Curcumin ameliorates high glucose‐induced acute vascular endothelial dysfunction in rat thoracic aorta

• 1 The aims of the present study were to explore the protective effect of curcumin against the acute vascular endothelial dysfunction induced by high glucose and to investigate the possible role of heme oxygenase (HO)‐1 in this protective action.
• 2 Thoracic aortic rings, with or without endothelium, obtained from male Sprague‐Dawley rats were mounted in an organ bath. Isometric contraction of the rings was recorded. After completion of the organ bath studies, rings were homogenized and centrifuged (30 000 g, 4°C, 15 min) and HO activity was determined in the supernatant.
• 3 After 2 h incubation of aortic rings in the presence of high glucose (44 mmol/L), the relaxation evoked by acetylcholine (3 × 10^?8 to 3 × 10^?5 mol/L) was significantly decreased only in rings with an intact endothelium. When rings were coincubated in the presence of curcumin (10^?13 to 10^?11 mol/L) and high glucose, curcumin reversed the vasodilator dysfunction induced by high glucose dose dependently.
• 4 Curcumin (10^?11 mol/L) increased HO activity in the aortic rings compared with activity in control rings (63.1 ± 3.6 vs control 43.2 ± 2.9 pmol/mg per h, respectively; P < 0.01). Protoporphyrin IX zinc (10^?6 mol/L), an inhibitor of HO‐1, offset the protective effects of curcumin. In addition, the non‐selective guanylate cyclase (GC) inhibitor methylene blue (10^?6 mol/L) completely abolished the protective effects of curcumin.
• 5 In conclusion, the results of the present study show that curcumin alleviates the acute endothelium‐dependent vasodilator dysfunction induced by high glucose in rat aortic rings. Increased HO‐1 activity and stimulation of GC may be involved in the protective effects of curcumin.

     

Trimebutine maleate relaxes the isolated rat thoracic aorta: The role of nitric oxide and L‐type calcium channels

Trimebutine maleate (TMB), a widely prescribed drug for functional gastrointestinal disorders, has been reported to regulate smooth muscle contractility by modulating multiple ion channel activities in the gastrointestinal tract. However, its action on isolated aorta has not yet been reported. The aim of the present study was to evaluate in vitro vasorelaxant properties and the underlying pharmacological mechanisms of TMB in isolated rat thoracic aortic rings. Vascular activity experiments were performed on thoracic aorta isolated from Sprague‐Dawley rats in vitro, including endothelium‐intact and endothelium‐denuded aortic rings. TMB (10^?10‐10^?5 mol/L) induced relaxation in endothelium‐intact aortic rings precontracted by phenylephrine with a potency similar to that of carbachol. TMB‐induced relaxation was not altered by glibenclamide and atropine in endothelium‐intact aortic rings. However, L‐NAME and endothelium denudation significantly reduced but not completely reversed the vasorelaxant effect of TMB. Also, TMB‐induced relaxation wasn't affected by diclofenac in endothelium‐intact aortic rings. TMB at 10^?5 mol/L significantly reduced the CaCl₂‐induced contractions in endothelium‐intact aortic rings stimulated with KCl, but not stimulated with phenylephrine under Ca²⁺free conditions. Moreover, TMB at 10^?5 mol/L effectively attenuated Bay‐K8644‐induced contractions in aortic rings. These results suggest that TMB‐induced relaxation was mediated by both endothelium‐dependent and endothelium‐independent manner in isolated rat thoracic aorta. The mechanism of TMB‐induced relaxation at low concentrations is partially related to NO‐ and endothelium‐dependent but unrelated to prostanoids formation. However, inhibition of Ca²⁺ influx through voltage‐operated calcium channels and L‐type Ca²⁺channel blocking effect appears to be involved in the mechanism of vasorelaxant effect of TMB at high concentrations.     

