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In type 2 diabetes, impaired insulin-induced Akt/endothelial nitric oxide synthase (eNOS) signaling may decrease the vascular relaxation response. Previously, we reported that this response was negatively regulated by G protein–coupled receptor kinase 2 (GRK2). In this study, we investigated whether/how in aortas from ob/ob mice (a model of type 2 diabetes) GRK2 and β-arrestin 2 might regulate insulin-induced signaling. Endothelium-dependent relaxation was measured in aortic strips. GRK2, β-arrestin 2, and Akt/eNOS signaling pathway proteins and activities were mainly assayed by Western blotting. In ob/ob (vs. control [Lean]) aortas: 1) insulin-induced relaxation was reduced, and this deficit was prevented by GRK2 inhibitor, anti-GRK2 antibody, and an siRNA specifically targeting GRK2. The Lean aorta relaxation response was reduced to the ob/ob level by pretreatment with an siRNA targeting β-arrestin 2. 2) Insulin-stimulated Akt and eNOS phosphorylations were decreased. 3) GRK2 expression in membranes was elevated, and, upon insulin stimulation, this expression was further increased, but β-arrestin 2 was decreased. In ob/ob aortic membranes under insulin stimulation, the phosphorylations of Akt and eNOS were augmented by GRK2 inhibitor. In mouse aorta, GRK2 may be, upon translocation, a key negative regulator of insulin responsiveness and an important regulator of the β-arrestin 2/Akt/eNOS signaling, which is implicated in diabetic endothelial dysfunction.

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, 17β-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.

In the present sutdy, we have examined the relationship between the CaMKII (Ca2+/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 Ser1177-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 Thr286-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 Thr320-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. Abbreviations: A23187, calcimycin A23187; ACh, acetylcholine; BAPTA/AM, 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester); BP, blood pressure; CA, cantharidic acid; CaMKII, Ca2+/calmodulin-dependent protein kinase II; EDHF, endothelium-derived hyperpolarizing factor; GK, Goto–Kakizaki; HDL, high-density lipoprotein; KHS, Krebs–Henseleit solution; L-NNA, NG-nitro-L-arginine; NA, noradrenaline; NOS, NO synthase; eNOS, endothelial NOS; NOx:, combined nitrate+nitrite; PE, phenylephrine; PKA, protein kinase A; PP, protein phosphatase; PPI2, PP inhibitor 2; SNP, sodium nitroprusside; TRAM, triarylmethane; vWF, von Willebrand factor

In type 2 diabetes, although Akt/endothelial NO synthase (eNOS) activation is known to be negatively regulated by G protein-coupled receptor kinase 2 (GRK2), it is unclear whether the GRK2 inhibitor would have therapeutic effects. Here we examined the hypotensive effect of the GRK2 inhibitor and its efficacy agonist both vascular (aortic) endothelial dysfunction (focusing especially on the Akt/eNOS pathway) and glucose intolerance in two type 2 diabetic models (ob/ob mice and nicotinamide+streptozotocin-induced diabetic mice). Mice were treated with a single injection of the GRK2 inhibitor or vehicle, and the therapeutic effects were compared by examining vascular function and by Western blotting. The GRK2 inhibitor lowered blood pressure in both diabetic models but not in their age-matched controls. The GRK2 inhibitor significantly improved clonidine-induced relaxation only in diabetic (ob/ob and DM) mice, with accompanying attenuations of GRK2 activity and translocation to the plasma membrane. These protective effects of the GRK2 inhibitor may be attributable to the augmented Akt/eNOS pathway activation (as evidenced by increases in Akt phosphorylation at Ser(473) and at Thr(308), and eNOS phosphorylation at Ser(1177)) and to the prevention of the GRK2 translocation and promotion of β-arrestin 2 translocation to the membrane under clonidine stimulation. Moreover, the GRK2 inhibitor significantly improved the glucose intolerance seen in the ob/ob mice. Our work provides the first evidence that in diabetes, the GRK2 inhibitor ameliorates vascular endothelial dysfunction via the Akt/eNOS pathway by inhibiting GRK2 activity and enhancing β-arrestin 2 translocation under clonidine stimulation, thereby contributing to a blood pressure-lowering effect. We propose that the GRK2 inhibitor may be a promising therapeutic agent for cardiovascular complications in type 2 diabetes.

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.

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 A2 (cPLA2) inhibitor; 2) both nucleotides induced increased release of PGE2 and PGF2α; 3) nucleotide-stimulated cPLA2 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 PGE2 and PGF2α, 3) suppressed UTP-stimulated cPLA2 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 cPLA2/COX pathway and, moreover, that losartan normalizes such contractions by a suppressing action within this pathway.

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 Thr³⁰⁸ 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.

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, β-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 Thr308) phosphorylation and eNOS (at Ser1177) phosphorylation, and also the β-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 β-arrestin2 inhibit insulin-induced stimulation of the Akt/eNOS pathway and that GRK2 overactivation may result from an increase in PKC activity.

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.

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 PPARγ than in that of PPARα. 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 PPARγ protein.

Endothelin (ET)-1 and angiotensin II (Ang II) are likely candidates for a key role in diabetic vascular complications. We demonstrated previously that an enhanced ET-1-induced contraction is present in mesenteric arteries from Goto-Kakizaki (GK) rats at the chronic stage of type 2 diabetes. Here, we investigated whether short-term treatment of such rats with losartan, an angiotensin type 1 receptor antagonist, might normalize the ET-1-induced contraction. In mesenteric arteries from GK rats at the chronic stage (34-38 weeks) (vs. those from age-matched control Wistar rats): (1) the ET-1-induced contraction was enhanced, (2) the levels of ET-1 and Ang II were increased, (3) ET-1-stimulated ERK2 phosphorylation was increased, and (4) the ACh-induced endothelium-dependent relaxation was reduced. Mesenteric arteries isolated from such GK rats following treatment with losartan (25mg/kg/day for 2 weeks) exhibited reduced ET-1- and Ang II-induced contractions, suppressed ET-1-stimulated ERK phosphorylation, and increased ACh-induced relaxation, while the rats exhibited normalized plasma NO metabolism and their mesenteric arteries exhibited increased basal NO formation. However, such losartan treatment did not alter the increased levels of ET-1 and Ang II seen in GK mesenteric arteries. Our data suggest that within the timescale studied here, losartan normalizes ET-1-induced mesenteric artery contraction through a suppression of ERK activities and/or by normalizing endothelial function.

