Nitric oxide involvement in the effect of acute lithium administration on the nonadrenergic noncholinergic-mediated relaxation of rat gastric fundus

INTRODUCTION: Lithium has largely met its initial promise as the first drug to be discovered in the modern era of psychopharmacology. However, the mechanism for its action remains an enigma. The aim of the present study was to verify the effect of acute lithium administration on the nonadrenergic noncholinergic (NANC)-mediated relaxation of rat isolated gastric fundus and to evaluate the role of nitric oxide pathway in this manner. MATERIALS AND METHODS: The isolated rat gastric fundus strips were precontracted with 0.5 microM serotonin and electrical field stimulation (EFS) was applied at 5 Hz frequency to obtain NANC-mediated relaxation in the presence or absence of lithium (0.1, 0.5, 1 and 5 mM). Also, effects of combining lithium (0.1 mM) with the NO synthase (NOS) inhibitor L-NAME (0.03 microM) or the guanylyl cyclase inhibitor ODQ (1 microM) on relaxant responses to EFS was investigated. Moreover, effects of combining lithium (1 mM) with 0.1 mM L-arginine (a precursor of NO) on neurogenic relaxation were assessed. Also, the effect of lithium (1 mM) on relaxation to sodium nitroprusside (SNP; 1 nM-0.1 mM) and glyceryltrinitrate (GTN; 0.1-10 microM) was investigated. RESULTS: The NANC-mediated relaxation was significantly (P<0.001) reduced by lithium in a dose- and time-dependent manner. Combination of lithium (0.1 mM) with L-NAME (0.03 microM), which separately had partial inhibitory effect on relaxations, significantly (P<0.001) reduced the NANC-mediated relaxation of gastric fundus. ODQ (1 microM) significantly inhibited the neurogenic relaxations in the presence or absence of lithium (0.1 and 1 mM). Although L-arginine at 0.1 mM had no effect on relaxation to EFS, it prevented the inhibition by lithium (1 mM) of relaxant responses to EFS. Also, SNP and GTN produced concentration-dependent relaxation in precontracted rat gastric fundus which was not altered by lithium incubation (1 mM). DISCUSSION: Our experiments indicated that lithium likely by interfering with L-arginine/NO pathway in nitrergic nerve can result in impairment of NANC-mediated relaxation of rat gastric fundus.     

Time-dependent alteration in cromakalim-induced relaxation of corpus cavernosum from streptozocin-induced diabetic rats

The purpose of the present study was to investigate the relaxant responses to the ATP-sensitive potassium (K(ATP)) channel opener cromakalim in corpus cavernosum strips from 1-, 2-, 4-, 6-, and 8-week streptozocin-induced diabetic rats. Cromakalim (1 nM-0.1 mM) produced concentration-dependent relaxation in phenylephrine (7.5 microM)-precontracted isolated rat corporal strips. Compared with age-matched control animals, a significant enhancement in cromakalim-induced relaxation of corpus cavernosum was observed in 2-week diabetic animals, whereas the relaxant responses to cromakalim were decreased in 6-and 8-week diabetic animals. However, the cromakalim-induced relaxation was not altered in either 1-week or 4-week rat corporal strips in comparison with corresponding age-matched non-diabetic groups. Preincubation with the K(ATP) channel blocker glibenclamide (10 microM) significantly inhibited the cromakalim-induced relaxation in both non-diabetic and diabetic rat corpus cavernosum, but neither the voltage-dependent K(+) channel (K(V)) antagonist 4-aminopyridine (1 mM) nor the calcium-activated K(+) channel (K(Ca)) antagonist charybdotoxin (0.1 microM) had significant effect on cromakalim-induced relaxation in both control and diabetic rat corporal strips. Relaxation responses to the nitric oxide donor sodium nitroprusside (1 nM-0.1 mM) in diabetic rat corpus cavernosum were similar to that of age-matched controls. These data demonstrated that the relaxant responses to cromakalim were altered in diabetic cavernosal strips in a time dependent manner, suggesting that the period of diabetes mellitus may play a key role in the K(ATP) channels function in rat corpus cavernosum.     

Identification of P1 and P2 purinoceptors in the aorta of the lizard (Agama sp.)

