bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Na+ influx mediated by ATP4—‐activated P2 purinoceptors in PC12 cells
Authors: Choi Se‐Young , Kim Kyong‐Tai
Published: July 2012

Micromolar levels of extracellular ATP increased cytosolic Na+ concentration ([Na+]i) as well as cytosolic Ca2+ concentration ([Ca2+]i) in PC12 cells.

Pretreatment of cells with tetrodotoxin, benzamil or thapsigargin did not alter the ATP‐induced Na+ influx.

Increased extracellular Mg2+ concentration decreased the ATP effect. Furthermore, when the extracellular ATP pool was treated to contain corresponding calculated concentrations of ATP4‐, the increase in [Na+]i stayed linked to the ATP4‐ concentration rather than to the total ATP concentrations in the stimulants.

Extracellular ATP does not create nonselective pores as shown by the fact that ethidium bromide does not enter the cells upon ATP stimulation.

Among the tested nucleotides, only adenosine 5′‐O‐(3‐thiotriphosphate), 2‐methylthio ATP and 2‐chloro ATP also caused Na+ influx.

Reactive Blue 2 specifically decreased the ATP effect in a concentration‐dependent manner.

The results suggest that extracellular ATP triggers Na+ influx through a P2 purinoceptor which is activated by ATP4‐ in PC12 cells.
Inhibition by amphetamine of testosterone secretion through a mechanism involving an increase of cyclic AMP production in rat testes
Authors: Tsai Shiow‐Chwen , Chiao Yu‐Chung , Lu Chien‐Chen , Doong Ming‐Luen , Chen Yen‐Hao , Shih Hsi‐Chang , Liaw Charlie , Wang Shyi‐Wu , Wang Paulus S.
Published: July 2012

The effect of amphetamine on the secretion of testosterone and the production of testicular adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) in rats was studied.

A single intravenous injection of amphetamine decreased the basal and human chorionic gonadotropin (hCG)‐stimulated levels of plasma testosterone. Plasma LH levels were not altered by the injection of amphetamine.

Administration of amphetamine in vitro resulted in a dose‐dependent inhibition of both basal and hCG‐stimulated release of testosterone.

Amphetamine enhanced the basal and hCG‐increased levels of cyclic AMP accumulation in vitro in rat testes.

These results suggest that amphetamine inhibits the spontaneous and hCG‐stimulated secretion of testosterone from the testes through a mechanism involving an increase in cyclic AMP production.
The vasoconstrictor effects of L‐NAME, a nitric oxide synthase inhibitor, in pregnant rabbits
Authors: Losonczy Gy. , Mucha I. , Müller V. , Kriston T. , Ungvári Z. , Tornóci L. , Rosivall L. , Venuto R.
Published: July 2012

We have used anaesthetized, acutely instrumented non‐pregnant (NP) and late pregnant (P) New Zealand white rabbits to examine the possible role of nitric oxide (NO) in the pregnancy‐induced fall of vascular tone and arterial pressure. Systemic, renal and pulmonary vascular resistance, as well as plasma concentrations of cyclic GMP (PcGMP) were compared before and after the inhibition of NO synthesis by NG‐nitro‐L‐arginine methyl ester (L‐NAME).

P rabbits had lower baseline total peripheral resistance (TPR), mean arterial pressure (MAP) and higher PcGMP than NP controls (all P<0.05 or less). L‐NAME (1, 10, 50 mg kg−1, i.v.) resulted in dose‐dependent elevation of TPR in both groups. However, the absolute, as well as percentage increases in TPR were greater (P<0.05) in NP than in P rabbits.

Cardiac output (CO) was reduced more (P<0.01) by NO inhibition in NP than P rabbits. Therefore, despite the smaller increase in TPR, the elevation of MAP was greater (P<0.001) in P than NP rabbits. After L‐NAME, NP rabbits developed more severe bradycardia and a greater increase of pulmonary vascular resistance which might have contributed to the more pronounced reduction of CO.

PcGMP increased in both groups following L‐NAME, but more (P<0.01) in NP than P rabbits.

Infusion of acetylcholine (ACh, 0.02 μmol l−1 kg−1) reduced MAP and TPR more (both P<0.05) in NP than P rabbits and L‐NAME reduced the ACh‐induced depressor response only in NP rabbits.

These results suggest that the low vascular tone and arterial pressure in pregnant rabbits is not mediated by NO.
Further investigation into the signal transduction mechanism of the 5‐HT4‐like receptor in the circular smooth muscle of human colon
Authors: McLean Peter G. , Coupar Ian M.
Published: July 2012

The nature of the receptor coupling mechanism of the 5‐hydroxytryptamine4 (5‐HT4) receptor in the circular smooth muscle of the human colon has been further investigated.

5‐HT stimulated cyclic AMP generation and caused a relaxation in a concentration‐dependent fashion, with EC50 values of 175.5 and 274.9 nM respectively. DAU 6236 increased cyclic AMP formation and caused a relaxant effect but was a partial agonist relative to 5‐HT.

The 5‐HT4 receptor antagonist, GR 113808, inhibited cyclic AMP formation and relaxation induced by 5‐HT with ‐log Ki values of 9.1 (cyclic AMP) and 8.9 (relaxation) and apparent pA2 values of 9.2 (cyclic AMP) and 9.5 (relaxation).

Ondansetron and methysergide failed to inhibit cyclic AMP formation or the relaxation induced by 5‐HT.

The phosphodiesterase inhibitor, IBMX, produced a concentration‐dependent relaxation (EC50 = 30 μm) and at 1 μm it enhanced the 5‐HT‐induced relaxation producing a leftward shift of the 5‐HT concentration‐effect curve with a concentration‐ratio of 4.1. Rolipram caused a concentration‐dependent relaxation (EC50 = 564.8 nM) and at 200 nM caused a leftward shift of the concentration‐effect curve to 5‐HT with a concentration‐ratio of 5.5.

Application of the adenylyl cyclase inhibitor, SQ 22536 (0.1 mM), and the protein kinase inhibitors, H7 (100 nM) and H89 (200 nM), inhibited the relaxant effect of 5‐HT inducing a rightward shift of the concentration‐effect curve with concentration‐ratios of 10.1, 2.7 and 4.2 respectively.