Background and purpose: Arteries from hypertensive subjects are reportedly hyperresponsive to 5-hydroxytryptamine (5-HT), but it remains unclear whether this is true in chronic type 2 diabetes. We have assessed responses to 5-HT shown by mesenteric arteries from type 2 diabetic ob/ob mice (27–32 weeks old) and have identified the molecular mechanisms involved. Experimental approach: Contractions of mesenteric rings to 5-HT were examined in vitro. Activation of mesenteric RhoA, Rho kinase and Src was measured by Western blotting or by modified enzyme-linked immunosorbent assay. Key results: Concentration-dependent contractions to 5-HT were greater in mesenteric rings from the ob/ob than in those from the age-matched control (‘Lean’) group. In each group, there was no significant change in the 5-HT-induced contractions after inhibition of nitric oxide synthase (with NG-nitro-L-arginine), of cyclooxygenase (with indomethacin) or of protein kinase C (with chelerythrine). However inhibition of the MEK/ERK pathway (with PD98059) decreased the response to 5-HT. Although the diabetes-related enhancement of the 5-HT response was preserved with each of these inhibitors, enhancement was abolished by a Rho kinase inhibitor (Y27632) and by Src kinase inhibitors (PP1 analogue or Src kinase inhibitor I). 5-HT-induced activation of RhoA, Rho kinase and Src kinase in mesenteric arteries was greater in the ob/ob than in the Lean group, but the expression of RhoA, Rho kinase isoforms and Src did not differ between these groups. Conclusions and implications: These results suggest that the enhancement of 5-HT-induced contraction in mesenteric arteries from ob/ob mice may be attributable to increased activation of RhoA/Rho kinase and Src kinase.

Ca2+-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]-1 H-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 acti-vator 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. Keywords:: angiotensin receptor antagonist, diabetes, endothelium-derived hyperpolarizing factor (EDHF), GK rat, potassium channel

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.

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.

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.

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 Ser1177, of Akt at Thr308, and of PDK-1 at Ser241) 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 Thr308)/NOS activities, and that reduced actions of the PDK-1/Akt (at Thr308) signal pathway may contribute to the impairments of insulin-induced endothelial functions seen in hyperinsulinemic 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)-κB 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-κB activation, and reducing COX-2 expression.

Type 2 diabetes mellitus is associated with high mortality and morbidity, mainly due to coronary artery disease and atherosclerosis, although female gender is a protective factor in the development of, for example, atherosclerosis and hypertension. Our main aim was to investigate gender differences in endothelial function in aortas from type 2 diabetic model mice. The nonfasting plasma glucose level was significantly elevated in diabetic mice (both males and females). The plasma insulin level was not different between controls and diabetics (either gender). The plasma adiponectin level was decreased by diabetes, and was lower in males. In control aortas (from males or females), the clonidine-induced relaxation was abolished by Akt-inhibitor treatment. In diabetic males (versus both control males and diabetic females): a) the clonidine- and insulin-induced endothelium-dependent aortic relaxations were impaired, but the acetylcholine (ACh)–induced and sodium nitroprusside (SNP)–induced aortic relaxations were not, b) the norepinephrine (NE)–induced aortic contractile response was enhanced, c) systemic blood pressure was elevated, and d) the clonidine-stimulated Ser-473 phosphorylation of Akt in the aorta was decreased. These results suggest that endothelial functions dependent on the Akt pathway are abrogated by type 2 diabetes only in male mice. Keywords:: diabetes, gender difference, endothelial cell, aorta

It has been shown that enhancement of vasoconstrictor prostanoids plays an important role in the development of cardiovascular diseases. The aim of the present study was to examine the effects of pyrrolidine dithiocarbamate (PDTC), a low-molecular-weight thiol antioxidant and a potent inhibitor of nuclear factor-κB (NF-κB), on both the response to and production of prostanoids in arterial vessels isolated from rats at the chronic stage of type 2 diabetes. Using aortas from type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats, control Long-Evans Tokushima Otsuka (LETO) rats, and LETO and OLETF rats treated with PDTC (30 mg/kg, s.c., daily, for 1 week), we measured the production of prostanoids and NF-κB activity. The arachidonic acid–induced contraction and the acetylcholine-induced endothelium-derived contracting factor (EDCF)-mediated contraction in mesenteric arteries were also compared among these groups. OLETF rats exhibited (vs. age-matched LETO rats) the following: increased responses to both arachidonic acid and EDCF and greater productions of PGE2 and TXA2. Treatment with PDTC resulted in the following: 1) reduced arachidonic acid– and EDCF-mediated contractions, 2) suppressed the production of prostanoids, and 3) normalized NF-κB activity. These results suggest that PDTC has beneficial effects against the abnormal vasoconstrictor prostanoid signaling present in rats at the chronic stage of type 2 diabetes. Keywords:: diabetes, endothelium-derived contracting factor (EDCF), nuclear factor-κB (NF-κB), pyrrolidine dithiocarbamate (PDTC), prostanoid

