bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacodynamics of the anticonvulsant effect of oxazepam in aging BN/BiRij rats
Authors: Stijnen Annemiek M. , Postel‐Westra Ineke , Langemeijer Mariska W.E. , Hoogerkamp Arendien , Voskuyl Rob A. , van Bezooijen Cornelis F.A. , Danhof Meindert
Published: July 2012

The purpose of this investigation was to examine the influence of increasing age on the pharmacokinetics and the time course of the anticonvulsant response of oxazepam in BN/BiRij rats as an animal model of aging.

Oxazepam was administered intravenously in a dose of 12 mg kg−1 body weight and the anticonvulsant effect intensity was measured as elevation above baseline of a threshold for induction of localized seizure activity (TLS). Direct cortical stimulation with ramp shaped electrical pulse trains of increasing intensity was used to determine this threshold.

The pharmacological effect vs. time profile showed in young rats an anticonvulsant component followed by proconvulsant component which is suggestive for the occurrence of acute tolerance and/or withdrawal syndrome. With increasing age the proconvulsant component disappeared, resulting in a monophasic effect profile (anticonvulsant effect only) at the age of 35 months with significantly higher anticonvulsant effect intensity immediately following drug administration. No age‐related changes in the pharmacokinetic parameters of oxazepam were observed.

In five animals of each age group, benzodiazepine receptor binding characteristics were determined in vitro with [3H]‐flunitrazepam as a ligand. Both receptor density and affinity did not show age‐related changes. Available literature data on post‐receptor events do not indicate conclusive age‐related changes.

It is concluded, that the observed change in the pharmacodynamics of anticonvulsant effect of oxazepam can be explained by the disappearance of the tolerance/withdrawal phenomenon. This is compatible with a decreased efficiency of homeostatic control mechanisms in the elderly.
Kinin‐induced chloride permeability changes in colony 29 epithelia estimated from 125I− efflux and MEQ fluorescence
Authors: MacVinish L.J. , Reancharoen T. , Cuthbert A.W.
Published: July 2012

The changes in apical Cl− permeability of Colony 29 human colonic epithelial monolayers were estimated from the rate constant of 125I− efflux from tissues loaded with the isotope.

Forskolin was used to increase intracellular concentrations of adenosine 3:5′ cyclic‐monophosphate (cyclic AMP), and A23187 to increase intracellular free Ca2+ (Cai). Both treatments increased the rate constant for 125I− efflux, indicating an increase in apical Cl− permeability.

Lysylbradykinin (LBK) also increased the rate constant for 125I− efflux, sometimes biphasically. Chelation of intracellular Ca2+ with BAPTA or prevention of prostaglandin formation with piroxicam, attenuated but did not eliminate the effect of LBK. It is concluded that LBK affects 125I− efflux through the agency of both cyclic AMP and Ca2+.

Ba2+ attenuated the effect of LBK and A23187 on 125I− efflux, but had no effect on the action of forskolin. It is concluded that Ca2+ has a major effect on K+ channels, the resulting hyperpolarization increasing the driving force for 125I− efflux. A secondary effect on Ca2+‐sensitive Cl− channels is possible. By contrast, cyclic AMP exerts it major effect on apical Cl− channels.

Using a Cl− sensitive fluorescent dye, MEQ, the intracellular chloride concentration, Cli was estimated to be around 30 mm, which was increased to around 50 mm by forskolin, suggesting cyclic AMP could activate the Na‐K‐2Cl co‐transporter.

MEQ fluorescence was used to estimate Cl− influx and efflux rates of epithelial cells. These were increased three fold by forskolin and dibutyryl cyclic AMP and two fold by LBK and histamine.

It is concluded that LBK increases electrogenic chloride secretion in Colony 29 epithelia through the generation of second messengers cyclic AMP and Ca2+, each of which may act on both apical and basolateral membranes.
Cibenzoline inhibits diazoxide‐ and 2,4‐dinitrophenol‐activated ATP‐sensitive K+ channels in guinea‐pig ventricular cells
Authors: Sato Toshiaki , Wu Bowei , Nakamura Sunao , Kiyosue Tatsuto , Arita Makoto
Published: July 2012

We have investigated the effects of diazoxide (a sulphonamide derivative) and cibenzoline (a class I antiarrhythmic drug) on ATP‐sensitive K+ currents in guinea‐pig ventricular cells, using whole‐cell clamp techniques.

Diazoxide (50 μm) produced a marked shortening of action potential duration which was antagonized by 1 μm glibenclamide, an ATP‐sensitive K+ channel blocker.

Diazoxide (50 μm) increased the quasi‐steady state outward current elicited by a ramp voltage protocol (−20 mV s−1) at potentials positive to about − 70 mV. This effect was completely prevented in the presence of glibenclamide (1 μm), thereby suggesting that diazoxide opens ATP‐sensitive K+ channels.

Cibenzoline (5 μm) depressed the diazoxide‐induced increases in the outward current and the pretreatment with this agent prevented the development of the diazoxide‐induced outward current.

Cibenzoline (10 μm) reversed the 2,4‐dinitrophenol (50 μm)‐induced shortening of the action potential duration partially but significantly.

These results suggest that diazoxide activates ATP‐sensitive K+ channels of guinea‐pig ventricular cells and that cibenzoline, at therapeutic concentrations, inhibits this channel.
Fendiline inhibits L‐type calcium channels in guinea‐pig ventricular myocytes: a whole‐cell patch‐clamp study
Authors: Tripathi O. , Schreibmayer W. , Tritthart H.A.
Published: July 2012

Fendiline, a diphenylalkylamine type of antianginal drug, was examined for its effects on L‐type calcium channels in guinea‐pig ventricular myocytes by the whole‐cell patch‐clamp technique.

Fendiline (0.3–100 μm) applied extracellularly inhibited the calcium channel current (ICa) in a concentration‐ and time‐dependent manner. The IC50 of fendiline was 17.0 ± 2.43 μm and the Hill slope was 1.39 ± 0.23.

