bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention by the tachykinin NK2 receptor antagonist, SR 48968, of antigen‐induced airway hyperresponsiveness in sensitized guinea‐pigs
Authors: Boichot Elisabeth , Germain Noella , Lagente Vincent , Advenier Charles
Published: July 2012

The involvement of tachykinins in antigen‐induced airway hyperresponsiveness (AHR) was characterized pharmacologically in guinea‐pigs sensitized to ovalbumin with antagonists of tachykinin NK1 and NK2 receptors, namely SR 140333 and SR 48968, respectively. AHR was illustrated by increased sensitivity to bronchoconstriction provoked by aerosolized acetylcholine in anaesthetized, ventilated animals, administrated 48 h after ovalbumin aerosol challenge. SR 48968 (1 mg kg−1, i.p.), when given once 30 min before the antigen challenge, prevented AHR, whereas SR 140333 did not. These findings suggest that the tachykinin NK2 receptor antagonist, SR 48968, may be useful for investigating mechanisms of tachykinins in the development of airway hyperresponsiveness.
5‐HT3 receptor antagonism by anpirtoline, a mixed 5‐HT1 receptor agonist/5‐HT3 receptor antagonist
Authors: Göthert M. , Hamon M. , Barann M. , Bönisch H. , Gozlan H. , Laguzzi R. , Metzenauer P. , Nickel B. , Szelenyi I.
Published: July 2012

The aim of this study was to provide evidence that anpirtoline, which is an agonist at 5‐HT1B and 5‐HT1D receptors and also displays submicromolar affinity for 5‐HT1A recognition sites, in addition, acts as an antagonist at 5‐HT3 receptors.

In radioligand binding studies on rat brain cortical membranes, anpirtoline inhibited specific binding of [3H]‐(S)‐zacopride to 5‐HT3 receptor recognition sites (pKi: 7.53).

In N1E‐115 neuroblastoma cells in which [14C]‐guanidinium was used as a tool to measure cation influx through the 5‐HT3 > receptor channel, the 5‐HT‐induced influx was concentration‐dependently inhibited by anpirtoline. In this respect, anpirtoline mimicked other 5‐HT3 receptor antagonists; the rank order of potency was ondansetron>anpirtoline>metoclopramide.

The concentration‐response curve for 5‐HT as a stimulator of [14C]‐guanidinium influx was shifted to the right by anpirtoline (apparent pA2: 7.78).

In urethane‐anaesthetized rats, anpirtoline inhibited (at lower potency than zacopride and tropisetron) the 5‐HT‐ or phenylbiguanide‐induced bradycardia (Bezold‐Jarisch reflex), but did not induce this reflex by itself.

Intravenous infusion of cisplatin in the domestic pig caused a consistent emetic response which was antagonized by anpirtoline.

It is concluded that anpirtoline, which was previously characterized as a 5‐HT1 receptor agonist also proved to be a 5‐HT3 receptor antagonist in several experimental models and, hence, exhibits a unique pattern of properties at different 5‐HT receptors.
Inhibition by dizocilpine (MK‐801) of striatal dopamine release induced by MPTP and MPP+: possible action at the dopamine transporter
Authors: Clarke P.B.S. , Reuben M.
Published: July 2012

The NMDA‐type glutamate receptor antagonist, dizocilpine (MK‐801) can protect against neurotoxicity associated with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and its principal metabolite, the 1‐methyl‐4‐phenylpyridinium ion (MPP+). It has been suggested that these neurotoxic effects may be mediated by release of excitatory amino acids, but possible alternative mechanisms have been little investigated.

MPTP and MPP+ (0.1–1000 μm) were tested in superfused rat striatal synaptosomes preloaded with [3H]‐dopamine. Both MPTP (10 μm and higher) and MPP+ (1 μm and higher) evoked an immediate and concentration‐dependent release of [3H]‐dopamine. The maximal effect exceeded that achievable with nicotine. For subsequent experiments, submaximal concentrations of MPTP (50 μm) and MPP+ (10 μm) were tested.

MK‐801 (0.1–100 μm) inhibited responses to MPTP (50 μm) and MPP+ (10 μm) in a concentration‐dependent manner. However, further tests of NMDA‐type glutamate receptor involvement proved negative. Responses to MPTP or MPP+ were unaffected by the omission of Mg2+ or Ca2+ and were not reduced by the NMDA receptor antagonists, AP‐7 (200 μm) and kynurenic acid (300 μm). In this assay, N‐methyl‐d‐aspartate (even in the absence of Mg2+ and with added glycine and strychnine) did not evoke [3H]‐dopamine release.

In crude membrane preparations of rat cerebral cortex, MPTP and MPP+ inhibited high‐affinity [3H]‐nicotine binding to nicotinic cholinoceptors (IC50 1.8 μm and 26 μm, respectively).

[3H]‐dopamine release evoked by nicotine (1 μm) was blocked by the nicotinic antagonists, mecamylamine and chlorisondamine, and by MK‐801 (all at 100 μm); K+‐evoked release was not affected. Release evoked by MPTP and MPP+ was significantly attenuated by MK‐801 but not by mecamylamine or chlorisondamine.

At a high concentration (10 μm), the selective dopamine uptake inhibitor, nomifensine, completely blocked [3H]‐dopamine release evoked by amphetamine 0.3 μm and MPP+ 10 μm, attenuated responses to MPTP 50 μm and did not affect responses to 12 mm K+. MK‐801 100 μm evinced a similar profile but was less effective.

MK‐801 inhibited [3H]‐dopamine uptake in striatal synaptosomes with an IC50 of 115 μm.

It is concluded that high concentrations of MK‐801 inhibit the acute dopamine release evoked by MPTP and MPP+ in synaptosomes. This antagonism may occur, at least in part, through inhibition of the cell membrane dopamine transporter. MPTP and MPP+ also appear to interact with brain nicotinic cholinoceptors but the functional consequences of this interaction are not yet clear.
Mechanism of activation of nonselective cation channels by putative M4 muscarinic receptor in guinea‐pig chromaffin cells
Authors: Inoue Masumi , Imanaga Itsusei
Published: July 2012

Mechanisms involved in the generation of nonselective cation currents (INS) by muscarinic agonists in the chromaffin cell were investigated by the perforated patch method.