Forskolin stimulated cyclic AMP production and caused a relaxation. The maximum relaxant effect of forskolin (6 μm, 13.8±1.9 cm.s) was not significantly different from the maximum relaxant effect of 5‐HT (10 μm, 12.7±4.9 cm.s). However, the cyclic AMP levels stimulated by forskolin (6 μm, 49.3±6.6 pmol mg−1) were markedly greater than those stimulated by 5‐HT (10 μm, 7.6±2.0 pmol mg−1).

In conclusion, these results indicate that the 5‐HT4 receptors of the circular smooth muscle of human colon mediate relaxation and inhibition of spontaneous contractions via activation of adenylyl cyclase, formation of cyclic AMP and activation of protein kinase A.
Neuroprotective properties of a protein kinase inhibitor against ischaemia‐induced neuronal damage in rats and gerbils
Authors: Satoh S. , Ikegaki I. , Suzuki Y. , Asano T. , Shibuya M. , Hidaka H.
Published: July 2012

. The neuroprotective properties of fasudil (HA1077), a novel protein kinase inhibitor, were evaluated in two animal models of cerebral ischaemia: transient bilateral carotid artery occlusion in Mongolian gerbils and cerebral microembolization in rats.

. The cytoprotective effect of fasudil on delayed neuronal death in gerbils was compared with the effects of nimodipine, a calcium channel antagonist and ozagrel, a thromboxane A2 synthetase inhibitor. The average of the neuronal cell density in the ischaemic control group was 17.8 ± 2.1 cells mm−1, whereas fasudil (30 mg kg−1) significantly diminished the loss of CA1 neurones with the average of the neuronal cell density of 101.0 ± 22.0 cells mm−1; nimodipine (10 mg kg−1) and ozagrel (30 mg kg−1) did not significantly protect against the ischaemia‐induced neuronal loss.

. In the rat model, the effects of fasudil on the histological and neurological consequences of cerebral microembolization produced via the injection of microspheres were examined. Twenty‐four hours after the injection of microspheres into the internal carotid artery, all animals in the control group showed typical symptoms of stroke. Neurological function was significantly improved in the fasudil‐treated animals. In the controls, the infarcted area in a cortical slice selected to include the hippocampal area was 0.25 ± 0.01 cm2 (mean ± s.e.mean) (43.9 ± 2.4% of cortical section of the half hemisphere); the difference was significant compared to the mean area of 32.7 ± 2.8 and 21.5 ± 4.8% observed in rats treated with fasudil (3, 10 mg kg−1), respectively. Fasudil (10 mg kg−1) significantly suppressed the increased water content in ischaemic brain tissues (saline‐treated rats, 82.4 ± 0.2% vs fasudil‐treated rats, 81.0 ± 0.4%).

. These results suggest that: (i) various protein kinases are involved in the pathogenesis of ischaemic injury; and (ii) the inhibition of protein kinases may be efficacious in preventing neuronal death, thus improving neurological function in the brain damage associated with ischaemic stroke.
Characterization of the PGE2 receptor subtype in bovine chondrocytes in culture
Authors: de Brum‐Fernandes Artur J. , Morisset Sophie , Bkaily Ghassan , Patry Caroline
Published: July 2012

. Prostaglandin E2 (PGE2) is an autacoid that decreases proteoglycan synthesis, increases metalloprotease production by cultured chondrocytes, and can modulate some of the actions of interleukin‐1 on cartilage. The objective of the present study was to characterize the subtype of prostaglandin E2 receptor present in bovine chondrocytes in culture.

. Primary cultures of articular chondrocytes were prepared from slices of bovine carpal cartilage by sequential digestion with type III hyaluronidase, trypsin, type II collagenase, followed by overnight incubation in Dulbecco's Modified Eagle's Medium (DMEM) with type II collagenase, washing, and seeding at a density of 2 × 105 cells cm−2 in DMEM with 10% foetal bovine serum.

. PGE2 and carbaprostacyclin induced dose‐dependent increases in intracellular cyclic AMP in bovine chondrocytes in culture. The potencies of these compounds were different, and maximal doses of PGE2 and carbaprostacyclin had an additive effect. PGD2 induced a small increase in intracellular cyclic AMP only at a high concentration (10−5 m).

. PGE2 was more potent that the EP2 agonist 11‐deoxy‐PGE1 at inducing increases in intracellular cyclic AMP. The EP2 agonist butaprost, however, induced only a small increase at a concentration of 10−5 m. 17‐Phenyl‐PGE2 (EP1 agonist), sulprostone and MB 28767 (15S‐hydroxy‐9‐oxo‐16‐phenoxy‐γ‐tetranorprost‐13E‐enoic acid) (EP3 agonists) did not induce an increase in intracellular cyclic AMP at concentrations up to 10−5 m.

. The EP4 antagonist AH 23848B ([1α(Z),2β,5α]‐(±)‐7‐[5‐[[(1,1′‐biphenyl)‐4‐yl]methoxy]‐2‐(4‐morpholinyl)‐3‐oxocyclopentyl]‐5‐heptenoic acid) antagonized PGE2 but not carbaprostacyclin effects on intracellular cyclic AMP. The Schild plot slope was different from 1 but this could be due to an interaction of PGE2 with IP receptors in high doses. The exact nature of the antagonism by compound AH 23848B could not be definitely established in these experimental conditions.

. Neither PGE2 nor any of its analogues inhibited the increase in intracellular cyclic AMP induced by forskolin, and pertussis toxin did not alter the response to PGE2, suggesting that no Gi‐coupled PGE2 receptors are present in these cells. Stimulation with PGE2 did not induce significant increases in intracellular inositol‐trisphosphate levels nor increases in intracellular free calcium as determined by confocal microscopy, suggesting the absence of phospholipase‐C‐coupled or of calcium channel‐coupled PGE2 receptors in bovine chondrocytes in these experimental conditions.

