Search

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

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.

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.

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.

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

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.

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

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.

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.

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.

The aim of the present study was to investigate the causal relationship between peroxisome proliferator-activated receptor (PPAR) and endothelium-dependent relaxation in streptozotocin (STZ)-induced diabetic rats. Acetylcholine (ACh)-induced endothelium-dependent relaxation was significantly weaker in diabetic rats than in age-matched controls. The decreased relaxation in diabetes was improved by the chronic administration of bezafibrate (30 mg kg−1, p.o., 4 weeks). The expressions of the mRNAs for PPARα and PPARγ were significantly decreased in STZ-induced diabetic rats (compared with the controls) and this decrease was restored partially, but not completely, by the chronic administration of bezafibrate. Superoxide dismutase activity in the aorta was not significantly different between diabetic rats and bezafibrate-treated diabetic rats. The expression of the mRNA for the p22phox subunit of NAD(P)H oxidase was significantly higher in diabetics than in controls, but it was lower in bezafibrate-treated diabetic rats than in nontreated diabetic rats. Although the expression of the mRNA for prepro ET-1 (ppET-1) was markedly increased in diabetic rats (compared with controls), this increase was prevented to a significant extent by the chronic administration of bezafibrate. These results suggest that downregulations of PPARα and PPARγ may lead to an increased expression of ppET-1 mRNA in diabetic states and this increment may trigger endothelial dysfunction. British Journal of Pharmacology (2003) 140, 23–32. doi:10.1038/sj.bjp.0705414

Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) are second messengers involved in the intracellular signal transduction of a variety of extracellular stimuli in several tissues. In the vascular system, these nucleotides play important roles in the regulation of vascular tone and in the maintenance of the mature contractile phenotype in smooth muscle cells. Given that cyclic nucleotide signaling regulates a wide variety of cellular functions, it is not surprising that cyclic nucleotide phosphodiesterases (PDEs). In paticular, the accumulating data showing that there are a large number of different PDE isozymes have triggered an equally large increase in interest about these enzymes. At least 11 different gene families of PDEs are currently known to exist in mammalian tissues. Most families contain several distinct genes, and many of these genes are expressed in different tissues as functionally unique alternative splice variants. This article reviews many of the important aspects about the structure, cellular localization, and regulation of each family of PDEs. Particular emphasis is placed on new information obtained in the last few years about vascular disease. The development of novel methods to deliver more potent and selective PDE inhibitors to individual cell types and subcellular locations will lead to new therapeutic uses for this class of drugs in diseases of the vascular system.

We previously reported that in the endothelium-denuded rat aorta, lysophosphatidylcholine (LPC) potentiates the contractile responses induced by high-K(+), UK14,304 (a selective alpha(2)-adrenoceptor agonist), and phorbol ester with an associated tyrosine-phosphorylation of proteins. To further investigate this phenomenon, we examined the effects of extracellular-signal-regulated protein kinase (ERK)-kinase (MEK) inhibitors on the LPC-induced potentiation of the contractile responses to high-K(+) and UK14,304 in this tissue. Although PD98059 (3 x 10(-)(5) M) did not affect the high-K(+)-induced contractile response itself, it selectively inhibited the potentiating effect of LPC on the contraction and strongly inhibited the LPC-induced augmentation of the associated increase in [Ca(2+)](i). PD98059 also attenuated the LPC-induced augmentations of the increases in [Ca(2+)](i) and contractile tension induced by UK14,304. U0126 (5 x 10(-)(5) M), another MEK inhibitor, also attenuated the potentiating effect of LPC on high-K(+)-induced contractions. Western blot analysis revealed that LPC produced an increase in ERK-phosphorylation, and that this was inhibited by PD98059. Nicardipine inhibited the contractile response to 15 mM K(+) in the LPC-treated aorta, but not the increase in ERK-phosphorylation induced by LPC. These results suggest that the LPC-induced augmentation of contractile responses in the rat aorta is due to activation of ERK, which in turn regulates Ca(2+) influx.