Bath application of muscarine (0.1–30 μm) produced an inward INS with or without a transient outward current at −40 mV, whereas oxotremorine (0.06–60 μm) induced INS alone. Rectangular hyperbolas with EC50S of 2.01 and 0.21 μm were fitted to muscarine‐ and oxotremorine‐induced INSs, respectively, and the maximal amplitude of the former was about 3.4 times larger than that of the latter.

In 36% of the cells exposed to Ca2+‐free solution, muscarine INS was suppressed, being 53% of control 20 min after the perfusion, and in four cells that were incubated with Ca2+‐free solution for 2 h or more, the INS averaged 44% of that induced subsequently in normal solution. In contrast, muscarine INS was enhanced by about 30% when A‐23187 was added to normal solution.

W‐7 and W‐5, calmodulin‐related agents, were almost equally potent in inhibiting muscarine INS, whereas compound 5, a potent inhibitor of calmodulin‐dependent kinase II (CaM kinase II), produced no evident inhibition.

HA 1004, a weak kinase C inhibitor, induced a reversible suppression of muscarine INS with an IC50 of 163 μm, whereas H‐8, another kinase inhibitor, produced an even small degree of inhibition. Administration of phorbol 12, 13‐dibutyrate did not mimic muscarinic stimulation of NS channels; rather, it led to a progressive inhibition of INS and this inhibition was almost complete within 20 min. An inactive phorbol ester had no such effect.

The muscarinic antagonists, pirenzepine and AF‐DX 116, shifted the dose‐response curve for the muscarine INS to the right in a parallel manner. The KDS for pirenzepine and AF‐DX 116 were estimated to be 13 nm (95% confidence interval, 11–16 nm) and 365 nm (283–470 nm), respectively.

These results suggest that muscarine efficiently produces INS, probably through binding to the M4 subtype, that intracellular Ca2+ has a facilitating, but not an essential role in the generation of INS, and that neither CaM kinase II nor protein kinase C is involved.
Necessity of protein kinase C activity for maintenance of acetylcholine receptor function at snake twitch fibre endplates
Authors: Hardwick Jean C. , Parsons Rodney L.
Published: July 2012

The extent of recovery of endplate sensitivity following a 5 or 10 min exposure to carbachol was determined from measurements of miniature endplate current (m.e.p.c.) amplitudes in voltage‐clamped snake twitch fibre endplates. M.e.p.c. amplitude recovery was dependent on the carbachol concentration (0.27–5.4 mm) and duration of application. Staurosporine pretreatment (0.5 μm for ∼15 min) further decreased the extent of m.e.p.c. amplitude recovery.

The decrease in m.e.p.c. amplitude at control endplates exposed to high concentrations of agonist (5.4 mm carbachol for 10 min) was due to an apparent decrease in postsynaptic receptor density, not to a change in the conductance of the acetylcholine (ACh)‐activated channels.

Pretreatment with either 1 μm lavendustin A or 50 μm KN‐62 had no effect on m.e.p.c. amplitude recovery, whereas pretreatment with either 0.5 μm staurosporine, 50 μm sphingosine, or 0.5 μm calphostin C significantly reduced m.e.p.c. amplitude recovery following carbachol exposure.

Sphingosine and staurosporine produced a concentration‐dependent decrease in the extent of m.e.p.c. amplitude recovery, but had no effect on m.e.p.c. characteristics in the absence of carbachol. In addition, this decrease in m.e.p.c. amplitude was not due to the presence of a subpopulation of small amplitude m.e.p.cs.

Prolonged treatment (18–20 h) of muscles with 200 nm phorbol 12‐myristate 13‐acetate (PMA), to down regulate protein kinase C, resulted in a significant reduction in m.e.p.c. amplitudes following exposure to carbachol. Conversely, treatment wtih 200 nm 4αPMA, an inactive analogue, had no effect on m.e.p.c. amplitude recovery.

Only large amplitude ACh‐activated channels (∼50 pS) were recorded from fibres either in the presence of 50 μm sphingosine or from fibres chronically exposed to PMA. However, following recovery from a 10 min exposure to 540 μm carbachol, both small conductance (∼25 pS) and large conductance ACh‐activated channels were recorded in both sphingosine‐ and phorbol‐treated preparations. The conductance of these two populations of channels was virtually identical to those seen in staurosporine‐treated fibres following carbachol exposure.

We conclude that protein kinase C is required for full recovery of AChR sensitivity following carbachol‐induced receptor inactivation. Exposure to high concentrations of agonist for prolonged periods appears to result in the inactivation of a subpopulation of receptors. These receptors must be replaced or reactivated by a process involving protein kinase C. When this phosphorylation step is inhibited, the AChRs remain in an activatable form, but with a reduced conductance.
Requirement of a colchicine‐sensitive component of the cytoskeleton for acetylcholine receptor recovery
Authors: Hardwick Jean C. , Parsons Rodney L.
Published: July 2012

The effect of colchicine treatment on acetylcholine receptor function was examined in potassium depolarized, voltage‐clamped snake twitch fibre endplates. Receptor function was assessed by analysis of miniature endplate currents (m.e.p.c.) as well as acetylcholine (ACh)‐induced single channel currents.

Pretreatment of snake muscle fibres with colchicine (10 μm to 100 μm) for 16–18 h had no effect on m.e.p.c. amplitude or decay rates. At higher concentrations (1 mm), there was a slight decrease in the average m.e.p.c. amplitude.

Colchicine produced a concentration‐dependent decrease in the extent of m.e.p.c. amplitude recovery following a 10 min exposure to 540 μm carbachol. Exposure of 100 μm colchicine‐treated preparations to 0.5 μm staurosporine further reduced the extent of m.e.p.c. amplitude recovery following carbachol exposure.

The decrease in m.e.p.c. amplitude following carbachol exposure was not due to a shift in the m.e.p.c. reversal potential. In addition, the distribution of m.e.p.c. amplitudes remained unimodal in both control and colchicine (100 μm)‐treated preparations following carbachol exposure.

