bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric oxide‐donating properties of mesoionic 3‐aryl substituted oxatriazole‐5‐imine derivatives
Authors: Kankaanranta H. , Rydell E. , Petersson A.‐S. , Holm P. , Moilanen E. , Corell T. , Karup G. , Vuorinen P. , Pedersen S.B. , Wennmalm ÅR. , Metsä‐Ketelä T.
Published: July 2012

The nitric oxide (NO)‐releasing properties of two new mesoionic 3‐aryl substituted oxatriazole‐5‐imine derivatives (GEA 3162 and GEA 3175) were characterized and compared with the known NO‐donors 3‐morpholino‐sydnonimine (SIN‐1) and S‐nitroso‐N‐acetylpenicillamine (SNAP).

GEA 3162, GEA 3175, SIN‐1 and SNAP inhibited adenosine 5′‐diphosphate‐induced platelet aggregation (IC50 values 0.18, 0.39, 3.73 and 2.12 μm, respectively). All four compounds induced a dose‐dependent and more than 4 fold increase in cyclic GMP in platelets. The increase in cyclic GMP concentration was potentiated more than 1.5 fold by a phosphodiesterase inhibitor, zaprinast (10 μm) and inhibited 38–97% by oxyhaemoglobin (10–45 μm).

All of the four compounds studied converted oxyhaemoglobin to methaemoglobin and formed a paramagnetic NO‐haemoglobin complex. All but GEA 3175 formed nitrite and nitrate in phosphate buffer. During a 40 min incubation, GEA 3162, SIN‐1 and SNAP (100 μm) produced 50–70 μm NO2−+NO3− as determined by high performance liquid chromatography. The release of NO and NO2 by GEA 3175 was increased 140 fold in the presence of human plasma (0.14 and 19.7 ppb in the absence and presence of 1% human plasma, respectively) as analyzed by ozone chemiluminescence.

The results suggest that the mesoionic 3‐aryl substituted oxatriazole‐5‐imine derivatives GEA 3162 and GEA 3175 as well as SIN‐1 and SNAP release nitric oxide.
Ontogenic aspects of liver and kidney catechol‐O‐ methyltransferase sensitivity to tolcapone
Authors: Vieira‐Coelho M.A. , Soares‐da‐Silva P.
Published: July 2012

The present work describes the catechol‐O‐methyltransferase (COMT) activities in the liver and kidney of developing and adult rats (aged 3, 6, 9, 18, 30 and 60 days; n = 5 per group) and evaluates the enzyme sensitivity to inhibition by tolcapone, a reversible COMT inhibitor.

COMT activity, evaluated by the ability to methylate adrenaline to metanephrine, was determined in liver and kidney homogenates prepared in 0.5 mM phosphate buffer (pH = 7.8) containing pargyline (0.1 mM), MgC12 (0.1 mM), EGTA (1 mM) and S‐adenosyl‐L‐methionine (0.1 mM). Vmax (in nmol mg−1 protein h−1) of liver COMT was found to decrease gradually with age, from 5.3±0.5 at the age of 3 days up to 2.9±0.2 at the age of 60 days; for the same age range, Km values (in μm; geometric means with 95% confidence limits) increased from 3.3 (1.0, 7.5) up to 13.1 (2.1, 24.1). At the age of 3 days, Vmax values for kidney COMT (2.6±0.1) were lower than those for the liver COMT. However, Vmax values for kidney COMT were found to increase up to 6.2±0.6 at the age of 18 days and then declined by 44% at the age of 30 and 60 days. In kidney, aging was also accompanied by an increase in Km values for COMT (from 2.7 [1.1, 4.3] up to 24.0 [11.7, 36.3]).

The sensitivity of liver and renal COMT activity to tolcapone was markedly dependent on the age, 3‐days old rats being more sensitive to tolcapone than older animals. The IC50 values (in nM) for inhibition of liver COMT by tolcapone increased gradually with age, from 41 (26, 65) at the age of 3 days up to 720 (640, 800) at the age of 60 days. As was found in the liver, IC50 values (in nM) for inhibition of kidney COMT by tolcapone also increased with age, from 8 (6, 10) at the age of 3 days up to 177 (131, 240) at the age of 60 days. In all experimental groups, the IC50 values for inhibition of liver COMT by tolcapone was higher than those for kidney COMT.

In conclusion, these results suggest that aging is accompanied by a decrease in liver and kidney COMT affinity for the substrate (evidenced by the increase in Km values) and a decrease in sensivity towards inhibition by tolcapone (evidenced by the increase in IC50 values). Furthermore, kidney COMT is shown to be more sensitive to inhibition by tolcapone than liver COMT, irrespective of the age of the animal.
Barbiturate interactions at the human GABAA receptor: dependence on receptor subunit combination
Authors: Thompson S.A. , Whiting P.J. , Wafford K.A.
Published: July 2012

Human GABAA receptors containing different α and β subunits with a γ2s subunit were expressed in Xenopus oocytes and the effects of pentobarbitone on these subunit combinations were examined by electrophysiological recording of GABA currents with the two‐electrode voltage‐clamp method.

Pentobarbitone has previously been shown to have three actions on GABAA receptors: a potentiation of GABA responses, a direct activation of GABAA receptors and, at high concentrations, a block of the GABA chloride channel. In this study pentobarbitone activity consisted of the above mentioned three components on all the subunit combinations tested. However, the affinities and efficacies varied with receptor subtype.

Potentiation of GABA by pentobarbitone occurred over the same concentration‐range for all the subunits with affinities in the range of 20–35 μm. The degree of potentiation obtained, however, varied from 236% of GABA EC20 on α1β2γ2s to 536% on α6β2γ2s.

Examination of the direct effect of pentobarbitone revealed that the type of α subunit present determines both the degree of affinity and efficacy obtained. Receptors containing an α6 subunit produced maximum direct responses to pentobarbitone larger than that obtainable with maximum GABA (150% to 170% of maximum GABA). The maximum direct pentobarbitone response obtainable with other α subunits ranged between 45% of maximum GABA for α5β2γ2s to 82% for α2β2γ2s. The affinity of the direct action of pentobarbitone on α6β2γ2s was 58 μm compared to affinities for the other α subunits ranging from 139 μm on α2β2γ2s to 528 μm on α5β2γ2s.