. These results show for the first time that bovine chondrocytes in culture present a functional PGE2 receptor that has some pharmacological characteristics of an EP4 subtype, as well as an IP receptor.
Mediation by 5‐HT3 receptors of an excitatory effect of 5‐HT on dorsal vagal preganglionic neurones in anaesthetized rats: an ionophoretic study
Authors: Wang Yun , Ramage Andrew G. , Jordan David
Published: July 2012

. Extracellular recordings were made from 141 vagal preganglionic neurones in the dorsal vagal nucleus (DVN). The effects of ionophoretic administration of 5‐hydroxytryptamine (5‐HT), the 5‐HT3 receptor agonist, phenylbiguanide (PBG) and the antagonists, granisetron and tropisetron (ICS 205–930) on these vagal preganglionic neurones were studied in pentobarbitone sodium anaesthetized rats.

. Ionophoretic application of 5‐HT at low currents (< 10 nA) increased the activity in 46 (73%) of 63 neurones tested. Application of granisetron (5–20 nA) or tropisetron (5–20 nA) abolished or attenuated the 5‐HT excitatory responses in 8 out of 11 and 5 out of 5 neurones respectively. At the currents used, neither antagonist had any effect on baseline firing rate.

. Ionophoresis of the selective 5‐HT3 receptor agonist, phenylbiguanide (0–40 nA) excited 54 (82%) of the 66 vagal neurones tested, whilst the remaining 12 neurones were unaffected.

. Granisetron applied either ionophoretically (8/11) or intravenously (3/3), abolished or attenuated the excitations evoked by PBG. Similarly, tropisetron administered either ionophoretically (2/3) or intravenously (2/2), attenuated the PBG excitation. In contrast, the PBG excitations were unaffected by the 5‐HT2 receptor antagonist, cinanserin (2/2), and the selective 5‐HT1A receptor antagonist, WAY‐100802 (6/6).

. In conclusion, excitation of vagal preganglionic neurones evoked by ionophoretic application of 5‐HT is mediated in part by 5‐HT3 receptors and activation of 5‐HT3 receptors on and/or in the vicinity of vagal motoneurones causes excitation of these neurones.
Interleukin‐1β stimulation of 45 Ca2+ release from rat striatal slices
Authors: Palmi Mitri , Frosini Maria , Sgaragli Gianpietro
Published: July 2012

. Previous observations that centrally injected interleukin‐1β (IL‐1β) into rabbits induces a sustained rise in cerebrospinal fluid (CSF) Ca2+ concentration ([Ca2+]) as well as fever, prompted us to undertake an in vitro study to corroborate the in vivo results and gain insight as to the source and mechanism of IL‐1β‐induced Ca2+ mobilization.

. IL‐1β treatment of rat striatal slices preloaded with 45Ca2+ elicited a rise in spontaneous 45Ca2+ release which was dose‐dependent, delayed in onset and of extended duration. At concentrations of 1, 5 and 10 ng ml−1, the 45Ca2+ efflux increased by 6.3 ± 1.3 (s.e.mean), 33.4 ± 5.0 and 159 ± 10.5% respectively.

. At 1 μg ml−1, the specific IL‐1 receptor antagonist, IRAP, antagonized the effect induced by, 10 ng ml−1 IL‐1.

. Caffeine 10 mM, which failed to release calcium on its own, potentiated IL‐1‐elicited 45Ca2+ release.

. Perfusion with a Ca2+‐free medium obtained by use of excess EGTA (3 mM) or in the presence of the Ca2+ channel blocker, nifedipine (3 × 10−8 m) abolished the potentiating effect of caffeine without affecting the IL‐1‐induced 45Ca2+ release.

. Preincubation of slices for 4 h with Bordetella pertussis toxin (PTX, 1.3 μg ml−1) did not change the pattern of Ca2+ efflux in response to IL‐1.

. In conclusion, these data indicate that IL‐1 stimulates calcium release from brain tissue by a specific, receptor‐mediated mechanism which partly depends on extracellular calcium but does not involve a PTX‐sensitive G protein as part of the transducing signal.
Analysis of variation in L‐365,260 competition curves in radioligand binding assays
Authors: Harper E.A. , Roberts S.P. , Shankley N.P. , Black J.W.
Published: July 2012

. For several years, we have used the cholecystokinin (CCK)B/gastrin receptor selective antagonist, L‐365,260, as a reference compound in a variety of studies in CCKB/gastrin receptor radioligand binding assays. Here, we have analysed the competition curve data sets obtained between L‐365,260 and [125I]‐BH‐CCK8S in guinea‐pig gastric gland and mouse and rat cerebral cortex preparations.

. Competition curves obtained for L‐365,260 in the mouse cortex assay were not different from rectangular hyperbolae (slope = 1.01 ± 0.02) implying the presence of a single population of binding sites (pKI = 8.41 ± 0.01; data from 47 experiments, slope constrained to unity). However, in the rat cortex and guinea‐pig gastric gland assays, the mean slope of the competition curves was significantly less than one and the mean apparent pKI significantly lower than that obtained in the mouse cortex (slope = 0.85 ± 0.03, 0.90 ± 0.03; apparent pKI = 7.98 ± 0.05, 8.07 ± 0.05; 48 and 45 experiments, in rat and guinea‐pig, respectively). The distribution of the individual pK1 and slope estimates of the competition curves in these two assays was consistent with expectations for the variable expression (in terms of absolute number and proportion) of two binding sites. The two sites were characterized by pK1 values for L‐365,260 of 8.50 ± 0.04 and 8.48 ± 0.04 for the high affinity site and 7.32 ± 0.04 and 7.22 ± 0.06 for the low affinity site in guinea‐pig and rat, respectively.

. The affinity estimates for L‐365,260, although obtained on different tissues, are consistent with data obtained from the analysis of L‐365,260 antagonism of pentagastrin‐stimulated responses in mouse and rat stomach (acid secretion) and guinea‐pig gastric muscle (isotonic contraction) assays. To this extent, these data suggest the existence of two CCKB/gastrin receptor subtypes.
Induction of calcium release from sarcoplasmic reticulum of skeletal muscle by xanthone and norathyriol
Authors: Kang J.J. , Cheng Y.W. , Ko F.N. , Kuo M.L. , Lin C.N. , Teng C.M.
Published: July 2012

. Effects of xanthone and its derivative, 1,3,6,7‐tetrahydroxyxanthone (norathyriol), on Ca2+ release and ryanodine binding were studied in isolated sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle.