In addition to the normal, large conductance (∼48 pS) ACh‐activated channels, a population of small conductance (∼29 pS) channels was observed in colchicine‐treated preparations following exposure to carbachol. In preparations treated with both colchicine and staurosporine and then exposed to carbachol, the conductance of these small channels was identical to that of colchicine or staurosporine alone.

We suggest that prolonged exposure of snake twitch fibre endplates to agonist results in the activation and desensitization of ACh receptors. Furthermore, we propose that for a subpopulation of the inactivated receptors, restoration of function requires both the integrity of a subsynaptic cytoskeletal component and phosphorylation by a staurosporine‐sensitive protein kinase. One plausible mechanism is that some receptors become destabilized in the membrane and phosphorylation of a cytoskeletal component, whose distribution may depend on an intact microtubular system, is required to re‐anchor these receptors. If this anchoring process is inhibited either by disruption of the cytoskeleton with colchicine, or inhibition of the kinase by staurosporine, these receptors remain activatable, but have a reduced conductance.
Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto‐ATPase
Authors: Crack B.E. , Pollard C.E. , Beukers M.W. , Roberts S.M. , Hunt S.F. , Ingall A.H. , McKechnie K.C.W. , IJzerman A.P. , Leff P.
Published: July 2012

FPL 67156 (6‐N,N‐diethyl‐β,γ‐dibromomethylene‐d‐ATP), is a newly synthesized analogue of ATP.

In a rabbit isolated tracheal epithelium preparation, measuring P2U‐purinoceptor‐dependent chloride secretion, FPL 67156 was discovered to potentiate the responses to UTP but not those to ATP‐γ‐S. UTP agonist‐concentration effect (E/[A]) curves were shifted to the left by 5‐fold in the presence of 100 μm FPL 67156. The differential effect of FPL 67156 on UTP and ATP‐γ‐S was hypothesized to be due to the greater susceptibility of UTP to enzymatic dephosphorylation and the ability of FPL 67156 to inhibit this process.

FPL 67156 was tested as an ecto‐ATPase inhibitor in a human blood cell assay, measuring [γ32P]‐ATP dephosphorylation. The compound inhibited [γ32P]‐ATP degradation with a pIC50 of 4.6.

FPL 67156 was then tested for its effects on ATP and α,β‐methylene‐ATP responses at P2X‐purinoceptors in the rabbit isolated ear artery. In the concentration range 30 μm‐1 mm, the compound potentiated the contractile effects of ATP but not those of α,β‐methylene‐ATP. At 1 mm, FPL 67156 produced a 34‐fold leftward shift of ATP E/[A] curves.

The effects of FPL 67156 on ATP E/[A] curves in the rabbit ear artery were analysed using a theoretical model (Furchgott, 1972) describing the action of an enzyme inhibitor on the effects of a metabolically unstable agonist. This analysis provided an estimate of the pK1 for FPL 67156 as an ecto‐ATPase inhibitor of 5.2.

Using appropriate assays, FPL 67156 was shown to have weak antagonist effects at P2X‐ and P2T‐purinoceptors (pA2 ≅ 3.3 and 3.5 respectively), and weak agonist effects at P2U‐purinoceptors (p[A50] ≅ 3.5).

The degree of potentiation of ATP and UTP effects elicited by FPL 67156 confirms previous results concerning the influence that ecto‐ATPase has on the position of E/[A] curves for metabolically unstable agonists. The magnitude of this influence is predicted to have a major effect on the agonist potency orders currently used to designate purinoceptors.

This study indicates FPL 67156 to be a potentially valuable probe in studies on the action of nucleotides and in the classification of purinoceptors.
Platelet‐activating factor: the effector of protein‐rich plasma extravasation and nitric oxide synthase induction in rat immune complex peritonitis
Authors: Steil Ana Angelica , Rodriguez Maria del Carmen Garcia , Alonso Andrés , Crespo Mariano Sanchez , Bosca Lisardo
Published: July 2012

The involvement of platelet‐activating factor (PAF) in immune complex‐induced/polymorphonuclear‐mediated tissue injury was studied by use of a reverse passive Arthus (RPA) model in the peritoneal cavity of rats.

Extravasation of protein‐rich plasma, accumulation of polymorphonuclear leukocytes (PMN), and the production of nitric oxide (NO) by resident peritoneal mononuclear phagocytes were assayed.

Treatment of rats with either UR‐12460 or BB‐823, two compounds which possess different chemical structures, but elicit the same antagonistic effect on the PAF receptor, abrogated protein‐rich plasma extravasation. In contrast, they did not show any effect on the accumulation of PMN.

Inhibition of NO production with both NG‐mono methyl‐l‐arginine and NG‐nitro‐l‐arginine failed to prevent protein‐rich plasma extravasation.

The production of NO by peritoneal adherent cells following RPA was measured in cells maintained for 2 to 28 h in culture, and it was significantly increased in cells removed as early as 15 min after RPA induction, as compared to controls.

Addition of 10 nm PAF to the culture medium reduced the generation of NO by peritoneal cells from RPA rats, whereas this mediator enhanced NO production in cells from naive control animals.

Treatment with either UR‐12460 or BB‐823 prior to the induction of RPA produced an almost complete inhibition of NO production.

Assay of nitric oxide synthase activity in cell homogenates from peritoneal cells showed that the activity was due to the inducible form of the enzyme.

Study by Northen blotting of mRNA coding for the inducible NO synthase (iNOS) showed transcription at 6 and 18 h after the induction of RPA, which was inhibited in UR‐12460‐treated rats.