Diabetes mellitus impairs endothelial function, an effect that can be considered a hallmark of the development of cardiovascular diseases in diabetics. Cilostazol, a selective phosphodiesterase 3 inhibitor, is currently used to treat patients with diabetic vascular complications. However, the effects of cilostazol on responses mediated by endothelium-derived relaxing [in particular, nitric oxide (NO) and hyperpolarizing factors (EDHF)] and contracting factors remain unclear. Here, we hypothesized that cilostazol could improve endothelial dysfunctions in mesenteric arteries isolated from type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Using cilostazol-treated (100 mg/kg/day for 4 weeks) or -untreated OLETF and control (Long Evans Tokushima Otsuka) rats, we examined the acetylcholine-induced endothelium-dependent responses and the cell-permeant cyclic adenosine monophosphate (cAMP) analog-induced relaxations in the superior mesenteric artery. We also determined blood parameters in these animals. In OLETF rats, chronic treatment with cilostazol reduced the blood levels of triglyceride, non-esterified fatty acids, and leptin, and increased antioxidant capacity, but did not alter the blood glucose or insulin levels. In studies on mesenteric arteries from cilostazol-treated OLETF animals, the cilostazol treatment improved: (a) the acetylcholine-induced EDHF-mediated relaxation and (b) the cAMP-mediated relaxation. However, cilostazol did not alter the NO-mediated relaxation or the endothelium-derived contracting factor-mediated contraction. These results suggest that cilostazol improves endothelial functions in OLETF mesenteric arteries by increasing EDHF signaling, and that it normalizes some metabolic abnormalities in OLETF rats. On that basis, cilostazol may prove to be a potent drug for the clinical treatment of diabetic vasculopathy.

Background and purpose: Mechanisms associated with the enhanced contractile response to endothelin-1 in hyperinsulinaemic diabetes have been examined using the rat aorta. Functions for angiotensin II, endothelin-1 receptor expression and extracellular signal-regulated kinase (ERK) have been investigated. Experimental approach: Streptozotocin-induced diabetic rats were infused with angiotensin II or, following insulin treatment, were treated with losartan, an angiotensin II receptor antagonist. Contractions of aortic strips with or without endothelium, in response to endothelin-1 and angiotensin II, were examined in vitro. Aortic ETA receptors and ERK/MEK expression were measured by western blotting. Key results: Insulin-treated diabetic rats exhibited increases in plasma insulin, angiotensin II and endothelin-1. The systolic blood pressure and endothelin-1-induced contractile responses in aortae in vitro were enhanced in insulin-treated diabetic rats and blunted by chronic losartan administration. LY294002 (phosphatidylinositol 3-kinase inhibitor) and/or PD98059 (MEK inhibitor) diminished the enhanced contractile response to endothelin-1 in aortae from insulin-treated diabetic rats. ETA and ETB receptors, ERK-1/2 and MEK-1/2 protein expression and endothelin-1-stimulated ERK phosphorylation were all increased in aortae from insulin-treated diabetic rats. Such increases were blunted by chronic losartan administration. Endothelin-1-induced contraction was significantly higher in aortae from angiotensin II-infused diabetic rats. angiotensin II-infusion increased ERK phosphorylation, but the expression of endothelin receptors and ERK/MEK proteins remained unchanged. Conclusions and implications: These results suggest that the combination of high plasma angiotensin II and insulin with a diabetic state induced enhancement of endothelin-1-induced vasoconstriction, ETA receptor expression and ERK expression/activity in the aorta. Losartan improved both the diabetes-related abnormalites and the diabetic hypertension. British Journal of Pharmacology (2008) 155, 974–983; doi:10.1038/bjp.2008.327; published online 18 August 2008

We previously reported that in mesenteric arteries from aged Otsuka Long-Evans Tokushima fatty (OLETF) rats (a type 2 diabetes model) endothelium-derived hyperpolarizing factor (EDHF)-type relaxation is impaired while endothelium-derived contracting factor (EDCF)-mediated contraction is enhanced (Matsumoto T, Kakami M, Noguchi E, Kobayashi T, Kamata K. Am J Physiol Heart Circ Physiol 293: H1480–H1490, 2007). Here we investigated whether acute and/or chronic treatment with metformin might improve this imbalance between the effects of the above endothelium-derived factors in mesenteric arteries isolated from OLETF rats. In acute studies on OLETF mesenteric arteries, ACh-induced relaxation was impaired and the relaxation became weaker at high ACh concentrations. Both metformin and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside [AICAR, an AMP-activated protein kinase (AMPK) activator that is also activated by metformin] 1) diminished the tendency for the relaxation to reverse at high ACh concentrations and 2) suppressed both ACh-induced EDCF-mediated contraction and ACh-stimulated production of prostanoids (thromboxane A2and PGE2). In studies on OLETF arteries from chronically treated animals, metformin treatment (300 mg·kg−1·day−1for 4 wk) 1) improved ACh-induced nitric oxide- or EDHF-mediated relaxation and cyclooxygenase (COX)-mediated contraction, 2) reduced EDCF-mediated contraction, 3) suppressed production of prostanoids, and 4) reduced superoxide generation. Metformin did not alter the protein expressions of endothelial nitric oxide synthase (eNOS), phospho-eNOS (Ser1177), or COX-1, but it increased COX-2 protein. These results suggest that metformin improves endothelial functions in OLETF mesenteric arteries by suppressing vasoconstrictor prostanoids and by reducing oxidative stress. Our data suggest that within the timescale studied here, metformin improves endothelial function through this direct mechanism, rather than by improving metabolic abnormalities.

The primary goal of this study was to investigate the effect of long-term (9 months) streptozotocin (STZ)-induced diabetes on the coronary vasoconstrictor responses to vasoactive agents such as high K(+), acetylcholine (ACh), endothelin-1 (ET-1), and the calcium-channel activator Bay K 8644. For this, we used isolated rat hearts perfused at constant flow rate. Each of the four agents caused dose-dependent increases in perfusion pressure in isolated hearts from age-matched control and STZ-induced diabetic rats. The dose-response curves for high K(+), ACh, and ET-1 were shifted to the left, so that at some lower doses of these agents the increased perfusion pressure was greater in coronary arteries obtained from diabetic rats than in those from control rats. On the other hand, the maximum contractile response induced by each of these agents was lower in the diabetic perfused heart. The Bay K 8644-induced contractile response was significantly greater in the coronary arteries of diabetic rats than in those of control rats. A threshold-constrictor concentration of Bay K 8644 (1 nM) potentiated the ACh-induced vasoconstriction in coronary arteries from both groups, and the potentiated responses were greater in diabetic rats than in controls at lower concentrations of ACh (100 nM and 1 microM). These findings suggest that the coronary artery contractile responses to lower concentrations of ACh or ET-1 are exaggerated in long-term STZ-induced diabetic hearts. These changes may be due to alterations in the activity of voltage-gated Ca(2+) channels.

