Nebivolol induces endothelium-dependent relaxations of canine coronary arteries.

Nebivolol is a new beta 1-antagonist that acutely reduces arterial blood pressure without depressing cardiac function. The present study was designed to determine the effect of nebivolol on coronary arteries. Rings of canine left anterior descending coronary (LAD) artery with or without endothelium were suspended in organ chambers and the isometric tension was recorded. In some experiments, the transmembrane potential of the smooth muscle cells was recorded by electrophysiological methods. During contractions to prostaglandin F2 alpha, nebivolol induced concentration-dependent relaxations of the coronary arteries. The enantiomer, l-nebivolol, also induced comparable relaxations; however, d-nebivolol induced smaller relaxations. The relaxations induced by nebivolol and its enantiomer were significantly larger in tissues with than in those without endothelium. The differences between tissues with and without endothelium were abolished by nitro-L-arginine (3 x 10(-5) M) or methylene blue (10(-5) M). The nebivolol-induced relaxations were not affected by indomethacin (10(-5) M), phentolamine (5 x 10(-6) M), propranolol (5 x 10(-6) M), or methysergide (3 x 10(-6) M). Nebivolol at a subthreshold concentration for inducing relaxation (3 x 10(-7) M) did not significantly affect endothelium-dependent relaxations to acetylcholine but potentiated ADP-induced endothelium-dependent relaxations. The potentiation is stereoselective for l-nebivolol. Nebivolol induced a small hyperpolarization of the coronary smooth muscle with endothelium (1 mV).(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-adrenoceptor agonist mediated relaxation of rat isolated resistance arteries: a role for the endothelium and nitric oxide.

1. Isoprenaline (10(-9)-10(-5) M) relaxed rat isolated mesenteric resistance arteries pre-contracted with K+ (30-60 mM) (p EC50 (M) 8.03 +/- 0.40; maximum relaxation 66.79 +/- 2.43%, n = 7). This relaxation was partially attenuated by the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 10(-4) M). 2. The beta 2-adrenoceptor agonist, salbutamol (10(-9)-10(-5) M), produced a modest maximum relaxation (35.93 +/- 2.93%), which was not sensitive to L-NAME. 3. The beta 1-adrenoceptor agonist, dobutamine (10(-9)-10(-5) M), relaxed arteries precontracted with K+. This relaxation was abolished by L-NAME (10(-4) M) and also by propranolol (10(-6) M), but not affected by D-NAME (10(-4) M). The inhibition by L-NAME was partially reversed by L-arginine (10(-3) M). Removal of the endothelium severely attenuated relaxation to dobutamine. 4. Contractile responses to depolarizing K+ solutions were enhanced by the addition of L-NAME, and also by removal of the endothelium. 5. The above findings demonstrate that beta 1-adrenoceptor causes relaxation via NO release from the endothelium of rat mesenteric resistance arteries. In addition, contraction to K+ is modified by release of NO from the endothelium, possibly in response to tension development.     

Nitric oxide mediated venodilator effects of nebivolol.

1. Nebivolol, a selective beta 1-adrenoceptor antagonist with antihypertensive effects, has haemodynamic effects suggestive of a direct vasodilator action. 2. The dorsal hand vein technique was used to determine whether nebivolol has venodilator action in vivo in man. 3. Nebivolol and atenolol were infused into the phenylephrine preconstricted superficial hand veins of 11 healthy male volunteers. In separate studies L-NMMA (0.1 microgram min-1) was pre- and co-infused with nebivolol to determine whether nitric oxide (NO) mediated mechanisms were present. Further studies with prostaglandin F2 alpha (PGF2 alpha) preconstriction were performed to exclude an alpha-adrenergic antagonistic effect of nebivolol. Effects of L-NMMA infusion on nitroglycerin venodilation were also determined. 4. Nebivolol produced a dose dependent venodilation, (72 +/- 18% maximum), whereas atenolol produced no significant venodilation. At doses of nebivolol producing plasma concentrations comparable with plasma levels achieved after standard oral dosing (10(-13)-10(-12) mol min-1) small (14 +/- 6% and 23 +/- 8%) but significant (P < 0.05) venodilation was observed. 5. The venodilator response to nebivolol was significantly reduced by infusion of L-NMMA (maximum dilation 18% vs 72%, P < 0.01). Venodilator responses to nitroglycerin were unaffected by L-NMMA infusion. A venodilator effect to nebivolol was also seen following preconstriction with PgF2 alpha (40 +/- 20% maximum). 6. Nebivolol has nitric oxide mediated, venodilator effects in man.     