The type of β subunit present did not influence the direct action of pentobarbitone to the same extent as the α subunit. There were no significant differences between affinity or efficacy on oocytes expressing α6 and γ2s with β1, β2 or β3. Affinities and efficacies on oocytes expressing α1 and γ2s with β1, β2 or β3 were significantly different with pentobarbitone having a higher affinity and efficacy on α1β3γ2s followed by α1β2γ2s and then α1β1γ2s.

The direct effect of pentobarbitone was blocked by picrotoxin but not by competitive antagonists, such as bicuculline or SR95531, indicating that the direct agonist activity of pentobarbitone was not mediated via the GABA binding site.

For the first time the influence of the various α and β subunits on the effects of pentobarbitone were demonstrated. The results indicate that GABAA receptors containing α6 subunits have both a higher affinity and efficacy for direct activation by pentobarbitone, and reveal that pentobarbitone binds to more than one site on the GABAA receptor, and these are dependent on receptor subunit composition.
Investigation into the 5‐hydroxytryptamine‐induced atropine‐resistant neurogenic contraction of guinea‐pig proximal colon
Authors: Kojima Shu‐Ichi , Shimo Yasuo
Published: July 2012

The aim of this study was to characterize the receptors mediating the atropine‐resistant neurogenic contraction to 5‐hydroxytryptamine (5‐HT) in the longitudinal muscle of the guinea‐pig proximal colon and to determine the type of tachykinin receptors involved in the contractile response to 5‐HT by the use of selective antagonists.

In the presence of atropine (0.3 μm), guanethidine (5 μm), hexamethonium (100 μm), ketanserin (0.1 μm) and indomethacin (3 μm), 5‐HT (0.01–3 μm) produced concentration‐dependent neurogenic contractions of colonic strips and at 0.3 μm produced a maximal effect (pEC50 = 7.39±0.09, n = 18). The 5‐HT4 receptor stimulant, 5‐methoxytryptamine (5‐MeOT, 0.03–10 μm) also produced neurogenic contractions with similar maximum effect to those of 5‐HT (pEC50 = 6.89±0.16).

The 5‐HT4 receptor antagonist, DAU 6285 (3 μm) shifted the concentration‐response curves to both 5‐HT and 5‐MeOT to the right without significant depression of the maximum, but the 5‐HT1/5‐HT2 receptor antagonist, metitepine (0.1 μm) and the 5‐HT3 receptor antagonist, ondansetron (0.3 μm) had no effect on the control curves to 5‐HT and 5‐MeOT.

The selective NK1 receptor antagonist, FK 888 (1 μm) markedly attenuated the contractions to 5‐HT and 5‐MeOT. In contrast, the selective NK2 receptor antagonist, SR 48968 (10 nM) and the selective NK3 receptor antagonist, SR 142801 (10 nM) had no effect on the contractions to 5‐HT and 5‐MeOT.

These results indicate that the 5‐HT‐induced atropine‐resistant neurogenic contraction of guinea‐pig proximal colon is due to activation of 5‐HT4 receptors, presumably located on excitatory motor neurones, innervating the longitudinal muscle. The contraction evoked by activation of the 5‐HT4 receptors is mediated primarily via NK1 receptors but not NK2 or NK3, suggesting that the 5‐HT4 receptor‐mediated contraction is evoked indirectly via tachykinin release from tachykinin‐releasing excitatory neurones.
Operational characteristics of somatostatin receptors mediating inhibitory actions on rat locus coeruleus neurones
Authors: Chessell I.P. , Black M.D. , Feniuk W. , Humphrey P.P.A.
Published: July 2012

In order to characterize somatostatin (SRIF) receptor inhibiting spontaneous firing of rat locus coeruleus neurones, and their transduction mechanism(S), extracellular recordings were obtained from a pontine slice preparation of rat brain containing the locus coeruleus (LC). LC neurones were identified by electrophysiological and pharmacological properties; spontaneous firing (characteristically 0.5‐5 Hz) was reversibly and concentration‐dependently inhibited by exogenously applied noradrenaline.

Spontaneous firing of LC neurones was reversibly and concentration‐dependently inhibited by SRIF and the N‐terminally extended form, somatostatin‐28 (SRIF‐28), with EC50 values of 15.1 and 19.4 nM, respectively. The synthetic SRIF analogues (octreotide, MK‐678, BIM‐23027 and L‐362,855) also caused concentration‐dependent inhibition of LC neurone firing with a rank order of agonist potencies compatible with actions at a receptor resembling the recombinant sst2 receptor. The putative sst3 selective agonist, BIM‐23056, was without agonist or antagonist effect.

Addition of 100 nM desipramine significantly increased the efficacy of exogenously applied noradrenaline (EC50 values, 2.96 and 0.13 μm, absence and presence of desipramine, respectively) but did not significantly affect SRIF‐induced inhibition (EC50 values, 15.6 and 8.0 nM, respectively). Furthermore, application of phenoxybenzamine (3 μm) abolished responses to NA, but did not affect responses to SRIF (EC50=14.1 nM).

Application of the cyclic AMP analogue, 8‐bromoadenosine‐cyclic monophosphate (8‐Br‐cyclic AMP; 500 μm), significantly increased the spontaneous firing rate of all neurones tested (223±24% over basal rate). Concentration‐effect curves for SRIF constructed in the absence and presence of 8‐Br‐cyclic AMP had similar threshold concentrations, maxima and EC50 values.

Incubation of pontine slices in a modified artificial CSF containing 500 ng ml−1 pertussis toxin (PTX) for 18 h prior to extracellular recording affected neither the spontaneous firing of LC neurones, nor the inhibitory responses to muscimol (EC50 2.2 and 1.2 μm, absence and presence of PTX). However, inhibitory responses to SRIF were markedly attenuated.