We investigated the effects of chronic simvastatin treatment on the impaired endothelium-dependent relaxation seen in aortas from type 2 diabetic mice. Starting at 8 weeks of diabetes, simvastatin (10 mg/kg per day) was administered to diabetic mice for 4 weeks. The significantly elevated systolic blood pressure in diabetic mice was normalized by simvastatin. Aortas from diabetic mice, but not those from simvastatin-treated diabetic mice, showed impaired endothelium-dependent relaxation in response to both clonidine and adrenomedullin. After preincubation with an Akt inhibitor, these relaxations were not significantly different among the three Akt inhibitor–treated groups (controls, diabetics, and simvastatin-treated diabetics). Although clonidine-induced NOx−(NO2−+NO3−) production was greatly attenuated in our diabetic model, it was normalized by simvastatin treatment. The expression levels of both total Akt protein and clonidine-induced Ser-473-phosphorylated Akt were significantly decreased in diabetic aortas, while chronic simvastatin administration improved these decreased levels. The expression level of clonidine-induced phosphorylated PTEN (phosphatase and tensin homolog deleted on chromosome ten) was significantly increased in diabetic aortas, but chronic simvastatin did not affect it. These results strongly suggest that simvastatin improves the endothelial dysfunction seen in type 2 diabetic mice via increases in Akt and Akt phosphorylation. Keywords:: diabetes, endothelial cell, statin, Akt, phosphatase and tensin homolog deleted on chromosome ten (PTEN)

Macro- and microvascular disorders currently represent the principal causes of morbidity and mortality in patients with diseases involving the cardiovascular system, such as atherosclerosis, hypertension, stroke, and diabetes. Abnormal vasomotor responses and impaired endothelium-dependent vasodilation have been demonstrated in a number of vessels in a variety of animal models and in humans with such diseases. Endothelial dysfunction plays a key role in the development of these diseases, yet the genesis of this endothelial dysfunction and its associated vasomotor abnormalities remain poorly understood. Peroxisome proliferator-activated receptor (PPAR)gamma is a nuclear receptor and transcription factor in the steroid superfamily, and PPARgamma agonists (the thiazolidinediones) are used clinically to treat type 2 diabetes. Recent studies have revealed that as well as being involved in adipogenesis and in increased sensitivity to insulin, PPARgamma plays critical roles in the vasculature. In the present review, we discuss the beneficial effects of PPARgamma agonists on vasomotor activities, focusing in particular on endothelium-dependent relaxation in vessels affected by cardiovascular diseases.

Lysophosphatidylcholine (LPC) is a bioactive proinflammatory lipid generated by pathological activities. LPC is also a major phospholipid component of oxidized low-density lipoprotein (Ox-LDL) and is implicated as a critical factor in the atherogenic activity of Ox-LDL. LPC is believed to play an important role in atherosclerosis and inflammatory diseases by altering various functions in a number of cell-types, including endothelial cells, smooth muscle cells, monocytes, macrophages, and T-cells. LPC activates several second messengers -- including protein kinase C, extracellular-signal-regulated kinases, protein tyrosine kinases, and Ca(2+) -- implicating the engagement of transduction mechanisms in its observed actions. Moreover, recent evidence suggests that in several cell-types, cloned orphan G-protein-coupled receptors may serve as the specific receptors via which LPC modulates second messenger pathways (although LPC may not be a direct ligand of such receptors). In addition, current evidence suggests that LPC impairs the endothelium-dependent relaxations mediated by endothelium-derived relaxing factors and directly modulates contractile responses in vascular smooth muscle. However, despite all this, and although elevated levels of LPC have been linked to the cardiovascular complications associated with atherosclerosis, ischemia, and diabetes, the precise pathophysiological roles played by LPC in several states remain to be established. In this review, we focus in some detail on the entirety of the signal-transduction system for LPC, its pathophysiological implications, and the vascular abnormalities associated with it.

We examined the responses of basilar arteries taken from Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a type 2 diabetes model. Both the nitric oxide (NO)-mediated relaxation and the cyclic 3',5'-guanosine monophosphate (cGMP) production elicited by acetylcholine (ACh) were much weaker in OLETF rats than in age-matched control Long Evans Tokushima Otsuka (LETO) rats. The contraction induced by an NO synthase (NOS) inhibitor [N(G)-nitro-L-arginine (L-NNA)] was weaker in the OLETF group. In that group, application of apocynin, an NAD(P)H oxidase inhibitor, normalized (i) ACh-induced relaxation, (ii) L-NNA-induced contraction, and (iii) ACh-induced cGMP production to the LETO levels. Superoxide anion production was greater in basilar arteries from OLETF rats than in those from LETO rats. The protein expression of gp91(phox), an NAD(P)H oxidase subunit, was upregulated in the OLETF arteries (versus LETO ones). These results suggest that the existence of endothelial dysfunction in basilar arteries in type 2 diabetes is related to increased oxidative stress mediated via NAD(P)H oxidase. Possibly, an impairment of NO-dependent relaxation responses and a basal impairment of NO signaling may be responsible for the increased risk of adverse cerebrovascular events in type 2 diabetes.

