Enhanced estradiol-induced vasorelaxation in aortas from type 2 diabetic mice may reflect a compensatory role of p38 MAPK-mediated eNOS activation.

Cardiovascular problems are a major cause of morbidity and mortality, mainly due to coronary artery disease and atherosclerosis, in type 2 diabetes mellitus. However, female gender is a protective factor in the development of, for example, atherosclerosis and hypertension. One of the female hormones, 17ß-estradiol (E2), is known to protect against the cardiovascular injury resulting from endothelial dysfunction, but the mechanism by which it does so remains unknown. Our hypothesis was that E2-mediated activation of Akt and mitogen-activated protein kinase (MAPK), and the subsequent endothelial NO synthase (eNOS) phosphorylation, might protect the aorta in diabetic mellitus. The experimental type 2 diabetic model we employed to test that hypothesis (female mice given streptozotocin and nicotinamide) is here termed fDM. In fDM aortas, we examined the E2-induced relaxation response and the associated protein activities. In control (age-matched, nondiabetic) aortas, E2 induced a vascular relaxation response that was mediated via Akt/eNOS and mitogen-activated/ERK-activating kinase (MEK)/eNOS pathways. In fDM aortas (vs. control aortas), (a) the E2-induced relaxation was enhanced, (b) the mediation of the response was different (via Akt/eNOS and p38 MAPK/eNOS pathways), and (c) E2 stimulation increased p38 MAPK and eNOS phosphorylations, decreased MEK phosphorylation, but did not alter estrogen receptor activity. We infer that at least in fDM aortas, E2 has beneficial effects (enhanced vascular relaxation and protection) that are mediated through Akt activation and (compensating for reduced MEK activation) p38 MAPK activation, leading to enhanced eNOS phosphorylation.

Protein kinase C delta contributes to increase in EP3 agonist-induced contraction in mesenteric arteries from type 2 diabetic Goto-Kakizaki rats.

Prostaglandin E(2) (PGE(2)), an important and ubiquitously present vasoactive eicosanoid, may either constrict or dilate systemic vascular beds. However, little is known about the vascular contractile responsiveness to and signaling pathways for PGE(2) at the chronic stage of type 2 diabetes. We hypothesized that PGE(2)-induced arterial contraction is augmented in type 2 diabetic Goto-Kakizaki (GK) rats via the protein kinase Cδ (PKCδ) pathway. Here, we investigated the vasoconstrictor effects of PGE(2) and of sulprostone (EP1-/EP3-receptor agonist) in rings cut from superior mesenteric arteries isolated from GK rats (37-44 weeks old). In arteries from GK rats (vs. those from age-matched Wistar rats), examined in the presence of a nitric oxide synthase inhibitor: 1) the PGE(2)- and sulprostone-induced vasocontractions (which were not blocked by the selective EP1 receptor antagonist sc19220) were enhanced, and these enhancements were suppressed by rottlerin (selective PKCδ inhibitor) but not by Gö6976 (selective PKCα/ß inhibitor); 2) the sulprostone-stimulated phosphorylation of PKCδ (at Thr(505)), which yields an active form, was increased and 3) sulprostone-stimulated caldesmon phosphorylations, which are related to isometric force generation in smooth muscle, were increased. The protein expression of EP3 receptor in superior mesenteric arteries was similar between the two groups of rats. Our data suggest that the diabetes-related enhancement of EP3 receptor-mediated vasocontraction results from activation of the PKCδ pathway. Alterations in EP3 receptor-mediated vasocontraction may be important factors in the pathophysiological influences over arterial tone that are present in diabetic states.

Pravastatin normalizes ET-1-induced contraction in the aorta of type 2 diabetic OLETF rats by suppressing the KSR1/ERK complex.