In the isolated Agama lizard aorta, acetylcholine (ACh; 3 nM-100 microM), noradrenaline (NA; 30 nM-0.3 mM), adrenaline (Adr; 30 nM-300 microM), adenosine 5'-triphosphate (ATP; 30 nM-1 mM), alpha,beta-methylene ATP (alpha,beta-meATP; 10 nM-10 microM), beta,gamma-methylene ATP (beta,gamma-meATP; 0.1-300 microM), 2-methylthio ATP (2-meSATP; 30 nM-30 microM) and high concentrations of uridine triphosphate (UTP; 1 microM-1 mM), all produced constriction. The P2 receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 30 microM), suramin (0.1 mM) and Reactive blue 2 (30 microM) all raised vascular tone and could not be utilized and the antagonist 2'-O-(trinitrophenyl) ATP (TNP-ATP; 0.1 microM) had no effect on responses to the ATP analogues. alpha,beta-MeATP (3 microMx3) desensitised responses to alpha,beta-meATP (10 microM) and beta,gamma-meATP (0.3 mM), but not to ATP (0.3 mM) or 2-meSATP (30 microM). On pre-constricted aorta (EC50 concentration of either ACh or Adr), adenosine (1 microM-1 mM), the A1-selective agonist N6-cyclopentyl adenosine (CPA; 1-300 microM) [but not the A2- and A3-selective agonists CGS 21680 and IB-MECA respectively (both up to 30 microM)] and sodium nitroprusside (10 nM-100 microM) produced vasodilatation. Adenosine vasodilatation was antagonised by 8-p-sulfophenyl-theophylline (8-pSPT; 30 microM) but not by N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.1 mM). ATP (up to 0.3 mM), 2-meSATP (up to 10 microM) and UTP (up to 1 mM) were not vasodilators. In summary, A1 receptors mediating relaxation and excitatory P2X1 receptors were identified in the smooth muscle of the lizard aorta. However, in contrast to mammalian aorta, P2Y receptors on endothelial cells mediating vasodilatation via nitric oxide do not appear to be present.     

Evidence of relaxant effect of omeprazole in rabbit corpus cavernosum in vitro

The present experiments were designed to investigate the effects of omeprazole, a H(+)-K+ ATPase inhibitor, on corporal smooth muscle tone in vitro. All spontaneous contractile activity in the corpus cavernosum was blocked following omeprazole (0.1 mM-1 mM) administration. However atropine (1 microM), Nw-nitro L-arginine methyl ester (L-NAME, 30 microM) or indomethacin (10 microM) did not affect the spontaneous contraction. Omeprazole (10 microM-1 mM) concentration-dependently induced relaxation in corporal smooth muscle precontracted with 10 microM phenylephrine or 80 mM KCl. Pretreatment of corporal tissue with L-NAME (30 microM), indomethacin (10 microM), ammonium chloride (7.5 mM), sodium acetate (7.5 mM), tetraethyl ammonium chloride (0.5 mM) or glibenclamide (1 microM) had no effect on the omeprazole induced relaxant responses. Nimodipine, an L-type Ca++ channel blocker, relaxed corporal strips precontracted with 80 mM KCl. Collectively, these results indicate that the inhibition of spontaneous contraction and the relaxation of precontracted corporal smooth muscle by omeprazole is probably mediated by the blockade of calcium channels. Further work is needed to determine the cellular mechanism(s) of action by which omeprazole acts on corpus cavernosum smooth muscle.     

Acetylcholine causes endothelium-dependent contraction of mouse arteries

The goal of this study was to determine whether acetylcholine evokes endothelium-dependent contraction in mouse arteries and to define the mechanisms involved in regulating this response. Arterial rings isolated from wild-type (WT) and endothelial nitric oxide (NO) synthase knockout (eNOS(-/-)) mice were suspended for isometric tension recording. In abdominal aorta from WT mice contracted with phenylephrine, acetylcholine caused a relaxation that reversed at the concentration of 0.3-3 microM. After inhibition of NO synthase [with N(omega)-nitro-l-arginine methyl ester (l-NAME), 1 mM], acetylcholine (0.1-10 microM) caused contraction under basal conditions or during constriction to phenylephrine, which was abolished by endothelial denudation. This contraction was inhibited by the cyclooxygenase inhibitor indomethacin (1 muM) or by a thromboxane A(2) (TxA(2)) and/or prostaglandin H(2) receptor antagonist SQ-29548 (1 microM) and was associated with endothelium-dependent generation of the TxA(2) metabolite TxB(2.) Also, SQ-29548 (1 microM) abolished the reversal in relaxation evoked by 0.3-3 microM acetylcholine and subsequently enhanced the relaxation to the agonist. The magnitude of the endothelium-dependent contraction to acetylcholine (0.1-10 microM) was similar in aortas from WT mice treated in vitro with l-NAME and from eNOS(-/-) mice. In addition, we found that acetylcholine (10 microM) also caused endothelium-dependent contraction in carotid and femoral arteries of eNOS(-/-) mice. These results suggest that acetylcholine initiates two competing responses in mouse arteries: endothelium-dependent relaxation mediated predominantly by NO and endothelium-dependent contraction mediated most likely by TxA(2).     

