Effect of Cu2+ on relaxations to the nitrergic neurotransmitter, NO and S-nitrosothiols in the rat gastric fundus

1. The effects of superoxide anion generators before and after treatment with inhibitors of Cu/Zn superoxide dismutase (Cu/Zn SOD) and the effects of thiol-modulating agents were investigated on nitrergic relaxations to electrical stimulation of non-adrenergic non-cholinergic (NANC) nerves of the rat gastric fundus and on relaxations to authentic nitric oxide (NO) and nitroglycerin. 2. The superoxide anion generators, pyrogallol (30 microM) and duroquinone (30-60 microM), significantly inhibited the relaxations to NO (0.03-3 microM) but not nitrergic relaxations to NANC nerve stimulation (0.5-8 Hz) or those to ATP (10 microM). Treatment of the rat gastric fundus with the inhibitors of Cu/Zn SOD, diethyldithiocarbamate (DETC, 1 mM for 2 h) or triethylenetetramine (TETA, 100 microM for 2 h) had no effect on the relaxations to NANC nerve stimulation (1-8 Hz), NO (0.03-3 microM) or on those to ATP (10 microM). 3. After treatment of the rat gastric fundus with DETC (1 mM) but not after treatment with TETA (100 microM), pyrogallol (30 microM) and duroquinone (30-60 microM) significantly inhibited the nitrergic relaxations to electrical stimulation (0.5-8 Hz) and those to NO (0.03-3 microM). This inhibitory effect of pyrogallol and duroquinone was prevented by addition of exogenous SOD (250 units ml-1). Pyrogallol but not duroquinone also inhibited the NO-independent relaxations to ATP (10 microM). 4. The thiol modulators, buthionine sulphoximine (1 mM for 2 h) and ethacrynic acid (30 microM for 2 h), significantly inhibited the relaxations to nitroglycerin (0.03-3 microM) but had no effect on the nitrergic relaxations to electrical stimulation (0.5-8 Hz) or on those to NO (0.03-10 microM) and ATP (10 microM). The thiol modulators, sulphobromophthalein (100 microM for 2 h) and diamide (30-100 microM for 2 h) did not affect the relaxations to nitroglycerin, or those to NANC nerve stimulation and NO. 5. In summary, thiol modulators significantly inhibited the thiol-dependent relaxations to nitroglycerin but not those to NANC nerve stimulation or NO. Relaxations to nitrergic stimulation were decreased by superoxide anion generators only after inhibition of Cu/Zn SOD. These results suggest that the nitrergic NANC neurotransmitter in the rat gastric fundus is not a nitrosothiol but more likely free NO, which is protected from breakdown by tissue SOD.     

Haemodynamic effects of dopexamine and nitric oxide synthase inhibition in healthy and endotoxaemic sheep

In endothelium-denuded guinea-pig isolated basilar artery preparations, hydroxocobalamin (30, 100 and 300 microM) concentration-dependently inhibited the vasodilator responses to exogenous nitric oxide (NO), whereas the vasodilator responses to nitrergic nerve stimulation were slightly reduced by high (100 and 300 microM) but not by the low (30 microM) concentration of hydroxocobalamin. Vasodilatation in response to sodium nitroprusside (10-100 nM) was totally abolished by 300 microM hydroxocobalamin. In endothelium-intact preparations, vasodilator responses to acetylcholine (0.3-3 microM) were significantly reduced or abolished by hydroxocobalamin (30-300 microM). The mean reduction by hydroxocobalamin of relaxations to acetylcholine was significantly greater than that of the equivalent response evoked by nitrergic nerve stimulation. The findings suggest that the nitrergic transmitter in the guinea-pig basilar artery may be quantitatively less susceptible than the endothelium-derived relaxing factor to the NO scavenger hydroxocobalamin.     