Characterization of relaxant mechanism of H2S in mouse corpus cavernosum

The aim of this study was to investigate the mechanism of H₂S‐induced relaxation in mouse corpus cavernosal tissue. l ‐cysteine (10^?6 × 10^?3 mol/L) and exogenous H₂S (NaHS; 10^?6 to 10^?3 mol/L) induced concentration‐dependent relaxation. l ‐cysteine‐induced relaxations was reduced by d,l ‐propargylglycine, a cystathionine gamma lyase (CSE) inhibitor but not influenced by aminooxyacetic acid, a cystathionine beta synthase (CBS) inhibitor. l ‐cysteine induced relaxations, but not of those of H₂S diminished in endothelium‐denuded tissues. N^ω‐nitro‐l ‐arginine (l ‐NA; 10^?4 mol/L), a nitric oxide synthase inhibitor, and ODQ (10^?4 mol/L), a guanylyl cyclase inhibitor, increased the H₂S‐induced relaxation. Zaprinast (5 × 10^?6 mol/L) and sildenafil (10^?6 mol/L), phosphodiesterase inhibitors, inhibited H₂S‐induced relaxation. Adenylyl cyclase inhibitors N‐ethylmaleimide (2.5 × 10^?5 mol/L) and SQ22536 (10^?4 mol/L) reduced relaxation to H₂S. Also, H₂S‐induced relaxation was reduced by KCl (50 mmol/L), 4‐aminopyridine (10^?3 mol/L), a K_v inhibitor, glibenclamide (10^?5 mol/L), a K_ATP inhibitor or barium chloride (10^?5 mol/L), a K_IR inhibitor. However, H₂S‐induced relaxation was not influenced by apamin (10^?6 mol/L), a SK_Ca²⁺ inhibitor, charybdotoxin (10^?7 mol/L), an IK_Ca²⁺ and BK_Ca²⁺ inhibitor or combination of apamin and charybdotoxin. Nifedipine (10^?6 mol/L), an L‐type calcium channel blocker and atropine (10^?6 mol/L), a muscarinic receptor blocker, inhibited H₂S‐induced relaxation. However, H₂S‐induced relaxation was not influenced by ouabain (10^?4 mol/L), a Na⁺/K⁺‐ATPase inhibitor. This study suggests that H₂S endogenously synthesizes from l ‐cysteine by CSE endothelium‐dependent in mouse corpus cavernosum tissue, and exogenous H₂S may cause endothelium‐independent relaxations via activation of K channels (K_ATP channel, K_V channels, K_IR channels), L‐type voltage‐gated Ca²⁺ channels, adenylyl cyclase/cAMP pathway and muscarinic receptor, and there is the interaction between H₂S and NO/cGMP.     

Unravelling the cardiovascular effects induced by α‐terpineol: A role for the nitric oxide–cGMP pathway

1. α‐Terpineol is a monoterpene found in the essential oils of several aromatic plant species. In the present study, we investigated the mechanisms underlying the cardiovascular changes induced by α‐terpineol in rats. 2. In normotensive rats, administration of α‐terpineol (1, 5, 10, 20 and 30 mg/kg, i.v.) produced a dose‐dependent hypotension (?10 ± 3, ?20 ± 8, ?39 ± 16, ?52 ± 21 and ?57 ± 23 mmHg, respectively; n = 5) followed by tachycardia. The hypotensive responses to 1, 5, 10, 20 and 30 mg/kg, i.v., α‐terpineol were significantly attenuated following the administration of N^G‐nitro‐l‐ arginine methyl ester (l ‐NAME; 20 mg/kg, i.v.; ?2 ± 1, ?5 ± 2, ?7 ± 3, ?22 ± 9 and ?22 ± 10 mmHg, respectively; P < 0.05; n = 5). 3. In 10 μmol/L phenylephrine (PE)‐precontracted mesenteric artery rings, α‐terpineol (10^?12 to 10^?5 mol/L) caused a concentration‐dependent relaxation (maximum relaxation 61 ± 6%; n = 7). After removal of the endothelium, the vasorelaxation elicited by α‐terpineol was attenuated (maximum relaxation 20 ± 1%; P < 0.05; n = 7). In addition, vasorelaxation induced by α‐terpineol in rings pretreated with 100 or 300 μmol/L l ‐NAME, 30 μmol/L hydroxocobalamin or 10 μmol/L 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one was attenuated (maximum relaxation 18 ± 3, 23 ± 3, 24 ± 7 and 21 ± 1%, respectively; n = 6; P < 0.05). 4. Furthermore, in a rabbit aortic endothelial cell line, 10^?6, 10^?5 and 10^?4 mol/L α‐terpineol induced concentration‐dependent increases in nitric oxide (NO) levels (12 ± 6, 18 ± 9 and 34 ± 12%Δ fluorescence, respectively; n = 3). 5. In conclusion, using combined functional and biochemical approaches in the present study, we were able to demonstrate that α‐terpineol‐induced hypotension and vasorelaxation are mediated, at least in part, by the endothelium, most likely via NO release and activation of the NO–cGMP pathway.     