Nitric oxide induces muscular relaxation via cyclic GMP-dependent and -independent mechanisms in the longitudinal muscle of the mouse duodenum.

The aim of this study was to investigate, in mouse duodenum, the role of nitric oxide (NO) in the relaxation of longitudinal muscle evoked by nerve activation and the coupled action mechanism. Electrical field stimulation (EFS; 0.5 ms, 10-s train duration, supramaximal voltage, at various frequencies) under nonadrenergic noncholinergic conditions evoked muscular relaxation occasionally followed, at the higher stimulus frequencies, by rebound contractions. Inhibition of the synthesis of NO by N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM) virtually abolished the evoked relaxation. The relaxation was reduced also by apamin (0.1 microM) and by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), a guanylyl cyclase inhibitor. The coadministration of apamin and ODQ produced additive effects on the responses to EFS. Sodium nitroprusside (0.1-100 microM) produced a concentration-dependent reduction of the phasic spontaneous activity and at the highest dose used suppressed phasic activity and induced muscular relaxation. These effects were tetrodotoxin and L-NAME resistant and were antagonized both by apamin and by ODQ. 8-Bromoguanosine 3',5'-cyclic monophosphate (0.1-100 microM) reduced in a concentration-dependent manner the spontaneous mechanical activity and at 100 microM suppressed the phasic activity and induced muscular relaxation, not antagonized by apamin. This study indicates that NO is the primary transmitter released by inhibitory nerves supplying the longitudinal muscle of mouse duodenum and that guanylate cyclase stimulation and opening of Ca(2+)-dependent K(+) channels are independent mechanisms working in parallel to mediate NO action.     

Nitric oxide-dependent and -independent mechanisms in the relaxation elicited by acetylcholine in fetal rat aorta

The aim of the present study was to analyze the mechanisms involved in the relaxation induced by 1 microM acetylcholine (ACh) in aortic segments from fetal rats at term precontracted with 3 microM prostaglandin F2alpha (PGF2alpha) and incubated with 1 microM indomethacin. The endothelium-dependent relaxation caused by ACh was reduced by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.1 mM), such an effect was reversed by 0.1 mM L-arginine (L-Arg). After precontraction of segments with 50 mM KCl the relaxant response to ACh was smaller than that after precontraction with PGF2alpha; this reduction was increased by L-NMMA, whereas L-NMMA plus L-Arg potentiated the relaxation. Thiopentone sodium (0. 1 mM), ouabain (10 microM), tetraethylammonium (TEA, 0.5 mM) and apamin (1 microM), inhibitors of cytochrome P450 monooxygenases, Na+ pump, Ca2+-activated (KCa) and small-conductance (SKCa) K+ channels, respectively, reduced the relaxation to ACh, which was unaffected by charybdotoxin (0.1 microM) and glibenclamide (1 microM), inhibitors of large-conductance BKCa and ATP-sensitive K+ channels. The L-NMMA/indomethacin-resistant relaxation to ACh was markedly reduced by thiopentone sodium, and similarly decreased by either ouabain or TEA. The endothelium-independent relaxation induced by exogenous NO (10 microM) in segments precontracted with PGF2alpha was unaltered by ouabain, glibenclamide, TEA and after precontraction with 50 mM KCl, and potentiated by L-NMMA. The potentiation of NO responses by L-NMMA was also observed in segments precontracted with KCl. These results suggest that ACh relaxes the fetal rat aorta by endothelial release of both NO and endothelium-derived hyperpolarizing factor (EDHF), a metabolite derived from cytochrome P450 monooxygenases, that hyperpolarizes smooth muscle cells by activation of KCa, essentially SKCa channels, and Na+ pump. It seems that when the effect of EDHF is abolished, the formation of NO could be increased.     