Inhibition of ICa by fendiline appeared with an onset of less than 3 s.

Fendiline inhibited ICa at all the membrane potentials tested and shifted the current‐voltage curve upwards. The overall calcium channel conductance (gCa) of the cell was reduced and conductance‐voltage curve was shifted to the left in the presence of fendiline.

Isoprenaline (0.5–1 μm), a β‐adrenoceptor agonist, partially reversed the inhibitory effect of fendiline on ICa.

It is suggested that fendiline applied extracellularly blocks L‐type calcium channels and reduces calcium channel conductance of the cell. The calcium channels thus inhibited are, nevertheless, still available for β‐adrenoceptor stimulation.
Nitric oxide affects mammalian distal colonic smooth muscle by tonic neural inhibition
Authors: Middleton S.J. , Cuthbert A.W. , Shorthouse M. , Hunter J.O.
Published: July 2012

The role of the l‐arginine‐nitric oxide (NO) pathway in tonic neural inhibition of spontaneous mechanical activity of distal colonic circular smooth muscle (DCCSM) was investigated in male Wistar rats.

Muscle strips were mounted in organ baths and spontaneous contractions recorded with isometric force transducers. They were characterized as low frequency (LFCs) 0.41 ± 0.03 N cm−2 or high frequency contractions (HFCs) 0.22 ± 0.04 N cm−2. The latter occurred intermittently to produce summation contractions (SCs) range 0.5–12 N cm−2.

Tetrodotoxin (100 nm) increased the forces of LFCs and SCs. Increase in force to tetrodotoxin did not occur after incubation of the muscle with NG‐monomethyl‐l‐arginine (l‐NMMA) 500 μm, an inhibitor of NO biosynthesis.

l‐NMMA but not its enantiomer d‐NMMA increased the force of LFCs (EC50:200 μm) and SCs (EC50:175 μm) in a concentration‐dependent manner which was reversed by l‐arginine but not by d‐arginine.

Muscle, precontracted by acetylcholine, relaxed to sodium nitroprusside (EC50:1.8 μm) NO gas (EV50:70 μl) and NO solutions (EC50:4 μm) in a concentration‐dependent manner. Guanosine 3′:5′‐cyclic monophosphate tissue concentrations (pmol mg−1 protein) were elevated in muscle after relaxation by sodium nitroprusside (500 μm) from 0.32 ± 0.06 to 1.2 ± 0.37 and by 1 ml of NO gas from 0.49 ± 0.05 to 1.54 ± 0.14.

These data suggest that DCCSM is under tonic neural inhibition mediated by NO biosynthesis.
Postnatal changes in N‐methyl‐D‐aspartate receptor binding and stimulation by glutamate and glycine of [3H]‐MK‐801 binding in human temporal cortex
Authors: Slater P. , McConnell S.E. , D'Souza S.W. , Barson A.J.
Published: July 2012

Homogenates of human infant and adult temporal cortex were used to measure [3H]‐TCP and [3H]‐MK‐801 binding to the N‐methyl‐d‐aspartate (NMDA)‐coupled ion channel phencyclidine site.

Both [3H]‐TCP and [3H]‐MK‐801 binding increased in infant cortex by> 100% between term and 26 weeks suggesting that the numbers of NMDA receptors increase during postnatal brain development.

[3H]‐MK‐801 binding was measured under non‐equilibrium conditions in temporal cortex homogenates with the addition of 100 μm of l‐glutamate plus a range of concentrations (0.05 μm−100 μm) of glycine. Glutamate and glycine increased [3H]‐MK‐801 binding by stimulating NMDA receptors and improving [3H]‐MK‐801 access to ion channel binding sites; maximum stimulation in adult and infant temporal cortex was achieved with 100 μm glutamate plus 5 μm glycine; a higher concentration of glycine (50 μm) reduced [3H]‐MK‐801 binding to below maximum.

The stimulation by 100 μm glutamate plus 5 μm glycine of [3H]‐MK‐801 binding in infant temporal cortex was affected by postnatal age. For example, although the stimulation of [3H]‐MK‐801 binding in 5–6 week infant cortex (236% of basal) was similar to adult cortex (230% of basal), in samples taken from infants aged 5–6 months glycine (plus glutamate) stimulation of [3H]‐MK‐801 binding (392% of basal) was substantially greater than that measured in adult temporal cortex.

The binding of [3H]‐glycine to the glycine modulatory site associated with the NMDA receptor in infant cortex also increased with postnatal age by > 100% between term and 26 weeks.

It is concluded that NMDA receptors in infant cortex increase to levels greater than those in adult cortex during postnatal development. The results do not exclude the possibility that the transiently increased NMDA receptor‐ion channel complex in infant cortex shows enhanced responses to agonists and modulators.
l‐Arginine exerts a dual role in nociceptive processing in the brain: involvement of the kyotorphin‐Met‐enkephalin pathway and NO‐cyclic GMP pathway
Authors: Kawabata Atsufumi , Umeda Nahoko , Takagi Hiroshi
Published: July 2012

Intracerebroventricular (i.c.v.) administration of l‐arginine (l‐Arg), at 10–100 μg per mouse, produced antinociception in mice, as assessed by the tail flick test; this antinociception was reversed by pretreatment (s.c.) with naltrindole (NTI), a δ‐selective opioid antagonist, and by co‐administered l‐leucyl‐l‐arginine (Leu‐Arg), a kyotorphin (endogenous Met‐enkephalin releaser) receptor antagonist.

l‐NG‐nitroarginine methyl ester (l‐NAME), a NO synthase inhibitor, but not d‐NG‐nitroarginine methyl ester, given i.c.v. at 3–10 μg per mouse, exhibited antinociceptive activity that was resistant to naloxone (s.c.), NTI (s.c.) and Leu‐Arg (i.c.v.).