No evidence for a significant non-nitrergic, hyperpolarising factor contribution to field stimulation-induced relaxation of the mouse anococcygeus

1. The aim of the study was to determine whether a nerve-derived hyperpolarizing factor (NDHF) might contribute to non-adrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus when low concentrations of contractile agent are used to raise tone and low frequencies of field stimulation applied; such a non-nitrergic NDHF has been proposed to contribute to NANC relaxations of the rat anococcygeus and guinea-pig taenia coli. 2. Phenylephrine (0.1-100 microM) produced concentration-related contractions of the mouse isolated anococcygeus muscle; 0.2 microM phenylephrine (EC26) was used to raise tone in subsequent experiments. 3. Field stimulation (0.5, 1.0 and 5.0 Hz) produced frequency-dependent relaxations of phenylephrine-induced tone. In the presence of the nitric oxide synthase inhibitor L-NG-nitro-arginine (L-NOARG; 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 microM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01-5 microM) were unaffected by L-NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8-Br-cyclic GMP (10 microM). 4. Nifedipine (1 microM) reduced the contractile response to 0.2 microM phenylephrine by 38%; however, it had no effect on NANC relaxations. 5. It is concluded that NANC relaxations of the mouse anococcygeus are purely nitrergic and that there is no significant contribution from a putative NDHF.     

Inhibition of relaxations to nitrergic stimulation of the mouse anococcygeus by duroquinone

1. The role of copper/zinc superoxide dismutase (Cu/Zn SOD) in protection of nitrergic neurotransmission in the mouse anococcygeus was investigated by use of duroquinone (DQ), which generates superoxide anions within tissues via reduction by flavoprotein enzymes. 2. In control anococcygeus muscles, DQ (10-100 microM) produced concentration-related inhibition (-log IC40 = 4.41) of relaxations to exogenous nitric oxide (NO; 15 microM). Nitrergic relaxations induced by field stimulation (10 Hz; 10 s train) were much less affected, 100 microM DQ reducing nitrergic relaxations by only 14 +/- 6%. 3. Following incubation with the Cu/Zn SOD inhibitor, diethyldithiocarbamate (DETCA; 3 mM; 45 min incubation; 10 min washout), the inhibitory effects of DQ on relaxations to NO were potentiated (-log IC40 = 5.22), and clear, concentration-related inhibitions of nitrergic relaxations were now observed (-log IC40 = 4.54). In both cases, these inhibitions were partially reversed by Cu/Zn SOD (250 u ml-1). In DETCA-treated tissues, DQ (100 microM) also reduced relaxations to sodium nitroprusside (1 microM) and S-nitroso-glutathione (30 microM), but potentiated those to 8-Br-cyclic GMP (100 microM). 4. Neither hydroquinone (HQ: 100 microM) nor 1,4-benzoquinone (BQ: 100 microM), both of which reduced responses to exogenous NO, inhibited relaxations induced by field stimulation in DETCA-treated tissues. Indeed, when added during DQ-induced inhibition of nitrergic relaxations, both HQ and BQ produced partial reversal of the block. 5. DQ had no effect on the detection of superoxide anions estimated via the xanthine:xanthine oxidase chemiluminescence assay, or of authentic NO as measured by a chemical microsensor. However, the detection of both superoxide anions and NO in these assays was inhibited by inclusion of either HQ or BQ. 6. The results support the proposal that nitrergic transmission in the peripheral nervous system is protected by Cu/Zn SOD activity in the region of the neuroeffector junction, and this may explain the lack of effect of superoxide anion generating drugs such as DQ. Such an explanation does not hold for either HQ or BQ, which appear to be acting directly as free radical scavengers in these experiments.     

Characterisation of excitatory and inhibitory transmitter systems in prostate glands of rats, guinea pigs, rabbits and pigs

Excitatory and inhibitory transmitter systems were investigated in strips of prostate glands from rats, guinea pigs, pigs and rabbits. In strips from all species, electrical field stimulation (1 ms pulses at 1-30 Hz for 10 s) produced frequency-dependent contractions which were abolished by tetrodotoxin (1 microM). In strips from rats, guinea pigs and rabbits, contractions were reduced by prazosin (1 microM), guanethidine (10 microM) and atropine (2 microM), indicating the presence of noradrenergic and cholinergic mechanisms. However, the smooth muscle in the pig prostate appears to have a non-(nor)adrenergic non-cholinergic (NANC) excitatory innervation for which the transmitter was not identified. When noradrenergic and cholinergic mechanisms were blocked by guanethidine and atropine, respectively, and tone was raised with noradrenaline or methoxamine, field stimulation produced relaxations only in strips of rabbit prostate, and these were greatly reduced by N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM), providing functional evidence for a nitrergic relaxant innervation. In accord with this, nitric oxide (NO) synthase activity was considerably higher in rabbit than in rat or pig prostates.     