Nitric oxide-mediated nonadrenergic noncholinergic relaxation of piglet pulmonary arteries decreases with postnatal age.

Nonadrenergic noncholinergic (NANC) vasodilator mechanisms may contribute to the maintenance of adult pulmonary and systemic vascular tone. However, their actions in the neonatal circulation have not been studied. We aimed to investigate NANC vasorelaxation in neonatal and 2-week-old piglet pulmonary and mesenteric arteries and to examine the potential role of nitric oxide (NO) in this phenomenon. Responses to electric field stimulation (EFS, 50V, 0.25-32 Hz) were investigated in pulmonary and mesenteric artery rings (external diameter 150-200 microm) precontracted with the thromboxane A2 mimetic U46619, in the presence of guanethidine (10 microM) and atropine (10 microM). Under these conditions, EFS resulted in a frequency dependent relaxation of newborn pulmonary (maximal relaxation of 53+/-9.1%), mesenteric (68.8.2+/-7.1%) and 2-wk-old mesenteric (46 6.3%) arteries but this relaxation was significantly reduced (4.5+/-2.2%) in 2-week-old pulmonary arteries. In neonatal pulmonary arteries, the neurotoxin tetrodotoxin (0.3 muM), the NO synthase inhibitor L-NAME (0.1 mM), and the guanylyl cyclase inhibitor ODQ (10 microM) abolished EFS-induced relaxations, suggesting that NANC relaxation of porcine neonatal pulmonary arteries is mediated by NO, which is probably neuronal in origin. However, The expression in pulmonary arteries of the neuronal NO synthase (nNOS), as determined by Western-blot analysis, increased with postnatal age whereas the expression of the endothelial NOS (eNOS) did not change. In conclusion, NANC relaxation is present in neonatal pulmonary and mesenteric arteries and it is, at least partially, mediated through NO. NANC relaxation of porcine pulmonary and mesenteric arteries decreases with postnatal maturation.     

Ursolic acid mediates the vasorelaxant activity of Lepechinia caulescens via NO release in isolated rat thoracic aorta

We have determined that the methanolic extract of L. caulescens (MELc) produced a significant vasodilator effect in a concentration-dependent and endothelium-dependent manner. This relaxation was blocked by N(omega)-nitro-L-arginine methylester (L-NAME), indicating that MELc vasodilator properties are endothelium mediated due to liberation of nitric oxide (NO). In this paper we aimed to corroborate its mode of action. MELc effects on noradrenaline (NA)-induced contraction in isolated rat aortic thoracic rings with endothelium (+E), in the presence of atropine (0.1 microM) and 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, 1 microM) were conducted. MELc relaxation curve was significantly shifted to the right in the presence of ODQ and atropine, thus confirming that its mode of action is related with activation of nitric oxide synthase (NOS) and the consequent increment in NO formation. Bio-guided study of MELc allowed the isolation of ursolic acid (UA, 50 mg) and ursolic-oleanolic acids mixture [UA/OA (7:3), 450 mg]. The relaxant effect of UA (0.038-110 microM) was evaluated in functional experiments. UA induced a significant relaxation in a concentration- and endothelium-dependent manner (IC(50)=44.15 microM) and did not produce a vasorelaxant effect on contraction evoked by KCl (80 mM). In addition, NA-induced contraction was significantly displaced to the right by UA (30 microM). In order to determine its mode of action, UA-induced relaxant effect was evaluated in the presence of atropine (0.1 microM), indomethacin (10 microM), L-NAME (100 microM) and ODQ (1 microM). Relaxation was blocked by L-NAME and ODQ. On the other hand, UA (3 microM) provoked a significant displacement to the left in the relaxation curve induced by sodium nitroprusside (SNP, 0.32 nM to 0.1 microM), but it was not significant in the presence of Carbamoyl choline (carbachol, 1 nM to 10 microM). These results indicate that UA-mediated relaxation is endothelium dependent, probably due to NO release, and the consequent activation of vascular smooth muscle soluble guanylate cyclase (sGC), a signal transduction enzyme that forms the second messenger cGMP.     

