Nociceptin inhibits non-adrenergic non-cholinergic contraction in guinea-pig airway

1. Electrical field stimulation (EFS) of guinea-pig isolated main bronchi induced a non-adrenergic non-cholinergic (NANC) contractile response. Nociceptin (0.01–1 μM) significantly inhibited the contractile response to EFS (P<0.01), but not to capsaicin (P>0.05).
2. The μ-, δ- and κ-opioid receptor antagonists, naloxone (0.3 μM), naltrindole (3 μM) and nor-binaltorphimine (1 μM), respectively, did not significantly affect the inhibitory effect of nociceptin (0.03 μM; P>0.05).
3. The novel nociceptin antagonist, [Phe¹ψ(CH₂-NH)Gly²]nociceptin(1–13)NH₂ (0.03–1 μM); the σ ligands, carbetapentane (30 μM), 3-phenylpiperidine (30–100 μM) and (+)-cyclazocine (10–100 μM) significantly reversed the inhibitory effect of nociceptin (0.03 μM, P<0.05). In contrast, rimcazole, did not significantly reverse the inhibitory effect of nociceptin (0.03 μM) at any concentration tested (P>0.05).
4. EFS of guinea-pig bronchial preparations significantly increased SP-LI release above basal SP-LI (P<0.05). In the presence of nociceptin (1 μM), EFS induced a significant increase in SP-LI release above basal SP-LI release (P<0.05). Nociceptin caused a 59±11% (n=5) inhibition of EFS-induced release of SP-LI.
5. Nociceptin reduces the release of sensory neuropeptides induced by EFS, but not capsaicin, from guinea-pig airways. These experiments provide further evidence for a role for nociceptin in regulating the release of sensory neuropeptides in response to EFS.

     

The effects of nifedipine and other calcium antagonists on the glibenclamide-sensitive K+ currents in smooth muscle cells from pig urethra

1. The effects of nifedipine on both levcromakalim-induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch-clamp techniques (conventional whole-cell configuration and cell-attached patches).
2. Nifedipine had a voltage-dependent inhibitory effect on voltage-dependent Ba²⁺ currents at −50 mV (K_i=30.6 nM).
3. In current-clamp mode, subsequent application of higher concentrations of nifedipine (⩾30 μM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately −82 mV by application of 100 μM levcromakalim.
4. The 100 μM levcromakalim-induced inward current (symmetrical 140 mM K⁺ conditions, −50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 μM). In contrast, Bay K 8644 (1 μM) possessed no activating effect on the amplitude of this glibenclamide-sensitive current.
5. When 100 μM nifedipine was included in the pipette solution during conventional whole-cell recording at −50 mV, application of levcromakalim (100 μM) caused a significant inward membrane current which was suppressed by 5 μM glibenclamide. On the other hand, inclusion of 5 μM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current.
6. The levcromakalim-induced K⁺ channel openings in cell-attached configuration were suppressed by subsequent application of 5 μM glibenclamide but not of 100 μM nifedipine.
7. These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide-sensitive 43 pS K⁺ channel activity mainly through extracellular blocking actions on the K⁺ channel itself.

     

The intravenous administration of tumor necrosis factor alpha,interleukin 8 and macrophage-derived neutrophil chemotactic factor inhibits neutrophil migration by stimulating nitric oxide production

1. The i.v. administration of tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8) and the recently described macrophage-derived neutrophil chemotactic factor (MNCF) inhibits the recruitment of neutrophils to the inflammatory site.
2. Pretreatment of mice with the NO synthase antagonist, N^G-monomethyl-L-arginine (L-NMMA, 15–60 mg kg⁻¹), but not the inactive enantiomer D-NMMA (30 mg kg⁻¹), prevented in a dose-dependent manner the TNF-α, IL-8 and MNCF-mediated inhibition of neutrophil migration into thioglycollate-challenged peritoneal cavities.
3. Treatment of the neutrophils with TNFα (10⁻⁷ M), IL-8 (10⁻⁷ M) or MNCF blocked their migration towards FMLP in the chemotaxis assay. The pretreatment of the neutrophils with L-NMMA (50–200 μM) prevented in a dose-dependent manner the inhibition of FMLP-induced chemotaxis by IL-8, but did not alter the inhibition caused by TNF-α or MNCF. Different concentrations of the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholino-sydnonimine (SIN-1), did not alter this chemotaxis.
4. Preincubating the neutrophils with L-NMMA (200 μM) significantly increased the TNF-α (10⁻⁷ M) and MNCF-mediated neutrophil adhesion to unstimulated endothelial cells, but had no effect on IL-8 (10⁻⁷ M)-mediated adhesion.
5. Although NO donors did not directly affect the mechanisms of neutrophil motility, NO is involved in the in vitro inhibitory action of IL-8 on chemotaxis. The TNF-α and MNCF-mediated inhibition of neutrophil migration seems to be indirect, by affecting the mechanisms of adhesion. It was concluded that TNF-α-, IL-8- and MNCF-mediated inhibition of neutrophil migration is associated with the stimulation of NO production.

