bluuebunny/crossref_metadata_2025 huggingface dataset:
NMDA antagonists increase recovery of evoked potentials from slices of rat olfactory cortex after anoxia
Authors: Yassin Maged , Scholfield C.N.
Published: July 2012

The role of glutamate in producing tissue damage during cerebral anoxia was investigated in brain slices using antagonists to the NMDA and AMPA receptor types.

Tissue function was assessed by field recordings of the synaptically evoked potentials elicited by stimulating the main afferent input to the olfactory cortex, the lateral olfactory tract. Anoxia was produced by bathing the slice in glucose‐free solution equilibrated with 95% N2/5% CO2.

The amount of recovery of the evoked potential was inversely dependent on the period of anoxia and temperature: at 24°C, 15 min of anoxia followed by reoxygenation produced a 14.6 ± 4.1% recovery whereas there was no recovery at 35°C.

Dizocilpine and ketamine had no effect on synaptic transmission in oxygenated media but following anoxia they produced an increased recovery of the responses: from 14.6 ± 4.1% to 48.3 ± 7.8% for dizocilpine (10 μm) and 21.6 ± 7.7% to 87.2 ± 7.1% for ketamine (200 μm); the tissue endurance to anoxia was increased by around 5 min.

Blockade of the AMPA receptors did not influence recovery in spite of the depressed synpatic transmission. A similar synaptic attenuation produced by lignocaine provided some increase in post‐anoxic recovery.

The NMDA receptor antagonist, AP5, antagonized NMDA at 50 μm by 3.7 fold and at 200 μm by 15 fold but only 200 μm increased post‐anoxic recovery. This suggests that a substantial degree of NMDA antagonist is required before anoxic tissue damage due to NMDA receptor activation can be nullified. The antagonist to the glycine binding site, 7‐chlorokynurenic acid also increased recovery.

These in vitro experiments confirm the idea that NMDA receptor activation makes a substantial contribution to cerebral tissue damage and that this can be reduced by a substantial blockade of these receptors.
Time‐dependent enhancement or inhibition of endotoxin‐induced vascular injury in rat intestine by nitric oxide synthase inhibitors
Authors: Laszlo F. , Whittle B.J.R. , Moncada S.
Published: July 2012

The effects of the nitric oxide (NO) synthase inhibitors, NG‐nitro‐l‐arginine methyl ester (l‐NAME) and NG‐monomethyl‐l‐arginine (l‐NMMA), on the vascular damage induced by the endotoxin, E. coli lipopolysaccharide (LPS), in the ileum and colon were investigated in the conscious rat over a 5 h period.

Administration of LPS (3 mg kg−1, i.v.) increased ileal and colonic vascular injury after a lag period of 2 h, as determined by the leakage of radiolabeled albumin.

Administration of l‐NAME (1–5 mg kg−1, s.c.) concurrently with LPS, produced a dose‐dependent increase in vascular albumin leakage in the intestinal tissues, when determined over a 5h period. Vascular albumin leakage with LPS and l‐NAME (5 mg kg−1) was substantially increased after 1 h, reached maximal levels 3 h after administration, and then slowly declined.

l‐NMMA (50 mg kg−1, s.c.), likewise elevated intestinal albumin leakage when administered concurrently with LPS, but this reached maximal levels after 1 h and rapidly declined over the subsequent 2 h.

In control rats, in the absence of LPS challenge, neither l‐NAME (5 mg kg−1, s.c.) nor l‐NMMA (50 mg kg−1, s.c.) increased intestinal vascular leakage of albumin over a 5h period.

By contrast, when l‐NAME (1–5 mg kg−1, s.c.) or l‐NMMA (12.5–50 mg kg−1, s.c.) was injected 3 h after LPS, a dose‐dependent reduction in the LPS‐provoked vascular albumin leakage was observed.

Pretreatment with l‐arginine (300 mg kg−1, s.c.) 15 min prior to the NO synthase inhibitors, reversed either the potentiation or the inhibition by l‐NAME (5 mg kg−1, s.c.) or l‐NMMA (50 mg kg−1, s.c.) of the LPS‐induced intestinal vascular damage.

These findings indicate that initial suppression of the constitutive NO synthase by l‐NAME or l‐NMMA following challenge with LPS aggravates the acute vascular injury in the ileum and colon, suggesting a defensive role of NO. By contrast, the delayed administration of NO synthase inhibitors, at a time of known expression of the inducible NO synthase, provides protection against the subsequent damage to the intestinal vasculature.
Activation of chloride current by P2‐purinoceptors in rat ventricular myocytes
Authors: Kaneda M. , Fukui K. , Doi K.
Published: July 2012

Rat ventricular myocytes were dissociated and their responses to extracellularly applied ATP were recorded using patch pipettes under the whole cell configuration.

ATP initially induced an inward current followed by an outward current at − 50 mV. With a Cs‐rich pipette solution the late outward current was blocked, leaving a sustained inward current (IATPs) suggesting that a K+conductance underlies the late response.

When the extracellular Cl−concentration was changed, the reversal potential of IATPs corresponded well to the shift of the Cl−equilibrium potential. IATPs was reversibly blocked by the chloride channel blocker, 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS).

The concentration‐response curve of IATPs had a Hill coefficient of 0.98 and an EC50 value of 5.2 × 10−6m.

ATP was more potent than ADP, while AMP and adenosine were ineffective, suggesting that P2‐purinoceptor activation induced IATPs.

The activation of IATPs was depressed by depleting the extracellular Mg2+and increased by adding Mg2+.

Our results strongly suggest that P2‐purinoceptor activation by ATP induces both a Cl−‐conductance (IATPs) and a K+‐conductance in rat ventricular myocytes.
Characterization and transduction mechanisms of purinoceptors in activated rat microglia
Authors: Langosch J.M. , Gebicke‐Haerter P.J. , Nörenberg W. , Illes P.
Published: July 2012

Purinoceptor agonist‐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.