. Both xanthone and norathyriol dose‐dependently induced Ca2+ release from the actively loaded SR vesicles which was blocked by ruthenium red, a specific Ca2+ release inhibitor, and Mg2+.

. Xanthone and norathyriol also dose‐dependently increased apparent [3H]‐ryanodine binding. Norathyriol, but not xanthone, produced a synergistic effect on binding activation when added concurrently with caffeine.

. In the presence of Mg2+, which inhibits ryanodine binding, both caffeine and norathyriol, but not xanthone, could restore the binding to the level observed in the absence of Mg2+.

. Xanthone activated the Ca2+‐ATPase activity of isolated SR vesicles dose‐dependently reaching 70% activation at 300 μm.

. When tested in mouse diaphragm, norathyriol potentiated the muscle contraction followed by twitch depression and contracture in either a Ca2+‐free bathing solution or one containing 2.5 mM Ca2+. These norathyriol‐induced effects on muscle were inhibited by pretreatment with ruthenium red or ryanodine.

. These data suggest that xanthone and norathyriol can induce Ca2+ release from the SR of skeletal muscle through a direct interaction with the Ca2+ release channel, also known as the ryanodine receptor.
Analysis of the variation in the action of L‐365,260 at CCKB/gastrin receptors in rat, guinea‐pig and mouse isolated gastric tissue assays
Authors: Roberts S.P. , Harper E.A. , Watt G.F. , Gerskowitch V.P. , Hull R.A.D. , Shankley N.P. , Black J.W.
Published: July 2012

. Since L‐365,260 was first described as a selective antagonist at cholecystokinin (CCK)B/gastrin receptors, we have used it periodically as a reference compound in isolated tissue assays of guinea‐pig gastric muscle and lumen‐perfused stomachs from mouse and immature rat. L‐365,260 behaved as a surmountable antagonist and produced parallel rightward shifts of pentagastrin con***ćentration‐effect curves in each of the replicate experiments. The experiments were performed by several different experimenters in the same laboratories over a five year period.

. In the isolated, lumen‐perfused, immature rat stomach assay, L‐365,260 behaved as a simple competitive antagonist (Schild plot slope = 1.00 ± 0.10, pKB = 7.54 ± 0.03 from a global analysis of the data) acting at a homogeneous population of receptors in five separate, highly‐reproducible, experiments. In contrast, the replicate data sets obtained from the interaction in the isolated, lumen‐perfused mouse stomach and guinea‐pig gastric muscle assays, over the same period, were not consistent with the presence of a single receptor population. The guinea‐pig gastric muscle data were relatively reproducible between experiments but some individual Schild plot slopes and the slope estimated from a global analysis of all the data were significantly less than unity (slope = 0.80 ± 0.07, pA2 = 8.56 ± 0.05 from the global analysis). The data obtained in the mouse stomach were significantly more variable than that obtained in the same assay, during the same period, from the interaction between histamine and the H2‐receptor antagonist, famotidine. The individual Schild plot slopes ranged from being very flat (0.20) to being not significantly different from unity (1.23) and the pA2 values ranged from 7.68 to 8.70.

. Overall, the data could be accounted for by assuming the variable expression of two receptor subtypes across the assays. The rat stomach appeared to express a single receptor characterized by a low affinity constant for L‐365,260 (pKB ∼ 7.5). The guinea‐pig gastric muscle and mouse stomach data could be explained by the presence of this receptor and a second one characterized by a high affinity constant for L‐365,260 (pKB ∼ 8.6). The activity of the two proposed receptor subtypes was consistent between experiments in the guinea‐pig and the high affinity receptor appeared to be predominant. In contrast, the mouse stomach data could only be simulated by assuming that the proportion and absolute number of each subtype varied significantly between the replicate experiments.

. The L‐365,260 affinity estimates at the inferred receptor subtypes were indistinguishable from those obtained in a corresponding analysis of the behaviour of L‐365,260 in CCKB/gastrin receptor radioligand binding experiments in guinea‐pig gastric gland and mouse and rat cerebral cortex preparations.
Potent block of volume‐activated chloride currents in endothelial cells by the uncharged form of quinine and quinidine
Authors: Voets Thomas , Droogmans Guy , Nilius Bernd
Published: July 2012

. The effects of quinine and quinidine on the volume‐activated chloride current (ICl(vol)) in cultured endothelial cells from bovine pulmonary artery were studied by use of the whole‐cell patch‐clamp technique.

. At pH 7.4 both quinine and quinidine induced a fast and reversible block of ICl(vol) with Ki values of 20 ± 4 μm and 30 ± 10 μm, respectively.

. The blocking efficiency of both drugs increased dramatically with increasing extracellular pH, indicating that the blockade is mediated by the uncharged form of quinine and quinidine.

. These results suggest a hydrophobic interaction with high affinity between volume‐activated chloride channels and uncharged quinine and quinidine within the membrane bilayer of endothelial cells.
Effect of opiates on transmitter release from visualized hypogastric boutons innervating the rat pelvic ganglia
Authors: Warren Darren A. , Lavidis Nickolas A.
Published: July 2012

The effect of opiates on neurotransmission between visualized hypogastric nerve boutons and postganglionic cell bodies has been examined using extracellular recording of nerve bouton impulses (NBIs) and excitatory postsynaptic currents (e.p.s.cs).

Morphine (10 to 40 μm) did not affect neurotransmission in the ganglia. Dynorphin‐A (4 μm) and U50488H (1 μm) decreased quantal transmitter release and naloxone (10 μm) reversed these effects.

Morphine (10 μm), dynorphin‐A (4 μm) and U50488H (1 μm) did not affect either the time course or consistency with which the NBI was recorded.

Dynorphin‐A (1 to 4 μm) and U50488H (1 μm) decreased the average amplitude of e.p.s.cs by increasing the number of failures to release quanta from single or small groups of 2 to 4 boutons during continuous nerve stimulation at 0.1 Hz.