     

Characterization of angiotensin II formation in human isolated bladder by selective inhibitors of ACE and human chymase: a functional and biochemical study

1. Functional recordings of smooth muscle tension and biochemical experiments on membrane fractions were performed to characterize angiotensin II (AII) formation in human isolated bladder smooth muscle.
2. A novel human chymase inhibitor CH 5450 (Z-Ile-Glu-Pro-Phe-CO₂Me) and a recently developed human chymase substrate Pro¹¹-,D-Ala¹²)-angiotensin I, claimed to be resistant to angiotensin converting enzyme (ACE) and carboxypeptidase, were used.
3. Angiotensin I (AI) (0.3 μM) induced a contractile response amounting to 58±5% (n=12) of the initial K⁺ (124 mM)-induced contractions. This response was reduced to 36±3% (n=8) by the ACE-inhibitor enalaprilat (10 μM), while pretreatment with soybean trypsin inhibitor (STI 200 μg ml⁻¹) or CH 5450 (10 μM) had no effect. However, the combination of enalaprilat and STI reduced the AI-induced contractions to 19±5% (n=6), and the combination of enalaprilat and CH 5450 caused an almost complete inhibition of the AI-induced contractions to 1±1% (n=6).
4. The substrate (Pro¹¹-,D-Ala¹²)-AI (3 μM) produced contractions which amounted to 57±4% (n=13) of the initial K⁺ (124 mM) contractions. These contractions were not affected by enalaprilat (10 μM). On the other hand, STI (200 μg ml⁻¹) and CH 5450 (10 μM) added separately, depressed the (Pro¹¹-,D-Ala¹²)-AI-induced contractions to 34±5% (n=6) and 24±4% (n=6), respectively. The combination of enalaprilat and STI or enalaprilat and CH 5450 did not produce any further inhibition.
5. Experiments with detrusor membrane fractions incubated with AI (50 μM) were performed. In the presence of enalaprilat (100 μM), carboxypeptidase inhibitor CPI (10 μg ml⁻¹) and aprotinin (15 μM), CH 5450 (10 nM–1 μM) caused a concentration-dependent inhibition of AII formation.
6. The results confirm that AII is a potent contractile agent in the human isolated detrusor muscle. They also indicate that the serine protease responsible for AII formation in the human bladder in vitro is human chymase or an enzyme similar to human chymase.

     

Vascular actions of octreotide in the portal hypertensive rat

1. We have investigated the actions of the somatostatin analogue octreotide in the portal hypertensive Wistar rat in vivo and in rat small mesenteric artery and aorta in vitro.
2. In small mesenteric artery, octreotide (0.1–0.3 μM) failed to produce any direct contraction, nor did it affect contractions to noradrenaline (NA, 10 μM) or endothelium-dependent relaxations to acetylcholine.
3. In rat aorta, octreotide (0.3 μM) and somatostatin (1 μM) failed to affect contractions to NA (1 μM), or concentration-contractile response curves to NA.
4. In rat vas deferens, octreotide and somatostatin significantly reduced contractile responses to electrical stimulation with pD₂ values (−log IC₅₀) of 8.19±0.10 (n=4) and 8.16±0.26 (n=4), respectively. Hence, the lack of effect of these agents in aorta or mesenteric artery was not due to lack of efficacy or inappropriate choice of concentration.
5. In the anaesthetized portal hypertensive rat, intravenous injection of octreotide (1–100 μg kg⁻¹) did not significantly affect systemic blood pressure, nor did it affect mesenteric vascular conductance as measured by laser doppler flow probes. However, octreotide (100 μg kg⁻¹) significantly reduced vascular conductance to 74.2±7.7% of control (n=6) in porto-systemic shunt vessels as measured by laser doppler flow probes.
6. Phenylephrine (1 μg kg⁻¹) significantly raised blood pressure and significantly decreased vascular conductance in both mesenteric (66.6±3.7% of control) and porto-systemic shunt vessels (58.7±10.0% of control).
7. It was concluded that octreotide has selective effects on porto-systemic shunt vessles in vivo in the portal hypertensive rat.

     

Ovalbumin-induced neurogenic inflammation in the bladder of sensitized rats

1. We have developed and characterized a model of immediate hypersensitivity/inflammation of the urinary bladder in vivo induced by local application of ovalbumin (OA) in OA- sensitive female rats. Two parameters of the inflammatory response were assessed following OA challenge: plasma protein extravasation (PPE) and changes in smooth muscle reactivity. The former was estimated by measurement of Evans blue extravasation at 0.5, 2, 4, 8 and 24 h time point following in vivo challenge. Changes in reactivity were determined by measurement of isotonic tension responses of urinary bladder strips following OA challenge in vitro.
2. Acute in vivo intravesical OA challenge (10 mg in 0.3 ml saline) in actively sensitized female Wistar rats caused a time-dependent PPE in the urinary bladder which was biphasic with peak responses at 2–4 and 24 h.
3. The PPE response to acute OA challenge, above base-line, at 2 h was abolished by systemic capsaicin pretreatment (50 mg kg⁻¹, s.c., 4 days before use) (P<0.05) whilst the response at 24 h was unaffected. The 2 h time point was then used for further studies.
4. Degranulation of mast cells, achieved by pretreatment with compound 48/80 (5 mg kg⁻¹, s.c. for 3 consecutive days), completely abolished the PPE response to OA challenge at the 2 h time point.
5. The tachykinin NK₁ receptor antagonist, SR 140333 (0.1 μmol kg⁻¹, i.v.), abolished the 2 h PPE response whilst the tachykinin NK₂ receptor antagonist MEN 11420 (0.1 μmol kg⁻¹, i.v.) appeared to reduce the response by approximately 50% but this did not reach significance. The bradykinin B₂ receptor antagonist, Hoe 140 (0.1 μmol kg⁻¹, i.v.), similarly to SR 140333, blocked the 2 h PPE response to OA, whereas the selective B₁ receptor antagonist B 9858 (0.1 μmol kg⁻¹, i.v.) had no significant effect. Inhibition of cyclo-oxygenase (COX) achieved by pretreatment with the COX inhibitor dexketoprofen (5.3 μmol kg⁻¹, i.v.) also blocked the PPE response, whilst the leukotriene receptor antagonist ONO 1078 (1 μmol kg⁻¹, i.v.) significantly reduced PPE by about 80%.
6. In the rat isolated urinary bladder OA (1 mg ml⁻¹) challenge produced a biphasic response with a rapidly achieved maximal contraction followed by a sustained contraction for approximately 25 min. In vitro capsaicin pretreatment (10 μM for 15 min) significantly attenuated the duration of the sustained contraction whilst having no effect on the maximum contractile response achieved. In vivo pretreatment of animals with compound 48/80 significantly attenuated (42%) the maximum contractile response. Combination of both treatments almost completely abolished the response. In vitro treatment with Hoe 140 (1 μM) had no significant effect on the response to OA and neither did ONO 1078 (1 μM).
7. These results show that both the early inflammatory response and alterations in smooth muscle reactivity to OA challenge in actively sensitized animals are dependent on mast cell degranulation and the activation of sensory C-fibres. Furthermore this model of allergic cystitis may be useful for investigating both the processes involved and potential novel therapies in the treatment of interstitial cystitis.