The l‐NAME (i.c.v.)‐induced antinociception was not reversed by l‐Arg (i.c.v.), which was antinociceptive by itself, but was abolished by combined injection of l‐Arg plus Leu‐Arg (i.c.v.) or by l‐Arg (i.c.v.) after NTI (s.c.).

Methylene blue (MB), a soluble guanylate cyclase inhibitor, at 0.1–1 μg per mouse, produced antinociception by i.c.v. administration. The antinociception induced by MB (i.c.v.) or l‐NAME (i.c.v.) was reversed by co‐administered dibutyryl cyclic GMP.

These findings suggest that l‐Arg plays a dual role in nociceptive processing in the brain, being antinociceptive via the kyotorphin‐Met‐enkephalin pathway and nociceptive via the NO‐cyclic GMP pathway.
The Leu13‐motilin (KW‐5139)‐evoked release of acetylcholine from enteric neurones in the rabbit duodenum
Authors: Kitazawa Takio , Ishii Akio , Taniyama Kohtaro
Published: July 2012

Involvement of cholinergic mechanisms in the contractile response to Leu13‐motilin (LMT, KW‐5139) was investigated in rabbit duodenal segments, and longitudinal muscle‐myenteric plexus (LM‐MP) preparations preincubated with [3H]‐choline.

Contractile response to LMT (0.1 nm −1 μm) consisted of an initial rapid (phasic) contraction and a tonic contraction slowly fading to a sustained plateau. LMT caused a concentration‐dependent phasic contraction of rabbit isolated duodenal segments. The EC50 value was 2.5 nm and the maximum amplitude of the contraction was 103% of the response induced by acetylcholine (ACh, 100 μm). Neither tetrodotoxin nor atropine changed the EC50 value or the maximum amplitude of the response to LMT.

Both atropine and tetrodotoxin decreased the amplitude and accelerated fading of the tonic contraction produced by LMT.

LMT (30 nm−3 μm) induced an increase of 3H‐outflow, in a concentration‐dependent manner. The LMT‐induced increase of 3H‐outflow was prevented by removal of external Ca2+ or by the presence of tetrodotoxin.

Porcine motilin (10 nm −1 μm) also stimulated the release of 3H at a similar concentration‐range to that seen with LMT.

Pretreatment with LMT (3 μm for 20 min) decreased LMT‐ and the porcine motilin‐evoked release of 3H but did not alter the high K+‐evoked release.

Our results suggest that LMT and porcine motilin stimulate the release of ACh from enteric neurones through the same receptor, and that the release of ACh plays a role in tonic components of contraction in the rabbit duodenum.
Blockade by calmodulin inhibitors of Ca2+ channels in smooth muscle from rat vas deferens
Authors: Nakazawa Ken , Higo Katsuya , Abe Kazuho , Tanaka Yoshio , Saito Hiroshi , Matsuki Norio
Published: July 2012

Effects of three compounds which are used as calmodulin inhibitors (trifluoperazine, W‐7 and calmidazolium) on Ca2+ channels were investigated in smooth muscle from rat vas deferens.

All three calmodulin inhibitors relaxed the smooth muscle precontracted by a high concentration of KCl (63.7 mm). The order of potency for the relaxation was trifluoperazine > W‐7 > calmidazolium.

In binding studies using a microsomal fraction of vas deferens, all these calmodulin inhibitors displaced specific [3H]‐nimodipine binding. Trifluoperazine and W‐7 inhibited the binding at concentrations that relaxed the smooth muscle whereas calmidazolium inhibited at concentrations much lower than those necessary for muscle relaxation.

Ba2+ current flowing through voltage‐gated Ca2+ channels was measured under whole‐cell voltage‐clamp conditions in isolated smooth muscle cells. The Ba2+ current was suppressed by the three calmodulin inhibitors in the concentration‐range where inhibition of [3H]‐nimodipine binding was observed. Neither voltage‐dependence nor the inactivation time course of Ba2+ current were affected by these compounds.

The results suggest that the calmodulin inhibitors directly block Ca2+ channels in the smooth muscle cells. The channel inhibition by trifluoperazine and W‐7, but perhaps not that by calmidazolium, may be responsible for the muscle relaxation observed with these compounds.
A non‐peptide NK1‐receptor antagonist, RP 67580, inhibits neurogenic inflammation postsynaptically
Authors: Moussaoui S.M. , Montier F. , Carruette A. , Blanchard J.C. , Laduron P.M. , Garret C.
Published: July 2012

The non‐peptide neurokinin NK1‐receptor antagonist, RP 67580 (3aR, 7a), a perhydroisoindolone derivative, powerfully reduced plasma extravasation in rat hind paw skin induced by local application of xylene (ID50 = 0.03 mg kg−1, i.v.) or capsaicin (ID50 = 0.06 mg kg−1, i.v.), or by i.v. injection of exogenous substance P (SP) or septide ([pGlu6,Pro9]SP(6–11)) (ID50 = 0.04–0.05 mg kg−1, i.v.). RP 67580 (1 mg kg−1, i.v.) also abolished capsaicin‐induced nasal fluid hypersecretion (by 82 ± 5%). These effects were found to be stereospecific, the enantiomer, RP 68651 (3aS, 7aS), being inactive at 1 mg kg−1, i.v.

In rats neonatally treated with capsaicin (50 mg kg−1, s.c.), plasma extravasation induced by SP was significantly increased (by 43 ± 7%). RP 67580 (1 mg kg−1, i.v.) completely inhibited the SP‐induced plasma extravasation in capsaicin neonatally treated‐animals, as it did in control animals. This result suggests that RP 67580 acts at the postsynaptic level for the inhibition of plasma extravasation.

