bluuebunny/crossref_metadata_2025 huggingface dataset:
Gabapentin potentiation of the antiepileptic efficacy of vigabatrin in an in vitro model of epilepsy
Authors: Lücke A , Mußhoff U , Köhling R , Osterfeld M , Mayer T , Wolf P , Schütte W , Speckmann E‐J
Published: January 2005

An enhancement of promoted release of γ‐aminobutyric acid (GABA) and a change in GABA‐metabolism have been suggested as mechanisms of action of gabapentin. Vigabatrin is supposed to act mainly via inhibition of GABA‐transaminase but it also interferes with GABA‐release and GABA‐uptake. On the basis of these mechanisms of action, a pharmacodynamic interaction of the two antiepileptic drugs could be supposed which might be of relevance in the sense of a rational polypharmacy.

To address the aforementioned hypothesis, experiments were carried out on hippocampal slices (n=107) of guinea‐pigs (n=70). Epileptiform field potentials (e.f.p.) were induced by omission of magnesium from the bath solution and recorded in the stratum pyramidale of the CA3 region. Gabapentin (30–600 μM; 5.1–102.72 μg ml−1), vigabatrin (50–200 μM, 6.45–25.8 μg ml−1) and the GABAA‐receptor antagonist bicuculline (100 μM) were added to the bath solution for 3 h.

Gabapentin, in concentrations up to 600 μM, failed to decrease the repetition rate or duration of e.f.p. (n=19). However, vigabatrin, evoked a dose‐dependent reduction of the repetition rate of e.f.p. For a concentration of 100 μM (12.9 μg ml−1) there was a reduction down to 48±5% (mean±s.e.mean) of the initial value within 3 h (n=11). With simultaneous administration of vigabatrin (100 μM) and gabapentin (60 μM) for 3 h (n=15), the repetition rate of e.f.p. decreased down to 8±3%, which is significantly different from the values obtained after administration of 100 μM vigabatrin alone (P<0.0001). Both, the antiepileptic effect of vigabatrin alone and the enhancement by gabapentin were blocked by the GABAA‐receptor antagonist bicuculline (100 μM, n=16).

These results demonstrate that gabapentin is able to augment the antiepileptic effects of vigabatrin significantly. It is possible that a change in the GABA‐release machinery is induced by vigabatrin which then can be augmented by gabapentin.
P2Y1‐receptors in human platelets which are pharmacologically distinct from P2YADP‐receptors
Authors: Fagura M S , Dainty I A , McKay G D , Kirk I P , Humphries R G , Robertson M J , Dougall I G , Leff P
Published: January 2005

In the present study we have classified the receptor(s) mediating increases in intracellular calcium concentration ([Ca2+]i) in human washed platelets and compared the pharmacological profile obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y1‐receptor.

The P2Y1‐receptor antagonist, adenosine‐3′‐phosphate‐5′‐phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar affinities (pKB of 5.8 and 6.0, respectively).

The selective P2YADP antagonist, AR‐C66096, exhibited partial agonism in the Jurkat cells with an affinity (pKA) of 4.9. This value is consistent with its known P2Y1‐receptor activity. In platelets, AR‐C66096 at a concentration (0.1 μM) approximately 100 fold greater than its known P2YADP receptor affinity, had no effect on ADP‐induced increases in [Ca2+]i.

The ability of adenine nucleotide analogues to elevate [Ca2+]i in the Jurkat cells was also determined. The rank order of agonist potency (p[A]50) was: 2‐MeSADP (8.3)>2‐ClATP (7.8)>ADP (7.5)=2‐MeSATP (7.4)>ATPγS (6.5)>ATP (6.2), with ATP appearing to be a partial agonist.

The same rank order of potency was observed when similar experiments were performed in platelets. However, the absolute potencies of all the agonists and the intrinsic activities of both ATPγS and ATP were lower in platelets.

The operational model of agonism was used to test whether the agonist concentration‐effect profiles obtained in these two cell types could be explained on the basis of differences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y1‐receptor system.

The hypothesis that the observed partial agonist behaviour of ATP was the result of receptor activation by contaminating ADP with concomitant receptor blockade by ATP, was tested in the platelet system. This hypothesis was supported by a theoretical analysis, which yielded an affinity value for ATP similar to that obtained previously at P2Y1‐receptors.

In summary, the results of this study indicate that human washed platelets contain P2Y1‐receptors which mediate increases in [Ca2+]i and that this receptor population is pharmacologically distinct from P2YADP‐receptors.
G‐protein activation at 5‐HT1A receptors by the 5‐ht1F ligand LY334370 in guinea‐pig brain sections and recombinant cell lines
Authors: Dupuis Delphine S , Colpaert Francis C , Pauwels Petrus J
Published: January 2005

G‐protein activation by the 5‐ht1F receptor agonist 5‐(4‐fluorobenzoyl)amino‐3‐(1‐methylpiperidin‐4‐yl)‐1H‐indole fumarate (LY334370) was investigated by use of autoradiography of receptor‐activated G‐proteins in guinea‐pig brain sections and [35S]‐GTPγS binding responses in cell lines stably expressing human 5‐HT1A (h 5‐HT1A) receptors.

LY334370 (10 μM) caused little or no stimulation of [35S]‐GTPγS binding in guinea‐pig brain regions enriched in 5‐ht1F binding sites (e.g., claustrum, caudate/putamen and thalamic nuclei), as identified by labelling with 10 nM [3H]‐sumatriptan plus 10 nM 5‐carboxamidotryptamine (5‐CT).