     

Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

1. Radioligand binding and patch-clamp techniques were used to study the actions of γ-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5α-pregnan-3α-ol-20-one on rat α1 and β3 GABA_A receptor subunits, expressed either alone or in combination.
2. Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [³⁵S]-tert-butyl bicyclophosphorothionate ([³⁵S]-TBPS) (<0.03 pmol mg⁻¹ protein). GABA (100 μM) applied to whole-cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents.
3. The membranes of cells expressing β3 cDNAs bound [³⁵S]-TBPS (∼1 pmol mg⁻¹ protein). However, the binding was not influenced by GABA (10 nM–100 μM). Neither GABA (100 μM) nor picrotoxin (10 μM) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABA^A receptors or spontaneously active ion channels.
4. GABA (10 nM–100 μM) modulated [³⁵S]-TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μM–1 mM) also dose-dependently activated inward currents with an EC₅₀ of 9 μM recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV.
5. Propofol (10 nM–100 μM), pentobarbitone (10 nM–100 μM) and 5α-pregnan-3α-ol-20-one (1 nM–30 μM) modulated [³⁵S]-TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μM), pentobarbitone (1 mM) and 5α-pregnan-3α-ol-20-one (10 μM) also activated currents recorded from cells expressing α1β3 receptors.
6. Propofol (1 μM–1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α-pregnan-3α-ol-20-one (10 μM) failed to activate detectable currents.
7. Propofol (100 μM)-activated currents recorded from cells expressing either α1β3 or β3 receptors reversed at the C1⁻ equilibrium potential and were inhibited to 34±13% and 39±10% of control, respectively, by picrotoxin (10 μM). 5α-Pregnan-3α-ol-20-one (100 nM) enhanced propofol (100 μM)-evoked currents mediated by α1β3 receptors to 1101±299% of control. In contrast, even at high concentration 5α-pregnan-3α-ol-20-one (10 μM) caused only a modest facilitation (to 128±12% of control) of propofol (100 μM)-evoked currents mediated by β3 homomers.
8. Propofol (3–100 μM) activated α1β3 and β3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 μM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a ‘surge'' current on cessation of propofol (300 μM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5α-pregnan-3α-ol-20-one (10 μM) to cells expressing α1β3 receptors.
9. These observations demonstrate that rat β3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABA_A receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

     

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor

1. DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone) was identified as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor.
2. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid-dependent production of prostaglandin E₂ (PGE₂) with at least a 1,000 fold selectivity for COX-2 (IC₅₀=41±14 nM) over COX-1 (IC₅₀>50 μM). Indomethacin was a potent inhibitor of both COX-1 (IC₅₀=18±3 nM) and COX-2 (IC₅₀=26±6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B₂ (TXB₂) by Ca²⁺ ionophore-challenged human platelets (IC₅₀>50 μM and 4.1±1.7 nM, respectively).
3. DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a K_i value of 140±68 μM for the initial reversible binding to enzyme and a k₂ value of 0.11±0.06 s⁻¹ for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62±26 μM and 0.06±0.01 s⁻¹, respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1 : 1 stoichiometry and to dissociate only very slowly (t_1/2=1–3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site.
4. Inhibition of purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC₅₀=63±5 μM at 0.1 μM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1.
5. DFU inhibited lipopolysaccharide (LPS)-induced PGE₂ production (COX-2) in a human whole blood assay with a potency (IC₅₀=0.28±0.04 μM) similar to indomethacin (IC₅₀=0.68±0.17 μM). In contrast, DFU was at least 500 times less potent (IC₅₀>97 μM) than indomethacin at inhibiting coagulation-induced TXB₂ production (COX-1) (IC₅₀=0.19±0.02 μM).
6. In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 μM), DFU inhibited COX-1 with an IC₅₀ value of 13±2 μM as compared to 20±1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac>indomethacin∼naproxen>nimesulide∼ meloxicam∼piroxicam>NS-398∼SC-57666>SC-58125>CGP 28238∼etodolac>L-745,337>DFU.
7. DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED₅₀ of 1.1 mg kg⁻¹ vs 2.0 mg kg⁻¹ for indomethacin) and hyperalgesia (ED₅₀ of 0.95 mg kg⁻¹ vs 1.5 mg kg⁻¹ for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED₅₀=0.76 mg kg⁻¹ vs 1.1 mg kg⁻¹ for indomethacin).
8. In a sensitive model in which ⁵¹Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg⁻¹, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg⁻¹), meloxicam (3 mg kg⁻¹) or etodolac (10–30 mg kg⁻¹). A 5 day administration of DFU in squirrel monkeys (100 mg kg⁻¹) did not affect chromium leakage in contrast to diclofenac (1 mg kg⁻¹) or naproxen (5 mg kg⁻¹).
9. The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX-1, a consistent high selectivity of inhibition of COX-2 over COX-1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX-1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti-inflammatory effect to gastropathy can be achieved with a selective COX-2 inhibitor.