We conclude that the inhibitory actions of SRIF on spontaneous firing of LC neurones are mediated directly by activation of somatodendritic SRIF receptors, and not indirectly by release of noradrenaline. The SRIF receptors involved appear to couple via a pertussis toxin sensitive G‐protein, and elicit their response by a mechanism apparently independent of inhibition of cyclic AMP formation. The agonist profile of several selective and novel SRIF analogues suggests the identity of this receptor to be similar to the recombinant sst2 receptor.
5‐HT3 and 5‐HT4 receptor‐mediated facilitation of the emptying phase of the peristaltic reflex in the marmoset isolated ileum
Authors: Tuladhar B.R. , Costall B. , Naylor R.J.
Published: July 2012

The patterns of intestinal motility and the effect of an increase in intraluminal pressure were studied in vitro on segments obtained from the marmoset small intestine.

Segments obtained from the distal half of the marmoset small intestine exhibited segmentation, consisting of narrow annular contractions (lasting for 2–3 s) interposed between the relaxed segments of varying length. The subsequent contractions occurred slightly distal to the previous contraction, with ring‐like contractions appearing to move in the aboral direction. Such segmentation was infrequent or absent in the segments obtained from the proximal half of the small intestine. An increase in intraluminal pressure inhibited segmentation and finally produced peristalsis in most of the tissues.

The influence of 5‐hydroxytryptamine (5‐HT) receptor agonists and antagonists on the threshold of the peristaltic reflex was investigated in the segments obtained from the distal half of the intestine after segmentation had subsided. The effect of drug application to the serosal surface was measured as a change in threshold pressure required to trigger the peristaltic reflex. A facilitation was defined in two ways (a) as a reduction in the threshold pressure required to trigger the reflex and (b) in those tissues that failed to respond with peristalsis on raising intraluminal pressure to the maximum attainable (1 kPa), as a reduction in threshold pressure compared to this value.

5‐HT (7.85±0.19), 5‐methoxytryptamine (7.79±0.24), 5‐carboxamidotryptamine (6.66±0.13) and 2‐methyl‐5‐HT (6.24±0.16) caused a concentration related facilitation of the peristaltic reflex, the pD2 values (mean±s.e.mean) being shown in parentheses.

The concentration‐response curves to both 5‐HT and 5‐methoxytryptamine were dextrally shifted in a surmountable manner in the presence of GR 113808 (30 nM). pD2 values for 5‐HT and 5‐methoxytryptamine were significantly decreased to 6.98±0.24 and 6.83±0.36 respectively in the presence of GR 113808 (30 nM).

In the presence of a high concentration of (10 μm) 5‐methoxytryptamine the subsequent addition of 2‐methyl‐5‐HT (3–10 μm) but not 5‐methoxytryptamine (10 μm) facilitated peristalsis; the effect of 3 μm 2‐methyl‐5‐HT was significantly decreased by 2 μm ondansetron.

It is concluded that the facilitation of the peristaltic reflex in the marmoset intestine induced by 5‐HT at submicromolar concentrations involves a 5‐HT4 receptor stimulation with an additional 5‐HT3 receptor activation at higher concentrations.
Differences in agonist and antagonist activities for two indices of metabotropic glutamate receptor‐stimulated phosphoinositide turnover
Authors: Mistry Rajendra , Challiss R.A. John
Published: July 2012

The abilities of the four diastereoisomers of 1‐aminocyclopentane‐1,3‐dicarboxylic acid (ACPD) to stimulate, and the metabotropic glutamate receptor (mG1uR) antagonist (±)‐α‐methylcarboxyphenylglycine (MCPG) to inhibit, phosphoinositide turnover in neonatal rat cerebral cortex have been studied. Two indices of phosphoinositide cycle activity were assessed; inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) mass accumulation, and total inositol phosphate [3H]‐InsPx accumulation (in the presence of Li+) in myo‐[3H]‐inositol prelabelled slices.

The diastereoisomers of ACPD stimulated each response with a rank order of potency of 1S, 3R > 1R, 3R > 1S, 3S > > 1R, 3S. The response to 1R, 3R‐ACPD was largely prevented by pre‐addition of the NMDA‐receptor antagonist, MK‐801, or omission of extracellular Ca2+, suggesting that this isomer acts indirectly on phosphoinositide responses through activation of NMDA‐type ionotropic glutamate receptors. In contrast, the responses to 1S, 3R‐ and 1S, 3S‐ACPD were unaffected by prior addition of MK‐801, but were blocked by MCPG.

The concentration of 1S, 3R‐ACPD required to half‐maximally stimulate the Ins(1,4,5)P3 response (‐log EC50 (M), −4.09 ± 0.10) was significantly higher than that required to exert a similar effect on [3H]‐InsPx accumulation (‐log EC50 (M), −4.87 ±0.07; P<0.01; n=4). A similar marked 8–9 fold discrepancy between these two values was observed for the 1S, 3S isomer, which elicited similar maximal responses to those caused by 1S, 3R‐ACPD.

Significant differences were also observed with respect to the ability of (±)‐MCPG (1 mM) to cause a rightward shift in the concentration‐response relationships for 1S, 3R‐ACPD‐stimulated Ins(1,4,5)P3 (5.59±0.24 fold shift) and [3H]‐InsPx (3.04±0.34 fold shift; P<0.01; n=4) responses, giving rise to Kd values of 218 and 490 μm for (±)‐MCPG antagonism of the respective responses.

The potency difference between the 1S, 3R‐ACPD‐stimulated Ins(1,4,5)P3 and [3H]‐InsPx responses was reduced when experiments were performed in nominally calcium‐free medium ([Ca2+]e = 2–5 μm) and EC50 values were almost identical when extracellular calcium was reduced further by EGTA addition ([Ca2+]e≤ 100 nM). Similarly, the Kd value for (±)‐MCPG antagonism of the 1S, 3R‐ACPD‐stimulated [3H]‐InsPx response decreased under [Ca2+]e‐free conditions, approaching those obtained for the 1S, 3R‐ACPD‐stimulated Ins(1,4,5)P3 response in the presence of normal [Ca2+]e.