We investigated the involvement of angiotensin II and vascular smooth muscle sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) function in the impaired NO-induced relaxation seen in established streptozotocin-induced diabetes. Plasma angiotensin II levels, which were elevated in untreated diabetic rats (vs age-matched controls), were improved by treatment with the angiotensin-converting enzyme inhibitor enalapril. Systolic blood pressure was significantly decreased in chronic enalapril-treated diabetics (vs the other two groups). Intact aortae from diabetic rats and chronic angiotensin II-infused control rats, but not those from diabetic rats treated with enalapril, showed impaired endothelium-dependent relaxations to acetylcholine (vs controls). The relaxation induced by Angeli's Salt (a NO donor) was significantly impaired in endothelium-denuded aortae from diabetic rats (vs controls) but it was normalised by enalapril treatment. After preincubation with the irreversible SERCA inhibitor, thapsigargin, the relaxation induced by Angeli's Salt was significantly impaired in endothelium-denuded aortae from the controls, but not from the diabetics, and there was no significant difference between the thapsigargin-treated groups. Nitrotyrosine, an indirect marker of peroxynitrite, was markedly increased in aortic smooth muscle from diabetic rats, while chronic enalapril administration reduced this increase. These results suggest that in streptozotocin-induced diabetic rats, excessive angiotensin II production may lead to the generation of peroxynitrite and that this may in turn trigger a dysfunction of vascular smooth muscle SERCA. Enalapril improved the diabetes-related impairments.

Background and purpose: The effect of lysophosphatidylcholine (LPC) on aortic contractions in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a type 2 diabetic model, was studied. Experimental approach: Using OLETF rats and control (Long Evans Tokushima Otsuka (LETO)) rats, the effects of LPC on the contractions induced by high-K+ (10-40 mM), UK14,304 (10∼100 nM; a selective α2-adrenoceptor agonist) and sodium orthovanadate (SOV; 10 μM∼3 mM) in endothelium-denuded aortae were compared. Aortic ERK activity and the mRNA expression for GPR4 (a putative LPC receptor) were also measured. Key results: OLETF rats exhibited (vs. age-matched LETO rats): (1) greater potentiation of high-K+-induced contraction by 10 μM LPC – a potentiation attenuated by 10 μM genistein, protein tyrosine kinase (PTK) inhibitor, (2) greater potentiation of UK14,304 (10∼100 nM)-induced contractions by LPC (1 μM∼10 μM) – a potentiation attenuated by 10 μM genistein, 50 μM tyrphostin A23 (PTK inhibitor) or 10 μM PD98059 (MEK 1/2 inhibitor), (3) greater basal and LPC (1 μM)-induced ERK activities, (4) greater basal and 100 nM UK14,304-stimulated ERK2 activities in both the absence and presence of 10 μM LPC, (5) greater SOV (10 μM∼3 mM)-induced contractions, (6) greater potentiation of SOV-induced contractions by 10 μM LPC – a potentiation suppressed by 10 μM PD98059 or 10 μM genistein, (7) upregulation of GPR4 mRNA. Conclusions and implications: These results suggest that the LPC-induced potentiation of contractions in the OLETF rat aorta may be attributable to increased PTKs or ERK activity and/or to receptor upregulation. British Journal of Pharmacology (2006) 149, 931–941. doi:10.1038/sj.bjp.0706937

AIM To clarify the time-related changes in cardiac function and the mechanism underlying the cardiac dysfunction present in diabetes mellitus, we studied mechanical responses induced by alpha(1)- and beta-adrenoceptors, the Ca(2+)-entry promoter Bay K 8644- and ryanodine (an agent known to inhibit Ca(2+) release from the sarcoplasmic reticulum) in papillary muscles from streptozotocin (STZ)-induced diabetic and age-matched control rats. METHODS Male Wistar rats received a single injection of STZ (60 mg kg(-1)) via the tail vein to induce diabetes. For the mechanical studies, papillary muscle preparations were suspended in an organ bath and isometric contractions were measured in 1-, 4-, and 10-week STZ-induced diabetic and age-matched control rats. RESULTS In 1-week diabetic rats, the contractions induced by isoproterenol, methoxamine and Bay K 8644 were unchanged (vs. age-matched controls). In 4-week diabetic rats, (a) the isoproterenol- and Bay K 8644-induced contractions were impaired, (b) sensitivity to ryanodine was reduced, whereas (c) the methoxamine-induced contraction was unchanged. In 10-week diabetic rats, the isoproterenol- and Bay K 8644-induced contractile responses were impaired and the sensitivity to ryanodine was reduced, but in sharp contrast the methoxamine-induced contraction was enhanced. Both the mRNA level for the alpha(1A) adrenoceptor (but not the alpha(1B) or alpha(1D) mRNAs) and alpha(1A) adrenoceptor protein were increased in 10-week diabetic rats (vs. age-matched controls). CONCLUSION These results suggest that impairments of beta-adrenergic and Ca(2+)-handling mechanisms occur early in the development of cardiomyopathy in STZ-induced diabetic rats, and that this is followed by augmentation of alpha(1A) adrenoceptor-mediated inotropy due to alpha(1A) adrenoceptor upregulation.

We studied the relationship among endothelial function, oxidative stress, and phenylephrine (PE; alpha(1)-adrenoceptor agonist)-induced contraction in mesenteric arteries from high-cholesterol (HC)-diet-fed mice. In HC mice (vs age-matched normal-diet-fed mice): (1) PE-induced contraction in endothelium-intact rings was enhanced (endothelial denudation increased contraction in "normal-diet" rings, but did not enhance it further in "HC" rings); (2) the enhanced PE-induced contraction was further enhanced in the presence of N(G)-nitro-L-arginine (L-NNA; nitric oxide synthase inhibitor) or L-NNA plus indomethacin (cyclooxygenase inhibitor) [to preserve endothelium-derived hyperpolarizing factor (EDHF)], but unchanged in the presence of charybdotoxin plus apamin (to block EDHF); (3) ACh-induced EDHF-type relaxation was reduced; and (4) oxidative stress [indicated by the plasma 8-isoprostane level (reliable systemic marker) and aortic superoxide production] was greater. In HC mice, PE-induced contraction was normalized by apocynin [NAD(P)H oxidase inhibitor] or tempol (superoxide dismutase mimetic), but enhanced by NADH [NAD(P)H oxidase substrate]. Oral dietary supplementation with apocynin (30 mg/kg/day for 4 weeks) corrected the above abnormalities. Hence: (1) PE-induced contraction is modulated by the endothelium, and the enhanced contractility in HC mice results from defective EDHF signaling and elevated oxidative stress, and (2) apocynin normalizes PE-induced contraction in HC mice by improving EDHF signaling.