     

Insulin-releasing action of the novel antidiabetic agent BTS 67 582

1. BTS 67 582 (1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate) is a novel antidiabetic agent with a short-acting insulin-releasing effect. This study examined its mode of action in the clonal B-cell line BRIN-BD11.
2. BTS 67 582 increased insulin release from BRIN-BD11 cells in a concentration-dependent manner (10⁻⁸ to 10⁻⁴ M) at both non-stimulating (1.1 mM) and stimulating (16.7 mM) concentrations of glucose.
3. BTS 67 582 (10⁻⁴ M) potentiated the insulin-releasing effect of a depolarizing concentration of K⁺ (30 mM), whereas the K⁺ channel openers pinacidil (400 μM) and diazoxide (300 μM) inhibited BTS 67 582-induced release.
4. Suppression of Ca⁺ channel activity with verapamil (20 μM) reduced the insulin-releasing effect of BTS 67 582 (10⁻⁴ M).
5. BTS 67 582 (10⁻⁴ M) potentiated insulin release induced by amino acids (10 mM), and enhanced the combined stimulant effects of glucose plus either the fatty acid palmitate (10 mM), or agents which raise intracellular cyclic AMP concentrations (25 μM forskolin and 1 mM isobutylmethylxanthine), or the cholinoceptor agonist carbachol (100 μM).
6. Inhibition of glucose-stimulated insulin release by adrenaline or noradrenaline (10 μM) was partially reversed by BTS 67 582 (10⁻⁴ M).
7. These data suggest that the insulin-releasing effect of BTS 67 582 involves regulation of ATP-sensitive K⁺ channel activity and Ca²⁺ influx, and that the drug augments the stimulant effects of nutrient insulin secretagogues and agents which enhance adenylate cyclase and phospholipase C. BTS 67 582 may also exert insulin-releasing effects independently of ATP-sensitive K⁺ channel activity.

     

Coexistence of purino- and pyrimidinoceptors on activated rat microglial cells

1. Nucleotide-induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml⁻¹) treated (non-proliferating) rat microglial cells were recorded by the whole-cell patch-clamp technique. Most experiments were carried out on non-proliferating microglial cells. ATP (100 nM–1 mM), ADP (10 nM–10 mM) and UTP (1 μM–100 mM), but not uridine (100 μM–10 mM) produced a slow outward current at a holding potential of 0 mV. The effect of UTP (1 mM) did not depend on the presence of extracellular Mg²⁺ (1 mM). The outward current response to UTP (1 mM) was similar in non-proliferating and proliferating microglia.
2. In non-proliferating microglial cells, the ATP (10 μM)-induced outward current was antagonized by suramin (300 μM) or reactive blue 2 (50 μM), whereas 8-(p-sulphophenyl)-theophylline (8-SPT; 100 μM) was inactive. By contrast, the current induced by UTP (1 mM) was increased by suramin (300 μM) and was not altered by reactive blue 2 (50 μM) or 8-SPT (100 μM).
3. The current response to UTP (1 mM) disappeared when K⁺ was replaced in the pipette solution by an equimolar concentration of Cs⁺ (150 mM). However, the effect of UTP (1 mM) did not change when most Cl⁻ was replaced with an equimolar concentration of gluconate⁻ (145 mM). The application of 4-aminopyridine (1 mM) or Cs⁺ (1 mM) to the bath solution failed to alter the UTP (1 mM)-induced current. UTP (1 mM) had almost no effect in a nominally Ca²⁺-free bath medium, or in the presence of charybdotoxin (0.1 μM); the inclusion of U-73122 (5 μM) or heparin (5 mg ml⁻¹) into the pipette solution also blocked the responses to UTP (1 mM). By contrast, the effect of ATP (10 μM) persisted under these conditions.
4. I-V relations were determined by delivering fast voltage ramps before and during the application of UTP (1 mM). In the presence of extracellular Cs⁺ (1 mM) and 4-aminopyridine (1 mM) the UTP-evoked current crossed the zero current level near−75 mV. Omission of Ca²⁺ from the Cs⁺ (1 mM)- and 4-aminopyridine (1 mM)-containing bath medium or replacement of K⁺ by Cs⁺ (150 mM) in the pipette solution abolished the UTP current.
5. Replacement of GTP (200 μM) by GDP-β-S (200 μM) in the pipette solution abolished the current evoked by UTP (1 mM).
6. When the pipette solution contained Cs⁺ (150 mM) instead of K⁺ and in addition inositol 1,4,5,-trisphosphate (InsP₃; 10 μM), an inward current absolutely dependent on extracellular Ca²⁺ was activated after the establishment of whole-cell recording conditions. This current had a typical delay, a rather slow time course and did not reverse its amplitude up to 100 mV, as measured by fast voltage ramps.
7. A rise of the internal free Ca²⁺ concentration from 0.01 to 0.5 μM on excised inside-out membrane patches produced single channel activity with a reversal potential of 0 mV in a symmetrical K⁺ solution. The reversal potential was shifted to negative values, when the extracellular K⁺ concentration was decreased from 144 to 32 mM. By contrast, a decrease of the extracellular Cl⁻ concentration from 164 to 38 mM did not change the reversal potential.
8. Purine and pyrimidine nucleotides act at separate receptors in rat microglial cells. Pyrimidinoceptors activate via a G protein the enzyme phospholipase C with the subsequent release of InsP₃. The depletion of the intracellular Ca²⁺ pool appears to initiate a capacitative entry of Ca⁺ from the extracellular space. This Ca²⁺ then activates a Ca²⁺-dependent K⁺ current.