In non‐proliferating microglia, adenosine (0.01–100 μm), 2‐methylthio ATP (3–3000 nm), ATP (0.1–1000 μm), and ATP‐γ‐S (1–10 μm), but not α,β‐methylene ATP (α,β‐MeATP; 100 μm) produced a slow outward current at a holding potential of 0 mV. When K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150 mm), the 2‐methylthio ATP‐ (300 nm) induced outward current disappeared. The effect of 2‐methylthio ATP (300 nm) did not depend on the presence of extracellular Mg2+ (1 mm). The outward current response to 2‐methylthio ATP (300 nm) was larger in proliferating than in non‐proliferating microglia.

ATP (1–1000 μm) evoked a fast inward current at a holding potential of − 70 mV in non‐proliferating microglia, while adenosine (100–1000 μm) was inactive. When the effects of ATP were compared at 0 and − 70 mV, it became evident that ATP is much more potent in evoking the outward current.

The 2‐methylthio ATP‐ (300 nm) induced outward current was blocked by suramin (300 μm), but not by 8‐(p‐sulphophenyl)‐theophylline (100 μm), while the adenosine‐ (1 μm) induced outward current had the reverse sensitivity to these antagonists.

The 2‐methylthio ATP‐ (300 nm) induced outward current was inhibited by inclusion of GDP‐β‐S (200 μm) into the pipette solution or by preincubation of microglial cells with pertussis toxin (50 ng ml−1) for 12 h. The 2‐methylthio ATP‐ (300 μm) induced inward current was not changed by intracellular GDP‐β‐S (200 μm). The outward current response to adenosine (1 μm) was also abolished after pretreatment with pertussis toxin (50 ng ml−1).

Rat microglia possess both ATP‐sensitive P2Y‐ and adenosine‐sensitive P1‐purinoceptors. The ATP‐evoked inward current is mediated by P2Y‐purinoceptors, while the ATP‐ and adenosine‐evoked outward currents are mediated by P2Y‐ and P1‐purinoceptors, respectively. The transduction mechanisms of the outward, but not the inward current activation involve a pertussis toxin‐sensitive G protein.
Neuropeptide Y in rat detrusor and its effect on nerve‐mediated and acetylcholine‐evoked contractions
Authors: Iravani M.M. , Zar M.A.
Published: July 2012

Immunohistochemical and isolated organ bath techniques were used to detect the presence of neuropeptide Y (NPY) in the rat urinary bladder and to determine its effect on tone, spontaneous activity and contractile responses of the detrusor muscle to electrical field stimulation, acetylcholine and α,β‐methylene ATP (α,β‐MeATP).

A very rich presence of NPY‐immunoreactive nerve fibres was found mainly within the bundles of detrusor muscle cells. Chronic treatment with 6‐hydroxydopamine did not affect the density of NPY‐positive nerve fibres.

NPY (> 1 nm) enhanced the force and frequency of spontaneous contractions and generated a rise in the resting tone of the detrusor. These effects of NPY on the tone and the spontaneous activity remained unaffected by atropine (3 μm), indomethacin (10 μm) and aspirin (100 μm) but were abolished by Ca2+‐withdrawal from the bathing medium.

The enhancing effects of NPY on the spontaneous contractions and the resting tone were not prevented by the induction of purinoceptor desensitization.

NPY (1–250 nm) potentiated electrical field stimulation (EFS, 1–64 Hz, 0.1 ms pulses duration, 10s train duration)‐evoked, tetrodotoxin (0.5 μm)‐sensitive contractions. The atropine (3 μm)‐resistant component of EFS‐evoked contractions was also potentiated by NPY. By contrast, the nifedipine (1 μm)‐resistant but atropine‐sensitive component of EFS‐evoked contraction was inhibited by NPY.

NPY (250 nm) did not affect acetylcholine‐evoked contractions, but potentiated α,β‐MeATP‐evoked contractions.

It is concluded that NPY‐innervation of rat urinary bladder is largely confined to the detrusor muscle and is abundant and mainly non‐adrenergic. It is further concluded that the enhancing effect of NPY on detrusor spontaneous activity and tone is caused by Ca2+ influx through nifedipine‐sensitive Ca2+ channels and is not mediated through acetylcholine or cyclo‐oxygenase‐sensitive eicosanoids or ATP.

The results are consistent with the hypothesis that intrinsic NPY in the rat detrusor innervation contributes to the motor transmission in two ways: by promoting non‐cholinergic motor transmission and by inhibiting prejunctionally the cholinergic transmission.
Characterization of the 5‐hydroxytryptamine receptor type involved in inhibition of spontaneous activity of human isolated colonic circular muscle
Authors: Tam F.S.‐F. , Hillier K. , Bunce K.T.
Published: July 2012

Experiments were carried out to characterize pharmacologically the 5‐hydroxytryptamine (5‐HT) receptor types which mediate inhibition of spontaneous contractions of the intertaenial circular muscle in human isolated colon.

5‐HT caused a reproducible concentration‐dependent inhibition of spontaneous contractions of the circular muscle of human colon in vitro with a mean EC50 value of 0.2 μm and 95% confidence limits of 0.1–0.5 μm. No evidence for a contractile action of 5‐HT was found. Tetrodotoxin (TTX, 1.5 μm) caused a rightward shift of the concentration‐response curve of 5‐HT with a concentration‐ratio of 2.9.

The inhibitory response to 5‐HT was mimicked by several indoles with the rank order of potency 5‐HT > 5‐methoxytryptamine = α‐methyl‐5‐HT > 5‐carboxamidotryptamine >> 2‐methyl‐5‐HT. 5‐Hydroxyindalpine was inactive.

The substituted benzamides were agonists with the following rank order of potency, 5‐HT > renzapride > zacopride > metoclopramide > cisapride.

The inhibitory responses to 5‐HT were not inhibited by methysergide (10 μm) or methiothepin (1 μm), which are antagonists selective for 5‐HT1‐like and 5‐HT2 receptors, nor by ondansetron (10 μ) which is an antagonist at 5‐HT3 receptors.

