盐酸贝尼地平对大鼠硝酸甘油耐受性的影响及机制

目的观察盐酸贝尼地平对硝酸甘油(NTG)耐受性的影响及其机制。方法通过描记大鼠舒张反应性曲线,测出各种NTG浓度时舒张反应百分率,检测血管组织中的丙二醛(MDA)含量、一氧化氮(NO)含量、一氧化氮合酶(NOS)活性和环磷酸鸟苷(cGMP)含量。结果通过对照NTG+盐酸贝尼地平组和NTG组发现,盐酸贝尼地平可以显著减少MDA含量[(7.83±1.41)比(14.37±0.88)μmol/g蛋白,P<0.05];增加NO的含量[(78.88±1.75)比(70.30±1.74)mmol/g蛋白,P<0.05];增加cGMP含量[(200±19)比(169±13)pmol/g蛋白,P<0.05];未增加NOS的活性[(310±20)比(290±30)U/g蛋白,P>0.05]。结论盐酸贝尼地平可以通过抗氧化作用,减少氧自由基的产生,使NTG源NO灭活减少和组织中NO含量增加;增加cGMP含量和活性,从而恢复NO信号传递系统完整性。     

Prevention of nitrate tolerance by long-term treatment with statins

Recent studies have shown that statins seem to upregulate the endothelial NO synthase pathway (eNOS) and may, therefore, enhance NO availability, a direct scavenger of O₂ ^– and an inhibitor of oxidative enzymes. Methods. To assess whether the oxidative stress produced by an in vivo exposure to nitroglycerin (NTG) is attenuated by statins, 4 groups of normocholesterolemic rats were treated; group 1 received pravastatin (20 mg/kg/d p.o) and group 2 atorvastatin (10 mg/kg/d) both for 5 weeks and the last 3 days, a cotreatment with the statin plus NTG (50 mg/kg/d, s.c. injections b.i.d.); group 3 (NTG) received only NTG (50 mg/kg/d, b.i.d. for 3 days) and group 4 served as control. Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM to 0.1 mM) were determined on phenylephrine-preconstricted rings and O₂ ^– production (counts/10 s/mg) was assessed by lucigenin chemiluminescence technique. Results. In vivo NTG exposure induced a rightward shift of the concentration-response curves to NTG: the pD₂ (–log NTG concentration evoking a half maximal relaxation) was 5.8 ± 0.3 (n = 7) vs. 7.2 ± 0.2 in the control group (not exposed to NTG, n = 7) and O₂ ^– production was enhanced (1259 ± 71 vs. 787 ± 76, (n = 5) P < .05). In contrast, groups 1 (n = 7) and 2 (n = 7) behaved as the control group (pD₂ values were 7.4 ± 0.1 (n = 7) and 6.9 ± 0.1 (n = 7); O₂ ^– production was 721 ± 109 and 647 ± 121). The protective effect on nitrate tolerance disappeared when L-NAME (an eNOS inhibitor, 100 mg/kg/d) was co-administered with NTG in groups 1 and 2. Incubation of aortic rings with NAD(P)H (100 M) also impaired the protective effect of both statins. Moreover, before NTG exposure, aortic cGMP content, reflecting EDNO availability, was significantly enhanced in group 1 (P < .05 vs. control). Conclusion. Long-term statin treatment protects against nitrate tolerance by counteracting NTG-induced increase in O₂ ^– production. Both eNOS pathway and NAD(P)H oxidases seem to be involved in this protective mechanism.     

Effect of nitrate tolerance and dipyridamole on the response to SIN1 in the human isolated saphenous vein