Effect of nebivolol on endothelial nitric oxide and peroxynitrite release in hypertensive animals: Role of antioxidant activity

We tested the activity of nebivolol, a beta1-selective blocker with respect to nitric oxide (NO) and peroxynitrite (ONOO) generation in the endothelium of normotensive Wistar Kyoto (WKY rats) and spontaneously hypertensive rats (SHR). The endothelial effects of nebivolol and its 2 optical enantiomers were correlated with its antioxidant activity and compared to another beta-blocker, atenolol, and 2 agonists of nitric oxide synthase (eNOS), calcium ionophore (CI) and acetylcholine (ACh). The effects of nebivolol on the bioavailability of NO and ONOO, indicators of endothelial function and dysfunction, respectively, were measured in vitro using nanosensors placed in mesenteric arteries. Compared with WKY rats, treatment of SHR vessels either with ACh (1 micromol/L) or CI (1 micromol/L) showed marked deficiencies (>40%, P < 0.01) in bioavailable NO concomitant with increased ONOO levels (>50%, P < 0.01). The [NO]/[ONOO] ratio measured after stimulation with CI was 2.77 +/- 0.05 in WKY rats and much lower (1.14 +/- 0.11) in SHR indicating significant eNOS uncoupling and endothelial dysfunction in hypertensive animals. Treatment with nebivolol (10 micromol/L) inhibited eNOS uncoupling and reduced endothelial dysfunction in SHR, as evidenced by an increase in the [NO]/[ONOO] ratio to 3.09 +/- 0.04. The basis for nebivolol activity is attributed to its unique membrane interactions as determined by small-angle x-ray diffraction, as well as its antioxidant activity at nanomolar to micromolar levels. The antioxidant effects of nebivolol and its enantiomers were not reproduced by atenolol. These results demonstrate that nebivolol inhibits endothelial dysfunction through a potent antioxidant mechanism attributed to its physicochemical interactions with the membrane, independent of beta1-blockade activity.     

Endothelial modulation of vasoconstrictor responses to endothelin-1 in human placental stem villi small arteries.

1. The aim of this study was to assess the role of endothelial cells in the modulation of vasocontractile responses to endothelin-1 (ET-1) of human placental vasculature. 2. Isolated stem villi small arteries (diameter = 170-250 microns) were obtained from healthy parturients who underwent caesarean surgery during the 39th week of pregnancy for cephalo-pelvic disproportion. Isometric tension was measured in vascular rings mounted in a myograph system and challenged with ET-1 (10(-12) to 10(-6) M). 3. The vasocontractile response to ET-1 was significantly (P < 0.001) increased in endothelial-denuded (active tension = 1156 +/- 214 mN mm-1) as compared with endothelial-preserved vascular rings (active tension = 458 +/- 48 mN mm-1). This difference was significantly (P < 0.05) but only partly abolished by the NO synthase inhibitor N omega-nitro-L-arginine (L-NOARG, 10(-4) M). 4. In endothelial-preserved rings submaximally precontracted with 5-hydroxytryptamine (10(-6) M), ET-1 (10(-12) to 10(-9) M) induced dose-dependent relaxation (maximum relaxation = 70 +/- 7%) at 10(-9) M, which was followed, at higher doses (10(-8) to 10(-6) M), by a contraction. In contrast, no relaxation was seen in endothelial-denuded rings. The relaxation in rings with endothelium was significantly (P < 0.001) reduced by L-NOARG (10(-4) M. Moreover, it was totally abolished by combined pretreatment with L-NOARG (10(-4) M) and the sulphonylurea glibenclamide (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of nitric oxide in the non-adrenergic non-cholinergic neurotransmission of horse deep penile arteries: role of charybdotoxin-sensitive K(+)-channels.