These data indicate that PAF is the main mediator of the early plasma leakage observed in RPA, and also that PAF is implicated in the triggering of long‐term changes via induction of specific genes, as judged from its ability to promote the expression of iNOS.
Reduction of vasoconstriction mediated by neuropeptide Y Y2 receptors in arterioles of the guinea‐pig small intestine
Authors: Neild T.O. , Lewis C.J.
Published: July 2012

Brief applications of a high‐K+ solution were used to evoke transient constrictions of arterioles from the guinea‐pig small intestine. Analogues of neuropeptide Y (NPY) selective for Y2‐receptors reduced the constrictions, whereas NPY or a Y1‐selective analogue potentiated the constrictions. We conclude that arteriolar smooth muscle has both Y1 and Y2 receptors, and suggest that Y2 receptors inhibit vasoconstriction by modulating the opening of voltage‐sensitive Ca2+ channels. This may be related to the role of NPY that is present in some vasodilator nerves.
Investigation of the subtype of α2‐adrenoceptor mediating prejunctional inhibition of cardioacceleration in the pithed rat heart
Authors: Smith Karen , Gavin Katherine , Docherty James R.
Published: July 2012

We have investigated the subtype of α2‐adrenoceptor mediating prejunctional inhibition of cardioacceleration in the pithed rat heart in comparison with α2‐adrenoceptor ligand binding sites.

In pithed rats, prejunctional α2‐adrenoceptors were investigated in terms of the ability of α2‐adrenoceptor antagonists to shift the inhibitory potency of the α2‐adrenoceptor agonist, xylazine, against the tachycardia to a single electrical stimulus given via the pithing rod.

Antagonist potency at prejunctional α2‐adrenoceptors in pithed rat heart was correlated with antagonist affinity at α2‐adrenoceptor ligand binding sites in membranes of rat kidney and submandibular gland labelled with [3H]‐yohimbine.

The correlation with the prejunctional α2‐adrenoceptor in pithed rat heart was best for the α2D‐adrenoceptor ligand binding site of rat submandibular gland (r = 0.98, n = 10, P < 0.0001), as compared to correlations with the α2A‐adrenoceptor ligand binding site of human platelet (r = 0.90, n = 9, P<0.001), the x2B‐adrenoceptor ligand binding site of rat kidney (r = 0.82, n = 10, P<0.01) and with published results for the α2C‐adrenoceptor ligand binding site (r = 0.48, n = 6, NS).

It is concluded that the functional prejunctional α2‐adrenoceptor of pithed rat heart closely resembles the α2D‐adrenoceptor ligand binding site of rat submandibular gland.
Kallikrein rK10‐induced kinin‐independent, direct activation of NO‐formation and relaxation of rat isolated aortic rings
Authors: Wassdal Irene , Hull Robert , Gerskowitch V. Paul , Berg Torill
Published: July 2012

rK10, a weak T‐kininogenase isolated from the rat submandibular gland, is a protein belonging to the rat kallikrein family. In the present work, we have studied the biological effects of rK10 with respect to its ability to alter vascular resistance, either directly like rK9, i.e., another kallikrein‐like protein, trypsin and thrombin, or through the release of kinins like tissue kallikrein (rK1). The direct effect was studied by its vasomotor activity on rat isolated aortic rings since this preparation was insensitive to the action of kinins. Its ability to induce altered vascular resistance through kinin‐generation was investigated by blood pressure studies in whole animals. The studies were performed in comparison to rK1.

Unlike rK1, which induces hypotension when administered intravenously to rats (ΔBP = − 56 ± 5 mmHg, 5 μg kg−1), rK10 did not have any: effect on systemic blood pressure (ΔBP= −3 ± 1, 5 μg kg−1, i.v.)

rK10 was without effect on uncontracted aortic rings, but showed a concentration‐dependent (10−8−10−6 m) relaxant effect on tissue precontracted with phenylephrine (10−6m). After removal of endothelial cells, no relaxation was observed. The relaxant response to rK10 was transient. rK1 (with and without endothelium), bradykinin and T‐kinin (with endothelium) had no effect on contracted or uncontracted aortic rings.

The relaxant effect of rK10 was dependent on its enzymatic activity since preincubation with aprotinin (1.02 mm) significantly reduced vasorelaxation from 74 ± 4% to 24 ±3%.

The relaxant effect was not inhibited by the kinin antagonist Hoe 140 (10−7 m; 34 ± 4% without, versus 30 ± 2% with Hoe 140), but was totally inhibited by the NO‐synthase inhibitor N.nitro‐L‐arginine methyl ester (l‐NAME) (2.5 × 10−4m; 27 ± 3% without and 2± 1% with l‐NAME).

These results show that rK10 has the ability to induce vascular relaxation by a specific, direct effect on endothelial cell NO‐synthesis, dependent on rK10 proteolytic activity, but independent of its ability to generate kinin. This effect, or its T‐kininogenase activity in blood, was not sufficient for rK10 to have an effect on peripheral vascular resistance since intravenous injections of rK10, unlike rK1, did not induce hypotension. Thus, rK10 does not seem to play a role in blood pressure homeostasis but may have a local effect on vascular resistance.
Thapsigargin‐ and cyclopiazonic acid‐induced endothelium‐dependent hyperpolarization in rat mesenteric artery
Authors: Fukao Mitsuhiro , Hattori Yuichi , Kanno Morio , Sakuma Ichiro , Kitabatake Akira
Published: July 2012

The present study was designed to determine whether putative, selective inhibitors of the Ca2+–pump ATPase of endoplasmic reticulum, thapsigargin (TSG) and cyclopiazonic acid (CPA), induce endothelium‐dependent hyperpolarization in the rat isolated mesenteric artery. The membrane potentials of smooth muscle cells of main superior mesenteric arteries were measured by the microelectrode technique.

In tissues with endothelium, TSG (10−8–10−5m) caused sustained hyperpolarization in a concentration‐dependent manner. In tissues without endothelium, TSG did not cause any change in membrane potential. CPA (10−5 m) also hyperpolarized the smooth muscle membrane, an effect that was endothelium‐dependent and long‐lasting.

The hyperpolarizing responses to these agents were not affected by indomethacin or NG–nitro‐L‐arginine (L‐NOARG).

In Ca2+–free medium, neither TSG nor CPA elicited hyperpolarization, in contrast to acetylcholine which generated a transient hyperpolarizing response.