The relaxant effects of nitroglycerin (NTG) and SIN1 on human vena saphena magna were studied in vitro. Nitrate tolerance was produced after incubation of the preparation with nitroglycerin (NTG 10 microM for 10 minutes). Vessels precontracted by serotonin (0.25 microM) and made tolerant to NTG exhibited a slight but significant shift (p less than 0.01) to the right of the dose-response curve to SIN1 (EC50 increased from 1.12 +/- 0.21 microM to 2.74 +/- 0.32 microM). The maximal relaxation was unaltered. On the contrary, there was a marked attenuation of the maximal relaxation to NTG in the nitrate-tolerant preparation (maximal relaxation decreased from 73 +/- 2% to 35 +/- 1%). Dipyridamole, a phosphodiesterase (PDe) inhibitor, significantly potentiated the responses to SIN1 on control rings (EC50 = 57.1 +/- 1.8 nM), and on NTG-tolerant rings it reversed the responsiveness to SIN1 (EC50 = 88.9 +/- 9.2 nM), which suggests that nitrate tolerance may be partially due to an increase in PDe activity. In conclusion we have demonstrated a slight cross-tolerance between SIN1 and NTG on human vena saphena magna. Nevertheless, after induction of in vitro NTG tolerance, the attenuation of responses to SIN1 is much less pronounced that the alteration of NTG relaxations.     

Relaxant properties mediated by nitroglycerin in aortic coarctation hypertensive rats

 Nitroglycerin-mediated vasorelaxation is chiefly attributed to the cyclic guanosine monophosphate (cGMP)-dependent pathway, and partly to the cGMP-independent pathway via calcium-activated K⁺ channels (K_Ca). To investigate whether chronic hypertension alters responses of vascular smooth muscle to vasoactive agonists, we determined nitroglycerin-mediated relaxation of aortic rings from coarctation hypertensive rats. Banding the abdominal aorta above the renal arteries for 4 weeks elevated blood pressure and caused cardiac hypertrophy by 49%. In response to nitroglycerin, the relaxation of aortic rings precontracted with 10⁻⁷ M norepinephrine was lower in the banded group than in the sham-operated group. Methylene blue, a guanylate cyclase inhibitor, suppressed a greater part of nitroglycerin-mediated relaxation and reached similar levels of relaxation in the two groups. Charybdotoxin, a specific K_Ca channel blocker, also suppressed the relaxation by about 40% in the aortic rings from sham-operated animals, but not in those from the banded group. The response to charybdotoxin was markedly diminished or virtually eliminated in the banded group in the presence or absence of methylene blue. The combination of charybdotoxin and methylene blue nearly abolished nitroglycerin-mediated relaxation in the sham-operated group, whereas nitroglycerin-mediated relaxation was seen to remain in the banded group. These results indicate that the involvement of cGMP-independent K_Ca channels in nitroglycerin-mediated relaxation disappeared after the development of hypertension produced by aortic coarctation. Received: May 20, 2002 / Accepted: July 27, 2002 Correspondence to K. Okumura     

Lipoproteins as factors in vessel tone and reactivity modulation

Summary Lipoprotein fractions purified from fresh canine plasma — very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) — produce relaxation of isolated ring segments of coronary arterics of canine (porcine and human) and rabbit aorta precon-tracted with high potassium, 30 mM, or phenylephrine, 10^–6 M, respectively. Lipoproteins do not possess high species specificity and produce relaxation of arteries of other species which differ quantitatively. There was no marked endothelium-dependent relaxation induced by animal lipoproteins although human LDL produced endothelium-dependent relaxation of rabbit aortic rings. Canine LDL and HDL decreased endothelium-dependent relaxation of rabbit aortic rings produced by acetylcholine (10^–9–10^–6 M), which was more pronounced when the LDL and HDL were present in the organ bath. In this case, LDL and HDL reduced the rate of development and amplitude of contraction produced by phenylephrine, 10^–6 M. We suggest that lipoprotein effects may result from blockade of some steps in electromechanical coupling or from inhibition of voltage- and receptor-operated Ca-channels of vessel smooth muscle cell membrane.     

Hydroxyl Radical: A Possible Mediator of Nitroglycerin-Induced Vascular Relaxation

The endothelium-derived relaxing factor, nitric oxide (NO·), is involved in acetylcholine (Ach) and nitroglycerin (NTG)-induced relaxation of vascular smooth muscle. It is known that NO· interacts with the superoxide anion (O₂·^–) at physiological pH and this interaction leads to the generation of hydroxyl radicals (·OH). Hydroxyl radicals are known to induce dilation and act as mediators in acetylcholine (Ach)-induced relaxation of rabbit aortic smooth muscle. Thus, it was hypothesized that ·OH may be involved in NTG-induced relaxation. To test this hypothesis we investigated the effect of NTG on norepinephrine (NE)-precontracted isolated rabbit aortic rings in the absence and/or presence of O₂·^– [superoxide dismutase (SOD)] and ·OH scavengers [dimethylthiourea (DMTU) or mannitol]. Superoxide dismutase, DMTU, and mannitol markedly reduced NTG-induced relaxation of isolated rabbit aortic rings. Superoxide dismutase produced a 32% reduction in NTG-induced vasodilation. Pretreatment with DMTU and/or mannitol reduced NTG-induced relaxation by 56% and 49%, respectively. These results suggest that ·OH may be a mediator involved in NTG-induced vascular relaxation of rabbit aorta.     