1. The involvement of nitric oxide (NO) and the signal transduction mechanisms mediating neurogenic relaxations were investigated in deep intracavernous penile arteries with an internal lumen diameter of 600-900 microns, isolated from the corpus cavernosum of young horses. 2. The presence of nitric oxide synthase (NOS)-positive nerves was examined in cross and longitudinal sections of isolated penile arteries processed for NADPH-diaphorase (NADPH-d) histochemistry. NADPH-d-positive nerve fibres were observed in the adventitia-media junction of deep penile arteries and in relation to the trabecular smooth muscle. 3. Electrical field stimulation (EFS) evoked frequency-dependent relaxations of both endothelium-intact and denuded arterial preparations treated with guanethidine (10(-5) M) and atropine (10(-7) M), and contracted with 10(-6) M phenylephrine. These EFS-induced relaxations were tetrodotoxin-sensitive indicating their non-adrenergic non-cholinergic (NANC) neurogenic origin. 4. EFS-evoked relaxations were abolished at the lowest frequency (0.5-2 Hz) and attenuated at higher frequencies (4-32 Hz) by the NOS inhibitor, NG-nitro-L-arginine (L-NOARG, 3 x 10(-3) M). This inhibitory effect was antagonized by the NO precursor, L-arginine (3 x 10(-3) M). NG-nitro-D-arginine (10(-4) M) did not affect the relaxations to EFS. 5. Incubation with either the NO scavenger, oxyhaemoglobin (10(-5) M), or methylene blue (10(-5) M), an inhibitor of guanylate cyclase activation by NO, caused significant inhibitions of the EFS-evoked relaxations, and while oxyhaemoglobin abolished the relaxations to exogenously added NO (acidified sodium nitrite, 10(-6) - 10(-3) M), there still persisted a relaxation to NO of 24.4 +/- 5.1% (n = 6) in the presence of methylene blue. 6. Glibenclamide (3 x 10(-6) M), an inhibitor of ATP-activated K(+)-channels, did not alter the relaxations to either EFS-stimulation or NO, while the blocker of Ca(2+)-activated K(+)-channels, charybdotoxin (3 x 10(-8) M), caused a significant inhibition of both the electrically-induced relaxations and the relaxations to exogenously added NO. Furthermore, charybdotoxin blocked relaxations induced by the cell permeable analogue of cyclic GMP, 8-bromo cyclic GMP (8 Br-cyclic GMP). 7. These results suggest that relaxations of horse deep penile arteries induced by NANC nerve stimulation involve mainly NO or a NO-like substance from nitrergic nerves. NO would stimulate the accumulation of cyclic GMP followed by increases in the open probability of Ca(2+)-activated K(+)-channels and hyperpolarization leading to relaxation of horse penile arteries.     

Impact of beta-adrenoceptor antagonists on myofilament calcium sensitivity of rabbit and human myocardium

Beta-adrenoceptor antagonists (beta-blockers) are commonly used in clinical pharmacotherapy of cardiovascular diseases. Carvedilol and nebivolol possess beneficial effects on myocardial function in situations of oxidative stress associated with intracellular calcium overload. This preservation of contractile function might be due to direct scavenging capacities or to compensation of the intracellular calcium overload through direct impact on myofilament calcium sensitivity. Accordingly, we measured the relation between calcium and force in the absence and in the presence of 10(-6) M carvedilol, nebivolol, or propranolol in skinned right ventricular trabeculae of rabbit hearts. In rabbit myocardium, nebivolol (10(-6) M) altered the pCa50% by a rightward shift (less sensitive) from 5.72 +/- 0.05 to 5.57 +/- 0.05 (p < 0.05). Maximal force development was reduced by nebivolol. In contrast, the same concentration of propranolol or carvedilol did not influence calcium sensitivity and force development. In additional experiments, we repeated this protocol in trabeculae from human failing hearts. As in rabbit trabeculae, nebivolol shifted the pCa50% by 0.16 +/- 0.04 pCa units to the right (p < 0.05). Experiments with intact rabbit trabeculae confirmed depressed contractility: when all beta-adrenoceptors were blocked by 10(-6) M propranolol, subsequent addition of 10(-6) M nebivolol reduced developed force of these muscles significantly from 3.1 +/- 0.9 to 1.7 +/- 0.4 mN/mm2. We conclude that nebivolol desensitizes cardiac myofilaments slightly, whereas neither propranolol nor carvedilol had an effect.     

Cocaine-induced relaxation of isolated rat aortic rings and mechanisms of action: possible relation to cocaine-induced aortic dissection and hypotension

Cocaine HCl is well known for its toxic effects on the cardiovascular system, but little is known about its effects on different regional blood vessels. We designed experiments to determine if cocaine HCl could influence the tension of isolated aortic rings, i.e., induce contraction or relaxation. Surprisingly, cocaine HCl (1 x 10(-5) to 6 x 10(-3) M) relaxed isolated aortic rings precontracted by phenylephrine in a concentration-dependent manner. No significant differences were found between intact or denuded isolated aortic rings (P>0.05). The maximal % relaxations of intact vs. denuded isolated aortic rings were 108.9+/-24.3% vs. 99.5+/-8.3% (P>0.05). Cocaine HCl, 2 x 10(-3) M, was found to inhibit contractions by phenylephrine; EC50s were increased (P<0.01) and Emax's were decreased (51.3+/-16.4% vs. 89.8+/-10.6%, P<0.01). A variety of amine antagonists could not inhibit the relaxant effects of cocaine HCl (P>0.05). The cyclooxygenase-1 inhibitor, indomethacin, also failed to inhibit relaxations induced by cocaine HCl (P>0.05). Neither L-arginine, NG-monomethyl-L-arginine (L-NMMA), nor methylene blue could inhibit the relaxations induced by cocaine HCl (P>0.05), suggesting cocaine HCl does not relax isolated aortic rings by inducing the synthesis or release of nitric oxide (NO) or prostanoids from either endothelial or vascular muscle cells. Inhibitors of cAMP, cGMP and protein kinase G (PKG) also failed to inhibit cocaine-induced relaxations. Cocaine HCl (1 x 10(-5) to 6 x 10(-3) M) could also relax isolated aortic rings precontracted by phenylephrine in high K+ depolarizing buffer. Surprisingly, calyculin A, an inhibitor of myosin light chain (MLC) phosphatase, inhibited cocaine-induced relaxations in a concentration-dependent manner, suggesting the probable importance of cocaine-induced MLC phosphatase activation in rat aortic smooth muscle cells. It was also found that cocaine HCl could dose-dependently inhibit Ca2+-induced contractions of isolated aortic rings in high K+-Ca2+-free buffer, suggesting that cocaine HCl may inhibit Ca2+ influx and/or intracellular release.     