Opioid receptor agonists, μ‐(morphine) and κ‐(PD‐117302) at 10 mg kg−1, s.c., in contrast to NK1‐receptor antagonists, did not inhibit plasma extravasation induced by exogenous SP. They were, however, partially effective against plasma extravasation induced by electrical nerve stimulation (74 ± 4% and 48 ± 9% inhibition at 10 mg kg−1, s.c. of morphine and PD‐117302, respectively, compared to 90 ± 3% inhibition obtained with RP 67580, 3 mg kg−1, s.c.). These results indicate the presynaptic action of opioid receptor agonists, in contrast to the postsynaptic action of NK1‐preceptor antagonists for the inhibition of plasma extravasation.

Ligature of the saphenous nerve distal to the point of electrical stimulation, local application of lignocaine to the saphenous nerve, neonatal capsaicin pretreatment, and colchicine at very low doses (120 μg kg−1 day−1 given for 3 days) were found to prevent plasma extravasation elicited by electrical nerve stimulation.

The foregoing results demonstrate that the non‐peptide NK1‐receptor antagonist, RP67580, is a potent inhibitor of plasma extravasation induced in skin by NK1‐receptor agonists, by local application of chemical irritants (capsaicin or xylene) or by electrical nerve stimulation. Moreover, opioid receptor agonists and colchicine inhibit plasma extravasation induced by electrical nerve stimulation but not that elicited by exogenous SP. Therefore, it is possible to inhibit neurogenic inflammation either at the presynaptic level with opioid receptor agonists and colchicine, or at the postsynaptic level with NK1‐receptor antagonists, and that the new non‐peptide NK1‐receptor antagonists may have a great potential for alleviation of inflammation in various pathological syndromes in man.
The diuretic action of 8‐cyclopentyl‐1,3‐dipropylxanthine, a selective A1 adenosine receptor antagonist
Authors: Knight R.J. , Bowmer C.J. , Yates M.S.
Published: July 2012

The diuretic effect of the selective A1 adenosine receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (CPX), was investigated in anaesthetized rats.

CPX (0.1 mg kg−1, i.v.) produced significant increases in urine flow, and the excretion rate and fractional excretion of both sodium and chloride. By contrast, CPX administration did not result in any significant change in the excretion of potassium.

The diuretic effect of CPX was accompanied by a transient increase in inulin clearance although p‐amino‐hippurate clearance was unaffected, indicating the CPX induced a temporary elevation of glomerular filtration rate but no change in renal blood flow.

The fractional excretion of lithium (a marker of delivery of fluid out of the proximal tubule) was also significantly increased by CPX. However, other measures of tubular function derived from lithium clearance indicated that there were no changes in the handling of sodium or water in the distal regions of the nephron.

CPX did not significantly alter the relationship between either free water reabsorption or free water clearance and the distal delivery of sodium, which suggests that CPX does not affect the renal concentration/dilution mechanism.

The results of this study show that the diuresis and increased excretion of sodium and chloride induced by CPX (0.1 mg kg−1) in the rat, occurs with only transient elevation in glomerular filtration rate and no change in renal blood flow. The primary reason for the diuresis appears to be inhibition of sodium reabsorption in the proximal tubule. Furthermore, the results provide evidence that production and release of endogenous adenosine modifies renal excretory function via stimulation of the A1 receptor subtype.
Thermodynamics of antagonist binding to rat muscarinic M2 receptors: antimuscarinics of the pridinol, sila‐pridinol, diphenidol and sila‐diphenidol type
Authors: Waelbroeck M. , Camus J. , Tastenoy M. , Lambrecht G. , Mutschler E. , Kropfgans M. , Sperlich J. , Wiesenberger F. , Tacke R. , Christophe J.
Published: July 2012

We studied the effect of temperature on the binding to rat heart M2 muscarinic receptors of antagonists related to the carbon/silicon pairs pridinol/sila‐pridinol and diphenidol/sila‐diphenidol (including three germanium compounds) and six structurally related pairs of enantiomers [(R)‐ and (S)‐procyclidine, (R)‐ and (S)‐trihexyphenidyl, (R)‐ and (S)‐tricyclamol, (R)‐ and (S)‐trihexyphenidyl methiodide, (R)‐ and (S)‐hexahydro‐diphenidol and (R)‐ and (S)‐hexbutinol]. Binding affinities were determined in competition experiments using [3H]‐N‐methyl‐scopolamine chloride as radioligand. The reference drugs were scopolamine and N‐methyl‐scopolamine bromide.

The affinity of the antagonists either increased or decreased with temperature, van 't Hoff plots were linear in the 278–310°K temperature range. Binding of all antagonists was entropy driven. Enthalpy changes varied from large negative values (down to −29 kJ mol−1) to large positive values (up to + 30 kJ mol−1).

(R)‐configurated drugs had a 10 to 100 fold greater affinity for M2 receptors than the corresponding (S)‐enantiomers. Enthalpy and entropy changes of the respective enantiomers were different but no consistent pattern was observed.

When silanols (R3SiOH) were compared to carbinols (R3COH), the affinity increase caused by C/Si exchange varied between 3 and 10 fold for achiral drugs but was negligible in the case of chiral drugs. Silanols induced more favourable enthalpy and less favourable entropy changes than the corresponding carbinols when binding. Organogermanium compounds (R4Ge) when compared to their silicon counterparts (R4Si) showed no significant difference in affinity as well as in enthalpy and entropy changes.

Exchange of a cyclohexyl by a phenyl moiety was associated with an increase or a decrease in drug affinity (depending on the absolute configuration in the case of chiral drugs) and generally also with a more favourable enthalpy change and a less favourable entropy change of drug binding.

Replacement of a pyrrolidino by a piperidino group and increasing the length of the alkylene chain bridging the amino group and the central carbon or silicon atom were associated with either an increase or a decrease of entropy and enthalpy changes of drug binding. However, there was no clear correlation between these structural variations and the thermodynamic effects.