Effects of a new 1,3-thiazole Derivative ZSY-39 on force of contraction and cyclic AMP content in canine ventricular muscle

Summary A newly synthesized 1,3-thiazole derivative ZSY-39 increased the force of contraction in a concentrationdependent manner in association with elevation of tissue cyclic AMP levels in the isolated canine ventricular trabeculae electrically driven at 0.5 Hz at 37°C. ZSY-39 shortened the duration of isometric contractions mainly by abbreviation of the relaxation time. The maximal response to and EC₅₀ of ZSY-39 were 0.7 (isoproterenol=1.0) and 4.6×10^–5 M. Bupranolol (3×10^–7 M) did not affect the positive inotropic effect of ZSY-39. The time course of increases in the force of contraction induced by ZSY-39 (10^–4 M) coincided with that of cyclic AMP accumulation. The concentration-response curve for the increase in the force of contraction produced by ZSY-39 was superimposable on that of the elevation of cyclic AMP levels. Carbachol (3×10^–6 M) shifted the concentration-response curve for the increase in force by ZSY-39 to the right and downward, and decreased the accumulation of cyclic AMP induced by ZSY-39 (10^–4 M). ZSY-39 (10^–5 M) enhanced significantly the positive inotropic effect of isoproterenol. The relationship between the force of contraction and cyclic AMP levels after the administration of ZSY-39 was not modified by the addition of carbachol or isoproterenol. These findings indicate that cyclic AMP plays an important role in the positive inotropic effect of ZSY-39 on canine ventricular muscle.     

Involvement of cyclo-oxygenase-generated vasodilating eicosanoid(s) in addition to nitric oxide in endothelin-1-induced endothelium-dependent vasorelaxation in guinea pig aorta

Summary This study investigates the vasodilatory effects of endothelin-1 (ET-1) in isolated guinea pig aortic rings in vitro. Cumulative dose-response curves to ET-1 were constructed and ET-1 actions on prostaglandin F₂ (PGF₂)-precontraction were studied in both endothelium-intact and endothelium-denuded preparations, in the presence or absence of a cyclooxygenase inhibitor (indomethacin) and/or nitric oxide inhibitors (N^G-nitro-L-arginine methyl ester and hemoglobin). In endothelium-intact preparations, pretreatment with indomethacin (10^–5M, 30 min), alone or in combination with N^G-nitro-L-arginine methyl ester (L-NAME, 10^–4M), significantly augmented the constrictive responses to ET-1, whereas indomethacin, L-NAME, and hemoglobin (10^–5M) had no significant effects in the endothelium-denuded preparations. Furthermore, in PGF₂-precontracted, endothelium-intact preparations, ET-1, at a dose of 10^–9M, induced initial relaxation followed by subsequent contraction, while it only contracted the endothelium-denuded preparations. The initial relaxation was abolished by indomethacin, but not by L-NAME or hemoglobin. In addition, this relaxation was not inhibited by a specific ET_A receptor antagonist, BQ-123 (6 × 10^–6M). In addition to the involvement of nitric oxide, these results show the involvement of cyclo-oxygenase-generated vasodilating eicosanoid(s) derived from endothelium in ET-1-induced vasorelaxation in guinea pig aorta in vitro. The results also indicate that this vasorelaxation is mediated by ET_B receptor activation.This study was financially supported by Toyobo Biotechnology Foundation, and by Grants-in-Aid for scientific research from the Ministry of Education, Science and Culture of Japan (project numbers: 3770533 and 04670571) and the Ministry of Health and Welfare of Japan.     