We investigated the relationship between the changes in vascular responsiveness and growth factor mRNA expressions induced by 1-wk treatment with high-dose insulin in control and established streptozotocin (STZ)-induced diabetes. Aortas from diabetic rats, but not those from insulin-treated diabetic rats, showed impaired endothelium-dependent relaxation in response to ACh (vs. untreated controls). The ACh-induced nitrite plus nitrate (NOx) level showed no significant difference between controls and diabetics. Insulin treatment increased NOx only in diabetics. In diabetics, insulin treatment significantly increased the aortic expressions of endothelial nitric oxide synthase (eNOS) mRNA and VEGF mRNA. The expression of IGF-1 mRNA was unaffected by diabetes or by insulin treatment. In contrast, the mRNA for the aortic IGF-1 receptor was increased in diabetics and further increased in insulin-treated diabetics. In aortic strips from age-matched control rats, IGF-1 caused a concentration-dependent relaxation. This relaxation was significantly stronger in strips from STZ-induced diabetic rats. These results suggest that in STZ-diabetic rats, short-term insulin treatment can ameliorate endothelial dysfunction by inducing overexpression of eNOS and/or VEGF mRNAs possibly via IGF-1 receptors. These receptors were increased in diabetes, perhaps as result of insulin deficiency.

We previously reported that while lysophosphatidylcholine (LPC) does not itself produce contraction, it significantly potentiates the contractile responses induced by high-K+, UK14,304 (a selective α2-adrenoceptor agonist) and phorbol ester in the endothelium-denuded rat aorta. To further investigate this phenomenon, we examined the effects of genistein and tyrphostin B42 (both tyrosine kinase inhibitors) on the LPC-induced potentiation of the contractile responses to high-K+ and UK14,304 in the endothelium-denuded rat aorta. Although genistein (3×10−6 M, 10−5 M) did not affect the high-K+-induced contractile response, it selectively inhibited the potentiating effect of LPC on the contraction and it strongly inhibited the LPC-induced augmentation of the associated increases in [Ca2+]i. Genistein also attenuated the LPC-induced augmentation effects on both the increase in [Ca2+]i and contractile response induced by the UK14,304. In contrast, daidzein (10−5 M) did not inhibit the potentiating effect of LPC. Tyrphostin B42 (3×10−5 M) attenuated the potentiating effect of LPC on high K+-induced contractions. Western blot analysis showed that LPC increased the tyrosine phosphorylation of a number of proteins, including 42 and 44 kDa proteins and 53 – 64 kDa proteins. These protein phosphorylations were inhibited by genistein. Sodium orthovanadate (10−4 M), a tyrosine phosphatase inhibitor, also markedly enhanced the high-K+-induced contractile responses. This enhancing effect was attenuated by genistein. These results suggest that the LPC-induced augmentation of contractile responses in the rat aorta is due to activation of tyrosine kinase, which in turn regulates Ca2+ influx. British Journal of Pharmacology (2002) 135, 789–799; doi:10.1038/sj.bjp.0704525

The hypothesis that the impaired endothelial function seen in streptozotocin (STZ)-induced diabetic rats may result from an increased nitric oxide (NO) metabolism was tested. Acetylcholine (ACh) increased the nitrite NO(2-) and nitrate (NO(3-)) levels in the perfusates from both control and diabetic aortic strips, although the level of NO(2-) was significantly lower in diabetic rats while the NO(3-) level was significantly higher. Both effects (decrease in NO(2-) and increase in NO(3-)) were ameliorated by chronic administration of insulin to diabetic rats but NOx (NO(2-) plus NO(3-)) was increased. The expression of endothelial nitric oxide synthase (eNOS) was significantly increased by chronic administration of insulin to diabetic rats. A decrease in NO(2-) and an increase in NO(3-) occurred following treatment of control aortae with hypoxanthine/xanthine oxidase. Incubating diabetic aortic strips with superoxide dismutase (SOD) normalized the production of both NO(2-) and NO(3-). Both the basal and the ACh-stimulated production of O(2)(-) were significantly higher in diabetic rats than in controls. These results demonstrate that the ACh-induced relaxation of aortic strips was significantly impaired in diabetic rats and that this impairment may be due to an abnormal oxidative metabolism of NO, rather than to a decrease in NOS mRNA and NO production.