The decrease in quantal release induced by dynorphin‐A and U50488H in 0.2 to 0.5 mM [Ca2+]o was readily reversed by increasing the extracellular calcium ion concentration to 1 mM.

It was concluded that k‐opioid receptors are located on the boutons of the hypogastric nerve and when activated by k‐opioid receptor agonists reduce quantal release without affecting the NBI.
Identification of potent P2Y‐purinoceptor agonists that are derivatives of adenosine 5′‐monophosphate
Authors: Boyer José L. , Siddiqi Suhaib , Fischer Bilha , Romero‐Avila Teresa , Jacobson Kenneth A. , Harden T. Kendall
Published: July 2012

A series of chain‐extended 2‐thioether derivatives of adenosine monophosphate were synthesized and tested as agonists for activation of the phospholipase C‐linked P2Y‐purinoceptor of turkey erythrocyte membranes, the adenylyl cyclase‐linked P2Y‐purinoceptor of C6 rat glioma cells, and the cloned human P2U‐receptor stably expressed in 1321N1 human astrocytoma cells.

Although adenosine monophosphate itself was not an agonist in the two P2Y‐purinoceptor test systems, eleven different 2‐thioether‐substituted adenosine monophosphate analogues were full agonists. The most potent of these agonists, 2‐hexylthio AMP, exhibited an EC50 value of 0.2 nM for activation of the C6 cell receptor. This potency was 16,000 fold greater than that of ATP and was only 10 fold less than the potency of 2‐hexylthio ATP in the same system. 2‐hexylthio adenosine was inactive.

Monophosphate analogues that were the most potent activators of the C6 cell P2Y‐purinoceptor were also the most potent activators of the turkey erythrocyte P2Y‐purinoceptor. However, agonists were in general more potent at the C6 cell receptor, and potency differences varied between 10 fold and 300 fold between the two receptors.

Although 2‐thioether derivatives of adenosine monophosphate were potent P2Y‐purinoceptor agonists no effect of these analogues on the human P2U‐purinoceptor were observed.

These results support the view that a single monophosphate is sufficient and necessary for full agonist activity at P2Y‐purinoceptors, and provide insight for strategies for development of novel P2Y‐purinoceptor agonists of high potency and selectivity.
Effects of intravenous anaesthetic agents on fast inhibitory oscillations in the rat hippocampus in vitro
Authors: Whittington M.A. , Jefferys TJ.G.R. , Traub R.D.
Published: July 2012

General anaesthetic agents prevent awareness of sensory input and subsequent recall of sensory events after administration. The mechanisms involved in higher sensory processing, including awareness and recall, are not fully elucidated. However, fast oscillations in neuronal activity in the 20–80 Hz (gamma) range have been strongly implicated. Here we have investigated the effects of two anaesthetic agents and a sedative/hypnotic drug on these oscillations.

Trains of fast oscillations, shown previously to be shaped by γ‐aminobutyric acid (GABAA) receptor activation, were evoked by pressure ejection of L‐glutamate (10 mM) onto the perisomatic region of hippocampal area CA1 in the presence of 3‐((R)‐2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (R‐CPP), 50 μm, 6‐nitro‐7‐sulphamoylbenzo[f]quinoxaline‐2,3‐dione (NBQX), 50 μm and 2‐hydroxysaclofen, 0.2 mM.

Thiopentone (10–200 μm) and propofol (0.5–10 μm) dose‐dependently decreased both the maximum oscillation frequency, by approx. 90%, and the incidence of evoked rhythmic oscillations by approx. 60%. Diazepam (0.05‐1 μm) decreased maximum oscillation frequency by about 40% but did not affect the incidence of evoked oscillations.

The similar effects of thiopentone and propofol were mediated by both a large (about 600%) increase in the decay constant (τD) of GABAA receptor‐mediated inhibitory postsynaptic currents (i.p.s.cs) and a bicuculline‐sensitive leak current. The two drugs had differing effects on i.p.s.c. amplitude. Diazepam caused a small increase in τD (about 170%) and did not alter leak currents at the doses used.

Effects of the anaesthetic agents were seen on the above measurements at similar concentrations to those estimated in the CNS during clinical and veterinary anaesthesia. We suggest that the effects on fast oscillations associated with cognition may contribute to the mechanism by which these agents produce general anaesthesia.
The effect of ovariectomy on depressed contractions to phenylephrine and KC1 and increased relaxation to acetylcholine in isolated aortic rings of female compared to male rabbits
Authors: Sánchez Alicia , Gómez María de Jesús , Dorantes Ana Luisa , Rosales José Luis , Pastelín Gustavo , Díaz Vicente , Posadas Francisco , Escalante Bruno
Published: July 2012

Differences in vascular responses to phenylephrine, acetylcholine (ACh) and potassium chloride (KC1) were studied in rabbit aorta from female and male rabbits, in the absence and presence of an inhibitor of nitric oxide (NO) production, NG‐nitro‐l‐arginine methyl ester (L‐NAME, 100 μm).

Phenylephrine and KC1‐induced contractions, were significantly reduced in amplitude (P < 0.01) in the rings from female rabbits compared to those from male rabbits.

ACh‐induced relaxation was greater (P < 0.01) in aortic rings from females than from males.

Incubation of the rings with L‐NAME abolished the phenylephrine‐induced contraction differences between rings from male and female rabbits.

Ovariectomy eliminated the differences in vascular responses to phenylephrine, KC1 and ACh of aortic rings from the female rabbits.

Both basal and ACh‐stimulated release of nitrites from aortic rings was greater (P < 0.01) in vascular tissue from female than male rabbits.

These results indicate that differences in vascular reactivity in aortic rings from male and female rabbits may be associated with a higher release of NO, resulting in an increased vasodilator response in the female rabbits.
Sources of calcium and α1‐adrenoceptor‐mediated contraction in rat tail artery
Authors: Tabernero Antonia , Vivas Nuria M. , Vila Elisabet
Published: July 2012

The relative importance of intracellular and extracellular Ca2+ on α1‐adrenoceptor‐mediated contraction by noradrenaline and St‐587 has been studied and correlated with the binding characteristics in intact tail artery from Sprague‐Dawley rats.

Noradrenaline and St‐587 behaved as full agonists inducing a concentration‐dependent vasoconstriction.