Inhibitory effect of 1,8-cineole on guinea-pig airway challenged with ovalbumin involves a preferential action on electromechanical coupling

• 1 1,8‐Cineole is a terpenoid constituent of essential oils with anti‐inflammatory properties. It reduces the neural excitability, functions as an antinociceptive agent and has myorelaxant actions in guinea‐pig airways. The aim of the present study was to investigate the mechanism underlying the myorelaxant effects of 1,8‐cineole in guinea‐pig isolated trachea from either naïve guinea‐pigs or ovalbumin (OVA)‐sensitized animals subjected to antigenic challenge.
• 2 Isometric recordings were made of the tone of isolated tracheal rings. Rings with an intact epithelium relaxed beyond basal tone in the presence of 1,8‐cineole (6.5 × 10^?6 to 2 × 10^?2 mol/L) in a concentration‐dependent manner (P < 0.001, anova ) with a pD₂ value of 2.23 (95% confidence interval 2.10–2.37). Removal of the epithelium or pretreatment of intact tissue for 15 min with 50 µmol/L N^G‐nitro‐l ‐arginine methyl ester, 5 mmol/L tetraethylammonium, 0.5 µmol/L tetrodotoxin or 5 µmol/L propranolol did not alter the potency (pD₂) or the maximal myorelaxant effect (E_max) of 1,8‐cineole.
• 3 1,8‐Cineole also significantly decreased the Schultz‐Dale contraction induced by OVA, mainly in preparations from OVA‐sensitized animals submitted to antigen challenge. 1,8‐Cineole decreased tracheal hyperresponsiveness to KCl and carbachol caused by antigen challenge and almost abolished the concentration–response curves to KCl, whereas it had little effect on the concentration–response curves to carbachol. Under Ca²⁺‐free conditions and in the presence of 10^?4 mol/L acetylcholine, neither 1,8‐cineole (6.5 × 10^?3 mol/L) nor verapamil (1 × 10^?5 mol/L) affected Ca²⁺‐induced contractions, but they almost abolished Ba²⁺‐induced contractions.
• 4 In conclusion, the findings of the present study show that 1,8‐cineole is a tracheal myorelaxant that acts preferentially on contractile responses elicited electromechanically.

     

Effects of magnolol on vascular contraction in rat aortic rings

1. Magnolol (5,5′‐diallyl‐2,2′‐dihydroxybiphenyl) is a major phenolic compound purified from Magnolia officinalis. The aim of the present study was to elucidate the effects of magnolol on vascular contractions. 2. Rat aortic rings were mounted in organ baths. Magnolol was added cumulatively (0.3–30 μmol/L) to elicit relaxation in endothelium‐intact and ‐denuded rat aortic rings precontracted with U46619 (30 nmol/L, 20 min), NaF (8.0 mmol/L, 40 min), phenylephrine (1.0 or 0.1 μmol/L, 15 min) or phorbol‐12,13‐dibutyrate (PDBu, 0.3 or 0.1 μmol/L, 40 min). In separate experiments, cumulative concentration?response curves were obtained for NaF (2.0–12 mmol/L), U46619 (1.0 nmol/L?1.0 μmol/L) or PDBu (1.0 nmol/L?1.0 μmol/L) after pretreatment with either magnolol or vehicle for 30 min in endothelium‐denuded aortic rings. After completion of the functional study, we measured the amount of guanosine triphosphate (GTP) RhoA by using a G‐LISA RhoA Activation Assay, as well as the phosphorylation of 20 kDa myosin light chain (MLC₂₀), myosin phosphatase‐targeting subunit 1 (MYPT1) and protein kinase C‐potentiated inhibitory protein for heterotrimeric myosin light‐chain phosphatase of 17 kDa (CPI‐17) by immunobloting. 3. Magnolol (0.3–30 μmol/L) reduced vascular tension induced by the thromboxane A₂ agonist U46619 (30 nmol/L), sodium fluoride (NaF) (8.0 mmol/L) and the α₁‐adrenoceptor agonist phenylephrine (1.0 or 0.1 μmol/L) in both endothelium‐intact and ‐denuded rings. The magnitude of the relaxation effects of magnolol on the contraction induced by each of the drugs were similar. The magnitude of the effect of magnolol in endothelium‐intact and ‐denuded rings were similar. 4. Pretreatment of rat aortic rings with 1.0, 3.0 or 10 μmol/L magnolol for 30 min dose‐dependently inhibited the maximum response on the concentration–response curves to NaF and U46619, but not to phorbol‐12,13‐dibutyrate (PDBu). 5. Magnolol (3.0 or 10 μmol/L) decreased RhoA activation, as well as the phosphorylation of MLC₂₀, MYPT1_Thr855 and CPI‐17_Thr38 induced by either 8.0 mmol/L NaF or 30 nmol/L U46619. In contrast, magnolol did not affect PDBu (0.1 μmol/L)‐induced phosphorylation of CPI‐17_Thr38. 6. In conclusion, magnolol reduces vascular contraction by inhibiting the RhoA/Rho kinase pathway in endothelium‐denuded rat aorta.     

