Kohsuke Uchida

Aldosterone impairs vascular endothelial cell function.

Aldosterone is a mineralocorticoid hormone that plays an important role in regulating electrolyte balance and blood pressure and also participates in endothelial dysfunction. We evaluated the direct effect of aldosterone on human umbilical vein cells (HUVEC). Levels of eNOS phosphorylation by vascular endothelial growth factor were diminished, and the amount of NO produced in response to vascular endothelial growth factor measured as NO2−+NO3− was significantly decreased in cells previously incubated with aldosterone. Incubation with aldosterone for 24 h dose-dependently increased Nox4 mRNA expression in HUVEC. Although NF-κB was not apparently activated by aldosterone, mRNA levels of vascular cell adhesion molecule-1, E-selectin, monocyte chemotactic protein-1, and intercellular adhesion molecule-1 in HUVEC were significantly increased after incubation with aldosterone. Thus, aldosterone directly causes the dysregulation of endothelial cell function, which may be partly responsible for high blood pressure and atherosclerosis.

Supplementation with tetrahydrobiopterin prevents the cardiovascular effects of angiotensin II-induced oxidative and nitrosative stress.

Objective The pteridine cofactor tetrahydrobiopterin (BH4) has emerged as a critical determinant of endothelial nitric oxide synthase (eNOS) activity. When BH4 availability is limited, eNOS does not produce nitric oxide (NO) but instead generates superoxide. BH4 may reverse endothelial dysfunction due to cardiovascular disease, including atherosclerosis, coronary artery disease and hypertension. In this study, the influence of BH4 on cardiovascular parameters and the production of free radicals following angiotensin II (Ang II) infusion was assessed. Methods BH4 (20 mg/kg per day in drinking water) was administered with Ang II (300 ng/kg per min subcutaneously, osmotic pump) for 7 days in Sprague–Dawley rats. In addition, BH4 was also given in vehicle-infused rats. Results Treatment with BH4 significantly prevented some of the effects of Ang II, such as impaired vascular responses to acetylcholine, hypertension and increases in heart weight index values. Treatment with BH4 also significantly reduced Ang II-induced increases in inducible NO synthase expression, nitrotyrosine immunostaining, NO production and superoxide anion formation in rats. Conclusion These results indicate that BH4 might prevent the development of hypertension and myocardial hypertrophy, as well as the Ang II-induced production of superoxide and NO, thereby reducing the production of peroxynitrite. Therefore, BH4 may protect against the cardiovascular manifestations of oxidative and nitrosative stress in this experimental model of Ang II-mediated hypertension.

Cilostazol Activates AMP-activated Protein Kinase and Restores Endothelial Function in Diabetes

BACKGROUND Endothelial dysfunction plays a key role in atherogenesis. We investigated whether AMP-activated protein kinase (AMPK) activity is a downstream mediator of the beneficial effects of cilostazol on vascular endothelial cells and whether cilostazol might reverse endothelial dysfunction in diabetic rats. METHODS AND RESULTS Treatment of human umbilical vein endothelial cells (HUVECs) with cilostazol resulted in time-dependent activation of AMPK, as monitored by phosphorylation of AMPK and its down-stream target, acetyl-CoA carboxylase (ACC). Activation of AMPK by cilostazol was through signaling pathway independent of cyclic AMP and caused phosphorylation of endothelial nitric oxide synthase (eNOS), leading to increased production of nitric oxide (NO), while inhibiting cytokine-induced nuclear factor-kappaB (NF-kappaB) activation, leading to suppression of VCAM-1 gene expression. Significantly reduced eNOS activity and NO production in response to cilostazol and attenuation of cilostazol-induced inhibition of NF-kappaB activation were observed in cells treated with AMPK siRNA. We also demonstrated that administration of cilostazol to diabetic rats significantly restored endothelium-dependent vasodilation. Furthermore, treatment of diabetic rats with cilostazol increased tetrahydrobiopterin (BH4) levels in the aorta. Thus, recovery of BH4 following administration of cilostazol might also contribute to restoration of endothelial function in diabetic rats. CONCLUSIONS Our findings suggest that the beneficial effects of cilostazol on endothelial function may be due to AMPK activation. Restoration of endothelial dysfunction in diabetic rats by cilostazol is at least partly attributed to amelioration of biopterin metabolism in the aorta.