Taken together, these results suggest that hydrogen bond‐forming OH groups and, to a lesser extent, polarizable phenyl groups contribute significantly to the thermodynamics of interactions between these classes of muscarinic antagonists and M2 muscarinic receptors.
Blockade by glibenclamide of the flow‐evoked endothelial release of ATP that contributes to vasodilatation in the pulmonary vascular bed of the rat
Authors: Hasséssian Haroutioun , Bodin Philippe , Burnstock Geoffrey
Published: July 2012

The effect of step augmentation of flow rate on the level of adenosine −5′‐triphosphate (ATP) measured in the Krebs perfusate was investigated, and the effect of glibenclamide on the release of ATP was tested in the rat pulmonary vascular bed.

For flow rates between 10.38 ± 1.18 and 28.88 ± 2.08 ml min−1(n= 8) 1 μmsuramin, a P2‐purinoceptor antagonist, significantly (P< 0.05) increased vascular resistance under conditions of step augmentation of flow rate. This suggests that endogenous ATP released during increases in flow rate dilates pulmonary vessels.

In response to a step augmentation in flow rate from 9.13 ± 0.97 to 18.3 ± 1.69 ml min−1(n= 4) ATP levels were up to 23 fold higher (P< 0.05) for 15 s, and gradually dropped to a level of about half the initial rise. Once the ATP levels had stabilized, another step augmentation of flow rate to 27.00 ± 3.49 ml min−1was able to evoke a corresponding increase of ATP release. The ability of the vascular bed to respond with increased ATP release after the initial ATP responses had tapered, demonstrates that the drop in ATP levels after the initial rise is not due to depletion of ATP. Furthermore, the maximal ATP response directly precedes the vasodilatation observed following each jump in perfusion pressure produced with each step increase in flow rate.

In response to two 3 fold step augmentations of flow rate (8.41–27.29 ml min−1) spaced 30 min apart there were two increases in the level of ATP which were not significantly different from each other. However, perfusion with 1 μmglibenclamide between the first and the second step augmentation of flow rate (8.08–24.67 ml min−1) significantly (P< 0.05;n= 6) blocked the increase in ATP release. This suggests that the release of intracellular ATP is mediated by glibenclamide‐sensitive K+channels.

A concentration of 1 μmglibenclamide perfused for 30 min was without effect on vascular pressure at constant flow. However, under conditions where flow was augmented in a stepwise manner (between 11.50 and 36.45 ml min−1) perfusing with 1 μmglibenclamide increased vascular resistance (P< 0.10).

It is concluded that flow‐induced ATP release is mediated by a glibenclamide‐sensitive K+channel, and that the release of ATP from endothelial cells probably functions to vasodilate the pulmonary vascular bed of the rat.
Reciprocal interaction of 5‐hydroxytryptamine and cholecystokinin in the control of feeding patterns in rats
Authors: Grignaschi Giuliano , Mantelli Barbara , Fracasso Claudia , Anelli Marina , Caccia Silvio , Samanin Rosario
Published: July 2012

The effect of the CCKA receptor antagonist, devazepide (100 mg kg−1) on meal parameters during the initial phase of the dark period was studied in free‐feeding rats by use of a procedure for continuously monitoring feeding patterns.

In a second experiment, the effect of devazepide on the reduction in meal parameters induced by the 5‐hydroxytryptamine (5‐HT) releaser and uptake inhibitor, (+)‐fenfluramine (1.5 mg kg−1) in 4 h food‐deprived rats was examined.

The hypophagic effect of an intraperitoneal injection of cholecystokinin (CCK‐8, 4 μg kg−1) was studied in rats treated with the 5‐HT receptor antagonist, metergoline (1 and 2 mg kg−1).

Devazepide increased the size of the first meal in free‐feeding, but not in 4 h food‐deprived rats and partially antagonized the effect of (+)‐fenfluramine on the size and duration of the first meal. The reduction in eating rate induced by (+)‐fenfluramine was not modified by devazepide. No changes in (+)‐fenfluramine or (+)‐norfenfluramine levels were found in the brain of rats treated with devazepide.

The effect of CCK‐8 on meal size was completely antagonized by 2 mg kg−1 metergoline. A significant interaction was also found between 2 mg kg−1 metergoline and CCK‐8 as regards their effect on the inter‐meal interval.

The results suggest a reciprocal interaction between 5‐HT and CCK‐8 in enhancing the satiating effect of food in rats.
Action of α‐dendrotoxin on K+ currents in nerve terminal regions of axons in rat olfactory cortex
Authors: McGivern J. , Scholfield C.N. , Dolly J.O.
Published: July 2012

In the rat olfactory cortex, unmyelinated axons give rise to synapses en passant. This tissue was used to study the pharmacology of axonal K+‐currents. Responses were measured from a group of these axons as unclamped field currents, with a polarizable suction electrode.

A single stimulus to the axons elicited a tetrodotoxin‐sensitive Na+‐dependent transient. K+‐currents were revealed by positive polarization of the suction electrode and were manifest as a negative current following the Na+‐component.

In the presence of tetraethylammonium (TEA, 5 mm) and Cd2+ (100 μm), the K+‐component was depressed by 3,4‐diaminopyridine (3,4‐DAP; 1 to 20 μm; IC50 2.0 ± 0.4 μm). α‐Dendrotoxin (DTX; 15–1500 nm) also attenuated the aminopyridine‐sensitive component (IC50 93 ± 4 nm). At the highest DTX concentration, depression of the K+‐current was incomplete, the residual K+ current being reduced by 3,4‐DAP (0.1 to 5 μm).

These results indicate the presence of two aminopyridine‐sensitive K+ currents in this preparation distinguished by their susceptibility to DTX.
Activation by Phoneutria nigriventer (armed spider) venom of tissue kallikrein‐kininogen‐kinin system in rabbit skin in vivo
Authors: Marangoni Rossana A. , Antunes Edson , Brain Susan D. , de Nucci Gilberto
Published: July 2012

The purpose of the present study was to investigate the mechanisms by which venom from Phoneutria nigriventer spider induces increases in vascular permeability in rabbit skin.