Endothelin (ET)-1 is a likely candidate for a key role in diabetic vascular complications. In the present study, we hypothesized that treatment with pravastatin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase) would normalize the ET-1-induced contraction in aortas isolated from type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats. Contractile responses were examined by measuring isometric force in endothelium-denuded aortic helical strips from four groups: Long-Evans Tokushima Otsuka (LETO; genetic control), OLETF (type 2 diabetic), pravastatin-treated LETO, and pravastatin-treated OLETF rats. Both immunoblot analysis and immunoprecipitation assays were used to examine Src, protein phosphatase (PP)2A, kinase suppressor of Ras (KSR)1, and ERK signaling pathway protein levels and activities. In endothelium-denuded aortas isolated from OLETF rats at the chronic stage of diabetes (56-60 wk) (vs. those from age-matched LETO rats), we found the following: 1) ET-1-induced contraction was enhanced, 2) ERK1/2 phosphorylation was increased, 3) phosphorylations of KSR1 and PP2A were reduced (i.e., enhancement of the kinase active state), 4) ERK1/2-KSR1 complexes were increased, and 5) Src tyrosine kinase activity was diminished. Endothelium-denuded aortas isolated from OLETF rats treated with pravastatin (10 mg/kg po, daily for 4 wk) exhibited normalized ET-1-induced contractions and suppressed ET-1-stimulated ERK phosphorylation, with the associated phosphorylated KSR1 and phosphorylated PP2A levels being increased toward normal levels. These results suggest that in type 2 diabetic rats, pravastatin normalizes ET-1-induced contraction in aortic smooth muscle via a suppression of PP2A/KSR1/ERK activities after an enhancement of Src kinase activity.

Involvement of CaM kinase II in the impairment of endothelial function and eNOS activity in aortas of Type 2 diabetic rats.

In the present sutdy, we have examined the relationship between the CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) pathway and endothelial dysfunction in aortas from GK (Goto-Kakizaki) Type 2 diabetic rats. The ACh (acetylcholine)-induced relaxation and NO production were each attenuated in diabetic aortas (compared with those from age-matched control rats). ACh-stimulated Ser(1177)-eNOS (endothelial NO synthase) phosphorylation was significantly decreased in diabetic aortas (compared with their controls). ACh markedly increased the CaMKII phosphorylation level within endothelial cells only in control aortas (as assessed by immunohistochemistry and Western blotting). ACh-stimulated Thr(286)-CaMKII phosphorylation within endothelial cells was significantly decreased in diabetic aortas (compared with their controls). The ACh-induced relaxations, NO production, eNOS phosphorylation, and CaMKII phosphorylation were inhibited by KN93 and/or by lavendustin C (inhibitors of CaMKII) in control aortas, but not in diabetic ones. Pre-incubation of aortic strips with a PP (protein phosphatase)-1 inhibitor, PPI2 (protein phosphatase inhibitor 2), or with a PP2A inhibitor, CA (cantharidic acid), corrected the above abnormalities in diabetic aortas. The expression of PP2A type A subunit was increased in diabetic aortas. The ACh-stimulated Thr(320)-phosphorylation level of PP1α was lower in diabetic aortas than in their controls, but the total PP1α protein level was not different. These results suggest that the aortic relaxation responses, NO production, and eNOS activity mediated by CaMKII phosphorylation are decreased in this Type 2 diabetic model, and that these impairments of CaMKII signalling may be, at least in part, due to enhancements of PP1α activity and PP2A expression.

Akt/eNOS pathway activation in endothelium-dependent relaxation is preserved in aortas from female, but not from male, type 2 diabetic mice.

Cardiovascular problems are major causes of morbidity and mortality, the main problems being coronary artery disease and atherosclerosis, in type 2 diabetes mellitus. However, female gender is a protective factor in the development of, for example, atherosclerosis and hypertension. Our aim was to investigate possible gender differences in the activation of Akt/eNOS signaling in aortas from a mouse type 2 diabetic model. Nonfasting plasma glucose was significantly above control in the diabetic mice (both males and females). Plasma insulin was not different between the age-matched controls and the diabetic mice (of either gender). In diabetic males (vs male controls and/or diabetic females): (a) systemic blood pressure was elevated, (b) the clonidine- and insulin-induced Akt-dependent aortic relaxations were impaired, but the ACh-induced Akt-independent and SNP-induced endothelium-independent aortic relaxations were not, (c) Akt and eNOS expression levels were lower, (d) both Akt phosphorylation at Ser(473) and eNOS phosphorylation at Ser(1177) in the aorta were lower under clonidine- or insulin-stimulation, but not under ACh-stimulation. These results suggest that in mice: (i) endothelial functions mediated via the Akt/eNOS pathway are abrogated in type 2 diabetes only in males and (ii) in females (vs males), eNOS expression is elevated and the endothelium resists dysfunction.

Angiotensin II type 2 receptor-dependent increase in nitric oxide synthase activity in the endothelium of db/db mice is mediated via a MEK pathway.