Nitric oxide and relaxation of pig lower urinary tract.

1. We studied the non-adrenergic, non-cholinergic (NANC) nerve-mediated relaxation induced by electrical stimulation in pig isolated lower urinary tract smooth muscle, and the possible involvement of the L-arginine (L-ARG)/nitric oxide (NO) pathway in this response. 2. Trigonal strips, precontracted by noradrenaline (NA), carbachol or endothelin-1 (ET-1), relaxed frequency-dependently in response to electrical stimulation. Maximum relaxation was obtained at 6-8 Hz, and amounted to 56 +/- 2%, 77 +/- 3% and 62 +/- 6% of the agonist-induced tension in preparations contracted by NA, carbachol, or ET-1, respectively. Exposure to NG-nitro-L-arginine (L-NOARG; 10(-7)-10(-5) M) concentration-dependently reduced the relaxant response in preparations contracted by NA. L-NOARG (10(-6) M) reduced the maximal response to 51 +/- 8% of control. L-NOARG (10(-5) M) abolished all relaxation, and unmasked a contractile component; D-NOARG had no effect. Also in trigonal preparations, where the tension had been raised by carbachol or ET-1, L-NOARG (10(-5) M) markedly reduced relaxations evoked by electrical stimulation. 3. In trigonal preparations contracted by NA, maximal relaxation was increased after pretreatment with L-ARG (10(-3) M), and the inhibitory effect of L-NOARG (10(-6) M) was prevented. Incubation of the trigonal strips with methylene blue had no effect on relaxations elicited at frequencies less than 6 Hz, but a small inhibition was observed at higher frequencies. 4. Administration of NO (present in acidified solution of NaNO2) induced concentration-dependent relaxations in trigonal preparations contracted by NA, carbachol, or ET-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protective effects of nebivolol and reversal of endothelial dysfunction in diabetes associated with hypertension

This study aims to decipher the potential effects of nebivolol in prevention and/or regression of renal artery dysfunction in diabetes associated with hypertension. Renal arteries were isolated from 80 male mice divided into four experimental groups: (i) group D: diabetics, at 2 months since streptozotocin injection; (ii) group Din: mice that at the initiation of streptozotocin diabetes were treated with 10 mg/kg b.w./day nebivolol for 2 months, to test for the potential prevention of vascular dysfunction; (iii) group Dfin: mice that after 2 months of diabetes were treated daily with 10 mg/kg b.w./day nebivolol for additional 2 months, in order to follow the possible regression of the dysfunction, and (iv) controls (C), age-matched healthy animals. The following measurements were performed: arterial blood pressure, plasma glucose concentration, and the vascular reactivity of the renal arteries in response to noradrenaline (10(-4) M), acetylcholine (10(-4) M) and sodium nitroprusside (10(-4) M). To assess the molecular mechanisms involved in the reactivity of the renal artery, the contribution of mitogen-activated protein kinase (MAP kinase) pathway and of L-type voltage gated Ca(2+) channels (in the contractile response to noradrenaline), of nitric oxide (NO) and Ca(2+) activated K(+) channels (in the endothelium-dependent vasodilator response), and of cGMP (in the endothelium-independent vasodilator response) was examined by exposing the arteries to corresponding inhibitors, and by using myograph and patch-clamp techniques, immunoblotting and NO assays. Results showed that, group D was characterized by hyperglycemia (blood glucose concentration: 136.66 +/- 4.96 mg/dl, a value approximately 65% increased compared to group C) and hypertension (systolic blood pressure: 145.66 +/- 5.96 mm Hg, a value approximately 34% increased compared to group C). Compared to group D, group Din was characterized by diminished blood glucose concentration ( approximately 1.6 fold), reduced systolic and diastolic blood pressure ( approximately 1.3 fold) and heart rate ( approximately 1.6 fold), as well as by increased contractile response of the renal artery to noradrenaline ( approximately 1.84 fold) and of the impeded vasodilator response to acetylcholine ( approximately 1.81 fold) and sodium nitroprusside ( approximately 1.42 fold). Together, these effects demonstrate that administration of 10 mg/kg b.w./day nebivolol at the moment of diabetes induction has preventive effects, ameliorating diabetes dysfunctions. Compared to group D, group Dfin was characterized by diminished glucose concentration ( approximately 1.3 fold), reduced systolic and diastolic blood pressure and heart rate (both approximately 1.2 fold), and by augmentation of contractile response of the renal artery to noradrenaline ( approximately 1.62 fold) and of vasodilator response to acetylcholine ( approximately 1.13 fold) and sodium nitroprusside ( approximately 1.19 fold). These effects assess that administration of 10 mg/kg b.w./day nebivolol after 2 months of diabetes contributes to regression of diabetes-associated dysfunctionalies. Nebivolol influenced the molecular mechanisms involved in renal artery reactivity in diabetic and hypertensive mice: it increased the NO production and endothelial NO synthase (eNOS) protein expression, decreased the expression of proportional, variant protein in L-type calcium channels and Ca(2+) activated K(+) channels, and diminished the MAP kinase activity. The reported data suggest that nebivolol may offer additional vascular protection for treating diabetes associated with hypertension.     