     

Effect of sodium rhein on electrically-evoked and agonist-induced contractions of the guinea-pig isolated ileal circular muscle

1. This study examined the effects of sodium rhein (0.03–30 μM) on the contractions of the isolated circular muscle of guinea-pig ileum induced by acetylcholine (100 nM), substance P (3 nM) and electrical stimulation (10 Hz for 0.3 s, 100 mA, 0.5 ms pulse duration). The effect of sodium rhein was also evaluated on the ascending excitatory reflex using a partitioned bath (oral and anal compartments). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 20 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the guinea-pig intestinal circular muscle in the oral compartment was recorded.
2. Sodium rhein (0.3, 3 and 30 μM) significantly potentiated (52±11% at 30 μM) acetylcholine-induced contractions. In the presence of tetrodotoxin (0.6 μM) or ω-conotoxin GVIA (10 nM) sodium rhein (3 and 30 μM) did not enhance, but significantly reduced (49±10% and 44±8%, respectively, at 30 μM) acetylcholine-induced contractions.
3. Sodium rhein (0.3, 3 and 30 μM) significantly increased (65±11% at 30 μM) substance P-induced contractions. In the presence of tetrodotoxin (0.6 μM), ω-conotoxin GVIA (10 nM) or atropine (0.1 μM), sodium rhein (3 and 30 μM) significantly reduced (50±10%, 55±8% and 46±10%, respectively, at 30 μM) substance P-induced contractions.
4. N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) abolished the potentiating effect of sodium rhein on acetylcholine and substance P-induced contractions. At the highest concentration (30 μM), sodium rhein, in presence of L-NAME, reduced the acetylcholine (30±6%)- or substance P (36±6%)-induced contractions.
5. Sodium rhein (30 μM) significantly potentiated (29±9%) the electrically-evoked contractions. L-NAME (100 μM), but not phentolamine, enhanced the effect of sodium rhein. Sodium rhein (30 μM) significantly increased (32±9%) the ascending excitatory reflex when applied in the oral, but not in the anal compartment.
6. These results indicate that sodium rhein (i) activates excitatory cholinergic nerves on circular smooth muscle presumably through a facilitation of Ca²⁺ entry through the N-type Ca²⁺ channel, (ii) has a direct inhibitory effect on circular smooth muscle and (iii) does not affect enteric ascending neuroneural transmission. Nitric oxide could have a modulatory excitatory role on sodium rhein-induced changes of agonist-induced contractions and an inhibitory modulator role on sodium rhein-induced changes of electrically-induced contractions.

     

Loss of endothelium-derived nitric oxide in rabbit aorta by oxidant stress: restoration by superoxide dismutase mimetics

1. Structurally distinct superoxide dismutase (SOD) mimetics were examined for their ability to protect nitric oxide (NO) from destruction by oxidant stress in rabbit aorta.
2. These were the spin traps, PTIYO (4-phenyl-2,2,5,5-tetramethyl imidazolin-1-yloxy-5-oxide), tempol (4-hydroxy 2,2,6,6,-tetramethylpiperidine-1-oxyl) and tiron (4,5-dihydroxy-1,3-benzene-disulphonic acid), the metal salts, CuSO₄ and MnCl₂, and the metal-based agents CuDIPS (Cu (II)-[diisopropylsalicylate]₂) and MnTMPyP (Mn (III) tetrakis [1-methyl-4-pyridyl]porphyrin).
3. Oxidant stress was generated in isolated aortic rings by inactivating endogenous Cu/Zn SOD with diethyldithiocarbamate (DETCA; 60 min) either alone at 3 mM or at 0.3 mM in combination with superoxide generation using xanthine oxidase (XO; 4.8 mu ml⁻¹) and hypoxanthine (HX; 0.1 mM).
4. Acetylcholine (ACh)-induced relaxation was inhibited by DETCA (3 mM, 60 min) and was not restored by exogenous SOD (250 u ml⁻¹), suggesting the oxidant stress was intracellular. MnTMPyP (600 μM and 1 mM) and MnCl₂ (100 μM) were the only agents to reverse the blockade of ACh-induced relaxation.
5. Addition of XO/HX to DETCA (0.3 mM)-treated tissues powerfully impaired ACh-induced relaxation and exogneous SOD (250 u ml⁻¹) fully reversed the blockade, suggesting the oxidant stress was extracellular. CuDIPS (0.1–3 μM), CuSO₄ (0.3–3 μM), MnCl₂ (1–100 μM) and MnTMPyP (100–600 μM) also reversed blockade powerfully, tempol (30 μM–1 mM) and tiron (0.3–10 mM) reversed blockade weakly and PTIYO (10–300 μM) enhanced the blockade.
6. Thus, MnTMPyP was the only SOD mimetic to restore NO-dependent relaxation in conditions of both extracellular and intracellular oxidant stress. This agent may, therefore, provide a lead in the development of SOD mimetics for the treatment of pathologies associated with oxidant stress.