Angiotensin II type 2 receptor (AT(2)R) stimulation may cause vasodilation. It could thereby contribute to the antihypertensive effects of angiotensin II type 1 receptor (AT(1)R) antagonists since AT(1)R blockade reportedly increases endogenous levels of Ang II, and this may then bind to the unblocked AT(2)R. Because this is potentially an important consideration in diabetes, we examined whether or not AT(2)R mediates vasorelaxation in db/db diabetic mice. We also examined if AT(2)R-mediated vasorelaxation is preserved after long-term treatment with the AT(1)R antagonist losartan. The effects of AT(2)R stimulation, with either Ang II or the selective agonist CGP-42112A, were studied in aortas from db/db mice (a type 2 diabetic model). CGP-42112A induced a concentration-dependent relaxation in db/db aortas (not in Lean aortas), and this was significantly weakened by the MEK-inhibitor PD98059. CGP-42112A-induced relaxations were increased by Ang II-stimulation (by the organ-culture method) or by AT(1)R blockade (by long-term losartan treatment) only in Lean aortas. Basal AT(2)R expression, and Ang II-stimulated MEK and eNOS phosphorylations were all increased in aortas from db/db (vs. Lean) mice. Long-term losartan treatment increased Ang II-stimulated MEK and eNOS phosphorylations in aortas from Lean, but not db/db, mice. Therefore, this study has provided evidence that AT(2)R-mediated NO production and vasorelaxation through a MEK pathway are enhanced (under basal conditions) in aortas from db/db (vs. Lean) mice. The preservation of such AT(2)R function during AT(1)R blockade needs to be considered in the search for a physiological role for AT(2)R.

Dysfunction of endothelium-dependent relaxation to insulin via PKC-mediated GRK2/Akt activation in aortas of ob/ob mice.

In diabetic states, hyperinsulinemia may negatively regulate Akt/endothelial nitric oxide synthase (eNOS) activation. Our main aim was to investigate whether and how insulin might negatively regulate Akt/eNOS activities via G protein-coupled receptor kinase 2 (GRK2) in aortas from ob/ob mice. Endothelium-dependent relaxation was measured in aortic rings from ob/ob mice (a type 2 diabetes model). GRK2, ß-arrestin2, and Akt/eNOS signaling-pathway protein levels and activities were mainly assayed by Western blotting. Plasma insulin was significantly elevated in ob/ob mice. Insulin-induced relaxation was significantly decreased in the ob/ob aortas [vs. age-matched control (lean) ones]. The response in ob/ob aortas was enhanced by PKC inhibitor or GRK2 inhibitor. Akt (at Thr(308)) phosphorylation and eNOS (at Ser(1177)) phosphorylation, and also the ß-arrestin2 protein level, were markedly decreased in the membrane fraction of insulin-stimulated ob/ob aortas (vs. insulin-stimulated lean ones). These membrane-fraction expressions were enhanced by GRK2 inhibitor and by PKC inhibitor in the ob/ob group but not in the lean group. PKC activity was much greater in ob/ob than in lean aortas. GRK2 protein and activity levels were increased in ob/ob and were greatly reduced by GRK2 inhibitor or PKC inhibitor pretreatment. These results suggest that in the aorta in diabetic mice with hyperinsulinemia an upregulation of GRK2 and a decrease in ß-arrestin2 inhibit insulin-induced stimulation of the Akt/eNOS pathway and that GRK2 overactivation may result from an increase in PKC activity.

Losartan improves aortic endothelium-dependent relaxation via proline-rich tyrosine kinase 2/Src/Akt pathway in type 2 diabetic Goto-Kakizaki rats.