The inhibitory responses induced by 5‐HT and 5‐methoxytryptamine were competitively antagonized by a weak 5‐HT4 receptor antagonist, tropisetron, with pKB values of approximately 6. Tropisetron had no significant effect on the inhibitory response curve produced by isoprenaline (0.01–100 μm).

The pharmacological profile of the 5‐HT‐evoked relaxations of human colon circular muscle are consistent with activation of a 5‐HT4‐like receptor.
A novel antagonist, phenylbenzene ω‐phosphono‐α‐amino acid, for strychnine‐sensitive glycine receptors in the rat spinal cord
Authors: Saitoh T. , Ishida M. , Maruyama M. , Shinozaki H.
Published: July 2012

3‐[2′‐Phosphonomethyl[1,1′‐biphenyl]‐3‐yl]alanine (PMBA) is a novel glycine antagonist at strychnine‐sensitive receptors. The chemical structure of PMBA, possessing both a glycine moiety and a phosphono group, is quite different from that of strychnine.

In the spinal motoneurone of newborn rats, glycine (100 μm‐1 mM) induced depolarizing responses in a concentration‐dependent manner. PMBA effectively inhibited depolarizing responses to glycine and other agonists, such as taurine and β‐alanine. The dose‐response curves for glycine were shifted to the right in an almost parallel manner (pA2 value: 5.30 ± 0.23, n = 5) by PMBA which was about 60 times less potent than strychnine (pA2 value: 7.08 ± 0.21, n = 5) as a glycine antagonist.

PMBA (1 − 100 μm) did not interact with modulatory glycine sites on N‐methyl‐D‐aspartate (NMDA) receptors, which suggests a high selectivity of PMBA for strychnine‐sensitive glycine receptors. At considerably high concentrations (0.1 mM‐1 mM), PMBA depressed responses to GABA (pA2 value: 3.57 ± 0.24, n = 3).

PMBA inhibited the binding of [3H]‐strychnine to synaptosomes from adult rat spinal cords; the IC50 values of PMBA, glycine and strychnine were 8 ± 2, 9 ± 3 and 0.08 ± 0.04 μm, respectively (n = 5) for [3H]‐strychnine (4.8 nM).

PMBA is a central excitant drug with relatively high potency and selectivity and should be useful as a pharmacological probe for analysing the mechanisms underlying physiological functions of glycine receptors.
Sensitization by dexamethasone of lymphocyte cyclic AMP formation: evidence for increased function of the adenylyl cyclase catalyst
Authors: Michel Martin C. , Knapp Jörg , Ratjen Hanna
Published: July 2012

Glucocorticoids and elevations of intracellular adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) may affect lymphocyte activation, proliferation and effector functions in similar ways. Therefore, we have investigated the effects of the glucorticoid, dexamethasone, on human lymphocyte cyclic AMP formation.

Treatment of resting human lymphocytes with the glucocorticoid, dexamethasone, sensitized prostaglandin E2‐stimulated cyclic AMP accumulation in a time‐ and concentration‐dependent manner.

In membranes of lymphocytes treated for 24 h with 100 nm dexamethasone, maximal adenylyl cyclase activity stimulated by prostaglandin E2, isoprenaline, guanosine 5′‐triphosphate (GTP), forskolin and MnCl2 was significantly enhanced; the EC50 for these agents was not significantly altered.

β2‐Adrenoceptor density, immunodetectable α‐subunits of the G‐proteins Gs and Gi, and pertussis toxin‐substrates were not significantly altered by dexamethasone treatment.

In dexamethasone‐treated lymphocytes, prostaglandin E2‐mediated inhibition of concanavalin A‐induced Ca2+ elevations was doubled compared to control cells.

Based on these data and the observation that enhancement of forskolin‐ and MnCl2‐stimulated adenylyl cyclase activity could quantitatively account for the enhancement of prostaglandin E2‐, isoprenaline‐ or GTP‐stimulated adenylyl cyclase activity, we conclude that dexamethasone treatment sensitizes cyclic AMP formation in resting human lymphocytes by altering the adenylyl cyclase catalyst rather than G‐proteins or hormone receptors. This results in an enhanced capability of cyclic AMP generating agonists to inhibit early steps of lymphocyte activation.
Binding and growth‐inhibitory effect of heparin and oligo‐heparin (2 kDa) in Balb/c 3T3 cells: lack of effect on PDGF‐ or serum‐induced inositol lipid turnover
Authors: Cavari Simone , Fiorelli Gianna , Vannucchi Simonetta
Published: July 2012

The ability of heparins (bovine heparin sm 1026, Av. mol. wt. 36.9 kDa and bovine heparin EP 756, Av. mol. wt. 12.9 kDa) and heparin fractions of different molecular weights (low molecular weight heparin, LMW 2123/OP, Av. mol. wt. 4.5 kDa and oligo‐heparin, Av. mol. wt. 2 kDa) to inhibit the proliferation and signalling of Balb/c 3T3 fibroblasts was investigated.

Heparin and heparin fractions of 4.5 and 2 kDa significantly inhibited DNA synthesis as monitored by [3H]‐thymidine incorporation.

3H‐labelled heparin fractions of 4.5 and 2 kDa were prepared by gel‐chromatography fractionation on Sephadex G‐75 of an 3H‐labelled commercial heparin after treatment with heparinase.

The binding of unfractionated and oligo‐heparin of 2 kDa to Balb/c 3T3 fibroblasts was studied; we determined the specificity of heparin and oligo‐heparin binding to the cells by means of displacement of bound 3H‐labelled compound in response to increasing concentrations of unlabelled compounds. Scatchard analysis of binding data obtained using [3H]‐heparin as ligand revealed the presence of a single class of high affinity binding sites (Kd = 28 nm) for heparin. Scatchard analysis of binding data obtained using [3H]‐oligo‐heparin as ligand revealed the presence of a single class of low affinity binding sites (Kd = 3.2 μm) for oligo‐heparin.

In addition heparin displaced [3H]‐oligo‐heparin at a concentration of approximately 100 fold of the Kd determined in displacement studies. Furthermore, oligo‐heparin significantly displaced [3H]‐heparin at a concentration of approximately 10 fold of the Kd determined by displacement studies.