Effects of nitric oxide on proliferation and differentiation of rat brown adipocytes in primary cultures

1. In the present work, we study the effect of NO on the proliferation and differentiation of brown fat cells in primary cultures. 2. Brown fat precursor cells isolated from rat brown adipose tissue were cultured for 8 days until confluence and treated daily with the NO donating agents, S-nitroso-acetyl penicillamine (SNAP) or S-nitroso-L-glutathione (GSNO). Both agents (300 microM) decreased cell proliferation approximately 8 fold on day 8. The inhibitory effect of NO was unlikely to be due to cytotoxicity since (i) cells never completely lost their proliferation capacity even after 8 days of exposure to repeated additions of SNAP or GSNO, and (ii) the inhibitory effect was reversible after removal of the media containing NO donors. 3. Daily treatment with nitric oxide synthase inhibitors, such as NG-nitro-L-arginine methyl ester (L-NAME, 300 microM), led to the stimulation of cell proliferation by 44+/-5%, n=3, suggesting that NO, endogenously produced in brown adipocytes, may be involved in modulating cell growth. 4. Daily treatment with both SNAP or GSNO induced significant mitochondriogenesis, measured as the mitochondrial conversion of 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT) to formazan, whilst daily treatment with L-NAME was without effect. 5. The inhibition of cell proliferation by NO donors was accompanied by the expression of two genes coding for peroxisome proliferator activated receptor-gamma and uncoupling protein-1, which are upregulated during differentiation. 6. Increasing cyclic GMP in cells by 8-bromo-cyclic GMP (100-1000 microM) did not reproduce the observed NO effects on either cell number or gene expression. On the other hand, chronic treatment with the inhibitor of the NO-stimulated guanylyl cyclase, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), reduced the expression of peroxisome proliferator activated receptor-gamma and uncoupling protein-1.     

Neocuproine, a selective Cu(I) chelator, and the relaxation of rat vascular smooth muscle by S-nitrosothiols

1. A study has been made of the effect of neocuproine, a specific Cu(I) chelator, on vasodilator responses of rat isolated perfused tail artery to two nitrosothiols: S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and S-nitroso-glutathione (GSNO). 2. Bolus injections (10 microl) of SNAP or GSNO (10(-7)-10(-3) M) were delivered into the lumen of perfused vessels pre-contracted with sufficient phenylephrine (1-7 microM) to develop pressures of 100-120 mmHg. Two kinds of experiment were made: SNAP and GSNO were either (a) pre-mixed with neocuproine (10(-4) M) and then injected into arteries; or (b) vessels were continuously perfused with neocuproine (10(-5) M) and then injected with either pure SNAP or GSNO. 3. In each case, neocuproine significantly attenuated vasodilator responses to both nitrosothiols, although the nature of the inhibitory effect differed in the two types of experiment. We conclude that the ability of exogenous nitrosothiols to relax vascular smooth muscle in our ex vivo model is dependent upon a Cu(I) catalyzed process. Evidence is presented which suggests that a similar Cu(I)-dependent mechanism is responsible for the release of NO from endogenous nitrosothiols and that this process may assist in maintaining vasodilator tone in vivo.     

Influence of a selective guanylate cyclase inhibitor, and of the contraction level, on nitrergic relaxations in the gastric fundus