Effects of 5-hydroxytryptamine and adenosine on the canine isolated cerebral resistance vessels

We investigated the effects of 5-hydroxytryptamine (5-HT), adenosine, and adenine nucleotides on the canine isolated cerebral resistance arteries ranging from 300 to 500 µm in diameter. Addition of 5-HT and 5-carboxami-dotryptamine (5-CT) produced concentration-dependent contractions in the resistance arteries. Methiothepin (10^–8 M-10^–6 M) caused a parallel shift to the right of the dose-response curves for 5-HT and 5-CT. Neither ketanserin (10^–7 M, 10^–6 M) nor [(1H, 5H)-8-methyl-8 azabicyclo [3,2,1] oct-3-y1]1H-indole-3-carboxylate hydrochloride (ICS 205–930 10^–7M, 10^–6M) caused any significant shift of the dose-response curve for 5-HT. Adenosine, ATP, ADP, and AMP caused concentration-dependent relaxations in the resistance arteries following contraction with PGF2. The adenosine analogs also caused a dose-dependent relaxation of the arterial segments contracted by PGF2. The decreasing order of potency of the agonists was 5-N-ethylcarboxamido adenosine (NECA)>adenosine L-diasteroisomers of phenylisopropyl adenosine (L-PIA). The relaxant response to NECA was competitively antagonized by 8-phenyltheophylline. The pretreatment with 3×10^–5 M N^G-monomethyl-L-arginine (L-NMMA) or 5×10^–5 M aspirin caused no significant effect on the adenosine-induced relaxation in the resistance arteries. These results suggest that 5-HT produces contraction of the canine isolated cerebral resistance arteries, which is mainly mediated via 5-HT₁-like receptors. Adenosine causes an endothelium-independent relaxation of the resistance arteries through the activation of adenosine A₂-receptors.     

Preclinical Profile of PF9404C,a Nitric Oxide Donor with β Receptor Blocking Properties

PF9404C ((2'S),(2S)‐3‐isopropylamine, 1‐[4‐(2,3‐dinitroxy)propoxymethyl]‐phenoxy‐2′‐propranol) is the S‐S diesteroisomer of a novel blocker of β‐adrenergic receptors with vasorelaxing properties. It causes a concentration‐dependent relaxation of rat aorta helical strips precontracted with 10^?6 M norepinephrine (NE; IC₅₀ 33 nM). It is equipotent to nitroglycerin (NTG; IC₅₀ 49 nM), but much more potent than isosorbide dinitrate (ISD; IC₅₀ 15,000 nM). In rat aorta smooth muscle cells, at 10 μM, PF9404C increased the formation of cGMP from 3 pmol/mg protein in basal conditions to 53 pmol/mg protein, suggesting that the mechanism of its vasorelaxing effects involves the slow generation of NO. This is supported by the facts that (i) ODQ (a blocker of guanylate cyclase) inhibited the vasodilatory effects of PF9404C; and (ii) PF9404C generates NO, as indirectly measured by the Griess reaction. In the electrically driven guinea pig left atrium, PF9404C blocks the inotropic effects of isoproterenol in a concentration‐dependent manner. Its IC₅₀ (30 nM) was similar to that of S‐propranolol (22.4 nM) and lower than that of metoprolol (120 nM) or atenolol (192 nM). The β adrenergic ligand (‐)‐[³H]‐CGP12177 (4‐[3‐[(1,1‐dimethylethyl)amino]‐2‐hydroxypropoxy]‐1,3‐dihydro‐2H‐benzimidazol‐2‐one hydrochloride) (0.2 nM) is displaced from its binding sites in rat brain membranes with a K_i of 7, 17, 170, and 1200 nM for PF9404C, S‐(‐)propranolol, metoprolol, and atenolol, respectively. PF9404C blocks ⁴⁵Ca²⁺ entry into bovine adrenal chromaffin cells induced by direct depolarization with 70 mM K⁺ or by the nicotinic agonist dimethylphenylpiperazinium (DMPP). PF9404C exhibits about 3‐fold higher potency than NTG to relax the majority of the vessels studied, especially when they were contracted with K⁺, and shows a certain selectivity of action for the renal artery. It produces auto‐tolerance that is ca. 20‐fold less pronounced than that observed with NTG. Cross‐tolerance in preparations pre‐exposed to PF9404C and later relaxed with NTG, was much greater than auto‐tolerance. This makes PF9404C a useful pharmacological tool for the development of novel NO‐donor compounds with a lesser degree of vascular tolerance than those currently available.     