Application of LY334370 (10 μM) to guinea‐pig brain sections resulted in an increase of [35S]‐GTPγS binding in hippocampus (123±17%), lateral septum (58±14%), dorsal raphe (57±10%), entorhinal (37±11%) and cingulate cortex (28±10%). This distribution fits with the G‐protein activation mediated by 5‐HT1A receptors as found with lisuride (10 μM), and labelling of 5‐HT1A receptors by 140 pM [125I]‐4‐(2′‐methoxy‐phenyl)‐1‐[2′‐(n‐2′′‐pyridinyl)‐p‐iodobenzamido]‐ethyl‐piperazine (p‐MPPI).

The LY334370‐mediated [35S]‐GTPγS response was antagonized by the selective, silent 5‐HT1A receptor antagonist N‐[2‐[4‐(2‐methoxyphenyl)1‐piperazinyl]ethyl]‐N‐(2‐pyridinyl)cyclohexanecarboxamide (WAY100635, 1 μM) in each of the brain structures investigated. The distribution pattern of the [35S]‐GTPγS binding response and the antagonist profile suggest that the LY334370‐induced response in guinea‐pig brain is mediated by 5‐HT1A receptors.

The maximal LY334370‐induced [35S]‐GTPγS binding response (83 to 94%) in membranes of recombinant C6‐glial/h 5‐HT1A and HeLa/h 5‐HT1A cells was close to that of 5‐HT, suggesting LY334370 to exert high intrinsic activity at h 5‐HT1A receptors.

In conclusion, in guinea‐pig brain sections and recombinant cell lines the 5‐ht1F receptor agonist LY334370 causes G‐protein activation that is mediated by 5‐HT1A receptors. Caution should be taken when employing this ligand as a putative selective 5‐ht1F agonist.
Modulation of cell adhesion molecule expression and function on human lung microvascular endothelial cells by inhibition of phosphodiesterases 3 and 4
Authors: Blease Kate , Burke‐Gaffney Anne , Hellewell Paul G
Published: January 2005

Expression of cell adhesion molecules (CAM) on the lung microvascular endothelium is believed to play a key role in the recruitment of leukocytes in pulmonary inflammation. Moreover, regulation of CAM expression may be an important mechanism through which this inflammation may be controlled. Experimental evidence has suggested that combined phosphodiesterase (PDE) 3 and 4 inhibitors increase cyclic AMP levels within cells greater than inhibition of either isoenzyme alone. In the present study we assessed the effect of combinations of rolipram (PDE4 inhibitor), ORG 9935 (PDE3 inhibitor) and salbutamol (β‐agonist) on CAM expression and neutrophil or eosinophil adhesion to human lung microvascular endothelial cells (HLMVEC).

Tumour necrosis factor‐α (TNF‐α)‐induced intercellular adhesion molecule (ICAM)‐1, vascular cell adhesion molecule (VCAM)‐1 and E‐selectin expression were measured on HLMVEC monolayers at 6 h by a specific ELISA technique in the presence of different combinations of medium, rolipram, ORG 9935 and salbutamol.

Rolipram in combination with salbutamol, but neither agent alone, inhibited TNF‐α‐induced E‐selectin expression, whilst ICAM‐1 and VCAM‐1 expression were not affected. ORG 9935 had no significant effect on CAM expression alone. However, in combination with rolipram a syngergistic inhibition of VCAM‐1 and E‐selectin, but not ICAM‐1, expression was observed. No further inhibition was seen in the additional presence of salbutamol.

Neutrophil adhesion to TNF‐α‐stimulated (6 h) HLMVEC was mainly E‐selectin dependent in this model, as ENA2 an anti‐E‐selectin monoclonal antibody (mAb) abrogated neutrophil adhesion. Eosinophil adhesion was E‐selectin‐, ICAM‐1‐ and VCAM‐1‐dependent, as assessed by the inhibitory activity of ENA2 and the ability of a mAb to the ICAM‐1 ligand, CD18, and a mAb to the VCAM‐1 ligand, VLA4, to attenuate adhesion.

Rolipram in the presence of salbutamol or ORG 9935 significantly inhibited neutrophil adherence to TNF‐α‐stimulated HLMVEC. Eosinophil adherence to monolayers was inhibited only when HLMVEC were activated in the presence of rolipram and ORG 9935.

Collectively, the findings presented in this manuscript suggest that inhibition of PDE4 with appropriate activation of adenylate cyclase is sufficient to inhibit induction of E‐selectin expression on HLMVEC to a level that has functional consequences for neutrophil adhesion. In contrast, combined inhibition of PDE3 and 4 isoenzymes is necessary to inhibit VCAM‐1 and to have inhibitory effects on eosinophil adhesion to activated HLMVEC. Upregulation of ICAM‐1 expression on HLMVEC does not appear to be modulated by PDE3 and 4 inhibition. These data may have implications for the use of selective PDE4 inhibitors in lung inflammation.
Different effects of gadolinium on IKR, IKS and IK1 in guinea‐pig isolated ventricular myocytes
Authors: Pascarel C , Hongo K , Cazorla O , White E , Le Guennec J‐Y
Published: January 2005

Using the whole cell configuration of the patch clamp technique, we studied the potential blocking effects of gadolinium (1 μM to 1 mM) on potassium currents: IKR, IKS and IK1. The study was performed on guinea‐pig isolated ventricular myocytes.

The background current, IK1 was insensitive to Gd3+. Thus, we found that no obvious screening of surface charges was visible with concentrations of Gd3+ up to 100 μM.

By use of three different protocols: tail currents fitting, analysis of envelope of tails and electrophysiological dissection, we found that IKR was the only component of IK that was sensitive to Gd3+. The sensitivity was apparently different depending on the protocol used.