REGIONAL DIFFERENCES IN THE MODULATORY ROLE OF THE EPITHELIUM IN SHEEP AIRWAY

• 1 The airway epithelium may modulate smooth muscle responsiveness via the release of biologically active substances, such as nitric oxide (NO) and prostaglandins. Based on regional differences in structure and function described for the airway epithelium, we performed a comparative study on the responsiveness of sheep isolated, epithelium‐intact or ‐denuded, first‐ to fourth‐order bronchi to acetylcholine (ACh).
• 2 We performed contractility studies using KCl or cholinergic stimuli in the presence or absence of NO or prostaglandin‐related drugs in epithelium‐intact and epithelium‐denuded bronchial strips obtained from all four airway regions. We also studied the expression of NO synthase (NOS), using the NADPH‐diaphorase staining technique, and the effect of airway epithelium removal on the synthesis of NO metabolites in the different bronchi orders.
• 3 There was no difference in the response of first‐ to fourth‐order epithelium‐intact bronchi to ACh (1 nmol/L–100 mmol/L) or KCl (5–100 mmol/L). Removal of the epithelium had no effect on ACh‐induced contractions of first‐ and second‐order bronchi, but increased responses of third‐ and fourth‐order bronchi to ACh. The NO synthase inhibitor N^G‐nitro‐l‐ arginine methyl ester (100 µmol/L) increased ACh‐induced contractions of fourth‐order epithelium‐intact bronchi only. The NO donor sodium nitroprusside (1 nmol/L–1 mmol/L) equally relaxed 1 µmol/L carbachol‐precontracted epithelium‐denuded first‐ and fourth‐order bronchi.
• 4 Although NAPDH–diaphorase staining demonstrated no regional differences in NOS expression, basal levels of NO metabolites were 4.5‐fold greater in fourth‐ compared with second‐order epithelium‐intact bronchi.
• 5 The cyclo‐oxygenase inhibitor indomethacin (10 µmol/L) had no effect on ACh‐induced contractions of first‐ to fourth‐order epithelium‐intact bronchi, but decreased responses of fourth‐order epithelium‐denuded bronchi to ACh. The contractile effect of the thromboxane A₂ mimetic U‐46619 (1 nmol/L–10 µmol/L) was greater in fourth‐ compared with first‐order epithelium‐denuded bronchi.
• 6 In conclusion, the sheep airway epithelium exhibits regional differences in its modulatory role and this is particularly apparent in small bronchi.

     

SUFENTANIL AND ALFENTANIL CAUSE VASORELAXATION BY MECHANISMS INDEPENDENT OF THE ENDOTHELIUM

1. The aim of these experiments was to determine if the vasorelaxation of the rat isolated aorta induced by sufentanil or alfentanil is mediated by the endothelium, and, if not, by α-adrenoceptor blockade, or a direct effect on the smooth muscle. 2. Both sufentanil (from 10^?7 mol/L to 10^?4 mol/ L) and alfentanil (from 10^?7 mol/ L to 3 × 10^?4 mol/L) relaxed rings, where endothelium was intact and precontracted with 40 mmol/L KC1, in a concentration-related manner. Similarly, sufentanil and alfentanil relaxed rings, in the presence or absence of endothelium, which had been precontracted with phenylephrine. 3. Naloxone (10^?4 mol/L) had no significant effect on the relaxation induced by either sufentanil or alfentanil. 4. In a similar manner as phentolamine, pretreatment with sufentanil protected α-adrenoceptors from blockade by phenoxybenzamine (PBZ) in both endothelium intact and denuded rings, but the estimated potency of sufentanil was approximately 100-fold less than that of phentolamine in α-adrenoceptor protection. Treatment with alfentanil did not produce any receptor protection. 5. We concluded that, in the rat aorta, vascular relaxation induced by sufentanil is mediated by both α-adrenoceptor blockade and a direct effect on smooth muscle, whilst the relaxant effect of alfentanil is caused by direct effects alone. We also concluded that the endothelium has little role in relaxation produced by either drug.     