In diabetic states, endothelial dysfunction is related to vascular complications. We hypothesized that insulin-induced relaxation and the associated proline-rich tyrosine kinase 2 (Pyk2)/Src/Akt pathway would be abnormal in aortas from the Goto-Kakizaki (GK) type 2 diabetic rat, which exhibits hyperglycemia/insulin resistance, and that losartan treatment of such rats (25 mg·kg(-1)·day(-1) for 2 wk) would correct these abnormalities. Endothelium-dependent relaxation was by measuring isometric force in helical strips of aortas from four groups, each of 30 rats: normal Wistar (control), GK (diabetic), losartan-treated normal, and losartan-treated GK. Pyk2, Src, and Akt/endothelial nitric oxide synthase (eNOS) signaling-pathway protein levels and activities were assayed mainly by Western blotting and partly by immunohistochemistry. In GK (vs. age-matched control) aortas, various insulin-stimulated levels [nitric oxide production and the phosphorylations of eNOS at Ser(1177), of Akt at Thr(308), of phosphoinositide-dependent kinase-1 (PDK1) at Ser(241), of Src at Tyr(416), and of Pyk2 at Tyr(579)] were all significantly decreased and unaffected by either Src inhibitor (PP2) or Pyk2 inhibitor (AG17), while the insulin-stimulated levels of insulin receptor substrate (IRS)-1 phosphorylation at Ser(307), total-eNOS, and total-Akt were significantly increased. Losartan treatment normalized these altered levels. The insulin-stimulated phosphorylation levels of Src/PDK1/Akt/eNOS, but not of Pyk2, were decreased by PP2 in control and losartan-treated GK, but not in GK, aortas. These results suggest that in the GK diabetic aorta increased phospho-IRS-1 (at Ser(307)) and decreased Pyk2/Src activity inhibit insulin-induced stimulation of the PDK/Akt/eNOS pathway. The observed increase in phospho-IRS-1 (at Ser(307)) may result from increased angiotensin II activity.

Mechanisms underlying altered extracellular nucleotide-induced contractions in mesenteric arteries from rats in later-stage type 2 diabetes: effect of ANG II type 1 receptor antagonism.

Little is known about the vascular contractile responsiveness to, and signaling pathways for, extracellular nucleotides in the chronic stage of type 2 diabetes or whether the ANG II type 1 receptor blocker losartan might alter such responses. We hypothesized that nucleotide-induced arterial contractions are augmented in diabetic Goto-Kakizaki (GK) rats and that treatment with losartan would normalize the contractions. Here, we investigated the vasoconstrictor effects of ATP/UTP in superior mesenteric arteries isolated from GK rats (37-42 wk old) that had or had not received 2 wk of losartan (25 mg·kg(-1)·day(-1)). In arteries from GK rats (vs. those from Wistar rats), 1) ATP- and UTP-induced contractions, which were blocked by the nonselective P2 antagonist suramin, were enhanced, and these enhancements were suppressed by endothelial denudation, by cyclooxygenase (COX) inhibitors, or by a cytosolic phospholipase A(2) (cPLA(2)) inhibitor; 2) both nucleotides induced increased release of PGE(2) and PGF(2α); 3) nucleotide-stimulated cPLA(2) phosphorylations were increased; 4) COX-1 and COX-2 expressions were increased; and 5) neither P2Y2 nor P2Y6 receptor expression differed, but P2Y4 receptor expression was decreased. Mesenteric arteries from GK rats treated with losartan exhibited (vs. untreated GK) 1) reduced nucleotide-induced contractions, 2) suppressed UTP-induced release of PGE(2) and PGF(2α), 3) suppressed UTP-stimulated cPLA(2) phosphorylation, 4) normalized expressions of COX-2 and P2Y4 receptors, and 5) reduced superoxide generation. Our data suggest that the diabetes-related enhancement of ATP-mediated vasoconstriction was due to P2Y receptor-mediated activation of the cPLA(2)/COX pathway and, moreover, that losartan normalizes such contractions by a suppressing action within this pathway.

Angiotensin II causes endothelial dysfunction via the GRK2/Akt/eNOS pathway in aortas from a murine type 2 diabetic model.

Nitric oxide (NO) production and endothelial function are mediated via the Akt/eNOS pathway. We investigated the reductions of these mechanism(s) in type 2 diabetes. Diabetic model (nicotinamide+streptozotocin-induced) mice were fed for 4 weeks on a normal diet either containing or not containing losartan, an AT1 R antagonist. Relaxations and NO productions were measured in isolated aortas. G-protein coupled receptor kinase 2 (GRK2) protein levels and activities in the Akt/eNOS signaling-pathway were mainly assayed by Western blotting. Clonidine- and insulin-induced relaxations and NO productions, all of which were significantly decreased in aortas isolated from the diabetics, were normalized by 4 weeks' losartan administration. Plasma angiotensin II (Ang II) and GRK2 protein levels were increased in diabetes, and each was normalized by 4 week's losartan administration. Additionally, there was a direct correlation between the plasma Ang II and aortic GRK2 protein levels. In the diabetics, the clonidine-induced responses (but not the insulin-induced ones) were enhanced by GRK2-inhibitor. Akt phosphorylation was markedly below control in the clonidine-stimulated diabetes. The phosphorylation of Akt at Thr³°8 was significantly normalized and the phosphorylation of eNOS at Ser¹¹77 tended to be increased by GRK2-inhibitor in the clonidine-stimulated diabetics. Our data suggest that (a) the Akt/eNOS pathway is downstream of GRK2, and that GRK2 inhibits Akt/eNOS activities, and (b) this pathway underlies the impaired NO production seen in type 2 diabetes, in which there are defective phosphorylations of Akt and eNOS that may be caused by an upregulation of GRK2 secondary to a high plasma Ang II level. Inhibitors of GRK2 warrant further investigation as potential new therapeutic agents in diabetes.