Comparison of the results obtained with the different protocols revealed that the rapid component of IKR is more sensitive to Gd3+ than the slow one.

Of the different protocols used to distinguish between IKR and IKS, the electrophysiological dissection seems to be the more accurate.
Investigation of the actions and antagonist activity of some polyamine analogues in vivo
Authors: Doyle Karen M , Shaw Graham G
Published: January 2005

The ability of three putative polyamine antagonists to antagonize behavioural changes induced by spermine was assessed.

Injection of an excitotoxic dose of spermine (100 μg, i.c.v.) in mice results in the development of a characteristic behavioural profile, which has two temporally distinct phases. The early events include clonic convulsions, and the later, more general excitation, includes tremor and culminates in the development of a fatal tonic convulsion.

Co‐administration of arcaine (25 μg, i.c.v.) potentiated the early phase effects after spermine injection, but antagonized the development of spermine‐induced tonic convulsions. A larger dose of arcaine (50 μg, i.c.v.) given alone resulted in the development of spermine‐like body tremor and convulsions. It therefore appears that arcaine is not a pure polyamine antagonist in vivo, but may be a partial agonist.

Similarly, 1,10‐diaminodecane appeared to act as a partial agonist in vivo, although it was less potent than arcaine.

In contrast, diethylenetriamine (DET) effectively inhibited the development of the early effects of spermine, but was ineffective against the spermine‐induced CNS excitation and tonic convulsions.

It is concluded that none of the putative polyamine antagonists tested behaved as effective polyamine antagonists in vivo, although each produced some antagonism.
Effect of inhibition of tyrosine phosphatases on voltage‐operated calcium channel currents in rabbit isolated ear artery cells
Authors: Wijetunge S , Lymn J S , Hughes A D
Published: January 2005

The effect of increasing cellular tyrosine phosphorylation by inhibiting endogenous tyrosine phosphatases was examined on voltage‐operated calcium channel currents in vascular smooth muscle cells.

In single ear artery smooth muscle cells of the rabbit, studied by the whole cell voltage clamp technique, intracellular application of the tyrosine phosphatase inhibitors, sodium orthovanadate (100 μM) and peroxyvanadate (100 μM orthovanadate+1 mM H2O2) increased voltage‐operated calcium channel currents by 56% and 83%, respectively.

Bath application of two other membrane permeant tyrosine phosphatase inhibitors, phenylarsine oxide (100 μM) and dephostatin (50 μM) also increased voltage‐operated calcium channel currents by 48% and 52%, respectively.

The selective tyrosine kinase inhibitor, tyrphostin‐23 (100 μM) reduced calcium channel currents by 41%. Pre‐incubation with tyrphostin‐23 abolished the effects of peroxyvanadate, phenylarsine oxide and dephostatin on calcium channels.

Western blot analysis of rabbit ear artery cell lysates showed increased tyrosine phosphorylation of several endogenous proteins following treatment with peroxyvanadate.

These results indicate that a number of structurally dissimilar inhibitors of tyrosine phosphatases increase voltage‐operated calcium channel currents in arterial smooth muscle cells presumably due to increased tyrosine phosphorylation.
Inhibition of NMDA‐gated ion channels by the P2 purinoceptor antagonists suramin and reactive blue 2 in mouse hippocampal neurones
Authors: Peoples Robert W , Li Chaoying
Published: January 2005

The action of suramin and reactive blue 2 on N‐methyl‐D‐aspartate (NMDA)‐activated ion current was studied in mouse hippocampal neurones in culture by use of whole‐cell patch‐clamp recording.

Suramin and reactive blue 2 inhibited steady‐state current activated by 25 μM NMDA with IC50 values of 68 and 11 μM, respectively.

Reactive blue 2 produced a gradual decline of NMDA‐activated current to a steady‐state, but this slow onset was not an indication of use‐dependence, as it could be eliminated by exposure to reactive blue 2 before NMDA application. In addition, NMDA‐activated current recovered completely from inhibition by reactive blue 2 in the absence of agonist.

The slow onset of inhibition by reactive blue 2 was not apparently due to an action at an intracellular site, as inclusion of 250 μM reactive blue 2 in the recording pipette did not alter inhibition by 25 μM reactive blue 2 applied externally.

Reactive blue 2 and suramin inhibited NMDA‐gated channels in a voltage‐independent manner.

Reactive blue 2, 25 μM, decreased the maximal response to NMDA from 1441 to 598 pA without changing its EC50. In contrast, 75 μM suramin increased the EC50 for NMDA from 13 to 35 μM, and decreased the maximal response to NMDA from 1822 to 1498 pA. Schild analysis of suramin inhibition of NMDA‐activated current yielded a nonlinear plot.

Both agents decreased the maximal response to glycine without altering its EC50.

Suramin and reactive blue 2 appear to inhibit NMDA receptor‐channels in a manner that is noncompetitive with respect to both NMDA and glycine. However, inhibition by suramin differed from that by reactive blue 2, in that suramin significantly increased the EC50 of NMDA.
Characterization of Ro 04‐6790 and Ro 63‐0563: potent and selective antagonists at human and rat 5‐HT6 receptors
Authors: Sleight Andrew J , Boess Frank G , Bös Michael , Levet‐Trafit Bernard , Riemer Claus , Bourson Anne
Published: January 2005

This study describes the in vitro characterization of two potent and selective 5‐HT6 receptor antagonists at the rat and human recombinant 5‐HT6 receptor.