VIP antagonists enhance excitatory cholinergic neurotransmission in the human airway

It has been reported that a low concentration of exogenously applied vasoactive intestinal peptide (VIP) suppresses the release of acetylcholine (ACh) from vagus nerve terminals in the ferret and feline trachea. There has been, however, no documentation of the prejunctional action of VIP in the human airway. We observed the effects of VIP and VIP antagonists on cholinergic excitatory neuro-effector transmission in the human bronchus to study the possible role of endogenous VIP on excitatory neurotransmission. In the human bronchus, VIP (10^–10 to 10^–7 M) showed no effect on either the contractions evoked by electrical field stimulation (EPS) or those evoked by ACh. To investigate the possible role of endogenous VIP on the human bronchus, we observed the effects of the VIP antagonists [4-Cl-D-Phe⁶,Leu¹⁷]-VIP and [Ac-Tyr¹,D-Phe²-]-GRF(1–29)-NH₂ on excitatory neuroeffector transmission. Both VIP antagonists (10^–8 M) significantly enhances the contractions evoked by EFS without affecting the ACh sensitivity of smooth muscle cells. These results indicate that VIP antagonists have a prejunctional action that enhances excitatory neurotransmission. This study suggests that endogenous VIP may suppresses ACh release from the vagus nerve terminals in the human airway. It is also suggested that exogenously applied VIP may be inactivated by some mechanism in the human airway. Offprint requests to: H. Aizawa.     

Attenuated inhibition of adrenergic contraction by nitric oxide in injured guinea pig femoral artery

Summary The present study aimed to examine the altered modulation of adrenergic contraction by nitric oxide and sensory neuropeptides in balloon-injured muscular artery. A guinea pig femoral artery (GPFA) was injured by a newly developed silastic microballoon catheter. The contralateral GPFA served as the control. The studied GPFAs consisted of six groups; control (C) and injured (I) GPFA, isolated at 0 days, and 2 and 8 weeks after injury (C0, I0, C2, I2, C8, and I8). Isometric tension was measured in the presence of indomethacin (10^–5M), to exclude effects of cyclo-oxygenase-generated eicosanoids. Endothelial removal with the catheter was confirmed by histological examination. In each group, except for I0, N^G-nitro-L-arginine methyl ester (l-NAME, 10^–6M) induced significant augmentation of perivascular nerve stimulation (PNS)-evoked adrenergic contraction, which was blocked byl-arginine (3 × 10^–4M). The degree ofl-NAME augmentation in I8 was significantly smaller than that in C8 and I2. Capsaicin (10^–6M) did not significantly affect PNS-contraction in any group, indicating that there was no sensory neuropeptide involvement in this contraction. In I8, acetylcholine (10^–6M)-induced relaxation after noradrenaline (10^–5M)-precontraction was significantly smaller than that seen in the other groups, except for I0, which was lacking in acetylcholine-induced relaxation. Histologically, injured GPFAs showed progressive intimal thickening. The present findings thus showed attenuated nitric oxide-mediated inhibition of adrenergic contraction, accompanying intimal thickening, in balloon-injured muscular artery, 8 weeks after injury.     

Cyclic GMP-Mediated Activation of a Glibenclamide-Sensitive Mechanism in the Rabbit Sphincter of Oddi

We investigated whether glibenclamide-sensitive potassium channels are involved in cyclic GMP (cGMP)-mediated relaxation of the rabbit Oddi's sphincter. Changes in isometric tension were measured in the presence of atropine (1 M) and guanethidine (4 M). Concentration–response curves for nitroglycerin, vasoactive intestinal polypeptide (VIP), and sodium nitroprusside (SNP) were shifted to the right in the presence of (p-chloro-d-Phe⁶, Leu¹⁷)-VIP (VIPa), a VIP receptor antagonist. Glibenclamide (1 M) attenuated the relaxations to VIP, nitroglycerin, or 8-bromo cGMP. In the presence of tetrodotoxin (TTX), glibenclamide attenuated relaxations to VIP without effect on those to nitroglycerin. Furthermore, nitroglycerin increased both cAMP and cGMP concentrations, however, it failed to increase the tissue cAMP concentration in the presence of TTX. VIPa also blocked the increase in content of either cyclic nucleotide. VIP increased cAMP with a TTX-sensitive increase in cGMP content. 8-Bromo cGMP (1 M) significantly increased the tissue cAMP content. This was blocked by either TTX or VIPa (both 1 M). We conclude that ATP-sensitive potassium channel (K_ATP) activation contributes to cGMP-mediated relaxation of the Oddi's sphincter of the rabbit. Activation of K_ATP results from a cyclic AMP-mediated process due to cGMP-dependent VIP release from neurons.     