CILOSTAMIDE PRODUCES HYPERPOLARIZATION ASSOCIATED WITH KATP CHANNEL ACTIVATION, BUT DOES NOT AUGMENT ENDOTHELIUM-DERIVED HYPERPOLARIZATION IN RAT MESENTERIC ARTERIES

• 1 Non‐nitric oxide/prostaglandin‐mediated endothelium‐derived hyperpolarization (EDH) is considered to be mediated, in part, by gap junctions and it has been suggested that cAMP increases endothelium‐derived hyperpolarizing factor (EDHF)‐mediated relaxation through the modulation of gap junctions. Cilostamide, which inhibits phosphodiesterase III, has been suggested to augment EDHF‐type relaxation by increasing the concentration of cAMP.
• 2 In the present study, we investigated the effect of cilostamide on EDH per se in mesenteric arteries of Wistar rats using a conventional microelectrode technique.
• 3 The resting membrane potential of the mesenteric arteries was significantly more negative in the presence of 10^?6 mol/L cilostamide compared with control conditions. Furthermore, EDH in response to 10^?6 mol/L acetylcholine (ACh) in the presence of 10^?5 mol/L indomethacin and 10^?4 mol/L N^G‐nitro‐L‐arginine was decreased in the presence of 10^?6 mol/L cilostamide by approximately 5 and 3.5 mV in proximal and distal arteries, respectively.
• 4 Glibenclamide (10^?5 mol/L), an ATP‐sensitive potassium channel (K_ATP) inhibitor, abolished the hyperpolarization to 10^?6 mol/L cilostamide. Furthermore, in the presence of glibenclamide, ACh‐induced EDH was unaffected by cilostamide, suggesting that the inhibition of ACh‐induced hyperpolarization by cilostamide in the absence of glibenclamide may be due to the smaller driving force for hyperpolarization because of the more negative membrane potential under such conditions.
• 5 The findings of the present study suggest that cilostamide produces hyperpolarization by activating K_ATP channels, presumably by increasing cAMP. However, cilostamide alone may not directly affect EDH.

     

CALCITONIN GENE-RELATED PEPTIDE MEDIATES THE CARDIOPROTECTIVE EFFECTS OF RUTAECARPINE AGAINST ISCHAEMIA–REPERFUSION INJURY IN SPONTANEOUSLY HYPERTENSIVE RATS

• 1 It has been shown that calcitonin gene‐related peptide (CGRP) plays an important role in mediating the cardioprotection exerted by rutaecarpine in normal animals. The aim of the present study was to determine whether rutaecarpine is able to decrease the susceptibility of hypertensive animals to ischaemia–reperfusion injury by stimulating CGRP release.
• 2 Spontaneously hypertensive rats (SHR) were pretreated with rutaecarpine (20 or 40 mg/kg per day, i.g.) for 18 days and then the heart and thoracic aorta were isolated for cardiac function and vascular relaxation analysis. Blood samples and coronary effluent were collected to measure CGRP levels and creatine kinase activity, respectively. The effect of 10 or 30 µmol/L rutaecarpine on CGRP release was also examined in isolated aortic rings set up in a homeothermal organ bath.
• 3 Rutaecarpine treatment resulted in a hypotensive effect in SHR concomitant with increases in plasma CGRP levels. In addition, rutaecarpine significantly stimulated the release of CGRP from aortic rings. Twenty minutes ischaemia and 30 min reperfusion resulted in a marked decrease in myocardial function and a significant increase in the release of creatine kinase in normal control (Wistar‐Kyoto) rats, an effect that was exacerbated in SHR. Similarly, the decreased vasodilator response to acetylcholine (3 ? 10^?9 to 10^?6 mol/L) in isolated aortic rings from Wistar‐Kyoto rats was also aggravated in SHR. Both cardiac function and vasodilator responses were significantly improved in SHR after pretreatment with rutaecarpine.
• 4 The results of the present study suggest that the increased cardiac susceptibility to ischaemia–reperfusion injury in SHR is related to decreased plasma CGRP levels and that antihypertensive therapy with rutaecarpine reverses cardiac susceptibility to reperfusion injury by stimulating CGRP release.

     

Metformin attenuates ventricular hypertrophy by activating the AMP-activated protein kinase-endothelial nitric oxide synthase pathway in rats