In binding assays with [3H]‐LSD, 4‐amino‐N‐(2,6 bis‐methylamino‐pyrimidin‐4‐yl)‐benzene sulphonamide (Ro 04‐6790) and 4‐amino‐N‐(2,6 bis‐methylamino‐pyridin‐4‐yl)‐benzene sulphonamide (Ro 63‐0563) had mean pKi values ±s.e.mean at the rat 5‐HT6 receptor of 7.35±0.04 and 7.83±0.01, respectively and pKi values at the human 5‐HT6 receptor of 7.26±0.06 and 7.91±0.02, respectively.

Both compounds were found to be over 100 fold selective for the 5‐HT6 receptor compared to 23 (Ro 04‐6790) and 69 (Ro 63‐0563) other receptor binding sites.

In functional studies, neither compound had any significant effect on basal levels of cyclicAMP accumulation in Hela cells stably expressing the human 5‐HT6 receptor, suggesting that the compounds are neither agonists nor inverse agonists at the 5‐HT6 receptor. However, both Ro 04‐6790 and Ro 63‐0563 behaved as competitive antagonists with mean ±s.e.mean pA2 values of 6.75±0.07 and 7.10±0.09, respectively.

In rats habituated to observation cages, Ro 04‐6790 produced a behavioural syndrome similar to that seen following treatment with antisense oligonucleotides designed to reduce the expression of 5‐HT6 receptors. This behavioural syndrome consisted of stretching, yawning and chewing.

Ro 04‐6790 and Ro 63‐0563 represent valuable pharmacological tools for the identification of 5‐HT6 receptors in natural tissues and the study of their physiological function.
Effects of the 5‐lipoxygenase activating protein inhibitor MK886 on voltage‐gated and Ca2+‐activated K+ currents in rat arterial myocytes
Authors: Smirnov Sergey V , Knock Greg A , Aaronson Philip I
Published: January 2005

The effects on the voltage‐gated (IK) and Ca2+ activated (IK,Ca) K+ currents in rat arterial myocytes of the 5‐lipoxygenase activating protein (FLAP) inhibitor MK886, and its inactive analogue L583,916 were evaluated.

In rat pulmonary arterial myocytes (RPAMs), MK886 caused a concentration‐dependent reduction of the IK, with little obvious change in the kinetics of the current. Half maximal current block was observed at 75 nM MK886.

MK886 application led to a concentration‐dependent increase in the amplitude of the TEA‐sensitive IK,Ca current and single channel activity in RPAMs in whole cell and inside‐out configurations, respectively. The threshold concentration for this effect was approximately 300 nM and a maximal 4–5 fold increase was observed at 10 μM MK886. MK886 also increased IK,Ca in rat mesenteric arterial myocytes (RMAMs).

L538,916, an analogue of MK886 which does not block FLAP, had no effect on either IK or IK,Ca at a concentration of 10 μM.

Leukotriene C4 (100 nM) had no effect on either IK or IK,Ca in RPAMs. MK886 produced its usual increase in IK,Ca and also blocked IK, in the presence of leukotriene C4. Similarly, leukotriene E4 (100 nM) did not alter the amplitude of IK. Also, the nonselective leukotriene receptor antagonist ICI 198,615 (3 μM) did not affect IK in RPAMs, and did not affect the response to MK886.

Arachidonic acid (10 μM) enhanced IK,Ca in both RPAMs and RMAMs.

The results show that MK886 markedly affects both IK and IK,Ca in a manner similar to that of arachidonic acid and independent of the endogenous production of leukotrienes. It is therefore possible that MK886, which is thought to compete with arachidonic acid for its binding to FLAP, may similarly occupy arachidonic acid binding sites on these K+ channels, and mimic its effects. Alternatively, MK886 might act via non‐selective effects on other arachidonic acid metabolites which could modify K+ channel function.
Effects of κ‐opioid receptor stimulation in the heart and the involvement of protein kinase C
Authors: Bian Jin‐Song , Wang Hong‐Xin , Zhang Wei‐Min , Wong Tak‐Ming
Published: January 2005

The role of protein kinase C (PKC) in mediating the action of κ‐receptor stimulation on intracellular Ca2+ and cyclic AMP production was determined by studying the effects of trans‐(±)‐3,4‐dichloro‐N‐methyl‐N‐(2‐[1‐pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective κ‐receptor agonist, and phorbol 12‐myristate 13‐acetate (PMA), a PKC agonist, on the electrically‐induced [Ca2+]i transient and forskolin‐stimulated cyclic AMP accumulation in the presence and absence of a PKC antagonist, staurosporine or chelerythrine, in the single rat ventricular myocyte.

U50,488H at 2.5–40 μM decreased both the electrically‐induced [Ca2+]i transient and forskolin‐stimulated cyclic AMP accumulation dose‐dependently, effects which PMA mimicked. The effects of the κ‐agonist, that were blocked by a selective κ‐antagonist, nor‐binaltorphimine, were significantly antagonized by the PKC antagonists, staurosporine and/or chelerythrine. The results indicate that PKC mediates the actions of κ‐receptor stimulation.

To determine whether the action of PKC was at the sarcoplasmic reticulum (SR) or not, the [Ca2+]i transient induced by caffeine, that depletes the SR of Ca2+, was used as an indicator of Ca2+ content in the SR. The caffeine‐induced [Ca2+]i transient was significantly reduced by U50,488H at 20 μM. This effect of U50,488H on caffeine‐induced [Ca2+]i transient was significantly attenuated by 1 μM chelerythrine, indicating that the action of PKC involves mobilization of Ca2+ from the SR. When the increase in IP3 production in response to κ‐receptor stimulation with U50,488H in the ventricular myocyte was determined, the effect of U50,488H was the same in the presence and absence of staurosporine, suggesting that the effect of PKC activation subsequent to κ‐receptor stimulation does not involve IP3. The observations suggest that PKC may act directly at the SR.