Mechanisms underlying the losartan treatment-induced improvement in the endothelial dysfunction seen in mesenteric arteries from type 2 diabetic rats.

It is well known that type 2 diabetes mellitus is frequently associated with vascular dysfunction and an elevated systemic blood pressure, yet the underlying mechanisms are not completely understood. We previously reported that in mesenteric arteries from established type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats, which exhibit endothelial dysfunction, there is an imbalance between endothelium-derived vasodilators [namely, nitric oxide (NO) and hyperpolarizing factor (EDHF)] and vasoconstrictors [contracting factors (EDCFs) such as cyclooxygenase (COX)-derived prostanoids]. Here, we investigated whether the angiotensin II receptor antagonist losartan might improve endothelial dysfunction in OLETF rats at the established stage of diabetes. In mesenteric arteries isolated from OLETF rats [vs. those from age-matched control Long-Evans Tokushima Otsuka (LETO) rats]: (1) the acetylcholine (ACh)-induced relaxation was impaired, (2) the NO- and EDHF-mediated relaxations were reduced, (3) the ACh-induced EDCF-mediated contraction and the production of prostanoids were increased, and (4) superoxide generation was increased. After such OLETF rats had received losartan (25 mg/kg/day p.o. for 4 weeks), their isolated mesenteric arteries exhibited: (1) improvements in ACh-induced NO- and EDHF-mediated relaxations, (2) reduced EDCF- and arachidonic acid-induced contractions, (3) suppressed production of prostanoids, (4) reduced PGE(2)-mediated contraction, and (5) reduced superoxide generation. Within the timescale studied here, losartan did not change the protein expressions of endothelial NO synthase, COX1, or COX2 in mesenteric arteries from either OLETF or LETO rats. Losartan thus normalizes vascular dysfunction in this type 2 diabetic model, and the above effects may contribute to the reduction of adverse cardiovascular events seen in diabetic patients treated with angiotensin II receptor blockers.

Losartan normalizes endothelium-derived hyperpolarizing factor-mediated relaxation by activating Ca2+-activated K+ channels in mesenteric artery from type 2 diabetic GK rat.

Ca(2+)-activated K(+) (K(Ca)) channels are important for endothelium-derived hyperpolarizing factor (EDHF) signaling. Since treatment with angiotensin II receptor blockers (ARBs) improves vasculopathies in type 2 diabetic patients, we asked whether the EDHF-type relaxation and its associated K(Ca) channels [small (SK(Ca))-, intermediate (IK(Ca))-, and large (BK(Ca))-conductance channels] are abnormal in mesenteric arteries isolated from Goto-Kakizaki (GK) rats at the chronic stage of type 2 diabetes (34 - 38 weeks) and whether an ARBs (losartan, 25 mg . kg(-1) . day(-1) for 2 weeks) might correct these abnormalities. Although the acetylcholine chloride-induced EDHF-type relaxation in mesenteric arteries from GK rats was reduced versus the Wistar controls, it was significantly restored by losartan treatment. The SK(Ca)-blocker apamin or the IK(Ca)-blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) inhibited such relaxations in the losartan-treated or -untreated Wistar groups and in the losartan-treated GK group, but not in the losartan-untreated GK group. The BK(Ca)-blocker iberiotoxin had a significant inhibitory effect in only one of these groups, the losartan-treated GK. The relaxations induced by the SK(Ca)/IK(Ca) activator NS309 and the BK(Ca) activator NS1619, which were impaired in GK rats, were normalized by losartan treatment. We conclude that losartan improves EDHF-type relaxation in GK rats at least partly by normalizing SK(Ca)/IK(Ca) activities and increasing BK(Ca) activity.