Local oedema formation, in response to intradermally‐injected agents, was measured in male New Zealand white rabbits as the local accumulation of i.v. injected 125I‐labelled human serum albumin into skin sites.

Phoneutria nigriventer venom (10–30 μg/site) increased vascular permeability, which was inhibited by trasylol (10 μg/site) and the bradykinin B2 receptor antagonists d‐Arg,[Hyp3,Thi5,8,d‐Phe7]‐BK (3 nmol/site) and Hoe 140 (0.3 nmol/site). In addition, the oedema induced by the venom was potentiated by the kinase II inhibitor, captopril (1 nmol/site). The lipoxygenase inhibitor, BWA4C (10 nmol/site) and the PAF antagonist, WEB 2086 (100 nmol/site) had no effect on the venom‐induced increase in vascular permeability.

Incubation of rabbit plasma with Phoneutria nigriventer venom in vitro did not cause bradykinin formation. Further, the plasma kallikrein inhibitor, soybean trypsin inhibitor (10 μg/site), had no effect on the venom‐induced increase in vascular permeability in rabbit skin.

These results indicate that the oedema produced by Phoneutria nigriventer venom is dependent on the activation of the tissue kallikrein‐kinin system.
Differential localization of endothelin ETa and ETB binding sites in human placenta
Authors: Rutherford Richard A.D. , Wharton John , McCarthy Andrew , Gordon Lee , Sullivan Mark H.F. , Elder Murdoch G. , Polak Julia M.
Published: July 2012

The localization and differential distribution of endothelin (ET) receptor subtypes (ETA and ETB) was investigated in sections of human placenta by use of quantitative in vitro autoradiography and receptor selective ligands.

Specific, high density [125I]‐ET‐1 binding sites were localized to the decidua and foetal membranes as well as to arteries and veins in the chorionic plate and throughout the villous tree. Moderate to low density binding was found in the extravillous and villous trophoblast respectively.

[125I]‐ET‐1 binding sites exhibited a rank order of inhibition by unlabelled peptide sequences (ET‐1 > ET‐3 >[Ala3,11,18Nle7]‐ET‐1 > BQ123 ≥ sarafotoxin 6c). However, in contrast to the monophasic inhibition curve of ET‐1, the other sequences produced a significantly better fit to a two component inhibition curve suggesting the presence of a heterogeneous population of ET binding sites.

ETA and ETB receptors were distinguished by competitive inhibition of [125I]‐ET‐1 binding with increasing concentrations of unlabelled ET‐3, [Ala3,11,18Nle7]‐ET‐1, sarafotoxin 6c and BQ123 and by incubating sections with the ETB agonist, [125I]‐BQ3020. ET receptor subtypes exhibited a differential distribution in the placenta. ETA type binding sites predominated (∼ 80% of the total) on veins and arteries in the chorionic plate. Veins in stem villi, blood vessels in distal regions of the villous tree and decidual cells displayed a high density (∼ 60–70% of the total) of the ETB receptor subtype.

No difference was detected in either the relative density of [125I]‐ET‐1 binding sites or the proportion of ETA to ETB sites in placentae from pregnancies complicated by pre‐eclampsia compared with normal term controls.

ET may have a local autocrine or paracrine role in the placenta, acting via specific receptors to influence foetoplacental blood flow and other aspects of placental function.
Kinetic study of the tubular dopamine outward transporter in the rat and dog kidney
Authors: Soares‐da‐Silva P.
Published: July 2012

The present study has determined the kinetic characteristics of the outflow of dopamine of renal origin in slices of rat and dog renal cortex loaded with exogenous l‐dihydroxyphenylalanine (l‐DOPA 5 to 5000 μm).

In both dog and rat renal tissues the production of dopamine was found to be dependent on the concentration of l‐DOPA used and reached its maximum at 2500 μm l‐DOPA. The decarboxylation of l‐DOPA in rat cortical slices (16.4 ± 2.6 to 1479.2 ± 85.2 nmol g−1) was 6 fold that in the dog (2.2 ± 0.4 to 252.1 ± 21.2 nmol g−1). In the rat kidney a large amount (approximately 50%) of the dopamine (5.2 ± 0.6 to 743.4 ± 58.3 nmol g−1) was found to escape into the incubation medium, whereas in dog renal slices the amount of newly‐formed dopamine escaping into the incubation medium (0.7 ± 0.2 to 46.5 ± 9.3 nmol g−1) was less than 25% of the total amount of the amine formed.

The application of the Michaelis‐Menten equation to the net transport of newly‐formed dopamine has allowed the identification of a saturable (carrier‐mediated transfer) and a non‐saturable component (diffusion). The Vmax (nmol g−1 15 min−1) and Km (nm) values for the saturable component were, respectively, 340 ± 41 and 396 ± 45 in the rat kidney and 112 ± 16 and 319 ± 35 in the dog kidney. In both rat and dog renal tissues, the magnitude of the non‐saturable component was found to be of minor importance up to a concentration of 250 nmol g−1 of dopamine to be transported. At high concentrations of the amine (greater then 250 nmol g−1), only attainable in rat kidney slices, most of the dopamine was found to leave the compartment where the synthesis did occur through a non‐saturable transport system.