Vascular alterations in diabetes cause or contribute to the etiology of microvascular complications such as nephropathy, neuropathy, and retinopathy. The endothelium controls the vascular smooth muscle tone through the production of vasodilator mediators such as nitric oxide, prostacyclin, and a still-elusive endothelium-derived hyperpolarizing factor (EDHF). Although EDHF is a prominent vasodilator, particularly in smaller arteries, little attention has been paid to the potential role of EDHF responses in diabetes. EDHF function may involve the participation of mediators, including several diffusible factors and non-diffusible factors, (e.g., conduction of hyperpolarization via myoendothelial gap junctions). Indeed, in several vessels, cyclic adenosine 3,5-monophosphate (cAMP) facilitates EDHF responses by enhancing electrotonic conduction via gap junctions. It has been demonstrated that the alterations in EDHF relaxation seen in mesenteric arteries from diabetic rats may be attributable to an increase in phosphodiesterase3 (PDE3) activity, leading to a reduction in the action of cAMP, and moreover the activity of protein kinase A (PKA) is decreased in such arteries. Although an improvement in EDHF responses has not been, as yet, the subject of any direct pharmaceutical effort, increasing cAMP/PKA signaling (e.g., by inhibiting PDE3 activity) has potential as an interesting therapeutic target in diabetic microvascular disease.

We investigated the involvement of ANG II and phosphatidylinositol 3-kinase (PI3-K) in the enhanced aortic contractile responses induced by hyperinsulinemia in chronic insulin-treated Type 1 diabetic rats. Plasma ANG II levels were elevated in untreated compared with control diabetic rats and further increased in insulin-treated diabetic rats. Aortic contractile responses and systolic blood pressure were significantly enhanced in chronic insulin-treated diabetic rats compared with the other groups. These insulin-induced increases were largely prevented by cotreatment with losartan (an ANG II type 1 receptor antagonist) or enalapril (an angiotensin-converting enzyme inhibitor). LY-294002 (a PI3-K inhibitor) diminished the increases in contractile responses in ANG II-incubated aortas and aortas from chronic insulin-treated diabetic rats. The norepinephrine (NE)-stimulated levels of p110δ-associated PI3-K activity and p110δ protein expression were increased in aortas from insulin-treated diabetic compared with control and untreated diabetic rats, and chronic administration of losartan blunted these increases. Contractions were significantly larger in aortas from diabetic rats incubated with a low concentration (inducing ∼10% of the maximum contraction) of ANG II or with NE or isotonic K+ than in aortas from nonincubated diabetic rats. NE-stimulated p110 PI3-K activity was elevated in aortas from diabetic rats coincubated with a noncontractile dose of ANG II. These results suggest that, in insulin-treated Type 1 diabetic rats with hyperinsulinemia, chronic ANG II type 1 receptor blockade blunts the increases in vascular contractility and blood pressure via a decrease in p110δ-associated PI3-K activity.

We previously reported that in mesenteric arteries from streptozotocin-induced diabetic rats, the endothelium-derived hyperpolarizing factor (EDHF)-type relaxation is impaired, possibly due to a reduced action of cAMP. Here, we observed an impairment of acetylcholine-induced EDHF-type relaxation in mesenteric arteries from a type 2 diabetic model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats [vs. age-matched control Long-Evans Tokushima Otsuka (LETO) rats], and we investigated the mechanism underlying this impairment. In the LETO group, this EDHF-type relaxation was attenuated by 18α-glycyrrhetinic acid (a gap-junction inhibitor) and by a protein kinase A (PKA) inhibitor. In both groups (OLETF and LETO), it was enhanced by 3-isobutyl-1-methylxanthine, a cAMP-phosphodiesterase (PDE) inhibitor, but following these enhancements it was still weaker in OLETF rats than in LETO rats. The relaxations induced by cilostamide (a selective PDE3 inhibitor) and 8-bromo-cAMP (a cell-permeant cAMP analog) were reduced in OLETF rats, as was PKA activity. The relaxations induced by two activators of Ca 2+ -activated K + channels (K Ca ) [1-ethyl-2-benzimidazolinone (1-EBIO), intermediate-conductance K Ca channel (IK Ca ) activator, and riluzole, small-conductance K Ca channel (SK Ca ) activator] were also impaired in OLETF rats. We conclude that the impairment of EDHF-type relaxation seen in OLETF rats may be attributable not only to a reduction in cAMP/PKA signaling, but also to reduced endothelial K Ca channel activities.

Anthocyanins, which are responsible for a variety of bright colors (including red, blue, and purple) in fruits, vegetables, and flowers, are consumed as dietary polyphenols. Anthocyanin-containing fruits are thought to decrease coronary heart disease and are used in anti-diabetic preparations. Diabetes is associated with a variety of cardiovascular complications that may be mediated by endothelial dysfunction, and so this study was designed mainly to characterize the influence of a synthesized anthocyanidin derivative (HK-008) over acetylcholine (ACh)-induced relaxation in mesenteric arterial beds isolated from rats. In a glucose-tolerance test in intact rats, HK-008 (30 mg/kg) reduced the glucose level as effectively as the same dose of glibenclamide. The aortic relaxation induced by pinacidil (an ATP-sensitive potassium channel opener) was greatly inhibited by glibenclamide (10 microM), and also significantly inhibited by HK-008 (10 microM). Interestingly, the ACh-induced relaxation in the perfused, preconstricted mesenteric arterial bed was significantly enhanced by HK-008 (10 microM), and this enhancement was significantly attenuated by indomethacin (10 microM). The ACh-induced mesenteric relaxation was impaired by an increase in oxidative stress, viz. superoxide-generating treatment [xanthine oxidase (XO; 0.1 U/ml) plus hypoxanthine (HX; 10 microM)]. However, this impairment was strongly suppressed by HK-008 (10 microM). These results suggest that HK-008 increases endothelium-induced relaxation by suppressing oxidative stress or modulating prostanoids signaling. This compound may therefore be useful against certain cardiovascular disorders.