1. The influence of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) on non-adrenergic non-cholinergic (NANC) relaxations and the possible role of a nerve-derived hyperpolarizing factor in NANC relaxation were investigated in the rat gastric fundus. 2. ODQ (10(-6) and 10(-5) M) concentration-dependently inhibited the short-lasting relaxations by NO (2 x 10(-6) M-10(-4) M) administered as a bolus without influencing the relaxation by 3 x 10(-8) M isoprenaline. The relaxation by an infusion of NO was reduced to the same extent by 10(-6) and 10(-5) M ODQ. 3. The electrically induced short-lasting and sustained relaxations (40 V, 1 ms, 0.5-16 Hz, 10 s trains at 2 min interval or cumulative increase in the frequency every 2 min) in NANC conditions were inhibited to a similar extent by 10(-6) and 10(-5) M ODQ, and by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M). 4. ODQ (10(-6) M) and L-NAME (3 x 10(-4) M), administered after 5, 10 or 20 min of long-term stimulation, reversed the relaxation to a similar extent (approximately 50% at 2 Hz and 20% at 8 Hz). 5. When the tissues were contracted to 40% of maximum by adapting the concentration of prostaglandin F2alpha (PGF2alpha), the inhibitory effect of 3 x 10(-4) M L-NAME on relaxations induced by train and cumulative stimulation was the same as when tissues were contracted with 3 x 10(-7) M PGF2alpha. 6. The findings of this study illustrate that the relaxation by exogenous and endogenous NO in the rat gastric fundus is due to activation of soluble guanylate cyclase. During long-term electrical stimulation, the partial contribution of NO to NANC relaxation is maintained but it is small at higher frequencies of stimulation. Evidence for the contribution of a nerve-derived hyperpolarizing factor to NANC relaxation was not obtained.     

Nitrergic neurotransmission mediates the non-adrenergic non-cholinergic responses in the clitoral corpus cavernosum of the rabbit

The corpus cavernosum is the erectile tissue in the penis and clitoris. Although nitrergic neurotransmission has been characterized in detail in the penile corpus cavernosum, functional studies on the inhibitory non-adrenergic non-cholinergic (NANC) transmission in the clitoral corpus cavernosum have been lacking. Here we demonstrate that electrical field stimulation (EFS) induces NANC relaxation responses in the clitoral corpus cavernosum of the rabbit. These responses were inhibited by NG-nitro-L-arginine methylester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) or tetrodotoxin. The inhibitory effect of L-NAME was partially reversed by L-arginine but not by D-arginine. EFS-induced relaxations were enhanced by an inhibitor of type V cyclic GMP phosphodiesterase, zaprinast. These results suggest that nitrergic neurotransmission is responsible for the NANC relaxation responses in the clitoral corpus cavernosum of the rabbit.     

Opposite effects of nitric oxide donors on DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide

1. The effects of three different NO donors on tert-butylhydroperoxide (tB-OOH)-induced DNA cleavage and toxicity were investigated in U937 cells. 2. Treatment with S-nitroso-N-acetyl-penicillamine (SNAP, 1-30 microM), while not in itself DNA-damaging, potentiated the DNA strand scission induced by 200 microM tB-OOH in a concentration-dependent fashion. The enhancing effects of SNAP were observed with two different techniques for the assessment of DNA damage. Decomposed SNAP was inactive. S-nitrosoglutathione (GSNO, 300 microM) and (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NO, 1 mM) also increased DNA cleavage generated by tB-OOH and these responses, as well as that mediated by SNAP, were prevented by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (PTIO). 3. SNAP neither inhibited catalase activity nor increased the formation of DNA lesions in cells exposed to H2O2. Furthermore, SNAP did not affect the rate of rejoining of the DNA single strand breaks generated by tB-OOH. 4. Under the conditions utilized in the DNA damage experiments, treatment with tB-OOH alone or associated with SNAP did not cause cell death. However, SNAP as well as GSNO markedly reduced the lethal response promoted by millimolar concentrations of tB-OOH and these effects were abolished by PTIO. Decomposed SNAP was inactive. 5. It is concluded that low levels of NO donors, which probably release physiological concentrations of NO, enhance the accumulation of DNA single strand breaks in U937 cells exposed to tB-OOH. This NO-mediated effect appears to (a) not depend on inhibition of either DNA repair (which would increase the net accumulation of DNA lesions by preventing DNA single strand break removal) or catalase activity (which would also enhance the net accumulation of DNA lesions since H2O2 is one of the species mediating the tB-OOH-induced DNA cleavage) and (b) be caused by enforced formation of tB-OOH-derived DNA-damaging species. In contrast to these results, similar concentrations of NO prevented cell death caused by millimolar concentrations of tB-OOH. Hence, DNA single strand breakage generated by tB-OOH in the absence or presence of NO does not represent a lethal event.     