These data suggest that estimates of the activities of mGluR agonists and antagonists, derived by measuring phosphoinositide turnover, can differ significantly depending on whether Ins(1,4,5)P3 mass or [3H]‐InsPx responses are measured. In particular, the possibility that the mGluR‐mediated [3H]‐InsPx response may not simply reflect direct receptor/G protein/phosphoinositidase C (PIC) activation, but may also be the consequence of stimulation of a facilitatory Ca2+‐influx pathway is discussed.
Possible role of nitric oxide in the development of L‐2‐chloropropionic acid‐induced cerebellar granule cell necrosis
Authors: Widdowson P.S. , Gyte A. , Simpson M.G. , Farnworth M. , Dunn D. , Moore R.B. , Wyatt I. , Lock E.A.
Published: July 2012

L‐2‐Chloropropionic acid (L‐CPA) produces selective neuronal cell necrosis in rat cerebellum when administered orally at 750 mg kg−1 that is mediated in part through activation of N‐methyl‐D‐aspartate (NMDA) receptors. Cerebellar granule cell death occurs between 30 and 36 h following L‐CPA administration exhibiting a number of features in common with excitatory amino acid‐induced cell death. We have used this in vivo model to examine the neurochemical processes following L‐CPA‐induced activation of NMDA receptors leading to neuronal cell death in the rat cerebellum.

The effects of a number of compounds which potently block nitric oxide synthase in vitro were examined on L‐CPA‐induced neurotoxicity 48 h following L‐CPA dosing, to discover whether the neuronal cell death is mediated in part by excessive nitric oxide generation. Four inhibitors were studied, NG‐nitro‐L‐arginine (L‐NOARG), NG‐nitro‐L‐arginine methyl ester (L‐NAME), NG‐iminoethyl‐L‐ornithine (L‐NIO) and 3‐bromo‐7‐nitroindazole (BrNI).

L‐NAME (50 mg kg−1, i.p. twice daily) and BrIN (50 mg kg−1, i.p. twice daily) administration prevented the L‐CPA‐induced loss of granule cells which can reach up to 80–90% of the total cell number in rats treated with L‐CPA alone. L‐NOARG (50 mg kg−1, i.p. twice daily) and L‐NIO administered at either 25 or 100 mg kg−1, twice daily did not produce any significant protection against L‐CPA‐induced neurotoxicity.

Both L‐NAME and BrIN also prevented the L‐CPA‐induced increase in cerebellar water content and sodium concentrations. L‐NIO when administered at the highest doses prevented the increase in cerebellar sodium concentration but not water content. L‐NIO and L‐NOARG were ineffective in preventing the L‐CPA‐induced increases in cerebellar water and sodium concentrations.

L‐CPA‐induced reductions in cerebellar aspartate and glutamate concentrations and increases in glutamine and GABA concentrations were prevented by L‐NAME and BrIN, but not by L‐NIO or L‐NOARG. Also reductions in L‐[3H]‐glutamate binding to glutamate ionotrophic and metabotrophic receptors in the granule cell layer of rat cerebellum was prevented by L‐NAME and BrIN, but not L‐NIO or L‐NOARG.

In conclusion, the neuroprotection offered by L‐NAME and BrIN suggests that L‐CPA‐induced cerebellar granule cell necrosis is possibly mediated by or associated with excessive generation of nitric oxide. The inability of nitric oxide synthase inhibitors, L‐NOARG and L‐NIO to afford protection may result from their limited penetration into the brain (L‐NIO) or rapid dissociation from the enzyme.
Synthesis and characterization of a selective peptide antagonist of neuropeptide Y vascular postsynaptic receptors
Authors: Lew Michael J. , Murphy Roger , Angus James A.
Published: July 2012

A cyclic dimeric nonapeptide neuropeptide Y (NPY) receptor antagonist, 1229U91, was synthesized by Fmoc chemistry and dimerised in solution. Its effects were assayed in mesenteric arteries from rats and mice, and in rat vas deferens.

Mesenteric arteries were cannulated and pressurised to 55 mmHg and the external diameters continuously measured. NPY, PYY, Leu31Pro34NPY and NPY(13–36) each caused concentration‐related contractions with the order of potency PYY≥Leu31Pro34NPY = NPY>NPY (13–36), consistent with the Y1 receptor subtype.

1229U91 had no agonist activity in the arteries but caused a concentration‐related rightward shift of NPY (mouse arteries) or Leu31Pro34NPY (rat) concentration‐response curves. The antagonism was competitive with pATBs of 7.69±0.15 and 7.47±0.13 in the mouse and rat arteries, respectively.

Sympathetic nerves in the vas deferens were stimulated with a single electrical field pulse every 20 s and the twitch responses recorded. NPY, PYY, Leu31Pro34NPY and NPY(13–36) inhibited the twitches with the order of potency PYY>NPY>NPY(13–36)> > Leu31Pro34NPY, consistent with the Y2 receptor subtype.

1229U91 inhibited the vas deferens twitch with a shallow concentration‐response curve and a time‐course of inhibition distinct from that of NPY. 1229U91 (30 μm) did not cause a rightward shift of the NPY concentration‐response curve. 1229U91 is at least 5 orders of magnitude less potent in the vas deferens than in rat brain Y2 binding assays reported by others, suggesting that the brain and vas deferens Y2 receptors are different.

It is concluded that 1229U91 is a competitive antagonist of NPY Y1 vascular receptors and has additional properties that inhibit the electrically evoked twitch of the rat vas deferens.
Effect of cyclo‐oxygenase inhibitors and modulators of cyclic AMP formation on lipopolysaccharide‐induced neutrophil infiltration in mouse lung
Authors: de Moraes Vera Lucia Gonçalves , Vargaftig B. Boris , Lefort Jean , Meager Anthony , Chignard Michel
Published: July 2012

The adult respiratory distress syndrome (ARDS) is an acute lung inflammation developed after direct or indirect contact with pathogenic agents. In the present study, a mouse model was developed to mimic this condition using aerosolized bacterial lipopolysaccharide (LPS) and to investigate the mechanisms involved in the lung inflammatory response.