We previously reported that in mesenteric arteries from streptozotocin (STZ)-induced diabetic rats that 1) endothelium-derived hyperpolarizing factor (EDHF)-type relaxation is impaired, possibly due to a reduced action of cAMP via increased phosphodiesterase 3 (PDE3) activity (Matsumoto T, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 285: H283–H291, 2003) and that 2) PKA activity is decreased (Matsumoto T, Wakabayashi K, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 287: H1064–H1071, 2004). Here we investigated whether chronic treatment with cilostazol, a PDE3 inhibitor, improves EDHF-type relaxation in mesenteric arteries isolated from STZ rats. We found that in such arteries 1) cilostazol treatment (2 wk) improved ACh-, A-23187-, and cyclopiazonic acid-induced EDHF-type relaxations; 2) the ACh-induced cAMP accumulation was transient and sustained in arteries from cilostazol-treated STZ rats; 3) the EDHF-type relaxation was significantly decreased by a PKA inhibitor in the cilostazol-treated group, but not in the cilostazol-untreated group; 4) cilostazol treatment improved both the relaxations induced by cAMP analogs and the PKA activity level; and 5) PKA catalytic subunit (Cat-α) protein was significantly decreased, but the regulatory subunit RII-β was increased (and the latter effect was significantly decreased by cilostazol treatment). These results strongly suggest that cilostazol improves EDHF-type relaxations in STZ rats via an increase in cAMP and PKA signaling.

We have investigated the mechanisms underlying the changes in vascular contractile responsiveness induced by insulin and IGF-I in established streptozotocin-induced diabetic rats. The contractile response to noradrenaline (NA) in organ-cultured diabetic rat aortae cultured with insulin or IGF-I was significantly greater than the corresponding responses in (a) diabetic rat aortae cultured in serum-free medium and (b) control rat aortae cultured with insulin or IGF-I. In aortae from which the endothelium was removed after organ culture the contractile response to NA was greater in those cultured with insulin or IGF-I than in those cultured in serum-free medium. This was not true of aortae endothelium denuded before organ culture. The IGF-I-induced enhancement was prevented by treatment with indomethacin (cyclo-oxygenase inhibitor), SQ29548 (thromboxane (TX) A2 receptor antagonist) or fregrelate (TXA2 synthase inhibitor). IGF-I-induced production of TXB2, a metabolite of TXA2, was greater in diabetic than in control aortae and was attenuated by endothelium denudation, indomethacin or AG1024 (IGF-I receptor inhibitor). The expression of the protein and mRNA for the IGF-I receptor (as assessed by RT-PCR and immunohistochemistry) was markedly increased within endothelial cells in diabetic aortae but only slightly increased within smooth muscle cells (versus control rat aortae). Thus, the NA-induced contractile response in aortae from diabetic rats was enhanced by both insulin and IGF-I and this enhancement may be mediated by sustained cyclo-oxygenase-dependent TXA2 production from endothelial cells. The observed enhancement of IGF-I receptor expression within endothelial cells may be causally related to the potentiation of vascular contractility and the increase in TXA2 production.

Protein tyrosine kinases and nitric oxide (NO) play important roles in several cardiovascular diseases. In this study, we examined the actions of two compounds, each has structure of genistein (a tyrosine kinase inhibitor) and an NO donor, on endothelium-independent relaxation responses in the isolated rat aorta. By rational drug design, genistein was modified to acquire an NO donor, and we synthesized two such compounds (G-II, G-VI). These compounds and genistein induced dose-dependent relaxation responses in endothelium-denuded aortic strips, the rank order of potencies being G-VIG-IIgenistein. Incubation of endothelium-denuded strips with 1H-[1,2,4] oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ, 10 microM), a guanylyl cyclase inhibitor, inhibited both the G-II- and G-VI-induced relaxations, but not the genistein-induced relaxation. The residual relaxations induced by these two compounds were similar to the genistein-induced relaxation. Incubation of endothelium-denuded strips with lysophosphatidylcholine (LPC, 20 microM)-which is a major atherogenic lysophospholipid component of oxidized low-density lipoprotein and is known to activate tyrosine kinase-caused a significant rightward shift in the dose-response curve for genistein. LPC also shifted the G-II- and G-VI-induced relaxation curves to the right; however, these relaxations in the presence of LPC were greater than that induced by genistein. The sodium nitroprusside-induced relaxation in endothelium-denuded strips was similar between in the absence and presence of LPC. These results suggest that each of our newly developed G-II and G-VI compounds has a dual action, as an NO donor and a tyrosine kinase inhibitor. These compounds may be useful against certain cardiovascular diseases.

Macro- and microvascular disease states currently represent the principal causes of morbidity and mortality in patients with type I or type II diabetes mellitus. Abnormal vasomotor responses and impaired endothelium-dependent vasodilation have been demonstrated in various beds in different animal models of diabetes and in humans with type I or type II diabetes. Several mechanisms leading to endothelial dysfunction have been reported, including changes in substrate avail ability, impaired release of NO, and increased destruction of NO. The principal mediators of diabetes-associated endothelial dysfunction are (a) increases in oxidized low density lipoprotein, endothelin-1, angiotensin II, oxidative stress, and (b) decreases in the actions of insulin or growth factors in endothelial cells. An accumulating body of evidence indicates that abnormal regulation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway may be one of several factors contributing to vascular dysfunction in diabetes. The PI3-K pathway, which activates serine/threonine protein kinase Akt, enhances NO synthase phosphorylation and NO production. Several studies suggest that in diabetes the relative ineffectiveness of insulin and the hyperglycemia act together to reduce activity in the insulin-receptor substrates (IRS)/PI3-K/Akt pathway, resulting in impairments of both IRS/PI3-K/Akt-mediated endothelial function and NO production. This article summarizes the PI3-K/Akt pathway-mediated contraction and relaxation responses induced by various agents in the blood vessels of diabetic animals.