Mechanisms underlying the relaxation induced by isokaempferide from Amburana cearensis in the guinea-pig isolated trachea

The present study examines possible mechanisms by which the flavonoid isokaempferide (IKPF; 5,7,4'-trihydroxy-3-methoxyflavone) from Amburana cearensis, a Brazilian medicinal plant popularly used as bronchodilator, induces relaxation of guinea-pig isolated trachea. In the trachea (with intact epithelium) contracted by carbachol, IKPF (1-1000 microM) caused a graded relaxation, and the epithelium removal increased the sensitivity of the airway smooth muscle to IKPF (EC50, in intact tissue: 77.4 [54.8-109.2] microM; in denuded epithelium: 15.0 [11.3-20.1] microM). The IKPF-induced relaxation was inhibited in 41% by the nitric oxide (NO) synthase inhibitor L-NAME (100 microM); in 31% and 50% by the soluble guanylate cyclase (sGC) inhibitor ODQ (3 and 33 microM); by propranolol (31%) and also by capsaicin (37%). In the trachea pre-contracted by 40 mM KCl the pre-incubation with glibenclamide (33 microM) or iberiotoxin (IbTX, 0.1 microM), selective K(+) channel inhibitors, inhibited the IKPF-induced relaxation by 39% and 38%, respectively. On the other hand, 4-aminopyridine (100 microM), a nonselective K(+) channel antagonist, did not significantly influence the effect of IKPF, while IbTX induced a rightward displacement of the IKPF concentration-response curve. However, in muscle pre-contracted with 120 mM KCl the relaxant effect of IKPF was significantly reduced and not affected by glibenclamide. In conclusion, these results indicate a direct and epithelium-independent relaxant effect of IKPF on smooth muscle fibers. Although this IKPF relaxant action seems to be multi-mediated, it occurs via both Ca(2+) and ATP-sensitive K(+) channels, but some other possible mechanisms unrelated to K(+) channels cannot be excluded.     

Increased free calcium in endothelial cells under stimulation with adenine nucleotides

The release of vasodilating substances from the vascular endothelium has been postulated to depend on a rise in the level of intracellular free calcium (Cai++). We measured Cai++ in intact monolayers of calf endothelial cells, grown in culture, that were loaded with the fluorescent calcium indicator quin 2. Fluorescence (excitation wavelength 340 nm, emission wavelength 492 nm) was calibrated by raising Cai++ to a maximum with the calcium ionophore ionomycin (0.1 microM) and by lowering it to a minimum with ionomycin plus manganese (0.4 mM), which quenches quin 2 fluorescence completely. Loss of fluorescent dye from the cells was calculated from fluorescence at the isosbestic excitation wavelength (365 nm). Resting Cai++ was 71 +/- 3 (SEM) nM. ATP (adenosine-5'-triphosphate) raised Cai++ dose-dependently and reversibly to 458 +/- 60 nM at a concentration of 10 microM, and at 0.1 mM to values close to those that occurred under ionomycin. ADP (A-5'-PP) and AMP (A-5'-P) had smaller effects with a maximal Cai++ of 287 +/- 72 nM at 30 microM ADP and 176 +/- 17 nM at 0.1 mM AMP. At these concentrations, ADP and AMP attenuated significantly the increase of Cai++ under ATP (10 microM). Adenosine (0.1 or 0.3 mM) and acetylcholine (0.1 to 30 microM) enhanced Cai++ inconsistently, by a maximum of 50 nM. These effects were abolished by theophylline and atropine, respectively. In the absence of extracellular calcium, ATP still raised Cai++, although endothelial responsiveness declined after repetitive stimulations. We conclude that activation of purinergic receptors increases intracellular free calcium in endothelial cells, and that this increase is probably an essential trigger for synthesis of prostacyclin and the labile endothelium-derived relaxant factor.     