In conclusion, the present study has provided evidence for the first time that PKC may be involved in the action of κ‐receptor stimulation on Ca2+ in the SR and cyclic AMP production, both of which play an essential role in Ca2+ homeostasis in the heart.
Role of hyperthermia in the protective action of clomethiazole against MDMA (‘ecstasy’)‐induced neurodegeneration, comparison with the novel NMDA channel blocker AR‐R15896AR
Authors: Colado M I , Granados R , O'Shea E , Esteban B , Green A R
Published: January 2005

The immediate effect of administration of 3,4‐methylenedioxymethamphetamine (MDMA or ‘ecstasy’) on rectal temperature and the effect of putative neuroprotective agents on this change has been examined in rats. The influence of the temperature changes on the long term MDMA‐induced neurodegeneration of cerebral 5‐hydroxytryptamine (5‐HT) nerve terminals was also examined.

The novel low affinity N‐methyl‐D‐aspartate (NMDA) receptor channel blocker AR‐R15896AR (20 mg kg−1, i.p.) given 5 min before and 55 min after MDMA (15 mg kg−1, i.p.) did not prevent the MDMA‐induced hyperthermia and did not alter either the MDMA‐induced neurodegenerative loss of 5‐HT and 5‐hydroxyindoleacetic acid (5‐HIAA) in cortex, striatum and hippocampus or loss of [3H]‐paroxetine binding in cortex 7 days later.

The neuroprotective agent clomethiazole (50 mg kg−1, i.p.) given 5 min before and 55 min after MDMA (15 mg kg−1) abolished the MDMA‐induced hyperthermic response and markedly attenuated the loss of 5‐HT, 5‐HIAA and [3H]‐paroxetine binding in the brain regions examined 7 days later.

When rats treated with MDMA plus clomethiazole were kept at high ambient temperature for 5 h post‐MDMA, thereby keeping their body temperature elevated to near that seen in rats given MDMA alone, the MDMA‐induced loss of 5‐HT, 5‐HIAA and [3H]‐paroxetine was still attenuated. However, the protection (39%) afforded by the clomethiazole administration was less than seen in rats kept at normal ambient temperature (75%).

These data support the proposals of others that NMDA receptor antagonists are neuroprotective against MDMA‐induced degeneration only if they induce hypothermia and further suggest that increased glutamate activity may not be involved in the neurotoxic action of MDMA.

These data further demonstrate that a proportion of the neuroprotective action of clomethiazole is due to an effect on body temperature but that, in addition, the compound protects against MDMA‐induced damage by an unrelated mechanism.
T‐type and L‐type calcium channel blockers exert opposite effects on renin secretion and renin gene expression in conscious rats
Authors: Wagner Charlotte , Krämer Bernhard K , Hinder Markus , Kieninger Martin , Kurtz Armin
Published: January 2005

This study aimed to investigate and to compare the effects of pharmacological T‐type calcium channel and of L‐type calcium channel blockade on the renin system. To this end, male healthy Sprague‐Dawley rats were treated with the T‐channel blocker mibefradil or with the L‐channel blocker amlodipine at doses of 5 mg kg−1, 15 mg kg−1 and 45 mg kg−1 per day for four days and their effects on plasma renin activity (PRA) and kidney renin mRNA levels were determined.

Whilst amlodipine lowered basal systolic blood pressure at 5 mg kg−1, mibefradil had no effect on basal blood pressure in the whole dose range examined. Amlodipine dose‐dependently induced up to 7 fold elevation of PRA and renin mRNA levels. Mibefradil significantly lowered PRA and renin mRNA levels at 5 mg kg−1 and moderately increased both parameters at a dose of 45 mg kg−1, when PRA and renin mRNA levels were increased by 100% and 30%, respectively. In primary cultures of renal juxtaglomerular cells neither amlodipine nor mibefradil (0.1–10 μM) changed renin secretion.

In rats unilateral renal artery clips (2K‐1C) mibefradil and amlodipine at doses of 15 mg kg−1 day−1 were equally effective in lowering blood pressure. In contrast mibefradil (5 mg kg−1 and 15 mg kg−1 day−1) significantly attenuated the rise of PRA and renin mRNA levels, whilst amlodipine (15 mg kg−1) additionally elevated the rise of PRA and renin mRNA levels in response to renal artery clipping.

These findings suggest that T‐type calcium channel blockers can inhibit renin secretion and renin gene expression in vivo, whilst L‐type calcium channel blockers act as stimulators of the renin system. Since the inhibitory effect of T‐type antagonists is apparent in vivo but not in vitro, one may infer that the effect on the renin system is indirect rather than directly mediated at the level of renal juxtaglomerular cells.
Differential regulation of β3‐adrenoceptors in gut and adipose tissue of genetically obese (ob/ob) C57BL/6J‐mice
Authors: Evans Bronwyn A , Papaioannou Maria , Anastasopoulos Frank , Summers Roger J
Published: January 2005

Levels of β3‐adrenoceptor (AR) mRNA were compared using reverse transcription‐polymerase chain reaction (RT–PCR) in white adipose tissue (WAT), brown adipose tissue (BAT), ileum and colon from genetically obese (ob/ob) and lean (+/+) C57BL/6J mice. Functional responses to the β3‐AR agonist CL 316243 were also characterized in ileal longitudinal smooth muscle from obese and lean mice.