In rings of mesenteric artery precontracted with phenylephrine, TSG and CPA produced endothelium‐dependent relaxations. L‐NOARG significantly inhibited the relaxations to these agents, but about 40–60% of the total relaxation was resistant to L‐NOARG. The L‐NOARG‐resistant relaxations were abolished by potassium depolarization.

These results indicate that TSG and CPA can cause endothelium‐dependent hyperpolarization in rat mesenteric artery possibly by releasing endothelium‐derived hyperpolarizing factor and that membrane hyperpolarization can contribute to the endothelium‐dependent relaxations to these agents. The mechanism of hyperpolarization may be related to increased Ca2+ influx into endothelial cells triggered by depletion of intracellular Ca2+ stores due to inhibition of endoplasmic reticulum Ca2+–pump ATPase activity.
Potentiation by viral respiratory infection of ovalbumin‐induced guinea‐pig tracheal hyperresponsiveness: role for tachykinins
Authors: Ladenius A. Rudolf C. , Folkerts Gert , van der Linde Henk J. , Nijkamp Frans P.
Published: July 2012

We investigated whether virus‐induced airway hyperresponsiveness in guinea‐pigs could be modulated by pretreatment with capsaicin and whether viral respiratory infections could potentiate ovalbumin‐aerosol‐induced tracheal hyperresponsiveness.

Animals were inoculated intratracheally with bovine parainfluenza‐3 virus or control medium 7 days after treatment with capsaicin (50 mg kg−1, s.c). Four days after inoculation, tracheal contractions were measured to increasing concentrations of substance P, histamine and the cholinoceptor agonist, arecoline.

In tracheae from virus‐infected guinea‐pigs, contractions in response to substance P, histamine and arecoline were significantly enhanced (P<0.01) by 144%, 46% and 77%, respectively. Capsaicin pretreatment inhibited the hyperresponsiveness to substance P partly (62%) and to histamine and arecoline completely.

In another series of experiments animals were first sensitized with ovalbumin (20 mg kg−1, i.p.). After 14 days animals were exposed to either saline or ovalbumin aerosols for 8 days. After 4 aerosol exposures (4 days) animals were inoculated with either parainfluenza‐3 virus or control medium. One day after the last ovalbumin aerosol, tracheal contraction in response to increasing concentrations of substance P, histamine and arecoline was measured.

Tracheae from ovalbumin‐aerosol‐exposed control inoculated animals showed a similar degree of airway hyperresponsiveness to saline‐aerosol‐exposed virus‐treated guinea‐pigs. Virus inoculation of ovalbumin‐treated animals significantly potentiated the tracheal contractions to substance P compared to either of the treatments alone. The contractions in response to histamine and arecoline were only slightly enhanced.

In conclusion, sensory nerves and/or tachykinins are involved in virus‐induced airway hyperresponsiveness in guinea‐pigs and viral respiratory infections can potentiate the increase in tracheal responsiveness to bronchoconstrictor agonists after ovalbumin exposure.
Inhibition by cromoglycate and some flavonoids of nucleoside diphosphate kinase and of exocytosis from permeabilized mast cells
Authors: Martin Matthew W. , O'Sullivan Antony J. , Gomperts Bastien D.
Published: July 2012

The anti‐allergic compound, cromoglycate, is reported to possess affinity for, and to suppress the autophosphorylation of a 72kDa protein having the sequence of nucleoside diphosphate kinase (NDPK).

We have tested the ability of cromoglycate, and a panel of ten structurally related flavonoids of plant origin, to inhibit the NDPK reaction and the exocytotic process of permeabilized mast cells. The conditions of permeabilization (use of an isotonic medium based on sodium glutamate) were selected to ensure that NDPK activity would be an essential component in the induction of Ca2+–induced exocytosis in which ATP is required for generation of GTP. For comparison, we also measured the inhibition of exocytosis induced by GTP‐γ‐S; this proceeds in the absence of ATP and bypasses the need for NDPK activity.

We found that cromoglycate does not discriminate between Ca2+and GTP‐γ‐S‐induced exocytosis and is a poor inhibitor of NDPK activity. Concentrations in the millimolar range are required for inhibition of all these functions. By comparison, many of the flavonoids are effective at concentrations in the micromolar range.

While we were unable to discern any systematic relationships between their ability to inhibit the three functions, two compounds, quercetin and genistein, inhibit Ca2+–induced, but not GTP‐γ‐S‐induced exocytosis. Inhibition of the late stages of the stimulus‐response pathway in mast cells by these compounds is therefore likely to be due to inhibition of NDPK and the consequent failure to generate GTP.
Effect of morphine on the nerve terminal impulse and transmitter release from sympathetic varicosities innervating the mouse vas deferens
Authors: Lavidis Nickolas A.
Published: July 2012

The effect of morphine on both the propagation of the nerve terminal impulse along the sympathetic varicose axons as well as the evoked and spontaneous transmitter release has been evaluated.

Morphine (1 μm) did not significantly change the shape or the regularity by which the nerve terminal impulse was recorded while evoked transmitter release was greatly reduced.

Morphine induced a uniform decrease in evoked transmitter release irrespective of the release probability of individual varicosities of their position along terminal branches.

Procedures which are thought to increase intracellular calcium concentration such as increasing the extracellular calcium concentration, stimulation of the nerve with trains of impulses and increasing the duration of the action potential with 4‐aminopyridine reduced the ability of morphine to decrease evoked transmitter release.

Morphine had to act directly on the varicosities to induce a decrease in evoked transmitter release.

The decrease in evoked quantal release does not involve an affect on the nerve terminal impulse or the vesicle release process and morphine may affect the dependence of the secretory process on calcium.
Inducible nitric oxide synthase activity and expression in a human colonic epithelial cell line, HT‐29
Authors: Kolios George , Brown Zarin , Robson Rachel L. , Robertson Duncan A.F. , Westwick John
Published: July 2012

We have determined which cytokines regulate the expression of human inducible nitric oxide synthase (iNOS) mRNA and nitrite generation in the human colonic epithelial cell line HT‐29.

Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin‐lα (IL‐lα), tumour necrosisfactor‐α (TNF‐α), interferon γ (IFN‐γ) or vehicle added alone or in combination. Human iNOS mRNA was determined by Northern blot analysis and nitrite generation by the use of a fluorometric assay.

Unstimulated cells produced a small time‐dependent increase in nitrite generation of 50 ± 4, 75 ± 8, and 103 ±8 nM per 106 cells at 24 h, 48 h, and 72 h respectively. This nitrite generation was unaffected by cycloheximide (5 μg ml−1) pretreatment and iNOS mRNA was not detected.

None of cytokines alone induced either iNOS mRNA expression or an increase in nitrite generation. The combination of IL‐lα/IFN‐γ produced a highly significant (P< 0.001) 4 fold increase in nitrite production at 48 h, compared to basal values, while no other pair of cytokines was effective.

Time course studies with IL‐lα/IFN‐γ combination revealed significant (P< 0.001) increases in nitrite at 24 h (153 ±7), 48 h (306 ±24), and 72 h (384 ±15) compared to basal values of 50 ±4, 75 ±8, and 103 ±8 nM per 106 cells respectively.

Studies with IL‐lα/IFN‐γ combination demonstrated a time dependent expression of iNOS mRNA, first observed at 6 h, peaked at 24 h and was undetectable by 72 h. IL‐lα (0.3–10 ng ml−1) and IFN‐γ (10–300 u ml−1) in combination induced a concentration‐dependent expression of iNOS mRNA at 24 h.

Pretreatment with cycloheximide before IL‐lα/IFN‐γ stimulation reduced nitrite levels to basal values. These data suggest that the IL‐lα/IFN‐γ‐induced nitrite production by HT‐29 cells is dependent on de novo protein synthesis, probably the iNOS enzyme.

The addition of TNF‐α produced a significant (P< 0.001) 3 fold increase of IL‐lα/IFN‐γ‐induced nitrite generation. In marked contrast the presence of TNF‐α had no effect on IL‐lα/IFN‐γ‐induced iNOS mRNA expression by HT‐29 cells. These findings suggest that the up‐regulation by TNF‐α of IL‐lα/IFN‐γ‐induced nitrite generation is at the post‐transcriptional level.

These data suggest that pro‐inflammatory cytokines induce NO production in colonic epithelial cells probably due to the induction of iNOS and these cells may be a major source of NO generation in inflammatory bowel disease.
Nitric oxide is a mediator of tachykinin NK3 receptor‐induced relaxation in rat mesenteric artery
Authors: Mizuta Aki , Takano Yukio , Honda Kenji , Saito Ryo , Matsumoto Takafumi , Kamiya Hiro‐O
Published: July 2012

The mechanism of vasodilatation induced by tachykinin peptides was studied in isolated mesenteric arteries of rats.

Senktide, a selective NK3 agonist, elicited potent endothelium‐dependent relaxation of arteries precontracted with phenylephrine (10‐5m), but an NK1 agonist did not.

A non‐peptide NK3 antagonist, SR 142801, inhibited senktide‐induced relaxation. However, a non‐peptide NK1 antagonist, CP‐96,345, and a peptide‐based NK2 antagonist, L‐659,877, had no effect on senktide‐induced relaxation.

Nω‐nitro‐L‐arginine (l‐NOARG), a nitric oxide synthesis inhibitor, markedly attenuated the relaxant response to senktide.

These results suggest that the endothelium of rat mesenteric arteries possesses tachykinin NK3 receptors, and that NK3 agonist‐induced vasodilatation is mediated by release of nitric oxide (NO) from the endothelium.
An endogenous protectant effect of cardiac cyclic GMP against reperfusion‐induced ventricular fibrillation in the rat heart
Authors: Pabla Ravinder , Bland‐Ward Philip , Moore Philip K. , Curtis Michael J.
Published: July 2012

. After a period of myocardial ischaemia, reperfusion of the myocardium can elicit cardiac arrhythmias. Susceptibility to these arrhythmias declines with time, such that a preceding period of more than approximately 40 min ischaemia is associated with few reperfusion‐induced arrhythmias. We have tested the hypothesis that this decline in susceptibility occurs, in part, because of protection by endogenous guanosine 3′:5′‐cyclic monophosphate (cyclic GMP).

Rat isolated hearts were subjected to 60 min left regional ischaemia followed by reperfusion (n= 10 per group). Methylene blue (20 μm), a soluble guanylate cyclase inhibitor, raised the incidence of reperfusion‐induced ventricular fibrillation (VF) from 10% in control hearts to 80% (P<0.05). This effect of methylene blue was abolished by co‐perfusion with zaprinast (100 μm), a phosphodiesterase inhibitor which, in the rat heart, is cyclic GMP‐specific (specific for the type‐V phosphodiesterase isozyme).

Methylene blue reduced cyclic GMP levels in the ischaemic, non‐ischaemic and reperfused myocardium (P<0.05) to 50±10, 52±12 and 70 ± 7 fmol mg−1 tissue wet weight, respectively from control values of 143 ± 38, 147±43 and 156±15 fmol mg−1. Co‐perfusion with zaprinast prevented this effect, and cyclic GMP levels were actually elevated (P<0.05) to 366±102, 396±130 and 293 ± 22 fmol mg−1 in ischaemic, non‐ischaemic and reperfused myocardium, respectively. Zaprinast by itself also elevated cyclic GMP content. Cyclic AMP levels were not affected by zaprinast or methylene blue.

In conclusion, when endogenous cardiac cyclic GMP synthesis is reduced, susceptibility to reperfusion‐induced VF after sustained ischaemia is substantially increased. The effect is prevented by inhibiting cyclic GMP degradation. Therefore cyclic GMP appears to be an endogenous intracellular cardioprotectant, and its actions may account for the low susceptibility to VF normally encountered in hearts reperfused after sustained ischaemia.
Effects of chronic vitamin E deficiency on vascular function ‐ a study of sympathetic nerves, smooth muscle and endothelium of the mesenteric arterial bed of the rat
Authors: Ralevic Vera , Milla Peter J. , Bumstock Geoffrey
Published: July 2012

Male rats were deprived as weanlings of dietary vitamin E for 2, 4, 6, 10 and 12 months. Mesenteric arterial beds from these rats and from age‐matched controls were isolated and perfused with Krebs solution at a constant flow rate (5 ml min−1). The function of perivascular sympathetic nerves, smooth muscle and endothelium was assessed.