Vasorelaxant effect of mexiletine in mesenteric resistance arteries of rats.

1. The vascular action of mexiletine, a class Ib antiarrhythmic agent, was investigated in the mesenteric resistance arteries of rats. 2. The second order branch of the mesenteric artery was cut into rings and changes in isometric tension were recorded. 3. Mexiletine (10(-6) -10(-3) M) evoked concentration-dependent, endothelium-independent relaxations in arteries contracted with noradrenaline. 4. Mexiletine (10(-4) M) did not affect the contraction induced by noradrenaline in Ca(2+)-free solution, while the compound inhibited the contraction induced by CaCl2 in noradrenaline-activated arteries. 5. The relaxation induced by mexiletine was less pronounced in arteries contracted with high KCl than in those contracted with noradrenaline. 6. Mexiletine induced identical relaxations in arteries contracted with noradrenaline in high KCl solution containing verapamil and in Krebs solution. 7. Thus, mexiletine induces relaxations by inhibiting transmembrane Ca2+ movement, but not Ca2+ release from the intracellular store site in mesenteric resistance arteries of rats. It is speculated that mexiletine possesses greater inhibitory effects against noradrenaline-activated, verapamil-insensitive (receptor-operated) Ca2+ channels than against verapamil-sensitive (voltage-dependent) channels.     

Nebivolol inhibits human aortic smooth muscle cell growth: effects on cell cycle regulatory proteins

An enhanced vasoconstriction and vascular smooth muscle cell proliferation are involved in pathogenesis of hypertension. Beta3-blockers are effective for treatment of hypertensive patients. Recently the new beta1-receptor blocker nebivolol showed a different hemodynamic profile from those of other classic beta-blockers. In this study we hypothesized that nebivolol may also have different effects on smooth muscle cell proliferation compared with other beta-blockers such as atenolol. Human aortic smooth muscle cells (SMCs) were cultured, and cell growth was determined by increase in cell number. Growth-signaling molecules such as mitogen-activated protein kinase (p42mapk) and S6-kinase (p70S6K) and cell-cycle regulatory proteins (i.e., Cdk2, p27Kip1, and pRb) were analyzed by immunoblotting. In cultured human aortic SMCs, cell number was markedly increased in response to 5% fetal calf serum (FCS) over 6 days (87 +/- 11 x 10(3)/well), which was inhibited by nebivolol (10(-8)-10(-5) M; 25 +/- 2 x 10(3)/well; n = 6; p < 0.05), but not by atenolol. 5% FCS activated p42mapk, S6K, and Cdk2, but downregulated p27Kip1 and hyperphosphorylated pRb. Nebivolol prevented Cdk2 activation without influencing p42mapk, S6K, pRB, and p27Kip1. Thus, the new beta1-blocker nebivolol exhibits antiproliferative effect on human SMC through inactivation of Cdk2. This effect of nebivolol may have advantages over other beta-blockers in treatment of patients with cardiovascular disease.     

Nonadrenergic, noncholinergic responses of the human colon smooth muscle and the role of K+ channels in these responses.