Nifedipine (1 μm and 10 μm) blocked by 50% (P < 0.001) and 75% (P < 0.001) respectively, the maximum contraction (Emax) induced by St‐587. Nevertheless, to reach 40% inhibition of Emax on noradrenaline responses (P < 0.01), 10 μm nifedipine was necessary.

Both agonists induced a concentration‐dependent accumulation of inositol phosphates. Noradrenaline behaved as a full agonist and St‐587 as a partial agonist for this response.

[3H]‐prazosin binding to intact tail artery rings was saturable and of high affinity (KD = 4.44 ± 0.46 nM; Bmax = 36.35 ± 4.22 fmol mg−1 tissue).

Competition curves for inhibition of specific [3H]‐prazosin binding by WB‐4101 suggest that the rat tail artery contains two α1‐adrenoceptor subtypes in an approximate ratio of 60:40.

After irreversible alkylation of α1B‐adrenoceptors with 100 μm chloroethylclonidine (CEC), nifedipine (1 μm) influenced to a greater extent the St‐587‐ than the noradrenaline‐induced contraction.

Our results indicate that the degree of participation of intracellular and extracellular Ca2+ sources, on the α1‐adrenoceptor‐mediated contraction, depends on the agonist used. The two α1‐adrenoceptor subtypes observed in binding experiments seem to be unrelated to the Ca2+ sources used for contraction.
Effects of agents which elevate cyclic AMP on guinea‐pig eosinophil homotypic aggregation
Authors: Teixeira M.M. , Rossi A.G. , Giembycz M.A. , Hellewell P.G.
Published: July 2012

Eosinophil recruitment and activation in inflamed tissue is thought to play an important role in the pathophysiology of allergic diseases. Experimental evidence suggests that elevating cyclic AMP is an effective means of reducing eosinophil recruitment in vivo and may therefore have therapeutic benefit. In the present study, we have assessed the capacity of cyclic AMP‐elevating agents to modulate guinea‐pig eosinophil homotypic aggregation, a CD18‐dependent process, which may be an important component of eosinophil function in vivo.

Prostaglandin E1 (PGE1, 10−10 to 10−6 m) inhibited platelet activating‐factor (PAF)‐ and C5a‐induced eosinophil aggregation in a concentration‐dependent manner. However, PAF‐induced responses were more potently and more effectively inhibited by PGE1. The inhibitory effects of PGE1 on PAF‐induced aggregation were reversed by pretreatment of eosinophils with the protein kinase A inhibitors H89 and KT5720.

The β2‐adrenoceptor agonists, salbutamol and salmeterol, concentration‐dependently inhibited eosinophil aggregation induced by C5a and PAF and, again, PAF‐induced responses were more effectively reduced. The inhibitory effect of salmeterol was mediated by β‐adrenoceptors, as assessed by the reversal after pretreatment with propranolol.

Rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, also attenuated PAF‐ and C5a‐induced aggregation and at a low concentration which did not affect aggregation per se, had a synergistic effect with PGE1 and salbutamol to suppress this response.

Activation of eosinophils with PAF or C5a induced a small but significant increase in the level of CD18 expression on the eosinophil surface. PGE1 (10−7 m) decreased PAF‐ and C5a‐induced upregulation of CD18 by 93% and 62%, respectively.

These results demonstrate that cyclic AMP‐elevating agents effectively inhibit eosinophil aggregation, a CD18‐dependent functional response. Because CD18 has been shown to be important for eosinophil recruitment to inflamed tissue in vivo, our findings may be of relevance to the efficacy of cyclic AMP‐elevating agents at inhibiting eosinophil trafficking.
Effects of caffeine and 3‐isobutyl‐1‐methylxanthine on voltage‐activated potassium currents in vertebrate neurones and secretory cells
Authors: Reiser Michelle A. , D'Souza Theresa , Dryer Stuart E.
Published: July 2012

The effects of caffeine and 3‐isobutyl‐1‐methylxanthine (IBMX) on voltage‐activated K+ currents were examined by use of patch clamp recording techniques in dissociated chick autonomic ganglion neurones, chick pineal cells and rat anterior pituitary cells.

In chick ciliary ganglion neurones, caffeine (0.1–10 mM) produced a robust blockade of delayed rectifier K+ currents (IDR). Blockade was rapid in onset and concentration‐ and voltage‐dependent. Caffeine produced greater inhibition with larger depolarizing voltage pulses. Similar inhibition of IDR was observed in excised outside‐out ‘maxi‐patches’ indicating a direct effect on the K+ channels. Caffeine also inhibited IDR in chick sympathetic neurones, chick pineal cells and rat anterior pituitary cells.

Application of 10 mM caffeine caused inhibition of transient A‐currents (IA) in chick ciliary ganglion neurones. Inhibition of IA was voltage‐dependent with greater inhibition observed at more positive command potentials. Application of 1 mM caffeine did not cause inhibition of IA.

Application of 1 mM IBMX, a structural analogue of caffeine, caused inhibition of IDR and IA in chick ciliary ganglion neurones. The voltage‐dependence of the inhibition of both currents was qualitatively different from that observed with caffeine. The inhibitory effects of 1 mM IBMX and 10 mM caffeine on IDR and IA were additive.

Direct inhibition of voltage‐activated K+ currents can potentially produce significant secondary effects on intracellular free Ca2+. These results indicate that caution must be used in the design and interpretation of experiments in which millimolar concentrations of caffeine or IBMX are used in pharmacological studies of intracellular Ca2+ dynamics or other second messenger mechanisms.
Delayed protection against ischaemia‐induced ventricular arrhythmias and infarct size limitation by the prior administration of Escherichia coli endotoxin
Authors: Song Wu , Furman Brian L. , Parratt James. R.
Published: July 2012

Bacterial endotoxin (lipopolysaccharide derived from Escherichia coli) was injected intraperitoneally in conscious rats in doses ranging from 0.5 to 2.5 mg kg−1. At various times afterwards the animals were anaesthetized and subjected to a 30 min period of left coronary artery occlusion.