     

The spasmogenic effects of vanadate in human isolated bronchus

1. Inhalation of vanadium compounds, particularly vanadate, is a cause of occupational bronchial asthma. We have now studied the action of vanadate on human isolated bronchus. Vanadate (0.1 μM–3 mM) produced concentration-dependent, well-sustained contraction. Its −logEC₅₀ was 3.74±0.05 (mean±s.e.mean) and its maximal effect was equivalent to 97.5±4.2% of the response to acetylcholine (ACh, 1 mM).
2. Vanadate (200 μM)-induced contraction of human bronchus was epithelium-independent and was not inhibited by indomethacin (2.8 μM), zileuton (10 μM), a mixture of atropine, mepyramine and phentolamine (each at 1 μM), or by mast cell degranulation with compound 48/80.
3. Vanadate (200 μM)-induced contraction was unaltered by tissue exposure to verapamil or nifedipine (each 1 μM) or to a Ca²⁺-free, EGTA (0.1 mM)-containing physiological salt solution (PSS). However, tissue incubation with ryanodine (10 μM) in Ca²⁺-free, EGTA (0.1 mM)-containing PSS reduced vanadate-induced contraction. A series of vanadate challenges was made in tissues exposed to Ca²⁺-free EGTA (0.1 mM)-containing PSS with the object of depleting intracellular Ca²⁺ stores. In such tissues cyclopiazonic acid (CPA; 10 μM) prevented Ca²⁺-induced recovery of vanadate-induced contraction.
4. Tissue incubation in K⁺-rich (80 mM) PSS, K⁺-free PSS, or PSS containing ouabain (10 μM) did not alter vanadate (200 μM)-induced contraction. Ouabain (10 μM) abolished the K⁺-induced relaxation of human bronchus bathed in K⁺-free PSS. This action was not shared by vanadate (200 μM). The tissue content of Na⁺ was increased and the tissue content of K⁺ was decreased by ouabain (10 μM). In contrast, vanadate (200 μM) did not alter the tissue content of these ions. Tissue incubation in a Na⁺-deficient (25 mM) PSS or in PSS containing amiloride (0.1 mM) markedly inhibited the spasmogenic effect of vanadate (200 μM).
5. Vanadate (200 μM)-induced contractions were markedly reduced by tissue treatment with each of the protein kinase C (PKC) inhibitors H-7 (10 μM), staurosporine (1 μM) and calphostin C (1 μM). Genistein (100 μM), an inhibitor of protein tyrosine kinase, also reduced the response to vanadate.
6. Vanadate (0.1–3 mM) and ACh (1 μM–3 mM) each increased inositol phosphate accumulation in bronchus. Such responses were unaffected by a Ca²⁺-free medium either alone or in combination with ryanodine (10 μM).
7. In human cultured tracheal smooth muscle cells, histamine (100 μM) and vanadate (200 μM) each produced a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i).
8. Intracellular microelectrode recording showed that the contractile effect of vanadate (200 μM) in human bronchus was associated with cellular depolarization.
9. It is concluded that vanadate acts directly on human bronchial smooth muscle, promoting the release of Ca²⁺ from an intracellular store. The Ca²⁺ release mechanism involves both the production of inositol phosphate second messengers and inhibition of Ca-ATPase. The activation of PKC plays an important role in mediating vanadate-induced contraction at values of [Ca²⁺]_i that are close to basal.

     

Selective inhibition of M-type potassium channels in rat sympathetic neurons by uridine nucleotide preferring receptors

1. UTP and UDP depolarize rat superior cervical ganglion neurons and trigger noradrenaline release from these cells. The present study investigated the mechanisms underlying this excitatory action of uridine nucleotides by measuring whole-cell voltage-dependent K⁺ and Ca²⁺ currents.
2. Steady-state outward (holding) currents measured in the amphotericin B perforated-patch configuration at a potential of −30 mV were reduced by 10 μM UTP in a reversible manner, but steady-state inward (holding) currents at −70 mV were not affected. This action of UTP was shared by the muscarinic agonist oxotremorine-M. In current-voltage curves between −20 and −100 mV, UTP diminished primarily the outwardly rectifying current components arising at potentials positive to −60 mV.
3. Slow relaxations of muscarinic K⁺ currents (I_M) evoked by hyperpolarizations from −30 to −55 mV were also reduced by 10 μM UTP (37% inhibition) and oxotremorine-M (81% inhibition). In contrast, transient K⁺-currents, delayed rectifier currents, fast and slow Ca²⁺-dependent K⁺ currents, as well as voltage-dependent Ca²⁺ currents were not altered by UTP.
4. In conventional (open-tip) whole-cell recordings, replacement of GTP in the pipette by GDPβS abolished the UTP-induced inhibition of I_M, whereas replacement by GTPγS rendered it irreversible.
5. The UTP-induced reduction of I_M was half maximal at 1.5 μM with a maximum of 37% inhibition; UDP was equipotent and equieffective, while ADP was less potent (half maximal inhibition at 29 μM). ATP had no effect at ⩽30 μM.
6. The inhibition of I_M induced by 10 μM UTP was antagonized by pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) at ⩾30 μM and by reactive blue 2 at ⩾10 μM, but not by suramin at concentrations up to 30 μM.
7. These results show that rat superior cervical ganglion neurons possess uridine nucleotide preferring P2Y receptors which inhibit K_M channels. This effect presumably forms the basis of the excitatory action of uridine nucleotides in rat sympathetic neurons.