Inhalation of LPS led to a time and dose‐dependent increase in tumour necrosis factor‐α (TNF‐α) production and neutrophil recruitment into the bronchoalveolar lavage fluid (BALF) of Balb/c mice. Under the same conditions, neutrophil infiltration was also found in the BALF of the LPS‐sensitive mouse strain C3H/HeN, but was absent in the LPS‐resistant strain C3H/HeJ. Intranasal administration of murine recombinant TNF‐α also triggered neutrophil recruitment.

One hour after inhalation of LPS, half of the maximal level of TNF‐α was measured in the BALF, but only a few neutrophils were detected at this time. The peak TNF‐α concentration was reached at 3 h, when the neutrophil amount started to increase. At 24 h, maximal neutrophil number was found in the BALF and TNF‐α was no longer present.

Pretreatment of mice under different experimental conditions demonstrated that: (a) cycloheximide almost completely blocks both neutrophil recruitment and TNF‐α production; (b) anti TNF‐α antibodies block neutrophil recruitment; (c) indomethacin or aspirin enhance by two fold neutrophil recruitment; (d) indomethacin significantly increases TNF‐α production 1 h after inhalation of LPS; (e) dibutyryl cyclic AMP and prostaglandin E2 (PGE2) block both neutrophil recruitment and TNF‐α production.

It is concluded that aerosolized LPS in mice triggers an acute lung inflammation which can be used as a potential model of inhalational ARDS and that, strategies leading to the elevation of cyclic AMP levels in vivo can be effective in modulating LPS‐induced TNF‐α synthesis and neutrophil recruitment.
Relative contribution of P2U‐ and P2Y‐purinoceptors to endothelium‐dependent vasodilatation in the golden hamster isolated mesenteric arterial bed
Authors: Ralevic Vera , Burnstock Geoffrey
Published: July 2012

P2‐purinoceptors were characterized pharmacologically in the constantly perfused isolated mesenteric arterial vascular bed of the golden hamster. Vasoconstrictor and vasodilator responses to the nucleotides ATP, ADP, 2 methylthio ATP (2MeSATP), α,β‐methylene ATP (αβ‐meATP) and uridine 5′‐triphosphate (UTP) and a role for ATP in sympathetic constriction were examined.

At basal tone nucleotides elicited dose‐dependent vasoconstriction with an observed rank order of potency of αβ‐meATP»2MeSATP>ATP=ADP>UTP (based on the doses required to elicit constrictor responses of 25 mmHg). Adenosine had no vasoconstrictor action at doses up to 5 μumol. After application of a single dose (0.5 μmol) of αβ‐meATP preparations were desensitized to constriction by subsequent application of nucleotides.

Electrical field stimulation (4–64 Hz, 90 V, 1 ms, 30 s) elicited frequency‐dependent constrictions which were abolished by guanethidine (5 μm) and by prazosin (1 μm).

The non‐selective P2‐purinoceptor antagonist suramin (100 μm) did not significantly affect vasoconstrictor responses to ATP. The P2X‐selective purinoceptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS, 3 μm), virtually abolished responses to ATP. When the endothelium was removed vasoconstrictor responses to ATP and noradrenaline were augmented.

In preparations with tone raised with methoxamine (10–80 μm) nucleotides elicited vasodilatation with an observed potency order of ATP=UTP>ADP>>adenosine. 2MeSATP had relatively minor vasodilator effects and at the highest dose tested (50 nmol) elicited only vasoconstriction. α,β‐meATP did not elicit vasodilatation but produced further constriction of the raised tone preparation. At the highest doses of ATP and ADP (0.5 μm) responses were biphasic with vasoconstriction preceding vasodilatation. After removal of the endothelium, with the exception of adenosine, vasodilator responses to purines and to UTP were abolished; vasoconstriction to ATP, ADP, UTP and 2MeSATP was evident at the highest doses.

Suramin (100 μm) inhibited vasodilatation to both ATP and UTP and abolished responses to 2MeSATP. PPADS (3 μm) inhibited relaxation to 2MeSATP but did not affect relaxation to ATP, UTP, adenosine and acetylcholine and ADP.

Reactive blue 2 (30 μm) blocked vasodilator responses to ATP, UTP, 2MeSATP and acetylcholine; it was without effect when used at 3 μm.

The results of this study show that ATP elicits vasoconstriction of mesenteric arteries of the golden hamster via P2X‐purinoceptors located on the smooth muscle, and vasodilatation via P2U‐receptors which are located on the endothelium. 2MeSATP has marginal vasodilator activity, suggesting that P2Y‐purinoceptors contribute minimally to relaxation to ATP in hamster mesenteric arteries.
Cerebrovascular responsiveness to NG‐nitro‐L‐arginine methyl ester in spontaneously diabetic rats
Authors: Fouyas Ioannis P. , Kelly Paul A.T. , Ritchie Isobel M. , Whittle Ian R.
Published: July 2012

There is evidence that endothelial dysfunction is associated with diabetes mellitus. The purpose of the present study was to assess local cerebral blood flow (LCBF) and cerebrovascular responsiveness to the NOS inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME) in spontaneously diabetic insulin‐dependent BioBred (BB) rats.

Diabetic rats, and non‐diabetic controls, were treated with L‐NAME (30 mg kg−1, i.v.) or saline, 20 min prior to the measurement of LCBF by the fully quantitative [14C]‐iodoantipyrine autoradiographic technique.

There were no significant differences in physiological parameters (blood pH, pH, PCO2, and PO2, rectal temperature, arterial blood pressure, or plasma glucose) between any of the groups of rats, and no difference in either the extent or the temporal characteristics of the hypertensive response to L‐NAME between diabetic and non‐diabetic rats.

In diabetic rats, a global reduction in basal LCBF was observed, although significant reductions (between −20 and −30%) were found in only 5 (mainly subcortical) out of the 13 brain regions measured. Following L‐NAME injection, significant reductions in LCBF (between −20 and −40%) were found in the non‐diabetic animals. In diabetic animals treated with L‐NAME, a significant reduction in LCBF was measured only in the hypothalamus (−33%).