Under these conditions the severity of ventricular arrhythmias was markedly suppressed, in comparison with saline‐injected controls, but this was particularly marked with the higher doses (1.5 and 2.5 mg kg−1); the number of ventricular premature beats was reduced from 1687 ± 227 over the 0.5 h coronary artery occlusion period to 190 ± 46 in those rats administered 2.5 mg kg−1 endotoxin 8 h previously (P < 0.05). The duration of ventricular tachycardia was also significantly reduced (138 ± 26 s to 8.9 ± 4.2 s; P < 0.01) and there was a reduction in the incidence of ventricular fibrillation (from 56% to 10%).

The time course of this protection was studied following the administration of a single dose of 2.5 mg kg−1 of endotoxin by anaesthetizing rats 4, 8 or 24 h later. Protection was apparent at each time but was particularly marked at 8 h.

No rat given the highest dose of endotoxin (32 in all) died as a result of ventricular fibrillation, or from any other cause, during an occlusion, in contrast to a 26% mortality in the controls (P < 0.01).

Infarct size, measured following a 30 min period of coronary artery occlusion followed by a 3 h reperfusion period, was reduced both 8 and 24 h after the administration of 2.5 mg kg−1 endotoxin (reductions of 24.3 and 23.1% respectively; P < 0.05). Endotoxin had no significant effect on the area at risk.

The beneficial effects of endotoxin on infarct size and on ventricular arrhythmias were markedly attenuated by the prior administration of dexamethasone, 3 mg kg−1 given 1 h prior to endotoxin administration. Dexamethasone itself reduced infarct size (P < 0.05) but had no direct effect on arrhythmia severity following coronary artery occlusion.

The mechanisms of this ‘cross‐tolerance’ induced by bacterial endotoxin against ischaemia‐reperfusion injury remain to be elucidated but the most likely mechanisms appear to be the induction of protective enzymes or proteins (e.g. nitric oxide synthase, cyclo‐oxygenase (COX) 2) probably mediated by cytokine release.
Substance P and capsaicin‐induced mechanical hyperalgesia in the rat knee joint; the involvement of bradykinin B1 and B2 receptors
Authors: Davis A.J. , Perkins M.N.
Published: July 2012

Substance P (SP) and capsaicin induced a mechanical hyperalgesia when injected into rat knee joints.

The NK1 receptor antagonists CP 99994 (10–100 nmol) and RP 67580 (0.1‐1 nmol) blocked the development of, and also reversed, SP‐induced hyperalgesia. Capsaicin (10 nmol)‐induced hyperalgesia was blocked by capsazepine (0.5‐5 nmol).

Capsaicin‐induced hyperalgesia was prevented and reversed by the NK1 receptor antagonists CP 99994 (100 nmol) and RP 67580 (1 nmol).

The bradykinin B2 receptor antagonist icatibant (5 pmol) blocked the development of both SP and capsaicin‐induced hyperalgesia. Icatibant (100 pmol kg−1, i.v.) also reversed an established SP and capsaicin‐induced hyperalgesia.

Both low dose SP (1 nmol) and capsaicin (1 nmol)‐induced hyperalgesia were potentiated by the kininase II inhibitor captopril (100 μg).

The B1 receptor antagonists desArg9Leu8‐bradykinin (BK) (0.5‐5 nmol) and desArg10[Hoe 140] (5–50 pmol) only blocked the development of SP‐induced hyperalgesia for 30 min after administration. desArg9Leu8‐BK (10 nmol kg−1 i.v.) did not reverse an established SP‐induced hyperalgesia.

Capsaicin‐induced hyperalgesia was blocked by desArg9Leu8‐BK (0.5 nmol) and this antagonist also reversed an established capsaicin‐induced hyperalgesia.

Interleukin‐1 receptor antagonist (IL‐1ra 0.1 μg) reduced the development of SP‐induced hyperalgesia up to 4 h after administration, but did not reverse an established hyperalgesia. IL‐1ra (0.1 μg) also blocked the development of and reversed an established capsaicin‐induced hyperalgesia.

Indomethacin pretreatment (1 mg kg−1, s.c.) did not reduce the development of either SP‐ or capsaicin‐induced hyperalgesia but following indomethacin‐pretreatment desArg9Leu8‐BK (10 nmol kg−1, i.v.) failed to reverse a capsaicin‐induced hyperalgesia.

In conclusion, both SP and capsaicin can induce behavioural hyperalgesia when injected into the knee joint of rats. In addition, blockade of NK1, bradykinin B1, B2 and IL‐1β receptors can substantially modulate this hyperalgesia.
Thromboxane A2 agonist modulation of excitatory synaptic transmission in the rat hippocampal slice
Authors: Hsu Kuei‐Sen , Kan Wai‐Ming
Published: July 2012

The effects of the selective thromboxane A2 (TXA2) receptor agonist I‐BOP on neuronal excitability and synaptic transmission were studied in the CA1 neurones of rat hippocampal slices by an intracellular recording technique.

Superfusion of I‐BOP (0.5 μm) resulted in a biphasic change of the excitatory postsynaptic potential (e.p.s.p.), which was blocked by pretreatment with SQ 29548, a specific antagonist of TXA2 receptors. The inhibitory phase of I‐BOP on the e.p.s.p. was accompanied by a decrease in neuronal membrane input resistance.

The sensitivity of postsynaptic neurones to glutamate receptor agonists, α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) or N‐methyl‐D‐aspartate (NMDA), was unchanged by I‐BOP (0.5 μm) pretreatment.

Bath application of Ba2+ (0.5 mM) prevented both the I‐BOP‐induced reduction of the neuronal membrane input resistance and the blockade of e.p.s.p. induced by I‐BOP.

Intracellular dialysis of the hippocampal CA1 neurones with GDP (10 mM) significantly attenuated the I‐BOP inhibition of e.p.s.p. and membrane input resistance. Incubation of the slices with either pertussis toxin (PTX, 5 μg ml−1 for 12 h) or cholera toxin (CTX, 5 μg ml‐1 for 12 h) did not affect the biphasic action of I‐BOP on the e.p.s.p. or the reduction of membrane input resistance induced by I‐BOP.