The aim of the present study was to investigate the possible role of nitric oxide (NO) as a nonadrenergic, noncholinergic (NANC) mediator in human colon smooth muscle in vitro and to examine its possible interactions with K+ channels. In the presence of atropine (10(-6) M) and guanethidine (10(-5) M), electrical field stimulation (EFS, 1-10 Hz, 0.3 msec, 50 V) for 10 sec induced relaxations which were inhibited by tetrodotoxin (10(-6) M). In the presence of NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), relaxations induced by EFS at 1, 2, 4, 8 and 10 Hz were reduced by 38.7 +/- 4.3, 31.5 +/- 3.8, 54.3 +/- 5.4, 59.8 +/- 4.5 and 68.6 +/- 5.3%, respectively. The relaxations inhibited by L-NAME were restored by the preincubation of L-arginine (L-ARG, 10(-3) M) at all frequencies tested. D-Arginine (D-ARG, 10(-3) M) had no effect. Tetraethylammonium (TEA, 10(-4) M) or glibenclamide (10(-6) M) significantly decreased the relaxations induced by EFS. Exogenously applied sodium nitroprusside caused concentration-dependent relaxation with maximum relaxation observed with 10(-3) M. TEA (10(-4) M) and glibenclamide (10(-6) M) significantly depressed the maximum response to sodium nitroprusside. In conclusion, our data indicate that NO is involved in NANC nerve-mediated relaxation in the human colon smooth muscle and the relaxant responses to endogenously released or exogenously applied NO are mediated, in part, by activation of calcium-dependent and ATP-sensitive K+ channels.     

NO and KATP channels underlie endotoxin-induced smooth muscle hyperpolarization in rat mesenteric resistance arteries

1 Smooth muscle membrane potential and tension measurements were made in isolated mesenteric resistance arteries from rats exposed to bacterial endotoxin (lipopolysaccharide, LPS; 10 mg kg(-1), i.p.) for 3 h to mimic septic shock syndrome. 2 Over this period, rats developed an endotoxaemic response, assessed in vivo as a 41+/-4 mmHg drop in mean blood pressure, vascular hyporeactivity to noradrenaline (1 microg kg(-1), i.v.) and a significant increase in core body temperature. 3 In mesenteric small resistance arteries from these rats (o.d. 180 - 240 microm), phenylephrine (0.01-3 microm)-evoked contraction was not altered when compared with arteries from sham-operated animals, but the concentration-relaxation curve to acetylcholine (ACh; 0.01 - 3 microm) displayed a small, but significant, shift to the right. 4 The smooth muscle resting membrane potential (-70.3+/-1.6 mV) in arteries from LPS-treated rats was significantly greater than in control arteries (-55.4+/-1.2 mV), but in both cases the smooth muscle was depolarized to a similar potential by the application of N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.3 mm; -54.1+/-2.3 vs -52.4+/-2.5 mV) or glibenclamide (10 microm; -55.0+/-2.1 vs -50.4+/-2.0 mV). 5 ACh (1 microm) elicited a maximal hyperpolarization, which ranged from -14.7+/-3.2 mV (in arteries from LPS-treated rats) to -20.6+/-2.4 mV (in arteries from sham-operated rats), and was not altered by the presence of L-NAME. Levcromakalim (1 microm) increased the smooth muscle membrane potential by around -24 mV in arteries from both sets of experimental animals. 6 These results indicate that at the level of the resistance vasculature, endotoxaemia is associated with pronounced smooth muscle hyperpolarization reflecting the action of NO on KATP channels. These changes were not associated with vascular hyporeactivity or depressed endothelial cell function in vitro, suggesting that mesenteric resistance arteries may not contribute to equivalent changes in vivo.     

Effect of glucagon-like peptide-1(7-36) and exendin-4 on the vascular reactivity in streptozotocin/nicotinamide-induced diabetic rats

We investigated the vascular effects of glucagon-like peptide-1 (GLP-1) and Exendin-4 in type 2 diabetic rat aortae. Studies were performed in a normal control group (NC) (0.2 ml i.p. saline, n = 10), streptozotocin (STZ)/nicotinamide diabetic control group (DC) (a single dose of 80 mg/kg STZ i.p. injection 15 min after administration of 230 mg/kg nicotinamide i.p.), GLP-1 (GLPC) control group (1 microg/kg twice daily i.p. for 1 month, n = 10), Exendin-4 control group (EXC) (0.1 microg/kg twice daily i.p. for 1 month, n = 10), GLP-1-treated diabetic group (GLPT) (1 microg/kg twice daily i.p. for 1 month, n = 10), and Exendin-4-treated diabetic group (EXT) (0.1 microg/kg twice daily i.p. for 1 month, n = 10). One month of GLP-1 and Exendin-4 treatment significantly decreased the blood glucose levels of diabetic rats (113 +/- 2 mg/dl, p < 0.001, and 117 +/- 1 mg/dl, p < 0.001, respectively versus 181 +/- 9 mg/dl in the DC group). Sensitivity (pD2) and maximum response (% Max. Relax) of acetylcholine-stimulated relaxations in the DC group (pD2: 6.73 +/- 0.12 and 55 +/- 6, respectively) were decreased compared with the non-diabetic NC group (pD2: 7.41 +/- 0.25, p < 0.05, and 87 +/- 4, p < 0.01). Treating diabetic rats with GLP-1, pD2 values and with Exendin-4, Max. Relax %values of aortic strips to acetylcholine returned to near non-diabetic NC values (pD2: 7.47 +/- 0.15, p < 0.05, and 87 +/- 3, p < 0.01, respectively). Maximal contractile responses (Emax) to noradrenaline in aortic strips from the diabetic DC group (341 +/- 27 mg tension/mg wet weight) were significantly decreased compared with the non-diabetic NC (540 +/- 66 mg tension/mg wet weight, p < 0.001) and the GLPT group (490 +/- 25 mg tension/mg wet weight, p < 0.05). There were no significant differences in pD2 values of aortic strips to noradrenaline from all groups. Emax to KCl in aortic strips from the DC group (247 +/- 10 mg tension/mg wet weight, p < 0.01) was significantly decreased compared with non-diabetic NC group (327 +/- 26 mg tension/mg wet weight). Treating diabetic rats with GLP-1 (GLPT), Emax values of aortic strips to KCl returned to near non-diabetic NC values (271 +/- 12 mg tension/mg wet weight). GLP-1 and (partially) Exendin-4 treatment could improve the increased blood glucose level and normalize the altered vascular tone in type 2 diabetic rats.     