In conclusion, the results presented here show that the outflow of newly‐formed dopamine in both dog and rat kidney slices loaded with exogenous l‐DOPA follows Michaelis‐Menten kinetics with a saturable component and a non‐saturable one, the latter assuming particular importance only at higher concentrations of the amine.
Impairment of pulmonary‐artery endothelium‐dependent relaxation in chronic obstructive lung disease is not due to dysfunction of endothelial cell membrane receptors nor to l‐arginine deficiency
Authors: Dinh‐Xuan A.T. , Pepke‐Zaba J. , Butt A.Y. , Cremona G. , Higenbottam T.W.
Published: July 2012

Endothelium‐dependent relaxation mediated by endothelium‐derived relaxing factor (EDRF) or nitric oxide (NO), is impaired in pulmonary arteries (PA) of hypoxic patients with chronic obstructive lung disease (COLD). To determine the mechanisms responsible for this impairment, we compared the response of rings of isolated PA from 12 COLD patients and 8 controls to the endothelium‐dependent vasodilators acetylcholine (ACh), adenosine diphosphate (ADP), and the calcium ionophore, A23187. The response of PA rings to the endothelium‐independent nitro‐vasodilator sodium nitroprusside (SNP) was also studied in both groups. The PA rings had been pre‐contracted by the α‐adrenoceptor agonist phenylephrine (PE).

Endothelium‐dependent relaxation was significantly reduced in PA rings from COLD patients as compared with controls when tested with ACh (37.8 ± 8.8% vs 73.4 ± 7.9%), ADP (38.4 ± 6.7% vs 80 ± 5.6%), and the calcium ionophore, A23187 (35.8 ± 6.1% vs 87 ± 6.6%). Relaxation with SNP was, however, significantly greater in PA rings from COLD patients (99.4 ± 0.6% vs 90.3 ± 3.1%), as was the contractile response to PE (1.91 ± 0.21 g vs 1.33 ± 0.15 g). Pretreatment with the specific inhibitor of NO formation, NG‐monomethyl‐l‐arginine (l‐NMMA; 10−4 m) significantly reduced the relaxation to ACh in all PA rings. This inhibition could be reversed by l‐arginine (10−3 m), the substrate for NO synthesis. Pretreatment with l‐arginine alone, however, did not restore the impaired endothelium‐dependent relaxation of PA rings from COLD patients.

We conclude that EDRF (NO) production is impaired in PA rings from COLD patients and that this impairment is neither due to endothelial receptors dysfunction nor a defect of l‐arginine availability and/or transport. Our hypothesis is that the abnormality must lie within the biosynthesis pathway of NO from l‐arginine, possibly involving the endothelial enzyme cell, NO synthase, the normal function of which might be altered by chronic hypoxia.
Differential properties of type I and type II benzodiazepine receptors in mammalian CNS neurones
Authors: Yakushiji Takashi , Shirasaki Tetsuya , Munakata Mitsutoshi , Hirata Akira , Akaike Norio
Published: July 2012

The effects of benzodiazepine receptor (BZR) partial agonists, Y‐23684 and CL218,872, were compared with its full agonist, diazepam, on γ‐aminobutyric acid (GABA)‐induced Cl− current (ICl) in acutely dissociated rat cerebral cortex (CTX), cerebellar Purkinje (CPJ) and spinal ventral horn (SVH) neurones, by the whole‐cell mode patch‐clamp technique.

The GABA‐induced responses were essentially the same in both SVH and CPJ neurones, but the KD value of the GABA response in CTX neurone was lower than those in the other two brain regions.

Enhancement of the GABA response by the two partial agonists was about one‐third of that by diazepam in the SVH neurones (where type II subtype of BZR, BZ2, is predominant), whereas these partial agonists potentiated the GABA response as much as diazepam in CPJ neurones (where the type I subtype of BZR, BZ1, is predominant). In CTX neurones where both type I and II variants are expressed, the augmentation ratio of the GABA response by diazepam was between the values in CPJ and SVH neurones.

In concentration‐response relationships of BZR partial agonists, the threshold concentrations, KD values and maximal augmentation ratio of the GABA response were similar in all CTX, CPJ and SVH neurones. Also, in all preparations, the threshold concentration and KD values of diazepam action were 10 fold less than those induced by partial agonists.

All BZR agonists shifted the concentration‐response relationship for GABA to the left without changing the maximum current amplitude, indicating that activation of both BZ1 and BZ2 increase the affinity of the GABAA receptor for GABA.

The results are important in clarifying the mechanism of anxiety and might explain the anxioselectivity of BZR partial agonists.
Caffeine‐induced decreases in the inward rectifier potassium and the inward calcium currents in rat ventricular myocytes
Authors: Varro Andras , Hester Simon , Papp Julius Gyula
Published: July 2012

The effects of high (20 mm) concentrations of caffeine were studied on the transmembrane voltage and currents in rat single ventricular myocytes by the whole cell configuration of the patch clamp technique. Rapid application of caffeine released Ca2+ from the sarcoplasmic reticulum and induced a Ni2+‐sensitive transient inward current with concomitant change of the transmembrane voltage from −72.6 ± 0.4 to −68.0 ± 0.6 mV (n = 4). Maintained application of caffeine lengthened the action potential duration (APD90) from 66.7 ± 16.9 to 135.1 ± 34.1 ms (n = 4) and depressed the amplitude of both the inward rectifier potassium and the inward calcium currents. It is concluded that these effects of caffeine should be recognized when it is used as a tool to study electromechnical coupling.
Different effects of aspirin on blood pressure of spontaneously hypertensive rats (SHR) with high and spontaneously low levels of blood pressure
Authors: Schirner Michael , Taube Christel
Published: July 2012

Spontaneously hypertensive rats (SHR) of the Okamoto strain with blood pressure above 161 mmHg and SHR with blood pressure levels of less than 160 mmHg were treated with oral doses of aspirin (100 mg kg−1) for three days. Whereas the blood pressure of SHR with blood pressure above 161 mmHg was decreased by aspirin, the blood pressure of SHR below 160 mmHg was increased by aspirin. The extent and direction of blood pressure change by aspirin was strongly correlated with the blood pressure of SHR before treatment (r = −0.88). The effect of aspirin supports an important role for endogenous prostanoids in the regulation of blood pressure of SHR.
Bronchodilatation of guinea‐pig perfused bronchioles induced by the H3‐receptor for histamine: role of epithelium
Authors: Burgaud J.L. , Oudart N.
Published: July 2012

The influence of epithelium on the effects of H3‐histamine receptor agonist (R)α‐methylhistamine [(R)α‐MeHist] on airways was investigated on the guinea‐pig perfused bronchioles.