1. Metformin is an activator of AMP‐activated protein kinase (AMPK). Recent studies suggest that pharmacological activation of AMPK inhibits cardiac hypertrophy. In the present study, we examined whether long‐term treatment with metformin could attenuate ventricular hypertrophy in a rat model. The potential involvement of nitric oxide (NO) in the effects of metformin was also investigated. 2. Ventricular hypertrophy was established in rats by transaortic constriction (TAC). Starting 1 week after the TAC procedure, rats were treated with metformin (300 mg/kg per day, p.o.), N^G‐nitro‐l‐ arginine methyl ester (l ‐NAME; 50 mg/kg per day, p.o.) or both for 8 weeks prior to the assessment of haemodynamic function and cardiac hypertrophy. 3. Cultured cardiomyocytes were used to examine the effects of metformin on the AMPK–endothelial NO synthase (eNOS) pathway. Cells were exposed to angiotensin (Ang) II (10^?6 mol/L) for 24 h under serum‐free conditions in the presence or absence of metformin (10^?3 mol/L), compound C (10^?6 mol/L), l ‐NAME (10^?6 mol/L) or their combination. The rate of incorporation of [³H]‐leucine was determined, western blotting analyses of AMPK–eNOS, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) were undertaken and the concentration of NO in culture media was determined. 4. Transaortic constriction resulted in significant haemodynamic dysfunction and ventricular hypertrophy. Myocardial fibrosis was also evident. Treatment with metformin improved haemodynamic function and significantly attenuated ventricular hypertrophy. Most of the effects of metformin were abolished by concomitant l ‐NAME treatment. l ‐NAME on its own had no effect on haemodynamic function and ventricular hypertrophy in TAC rats. 5. In cardiomyocytes, metformin inhibited AngII‐induced protein synthesis, an effect that was suppressed by the AMPK inhibitor compound C or the eNOS inhibitor l ‐NAME. The improvement in cardiac structure and function following metformin treatment was associated with enhanced phosphorylation of AMPK and eNOS and increased NO production. 6. The findings of the present study indicate that long‐term treatment with metformin could attenuate ventricular hypertrophy induced by pressure overload via activation of AMPK and a downstream signalling pathway involving eNOS–NO.     

Pharmacological characterization of mechanisms involved in the vasorelaxation produced by rosuvastatin in aortic rings from rats with a cafeteria‐style diet

The present study aimed to investigate the possible influence of several inhibitors and blockers on the vascular effect produced by the acute in vitro application of rosuvastatin to phenylephrine‐precontracted aortic rings from rats with a semi‐solid, cafeteria‐style (CAF) diet. It also aimed to examine the effects of rosuvastatin on the expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase in aortic rings from rats with a CAF diet. From comparisons of the effect on phenylephrine‐precontracted aortic rings extracted from rats with two different diets (a standard and a CAF diet), it was found that 10^?9–10^?5‐mol/L rosuvastatin produced lower concentration‐dependent vasorelaxation on rings from the CAF diet group. The vasorelaxant effect was unaffected by the vehicle, but it was significantly attenuated by 10^?5‐mol/L N^G‐nitro‐l ‐arginine methyl ester, 10^?2‐mol/L tetraethylammonium, 10^?3‐mol/L 4‐aminopyridine, 10^?7‐mol/L apamin plus 10^?7‐mol/L charybdotoxin, 10^?5‐mol/L indomethacin, or 10^?5‐mol/L cycloheximide. Moreover, in aortic rings from rats with a CAF diet, rosuvastatin enhanced the expression of eNOS, inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase. The acute in vitro application of rosuvastatin to phenylephrine‐precontracted aortic rings from rats with a CAF diet had a vasorelaxant effect. Overall, the present results suggest that the stimulation of eNOS, the opening of Ca²⁺‐activated and voltage‐activated K⁺ channels, the stimulation of prostaglandin synthesis and enhanced protein levels of eNOS, inducible nitric oxide synthase, constitutive cyclooxygenase, and inducible cyclooxygenase are involved in this relaxant effect.     

Effect of orchiectomy on renal function in control and diabetic rats with chronic inhibition of nitric oxide

• 1 Male gender is associated with higher blood pressure (BP) and more rapid loss of renal function in a spectrum of clinical and experimental renal diseases, including diabetic nephropathy. Consequently, modulation of testosterone levels could exert beneficial effects in the diabetic kidney.
• 2 The aim of the present study was to determine whether testosterone deficiency (orchiectomy) could influence BP and renal function in streptozotocin‐diabetic rats, with or without accelerated endothelial dysfunction achieved by chronic inhibition of nitric oxide (NO) synthesis using N^G‐nitro‐l‐ arginine methyl ester (l ‐NAME; 40–100 mg / L in the drinking water for 2 weeks), as well as in age‐matched non‐diabetic rats subjected to the same interventions.
• 3 Orchiectomy did not affect l ‐NAME‐induced increases in BP in non‐diabetic or diabetic rats. In non‐diabetic rats, orchiectomy prevented l ‐NAME‐induced increases in proteinuria. These effects on proteinuria were not observed in diabetic rats. In non‐diabetic rats, orchiectomy had no effect on renal haemodynamics in animals receiving vehicle and did not affect l ‐NAME‐induced changes in renal haemodynamics, characterized by reductions in renal plasma flow (RPF) and higher filtration fractions (FF). In intact diabetic rats, l ‐NAME treatment resulted in lower RPF. This difference was not observed in diabetic rats subjected to orchiectomy, although l ‐NAME‐treated diabetic orchiectomized rats had lower RPF and higher FF compared with vehicle‐treated intact diabetic rats.
• 4 In conclusion, we report modest beneficial effects of orchiectomy on proteinuria in normal, but not in diabetic, rats with inhibition of NO production. This suggests that testosterone reduction does not attenuate the deleterious impact of the diabetic metabolic milieu in the kidney.