The cerebrovascular response to acute L‐NAME is attenuated in spontaneously diabetic insulin‐dependent BB rats. This would be consistent with the endothelial dysfunction in cerebral vessels, known to be associated with diabetes mellitus and it is possible that a loss of NO‐induced dilator tone, amongst other factors, may underlie the observed reductions of basal LCBF in these animals.
The effect of a tachykinin NK1 receptor antagonist, SR140333, on oedema formation induced in rat skin by venom from the Phoneutria nigriventer spider
Authors: Palframan R.T. , Costa S.K.P. , Wilsoncroft P. , Antunes E. , de Nucci G. , Brain S.D.
Published: July 2012

The possibility that tachykinin NK1 receptors are involved in the plasma extravasation evoked by intradermal (i.d.) injection of Phoneutria nigriventer venom (PNV) in rat dorsal skin in vivo has been investigated.

Local oedema formation induced by the i.d. injection of test agents was measured by the extravascular accumulation of intravenously (i.v.) injected 125I‐labelled human serum albumin over a 30 min period.

The tachykinin NK1 agonist, GR73632 (30 pmol per site), induced local oedema formation which was potentiated by co‐injection with the neuropeptide vasodilator, calcitonin gene‐related peptide (CGRP, 10 pmol per site). The non‐peptide tachykinin NK1 receptor antagonist, SR140333 (0.03‐1 nmol per site co‐injected, i.d.) significantly inhibited (0.3 nmol per site, P<0.05; 1 nmol per site, P<0.001) local oedema formation induced by GR73632 with CGRP but not that induced by histamine (10 nmol per site) with CGRP.

PNV (0.03‐0.3 μg per site) injected i.d. induced dose‐dependent local oedema formation. SR140333 (1 nmol per site, co‐injected i.d.) inhibited oedema formation; with complete inhibition observed at doses of 0.03 μg (P<0.05) and 0.1 μg (P<0.001); and partial inhibition (50%) observed with the highest dose of PNV, 0.3 μg (P<0.05).

Local oedema formation induced by PNV was not affected by systemic pretreatment with the bradykinin B2 receptor antagonist, Hoe 140 (80 nmol kg−1, i.v.), which was used at a dose which significantly inhibited oedema formation by bradykinin (1 nmol per site).

Local oedema formation induced by PNV was significantly inhibited (P<0.01) by co‐injection of the histamine H1 receptor antagonist, mepyramine (2.5 nmol per site), together with the 5‐hydroxytryptamine (5‐HT) antagonist, methysergide (2.8 nmol per site).

In the presence of all three antagonists (mepyramine 2.5 nmol per site; methysergide, 2.8 nmol per site and SR140333 1 nmol per site), the plasma extravasation induced by PNV was further significantly inhibited (P<0.001, when compared with PNV injected i.d. alone; P<0.05 when compared with PNV co‐injected with mepyramine and methysergide and P<0.01, when compared with PNV co‐injected with SR140333).

These results suggest that oedema formation evoked by i.d. PNV in rat skin may be partially mediated via a mechanism involving tachykinin NK1 receptors and that this effect is independent of histamine and 5‐HT.
Effects of dexamethasone and cyclosporin A on the accumulation of eosinophils in acute cutaneous inflammation in the guinea‐pig
Authors: Teixeira M.M. , Williams T.J. , Hellewell P.G.
Published: July 2012

Eosinophils are thought to play an important role in the pathophysiology of allergic diseases and pharmacological suppression of their recruitment is considered to be of therapeutic benefit. In the present study we have assessed and compared the effects of treatment with dexamethasone and cyclosporin A on the accumulation of 111In‐labelled eosinophils and local oedema formation in sites of acute inflammation in guinea‐pig skin.

When injected locally 150 min prior to i.d. administration of antigen in a passive cutaneous anaphylactic (PCA) reaction, dexamethasone (10−9 to 3 × 10−7 mol per site) dose‐dependently inhibited oedema formation by up to 50%. Similarly, oedema formation induced by PAF and lipopolysaccharide (LPS), but not by zymosan‐activated plasma (ZAP), was significantly inhibited by dexamethasone. In contrast, 111In‐eosinophil accumulation measured in response to i.d. injection of PAF, LPS and ZAP or in the PCA reaction was not altered.

Systemic treatment with dexamethasone (4 mg kg−1, i.v., 150 min pretreatment period) inhibited both oedema formation and 111In‐eosinophil accumulation induced by PAF, ZAP, LPS and in the PCA reaction.

The effects of i.d. injection of cyclohexamide (2 × 10−7 mol per site) on 111In‐eosinophil accumulation and oedema formation induced by PAF, ZAP or in a PCA reaction were evaluated in order to assess the dependency of these responses on protein synthesis. Cycloheximide had no effect on the responses measured. In contrast, 111In‐eosinophil accumulation, but oedema formation, induced by LPS was inhibited by 30%.

Acute (10 mg kg−1, i.v., 15 min pretreatment) or prolonged (10 mg kg−1, s.c. daily for 3 days) systemic treatment with cyclosporin A had no effect on 111In‐eosinophil accumulation or oedema formation induced by PAF, ZAP, LPS or in the PCA reaction.

In conclusion, we demonstrate preferential inhibitory effects of dexamethasone on 111In‐eosinophil accumulation according to its site of administration. In addition we show that dexamethasone inhibits protein synthesis‐independent acute inflammation in guinea‐pig skin. Finally, our results do not support the concept that eosinophils are an important cellular site of action for the inhibitory effects of cyclosporin A in a guinea‐pig model of allergic inflammation.
Pharmacokinetic‐haemodynamic relationships of 2‐chloroadenosine at adenosine A1 and A2a receptors in vivo
Authors: Mathôt R.A.A. , Soudijn W. , Breimer D.D. , IJzerman A.P. , Danhof M.
Published: July 2012

The purpose of the present study was to develop an experimental strategy for the quantification of the cardiovascular effects of non‐selective adenosine receptor ligands at the adenosine and A1 and A2a receptor in vivo. 2‐Chloroadenosine (CADO) was used as a model compound.