Gender differences in vascular reactivity of aortas from streptozotocin-induced diabetic mice.

The aim of the present study was to assess gender differences in diabetes-related vascular reactivity in murine aortas. Diabetes is a risk factor for ischemic heart disease, cerebral ischemia, and atherosclerosis, conditions in which endothelial dysfunction plays a pathogenetic role. We examined vascular responses in aortas isolated from streptozotocin (STZ)-induced type 1 diabetic mice and age-matched control mice, and looked for gender differences in the diabetes-induced changes in these responses. For each gender, the plasma adiponectin levels were lower in diabetic mice than in the controls, and they were significantly higher in females than in males. The acetylcholine (ACh)-induced endothelium-dependent relaxation of aortic rings was impaired (vs. that in the age-matched controls) in diabetic male mice, but not in diabetic female mice. The sodium nitroprusside-induced endothelium-independent aortic relaxation was not altered by diabetes in either male or female mice. The norepinephrine-induced aortic contraction was enhanced (vs. that in the control group) in diabetic female mice, but not in diabetic male mice, whereas in the presence of N(G)-nitro-L-arginine neither gender exhibited a significant diabetes-induced change in this contraction. The clonidine-induced and insulin-induced endothelium-dependent aortic relaxations were impaired only in the diabetic female group (vs. the age-matched controls). These results suggest that: a) in male diabetic mice, which exhibited low adiponectin levels, these were impairments of both the aortic relaxation and nitric oxide (NO) production induced by ACh, whereas b) in female diabetic mice, there were impairments of the aortic relaxations induced by both insulin and clonidine.

Vasodilator effect of Cassiarin A, a novel antiplasmodial alkaloid from Cassia siamea, in rat isolated mesenteric artery.

The aim of this study was to investigate the vasorelaxant effect induced by cassiarin A, a novel antiplasmodial alkaloid from Cassia siamea, in rings cut from rat superior mesenteric arteries. In rings precontracted with phenylephrine, cassiarin A induced a concentration-dependent relaxation. This relaxation was attenuated: 1) after removal of the endothelium or after pretreatment of rings with 100 microM of N(G)-nitro-L-arginine (nitric oxide synthase inhibitor) or 10 microM of 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (guanylyl cyclase inhibitor), but not after pretreatment with 10 microM of indomethacin (cyclooxygenase inhibitor); and 2) after pretreatment of preparations with either a nonselective or selective inhibitor of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels [1 mM of tetraethylammonium or 100 nM of iberiotoxin, respectively]. The cassiarin A-induced relaxation was also attenuated by these BK(Ca) inhibitors in endothelium-denuded preparations. The cassiarin A-induced relaxation was not altered by treatment with the ATP-sensitive K(+)-channel inhibitor glibenclamide (10 microM) or with the voltage-dependent K(+)-channel inhibitor 4-aminopyridine (1 mM). In isolated mesenteric artery rings, cassiarin A tended to increase nitric oxide (NO) levels. These results suggest that in the rat mesenteric artery, cassiarin A-induced relaxation may be mediated by endothelial NO and may occur partly via BK(Ca)-channel activation.

Vasorelaxant activity of Sappan Lignum constituents and extracts on rat aorta and mesenteric artery.

We investigated the vasorelaxant activity of the methanolic extracts of Sappan Lignum (CSE) and its constituents, brazilin, sappanchalcone, and protosappanins A-E, on rat aorta and mesenteric artery. By comparing the vasorelaxant activity of CSE and brazilin on both blood vessels, we found that CSE contained active constituents other than brazilin. When added to brazilin, sappanchalcone and protosappanin D showed vasorelaxant activity on both blood vessels precontracted with phenylephrine. We clarified that the vasorelaxant activity of brazilin was endothelium-independent, while that of sappanchalcone was endothelium-dependent, on both blood vessels. On the other hand, the vasorelaxant activity of protosappanin D was independent of the endothelium of the aorta and dependent on the endothelium of the mesenteric artery. Experiments on sappanchalcone and protosappanin D using NG-nitro-L-arginine and indomethacin revealed the involvement of nitric oxide and prostaglandin as endothelium-derived relaxation factors (EDRFs). The anti-oketsu effect of Sappan Lignum might be attributable to the interaction of those compounds. We could partly evaluate the anti-oketsu activity of Sappan Lignum using both the aorta and the mesenteric artery. Through this study, we showed the importance of comparing the effects on the aorta and the mesenteric artery as we found that natural compounds showed different mechanisms of action on the two blood vessels.