Our goals were to determine whether the response of the rat isolated basilar artery to activation of ATP-sensitive potassium (KATP) channels is altered in diabetes mellitus, and to determine the effect of chronic insulin treatment on this response in established diabetic rats. The relaxation induced by cromakalim, an activator of KATP channels, was significantly weaker in insulin-untreated streptozotocin-induced diabetic rats than in the controls. This impairment was significantly improved following chronic administration of insulin. The relaxations induced by two Ca2+-activated K+-channel activators [1-ethyl-2-benzimidazolinone (1-EBIO) or 1, 3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619)] were not significantly different between control and insulin-untreated diabetic rats. The sodium nitroprusside-induced relaxation was similar among the three groups (control, diabetic, and insulin-treated diabetic). These results suggest that: (a) the impaired cromakalim-induced relaxation seen in diabetic rats is not due to a nonspecific effect of diabetes mellitus on vasorelaxation, but at least partly to an effect on KATP channels, and (b) that this impaired relaxation can be restored by chronic insulin treatment.

Using superior mesenteric artery rings isolated from age-matched controls and streptozotocin (STZ)-induced diabetic rats, we recently demonstrated that EDHF-type relaxation is impaired in STZ-induced diabetic rats, possibly due to a reduced action of cAMP via increased phosphodiesterase (PDE) activity (Matsumoto T, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 285: H283–H291, 2003). Here, we investigated the activity and expression of cAMP-dependent protein kinase (PKA), an enzyme that is produced by a pleiotropic and plays key roles in the transduction of many external signals through the cAMP second messenger pathway and in cAMP-mediated vasorelaxation. The relaxation induced by cilostamide, a selective PDE3 inhibitor, was significantly weaker in superior mesenteric artery rings from STZ-induced diabetic rats than in those from age-matched controls. The relaxation responses to 8-bromo-cAMP (8Br-cAMP) and N6, O2-dibutyryl-adenosine-cAMP (db-cAMP), a cell-permeant cAMP analog, were also impaired in the STZ diabetic group. PKA activity in the db-cAMP-treated mesenteric artery was significantly lower in the STZ diabetic group. The expression levels of the mRNA and protein for PKA catalytic subunit Cat-α were significantly decreased in the STZ diabetic group, but those for PKA regulatory subunit isoform RII-β were increased. We conclude that the abnormal vascular relaxation responsiveness seen in STZ-induced diabetic rats may be attributable not only to increased PDE activity but also to decreased PKA activity. Possibly, the decreased PKA activity may result from an inbalance between PKA catalytic and regulatory subunit expressions.

The aim of the present study was to compare vascular dysfunction between the early (12 wk old) and later (36 wk old) stages of spontaneous diabetes in Goto-Kakizaki (GK) rats. We also evaluated the aortic expression of the α2D-adrenoceptor and endothelial nitric oxide synthase (eNOS). Vascular reactivity was assessed in thoracic aortas from age-matched control rats and 12- and 36-wk GK rats. Using RT-PCR and immunoblots, we also examined the changes in expression of the α2D-adrenoceptor and eNOS. In aortas from GK rats (vs. those from age-matched control rats): 1) the relaxation response to ACh was enhanced at 12 wk but decreased at 36 wk; 2) the relaxation response to sodium nitroprusside was decreased at both 12 and 36 wk, 3) norepinephrine (NE)-induced contractility was decreased at 12 wk but not at 36 wk, 4) the expressions of α1B- and α1D-adrenoceptors were unaffected, whereas those of α2D-adrenoceptor and eNOS mRNAs were increased at both 12 and 36 wk; and 5) NE- and ACh-stimulated NOx (nitrite and nitrate) levels were increased at 12 wk, although at 36 wk ACh-stimulated NOx was lower, whereas NE-stimulated NOx showed no change. These results clearly demonstrate that enhanced ACh-induced relaxation and impaired NE-induced contraction, due to NO overproduction via eNOS and increased α2D-adrenoceptor expression, occur in early-stage GK rats and that the impaired ACh-induced relaxation in later-stage GK rats is due to reductions in both NO production and NO responsiveness (but not in eNOS expression).

1. We investigated the mechanisms underlying the changes in vascular contractile responsiveness induced by chronic treatment with insulin in controls and established streptozotocin (STZ)-induced diabetic rats. 2. The aortic contractile response to noradrenaline (NA) showed no significant difference between controls and diabetics, but it was significantly greater in insulin-treated diabetic rats than in the other groups. To investigate the mechanism, we examined the changes in NA-induced contractility following treatment with insulin and insulin-like growth factor-1 (IGF-1) in organ-cultured control and diabetic aortas. 3. The contractile response to NA in organ-cultured diabetic rat aortas treated with insulin (500 ng ml-1, 16 h) or IGF-1 (20 ng ml-1, 16 h) was significantly greater than the corresponding values for (a) diabetic rat aortas cultured in serum-free medium, and (b) control aortas incubated with insulin or IGF-1. Incubating control aortas with insulin or IGF-1 had no significant effect on the contraction induced by NA. 4. The expressions of the IGF-1 receptor mRNA and protein were increased in STZ-induced diabetic aortas and further increased in insulin-treated diabetics. The mRNA expressions of IGF-binding protein (IGFBP)-2 and IGFBP-3 were normal in diabetic aortas. In contrast, those of IGFBP-4 and IGFBP-5 were significantly decreased in diabetic aortas, and not restored by insulin treatment. 5. These results suggest that the insulin deficiency and chronic hyperinsulinemia in diabetes upregulate the IGF-1 receptor and downregulate IGFBP-4 and IGFBP-5 in the aorta. This may be a major cause of the increased vascular contractility induced by insulin administration and by hyperinsulinemia in established diabetes, resulting in hypertension.