Three groups of normotensive conscious rats received an short intravenous infusion of 1.4 mg kg−1 CADO during constant infusions of the A1‐selective antagonist, 8‐cyclopentyltheophylline (CPT; 20 μm min−1 kg−1), the A2a‐selective antagonist, 8‐(3‐chlorostyryl)caffeine (CSC; 32 μm min−1 kg−1) or the vehicle. The heart rate (HR) and mean arterial blood pressure (MAP) were recorded continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentrations. The ratio MAP/HR was also calculated, which may reflect changes in total peripheral resistance on the assumption that no changes in stroke volume occur.

During the infusion of CPT, CADO produced a reduction in both blood pressure and MAP/HR by activation of the A2a receptor. The concentration‐effect relationships were described according to the sigmoidal Emax model, yielding potencies based on free drug concentrations (EC50,u) of 61 and 68 ng ml−1 (202 and 225 nM) for the reduction of blood pressure and MAP/HR, respectively. During the infusion of CSC, an EC50,u value of 41 ng ml−1 (136 nM) was observed for the A1 receptor‐mediated reduction in heart rate. The in vivo potencies correlated with reported receptor affinities (Ki(A1)=300 nM and Ki(A2a)=80 nM). The maximal reductions in MAP/HR and heart rate were comparable to those of full agonists, with the values of −12 ± 1 × 1O−2 mmHg b.p.m.−1 and −205 b.p.m. respectively.

It is concluded that this integrated pharmacokinetic‐pharmacodynamic approach can be used to obtain quantitative information on the potency and intrinsic activity of new non‐selective adenosine receptor agonists at different receptor subtypes in vivo.
Vasoconstrictor responsiveness of the rat mesenteric arterial bed in cirrhosis
Authors: Ralevic Vera , Mathie Robert T. , Moore Kevin P. , Burnstock Geoffrey
Published: July 2012

The effects of cirrhosis on mesenteric vascular reactivity were assessed in constantly perfused mesenteric arterial beds isolated from cirrhotic rats (carbon tetrachloride with phenobarbitone, n=6), and from phenobarbitone‐treated and untreated age‐matched controls (n=4,5).

At a constant flow rate of 5 ml min−1 there was no difference in basal perfusion pressure between the groups. Electrical field stimulation (EFS; 4–32 Hz, 90V, 1 ms, 30 s) of perivascular nerves caused frequency‐dependent increases in perfusion pressure which were not different between the groups. Dose‐dependent vasoconstrictor responses to exogenous noradrenaline (NA), methoxamine (an α1‐adrenoceptor agonist), adenosine 5′‐triphosphate (ATP) and vasopressin were also similar between the groups.

The nitric oxide (NO) synthesis inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME; 30 μm) augmented constrictor responses to NA, EFS, methoxamine and vasopressin in all groups, and as shown for EFS and NA, this was reversed by L‐arginine (300 μm). However, the maximum constrictor responses of cirrhotic preparations in the presence of L‐NAME were significantly lower than those of both groups of control animals at the highest frequency of EFS (32 Hz) and highest doses of NA (0.15 and 0.5 μmol) and, compared to phenobarbitone‐treated controls, methoxamine (5 μmol). Responses to ATP were significantly augmented by L‐NAME only in the cirrhotic group.

A step‐wise increase in perfusate flow to 10, 15 and 20 ml min−1 produced a broadly similar increase in perfusion pressure within each group. At increased flow rates, cirrhotic preparations were hyporesponsive to NA (15 nmol) compared to the phenobarbitone‐treated animals but not the untreated controls. Glibenclamide (5 μm) or L‐NAME (30 μm) had no significant effect on the relationship between flow and perfusion pressure or on responses to NA at the different flow rates.

We conclude that sympathetic neurotransmission is unchanged in cirrhosis. Endogenous NO is important in modulation of constriction in both normal and cirrhotic states. Changes in NO may occur in cirrhosis, although the role of this in hyporesponsiveness of cirrhotic preparations to NA at higher flow rates and to the greater potentiation of ATP‐mediated constriction in the presence of L‐NAME, together with the impact of factors such as changes in calcium and potassium channels, is not entirely clear.
Comparison of the autoradiographic binding distribution of [3H]‐gabapentin with excitatory amino acid receptor and amino acid uptake site distributions in rat brain
Authors: Thurlow Richard J. , Hill David R. , Woodruff G.N.
Published: July 2012

Gabapentin is a novel anticonvulsant with an unknown mechanism of action. Recent homogenate binding studies with [3H]‐gabapentin have suggested a structure‐activity relationship similar to that shown for the amino acid transport system responsible for the uptake of large neutral amino acids (LNAA).

The autoradiographic binding distribution of [3H]‐gabapentin in rat brain was compared with the distributions for excitatory amino acid receptor subtypes and the uptake sites for excitatory and large neutral amino acids in consecutive rat brain sections.

Densitometric measurement of the autoradiographic images followed by normalisation with respect to the hippocampus CA1 stratum radiatum, was carried out before comparison of each binding distribution with that of [3H]‐gabapentin by linear regression analysis. The correlation coefficients observed showed no absolute correlation was observed between the binding distributions of [3H]‐gabapentin and those of the excitatory amino acid receptor subtypes. The acidic and large neutral amino acid uptake site distributions demonstrated a much closer correlation to the [3H]‐gabapentin binding site distribution. The correlation coefficients for D‐[3H]‐aspartate, L‐[3H]‐leucine and L‐[3H]‐isoleucine binding site distributions were 0.76, 0.90 and 0.88 respectively.

Concentration‐dependent inhibition by unlabelled gabapentin of autoradiographic binding of L‐[3H]‐leucine and L‐[3H]‐isoleucine was observed, with non‐specific binding levels being reached at concentrations between 10 and 100 μm.

Excitotoxic quinolinic acid lesion studies in rat brain caudate putamen and autoradiography were carried out for the amino acid uptake sites mentioned above. The resulting glial infiltration of the lesioned areas was visualized by autoradiography using the peripheral benzodiazepine receptor specific ligand [3H]‐PK11195. A significant decrease in binding density in the lesioned area compared with sham‐operated animals was observed for D‐[3H]‐aspartate, L‐[3H]‐leucine, L‐[3H]‐isoleucine and [3H]‐gabapentin, whilst [3H]‐PK11195 showed a significant increase in binding density indicative of glial infiltration into the lesioned area. These results suggest that the gabapentin binding site and the acidic and LNAA uptake site may be present on cell bodies of a neuronal population of cells.