β3‐AR mRNA levels were significantly higher in WAT (100±16%) and BAT (100±13%) from lean compared to WAT (21.0±0.9%; n=4; P<0.005) and BAT (14.1±2.2%; n=5; P<0.01) from obese mice. In contrast, β3‐mRNA levels were not significantly different in ileum (100±15%) and colon (100±22%) from lean mice, compared to ileum (78±13%; n=4; P=0.31) or colon (82±15%; n=4; P=0.52) from obese mice.

Concentration‐response curves to CL 316243 did not differ significantly in slope or position in ileal longitudinal smooth muscle from obese or lean mice. pEC50 (±s.e.mean) values were not significantly different (P=0.59) between obese (7.90±0.13, n=7) and lean (7.77±0.20, n=7) mice.

pKB values for the β1‐AR and β2‐AR selective antagonist propranolol or the β3‐AR selective antagonist SR 58894 against relaxations to CL 316243 were similar in ileum of genetically obese (propranolol 6.31±0.22 and 6.13±0.12; SR 58894 8.22±0.06) and lean mice (propranolol 6.40±0.08 and 6.60±0.13; SR 58894 8.27±0.12) and were consistent with values previously found at β3‐AR.

Treatment of lean C57BL/6J mice with dexamethasone (1 mg kg−1, i.p.) significantly reduced β3‐AR mRNA levels after 4 h in WAT (100±6.1 to 41.4±4.3; n=16–18; P<0.0001) and BAT (100±8.0 to 35.1±5.8; n=17; P<0.0001), but caused no change in ileum (100±6.1 to 101±17; n=10–11; P=0.95) or colon (100±11 to 101±11; n=11; P=0.94). β3‐mRNA levels in ileum and colon also did not change significantly when examined over 24 h or after the administration of a higher dose of dexamethasone (5 mg kg−1).

In summary, β3‐AR mRNA levels were considerably lower in WAT and BAT of obese compared to lean mice whereas the levels in ileum and colon were not significantly different. The similar β3‐mRNA levels in ileum of obese and lean mice were associated with indistinguishable responses of carbachol‐contracted ileum to a β3‐agonist and similar affinity for β‐antagonists. Administration of glucocorticoids to lean mice reduced β3‐AR mRNA levels in WAT and BAT but not in ileum or colon. These studies show that in mice, β3‐ARs are differentially regulated in ileum and colon compared to adipose tissues.
Selectivity of action of 8‐alkylamino analogues of N6‐cyclopentyladenosine in vivo: haemodynamic versus anti‐lipolytic responses in rats
Authors: Van Schaick E A , Tukker H E , Roelen H C P F , IJzerman A P , Danhof M
Published: January 2005

A1 adenosine receptor agonists with reduced intrinsic activity may be therapeutically useful as result of an increased selectivity of action. In this study the tissue selectivity of three 8‐alkylamino substituted analogues of N6‐cyclopentyladenosine (CPA) was investigated for haemodynamic and anti‐lipolytic effects using an integrated pharmacokinetic‐pharmacodynamic approach.

Chronically instrumented male Wistar rats received intravenous infusions of 4.0 mg kg−1 8‐methylaminoCPA (8MCPA), 12.0 mg kg−1 8‐ethylaminoCPA (8ECPA), 20.0 mg kg−1 8‐butylaminoCPA (8BCPA) or vehicle during 15 min. During experimentation, serial arterial blood samples were drawn for the determination of agonist concentrations and plasma non‐esterified fatty acid (NEFA) levels. Blood pressure and heart rate were monitored continuously. In addition to the CPA analogues, each rat received a rapid bolus infusion of CPA to determine the maximal effects of the full agonist.

The concentration‐time profiles of the CPA analogues could be described by a bi‐exponential function. Values for clearance, volume of distribution at steady state and elimination half‐life were 44±5, 48±6 and 39±2 ml min−1 kg−1, 0.97±0.09, 0.84±0.10 and 1.05±0.07 1 kg−1 and 25±2, 28±2 and 40±2 min for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8).

Different models were used to derive the concentration‐effect relationships for heart rate and NEFA, yielding estimates of potency (EC50) and instrinsic activity (Emax) for both effects of the compounds in vivo. On heart rate the compounds acted as partial agonists, with Emax values of −173±14, −131±11 and −71±6 beats min−1 for 8MCPA, 8ECPA and 8BCPA, respectively. These Emax values were significantly lower than the maximal effect of CPA (−208±8 beats min−1). With regard to the anti‐lipolytic effect all three compounds were full agonists and lowered NEFA levels to the same extent as CPA (69%). The estimated Emax values were 63±5, 63±4 and 68±2%, respectively.

Furthermore, the compounds were more potent in causing anti‐lipolytic than cardiovascular effects. The EC50 values for the NEFA and heart rate lowering effects were 37±15, 68±22 and 659±108 ng ml−1 and 164±22, 341±76 and 975±190 ng ml−1 for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8).

This study demonstrates that partial agonists for the A1 adenosine receptor have increased selectivity of action in vivo. The 8‐alkylamino analogues of CPA may be useful anti‐lipolytics with less pronounced haemodynamic side effects.
Depression of NMDA receptor‐mediated synaptic transmission by four α2 adrenoceptor agonists on the in vitro rat spinal cord preparation
Authors: Faber E S L , Chambers J P , Evans R H
Published: January 2005

α2‐Adrenoceptor agonists have a spinal site of analgesic action. In the current study the synaptic depressant actions of xylazine, detomidine, romifidine and dexmedetomidine have been compared on segmental reflexes containing NMDA receptor‐mediated components in the neonatal rat hemisected spinal cord preparation in vitro.