This study investigated the influence of superoxide anion on the norepinephrine (NE)-induced contractile response in spontaneously diabetic mice. In aortic rings with intact endothelium, NE elicited only a slight increase in tension in nondiabetic mice (db/+M), but a much greater dose-dependent contraction in spontaneously diabetic mice (db/db mice). The NE-induced contractile response was significantly reduced by pretreatment with SOD (180 U/ml) in diabetic mice, but not in control mice. The NE-induced contraction was significantly enhanced by pretreatment with diethyldithiocarbamic acid (DETCA, 10(-3) M), a Cu/Zn SOD inhibitor, in control mice, but not in diabetic mice. The dose-response curve for the acetylcholine-induced relaxation was slightly, but significantly attenuated in diabetic mice. When aortic rings from control mice were incubated with a mixture of hypoxanthine (10(-5) M), xanthine oxidase (0.1 U/ml) and catalase (1000 U/ml) in control mice, they gradually contracted. This contraction was abolished by pretreatment with SOD (180 U/ml) or indomethacin (10(-5) M) or by removal of the endothelium. The enhanced NE-induced dose-dependent contraction seen in diabetic mice was markedly attenuated by indomethacin. These results suggest that in db/db diabetic mice, superoxide anion, perhaps via vasoconstrictor prostanoids, may enhance the contraction induced by NE.

We investigated the effects of chronic pravastatin treatment on the impaired endothelium-dependent relaxation seen in aortae from established streptozotocin (STZ)-induced diabetic rats. Starting at 6 weeks of diabetes, pravastatin (10 mg kg−1) was administered to STZ-induced diabetic rats for 4 weeks. The increased total cholesterol and low-density lipoprotein (LDL) cholesterol levels seen in STZ-induced diabetic rats were not restored to normal by pravastatin. Aortae from pravastatin-treated diabetic rats did not show an impaired endothelium-dependent relaxation to acetylcholine. The expression of the mRNA for endothelial nitric oxide synthase was unaffected by diabetes or pravastatin. The enhanced level of malondialdehyde (MDA)-modified LDL seen in STZ-induced diabetic rats was normalized by pravastatin treatment. The resistance of LDL to oxidation was assessed by measuring the amount of MDA or conjugated dienes generated by incubation with copper ions. LDL isolated from diabetic rats, but not those from pravastatin-treated diabetics, showed enhanced the susceptibility to oxidation, but incubation in vitro with pravastatin had no effect on LDL oxidation. Following incubation of control aortae for 6 h with LDL (0.1 mg protein ml−1) isolated from diabetic rats, the endothelium-dependent relaxation to acetylcholine or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 was impaired, but LDL isolated from control or pravastatin-treated rats had no such effect. This inhibitory effect of diabetic LDL was prevented by superoxide dismutase (SOD), a superoxide scavenger. These results suggest that pravastatin preserves endothelial function in aortae from STZ-induced diabetic rats without lowering plasma cholesterol, and its effect may be due to decreased LDL oxidation. Keywords: Pravastatin, diabetes, endothelium, LDL, oxidized LDL, relaxation, streptozotocin Introduction The relaxation responses of aortic strips to endothelium-dependent agents are weaker in streptozotocin (STZ)-induced diabetic rats than in normal rats (Oyama et al., 1986; Kamata et al., 1989; 1996; Poston & Taylor, 1995; Kamata & Kobayashi, 1996; Pieper, 1998). Oxidized LDL (ox-LDL) is thought to promote atherogenesis by a multitude of different mechanisms. Indeed, ox-LDL acts more potently than native LDL in markedly inhibiting the endothelium-dependent relaxation of the rabbit aorta (Kugiyama et al., 1990) and pig coronary artery (Simon et al., 1990). Thus, the circulating modified-LDL in diabetes may be responsible for the impaired endothelium-dependent relaxation. However, few studies of endothelial dysfunction in diabetes have directly assessed the relationship between circulating modified LDL and endothelium-dependent relaxation. Pravastatin, an 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has been shown to reduce adverse cardiovascular events in patients suffering from coronary artery disease with or without hypercholesterolemia (Sacks et al., 1996). Furthermore, It has been shown that HMG-CoA reductase inhibitors improve the defective endothelium-dependent vasodilation of atherosclerotic vessels in humans or animals by lowering plasma cholesterol (Kroon, et al., 1993; John et al., 1998). Although the mechanism by which HMG-CoA reductase inhibitors preserve vascular function is primarily attributed to their inhibition of hepatic HMG-CoA reductase and the subsequent lowering of plasma cholesterol levels, some recent studies have questioned the exclusive involvement of the decrease in plasma LDL levels in the effects of HMG-CoA reductase inhibitors (Hussein et al., 1997; O'Driscoll et al., 1997; Essig et al., 1998; Laufs et al., 1998). Thus, it remains unclear how an early improvement in endothelial function might occur during treatment with an HMG-CoA reductase inhibitor. The aims of the present study were (1) to investigate the influence of chronic pravastatin treatment on the impairment of endothelium-dependent relaxation seen in aortae from rats with established STZ-induced diabetes and (2) to evaluate the effects of diabetes-modified LDL on the endothelium-dependent relaxation of the rat aorta.