L-citrulline recycling in opossum internal anal sphincter relaxation by nonadrenergic, noncholinergic nerve stimulation

BACKGROUND & AIMS: L-citrulline formed stoichiometrically along with nitric oxide (1:1) from L-arginine may be enzymatically converted to L-arginine. The possibility of L-citrulline recycling in the maintenance of nitrergic neurotransmission in the opossum internal anal sphincter (IAS) smooth muscle strips was investigated. METHODS: Responses to nonadrenergic, noncholinergic (NANC) nerve stimulation by electrical field stimulation (EFS) (either short-train or continuous stimulation) on the basal IAS tension were recorded before and after the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA), L-NNA plus L-citrulline, or L-arginine. During continuous EFS, when the basal IAS tone after the initial relaxation had recovered to almost pre-EFS levels, the effects of L-citrulline or L-arginine were examined before and after L-glutamine, which is a putative blocker of L-citrulline uptake. RESULTS: Inhibition of NANC nerve-mediated IAS relaxation by L-NNA was reversed by L-citrulline as well as L-arginine. L-Citrulline and L-arginine caused concentration-dependent relaxation of the IAS tone recovered during the prolonged EFS. L-Glutamine blocked the responses of L-citrulline but not of L-arginine. Furthermore, L-glutamine increased the speed of recovery of IAS tone during continuous EFS. CONCLUSIONS: L-citrulline recycling may be responsible for the maintenance of IAS relaxation during frequent short-train and prolonged NANC nerve stimulation.     

Evidence that mechanisms dependent and independent of nitric oxide mediate endothelium-dependent relaxation to bradykinin in human small resistance-like coronary arteries

1. The effects of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NOARG), the NO scavenger, oxyhaemoglobin (HbO) and high extracellular K+ upon endothelium-dependent relaxation to bradykinin were investigated in human isolated small coronary arteries. 2. Endothelium-dependent relaxations to bradykinin were compared in vessels contracted to approximately 50% of their maximum contraction to 124 mM KCl Krebs solution, regardless of treatments, with the thromboxane A2 mimetic, U46619 and acetylcholine. All relaxations were expressed as percentage reversal of the initial level of active force. 3. L-NOARG (100 microM) caused a small but significant, 12% (P < 0.01), decrease in the maximum relaxation (Rmax: 91.5 +/- 5.4%) to bradykinin but did not significantly affect the sensitivity (pEC50: 8.08 +/- 0.17). Increasing the concentration of L-NOARG to 300 microM had no further effect on the pEC50 or Rmax to bradykinin. HbO (20 microM) and a combination of HbO (20 microM) and L-NOARG (100 microM) reduced Rmax to bradykinin by 58% (P < 0.05) and 54% (P < 0.05), respectively. HbO (20 microM) and L-NOARG (100 microM, combined but not HbO (20 microM) alone, caused a significant 11 fold (P < 0.05) decrease in sensitivity to bradykinin. HbO (20 microM) decreased the sensitivity to the endothelium-independent NO donor, S-nitroso-N-acetylpenicillamine (SNAP), approximately 17 fold (P < 0.05). 4. Raising the extracellular concentration of K+ isotonically to 30 mM, reduced the Rmax to bradykinin from 96.6 +/- 3.1% to 43.9 +/- 10.1% (P < 0.01) with no significant change in sensitivity. A combination of HbO, L-NOARG and high K+ (30 mM) abolished the response to bradykinin. High K+ did not change either the sensitivity or maximum relaxation to SNAP. 5. In conclusion, L-NOARG does not completely inhibit endothelial cell NO synthesis in human isolated small coronary arteries. By comparison, HbO appeared to block all the effects of NO in this tissue and revealed that most of the relaxation to bradykinin was due to NO. The non-NO -dependent relaxation to bradykinin in the human isolated small coronary arteries appeared to be mediated by a K(+)-sensitive vasodilator mechanism, possibly endothelium-derived hyperpolarizing factor (EDHF).     