Heterogeneity of muscarinic receptors in the guinea pig esophageal muscularis mucosae and ileal longitudinal muscle

Pharmacologic characteristics of muscarinic receptors in the muscularis mucosae of the guinea pig esophagus were examined in vitro and compared with those of the longitudinal muscle of the guinea pig ileum. All cholinomimetics tested produced a sustained contraction of the muscularis mucosae, in a concentration-dependent manner, which was associated with a small biphasic change in membrane potential, initially a hyperpolarization followed by a depolarization. The contractile response was hardly modified by verapamil, but was depressed by calcium removal from the bathing medium. Both atropine and pirenzepine antagonized the contractile response in a competitive manner, with higher pA2 values than those in the ileum. These results indicate that the muscarinic receptors of the muscularis mucosae of the guinea pig esophagus mainly link with calcium ion channels which are independent of changes in membrane potential and that their subtype populations are probably pharmacologically distinct from those in the ileal longitudinal muscle.

Glimepiride upregulates eNOS activity and inhibits cytokine‐induced NF‐κB activation through a phosphoinoside 3‐kinase–Akt‐dependent pathway

Aims:  Several studies suggest increased mortality postcoronary angioplasty in patients on sulphonylureas. However, a theoretical reduction in cardiac risk has been suggested with the newer sulphonylurea agents, which differ from the first‐generation agents. In the present study, we investigated whether a third generation of sulphonylurea, glimepiride, might stimulate nitric oxide (NO) production and thereby inhibit cytokine‐induced nuclear factor (NF)‐κB activation in endothelial cells compared with the classical sulphonylurea glibenclamide.

Pharmacological characterization of endothelin-induced contraction in the guinea-pig oesophageal muscularis mucosae.

1 In the oesophageal muscularis mucosae, we examined the effects of endothelin‐1 (ET‐1), endothelin‐2 (ET‐2), endothelin‐3 (ET‐3) and sarafotoxin S6c (SX6c) as agonists, and FR139317, BQ‐123 and RES‐701‐1 as endothelin receptor antagonists. 2 All of the endothelins produced tonic contractions which were frequently superimposed on rhythmic motility in a concentration‐dependent manner. The order of potency (−log EC50) was ET‐1 (8.61)=SX6c (8.65)>ET‐2 (8.40)>ET‐3 (8.18). 3 FR139317 (1–3 μM) and BQ‐123 (1 μM) caused parallel rightward shifts of the concentration‐response curve to ET‐1, but at higher concentrations caused no further shift. RES‐701‐1 (3 μM) caused a rightward shift of the concentration‐response curve to ET‐1, while RES‐701‐1 (10 μM) had no additional effect. RES‐701‐1 (0.1–1 μM) concentration‐dependently caused a rightward shift of the concentration‐response curve to SX6c. The contraction to ET‐1 (10 nM) in preparations desensitized to the actions of SX6c was greatly inhibited by pretreatment with FR139317 (10 μM). 4 Modulation of the Ca2+ concentration in the Krebs solution caused the concentration‐response curve to ET‐1 or SX6c to shift to the right and downward as external Ca2+ concentrations decreased. Verapamil (30 μM) abolished rhythmic motility induced by ET‐1 or SX6c. Ni2+ (0.1 mM) weakly inhibited ET‐1‐ or SX6c‐induced tonic contraction. SK&F 96365 (60 μM) completely inhibited ET‐1‐induced contractions. 5 We conclude that there are two types of ET‐receptors, excitatory ETA‐ and ETB‐receptors in the oesophageal muscularis mucosae. These receptors mediate tonic contractions predominantly by opening receptor‐operated Ca2+ channels (ROCs) and partly by opening T‐type Ca2+ channels, and mediate rhythmic motility by opening L‐type Ca2+ channels.

INHIBITORY ACTIONS OF CATECHOLAMINES ON ELECTRICALLY INDUCED CONTRACTIONS OF THE SUBMUCOUS PLEXUS‐LONGITUDINAL MUSCULARIS MUCOSAE PREPARATION OF THE GUINEA‐PIG OESOPHAGUS

1 The submucous plexus‐longitudinal muscularis mucosae preparation of the guinea‐pig oesophagus was used to study the actions of catecholamines on the twitch responses to electrical stimulation. 2 When the preparation was stimulated coaxially (0.1 Hz, 0.5 ms, supramaximal voltage), stable twitch‐like contractions were obtained. These were abolished by tetrodotoxin (0.1 μm) and atropine (0.1 μm), potentiated by physostigmine (0.1 μm), and were mediated presumably by stimulation of intramural cholinergic nerves. 3 The twitch contractions of the muscularis mucosae were inhibited by catecholamines, in a concentration‐dependent manner. The order of potency was isoprenaline > adrenaline > noradrenaline > dopamine. 4 The inhibitory actions of noradrenaline (1 μm) and adrenaline (1 μm) were partly reversed by phentolamine (1 μm) or by propranolol (1