We examined endothelium-dependent and -independent hyperpolarizations and endothelium-dependent relaxation responses in carotid arteries isolated from streptozotocin-induced diabetic rats and age-matched controls. The resting membrane potentials were not significantly different between control and diabetic carotid arteries. The endothelium-dependent hyperpolarization induced by acetylcholine, which was inhibited by TEA but not by glibenclamide or by treatment with either a high concentration of glucose or pertussis toxin, was significantly weaker in diabetic arteries than in the controls. The relaxation responses to acetylcholine in carotid artery rings were significantly decreased in streptozotocin-diabetic rats. Treatment with NG-nitro-L-arginine (L-NOARG) inhibited the acetylcholine-induced maximal relaxation by 80% and 30% in control and streptozotocin-diabetic rats, respectively, and the simultaneous application of L-NOARG and indomethacin had a more potent inhibitory effect on this relaxation in both groups. The release of 6-keto-prostaglandin F1alpha and that of thromboxane A2 in response to methoxamine or methoxamine plus acetylcholine were both markedly decreased in diabetic rats. The cromakalim-induced hyperpolarization of the carotid artery, which was completely prevented by glibenclamide, was also significantly weaker in diabetic arteries than in the controls. These results suggest that changes in (1) various K+ channels on smooth muscle, (2) the biosynthesis of cyclooxygenase products and (3) endothelium-dependent relaxation may be important factors in the development of diabetic complications in the carotid artery.

Experiments were designed to investigate the mechanisms underlying the diabetes-related impairment of the vasodilatations of the perfused mesenteric arterial bed induced by acetylcholine (ACh) and K(+). In streptozotocin (STZ)-diabetic rats, the ACh-induced endothelium-dependent vasodilatation was attenuated. The dose-response curves for ACh in control and diabetic rats were each shifted to the right by N(G)-nitro-L-arginine (L-NOARG) and by isotonic high K(+) (60 mM). The ACh dose-response curves under isotonic high K(+) were not different between control and diabetic rats. We also examined the vasodilatation induced by K(+), which is a putative endothelium-derived hyperpolarizing factor (EDHF). The mesenteric vasodilatation induced by a single administration of K(+) was greatly impaired in STZ-induced diabetic rats. Treatment with charybdotoxin plus apamin abolished the ACh-induced vasodilatation but enhanced the K(+)-induced response in controls and diabetic rats. After pretreatment with ouabain plus BaCl(2), the ACh-induced vasodilatation was significantly impaired and the K(+)-induced relaxation was abolished in both control and diabetic rats. The impairment of the endothelium-dependent vasodilatation of the mesenteric arterial bed seen in STZ-induced diabetic rats may be largely due to a defective vascular response to EDHF. It is further suggested that K(+) is one of the endothelium-derived hyperpolarizing factors and that the vasodilatation response to K(+) is impaired in the mesenteric arterial bed from diabetic rats.