Both heparin and oligo‐heparin exert their inhibitory effects on Balb/c 3T3 DNA synthesis stimulated by PDGF or serum. However these molecules did not affect the inositol lipid turnover triggered by PDGF at a concentration which did not produce maximal response. The increase of inositol phosphate metabolism produced by 20% serum was also unaffected by heparin. This concentration of serum elicited a response comparable to that induced by a submaximal concentration of PDGF.
Effect of dehydration and hyperosmolal hydration on lignocaine and metabolites disposition in conscious rabbits
Authors: Chamelian M. , Lécrivain A. , Robichaud A. , du Souich P.
Published: July 2012

The present study aimed to investigate the effect of dehydration and hyperosmolal hydration on the disposition of lignocaine and two of its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX).

Lignocaine was infused to three groups of conscious rabbits: controls, rabbits previously deprived of water for 48 h and rabbits receiving an infusion of 2.5% NaCl.

In dehydrated and hyperosmolal‐hydrated rabbits, plasma osmolality was 321 ± 1 and 313 ± 1 mOsm kg−1, respectively (P<0.01 compared to controls, 285 ± 1 mOsm kg−1). In dehydrated animals, baseline values of plasma arginine vasopressin (AVP) concentrations and plasma renin activity (PRA) were higher than in controls, i.e. 12.4 ± 1.4 pg ml−1 and 15.4 ± 1.7 ng AI ml−1 h−1 vs. 3.4 ± 0.2 pg ml−1 (P<0.01), and 5.1 ± 0.6 ng AI ml−1 h−1 (P<0.01), respectively; atrial natriuretic peptide (ANP) decreased from 55 ± 11 to 32 ± 4 pg ml−1 (P<0.05). Compared to controls, hyperosmolal hydration only increased AVP to 15.5 ± 0.7 pg ml−1 (P<0.01).

Under both experimental conditions, lignocaine plasma concentrations were almost double (P<0.01) those in controls, due to a lower systemic clearance, e.g. 54 ± 3 and 59 ± 1 vs. 96 ± 5 ml min−1 kg−1, respectively. Plasma levels of MEGX increased (P<0.01) only in dehydrated animals, although GX plasma concentrations were augmented (P<0.01) about three fold in both groups of animals. The changes in lignocaine plasma concentrations were correlated with AVP levels (R2 = 0.5168, P<0.001).

To document the effect of AVP on hepatic plasma flow, another group of rabbits received on separate occasions two doses of AVP (17 and 84 ng kg−1) while receiving an infusion of indocyanine green. AVP reduced hepatic plasma flow from 38.9 ± 2.7 ml min−1 to 19.6 ± 2.5 ml min−1 (P<0.01). The predicted maximal AVP‐induced decrease in hepatic plasma flow was 19.6 ml min−1 kg−1 (Emax), and AVP concentration eliciting 50% of Emax (ED50) was 28.7 pg ml−1.

It is concluded that both dehydration and hyperosmolal hydration alter the disposition of lignocaine and two of its metabolites.
Endothelium‐derived relaxing factor released by 5‐HT: distinct from nitric oxide in basilar arteries of normotensive and hypertensive rats
Authors: Yokota Yoshihiro , Imaizumi Yuji , Asano Masahisa , Matsuda Tomohiro , Watanabe Minoru
Published: July 2012

The role of the endothelium in cerebrovascular responses to 5‐hydroxytryptamine (5‐HT) was investigated in spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto rats (WKY) in vitro.

Cumulative addition of 5‐HT caused concentration‐dependent contractions in ring preparations of SHR basilar arteries; the contractile response was smaller in WKY basilar arteries.

Removal of the endothelium enhanced markedly the contractile responses to 5‐HT in WKY arteries but had only a slight effect in SHR arteries. The responsiveness to 5‐HT in WKY arteries after removal of endothelium was comparable to that in SHR arteries.

The endothelium‐dependent relaxation induced by acetylcholine in WKY basilar arteries was almost abolished by treatment with 10 μm methylene blue or 10 μm NG‐nitro‐l‐arginine (l‐NOARG). However, the response to 5‐HT was not affected by treatment with methylene blue, l‐NOARG or indomethacin.

Application of 10–20 mm K+ or 3.2 mm tetraethylammonium (TEA) did not change significantly, or only increased slightly, the resting tension, but markedly enhanced the contractile response to 5‐HT in WKY arteries with endothelium. In contrast, the submaximal response to 5‐HT in SHR arteries with endothelium was significantly enhanced by 0.3 mm TEA.

In the presence of 1 mm TEA, the application of 10 μm l‐NOARG further enhanced the responses of 5‐HT in WKY arteries with endothelium. In SHR arteries with endothelium, 10 μm l‐NOARG per se enhanced slightly but significantly the responses to 5‐HT.

These results indicate that 5‐HT‐induced contraction in basilar arteries is substantially attenuated by an endothelium‐dependent mechanism in WKY, but to a much lesser extent in SHR. The major relaxing factor released by 5‐HT from endothelium in WKY is distinct from NO and may exert its effect by activating K+ channels.
Increased striatal dopamine efflux in vivo following inhibition of cerebral nitric oxide synthase by the novel monosodium salt of 7‐nitro indazole
Authors: Silva M.T. , Rose S. , Hindmarsh J.G. , Aislaitner G. , Gorrod J.W. , Moore P.K. , Jenner P. , Marsden C.D.
Published: July 2012

The role of nitric oxide (NO) in striatal dopamine release has been controversial. Most NO synthase inhibitors affect more than one isoform of the enzyme and exert vasoconstrictor effects which may also affect striatal dopamine function. We now report on the effect of a soluble monosodium salt of the selective brain NO synthase inhibitor 7‐nitro indazole (7‐NINA). Using 7‐NINA the first study of selective inhibition of the brain isoform of NO synthase on dopamine efflux in rat striatum was undertaken by use of in vivo microdialysis. Perfusion with 7‐NINA (1 mm) increased striatal dopamine efflux. The effect of 7‐NINA was partially antagonized (67%) by co‐perfusion with l‐arginine (1 mm), the precursor of NO formation in vivo. This suggests that 7‐NINA induces a competitive inhibition of NO synthase activity. These data show that endogenous NO has an inhibitory effect on striatal dopamine efflux in vivo.
Contribution of a cholinergic reflex mechanism to allergen‐induced bronchial hyperreactivity in permanently instrumented, unrestrained guinea‐pigs
Authors: Santing Ruud E. , Pasman Yfke , Olymulder Clemens G. , Roffel Ad F. , Meurs Herman , Zaagsma Johan
Published: July 2012

In conscious, permanently instrumented, unrestrained, ovalbumin‐sensitized guinea‐pigs the development of allergen‐induced bronchial hyperreactivity to histamine‐ and methacholine‐inhalation was investigated after the early as well as after the late asthmatic response.