Ontogeny of epithelial modulation of airway smooth muscle function in the guinea pig

The purpose of this study was to investigate the ontogeny of guinea pig airway smooth muscle (ASM) responses and the epithelial modulation of these responses. Paired tracheal rings from fetal, newborn, and adult guinea pigs were studied. One of each pair was denuded of airway epithelium (AE) by gentle rubbing. Isometric tension was measured in rings mounted in organ baths filled with ‘Krebs’ solution. Cumulative dose-response curves were generated by adding either acetylcholine (ACh) or histamine over a concentration range of 1 0^?8?10^?4 M. Significant agent-specific, age-related differences in maximal contraction were seen for both ACh and histamine in intact tissues (Ach: for fetus 66.7 ± 6.2 × 10^?2 g/mg wet wt, for newborn 51.4 ± 6.2, for adult 29.3 ± 2.6; histamine: for fetus 46.1 ± 5.1, for newborn 72.9 ± 6.0, for adult 25.3 ± 3.2). Similar differences in sensitivity to both agents were observed (EC₅₀ with ACh: for fetus 0.80 ± 0.11×10^?6 M; for newborn 0.85 ± 0.26 × 10^?6 M; for adult 1.7 ± 0.20 × 10^?6M; EC₅₀ with histamine: for fetus 1.88 ± 0.50 × 10^?6 M; for newborn 1.34 ± 0.16 × 10^?6M; for adult 3.78 ± 0.75 × 10^?6 M). Removal of AE caused a significant decrease in maximal responses to ACh in fetal tissue, a smaller, insignificant one for newborn and a nonsignifcant alteration for adult tissues. Age-related sensitivity difference was abolished with removal of AE to ACh but not to histamine. In conclusion, during development (1) maximum tension, expressed as force per tissue weight, when generated follows the pattern, fetus > newborn > adult with ACh and newborn > fetus > adult with histamine; (2) the EC₅₀ pattern for both ACh and histamine was, fetus = newborn < adult; (3) for denuded tissue maximum tension by ACh was significantly decreased only in fetal trachea; and (4) removal of AE abolished the age-dependent difference in sensitivity to ACh, but not to histamine. © 1993 Wiley-Liss, Inc.     

In-vitro study of the effect of clentiazem on rabbit aorta and on myocardium

Summary We evaluated the pharmacologic action of clentiazem, a newly synthesized diltiazem derivative, on isolated rabbit aorta and myocardium. In addition to its Ca²⁺ blocking action, clentiazem demonstrated both vasorelaxing and negative inotropic actions similar to diltiazem. The vasorelaxant action of clentiazem on the tonic phase of KCl-induced contraction (EC₅₀=6.13×10^–8 M) was 4.5 times more potent than diltiazem (EC₅₀=2.77×10^–7 M). However, the negative inotropic action of clentiazem (IC₅₀=4.34×10^–5 M) was similar to diltiazem (IC₅₀=3.94×10^–5 M). Selectivity ratios, comparing the effectiveness in cardiac muscle with aorta, were clentiazem (708) > diltiazem (142). In conclusion, clentiazem is a Ca²⁺ antagonist and demonstrates greater vasoselectivity than diltiazem. It also has a longer lasting action than diltiazem.     