The aim of this study was to elucidate the efficiency of soybean-derived sterylglucoside (SG) and its main component beta-sitosterol beta-D-glucoside (Sit-G), as nasal absorption enhancers. Nasal administration of verapamil with SG and Sit-G showed the higher bioavailabilities (60.4 and 90.7%, respectively) than that with lactose (39.8%). It was clear that SG and Sit-G promoted the absorption of verapamil through nasal mucosa. To elucidate the mechanism, we measured the calcein leakage from liposomes by incubation with SG, Sit-G, oleic acid, soybean-derived sterol, and beta-sitosterol to investigate transcellular absorption and measured the changes in intracellular Ca2+ concentrations ([Ca2+]i) by Sit-G to analyze paracellular absorption. The large amount of calcein leakage induced by enhancers was consistent with an enhancement of bioavailability of verapamil and insulin following nasal administration (oleic acidSGSit-G). Moreover, Sit-G increased [Ca2+]i in the medium containing Ca2+, but not in Ca2+ free medium. This result suggested that Sit-G increases the fluidity of the mucosal membrane and facilitates Ca2+ influx from extracellular sources. In conclusion, a possible explanation for SG and Sit-G to promote drug absorption, is that they may affect both paracellular pathway and transcellular pathways caused by pertubation of lipid.

The neuromedin B-preferring receptor (NMB-R) is one of the members of the bombesin (BN)-like peptide receptor subfamily in mammals. Previously, we have generated and characterized mice with targeted disruption of the two other BN-like peptide receptors, bombesin receptor subtype-3 (BRS-3) and gastrin-releasing peptide-preferring receptor (GRP-R). Here we describe the generation and analysis of NMB-R-deficient mice to investigate how NMB-R differs from BRS-3 and GRP-R. Compensation for NMB-R deficiency by overexpression of GRP-R and/or BRS-3 was not detected. Although the hypothermic effect of NMB was reduced by 50% in NMB-R-deficient mice, the effect of GRP infusion was comparable to the wild-type mice. In contrast, fundic smooth muscle contraction on stimulation with NMB or GRP was normal in NMB-R-deficient mice. Administration of GRP but not NMB suppressed glucose intake in both normal and NMB-R-deficient mice. These results suggest that the NMB-R has an essential role in thermoregulation, but not for smooth muscle contraction of the fundus or for the suppression of feeding behavior. In addition, the behavioral phenotypes of GRP-R-deficient mice were not observed in NMB-R-deficient mice. These data show that the functions of NMB-R and GRP-R are distinct, with only partial overlap.

High concentration of acetylcholine (ACh) caused a rapid and long lasting relaxation response in age-matched controls, whereas this response was significantly weaker in streptozotocin (STZ)-diabetic and cholesterol fed mice. The levels of basal and ACh-stimulated cyclic GMP in the aorta was also significantly smaller in STZ-diabetic and cholesterol-fed mice. The attenuated relaxation responses to ACh in both STZ-diabetic and cholesterol-fed mice were ameliorated by the chronic administration of cholestyramine. A prior incubation of aortic strips with superoxide dismutase (SOD, 60 U/ml) improved the recovery phase of the relaxation of diabetic aorta after single administration of ACh, whereas SOD had no effects on ACh-induced relaxation of aortic strips from cholesterol fed mice. These results suggest that superoxide anion may be responsible for an impairment of endothelium-dependent relaxation of aorta from STZ induced diabetic mice. It is further suggested that impairment of endothelium-dependent relaxation in STZ-diabetic and cholesterol fed mice may be caused by different mechanisms.