     

Exogenous L‐arginine attenuates the effects of angiotensin II on renal hemodynamics and the pressure natriuresis–diuresis relationship

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Effect of 17β-oestradiol replacement on vascular responsiveness in ovariectomized diabetic rats

1. Women with functional ovaries exhibit a gender advantage in terms of the prevalence of cardiovascular diseases. However, whether this gender bias pertains in diabetes is unknown. 2. The aim of the present study was to examine the effects of 17β‐oestradiol (E2) on vascular responsiveness in normal and diabetic ovariectomized (OVX) rats. Aged‐matched female rats were divided into four groups as follows: (i) OVX; (ii) OVX + E2 treated; (iii) diabetic OVX; and (iv) diabetic OVX + E2 treated. Bilateral ovariectomy was performed and streptozotocin was used to induce experimental diabetes. Rats were treated with 1 mg/kg per day, p.o., E2 for 8 weeks. 3. Although E2 treatment had no effect on blood glucose levels in normal and diabetic OVX rats, it significantly reduced systolic blood pressure and prevented diabetes‐induced loss of bodyweight gain. 4. In segments of the thoracic aorta, concentration‐dependent vasoconstrictor responses to KCl and phenylephrine were significantly attenuated following E2 treatment in both the normal and diabetic groups. The sarcoplasmic/endoplasmic reticulum calcium ATPase inhibitor thapsigargin (10^?6 mol/L) and the Ca²⁺ channel blocker nifedipine (10^?6 mol/L) inhibited the transient vasoconstriction to PE in all groups. The constrictor effect of PE was increased by the nitric oxide synthase inhibitor N^G‐nitro‐l‐ arginine methyl ester (l ‐NAME; 10^?6 mol/L), but was reduced by superoxide dismutase (SOD; 100 U/mL) and the cyclo‐oxygenase inhibitor indomethacin (10^?6 mol/L) in all groups. Responses to acetylcholine (ACh; 10^?6 mol/L) demonstrated reduced endothelium‐dependent relaxation in non‐E2‐treated groups. Relaxation responses to ACh were increased by 100 U/mL SOD and 10^?6 mol/L indomethacin, but were reduced by 10^?6 mol/L l ‐NAME in all groups. There were no differences among the four groups in terms of relaxation responses to sodium nitroprusside (10^?11 to 10^?6 mol/L). 5. In conclusion, the results of the present study suggest that oestrogen treatment has beneficial effects on vascular function in both diabetic and non‐diabetic OVX rats due to Ca²⁺ regulation and anti‐oxidation.     

Angiotensin‐(1–7) treatment ameliorates angiotensin II‐induced apoptosis of human umbilical vein endothelial cells

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Cardiovascular effects elicited by milonine, a new 8,14-dihydromorphinandienone alkaloid

Abstract: The mechanisms underlying the cardiovascular responses evoked by milonine (i.v.), an alkaloid, were investigated in rats. In normotensive rats, milonine injections produced hypotension and tachycardia, which were attenuated after N^w‐nitro‐l ‐arginine methyl esther (l ‐NAME; 20 mg/kg, i.v.). In phenylephrine (10 μM), pre‐contracted mesenteric artery rings, milonine (10^?10 M to 3 × 10^?4 M) caused a concentration‐dependent relaxation (EC₅₀ = 1.1 × 10^?6 M, E_max = 100 ± 0.0%) and this effect was rightward shifted after either removal of the vascular endothelium (EC₅₀ = 1.6 × 10^?5, p < 0.001), or after l ‐NAME 100 μM (EC₅₀ = 6.2 × 10^?5, p < 0.001), hydroxocobalamin 30 μM (EC₅₀ = 1.1 × 10^?4, p < 0.001) or ODQ 10 μM (EC₅₀ = 1.9 × 10^?4p < 0.001). In addition, in rabbit aortic endothelial cells, milonine increased NO₃^? levels. The relaxant effect induced by milonine was attenuated in the presence of KCl (20 mM), a modulator efflux K⁺ (EC₅₀ = 1.2 × 10^?5, p < 0.001), or different potassium channel blockers such as glibenclamide (10 μM) (EC₅₀ = 6.3 × 10^?5, p < 0.001), TEA (1 mM) (EC₅₀ = 2.3 × 10^?5 M, n = 6) or Charybdotoxin (0.2 μM) plus apamin (0.2 μM) (EC₅₀ = 3.9 × 10^?4 M, n = 7). In addition, pre‐contraction with high extracellular potassium concentration prevented milonine‐induced vasorelaxation (EC₅₀ = 1.0 × 10^?4, p < 0.001). Milonine also reduced CaCl₂‐induced contraction in Ca²⁺‐free solution containing KCl (60 mM). In conclusion, using combined functional and biochemical approaches, we demonstrated that the hypotensive and vasorelaxant effects produced by milonine are, at least in part, mediated by the endothelium, likely via nitric oxide release, activation of nitric oxide‐cGMP pathway and opening of K⁺ channels.     