The allergen‐induced increase in bronchial reactivity to histamine was significantly higher than to methacholine.

The muscarinic receptor antagonist, ipratropium bromide (1.0 mm, 3 min inhalation), blocked methacholine‐induced bronchoconstriction and caused a significant 1.7 fold inhibition of the histamine‐induced bronchoconstriction of control animals.

A lower dose of ipratropium bromide (0.1 mm, 3 min inhalation) had no significant effect on histamine‐induced bronchoconstriction in control animals, but significantly reduced the allergen‐induced increase in bronchial reactivity to histamine between the early and late asthmatic response. At 1.0 mm ipratropium bromide, no further reduction was observed.

These results clearly indicate that an exaggerated cholinergic reflex mechanism contributes to allergen‐induced bronchial hyperreactivity to histamine.
Inhibition by wortmannin of M‐current in bullfrog sympathetic neurones
Authors: Tokimasa Takayuki , Ito Masaaki , Simmons Mark A. , Schneider Carla R. , Tanaka Toshio , Nakano Takeshi , Akasu Takashi
Published: July 2012

The actions of wortmannin, an inhibitor of myosin light chain kinase (MLCK), on M‐type potassium current of dissociated bullfrog sympathetic neurones have been examined.

The amplitude of M‐current was measured by whole cell recordings from cells pretreated with wortmannin (0.01–10 μm) or the wortmannin vehicle, dimethylsulphoxide (0.0001–0.1 vol%), for 30 min. Internal (recording pipette) solutions having three different pCa values (6, 7 and 8) were used for the measurements.

Irrespective of the pCa, M‐current was not detectable when the cells were pretreated with 10 μm wortmannin. Wortmannin, 3 μm, produced 85–95% inhibition of the M‐current. Pretreatment with 10–30 nm wortmannin was without effect on M‐current.

The M‐current inhibition by wortmannin at concentrations of 0.1–1 μm depended on the pCa of the internal solution. Inhibition occurred only when the calcium‐rich (pCa = 6) internal solution was used.

Pre‐treatment of the cells with wortmannin (10 μm) did not affect rapidly‐inactivating A‐type or delayed rectifier‐type potassium currents nor did it alter inwardly rectifying sodium‐potassium current (IH).

These observations show that M‐current inhibition by wortmannin has two pharmacological profiles. One is calcium‐dependent and occurs at lower concentrations (0.1–1 μm), and is attributed to inhibition of MLCK by wortmannin. At higher concentrations (3–10 μm), wortmannin has an additional, calcium‐independent action, inhibiting the M‐current by an unknown mechanism.
Isothioureas: potent inhibitors of nitric oxide synthases with variable isoform selectivity
Authors: Southan Garry J. , Szabó Csaba , Thiemermann Christoph
Published: July 2012

The induction of a calcium‐independent isoform of nitric oxide (NO) synthase (iNOS) and a subsequent enhanced formation of NO has been implicated in the pathophysiology of a variety of diseases including inflammation and circulatory shock. Here we demonstrate that the S‐substituted isothioureas, S‐methylisothiourea (SMT), S‐(2‐aminoethyl)isothiourea (aminoethyl‐TU), S‐ethylisothiourea (ethyl‐TU) and S‐isopropylisothiourea (isopropyl‐TU) potently inhibit iNOS activity in J774.2 macrophages activated with bacterial endotoxin with EC50 values 8–24 times lower than that of NG‐methyl‐l‐arginine (MeArg) and 200‐times lower than that of NG‐nitro‐l‐arginine (l‐NO2Arg).

The inhibition of iNOS activity by these S‐substituted isothioureas is dose‐dependently prevented by excess of l‐arginine suggesting that these isothioureas are competitive inhibitors of iNOS at the l‐arginine binding site.

Ethyl‐TU and isopropyl‐TU are 4–6 times more potent than MeArg in inhibiting the constitutive NOS activity in homogenates of bovine aortic endothelial cells (eNOS) and are more potent pressor agents than MeArg in the anaesthetized rat. SMT is equipotent with MeArg, whereas aminoethyl‐TU is 6‐times less potent in inhibiting eNOS activity in vitro. Both SMT and aminoethyl‐TU, however, elicit only weak pressor responses (approximately 15mmHg at 10 mg kg−1, i.v.) in vivo.

A comparison of the potencies of ethyl‐, iso‐propyl‐, n‐propyl‐, t‐butyl‐ and n‐butyl‐isothioureas on iNOS activity shows that the inhibitory activity of S‐substituted isothioureas declines sharply if the side chain exceeds 2 carbon atoms in length. Similarly, substitution of the ethylene side chain of ethyl‐TU also results in a diminished potency. Substitution of either one or both nitrogens of SMT with either amino or alkyl groups also substantially reduces its NOS inhibitory potency.