Biotransformation and cytotoxic properties of NO-donors on MCF7 and U251 cell lines

Previous studies have shown a role for nitric oxide (NO) as a cytotoxic effector. In the present work, two chemically different NO-donors such as glyceryl trinitrate (GTN) and S-nitroso-N-acetylpenicillamine (SNAP) were evaluated for both NO release and cytostatic/cytotoxic properties. Nitrite accumulation in the supernatant of MCF-7 and U251 cell lines indicated a greater and quickly release of NO derived from SNAP. A time-course of hemoglobin absorption spectral changes showed a greater release of NO derived from GTN in presence of cells compared to the values observed in the media, confirming that the release of NO by GTN can be enzymatic and non-enzymatic. On the contrary, SNAP generated NO without contribution of cellular components and saturated oxyhemoglobin quickly, within 2 hours. Both NO-donors inhibited thymidine incorporation in a similar manner and dose-dependently in U251 cells, but not in MCF-7 cells, where SNAP at the highest tested dose of 1000 microM induced only a 33% cytostatic effect. About trypan blue exclusion test, after 24 h GTN and SNAP, releasing similar amounts of NO, showed comparable cytotoxic effects on U251 cells (50% dead cells), but not on MCF-7 cells, where GTN resulted more cytotoxic. From our data, the "in vitro" antitumoral activity of NO-donors seems to be related to the type of tumor cell lines, to the amount and duration of NO release.     

Excitatory transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of cannabinoid CB1 receptors

1. The effect of cannabinoid drugs has been investigated on cholinergic and non-adrenergic non-cholinergic (NANC) contractile responses to the circular smooth muscle of guinea-pig ileum elicited by electrical field stimulation (EFS). 2. The cannabinoid receptor agonist WIN 55,212-2 (1-1000 nM) and the putative endogenous ligand anandamide (0.1-100 microM) both produced a concentration-dependent inhibition of the cholinergic (9-57% and 1-51% inhibition) and NANC (9 55% and 2-57% inhibition) contractile responses. WIN 55,212-2 and anandamide did not modify the contractions produced by exogenous acetylcholine or substance P. 3. Apamin (30 nM), a blocker of Ca2+-activated K+ channels, reduced the inhibitory effect of WIN 55,212-2 on cholinergic, but not NANC, contractile response. NG-nitro-L-arginine methyl ester (100 microM), an inhibitor of nitric oxide synthase, or naloxone (1 microM), an opioid receptors antagonist, did not modify the inhibitory effect of WIN 55,212-2 on both cholinergic and NANC contractions. 4. The inhibitory effects of WIN 55,212-2 and anandamide on both cholinergic and NANC contractile response was competitively antagonized by the cannabinoid CB1 receptor antagonist SR 141716A (10-1000 nM). 5. In absence of other drugs, SR 141716A (1-1000 nM) enhanced cholinergic (1-45% increase) and NANC (2-38% increase) contractile responses elicited by electrical stimulation, but did not modify the contractions produced by acetylcholine or substance P. 6. It is concluded that activation of prejunctional cannabinoid CB1 receptors produces inhibition of cholinergic and NANC excitatory responses in the guinea-pig circular muscle. The inhibition of cholinergic (but not NANC) transmission involves activation of apamin-sensitive K+ channels. In addition, an endogenous cannabinoid ligand could inhibit cholinergic and NANC transmission in the guinea-pig ileal circular muscle.     

Coronary endothelial dysfunction after Kawasaki disease: evaluation by intracoronary injection of acetylcholine

1. Caffeine (Cf) enhances the DNA cleavage induced by tert-butylhydroperoxide (tB-OOH) in U937 cells via a mechanism involving Ca2+-dependent mitochondrial formation of DNA-damaging species (Guidarelli et al., 1997b). Nitric oxide (NO) is not involved in this process since U937 cells do not express the constitutive nitric oxide synthase (cNOS). 2. Treatment with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP, 10 microM), or S-nitrosoglutathione (GSNO, 300 microM), however, potentiated the DNA strand scission induced by 200 microM tB-OOH. The DNA lesions generated by tB-OOH alone, or combined with SNAP, were repaired with superimposable kinetics and were insensitive to anti-oxidants and peroxynitrite scavengers but suppressed by iron chelators. 3. SNAP or GSNO did not cause mitochondrial Ca2+ accumulation but their enhancing effects on the tB-OOH-induced DNA strand scission were prevented by ruthenium red, an inhibitor of the calcium uniporter of mitochondria. Furthermore, the enhancing effects of both SNAP and GSNO were identical to and not additive with those promoted by the Ca2+-mobilizing agents Cf or ATP. 4. The SNAP- or GSNO-mediated enhancement of the tB-OOH-induced DNA cleavage was abolished by the respiratory chain inhibitors rotenone and myxothiazol and was not apparent in respiration-deficient cells. 5. It is concluded that, in cells which do not express the enzyme cNOS, exogenous NO enhances the accumulation of DNA single strand breaks induced by tB-OOH via a mechanism involving inhibition of complex III.     