Relationship among superoxide-related enzyme, PPARs, and endothelium-dependent relaxation in murine aortas previously organ-cultured in high-glucose conditions.

The aim of the present study was to investigate the relationship among superoxide anion, peroxisome proliferator-activated receptors (PPARs), and endothelium-dependent relaxation in murine aortas organ-cultured in a high-glucose condition. Aortas organ-cultured with a high concentration of glucose (40 mmol/L, 20 h; HG group) exhibited the following characteristics (versus aortas cultured in serum-free medium): (i) significantly weaker relaxation to acetylcholine, but unchanged relaxation to SNP and unchanged contractions to norepinephrine and isotonic K+, (ii) significantly greater superoxide generation (indicated by the amount of nitroblue tetrazolium reduced, (iii) significantly higher protein expression levels of gp91phox, NAD(P)H oxidase subunits, and endothelial NO synthase, (iv) significantly lower protein expression level of Mn-superoxide dismutase (SOD), and (v) markedly greater reduction in the protein expression of PPARgamma than in that of PPARalpha. The HG-induced impairment of endothelium-dependent relaxation was prevented by cotreatment with tempol (a SOD mimetic). These results suggest that in the mouse aorta, exposure to high glucose levels may lead to an excessive generation of superoxide via increased gp91phox and decreased Mn-SOD protein expression and that this may in turn trigger an impairment of endothelium-dependent relaxation. Moreover, such protein changes in gp91phox and Mn-SOD may be secondary to a decreased expression of PPARgamma protein.

Activation of the PDK-1/Akt/eNOS pathway involved in aortic endothelial function differs between hyperinsulinemic and insulin-deficient diabetic rats.

In diabetic states, altered plasma insulin is likely to play key roles in 3-phosphoinositide-dependent protein kinase (PDK)/Akt pathway activation, in insulin resistance and in endothelial dysfunction. Since the molecular mechanism(s) remains unclear, we examined the relationship between the PDK/Akt/endothelial nitric oxide synthase (NOS) pathway and endothelial function in aortas from diabetic rats that were either insulin deficient or hyperinsulinemic. Untreated diabetic (diabetic) rats exhibited hyperglycemia and hypoinsulinemia, whereas high-insulin-treated diabetic (HI-diabetic) rats exhibited hyperinsulinemia. Aortas from the diabetic group displayed impaired endothelium-dependent relaxation in response to ACh, whereas the insulin-induced relaxation was increased. In HI-diabetic aortas, the ACh-induced relaxation was normal, but that induced by insulin was impaired. The insulin-induced relaxation was inhibited by treatment with an Akt inhibitor in control and diabetic aortas, but not in the HI-diabetic aorta. This inhibitory effect on insulin-induced relaxation was greater in diabetic aortas than in control aortas. In all groups, ACh-induced relaxation was unaffected by the above inhibitor. In the diabetic group, various insulin-stimulated levels (nitric oxide production, phosphorylation of endothelial NOS at Ser(1177), of Akt at Thr(308), and of PDK-1 at Ser(241)) were significantly increased, whereas, in the HI-diabetic group, these levels were all decreased (vs. control aortas). These results suggest that the plasma insulin level has a close relation to the level of aortic PDK-1/Akt (at Thr(308))/NOS activities, and that reduced actions of the PDK-1/Akt (at Thr(308)) signal pathway may contribute to the impairments of insulin-induced endothelial functions seen in hyperinsulinemic diabetes.

Eicosapentaenoic acid improves imbalance between vasodilator and vasoconstrictor actions of endothelium-derived factors in mesenteric arteries from rats at chronic stage of type 2 diabetes.