At 12 months vitamin E deficient rats exhibited the characteristic symptoms of vitamin E deficiency, namely poor coat condition, muscle wasting, kyphoscoliosis and impaired gait. In the isolated mesenteric arterial bed electrical field stimulation (EFS) of perivascular nerves (4–32 Hz, 90 V, 1 ms, for 30 s) elicited frequency‐dependent vasoconstrictor responses which were unaffected by vitamin E deficiency except at 12 months, at which age responses were significantly greater than those of the controls at 24 and 32 Hz (P<0.01).

Exogenous noradrenaline (NA; 0.15–500 nmol) elicited dose‐dependent vasoconstriction which was similar in vitamin E‐deficient and control preparations at all ages. Potassium chloride (0.15 mmol) also produced similar vasoconstrictor responses in vitamin E‐deficient and control preparations at each age.

Tone of the preparations was raised by continuous perfusion with methoxamine (4–70 μm), producing similar increases in perfusion pressure in vitamin E‐deficient and control preparations at each age. Endothelium‐dependent dose‐dependent vasodilatation to adenosine 5′‐triphosphate was significantly impaired in mesenteric arterial beds from 12 month‐old vitamin E‐deficient rats compared with the controls (P<0.05). Relaxation to acetylcholine was not significantly different at any age.

Endothelium‐independent vasodilatation to sodium nitroprusside was similar in vitamin E‐deficient rats and age‐matched controls.

These results suggest that long term (12 months) deprivation of dietary vitamin E may impair endothelial function in mesenteric arteries of the rat. Sympathetic perivascular nerve constrictor function was increased at 12 months. There were no functionally expressed changes in the vascular smooth muscle, which appears to be more resilient to the effects of oxidative stress in vitamin E deficiency.
Pentobarbitone modulation of NMDA receptors in neurones isolated from the rat olfactory brain
Authors: Charlesworth P. , Jacobson L. , Richards C.D.
Published: July 2012

The action of pentobarbitone on the N‐methyl‐D‐aspartate (NMDA) receptors of neurones freshly dissociated from the olfactory bulb and olfactory tubercle has been studied using patch‐clamp techniques.

Pentobarbitone produced a concentration‐dependent depression of the currents evoked by NMDA with an IC50 value of c. 250 μm.

Analysis of the NMDA‐evoked noise produced power spectra that could be fitted by the sum of two Lorentzians with comer frequencies of 17 and 82 Hz. Pentobarbitone increased the comer frequency of the high frequency component but did not alter the apparent single channel conductance estimated from the noise.

Single channel recordings in either the cell‐attached or outside‐out patch configurations revealed that NMDA (20 or 50 μm) opened channels with a main conductance level around 55 pS and a principal subconductance around 44 pS. The uncorrected mean open time of the channels was 3.4 ms and mean burst length was 6.0 ms. Mean cluster length was about 12 ms.

Pentobarbitone produced a concentration‐dependent reduction in both mean open time and burst length. Mean cluster length was much less affected. Pentobarbitone did not decrease unitary current amplitude or bias the open‐state current amplitude distribution in favour of a particular substate.

From these data it appears that pentobarbitone depresses the inward current evoked by NMDA by reducing the probability of channel opening and this results from a shortening of the lifetime of the channel open state and by decreasing burst length.
Identification of a prostanoid FP receptor population producing endothelium‐dependent vasorelaxation in the rabbit jugular vein
Authors: Chen J. , Champa‐Rodriguez M.L. , Woodward D.F.
Published: July 2012

Prostaglandin F2α (PGF2α) and its synthetic analogue, fluprostenol, potently relaxed the precontracted isolated jugular vein of the rabbit (RJuV). The vasorelaxant activity of PGF2α and fluprostenol was dependent upon an intact vascular endothelium. Although removal of the vascular endothelium abolished activity associated with PGF2α‐like agonists, it did not significantly alter the relaxant effects of prostaglandin E2 (PGE2).

The nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine methyl ester (l‐NAME), at 100 μm significantly inhibited the endothelium‐dependent relaxations induced by PGF2α. Lower doses (1 μm, 10 μm) of l‐NAME had little or no effect. The relaxant effects of PGE2 were not affected by l‐NAME (1–100 μm). D‐NAME at 100 μm was without effect on the vasorelaxant responses to either PGF2α or PGE2.

The potassium (K)‐channel blockers tetraethylammonium (TEA, 1 mm), barium (1 mm) and quinine (100 μm), each tested in the presence of the inactive enantiomer D‐NAME (100 μm) did not significantly affect the response to PGF2α. Unexpectedly, both TEA and barium significantly and partially reversed the inhibitory effects of 100 μm l‐NAME, whereas quinine had no effect. In similar studies, none of the three potassium channel blockers had any effect on relaxations elicited by PGE2 when given with D‐NAME or l‐NAME.

These results indicate that the PGF2α‐sensitive prostanoid receptors found in the vascular endothelium of the rabbit jugular vein are of the FP‐receptor subtype. Nitric oxide (NO) appears to be the predominant messenger involved in PGF2α‐induced relaxation of the rabbit jugular vein. Potassium channels may have a minor role in mediating the vasorelaxation response to PGF2α. When both NO synthesis and K‐channels are simultaneously blocked, inhibition of PGF2α‐induced vasorelaxation by l‐NAME is opposed by K‐channel blockers. This diminution of the inhibitory effect of l‐NAME by TEA and barium suggests that K‐channels may possibly serve a compensatory role via the NO pathway.
Discrimination between UTP‐ and P2‐purinoceptor‐mediated depolarization of rat superior cervical ganglia by 4, 4′‐diisothiocyanatostilbene‐2, 2′‐disulphonate (DIDS) and uniblue A
Authors: Connolly G.P. , Harrison P.J.
Published: July 2012

Using a grease‐gap recording technique we have investigated the effects of some antagonists of P2‐purinoceptors on the depolarization of the rat isolated superior cervical ganglion evoked by 100 μMα, β‐methylene‐adenosine 5′‐triphosphate (α, β‐MeATP) or uridine 5′‐triphosphate (UTP). The effects of the putative P2Z‐purinoceptor antagonist, coomassie brilliant blue G, putative P2X‐purinoceptor antagonist, 4, 4′‐diisothiocyanatostilbene‐2, 2′‐disulphonate (DIDS) and uniblue A (an analogue of the P2Y‐ and P2X‐purinoceptor antagonist reactive blue 2) were investigated.