     

Pharmacologically distinct GABAB receptors that mediate inhibition of GABA and glutamate release in human neocortex

1. The release of endogenous γ-aminobutyric acid (GABA) and glutamic acid in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply located tumours.
2. The basal outflows of GABA and glutamate from superfused synaptosomes were largely increased during depolarization with 15 mM KCl. The K⁺-evoked overflows of both amino acids were almost totally dependent on the presence of Ca²⁺ in the superfusion medium.
3. The GABA_B receptor agonist (−)-baclofen (1, 3 or 10 μM) inhibited the overflows of GABA and glutamate in a concentration-dependent manner. The inhibition caused by 10 μM of the agonist ranged from 45–50%.
4. The effect of three selective GABA_B receptor antagonists on the inhibition of the K⁺-evoked GABA and glutamate overflows elicited by 10 μM (−)-baclofen was investigated. Phaclofen antagonized (by about 50% at 100 μM; almost totally at 300 μM) the effect of (−)-baclofen on GABA overflow but did not modify the inhibition of glutamate release. The effect of (−)-baclofen on the K⁺-evoked GABA overflow was unaffected by 3-amino-propyl (diethoxymethyl)phosphinic acid (CGP 35348; 10 or 100 μM); however, CGP 35348 (10 or 100 μM) antagonized (−)-baclofen (complete blockade at 100 μM) at the heteroreceptors on glutamatergic terminals. Finally, [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic aid (CGP 52432), 1 μM, blocked the GABA_B autoreceptor, but was ineffective at the heteroreceptors. The selectivity of CGP 52432 was lost at 30 μM, as the compound, at this concentration, inhibited completely the (−)-baclofen effect both on GABA and glutamate release.
5. It is concluded that GABA and glutamate release evoked by depolarization of human neocortex nerve terminals can be affected differentially through pharmacologically distinct GABA_B receptors.

     

Inhibition of glycine currents by dextromethorphan in neurones dissociated from the guinea-pig nucleus tractus solitarii

1. The effect of dextromethorphan (DM) on the current induced by glycine in acutely dissociated nucleus tractus solitarii (NTS) neurones of guinea-pigs was studied by use of the whole-cell patch clamp technique. The effect of DM on γ-aminobutyric acid (GABA)-induced currents (I_GABA) was also examined.
2. DM inhibited 30 μM glycine-induced current (I_Gly), without affecting the current caused by 30 μM GABA. The action of DM was concentration-dependent, with a maximum effect at 100 μM, and reversible. The half-maximum inhibitory concentration (IC₅₀) of DM was 3.3 μM, about 85 times higher than that of strychnine.
3. DM 3 μM shifted the concentration-response curve for glycine to the right without affecting the maximum value. DM 10 μM shifted the curve even more to the right, although it was not a parallel shift. Strychnine at a concentration of 0.1 μM shifted the curve for glycine in a nearly parallel fashion.
4. The effect of 10 μM DM was slightly weak voltage-dependency, but the lower concentration of DM, 3 μM, inhibited I_Gly equally at −50 mV and +50 mV. The effect of 3 μM DM on I_Gly showed no use-dependence. Blockade by strychnine 0.1 μM showed no voltage- or use-dependence.
5. The results indicate that DM inhibits I_Gly in single neurones of NTS, and further suggest that DM at a low concentration may act on the glycine receptor-ionophore complex, but not on the Cl⁻ channel of the complex. However, a relatively high concentration of DM may at least partly affect the Cl⁻ channel of the complex.

     

The signalling pathway which causes contraction via P2-purinoceptors in rat urinary bladder smooth muscle

1. The signalling pathway which causes contractions to adenosine 5′-O-2-thiodiphosphate (ADPβS) and α,β-methylene adenosine 5′-diphosphate (α,β-Me ADP) was investigated in rat urinary bladder smooth muscle by measuring isotonic tension.
2. The responses to 10 μM α,β-methylene adenosine 5′-triphosphate (α,β-Me ATP) in 0 and 3.6 mM Ca²⁺ were 5.9±1.3 (n=10) and 122.2±6.4 (n=8) % respectively of those obtained in 1.8 mM Ca²⁺, whereas those to 100 μM ADPβS were 34.6±3.3 (n=8) and 96.8±7.2 (n=8) %, in 0 and 3.6 mM Ca²⁺, respectively. In both experimental conditions, the responses to the two agonists expressed as % of the control responses were significantly different (P<0.01).
3. Indomethacin at high concentrations (>1 μM) decreased the responses to α,β-Me ATP (10 μM), ADPβS (100 μM) and α,β-Me ADP (100 μM). However, no significant difference was obtained between the responses to all the agonists at 30 μM indomethacin.
4. 2-Nitro-4-carboxphenyl n,n-diphenylcarbamate (NCDC) at concentrations between 1 μM and 100 μM concentration-dependently decreased the responses to ADPβS (100 μM) and α,β-Me ADP (100 μM) and almost completely inhibited them at 100 μM. Although the responses to α,β-Me ATP (10 μM) were also inhibited by the drug, at 50 and 100 μM NCDC the responses to α,β-Me ATP were significantly larger than those to ADPβS and α,β-Me ADP (P<0.01).
5. NCDC 100 μM significantly inhibited the KCl-induced contraction to 65.9±4.9% (n=6) of the control (P<0.01).
6. It is suggested that the contraction via ADPβS-sensitive receptors in the rat urinary bladder smooth muscle mainly depends on Ca²⁺ ions liberated from intracellular Ca²⁺ stores, though the contribution of Ca²⁺ ions from the extracellular space cannot be neglected. The release of Ca²⁺ ions from stores is mainly mediated by the production of inositol trisphosphate (IP₃) via the activation of phospholipase C.