Reflexes were evoked in the ventral root following either supramaximal electrical stimulation of the corresponding ipsilateral lumbar dorsal root to evoke the high intensity excitatory postsynaptic potential (e.p.s.p.) involving all primary afferent fibres, or low intensity stimulation to evoke the solely A fibre‐mediated low intensity e.p.s.p. The high intensity e.p.s.p. contains a greater NMDA receptor‐mediated component.

Xylazine, romifidine, detomidine and dexmedetomidine all depressed both the high intensity e.p.s.p. and the low intensity e.p.s.p. giving respective EC50 values of 0.91±0.2 μM (n=12), 23.4±3 nM (n=12), 37.7±7 nM (n=8) and 0.84±0.1 nM (n=4) for depression of the high intensity e.p.s.p. and 0.76±0.1 μM (n=12), 22.0±3 nM (n=12), 24.9±6 nM (n=4) and 2.7±0.6 nM (n=4) for depression of the low intensity e.p.s.p., respectively. Unlike the other three drugs, the two values for dexmedetomidine, showing a greater selectivity for the high intensity e.p.s.p., are significantly different.

Each of these depressant actions was reversed by the selective α2‐adrenoceptor antagonist atipamezole (1 μM).

In contrast to previous reports of the actions of α2‐adrenoceptor agonists on the in vitro spinal cord preparation, at concentrations ten fold higher than the above EC50 values xylazine, romifidine, detomidine and dexmedetomidine depressed the initial population spike of motoneurons (MSR). This depression was not reversed by atipamezole.

Comparison of the rank order of the present EC50 values for depression of the high intensity e.p.s.p. with potency ratios from in vivo analgesic tests in previous studies show a close correlation between the present in vitro tests and analgesic potency. There is no correlation between the present data and previously obtained affinities of the agonists at non‐adrenergic imidazoline binding sites.

The current findings therefore suggest that xylazine, romifidine, detomidine and dexmedetomidine are exerting their central analgesic actions at the spinal level principally through α‐2‐adrenoceptors. All four agonists showed the same profile of selective depression of the NMDA receptor‐mediated component of reflexes similar to that reported previously for clonidine. However dexmedetomidine, unlike the other ligands, selectively depressed the high intensity e.p.s.p.
Impairment in connective tissue mast cells degranulation in spontaneously hypertensive rats: stimulus dependent resistance
Authors: Kwasniewski F H , Tavares de Lima W , Bakhle Y S , Jancar S
Published: January 2005

Microvascular permeability in the mesentery and consequent leakage of protein into the peritoneum of spontaneously hypertensive rats (SHR) and normotensive rats (NTR) was measured in vivo by the extravasation of Evans blue dye.

In sensitized NTR, challenge with antigen produced extensive increases in dye extravasation in the mesentery and in peritoneal lavage fluid within 10 min.

In sensitized SHR there was no increase in the permeability of the mesentery and a very weak increase in dye extravasation in the peritoneal cavity following challenge.

The glucocorticoid antagonist RU38486 did not change the permeability response induced by antigen in sensitized NTR and SHR.

However, compound 48/80 was equally effective in either NTR or SHR in causing increased vasopermeability.

Mesenteric mast cells in the NTR were degranulated after immunological challenge, whereas those in the SHR were resistant, as measured histologically.

Similarly, challenge ex vivo of mesentery from sensitized NTR induced contraction of guinea‐pig ileum in co‐incubation experiments, whereas SHR mesentery was unresponsive.

Plasma levels of antigen‐specific IgE and IgG2a in sensitized NTR and SHR were identical.

Immune serum from SHR was unable to induce a passive cutaneous anaphylaxis (PCA) reaction in the skin of NTR and SHR did not develop a PCA reaction upon passive sensitization with NTR immune serum.

We conclude that the mast cells of SHR are resistant to degranulation following immunological challenge, although the relevant antibodies are present.
The effects of propofol on macroscopic and single channel sodium currents in rat ventricular myocytes
Authors: Saint David A
Published: January 2005

The effects of the injectable anaesthetic agent propofol (di‐isopropyl phenol) were examined on sodium currents and single sodium channels by use of patch‐clamp techniques in ventricular myocytes isolated from rat hearts.

Propofol dose‐dependently blocked the whole cell sodium currents evoked by a voltage step to −30 mV from a holding potential of −90 mV with an EC50 of 14.8±2.3 μM (mean±s.e.mean).

Propofol caused a substantial hyperpolarizing shift in the voltage‐dependence of inactivation of sodium currents (168 μM (30 μg ml−1) propofol caused a −14 mV shift (P<0.01); 56 μM caused a −8 mV shift (P<0.05)). A smaller shift in the voltage‐dependence of activation was produced (4 mV by 168 μM (not statistically significant)), but this was to more depolarized potentials. The maximal sodium conductance, as judged from the activation and inactivation curves, was reduced by 13% by 168 μM propofol (not statistically significant), but propofol did not affect the reversal potential of the current ‐ voltage relationship.

The macroscopic rate of inactivation, as measured by the time constant of the exponential fall of current amplitude from the peak current, was also slowed by propofol, from a control time constant of 1.78±0.31 ms to 2.93±0.47 ms (mean±s.e.mean, n=8, P<0.05) by 168 μM propofol. Despite the increase in the time constant, the macroscopic inactivation remained well fitted by a single exponential. The macroscopic rate of activation was also slowed, but to a lesser degree (<10%, not statistically significant) by 168 μM propofol.