Effects of oxidizing and reducing agents on ovine pulmonary artery responses to nitric oxide donors, sodium nitroprusside and 3-morpholino-sydnonimine

Nitrovasodilators-sodium nitroprusside (SNP; 10(-9)-10(-4) M) and 3-morpholino-sydnonimine (SIN-1; 10(-9)-10(-4) M) produced concentration-dependent relaxation of the fourth generation sheep pulmonary artery, preconstricted with 5-hydroxytryptamine (1 microM). Oxidizing agents [oxidized glutathione (GSSG, 1 mM) and CuSO4 (5 and 20 microM)] and reducing agents [dithiothreitol (DTT, 0.1 mM), ascorbic acid (1 mM) and reduced glutathione (GSH, 1 mM)] caused opposite effects on nitric oxide (NO)-induced vasodilation in the artery. Ascorbic acid and GSH potentiated the NO responses, while GSSG and CuSO4 inhibited relaxation caused by the nitrovasodilators. DTT, however, reduced the relaxant potency and efficacy of SNP and SIN-1. Pretreatment of the pulmonary artery strips with DTT (0.1 mM) inhibited SNP (10 microM)-induced Na(+)-K(+)-ATPase activity, while ascorbic acid (1 mM) and GSH (1 mM) had no effect either on basal or SNP (10 microM)-stimulated 86Rb uptake, an index of Na(+)-K(+)-ATPase activity, in ovine pulmonary artery. The results suggest that reducing agents like ascorbic acid may have beneficial effect in improving the vascular function under oxidative stress.     

The role of nitric oxide in mediating non-adrenergic non-cholinergic relaxation in longitudinal muscle of human taenia coli.

Electrical field stimulation (EFS) of isolated longitudinal muscle of human taenia coli at 4Hz produced relaxation which was abolished by tetrodotoxin but not adrenergic and cholinergic blockade (NANC-relaxation). NG-nitro L-arginine (L-NOARG; 1-100 microM), an NO synthesis inhibitor, produced a concentration-dependent partial inhibition of the NANC response; 10 microM L-NOARG inhibited EFS-induced relaxation by 48.6 +/- 5.20% and 100 microM L-NOARG by 54.2 +/- 10.1%. L-Arginine (1mM), but not D-arginine (1mM) partially reversed the inhibitory effect and this was inversely proportional to the concentration of L-NOARG used. Cumulative administration of NO (acidified sodium nitrite solution; 1-100 microM) produced a concentration-dependent relaxation of the strips. L-NOARG (1 mM) did not affect either NO or isoprenaline-induced relaxations. These results provide the first preliminary evidence that NO is partially responsible for the NANC inhibitory transmission in the longitudinal muscle of the taenia coli of human colon.     

Purinergic and nitrergic junction potential in the human colon

The aim of the present work is to investigate a putative junction transmission [nitric oxide (NO) and ATP] in the human colon and to characterize the electrophysiological and mechanical responses that might explain different functions from both neurotransmitters. Muscle bath and microelectrode techniques were performed on human colonic circular muscle strips. The NO donor sodium nitroprusside (10 microM), but not the P2Y receptor agonist adenosine 5'-O-2-thiodiphosphate (10 microM), was able to cause a sustained relaxation. NG-nitro-L-arginine (L-NNA) (1 mM), a NO synthase inhibitor, but not 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) (10 microM), a P2Y antagonist, increased spontaneous motility. Electrical field stimulation (EFS) at 1 Hz caused fast inhibitory junction potentials (fIJPs) and a relaxation sensitive to MRS 2179 (10 microM). EFS at higher frequencies (5 Hz) showed biphasic IJP with fast hyperpolarization sensitive to MRS 2179 followed by sustained hyperpolarization sensitive to L-NNA; both drugs were needed to fully block the EFS relaxation at 2 and 5 Hz. Two consecutive single pulses induced MRS 2179-sensitive fIJPs that showed a rundown. The rundown mechanism was not dependent on the degree of hyperpolarization and was present after incubation with L-NNA (1 mM), hexamethonium (100 microM), MRS 2179 (1 microM), and NF023 (10 microM). We concluded that single pulses elicit ATP release from enteric motor neurons that cause a fIJP and a transient relaxation that is difficult to maintain over time; also, NO is released at higher frequencies causing a sustained hyperpolarization and relaxation. These differences might be responsible for complementary mechanisms of relaxation being phasic (ATP) and tonic (NO).     