In preparations under resting tone, removal of the bronchiolar epithelium or treatment with the cyclo‐oxygenase inhibitor indomethacin (10−5 m) increased the constriction induced by histamine and acetylcholine in a concentration‐dependent manner without an alteration of the K+‐induced contraction.

In this preparation (R)α‐MeHist induced a concentration‐dependent bronchodilatation which was antagonized in a competitive manner by thioperamide (an H3‐antagonist) with a pA2 value of 8.6.

This bronchodilatation was reversed to a low concentration‐dependent constriction after either removal of the epithelium or treatment with indomethacin (10−5 m) but was unaffected by both 10−5 m tranylcypromine (an inhibitor of PGI2 synthesis) and 5 × 10−5 m NG‐nitro‐l‐arginine methyl ester (an inhibitor of NO synthesis).

It is suggested that, in guinea‐pig perfused bronchioles (R)α‐MeHist induces an epithelium‐dependent relaxation via the release of metabolite(s) of arachidonic acid.
Demonstration of the suitability of CGP 12177 for in vivo studies of β‐adrenoceptors
Authors: Law Marilyn P.
Published: July 2012

Positron emission tomography (PET) with appropriate radioligands offers the possibility of studying receptors non‐invasively in man. The suitability of CGP 12177, a hydrophilic non‐selective β‐adrenoceptor antagonist which can be labelled with the positron emitter 11C, as a ligand for in vivo studies of β‐adrenoceptors was assessed in rats.

[3H]‐CGP 12177 was injected into the tail veins of restrained conscious rats. Serial blood samples were taken from tail arteries to determine clearance from plasma. Rats were killed and tissues removed to determine tissue uptake. Radioactivity was assessed by liquid scintillation counting.

The uptake of (−)‐[3H]‐CGP 12177 in various tissues was compared to that of (±)‐[3H]‐CGP 12177. Maximum tissue:plasma ratios obtained for the (−)‐enantiomer in lung, heart and liver were 170, 42 and 13 compared with 60, 15 and 12 for the racemate. Prior injection of excess unlabelled (±)‐CGP 12177 blocked the uptake of both (−)‐ and (±)‐[3H]‐CGP 12177 in lung and heart but not liver, tissue:plasma ratios for both tracers being reduced to 7, 3 and 7 respectively.

Clearance of (−)‐[3H]‐CGP 12177 from plasma was rapid during the first 5 min but showed only small changes during 5 to 90 min. Uptake in lung and heart reached a maximum at 1 to 5 min and showed a slow decrease during 5–90 min. Prior injection of unlabelled (±)‐CGP 12177 reduced uptake in lung and heart to 10% and 20% respectively. Injection of unlabelled (±)‐CGP 12177 at 15 min displaced ∼75% of the radioactivity by 90 min.

(±)‐Propranolol had a similar effect to that of unlabelled (±)‐CGP 12177. Prior injection reduced uptake of radioactivity in lung and heart to 15% and 20% respectively. Injection of (±)‐propranolol at 15 min displaced ∼60% of the radioactivity by 90 min indicating that the tracer binds to β‐adrenoceptor sites in vivo.

In vivo saturation curves, obtained by injection of (−)‐[3H]‐CGP 12177 with increasing amounts of unlabelled (−)‐CGP 12177, gave values of Bmax for lung of ∼45 pmol per g wet weight of tissue and for heart of ∼ 6 pmol per g wet weight of tissue. KD could only be expressed as nmol injected per kg body weight, that for lung (2.5 nmol kg−1) being greater than that for heart (1.3 nmol kg−1).

Competition studies carried out by co‐injecting (−)‐[3H]‐CGP 12177 with unlabelled (±)‐CGP 12177 or (−)‐propranolol gave similar values for Bmax (lung 44 pmol g−1, heart 6 pmol g−1,). Values of KD for (±)‐CGP 12177 (lung 4.7 pmol kg−1, heart 2.6 pmol kg−1) were approximately twice those for (−)‐CGP 12177. Values of KD for (−)‐propranolol (lung 38 nmol kg−1, heart 104 nmol kg−1) were greater.

The results show that (−)‐[3H]‐CGP 12177 is a suitable ligand for assessing β‐adrenoceptors in vivo.
Pharmacological characterization of acetylcholine‐stimulated [35S]‐GTPγS binding mediated by human muscarinic m1–m4 receptors: antagonist studies
Authors: Lazareno S. , Birdsall N.J.M.
Published: July 2012

We have used dose‐ratio analysis to estimate functionally the affinity constants (pKb) and Schild slope factors of a range of selective or atypical antagonists at human muscarinic m1–m4 receptors.

The functional response was the stimulation by acetylcholine of [35S]‐GTPγS binding to membranes from Chinese hamster ovary (CHO) cells stably expressing individual receptor subtypes.

A novel experimental design and analysis was used which allowed the estimation of affinity and Schild slope factor from a single antagonist inhibition curve, and the results were compared with other methods of analysis, both theoretically valid and invalid.

In general, the affinity estimates were very similar to previously reported values obtained in binding studies with animal tissues and cloned human receptors and the Schild slope factors were close to unity.

These results demonstrate the validity of the assay and provide no evidence for species differences in antagonist affinity for muscarinic receptor subtypes.

The results confirm both the utility of himbacine in distinguishing between m1 and m4 receptors and a previously reported modest m4‐selectivity for tropicamide and secoverine.

The cholinesterase inhibitor, tacrine (THA), had a potency profile similar to that of gallamine but with less selectivity. Its affinity could not be determined since it had Schild slope factors of about 2 at all subtypes.

o‐Methoxy‐sila‐hexocyclium had only a modest selectivity for the ml subtype.