Experiments were performed to compare Ca2+ mobilization in the aortic endothelium in streptozotocin (STZ)-induced diabetic and cholesterol-fed mice with that in age-matched controls. The intracellular free Ca2+ ([Ca2+]i) in the fura PE-3 loaded endothelium of aortic rings was dose-dependently increased by cumulative administration of acetylcholine (ACh). ACh caused a transient rise in [Ca2+]i in Ca2+-free medium. The ACh-induced increase in [Ca2+]i in normal or Ca2+-free medium was significantly weaker in both STZ-induced diabetic and cholesterol-fed mice. The weaker [Ca2+]i response in Ca2+-containing medium in STZ-induced diabetic and cholesterol-fed mice was normalized by chronic administration of cholestyramine. The increased low density lipoprotein (LDL) levels seen in both STZ-induced diabetic and cholesterol-fed mice were normalized by the same chronic administration of cholestyramine (300 mg kg−1, p.o. daily for 10 weeks). Chronic administration of cholestyramine had no effect on the plasma glucose level. Lysophosphatidylcholine (LPC) decreased the [Ca2+]i responses to ACh in the aortic endothelium from normal mice. These results suggest that ACh increases both Ca2+ influx and Ca2+ release from storage in the aortic endothelium. The weaker [Ca2+]i influx seen in the endothelium of aortae from both STZ-induced diabetic and cholesterol-fed mice was improved by the chronic administration of cholestyramine, and we suggest that this improvement is due, at least in part, to a lowering of the plasma LDL level. It is further suggested that LPC may have an important influence over Ca2+ mobilization in the endothelium. British Journal of Pharmacology (1998) 123, 1509–1516; doi:10.1038/sj.bjp.0701754

In thoracic aortic strips with intact endothelium, the first and second (1 h later) dose-response curves obtained with methoxamine were almost the same. Methoxamine caused a dose-dependent increase in perfusion pressure in the rat isolated mesenteric arterial bed, but the second (1 h later) dose-response curve for methoxamine showed a significant attenuation of the response in comparison with the first. The attenuation shown by the second dose-response curve for methoxamine was significantly reduced, but not abolished, in mesenteric arterial beds without endothelium. Incubating endothelium-intact mesenteric arterial beds with NG-nitro-l-arginine (l-NOARG) caused a significant, but not complete, reversal of the attenuation shown in the second dose-response curve. Incubating the mesenteric arterial bed with capsaicin, tetrodotoxin, indomethacin or with isotonic high k+ (60 mm) plus nicardipine did not affect the above attenuation seen in the second dose-response curve. The guanosine 3′:5′-cyclic monophosphate (cyclic GMP) level in the effluent from the perfused mesenteric arterial bed was significantly increased after the second exposure to methoxamine. This effect was significantly smaller after removal of the endothelium or pretreatment with l-NOARG. These results suggest that a desensitization to methoxamine develops rapidly in the mesenteric arterial bed, but not in the aorta, and that release of nitric oxide from the endothelium plays a major role in this desensitization. British Journal of Pharmacology (1997) 120, 1221–1228; doi:10.1038/sj.bjp.0701031

We examined the characteristics of the non-adrenergic, non-cholinergic (NANC) inhibitory response of the circular muscle of the rat stomach fundus to transmural nerve stimulation or high K+. Treatments with isotonic high K+ (20 mM), nitric oxide (NO) and sodium nitroprusside (SNP) all elevated cyclic GMP levels in the rat stomach fundus in the presence of atropine and guanethidine. Isotonic high K+-induced formation of cyclic GMP was completely inhibited by tetrodotoxin (TTX) or NG-nitro-L-arginine (L NNA). The K+ also increased cyclic AMP levels and this response was completely inhibited by TTX. Dose-dependent relaxation of the fundus in response to SNP was shifted to the right by a prior incubation with high concentration of SNP (10(-4) M) for 2 hrs. Incubating the fundus with SNP for 2 hrs significantly inhibited NO induced cyclic GMP formation. Relaxation responses to transmural stimulation (1 Hz or 30 Hz), isotonic high K+ and NO were significantly reduced by a prior incubation with SNP. Isotonic high K+ (20 mM)-induced relaxation of circular muscle strips was not completely inhibited by combined treatment with 10(-5)M L-NNA, 5 x 10(-5)M oxyhemoglobin and anti VIP (1:200). These results suggest that NO as well as VIP is possible transmitter from NANC nerves in the circular muscle of the rat stomach fundus and there should be one or more inhibitory mediators other than VIP and NO.