Modulation of contractions to ergonovine and methylergonovine by nitric oxide and thromboxane A2 in the human coronary artery

This study explored the modulatory effects of nitric oxide and thromboxane A2 on contractions to ergonovine and methylergonovine in human coronary arteries. To elucidate the different role of nitric oxide synthase in the response to the ergot alkaloids, the serotonin (5-HT) receptors involved in nitric oxide synthase in the response to the ergot alkaloids, the 5-HT receptors involved in nitric oxide release and the contraction of the vascular smooth muscle were characterized with more selective 5-HT-receptor agonists and antagonists. Rings of human coronary arteries from explanted hearts were suspended in organ chambers for isometric tension recording. After testing for contractile (potassium chloride, 60 mM) and endothelial function (substance P, 10(-8) M), respectively, they were exposed to ergot alkaloids or other agonists in the absence or presence of U 46619 (10(-9) M), or nitro-L-arginine (10(-4) M), or both. Ergonovine and methylergonovine were comparable, weak vasoconstrictors in untreated preparations. Contractions to ergonovine were augmented by U 46619, but not by nitro-L-arginine. Contractions to methylergonovine were augmented only by combining U 46619 and nitro-L-arginine. Serotonin and methylergonovine, but not ergonovine, elicited endothelium-dependent, nitric oxide-mediated relaxations. Nonselective 5-HT(1B/1D)-receptor stimulation caused both contractions and relaxations; selective 5-HT1B stimulation caused relaxations only. In the human coronary artery, contractions to ergonovine are not dependent on NO release but are synergistically augmented by thromboxane. Methylergonovine causes similar effects on the vascular smooth muscle, but contractions are inhibited by the release of NO from the endothelium. The 5-HT receptor on the endothelium appears to be different from the receptor on the vascular smooth muscle, which mediates the contractile response to the ergot alkaloids.     

Sodium nitroprusside regulates the relaxation of the longitudinal muscle in the gut

Nitric oxide (NO) has been shown to mediate nonadrenergic-noncholinergic relaxation in gastrointestinal (GI) smooth muscle cells. As GI smooth muscles relaxations are partly dependent on NO, we decided to investigate the effect of sodium nitroprusside (SNP) on the longitudinal muscle contraction of the isolated guinea pig ileum. Increasing concentrations of SNP (10(-10)M, 10(-9)M, 10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M) reduced ileum contractions stimulated by electrical stimulation (ES) (8-76%; p < 0.05) and by acetylcholine (Ach) (23-62%; p < 0.05) significantly and in a concentration-dependent manner. Furthermore, treatment with an inhibitor of the soluble guanylate cyclase, methylene blue (10 mM), antagonized significantly the relaxing effect of SNP (0-39%; p < 0.05, p < 0.01, p < 0.001 for ES- and 4-27%; p < 0.05 for Ach-induced contractions). The results show that treatment with 1 microM manganese-containing superoxide dismutase (MnSOD) and 10 microM L-arginine (L-arg) caused a significant decrease in SNP induced relaxations (6-55%; p < 0.05, p < 0.001 and 2-46%; p < 0.05, p < 0.01 for ES- and 15-28%; p < 0.05, p < 0.01, p < 0.001 and 12-32%; p < 0.05, p < 0.01 for Ach-induced contractions, respectively). In conclusion, our data suggest that SNP, which releases NO, is able to depress longitudinal muscle contraction of the isolated guinea pig ileum, suggesting that exogenous application of NO inhibits intestinal contractions of smooth muscle cells and that cGMP mediates the response to NO. In addition, MnSOD and L-arg decreased the relaxing effect of SNP on the isolated ileum of the guinea pig.     