Warifteine,a bisbenzylisoquinoline alkaloid,induces relaxation by activating potassium channels in vascular myocytes

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ENHANCEMENT OF ENDOTHELIUM-DEPENDENT RELAXATION IN THE AORTA FROM STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS AT DEVELOPMENTAL STAGES OF HYPERTENSION

1. Endothelium-dependent relaxation (EDR) in aortic rings from young (8 weeks) and adult (16 weeks and 20 weeks) stroke-prone spontaneously hypertensive rats (SHRSP) was investigated in comparison with age-matched Wistar-Kyoto rats (WKY). 2. At 8 weeks, acetylcholine (3×10^?-⁹-^?10^?-⁵ mol/L) and ionomycin (4×10^?8-10^?6 mol/L)-induced EDR in SHRSP aortae was significantly enhanced compared to that in WKY aortae. Mechanical denudation of the endothelium completely abolished, and pretreatment of aortae with N^G-monomethyl L-arginine (1 mmol/L), an inhibitor of nitric oxide formation, greatly reduced the relaxation in both strains. Indomethacin (10^?5 mol/L), a cyclo-oxygenase inhibitor that blocks the production of endothelium-derived contracting factors, did not significantly alter the relaxation by acetylcholine at this age. There was no difference in endothelium-independent relaxation of denuded aortae by sodium nitroprusside (10^?9-10^?6 mol/L) and 8-bromoguanosine 3‘, 5‘-cyclic monophos-phate (10^?6-10^?3 mol/L). 3. In adult SHRSP with established hypertension, however, the acetylcholine (10^?8-^?10^?5 mol/L)-induced relaxation markedly diminished at any of the concentrations tested compared to that observed in 8 week old SHRSP and WKY at 8–20 weeks of age. This finding differed from other observations where the relaxation in SHRSP was impaired only at higher concentrations of acetylcholine. Indomethacin pretreatment of aortae from 20 week old SHRSP restored acetylcholine-induced EDR to a level comparable with that in age-matched WKY. 4. These results suggest that the aorta from young SHRSP releases more endothelium-derived relaxing factors in response to acetylcholine and ionomycin, but the relaxation greatly diminishes in adult SHRSP with established hypertension. This may be due to counteraction of endothelium-derived contracting factors released from the endothelium with functional changes resulting from the long duration of the hypertension.     

ROLE OF EXTRACELLULAR Na+, Ca2+-ACTIVATED Cl- CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE-DEPLETED TRACHEAL SMOOTH MUSCLE

• 1 In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re‐addition of Ca²⁺ in an in vitro experimental model in which Ca²⁺ stores had been depleted and their refilling had been blocked by thapsigargin.
• 2 Mean (±SEM) contraction was diminished by: (i) inhibitors of store‐operated calcium channels (SOCC), namely 100  µ mol/L SKF‐96365 and 100  µ mol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca²⁺ channels Ca_V1.2 channels, namely 1  µ mol/L nifedipine and 10  µ mol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20  µ mol/L niflumic acid, a non‐selective inhibitor of Ca²⁺‐dependent Cl^? channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3‐fold by 100 nmol/L iberiotoxin, a blocker of the large‐conductance Ca²⁺‐activated K⁺ (BK) channels.
• 3 Furthermore, contraction was significantly inhibited when Na⁺ in the bathing solution was replaced by N‐methyl–d ‐glucamine (NMDG⁺) to 39.9 ± 7.2% of control, but not when it was replaced by Li⁺ (114.5 ± 24.4% of control). In addition, when Na⁺ had been replaced by NMDG⁺, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na⁺ had been replaced by Li⁺ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control).
• 4 In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca_V1.2 channels in the contractions induced by the re‐addition of Ca²⁺ to the solution bathing guinea‐pig tracheal rings under conditions of Ca²⁺‐depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na⁺, suggesting a role for SOCC in mediating the Na⁺ influx.