Mechanism of bradykinin-induced cyclic GMP accumulation in bovine tracheal smooth muscle

Bradykinin (10^–8-10^–5M) caused a concentration-dependent increase in cyclic GMP (cGMP) production in bovine tracheal smooth muscle in the absence of epithelium. The effect was calcium-dependent and was inhibited by pyrogallol (10 M) and methylene blue (10 M). The inhibition of pyrogallol was reversed by superoxide dismutase (100 Usnowml). Nitric oxide (NO) synthase inhibitors, N ^G-methyl-l-arginine (10–100 M) and N ^G-nitro-l-arginine (10–100 M) reduced cGMP accumulation induced by bradykinin in a concentration-dependent fashion, and the inhibition was reversed by l-arginine. Immunohistochemistry with a specific antibody against neuronal NO synthase from rat cerebellum showed positive staining localized in some nerve fibers. Bradykinin-induced cGMP accumulation appears to be related to the release of NO, part of which is probably synthesized in nonadrenergic noncholinergic nerve in bovine trachea.Offprint requests to: Dr. Hong Sheng     

Putative Nox2 inhibitors worsen homocysteine-induced impaired acetylcholine-mediated relaxation

Background and aimIncreased homocysteine (Hcy) is associated with coronary artery disease (CAD). Hcy increases reactive oxygen species (ROS) via NADPH oxidases (Nox), reducing acetylcholine-mediated vasorelaxation. We aimed to determine if putative Nox2 inhibitors prevent Hcy-impaired acetylcholine-mediated vasorelaxation.Methods and resultsNew Zealand White rabbit and wild-type (C57BL/6) and Nox2^−/− (NOX) mice aortic rings were mounted in organ baths. Rabbit rings were incubated with either apocynin (10 μM), gp91ds-tat (GP, 1 μM) or PhoxI2 (1 μM) and mice rings GP (1 μM) only. Some rabbit rings were incubated with 3 mM Hcy, before pre-contraction, followed by dose–response relaxation to acetylcholine (ACh; 0.01μM-10μM). In rabbit rings treated with Hcy and GP, O₂‾ donor pyrogallol (1 μM) or Akt activator SC79 (1 μM) was added 5 min before ACh. Mice rings were used to compare Nox2 deletion to normal acetylcholine-mediated relaxation.In rabbits, Hcy reduced acetylcholine-mediated relaxation vs. control (p < 0.0001). Treatment + Hcy reduced relaxation compared with treatment alone (p < 0.0001). Pyrogallol and SC79 reversed the response of GP + Hcy (p = 0.0001). In mice, Nox2 deletion reduced acetylcholine-mediated vasorelaxation. Rabbit tissue analysis revealed that Hcy reduced eNOS phosphorylation at Thr⁴⁹⁵ and increased eNOS phosphorylation at Ser¹¹⁷⁷; no further alteration at Thr⁴⁹⁵ was observed with GP. In contrast, GP prevented increased phosphorylation at Ser¹¹⁷⁷.ConclusionsApocynin, GP and PhoxI2 worsens acetylcholine-mediated vascular relaxation in rabbit aorta, which is supported by results from mouse Nox2 deletion data. These inhibitors worsen Hcy-induced vascular dysfunction, suggesting that current putative Nox2 inhibitors might not be useful in treating HHcy.     

Influence of endothelin 1 on human atrial myocardium—myocardial function and subcellular pathways

The influence of endothelin 1 on isometrically contracting human atrial muscle strip preparations was investigated under physiological conditions (37°C, 1 Hz, Ca²⁺ 2.5 mM). Endothelin dose-dependently increased isometric tension from 3×10^–10M to 1×10^–7M. At 1×10^–7M the inotropic effect of endothelin was maximum with isometric tension being increased by 32±6% (n=11, p<0.05). At 1×10^–7M endothelin the positive inotropic effect was preceded by a transient negative inotropic effect with a decline in tension by –5±1% (n=11, p<0.05). Endothelin prolonged time from peak tension to 50% relaxation (RT50) by 29±5%. With BQ123 a competitive antagonist of the ET_A receptor positive inotropic effect and the prolongation of relaxation was significantly reduced and initial negative inotropic effect was abolished, indicating a ET_A receptor mediated effect. Preincubation with phorbolmyristateacetate (10^–5M) to downregulate proteinkinase C (PKC) eliminated the positive inotropic effect of endothelin. Similarly, N-5,5-dimethylamiloride (10^–5M) which inhibits Na⁺/H⁺-exchanger activity, abolished the positive inotropic effect of ET. However, with either PMA or DMA the initial transient negative inotropic effect was still present (–13±7%, n=9, p<0.05 and –3±1%, n=6, p<0.05). Furthermore, both substances did not abolish the prolongation of twitch time parameters observed under endothelin. After preincubation with PMA, endothelin prolonged RT50 by 18±6% and with DMA by 11±2%. Using the photoprotein aequorin as an indicator for intracellular calcium concentrations showed that the positive inotropic effect was mainly mediated by an increase of systolic intracellular calcium concentrations. Thus, the present data indicate that the positive inotropic effect of endothelin in human atrial myocardium results from activation of PKC with a subsequent activation of the Na⁺/H⁺-exchanger. However, the initial negative inotropic effects as well as the prolongation of relaxation seem to result from a different intracellular mechanism of endothelin.     