Pretreatment of the slices with the protein kinase C (PKC) inhibitor, NPC‐15437 (20 μm), abolished the biphasic modulation by I‐BOP (0.5 μm) of the e.p.s.p. Intracellular application of a specific PKC inhibitor, PKCI 19–36 (20 μm), completely inhibited the I‐BOP reduction of e.p.s.p. The specific cyclic AMP‐dependent protein kinase (PKA) inhibitor, Rp‐cyclic adenosine 3′,5′‐monophosphate (Rp‐cyclic AMPS, 25 μm), had no effect on the I‐BOP action.

In this study we have demonstrated, for the first time, the existence of functional TXA2 receptors in the hippocampus which mediate the effects of a TXA2 agonist on neuronal excitability and synaptic transmission. Activation of the presynaptic TAX2 receptors may stimulate the release of glutamate. Conversely, activation of postsynaptic TXA2 receptors leads to inhibition of synaptic transmission resulting from a decrease in the membrane input resistance of the neurones. The pre‐ and postsynaptic actions of the TXA2 agonist are both mediated by PTX‐ and CTX‐insensitive G‐protein‐coupled activation of PKC pathways.
Stimulation of the hypothalamo‐pituitary‐adrenal axis in the rat by the type 4 phosphodiesterase (PDE‐4) inhibitor, denbufylline
Authors: Hadley A.J. , Kumari M. , Cover P.O. , Osborne J. , Poyser R. , Flack J.D. , Buckingham J.C.
Published: July 2012

Preliminary studies in our laboratories showed that the synthetic xanthine analogue denbufylline, a selective type 4 phosphodiesterase (PDE‐4) inhibitor, is a potent activator of the hypothalamo‐pituitary‐adrenal (HPA) axis when given orally to adult male rats. This paper describes the results of experiments in which well established in vivo and in vitro models were used to (a) examine further the effects of denbufylline on HPA function and (b) identify the site and mode of action of the drug within the axis.

In vivo, administration of denbufylline (0.1–2.5 mg kg−1, i.p.) produced a significant increase in the serum corticosterone concentration; maximal responses were attained at a dose of 1.0 mg kg−1 (P < 0.01 vs. vehicle control, Scheffe's test). However, when denbufylline was administered by intracerebroven‐tricular injection (0.05‐1 μg kg−1) it failed to influence significantly the serum corticosterone concentration (P > 0.05 vs. vehicle control, Scheffe's test). The adrenocortical responses to peripheral injections of denbufylline (1 mg kg−1, i.p.) were reduced in rats in which the secretion of endogenous corticotrophin releasing factors (CRFs) from the hypothalamus was blocked pharmacologically (P < 0.01 vs. controls, Scheffe's test). However, denbufylline (0.1 mg kg−1, i.p.) potentiated the significant (P < 0.01) increases in serum corticosterone concentration provoked in ‘CRF blocked rats’ by hypothalamic extract (5 hypothalamic extracts kg−1, i.v.) although it failed to influence (P > 0.05) the relatively moderate increases in corticosterone secretion evoked by CRH‐41 (2 mg kg−1, i.v.).

In vitro, denbufylline (0.01‐1 mM) evoked small but significant (P < 0.05) increases in the release of ACTH from rat anterior pituitary segments; furthermore, at these and lower concentrations (0.01 μm‐1 mM), it potentiated the adrenocorticotrophic responses to sub‐maximal concentrations of hypothalamic extract (P < 0.01) and forskolin (0.1 mM, P < 0.01) but not those to CRH‐41 (10 nM) or 8‐bromo‐cyclic AMP (1–100 μm). In addition, denbufylline (0.1 mM) increased the anterior pituitary cyclic AMP content (P < 0.05) and potentiated the rises in tissue content of the cyclic nucleotide induced by hypothalamic extract (0.1 hypothalamic equivalents ml−1, P < 0.01) and forskolin (0.1 mM, P < 0.01) but not by CRH‐41 (10 nM, P < 0.05). By contrast, denbufylline (1 μm‐1 mM) failed to influence the release of AVP from rat isolated hypothalami and stimulated the secretion of CRH‐41 (P < 0.01) release only at the highest concentration tested (1 mM).

The results suggest that the stimulatory actions of denbufylline on the hypothalamo‐pituitary‐adrenocortical axis are exerted predominantly at the level of the anterior pituitary gland and that they may be attributed, at least in part, to inhibition of type 4 phosphodiesterase enzymes.
Inhibition by fenamates of calcium influx and proliferation of human lymphocytes
Authors: Kankaanranta H. , Luomala M. , Kosonen O. , Moilanen E.
Published: July 2012

Flufenamic and tolfenamic acids have recently been shown to inhibit receptor‐mediated calcium influx in human neutrophils. The present work was designed to study the effects of these two nonsteroidal anti‐inflammatory drugs on human peripheral blood lymphocyte activation.

Peripheral blood mononuclear cells (PBMNCs; containing 90% lymphocytes) were stimulated by mitogen concanavalin A (Con A) or by a combination of an inhibitor of microsomal Ca2+‐adenosine triphosphatase thapsigargin (TG) and phorbol myristate acetate (PMA). The effects of the two fenamates on cell proliferation were compared with respective changes in calcium metabolism.

Flufenamic and tolfenamic acids (10–100 μm) inhibited both Con A and TG+PMA‐induced [3H]‐thymidine incorporation in a dose‐dependent manner. At the same concentration range, the two fenamates inhibited the increase in intracellular free calcium concentration induced by Con A or TG+PMA. This effect was due to inhibition of calcium influx whereas calcium release from intracellular stores remained unaltered.

The inhibition of divalent cation influx was confirmed by showing that fenamates inhibited TG+PMA‐induced Mn2+ influx.

The inhibitory effects of fenamates on PBMNC proliferation and Ca2+ influx were qualitatively similar with those of SK&F 96365, an earlier known inhibitor of receptor‐mediated calcium entry. Ketoprofen, a chemically different prostaglandin synthetase inhibitor did not show similar suppressive effects on PBMNCs.

The data suggest that flufenamic and tolfenamic acids suppress proliferation of human peripheral blood lymphocytes by a mechanism which involves inhibition of Ca2+ influx and is not related to inhibition of prostanoid synthesis.