Angiotensin II increases neurogenic nitric oxide metabolism in mesenteric arteries from hypertensive rats

We investigated, in mesenteric arteries from hypertensive rats (SHR), the possible changes in neurogenic nitric oxide (NO) release produced by angiotensin II (AII), and the possible mechanisms involved in this process. In deendothelialized segments the NO synthase inhibitor N(G)-nitro-L-arginine (L-NAME, 10 microM) increased the contractions caused by electrical field stimulation (EFS, 200 mA, 0.3 ms, 1-16 Hz, for 30 s). AII (0.1 nM) enhanced the response to EFS, which was unmodified by the subsequent addition of L-NAME. The AII antagonist receptor saralasine (0.1 microM) prevented the effect of AII, and the subsequent addition of L-NAME restored the contractile response. SOD (25 u/ml) decreased the reponse to EFS and the subsequent addition of L-NAME increased this response. AII did not modify the decrease in EFS response induced by SOD, and the addition of L-NAME increased the response. None of these drugs altered the response to exogenous noradrenaline (NA) or basal tone except SOD, which increased the basal tone, an effect blocked by phentolamine (1 microM). In arteries pre-incubated with [3H]-NA, AII did not modify the tritium efflux evoked by EFS, which was diminished by SOD. AII did not alter basal tritium efflux while SOD significantly increased it. These results suggest that EFS of SHR mesenteric arteries releases neurogenic NO, the metabolism of which is increased in the presence of AII by the generation of superoxide anions.     

The role of nitric oxide in mediating nonadrenergic, noncholinergic relaxation in rat pulmonary artery.

Experiments were undertaken to investigate the existence of inhibitory nonadrenergic, noncholinergic (i-NANC) nerve activity by using in vitro functional and immunohistochemical techniques in rat main pulmonary arterial rings. Vessels precontracted with phenylephrine (3 microM) relaxed in response to electrical field stimulation (EFS) (50 V, 0.2 ms, 0.1-10 Hz for 5 s) in the presence of atropine (1 microM) and guanethidine (1 microM). Tetrodotoxin (0.3 microM) abolished this response, indicating that it is neuronal in origin. l-NAME (30 microM), methylene blue (10 microM), and removal of endothelium significantly reduced the EFS-induced relaxations. The inhibitory action of l-NAME was completely reversed by l-arginine (1 mM) but not by d-arginine (1 mM). Moreover l-arginine alone potentiated the magnitude of the relaxations elicited by EFS. On the other hand, immunohistochemical work clearly demonstrated the existence of neuronal nitric oxide synthase in the pulmonary artery vessel wall. All these results are consistent with the suggestion that nitric oxide is the likely mediator of this vasodilatation. However, the incomplete blockade of the responses by l-NAME gives evidence of an additional inhibitory NANC neurotransmitter(s) mediating the residual relaxation, which requires further experiments to clarify its nature.     

Reactivity of mesenteric and aortic rings from trained rats fed with high caloric diet

A strong association between the benefits of physical exercise on the cardiovascular disease with an improvement of the endothelium-derived relaxing factor production has been consistently shown. The purpose of this study was to evaluate the effect of exercise training associated with high caloric diet in the reactivity of rat mesenteric and aortic rings. Experimental protocol consisted of 4 weeks of high caloric diet consumption previous to 4 weeks of run training (1.2 km/h, 0% grade, in sessions of 60 min, 5 days/week). Concentrations of triglycerides, glucose, insulin and nitrite/nitrate levels were measured and atherogenic index was calculated. Concentration-response curves to acetylcholine (10 nM-100 microM), sodium nitroprusside (100 pM-100 nM) and phenylephrine (1 nM-3 microM) were obtained. Exercise training reduced body mass (6%) and triglyceride levels (about 54%), without changes in glucose and insulin concentrations. An improvement of endothelium-dependent relaxation responses to acetylcholine in mesenteric and aortic rings was observed in trained group. No changes were seen for sodium nitroprusside and phenylephrine. In conclusion, our study is the first to show clearly that run training promotes an improvement of the endothelium-dependent relaxing response in aorta and mesenteric rings from rats fed with high caloric diet and that is associated with increase of NO production.     