Neurotransmitters partially restore glucose sensitivity of insulin and glucagon secretion from perfused streptozotocin-induced diabetic rat pancreas

Summary To elucidate the mechanisms of insensitivity of hormone secretion to glucose in streptozotocin-induced diabetic rat islets, we investigated the effects of acetylcholine (ACh) and norepinephrine on insulin and glucagon secretion in response to changes in glucose concentration, using perfused pancreas preparations. Basal insulin secretion at a blood glucose level of 5.6 mmol/l was significantly higher and basal glucagon secretion significantly lower in streptozotocin-induced diabetic rats than in controls, and neither high (16.7 mmol/l) nor low (1.4 mmol/l) blood glucose concentrations influenced insulin or glucagon secretion. Addition of 10^–6 mol/l ACh to the perfusate increased glucose-stimulated insulin secretion. Also, 10^–6 mol/l ACh, 10^–7 mol/l norepinephrine, as well as a combination of both, induced marked glucagon secretion, this was suppressed by high blood glucose level. Although simultaneous addition of 10^–6 mol/l ACh and 10^–7 mol/l norepinephrine induced only a slight increase in glucagon secretion in response to glucopenia, there was a significant increase in glucagon secretion in conjunction with an ambient decrease in insulin. Histopathological examination revealed a marked decline in acetylcholinesterase and monoamine-oxidase activities in the islets of streptozotocin-induced diabetic rats. We speculate that reduction of the potentiating effects of ACh and norepinephrine lessens glucose sensitivity of islet beta and alpha cells in this rat model of diabetes.Abbreviations STZ Streptozotocin - STZD streptozotocin-induce diabetic - ACh acetylcholine - AChE acetylcholinesterase - NE norepinephrine - MAO monoamine-oxidase     

Endothelium-Dependent Tolerance to Ethanol-Induced Contraction of Rat Aorta: Effect of Inhibition of EDRF Action and Nitric Oxide Synthesis

In vitro ethanol induces a dose-dependent contraction of the aorta. Tolerance to this effect of ethanol is expressed by a rightward shift of the dose-response curve in aorta from animals intoxicated with ethanol for 2 days. The expression of tolerance in the aorta is dependent upon the presence of functional endothelial cells which suggests that tolerance is mediated, in part, by endothelium-derived relaxing factor (EDRF). To test this hypothesis, three inhibitors of EDRF action and an inhibitor of nitric oxide synthesis were studied for their ability to alter tolerance to ethanol-induced contraction of the aorta. In vitro pretreatment of aortic rings with gossypol (10^-6– 10^-5 M), pyrogallol (10^-5 M), hemoglobin (10^-6 M), and N^G-nitro-L-arginine (NOARG, 10^-4 M) inhibited endothelium-dependent relaxation induced by carbachol. The inhibition of carbachol-induced relaxation produced by pyrogallol was reversed by superoxide dismutase (SOD, 45 units/ml). In vitro pretreatment of rings obtained from ethanol-treated rats with gossypol, pyrogallol, hemoglobin, or NOARG inhibited the expression of ethanol tolerance, shifting the ethanol dose-response curve to control values. SOD reversed the effect of pyrogallol pretreatment. None of the antagonists significantly altered the ethanol dose-response curve of aortic rings obtained from control animals. These data support the hypothesis that tolerance to ethanol-induced contraction of the aorta is mediated by the release of EDRF from endothelial cells.