strains. The reduction by Rho kinase inhibitors of endothelium-dependent contractions is mainly because of their direct effect on the vascular smooth muscle cells.
Subject(s)
Antihypertensive Agents/pharmacology , Aorta, Thoracic/drug effects , Endothelium, Vascular/drug effects , Isometric Contraction/drug effects , Vasoconstriction/drug effects , rho-Associated Kinases/antagonists & inhibitors , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/chemistry , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Amides/chemistry , Amides/pharmacology , Animals , Antihypertensive Agents/chemistry , Aorta, Thoracic/enzymology , Aorta, Thoracic/physiopathology , Blotting, Western , Dose-Response Relationship, Drug , Endothelium, Vascular/enzymology , Endothelium, Vascular/physiology , Hypertension/drug therapy , Hypertension/enzymology , Hypertension/physiopathology , In Vitro Techniques , Male , Pyridines/chemistry , Pyridines/pharmacology , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Species Specificity , rho-Associated Kinases/biosynthesisABSTRACT
OBJECTIVE: To explore the effects of different estrogens on vascular function, we compared the vasorelaxant effects of 17beta-estradiol, 17alpha-estradiol, estrone, and the synthetic estrogen diethylstilbestrol (DES) on porcine coronary arterial segments. DESIGN: Porcine coronary arterial rings were contracted with the stable thromboxane A2 analogue U46619 (3 x 10(-8) M), and direct relaxation was examined by the addition of increasing concentrations of 17beta-estradiol, 17alpha-estradiol, estrone, or DES (10(-9) to 10(-4) M). Modulation of agonist-induced contraction and relaxation was studied in coronary arterial rings incubated for 20 minutes with DES or estrone (10(-10)-10(-6) M) with 17beta-estradiol (10(-9) M) as comparison. RESULTS: Direct relaxation of arterial rings potentiated by these estrogens was recorded with a rank order potency of DES > 17beta-estradiol > estrone > 17alpha-estradiol. 17beta-Estradiol potentiated relaxation responses to sodium nitroprusside and levcromakalim but not bradykinin or A23187 while reducing contractions to 5-hydroxytryptamine and U46619. DES and estrone, both at 10(-6) M, mimicked the 17beta-estradiol-potentiated sodium nitroprusside and levcromakalim relaxation responses. Additionally, the inhibitory effects of 17beta-estradiol (10(-9) M) on 5-hydroxytryptamine- and U46619-induced contractions were partially reproducible by DES (10(-6) M) and estrone (10(-6) M). CONCLUSIONS: Although DES is the most potent among the tested estrogenic compounds in eliciting relaxation, 17beta-estradiol is more effective than estrone and DES at enhancing endothelium-independent relaxation and reducing vascular contraction in porcine coronary arteries.
Subject(s)
Coronary Vessels/drug effects , Diethylstilbestrol/pharmacology , Estrone/pharmacology , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Animals , Cromakalim/pharmacology , Drug Synergism , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Estradiol/pharmacology , Muscle Contraction/drug effects , Muscle Relaxation/drug effects , Nitroprusside/pharmacology , Swine , Vasoconstrictor Agents/pharmacologyABSTRACT
Timosaponin A-III (TAIII), a saponin isolated from the rhizome of Anemarrhena asphodeloides, exhibits potent cytotoxicity and has the potential to be developed as an anticancer agent. Here, we provide evidence that TAIII induces autophagy in HeLa cells followed by apoptotic cell death. TAIII-induced autophagy was morphologically characterized by the formation of membrane-bound autophagic vacuoles recognizable at the ultrastructural level. TAIII-treated cells expressing green fluorescent protein (GFP)-labeled microtubule-associated protein 1 light chain 3 (LC3) displayed punctate fluorescence indicative of LC3 recruitment to the autophagosome. This was associated with the conversion of LC3-I (the cytosolic form) into LC3-II (the lipidated form located on the autophagosome membrane). TAIII treatment also induced mitochondrial dysfunction involving overproduction of reactive oxygen species and reduction of mitochondrial membrane potential accompanied by induction of mitochondrial permeability transition. Prolonged exposure to TAIII resulted in cytochrome c release and caspase-3 activation, events that signified the onset of apoptotic cell death. TAIII-induced autophagy preceded apoptosis, as evidenced by early autophagic vacuole formation, GFP-LC3 translocation, and LC3-II increase in the absence of caspase-3 cleavage. Notably, TAIII-mediated apoptotic cell death was potentiated by treatment with autophagy inhibitor 3-methyladenine or small interfering RNA against the autophagic gene beclin 1. These findings suggest that TAIII-elicited autophagic response plays a protective role that impedes the eventual cell death. In terms of structure-activity relationship, the sugar chain in TAIII is indispensable to the drug action, as the sugar-lacking aglycone sarsasapogenin did not induce autophagy and exhibited weaker cytotoxicity.