Endothelium-dependent sensory non-adrenergic non-cholinergic vasodilatation in rat thoracic aorta: involvement of ATP and a role for NO

The involvement of non-adrenergic non-cholinergic (NANC) transmitters, such as adenosine 5'-triphosphate (ATP) and nitric oxide (NO), in the neurogenic relaxation of rat thoracic aorta was investigated in vessel segments suspended for isometric tension recording by polygraph. Responses to electrical field stimulation (EFS) and exogenous vasodilator were investigated in vessels precontracted with 5-hydroxytryptamine. EFS (100 V, 2-16 Hz, for 10 s at 3-min intervals), in the presence of guanethidine (10 microM) and atropine (10 microM) produced frequency-dependent relaxations. Pretreatment with tetrodotoxin (1 microM) markedly reduced the relaxation and desensitization with capsaicin (10 microM) significantly inhibited the relaxation. Exogenously added ATP caused concentration-dependent relaxations. Mechanical removal of the endothelium significantly inhibited EFS- and ATP-induced relaxation by 30+/-3% and 37+/-2%, respectively. Pretreatment with a P1-purinoceptor antagonist, 8-phenyltheophylline (10 microM) or P2X-purinoceptor antagonist, Evans blue (10 microM) did not influence the relaxations to EFS and exogenously added ATP. In contrast, the P2Y-purinoceptor antagonist, basilen blue (100 microM) markedly reduced the relaxations to EFS by 52+/-4% in the endothelium-intact preparations. However, in the endothelium-denuded preparations and capsaicin-pretreated preparations, basilen blue did not change relaxations elicited by EFS. The NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) also significantly inhibited the relaxations to EFS and ATP by 40+/-6% and 30+/-2%, respectively, in the endothelium-intact preparations but had no effect on the relaxations in the endothelium-denuded preparations or capsaicin-pretreated preparations. In addition, the EFS-induced relaxations were also inhibited 43+/-7% by pretreatment with 1H-[1,2,4]-oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ; 1 microM), soluble guanylate cyclase inhibitor. This study suggests that the NANC nerve system is present in the thoracic aorta of rat, mediating vasodilatation by sensory nerves. ATP, as a neurotransmitter released from sensory nerves, activates P2Y-purinoceptors located on the endothelium and stimulates the NO/cyclic GMP pathway, resulting in vasodilatation.     

Alterations of cyclo-oxygenase products and NO in responses to angiotensin II of resistance arteries from the spontaneously hypertensive rat.

1. The involvement of cyclo-oxygenase (COX) products and nitric oxide (NO) in contractile responses of resistance arteries to angiotensin II (AII) were investigated in small mesenteric arteries from spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats. 2. In endothelium intact vessels, AII induced concentration-dependent responses without any significant difference between the two strains. However, removal of functional endothelium resulted in enhanced sensitivity to AII, the pD2 value increasing from 8.4 +/- 0.2 to 8.9 +/- 0.2 (P < 0.05) in WKY and from 8.2 +/- 0.1 to 8.6 +/- 0.1 (P < 0.05) in SHR (not significantly different between strains, n = 9 - 12). In addition, endothelium removal enhanced maximal contractions elicited by AII in SHR (1.4 +/- 0.1 to 2.1 +/- 0.2 mN mm-1, n = 5; P < 0.05) but not in WKY (1.0 +/- 0.1 to 1.2 +/- 0.1 mN mm-1, n = 5) vessels. 3. In the absence of functional endothelium, the COX inhibitor indomethacin (10(-5) M) reduced contractile responses elicited by AII in SHR arteries, resulting in 33 +/- 5% (n = 5) decrease in maximal contraction. However, it produced minimal if any, effect on responses of WKY vessels. In both strains, the TP receptor antagonist GR32191 B (3 x 10(-6) M) did not modify contractions elicited by AII in these conditions. 4. In the presence of functional endothelium, indomethacin (10(-5) M) almost abolished the responses to AII in both strains. GR32191 B (3 x 10(-6) M) reduced the sensitivity of WKY arteries to AII (pD2 = 8.1 +/- 0.1, P < 0.01) without any effect on maximal contraction. In SHR arteries, it markedly reduced maximal contraction (47 +/- 3.5%). 5. In both strains, the NO synthase inhibitor NG-nitro-L-arginine methy lester (L-NAME; 10(-4) M) had no effect in the absence of functional endothelium but it markedly reduced the inhibitory influence of endothelium on contractile responses to AII. Furthermore, in arteries with endothelium, it reduced the effect of both indomethacin and GR32191 B to the same level as observed in vessels without functional endothelium. 6. The results suggest that enhanced contraction caused by COX products was counteracted by enhanced relaxation caused by endothelium-derived NO in resistance mesenteric arteries of the SHR exposed to AII, compared to WKY arteries. The COX products involved in alterations of SHR responses comprised an endothelium-derived prostaglandin activating TP receptors and another nonendothelial unidentified vasoconstrictor compound which did not activate these receptors.