1. Experiments were designed to investigate the involvement of superoxide anions in the attenuated endothelium-dependent relaxation of the rat aorta from streptozotocin (STZ)-induced diabetic rats. 2. The endothelium-dependent relaxation responses to acetylcholine (ACh, 10(-7) M) in helical strips of the aorta precontracted with noradrenaline (NA, 5 x 10(-3) approximately 3 x 10(-7) M) were significantly decreased in STZ-induced diabetic rats. The recovery phase of the relaxation after single administration of ACh in the STZ-induced diabetic rats was more rapid than those in control vessels. 3. Preincubation of aortic strips with superoxide dismutase (SOD, 60 u ml-1) normalized the recovery phase of the relaxation of diabetic aorta after single administration of ACh, whereas catalase (150 u ml-1) or indomethacin (10(-5) M) had no effects on the relaxation. 4. SOD (180 u ml-1) caused relaxation in NA precontracted aortic strips and the degree of the SOD-induced relaxation was significantly greater in diabetic aorta as compared with age-matched control vessels. 5. When the changes in mRNA expressions of Mn-SOD or Cu-Zn-SOD were observed, Mn-SOD mRNA expression was markedly decreased, and Cu-Zn-SOD was slightly decreased in diabetic aorta. 6. These results suggest that the rapid destruction of NO by superoxide anions may occur in the STZ-induced diabetic rats, and this may be due to a decrease in mRNA expression of Mn-SOD or Cu-Zn-SOD.

1. Administration of VA-045 [2-(nitrooxy)ethyl apovincaminate] and thyrotropin-releasing hormone (TRH) led to improvement in the closed head injury (CHI)-induced neuronal dysfunction such as the loss of righting reflex and disruption of spontaneous movement in rats. 2. The improvement seen with effect of VA-045, but not TRH, was abolished in rats pretreated with N-2-chloroethyl-N-ethyl-2-bromobenzylamine (DSP4), a selective noradrenaline (NA) neurotoxin. DSP4 reduced endogenous NA levels in all central nervous system (CNS) regions analyzed. 3. The extracellular concentrations of NA in the frontal cortex (FC) and in the locus coeruleus (LC) of urethane-anesthetized rats were measured using in vivo microdialysis coupled with high-performance liquid chromatography (HPLC) with electrochemical detection. VA-045 had no effect on extracellular concentrations of NA, in both FC and LC. Perfusion with clonidine, and alpha 2 adrenoceptor agonist, led to inhibition in NA output in both FC and LC, and VA-045 antagonized the effect of clonidine. 4. These findings indicate that the mode of action of VA-045 may be, at least in part, related to central NA neuronal systems.

We examined the effects of taurine on levels of low-density lipoprotein (LDL) cholesterol and glucose, and an endothelium-dependent relaxation in response to acetylcholine in cholesterol-fed or streptozotocin-induced diabetic mice. The acetylcholine-induced concentration-dependent relaxation was significantly attenuated in aortic rings from cholesterol-fed and streptozotocin-induced diabetic mice. The attenuated vasodilation in both cholesterol-fed and streptozotocin-induced diabetic mice was normalized by the chronic administration of taurine. The endothelium-independent relaxation of aortic rings induced by sodium nitroprusside was not significantly different between control, cholesterol-fed and streptozotocin-induced diabetic mice. The increased serum levels of LDL cholesterol in cholesterol-fed and diabetic mice were returned to normal by the chronic administration of taurine. The chronic administration of taurine had no effects on serum glucose levels. These results suggest that the impaired endothelium-dependent vasodilation seen in both cholesterol-fed and streptozotocin-diabetic mice can be normalized by the chronic administration of taurine and this effect may be, at least in part, due to lowering of serum LDL levels.