In conclusion, isothioureas represent a new class of NOS inhibitors which includes the most potent inhibitors of iNOS activity reported to date. Some members of this class (ethyl‐TU and isopropyl‐TU) are potent inhibitors of eNOS and iNOS with little selectivity towards either isoform, while others (SMT and aminoethyl‐TU) are relatively selective inhibitors of iNOS activity. These latter agents may become useful tools for studying the role of iNOS in various disease models and may be useful in the therapy of diseases that are associated with an enhanced formation of NO due to iNOS induction, such as inflammation, circulatory shock or cancer.
Modulation of Ca2+‐dependent currents in metabolically stressed cultured sensory neurones by intracellular photorelease of ATP
Authors: Stapleton S.R. , Bell B.A. , Wootton J.F. , Scott R.H.
Published: July 2012

The whole cell recording technique was used to study high voltage‐activated Ca2+ currents and Ca2+‐activated Cl− tail currents from cultured neonatal dorsal root ganglion neurones of the rat which were metabolically stressed. The neurones were metabolically stressed with 2‐deoxy‐d‐glucose (5 mm) for 30 min to 3 h. The aim of the project was to examine the actions of intracellular photorelease of ATP on the properties of Ca2+‐dependent currents and determine if the effects of metabolic stress could be reversed.

The mean duration of Ca2+‐activated Cl− tail currents was significantly increased by metabolic stress and this effect was reversed by intracellular photorelease of approximately 300 μm ATP. Intracellular photolysis of ‘caged’ photolabile compounds was achieved with a xenon flash lamp.

Intracellular photorelease of ATP and adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) (about 40 μm) also accelerated the inactivation of high voltage‐activated Ca2+ currents evoked by 500 ms depolarizing step commands from −90 mV to 0 mV. This effect was prevented by intracellular application of the calcineurin (protein phosphatase‐2B) inhibitor cyclosporin A (14 nm) and cyclophihn A (50 nm) either applied together or individually. In contrast the protein phosphatase 1 and 2A inhibitor, calyculin A, increased voltage‐activated Ca2+ currents, but failed to prevent enhanced inactivation induced by intracellular photorelease of ATP. Intracellular photorelease of ATP had no effect on Ca2+ currents recorded from control neurones which were not metabolically stressed and supplied with glucose and ATP in the extracellular and patch pipette solutions respectively.

In conclusion, intracellular photorelease of ATP increases the decay of Ca2+‐activated Cl− tail currents in metabolically stressed neurones suggesting that the efficiency of intracellular Ca2+ buffering was improved. Additionally, an ATP/cyclic AMP‐dependent component of high voltage‐activated Ca2+ current inactivation which is mediated by calcineurin is revealed following photolysis of ‘caged’ ATP or cyclic AMP in metabolically stressed neurones.
Secretion of endothelin‐1 and endothelin‐3 by human cultured vascular smooth muscle cells
Authors: Yu Julie C.M. , Davenport Anthony P.
Published: July 2012

It is generally accepted that endothelial cells secrete endothelin (ET) to the underlying media which mediates the contractile effects of ET. However, there is some evidence that animal vascular smooth muscle cells (VSMCs) also secrete ET. We cultured VSMCs from human vessels representative of a number of different vascular beds to determine whether human VSMCs endogenously secrete ET.

VSMCs explanted from adult arterial vessels secrete picomolar quantities of immunoreactive mature ET: coronary artery 226.6 ± 58.8 pm/106 cells (n = 7), thoracic aorta 169.5 ± 105.4 pm/106 cells (n = 3), left internal mammary artery 102.4 ± 23.1 pm/106 cells (n = 3) and saphenous vein 69.4 ± 19.9 pm/106 cells (n = 3), as well as from umbilical vein (HUVSMCs) 38.3 ± 4.3 pm/106 cells (n = 3). Secretion of immunoreactive big ET‐1 was also detected: coronary artery 249.1 ± 59.4 pm/106 cells (n = 7), thoracic aorta 120.0 ± 13.4 pm/106 cells (n = 3), left internal mammary artery 170.0 ± 68.2 pm/106 cells (n = 3), saphenous vein 105.1 ± 30.7 pm/106 cells (n = 3) and from umbilical vein 146.3 ± 7.4 pm/106 cells (n = 3). Comparable, intracellular levels of immunoreactive big ET‐1 and mature ET were also detected in cultured VSMCs.

Since enzyme‐dispersed VSMCs are thought to be more differentiated and more closely resemble their in vivo counterparts, and these enzyme‐dispersed VSMCs from human umbilical vein (HUVSMCs) also secreted the greatest levels of immunoreactive peptides, they were characterized further. Reverse transcription‐polymerase chain reaction assay demonstrated that HUVSMCs express ET‐1 mRNA. High performance liquid chromatography coupled to radioimmunoassay revealed that HUVSMCs secrete ET‐1 and ET‐3, in addition to big ET‐1. However, levels of ET are not altered by 100 μm phosphoramidon, an inhibitor of metalloproteases or by 100 μm pepstatin A, an aspartyl protease inhibitor.

In concordance, KD and Bmax values for [125I]‐ET‐1 saturation binding are not altered in HUVSMC cultures incubated for 24 h with 100 μm phosphoramidon (431 ± 218 pm and 31.1 ± 12.7 fmol mg−1; mean ± s.e. mean, n = 3) or 100 μm pepstatin A (381 ± 169 pm and 19.9 ± 7.8 fmol mg−1, n = 3) as compared to controls (355 ± 99 pm and 33.3 ± 9.3 fmol mg−1; n = 3). This observation indicates the absence of an autocrine ‘unmasking’ effect for ET receptors.

HUVSMCs synthesize and secrete immunoreactive ET‐1, ET‐3 and big ET‐1, and possess intracellular levels of immunoreactive mature ET and big ET‐1. There is some evidence of common cellular mechanisms between growth factors and vasoconstrictor peptides, suggesting a close relationship between contraction and proliferation. Since the development of various vascular pathologies such as atherosclerosis, hypertension and after vessel injury has been attributed to alterations in the normal growth pattern of VSMCs, the role of ET in these diseases may be of significance.
Respiratory effects of baclofen and 3‐aminopropylphosphinic acid in guinea‐pigs
Authors: Hey John A. , Mingo Garfield , Bolser Donald C. , Kreutner William , Krobatsch Dawn , Chapman Richard W.
Published: July 2012

The effects of the GABAB receptor agonists, baclofen and 3‐aminopropylphosphinic acid (3‐APPi) given by the subcutaneous or intracerebroventricular (i.c.v.) route were examined on minute ventilation (), tidal volume (VT) and respiratory rate (f) due to room air and carbon dioxide (CO2)‐enriched gas hyperventilation in conscious guinea‐pigs.