The relaxant effect of urocortin in rat pulmonary arteries

Urocortin is a potent vasodilator, which plays physiological or pathophysiological roles in systemic circulation. However, little is known about its action on pulmonary circulation. The present study was aimed to characterize some cellular mechanisms underlying the relaxant effect of urocortin in isolated rat pulmonary arteries. Changes in isometric tension were measured on small vessel myographs. Urocortin inhibited U46619-induced contraction with reduction of the maximal response. Urocortin-induced relaxation was independent of the presence of endothelium. Inhibitors of nitric oxide (NO)-dependent dilator, NG-nitro-L-arginine methyl ester or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one, did not affect the relaxation. Astressin (100-500 nM), a corticotropin-releasing factor (CRF) receptor antagonist and KT5720, a protein kinase A (PKA) inhibitor reduced urocortin-induced relaxation. Urocortin produced less relaxant effect in 30 mM K+- than U46619-contracted arterial rings. Urocortin did not reduce CaCl2-induced contraction in 60 mM K+-containing solution. Ba2+ (100-500 microM) but not other K+ channel blockers reduced the relaxant responses to urocortin. Urocortin also relaxed the rings preconstricted by phorbol 12,13-diacetae in normal Krebs solution while this relaxation was less in a Ca2+-free solution. Our results show that urocortin relaxed rat pulmonary arteries via CRF receptor-mediated and PKA-dependent but endothelium/NO or voltage-gated Ca2+ channel-independent mechanisms. Stimulation of Ba2+-sensitive K+ channel may contribute to urocortin-induced relaxation. Finally, urocortin relaxed pulmonary arteries partly via inhibition of a PKC-dependent contractile mechanism.     

Inhibition of the acetylcholine-induced relaxation of canine isolated basilar artery by potassium-conductance blockers

Canine basilar artery rings precontracted with 5-hydroxytryptamine (0.1-0.5 microM) relaxed in the presence of acetylcholine (25-100 microM), sodium nitroprusside (0.1 microM), or stimulation of the electrogenic sodium pump by restoration of extracellular K+ (4.5 mM) after K(+)-deprivation. Acetylcholine-induced relaxation is believed to be caused by the release of endothelium-derived relaxing factor (EDRF) and is prevented by mechanical removal of the endothelium, while relaxations induced by sodium nitroprusside or restarting of the sodium pump are endothelium-independent. Acetylcholine-induced relaxation was selectively blocked by pretreatment of the tissue with the nonselective K+ conductance inhibitors, 4-aminopyridine (4-AP, 3 mM), Ba2+ (1 mM), and tetraethylammonium (20 mM), 4-AP also blocked ACh-mediated relaxation in muscles contracted with elevated external K+. Relaxation of 5-hydroxytryptamine-induced contraction by sodium nitroprusside, or by addition of K+ to K(+)-deprived muscle, was not affected by 4-AP. Relaxation of basilar artery with acidified sodium nitrite solution (containing nitric oxide) was reduced by 4-AP. These results suggest that 4-AP and possibly Ba2+ inhibit acetylcholine-induced endothelium-dependent relaxation by inhibition of the action of EDRF on the smooth muscle rather than through inhibition of release of EDRF. The increase in K+ conductance involved in acetylcholine-induced relaxation is not due to ATP-inhibited K+ channels, as it is not blocked by glyburide (10(-6) M). Endothelium-derived relaxant factor(s) may relax smooth muscle by mode(s) of action different from that of sodium nitroprusside or by hyperpolarization due to the electrogenic sodium pumping.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasorelaxant effect of the flavonoid galangin on isolated rat thoracic aorta

Here we investigated the effect of the flavonoid galangin in isolated rat thoracic aortic rings. Galangin (0.1-100 microM) induced relaxation in rings pre-contracted with phenylephrine (PE 1 microM) or with KCl (100 mM) or pre-treated with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 100 microM), the cyclooxygenase inhibitor indomethacin (10 microM) and the adenylate cyclase inhibitor, SQ 22,536 (100 microM). In another set of experiments, rat aortic rings were incubated with galangin (1-100 microM) and the contractile responses to PE (0.001-3 microM) or to KCl (60 mM) were evaluated. We also evaluated the effect of galangin (100 microM) on PE (10 microM)-induced contraction in a Ca2+-free medium. Galangin relaxed aortic rings with or without endothelium. Galangin effect was significantly inhibited by L-NAME. Galangin inhibited the contractile response to PE, either in presence or in absence of external calcium, and to KCl. In the end, we also found that galangin caused nitric oxide (NO) release from aortic rings and abolished the increase in [Ca2+]i triggered by PE or KCl in aortic smooth muscle cells, either in presence and in absence of external Ca2+. Our results suggest that galangin reduces the contractility of rat aortic rings through an endothelium-dependent mechanism, involving NO, and also through an endothelium-independent mechanism, inhibiting calcium movements through cell membranes.