Anesthetic management in epidermolysis bullosa: review of 129 anesthetic episodes in 32 patients

Non-adrenergic non-cholinergic (NANC) inhibitory nerves have been described in all regions of the gastrointestinal tract, but have not been shown previously in the human gall bladder. Electrical field stimulation was used in the presence of various agonists and antagonists to show NANC inhibitory innervation in strips of human gall bladder muscle. Gall bladder strips were set up isometrically in an organ bath containing oxygenated Krebs's solution. Electrical field stimulation was applied at 10 Hz, pulse width 0.3 ms and supramaximal voltage at intervals of 3 to 5 minutes. Of 60 strips that contracted in response to electrical field stimulation, 30 showed relaxation on electrical field stimulation in the presence of either carbachol (5-10 microM) or else atropine (0.5-2 microM) plus cholecystokinin octapeptide (0.01-0.1 microM) or caerulein (0.1 nM) or histamine (5-10 microM). In 22 strips this relaxation was not abolished by guanethidine (2-5 microM) showing the NANC nature of this response. The NANC relaxation was abolished by L-nitroarginine (100 microM) and this effect was partly reversible by L arginine (200 microM). All responses to electrical field stimulation were abolished by tetrodotoxin (0.2-2 microM). These results show for the first time a NANC inhibitory innervation in human gall bladder muscle. The probable neurotransmitter is nitric oxide.     

Cocaine exposure prebreeding to weaning: maternal and offspring effects

1. The distribution of NADPH-diaphorase positive and catecholamine-containing nerve structures, and functional noradrenergic-nitrergic interactions, were studied in the rat anococcygeus muscle. 2. The morphological findings demonstrated NADPH-diaphorase positive neurons mostly as aggregates in intramural ganglia, nerve tracts and few single nerve fibres forming plexus-like structures. 3. The nitric oxide synthase inhibitor NG-nitro-L-arginine (L-NOARG) inhibited concentration-dependently the nitrergic relaxation, an effect reversed by L-arginine. The drug had dual effects on noradrenergic contractile responses: at lower concentrations (0.1-10 microM) it decreased the amplitude of contractions and this was not affected by L-arginine; higher concentrations (50-500 microM) potentiated the contractions, an effect that was prevented by L-arginine. 4. The electron acceptor, nitro blue tetrazolium (NBT) produced a rapid inhibition of the noradrenergic contractile responses (EC50 0.178 +/- 0.041 microM). The drug decreased the tone of the preparations. However, it potentiated concentration-dependently the nitrergic relaxations. 5. NBT (1 microM) had no significant effect on the relaxations induced by exogenously applied nitric oxide (NO)-donor sodium nitroprusside (SNP, 0.01-50 microM). However, the effect of NBT (0.1-10 microM) on the electrically induced relaxation was significantly decreased by L-NOARG (10 and 50 microM). The inhibition was of a non-competitive type. 6. Neither L-NOARG (100 microM) nor NBT (1 microM) had any effect on the spontaneous or electrically-induced release of 3H-radioactivity from the tissues preincubated in [3H]-noradrenaline. 7. It is concluded that L-arginine-NO pathway can modulate noradrenergic transmission in the rat anococcygeus muscle at postjunctional, but not prejunctional site(s).     

The adrenergic, cholinergic and NANC nerve-mediated contractions of the female rabbit bladder neck and proximal, medial and distal urethra