From these studies it appears that [3H]‐gabapentin, L‐[3H]‐leucine and L‐[3H]‐isoleucine bind to the same site in rat brain. The inhibition of [3H]‐gabapentin binding by the LNAA uptake system‐specific ligand, BCH, suggests that [3H]‐gabapentin may label this uptake site, termed system‐L. Conversely these ligands could be labelling a novel site that coincidentally has a similar structure‐activity relationship to this uptake site. These results suggest a novel mechanistically relevant site of action for gabapentin and may enable further anti‐epileptic agents of this type to be developed.
VIP‐induced relaxation of guinea‐pig intestinal smooth muscle cells: sequential involvement of cyclic AMP and nitric oxide
Authors: Rekik Moez , Delvaux Michel , Tack Ivan , Frexinos Jacques , Bueno Lionel
Published: July 2012

A possible interaction between cyclic AMP and nitric oxide (NO) in mediating the relaxant effect of vasoactive intestinal polypeptide (VIP) on intestinal smooth muscle cells has been investigated. The effects of the inhibitor of NO synthesis, NG‐nitro‐L‐arginine methyl ester (L‐NAME), have been studied on VIP‐, forskolin‐, and 8 bromo‐cyclic AMP‐ induced relaxation of cells, dispersed by enzymatic digestion of muscle strips from the circular layer of guinea‐pig ileum.

VIP alone did not modify the length of isolated muscle cells. By contrast, when the cells were contracted by cholecystokinin octapeptide, CCK8 (10 nM), VIP inhibited this contraction, inducing a concentration‐dependent relaxation of the cells. Maximal relaxation was induced by 1 μm VIP (EC50 = 408.2 ± 16.7 pM).

N‐ethylmaleimide, inhibitors of adenylate cyclase or somatostatin, abolished the relaxing effect of VIP. (R)‐p‐cAMPs, an antagonist of cyclic AMP on protein kinase A also inhibited the VIP‐induced relaxation by 92.1 ± 6.3%. Inhibitors of nitric oxide synthase (NOS), L‐NAME and L‐NMMA, partially inhibited VIP‐induced relaxation. The effect of L‐NAME was reversed by L‐arginine but not by D‐arginine.

(R)‐p‐cAMPS and L‐NAME also inhibited the cell relaxation induced either by forskolin which directly stimulates adenylate cyclase activity or 8‐bromo‐cyclic AMP, an analogue of cyclic AMP.

When cells were incubated for 30 min with dexamethasone 10 μm, a glucocorticoid known to decrease the synthesis of iNOS, the relaxing effect of a maximal concentration of VIP was decreased by 52 ± 4% and L‐NMMA had no further effect on this residual VIP‐induced relaxation. Milrinone, a phosphodiesterase type III inhibitor, potentiated the relaxant effect of VIP.

These data demonstrate that the intracellular pathway mediating the relaxant effect of VIP in intestinal smooth muscle cells includes the sequential activation of adenylate cyclase, protein kinase A, activation of NOS and finally production of NO and cyclic GMP. NO could in turn regulate the cyclic AMP‐dependent pathway of cell relaxation.
Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes
Authors: Downie D.L. , Hall A.C. , Lieb W.R. , Franks N.P.
Published: July 2012

Glycine responses were studied under voltage clamp in Xenopus oocytes injected with cDNA encoding mammalian glycine receptor subunits and in rat medullary neurones. Bath application of glycine gave strychnine‐sensitive currents which reversed close to the expected equilibrium potentials for chloride ions. The peak currents for the receptors expressed in oocytes fitted a Hill equation with EC50 = 215 ± 5 μm and Hill coefficient nH = 1.70 ± 0.05 (means ± s.e.means). The peak currents from the receptors in medullary neurones fitted a Hill equation with EC50 = 30 ± 1 μm and Hill coefficient nH = 1.76 ± 0.08. The current‐voltage relationship for the receptors expressed in oocytes showed strong outward rectification (with Vrev = −21 ± 2 mV), while that for the glycine responses from the medullary neurones in symmetrical Cl− was linear (with Vrev = 3.2 ± 0.6 mV).

Inhalational general anaesthetics, at concentrations close to their human minimum alveolar concentrations (MACs), potentiated responses to low concentrations of glycine. The potentiation observed with the recombinant receptors (between 60–220%) was approximately twice that found with the medullary neurones (between 40–80%). For both the recombinant receptors and the receptors in medullary neurones, the degree of potentiation increased in the order of methoxyflurane ≅ sevoflurane < halothane ≅ isoflurane ≅ enflurane. There was no significant difference between the potentiations observed for the two optical isomers of isoflurane.

For both the recombinant and native receptors, isoflurane potentiated the currents in a dose‐dependent manner at low concentrations of glycine, although at high glycine concentrations the anaesthetic had no significant effect on the glycine‐activated responses. The major effect of isoflurane was to cause a parallel leftward shift in the glycine concentration‐response curves. The glycine EC50 concentration for the recombinant receptors decreased from a control value of 215 ± 5 μm to 84 ± 7 μm glycine at 610 μm isoflurane, while that for the medullary neurones decreased from a control value of 30 ± 1 μm to 18 ± 2 μm glycine at the same concentration of isoflurane. The potentiation was independent of membrane potential.

Isoflurane also potentiated responses to taurine, a partial agonist at the glycine receptor. This was observed for receptors expressed in oocytes at both low and saturating concentrations of taurine. The EC50 concentration decreased from a control value of 1.6 ± 0.2 to 0.9 ± 0.1 mM taurine in the presence of 305 μm isoflurane, while the maximum response to taurine increased from 47 ± 2 to 59 ± 2% of the maximum response to glycine.

Glycine receptors, like other members of the fast ligand‐gated receptor superfamily, are sensitive to clinically relevant concentrations of inhalational general anaesthetics. Effects at these receptors may, therefore, play some role in the maintenance of the anaesthetic state.