At the highest concentration examined uniblue A (300 μM) depressed α, β‐MeATP‐induced depolarization and at 100 and 300 μM enhanced UTP‐evoked depolarizations. Coomassie brilliant blue G (1 and 10 μM) did not affect depolarizations evoked by α, β‐MeATP or UTP. Depolarizations evoked by potassium (5 mM) or muscarine (100 nM) were unaltered by either coomassie brilliant blue G or uniblue A. Uniblue A (100 and 300 μM) produced a concentration‐dependent depression of hyperpolarizations evoked by adenosine (100 μM) whereas coomassie brilliant blue G at up to 10 μM, did not alter adenosine‐induced hyperpolarizations.

DIDS (30 and 100 μM) did not alter adenosine‐evoked hyperpolarizations, or depolarizations evoked by potassium or UTP. DIDS at 100 μM did not alter depolarizations evoked by muscarine. In contrast DIDS produced a concentration‐dependent depression of α, β‐MeATP‐evoked depolarizations.

These results are consistent with the proposal that uniblue A and DIDS but not coomassie brilliant blue G are antagonists of P2‐purinoceptors and that uniblue A is also an antagonist at P1 ‐purinoceptors present on the rat superior cervical ganglion.

The ability of uniblue A and DIDS to distinguish between depolarizations evoked by UTP and α, β‐MeATP provides further justification for the proposal that these nucleotides activate separate receptors present on the rat superior cervical ganglion, i.e. pyrimidinoceptors and P2‐purinoceptors respectively.
Attenuation by nitrosothiol NO donors of acute intestinal microvascular dysfunction in the rat
Authors: László F. , Whittle B.J.R. , Moncada S.
Published: July 2012

The effects of the nitric oxide (NO) donors, S‐nitroso‐glutathione (SNG) and S‐nitroso‐N‐acetyl‐penicillamine (SNAP), on the acute intestinal microvascular dysfunction induced by NG‐nitro‐L‐arginine methyl ester (L‐NAME) in combination with low doses of endotoxin were investigated in the anaesthetized rat.

Administration of L‐NAME (5mg kg−1, s.c.) concurrently with E. coli lipopolysaccharide (LPS, 3 mg kg−1, i.v.) provoked the leakage of radiolabelled albumin in the ileum and colon, as a measure of microvascular damage, determined 1 h after challenge.

Intravenous infusion of SNOG or SNAP (1–10 μg kg−1 min−1) dose‐dependently attenuated the microvascular leakage induced by L‐NAME and LPS.

Infusion of the lowest doses of SNOG or SNAP (1 μg kg−1 min−1, i.v.) that significantly reduced the albumin leakage, did not affect the increase in blood pressure in response to L‐NAME in LPS‐treated rats. Higher doses of SNOG or SNAP (5–10 μg kg−1 min−1, i.v.) dose‐dependently reduced this increase in blood pressure.

In control studies, intravenous infusion of glutathione (10 μg kg−1 min−1) or N‐acetyl‐penicillamine (10 μg kg−1 min−1) had no effect on microvascular leakage in the ileum and colon induced by LPS and L‐NAME.

Pretreatment with rabbit anti‐rat neutrophil serum (0.4 ml kg−1, i.p., 4h before challenge), which reduced the neutrophil count in peripheral arterial blood, also inhibited the microvascular leakage in the ileum and colon.

The protective effects of the nitrosothiol NO donors in this model may reflect, in part, modulation of neutrophil interactions within the microcirculation or actions on endothelial cell integrity, in addition to any local vasodilator action.
The use of microdialysis for the study of drug kinetics: some methodological considerations illustrated with antipyrine in rat frontal cortex
Authors: Patsalos P.N. , Abed W.T. , Alavijeh M.S. , O'Connell M.T.
Published: July 2012

The neuropharmacokinetics of antipyrine, a readily dialysable drug, in rat frontal cortex were studied and the effect of sampling time and contribution of period sampling and dialysate dead volume investigated in relation to tmax, Cmax, AUC and t1/2 values.

After i.p. administration, antipyrine (35 mg kg−1, n = 5) concentrations rose rapidly in rat frontal cortex (tmax, 12 min) and then declined exponentially. tmax, Cmax, AUC and t1/2 values were determined after 2 min dialysate sampling and compared to values obtained from simulated sampling times of 4, 6, 8, 10 and 20 min.

Antipyrine tmax and Cmax values were directly dependent on sampling frequency. Thus, mean 2 min sampling tmax and Cmax values were 63% lower and 27% higher, respectively, compared to 20 min sampling values. AUC and t1/2 values were unaffected.

Adjustment for dialysate dead volume (the volume of dialysate within the dialysis probe and sampling tube) reduced tmax values significantly but did not affect the other neuropharmacokinetic parameters.

Contribution of period sampling on neuropharmacokinetic parameters were investigated by comparing plots of antipyrine concentration data at midpoint and at endpoint of sampling time interval. Only tmax values were affected with values decreasing with increasing sampling time interval.

In conclusion, although microdialysis is a useful method for monitoring events at the extracellular level and for kinetic studies, it is important to understand its inherent characteristics so that data can be interpreted appropriately. Sampling frequency, particularly during monitoring of periods of rapid change, is very important since Cmax and tmax values will be significantly underestimated and overestimated respectively, if sampling time is longer rather than shorter. These considerations are particularly important in relation to microdialysis studies of pharmacokinetic‐pharmacodynamic interrelationships and modelling.