1. The nerve-mediated contraction of the female rabbit bladder neck and different portions of the urethra (proximal, medial and distal) was studied in vitro by electrical stimulation (50 V, 30 Hz, 0.05 ms width, trains of 5 s every 5 min) by use of a superfusion system. 2. The amplitude (Emax) and the duration (Dmax) of the stimulated contraction were studied in the four tissues. The Emax value was significantly higher in distal urethra (2.07+/-0.15 g) compared to the bladder neck (1.08+/-0.10 g), proximal urethra (0.73+/-0.07 g) and medial urethra (0.87+/-0.07 g). In contrast, the Dmax value appeared slightly but significantly lower (P<0.05) in distal urethra (68.5+/-2.3 s) than in bladder neck (76.7+/-6.0 s), proximal urethra (84.5+/-5.0 s) and medial urethra (81.3+/-3.5 s). 3. Cocaine (1 microM) significantly increased the basal Emax values in medial and distal urethra and the basal Dmax values in the four tissues. 4. Prazosin (1 microM) significantly reduced E max value in proximal, medial and distal urethra and Dmax value in bladder neck and proximal urethra. Atropine (1 microM) also significantly reduced Emax values in bladder neck and proximal urethra and reduced Dmax value in bladder neck, but not in other tissues. Yohimbine (0.1 microM) was devoid of effect in the four tissues. 5. The association of prazosin (1 microM) and atropine (1 microM) did not modify the Emax and the Dmax values of the electrically-induced contractions, except in proximal urethra and in bladder neck where an additive inhibitory effect (on Emax only) was observed compared to prazosin and atropine alone. 6. The residual contractile response after combined treatment with prazosin and atropine was significantly diminished by tetrodotoxin (TTX; 1 microM) but not completely abolished. These NANC contractions were insensitive to P2X-purinoceptor desensitization by continuous tissue perfusion with alpha,beta-methylene ATP (30 microM). 7. These results demonstrate that bladder neck and proximal urethra are mainly innervated by the parasympathetic nervous system, whereas medial and distal urethras are to a greater extent under the control of the sympathetic innervation. The residual responses, insensitive to prazosin and atropine, may indicate a NANC innervation in the four tissues. However, the nature of the NANC neurotransmitter remains to be identified.     

Vitamin C attenuates nitrate tolerance independently of its antioxidant effect

In LLC-PK1 kidney epithelial cells, a 5-h pretreatment with glyceryl trinitrate (GTN) resulted in substantial desensitization of the intracellular cyclic GMP response to a subsequent 10-min challenge with GTN (1 microM). GTN-tolerant cells were fully sensitive to the spontaneous nitric oxide (NO) donor spermine NONOate, which does not require enzymatic bioactivation. Cyclic GMP stimulation by GTN was up to 3.1-fold higher when vitamin C (1-10 mM) was present during the pretreatment period. In contrast, other oxygen radical scavengers such as tiron or dimethylsulfoxide and the NO scavenger PTIO left tolerance induction unaltered. Together, our results suggest that reactive oxygen species or NO do not contribute to the development of nitrate tolerance. Tolerance reduction by vitamin C may be due to a stabilizing effect on enzymes involved in the bioconversion of GTN to NO.     

Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-alpha-induced apoptosis by inducing heat shock protein 70 expression

Nitric oxide (NO) and tumor necrosis factor-alpha (TNFalpha) play important roles in the pathogenesis of liver disease during acute inflammation. The present study was designed to elucidate the effect of NO pre-exposure on TNFalpha-induced hepatotoxicity. Pretreatment of primary cultures of rat hepatocytes with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced the expression of heat shock protein 70 (HSP70) mRNA and protein, which was associated with thermotolerance and cytoprotection from TNFalpha+actinomycin D-induced hepatotoxicity and apoptosis. SNAP transiently changed the intracellular redox state by inducing glutathione (GSH) oxidation associated with the formation of S-nitrosoglutathione (GSNO). HSP70 mRNA was also induced by the GSH-oxidizing agent diamide and the GSH-conjugating agent N-ethylmaleimide, suggesting that NO induces HSP70 expression through GSH oxidation. The protective effect of SNAP pretreatment on TNFalpha-induced apoptosis correlated with the level of HSP70 expression. SNAP pretreatment inhibited reactive oxygen intermediate generation and lipid peroxidation effects that were reversed by blocking HSP70 expression using an antisense oligonucleotide to HSP70. Finally, endogenous NO formation, induced in hepatocytes stimulated with interferon-gamma and interleukin-1beta, led to the formation of GSNO and GSSG, induced HSP70, and attenuated TNFalpha-mediated cytotoxicity. These findings demonstrated that NO can induce resistance to TNFalpha-induced hepatotoxicity, possibly through the stimulation of HSP70 expression.