     

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2

1. In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels.
2. Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 μM, the highest concentration tested, increasing the rate to 153±4% of control (n=9).
3. Repetitive 5 min applications of substance P (1 μM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively.
4. The competitive antagonist of tachykinin receptors, spantide (5 μM) and the specific NK₁ receptor antagonist, WIN51708 (10 μM) both prevented the enhancement of constriction rate induced by 1 μM substance P.
5. Endothelial cells loaded with the Ca²⁺ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca²⁺]_i upon application of 1 μM substance P.
6. Inhibition of nitric oxide synthase by N^G nitro-L-arginine (L-NOARG; 100 μM) had no significant effect on the response induced by 1 μM substance P.
7. The enhancement of constriction rate induced by 1 μM substance P was prevented by the cyclo-oxygenase inhibitor, indomethacin (3 μM), the thromboxane A₂ synthase inhibitor, imidazole (50 μM), and the thromboxane A₂ receptor antagonist, SQ29548 (0.3 μM).
8. The stable analogue of thromboxane A₂, U46619 (0.1 μM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μM).
9. Treatment with pertussis toxin (PTx; 100 ng ml⁻¹) completely abolished the response to 1 μM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate.
10. Application of the phospholipase A₂ inhibitor, antiflammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 μM), without inhibiting the response to either U46619 (0.1 μM) or acetylcholine (10 μM).
11. The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK₁ receptors coupled by a PTx sensitive G-protein to phospholipase A₂ and resulting in generation of the arachidonic acid metabolite, thromboxane A₂, this serving as the diffusible activator.

     

Comparison of the acute cardiotoxicity of the antimalarial drug halofantrine in vitro and in vivo in anaesthetized guinea-pigs

1. Several unrelated drugs have pro-arrhythmic activity associated with an ability to prolong the QT interval of the ECG. The aim of this work was to examine the effects of the antimalarial drug halofantrine in vivo and in vitro.
2. In anaesthetized guinea-pigs consecutive bolus doses of halofantrine (0.3, 1, 3, 10 and 30 mg kg⁻¹, i.v.) at 25 min intervals caused dose-dependent prolongation of the rate corrected QTc interval and bradycardia. The change in heart rate became significant after administration of 10 mg kg⁻¹ halofantrine (−23±9 beats min⁻¹) whereas the increase in QTc was significant with only 1 mg kg⁻¹ halofantrine (22±10 ms). It was only with the highest dose of halofantrine that the PR interval was increased (from 52±3 to 67±4 ms) and second degree atrioventricular (AV) block (type 1 Mobitz) occurred in all animals. No changes were observed in any parameters in a separate group of guinea-pigs which received vehicle (dimethylacetamide 60% propylene glycol 40%) at equivalent time points.
3. The blood concentrations of halofantrine ranged from 0.26±0.17 μM after administration of 0.3 mg kg⁻¹ to 2.79±0.87 μM after 30 mg kg⁻¹, i.v. There was a significant correlation between the blood concentrations of halofantrine and the changes in QTc interval.
4. In guinea-pig left papillary muscles the effective refractory period was increased significantly 60 min after addition of halofantrine; from 161±4 to 173±6 ms with 10 μM, 156±8 to 174±6 ms with 30 μM and 165±6 to 179±5 ms with 100 μM halofantrine. However, the vehicle (0.1% Tween 80 in DMSO; final concentration of vehicle in Krebs, 1%) also increased the effective refractory period from 164±5 to 173±6 ms. Similar results were obtained in right ventricular strips but left atrial effective refractory periods were not altered by either the vehicle or halofantrine.
5. The results of these experiments suggest that any direct effects that halofantrine may have had on the effective refractory period of cardiac muscle cannot be separated from those of the vehicle. The prolongation of QTc and consistent observation of AV block with halofantrine in anaesthetized guinea-pigs suggest that in vivo models may be more useful for further studies investigating the mechanisms underlying the cardiotoxicity of halofantrine.

     

Release of the antioxidants ascorbate and urate from a nitrergically-innervated smooth muscle

1. The main object of the present study was to determine whether ascorbate, an antioxidant which has been shown to protect nitric oxide (NO) from attack by scavenger molecules, might be released from nitrergically-innervated smooth muscle; ascorbate release from the rat anococcygeus was measured by use of h.p.l.c. with electrochemical detection.
2. Incubation of rat anococcygeus muscles in normal physiological salt solution (PSS; 30 min) resulted in release of ascorbate into the bathing medium (7.7±0.9 nmol g⁻¹ tissue). This release was increased by 96% when muscles were incubated in high K⁺ (70 mM) PSS. The resting release of ascorbate was unaffected by tetrodotoxin (TTX; 1 μM), ω-conotoxin GVIA (10 nM) or omission of calcium ions from the PSS (with addition of 0.2 mM EGTA), but all three procedures attenuated the increased release observed under depolarizing conditions. Resting release of ascorbate was unaffected by glutamate (100 μM), aspartate (100 μM), γ-aminobutyric acid (100 μM) or carbachol (50 μM).
3. A second h.p.l.c. peak, which always preceded the ascorbate peak, was identified as urate. Urate release from the anococcygeus, following 30 min incubation in normal PSS, was 64.6±12.7 nmol g⁻¹ tissue but, unlike ascorbate, urate release was unchanged in high K⁺ PSS. In functional experiments, urate (100–400 μM) partially protected NO (15 μM)-induced relaxations of the rat anococcygeus from inhibition by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; 50 μM), but not from inhibition by hydroquinone or duroquinone (both 100 μM).
4. Muscles chemically sympathectomized with 6-hydroxydopamine (6-OHDA, 500 μM; 2 h) still exhibited release of ascorbate (2.5±0.4 nmol g⁻¹ tissue) and urate (22.2±2.9 nmol g⁻¹ tissue); in both cases the release was similar to that observed in time-matched control tissues not exposed to 6-OHDA. High K⁺ PSS produced a TTX-sensitive increase in release of ascorbate, but not urate, from 6-OHDA-treated muscles.
5. The results demonstrate that significant amounts of ascorbate and urate are released from the rat anococcygeus muscle. Ascorbate, but not urate, release appears to be enhanced by activation of nerves which are resistant to 6-OHDA pretreatment. Since both antioxidants can protect NO from attack by scavenger molecules, their release in nitrergically-innervated tissues may be important for the provision of the correct redox environment to allow NO to fulfill its proposed neurotransmitter role.