Accumulating evidence demonstrates that dietary intake of n-3 polyunsaturated fatty acids (PUFAs) is associated with a reduced incidence of several cardiovascular diseases that involve endothelial dysfunction. However, the molecular mechanism remains unclear. We previously reported that mesenteric arteries from type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats exhibit endothelial dysfunction, leading to an imbalance between endothelium-derived vasodilators [namely, nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)] and vasoconstrictors [endothelium-derived contracting factors (EDCFs)] [namely cyclooxygenase (COX)-derived prostanoids] (Am J Physiol Heart Circ Physiol 293:H1480-H1490, 2007). We hypothesized that treating OLETF rats with eicosapentaenoic acid (EPA), a major n-3 PUFA, may improve endothelial dysfunction by correcting this imbalance. In OLETF rats [compared with age-matched control Long-Evans Tokushima Otsuka (LETO) rats]: 1) acetylcholine (ACh)-induced (endothelium-dependent) relaxation was impaired, 2) NO- and EDHF-mediated relaxations and nitrite production were reduced, and 3) ACh-induced EDCF-mediated contraction, production of prostanoids, and the protein expressions of COX-1 and COX-2 were all increased. When OLETF rats received chronic EPA treatment long-term (300 mg/kg/day p.o. for 4 weeks), their isolated mesenteric arteries exhibited: 1) improvements in ACh-induced NO- and EDHF-mediated relaxations and COX-mediated contraction, 2) reduced EDCF- and arachidonic acid-induced contractions, 3) normalized NO metabolism, 4) suppressed production of prostanoids, 5) reduced COX-2 expression, and 6) reduced phosphoextracellular signal-regulated kinase (ERK) expression. Moreover, EPA treatment reduced both ERK2 and nuclear factor (NF)-kappaB activities in isolated OLETF aortas. We propose that EPA ameliorates endothelial dysfunction in OLETF rats by correcting the imbalance between endothelium-derived factors, at least partly, by inhibiting ERK, decreasing NF-kappaB activation, and reducing COX-2 expression.

Synthesis and structure-activity relationships of cassiarin A as potential antimalarials with vasorelaxant activity.

Cassiarin A 1, a tricyclic alkaloid, isolated from the leaves of Cassia siamea (Leguminosae), shows powerful antimalarial activity against Plasmodium falciparum in vitro as well as P. berghei in vivo, which may be valuable leads for novel antimalarials. Interactions of parasitized red blood cells (pRBCs) with endothelium in aorta are especially important in the processes contribute to the pathogenesis of severe malaria. Nitric oxide (NO) reduces endothelial expression of receptors/adhesion molecules used by pRBC to adhere to vascular endothelium, and reduces cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 showed vasorelaxation activity against rat aortic ring, which may be related with NO production. A series of a hydroxyl and a nitrogen-substituted derivatives and a dehydroxy derivative of 1 have been synthesized as having potent antimalarials against P. falciparum with vasodilator activity, which may reduce cytoadherence of pRBC to vascular endothelium. Cassiarin A 1 exhibited a potent antimalarial activity and a high selectivity index in vitro, suggesting that the presence of a hydroxyl and a nitrogen atom without any substituents may be important to show antimalarial activity. Relative to cassiarin A, a methoxy derivative showed more potent vasorelaxant activity, although it did not show improvement for inhibition of P. falciparum in vitro. These cassiarin derivatives may be promising candidates as antimalarials with different mode of actions.

Gender differences in vascular reactivity to endothelin-1 (1-31) in mesenteric arteries from diabetic mice.

Endothelin-1 (1-31) [ET-1 (1-31)], a novel member of the ET family, comprises 31 amino acids and is derived from the selective hydrolysis of big ET-1 by chymase. Although ET-1 (1-31) reportedly exerts biological effects by direct or indirect [via its conversion to ET-1 (1-21)] mechanisms, it is unclear whether in diabetes the vascular effects of ET-1 (1-31) display gender differences. We investigated this question by exposing mesenteric artery rings to ET-1 (1-31), using arteries from mice in the early or chronic phase of diabetes. In the early stage of diabetes, the ET-1 (1-31)-induced contraction was similar between age- and sex-matched control and streptozotocin (STZ)-induced diabetic mice. In the chronic stage of diabetes, the ET-1 (1-31)-induced contraction was enhanced in diabetic female mice, but not in diabetic male mice (vs. both age-matched control and early-stage diabetic mice). This enhancement was largely prevented by Y27632 (Rho kinase inhibitor), PD98059 [inhibitor of extracellular signal related kinases 1 and 2 (ERK1/2)], or SP600125 [C-jun terminal kinase (JNK) inhibitor]. These data indicate that the ET-1 (1-31)-induced vasoconstriction in the mesenteric artery may be specifically enhanced in established diabetic female mice, and that this enhancement may be due to alterations in the activities of Rho/Rho kinase or mitogen-activated protein kinase.