Baclofen (0.3–10 mg kg−1, s.c.) produced a dose‐dependent inhibition of and f due to room air and CO2 inhalation. The maximum inhibition of room air breathing was 85% ± 3 and f was 74% ± 3 at 10 mg kg−1, s.c. The maximum effects on CO2‐induced hyperventilation were 68% ± 9 and 51% ± 6, for and f respectively. Only the highest dose of baclofen studied (10 mg kg−1) produced a significant inhibition of VT due to room air breathing (46% ± 6) and CO2 breathing (38% ± 11).

3‐aPPi (0.3–100 mg kg−1, s.c.) did not affect , VT or f due to room air breathing or CO2 inhalation at any dose tested. Also, i.c.v. administration of 3‐aPPi (100 μg) did not affect ventilatory responses due to room air breathing or CO2 inhalation.

Pretreatment with the GABAB antagonist, CGP 35348 3‐aminopropyl‐(diethoxymethyl) phosphinic acid (3–30 mg kg−1, s.c.) blocked the respiratory depressant effects of baclofen (3 mg kg−1, s.c.) in a dose‐related fashion.

Intracerebroventricular (i.c.v.) administration of CGP 35348 (50 μg) blocked the respiratory depressant effects of baclofen. CGP 35348 given alone either i.c.v. or s.c. had no effects on respiration due to room air or CO2 inhalation.

Pretreatment with either the GABAA antagonist bicuculline (30 mg kg−1, s.c.) or the opioid antagonist, naloxone (1 mg kg−1, s.c.) had no effect on the respiratory depressant action of baclofen (3 mg kg−1, s.c).

These results show that baclofen inhibits ventilation due to room air breathing, and attenuates the hyperventilation response to CO2 inhalation. The peripherally acting GABAB agonist, 3‐APPi had no effect on ventilation. These findings demonstrate that the respiratory depressant effects of baclofen are due to activation of CNS GABAB receptors and indicates that only GABAB receptor agonists that penetrate into the CNS may cause respiratory depression.
Effects of Bordetella pertussis toxin pretreatment on the antiarrhythmic action of ischaemic preconditioning in anaesthetized rats
Authors: Piacentini L. , Wainwright C.L. , Parratt J.R.
Published: July 2012

Bordetella pertussis toxin, which catalyses the ADP‐ribosylation of certain guanine nucleotide binding proteins (G proteins), thus functionally uncoupling them from associated receptors, was examined to determine whether it modified the antiarrhythmic effect of ischaemic preconditioning in anaesthetized rats.

Pertussis toxin (25 μg kg−1, i.p., 48 h prior to heart isolation) attenuated the negative chronotropic effect of acetylcholine (ACh) in rat isolated Langendorff perfused hearts. ACh (10 μm) reduced heart rate by 4% in hearts taken from pertussis toxin‐treated animals, compared to a reduction of 57% in hearts taken from animals treated only with vehicle.

In anaesthetized rats, ischaemic preconditioning (a single 3 min occlusion of the left main coronary artery followed by 10 min reperfusion) had a pronounced antiarrhythmic effect during a subsequent 30 min period of regional myocardial ischaemia. Compared to hearts receiving only a 30 min period of left coronary occlusion, there was a reduced mortality (67% and 0% for control and preconditioned groups, respectively; P<0.01) and decreased incidences of ventricular tachycardia (VT) and ventricular fibrillation (VF). Pretreatment with pertussis toxin (25 μg kg−1, i.p., 48 h previously) did not modify the arrhythmias associated with a 30 min period of regional myocardial ischaemia, neither did it modify the reduction in mortality (from 56% to 0%; P<0.05) associated with preconditioning. Furthermore, the decrease in total ventricular premature beat count induced by preconditioning seen in controls (from 427 ± 130 to 95 ± 45) was also seen in pertussis toxin‐treated rats (from 252 ± 190 to 57 ± 25).

These results suggest that receptor coupling to pertussis toxin‐sensitive G proteins is not necessary for the antiarrhythmic effect of ischaemic preconditioning in this model.
Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle
Authors: Masuda Yutaka , Yamahara Natsuki S. , Tanaka Masako , Ryang Shinwon , Kawai Tomoyuki , Imaizumi Yuji , Watanabe Minora
Published: July 2012

The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined.

Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 μm or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN‐A‐343 induced mainly relaxation but no substantial contraction.

After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN‐A‐343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction.

pA2 values of pirenzepine, methoctramine, AF‐DX 116, himbacine, and 4‐DAMP for antagonism to pilocarpine‐induced relaxation in normal dilator muscles and those for antagonism to ACh‐induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3‐type muscarinic receptors.

Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3‐like receptors which have a very low affinity for methoctramine.

In conclusion, ACh‐induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only.
Modulation of NMDA effects on agonist‐stimulated phosphoinositide turnover by memantine in neonatal rat cerebral cortex
Authors: Mistry Rajendra , Wilke Rolf , Challiss R.A. John
Published: July 2012

The ability of memantine (1‐amino‐3,5‐dimethyladamantane) to antagonize the modulatory effects of N‐methyl‐d‐aspartate (NMDA) on phosphoinositide turnover stimulated by muscarinic cholinoceptor‐and metabotropic glutamate receptor‐agonists has been examined in neonatal rat cerebral cortex slices.

Memantine antagonized the inhibitory effect of NMDA (100 μm) on both total [3H]‐inositol phosphate ([3H]‐InsPx) and inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) mass accumulations stimulated by carbachol (1 mm) with EC50 values of 21 and 16 μm respectively.

Memantine concentration‐dependently antagonized (IC50 24 μm) the ability of NMDA (10 μm) to potentiate [3H]‐InsPx accumulation in response to a sub‐maximal concentration of the metabotropic glutamate receptor agonist, 1S,3R‐ACPD (10 μm).