Histamine responses mediated via H1 and H2−receptors in the isolated portal vein of the dog

mUM), and completely abolished by both antagonists together. The inhibitory effect of dopamine (300 μm)> was largely reversed by phentolamine (1 μm), but not by propranolol (1 μm), while the inhibitory action of isoprenaline was competitively antagonized only by propranolol (pA2 of 7.6). 5 The contraction of the muscularis mucosae to exogenously applied acetylcholine (ACh, 20 nM) which was comparable in magnitude with that to electrical stimulation was also inhibited by isoprenaline (0.1 μm), adrenaline (1 μm) and noradrenaline (1 μm), but not by dopamine (300 μm). In the presence of propranolol (1 μm), noradrenaline, adrenaline and dopamine potentiated the ACh‐induced contraction, while the effect of isoprenaline was mainly antagonized. The potentiating effects were antagonized by further treatment with phentolamine (1 μm). 6 Adrenaline, noradrenaline and dopamine but not isoprenaline, produced a weak contraction of the longitudinal muscularis mucosae in the presence of propranolol (3 μm). The contractile responses were completely inhibited by phentolamine (3 μm). Tone in the muscularis mucosae induced by carbachol (3 μm) in the presence of phentolamine (10 μm) was inhibited by catecholamines, in a concentration‐dependent manner, an effect that was competitively antagonized by propranolol. 7 In the submucous plexus‐longitudinal muscularis mucosae preparation of the guinea‐pig oesophagus there are three types of adrenoceptor, inhibitory prejunctional α‐adrenoceptors, excitatory postjunctional α‐adrenoceptors and inhibitory postjunctional β‐adrenoceptors, and cholinergic neurotransmission is inhibited by catecholamines acting at both prejunctional α‐ and postjunctional β‐adrenoceptors.

Time-dependent augmentation of the contractile responses to adrenaline and noradrenaline of the guinea-pig esophageal muscularis mucosae in vitro

The effects of histamine were studied on the isolated circular muscle strip and longitudinal muscle strip of the dog portal vein. Histamine‐induced contractions of the circular muscle were inhibited by H1‐receptor antagonist pyrilamine but increased by H2‐receptor antagonist ranitidine. When the tissues were contracted with PGF2α in the presence of pyrilamine, histamine produced the relaxation of the circular muscle but not longitudinal muscle. The relaxation of the circular muscle was inhibited by ranitidine in a concentration dependent manner, the pA2 value for ranitidine being 6·97 (6·55–7·39). It is concluded that the response of the circular muscle to histamine is the sum of two components, H1‐receptor mediated contraction and H2‐receptor mediated relaxation.

The role of receptor-operated CA2+ influx in endothelin-induced contraction of the muscularis mucosae.

SummaryThe effects of long-term incubation and of indomethacin on contractile responses to adrenaline and noradrenaline of the guinea-pig esophageal muscularis mucosae were studied in vitro.In the presence of propranolol (3 μM), contractions of the isolated muscularis mucosae of the guinea-pig esophagus induced by adrenaline (10 μM) and noradrenaline (10 μM) gradually increased in magnitude with incubation time, and after 7.5 h reached a peak which was about 3 times the initial response. Contractions to adrenaline and noradrenaline observed at 2.5 h after the incubation was started were fully inhibited by phentolamine (3 μM) and prazosin (3 μM), and slightly by yohimbine (3 μM). These responses were also inhibited by polyphloretin phosphate (PPP, 30 μg/ml), indomethacin (1 μM) and aspirin (100 μM). The augmented contractions to adrenaline and noradrenaline observed at 7.5 h after the incubation was started were also inhibited by PPP, indomethacin and aspirin, while they were rather resistant to the blocking actions of phentolamine, prazosin and yohimbine.The effect of matched concentrations of carbachol (0.1 μM), prostaglandin E2 (PGE2, 0.01 μM) and arachidonic acid (0.2 μM), which produced the same degree of contraction induced by catecholamines (10 μM), was gradually increased with incubation time. When the strip was treated with indomethacin (1 μM) throughout the experiment, contraction to arachidonic acid was abolished, that to carbachol was almost constant during long-term incubation but that to PGE2 was still increased after long-term incubation. Moreover, both concentration-response curves to carbachol and PGE2 examined at 7.5 h after the incubation was started were located to the left to those examined at 1.5 h after the incubation was started. In the presence of indomethacin (1 μM), the curve to PGE2, but not to carbachol, still shifted to the left after long-term incubation.The present results demonstrated that the contractile responses of the guinea-pig esophageal muscularis mucosae were increased in magnitude depending on the incubation time. The mechanism of this augmentation may involve the development of atypical adrenoceptors, the stimulated arachidonic acid cascade and the increased responsiveness to PGs.