We found that vasodilator effects of platelet-activating factor (PAF) on the mesenteric arterial bed of the rat were significantly attenuated in spontaneously hypertensive rats (SHR) and renal hypertensive rats (RHR). Perfusion of the mesentery with acetylcholine and PAF caused endothelium-dependent vasodilatation accompanied by an increase in cyclic GMP levels in the mesentery from normotensive Wistar Kyoto rats (WKY). Acetylcholine caused a significant increase in cyclic GMP levels in the effluent in both SHR and RHR, whereas PAF could not increase cyclic GMP levels in SHR and slightly increased cyclic GMP in RHR. Incubating the mesentery with PAF markedly inhibited the vasodilatation induced by PAF, but not acetylcholine or sodium nitroprusside. The cyclic GMP accumulation in the effluent was impaired in the mesenteric arterial bed pretreated with PAF and in that obtained from rats given islet-activating protein (IAP). The PAF-induced vasodilatation was completely reversed by the PAF receptor antagonist, CV-6209 (2-[N-acetyl-N-(2-methyl-3-octadecylcarbamoyl-oxypropoxycarbonyl)aminomethyl]-1-ethylpyridinium chloride). There results suggest that (1) attenuated vasodilator effects of PAF and decreased cyclic GMP levels in the mesentery from SHR and RHR are due to desensitization but not to impairment of the endothelium; (2) GTP-binding protein, which is IAP-sensitive, may be involved in PAF-induced vasodilatation and cyclic GMP accumulation; (3) desensitization of the mesentery to PAF in SHR and RHR may be due to PAF receptor and GTP-binding protein uncoupling.

A potent vasodilator substance (compound III), [alpha]D +141 degrees, was isolated from salviae miltiorrhizae radix (dan-shen). This substance was determined to be des(alpha-carboxy-3,4-dihydroxyphenethyl)lithospermic acid on the basis of spectrometric and chemical evidence, and was identified with an authentic sample of 8-epiblechnic acid. However, comp. III seemed to be formed from lithospermic acid (LSA) and LSA-B during a chemical procedure to separate active ingredients. It caused a sustained, slowly developing relaxation of rat aortic strips precontracted with norepinephrine (NE) in preparations with or without endothelium. The NE-induced concentration-dependent contraction of aortic strips was significantly attenuated by pretreatment with comp. III. Concentration-response curves for Ca(2+)-induced contracture of depolarized aortic strips with isotonic high K+ (60 nM) were not affected by comp. III. Ca(2+)-induced contraction of aortic strips, preincubated with 10(-6) M NE in the presence of 10(-6) M nicardipine and 0.01 mM EGTA in Ca(2+)-free solution, was slightly inhibited by comp. III. Pretreatment of aortic strips with comp. III slightly inhibited the phorbol ester (PMA)-induced contraction. These results suggest that comp. III inhibits NE-induced contraction of the aortic strips through reduction in Ca2+ mobilization. Since comp. III inhibits NE-induced sustained contraction, this agent may be useful in the treatment of hypertension.

The effects of phorbol ester, phorbol 12-myristate 13-acetate (PMA), on vasodilation induced by endothelium-dependent or independent vasodilators in the mesenteric arterial bed were examined. In mesentery precontracted with methoxamine, acetylcholine (ACh) produced a concentration-dependent vasodilation, but ACh-induced vasodilation was significantly reduced when the tonus of the mesentery was raised by an equieffective concentration of PMA. Sodium nitroprusside (SNP) and forskolin also caused a concentration-dependent relaxation in the mesenteric arterial bed pre-contracted with methoxamine, but could not induce relaxation in mesentery precontracted with PMA. In mesentery precontracted with PMA or methoxamine, ACh-induced vasodilation was significantly inhibited by tetraethylammonium (TEA), but not by ouabain, glibenclamide, or apamin. ACh-induced vasodilation was significantly inhibited by NG-nitro-L-arginine (L-NNA), whereas L-NNA was not capable of effectively inhibiting the ACh-induced vasodilation of the mesentery precontracted with PMA. These results suggest that stimulation of protein kinase C (PKC) by phorbol ester (PMA) in the mesenteric arterial bed inhibits the relaxation of vascular smooth muscle (VSM) in response to cyclic nucleotides. Furthermore, the endothelium of the mesenteric arterial bed may release endothelium-derived hyperpolarizing factor (EDHF), in addition to nitric oxide (NO), into the mesentery.