The small (approx. 3 fold), concentration‐dependent increase in [3H]‐InsPx accumulation stimulated by NMDA was completely antagonized by the prototypic NDMA receptor‐channel blocker, MK‐801 (1 μm) at all concentrations of NDMA studied (1 – 1000 μm). In contrast, antagonism by memantine (100 μm) was observed only at low concentrations of NMDA (1 – 10 μm), whilst [3H]‐InsPx accumulation stimulated by high concentrations of NMDA (300 – 1000 μm) was markedly enhanced by memantine.

Assessment of the incorporation of [3H]‐inositol into inositol phospholipids revealed that memantine (100 μm) caused an approximate 2 fold increase in the labelling of phosphatidylinositol, phos‐phatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate.

H.p.l.c. separation of [3H]‐inositol (poly)phosphates demonstrated that whilst memantine (100 μm) alone had no significant effect on the accumulation of any isomer, it substantially altered the profile of accumulation stimulated by NMDA (1 mm), greatly facilitating accumulation of Ins(1,4,5)P3 and inositol 1,3,4,5‐tetrakisphosphate (Ins(1,3,4,5)P4).

These data provide evidence that memantine can antagonize the actions of NMDA in neonatal rat cerebral cortex slices in a manner consistent with this agent acting as a NMDA receptor‐channel blocker. In addition, at least two further actions of memantine can be proposed. Memantine increases the rate of [3H]‐inositol incorporation into the cellular inositol phospholipid fraction, without significantly stimulating phosphoinositide turnover. Furthermore, memantine can substantially alter patterns of inositol (poly)phosphates stimulated by NMDA, promoting the accumulation of the established and putative second messengers Ins(1,4,5)P3 and Ins(1,3,4,5)P4 which are not increased by NMDA in the absence of memantine. It is unknown whether these latter loci of memantine action contribute to known therapeutic actions of this agent.
Rapid degradation of endothelin‐1 by an enzyme released by the rat isolated perfused mesentery
Authors: Pérez‐Vizcaíno Francisco , Cooper Andrea C. , Corder Roger , Fournier Alain , Warner Timothy D.
Published: July 2012

In vivo the effects of endothelin‐1 (ET‐1) are limited by its rapid removal from the circulation and possibly by its metabolism by enzymes such as neutral endopeptidase 24.11, deamidase or carboxypeptidase A. Here, using as a model the isolated perfused mesenteric arterial bed of the rat, we have examined the involvements of these enzymatic activities in the vascular responses to ET‐1.

Samples of Krebs buffer which had been recirculated through the mesenteric arterial bed for 30 min rapidly destroyed the activity of ET‐1 as assessed either by bioassay on rings of rat thoracic aorta or by high performance liquid chromatography (h.p.l.c). For instance, after 15 min incubation with the recirculated‐Krebs solution (recirc‐K) the contraction induced by 3 times 10−9 m ET‐1 was reduced by more than 90%. Contractions induced by sarafotoxin 6b (3 times 10−9 m) were similarly suppressed by preincubation with recirc‐K whereas those to Arg‐vasopressin (3 times 10−9 m) were unaffected.

The degradation of ET‐1 by recirc‐K was prevented by 1,10‐phenanthroline (10−3 m), abolished by heating the recirc‐K solution to 90°C for 15 min, and reduced by EGTA (5 times 10−3 m) or ET‐1(16–21) (10−5 m). For instance, in the presence of ET‐1(16–21) (n = 6) the contraction induced by ET‐1 was reduced by only 40% after 15 min incubation with recirc‐K buffer. Leupeptin (3 times 10−4 m), dichloroisocoumarin (5 times 10−5 m), phenylmethyl‐sulphonyl fluoride (10−3 m), a combination of bacitracin (300 mg ml−1), bestatin (10−5 m), captopril (10−5 m), phosphoramidon (10−4 m) and thiorphan (10−4 m) or Polypep (a proprietary protein digest) did not inhibit the degradation of ET‐1 by recirc‐K.

In experiments examining directly the vascular responses of the isolated perfused mesentery of the rat, the addition of cumulative concentrations of ET‐1 to the recirculating Krebs solution caused small concentration‐dependent increases in perfusion pressure. The inclusion of ET‐1(16–21), ET‐1(17–21), or ET‐1(18–21) (10−5 m) greatly potentiated these responses, but not those to Arg‐vasopressin or methoxamine. The effects of 1, 10‐phenanthroline or EGTA could not be examined in this system because these agents both depressed non‐specifically the vasoconstrictor responses of the mesenteric vascular bed.

Thus, the rat mesentery releases an enzyme that very rapidly destroys ET‐1 or the very closely related peptide, sarafotoxin 6b but not Arg‐vasopressin. This enzyme is most probably a metallo‐peptidase because of its sensitivity to inhibition by 1,10‐phenanthroline or EGTA. It is particularly interesting that a simple vascular bed such as the mesentery produces such a powerful endothelin metabolising enzyme. It is tempting, therefore, to speculate that the endothelin degrading enzyme active at neutral pH that we have found is important in the metabolism of ET‐1 throughout the vasculature.
SPECIAL REPORT Endothelium‐dependent relaxation to the B1 kinin receptor agonist des‐Arg ‐bradykinin in human coronary arteries
Authors: Drummond G.R. , Cocks T.M.
Published: July 2012

Des‐Arg9‐bradykinin (des‐Arg9‐BK) caused endothelium‐dependent relaxations in human, isolated coronary arteries which upregulated with in vitro incubation time. Relaxations to des‐Arg9‐BK were inhibited by the B1 receptor antagonist, des‐Arg9‐[Leu8]‐BK (pKB, 6.14 ± 0.11) but were unaffected by the B2 receptor antagonist, Hoe‐140. Therefore, this is the first demonstration that human coronary arteries possess endothelial B1 receptors which mediate endothelium‐dependent relaxation and appear to be synthesized de novo during the incubation period.