Propofol slowed the rate of recovery from inactivation of the sodium current, as measured by a two pulse protocol. Propofol (168 μM) increased the time constant of recovery, measured at −100 mV and room temperature, from a control value of 55±5.9 ms to 141±24.2 ms (mean±s.e.mean, n=8, P<0.01). Although the time constant was increased at all voltages measured, the intrinsic voltage‐dependence of the rate of recovery was not changed.

Single channel recordings showed that the mean open time of single sodium channels was dramatically reduced by propofol (from 0.50±0.02 ms in control to 0.28±0.01 ms by 56 μM propofol and to 0.24±0.01 ms by 168 μM, both significantly different from control, P<0.01). Single channel conductance was not changed by either concentration of propofol.
Effects of cetirizine on the delayed K+ currents in cardiac cells: comparison with terfenadine
Authors: Carmeliet Edward
Published: January 2005

The aim of the present experiments was to analyse the effect of the H1‐histamine antagonist, cetirizine, on the delayed K+ currents in cardiac cells and to compare its effects with another H1‐histamine antagonist terfenadine, known to possess proarrhythmic effects.

Whole cell currents were measured by use of the single electrode patch‐clamp technique in rabbit and guinea‐pig myocytes.

The activation relationship for the IKr current in rabbit ventricular myocytes was depressed and its voltage‐dependence shifted in the negative direction with a V1/2 value −13.4±2.4 mV under control conditions which changed to −19.1±1.9 mV (n=4) in the presence of 0.1 mM cetirizine.

In rabbit ventricular myocytes the IC50 for block of IKr was 108±8 μM (n=5); in guinea‐pig ventricular myocytes this concentration of cetirizine reduced the rapidly activating component IKr to 49±4.5% (n=5), while the slowly activating IKs was less affected and only inhibited to 79±2.3% (n=5).

The block of IKr did not show use‐dependence and the time course of the tail current was not changed, suggesting rested‐state block or fast activated‐state block and no rapid recovery on deactivation. No important difference was found in the activity of the two enantiomers of cetirizine.

Terfenadine in comparison was more potent in blocking IKr, the IC50 being 96±15 nM (n=6).

Based on the present results and information in the literature on binding, it was concluded that cetirizine is a relatively selective H1‐histamine receptor antagonist, with minor effects on K+ currents. The IC50 concentration for IKr block in heart cells was 1.000 times higher than the concentrations needed to block H1 histamine receptors. The occurrence of cardiac arrhythmias due to K+ current blockade is therefore unlikely with this drug.
ATP and adenosine inhibit transmitter release at the frog neuromuscular junction through distinct presynaptic receptors
Authors: Giniatullin R A , Sokolova E M
Published: January 2005

The effects of exogenous ATP or adenosine on end‐plate currents (e.p.cs; evoked by simultaneous action of a few hundred quanta of ACh) or on miniature e.p.cs (m.e.p.cs) were studied under voltage clamp conditions on frog sartorius muscle fibres.

ATP or adenosine (100 μM–1 mM) reduced the e.p.c. amplitude but did not affect m.e.p.c. amplitude, decay time constant and voltage‐dependence of m.e.p.c., suggesting that e.p.c. depression induced by these purines had presynaptic origin only.

The action of ATP, unlike that of adenosine, was prevented by the P2‐purinoceptor antagonist suramin (100 μM). The stable ATP analogue α,β‐methylene ATP (100 μM), known to be desensitizing agent on P2X receptors, also abolished the depressant effect of ATP while sparing the action of adenosine. Concanavalin A, an inhibitor of ecto‐5′‐nucleotidase, did not affect the presynaptic action of exogenously applied ATP.

The presynaptic action of adenosine was prevented by theophylline (1 mM), a blocker of adenosine receptors, while the effect of ATP was not changed under these conditions. The selective blocker of A1 adenosine receptors, 8‐cyclopentyl‐1,3,dipropylxanthine (DPCPX; 0.1 μM), abolished the presynaptic action of adenosine but did not prevent the depressant effect of ATP.

The effects of ATP and adenosine (at nearly saturating concentration) were additive suggesting that these purines activated not only distinct receptors but also different intracellular signalling mechanisms.

In contrast to the hypothesis that at the neuromuscular junction ATP reduces transmitter release via enzymatic degradation to presynaptically active adenosine, our data suggest that ATP (through its own presynaptic receptors) directly inhibits ACh release. Thus, ATP and adenosine might be almost equipotent as endogenous prejunctional neuromodulators at the neuromuscular junction.
Effects of the cysteinyl leukotriene receptor antagonists pranlukast and zafirlukast on tracheal mucus secretion in ovalbumin‐sensitized guinea‐pigs in vitro
Authors: Liu Yu‐Chih , Khawaja Aamir M , Rogers Duncan F
Published: January 2005

We investigated the inhibitory effects of the cysteinyl leukotriene (CysLT1) receptor antagonists, pranlukast and zafirlukast, on 35SO4 labelled mucus output, in vitro, in guinea‐pig trachea, induced by leukotriene D4 (LTD4) or by antigen challenge of sensitized animals. Agonists and antagonists were administered mucosally, except in selected comparative experiments where drugs were administered both mucosally and serosally to assess the influence of the epithelium on evoked‐secretion.

LTD4 increased 35SO4 output in a concentration‐related manner with a maximal increase of 23 fold above controls at 100 μM and an approximate EC50 of 2 μM. Combined mucosal and serosal addition of LTD4 did not significantly affect the secretory response compared with mucosal addition alone. Neither LTC4 nor LTE4 (10 μM each) affected 35SO4 output. Pranlukast or zafirlukast significantly inhibited 10 μM LTD4‐evoked 35SO4 output in a concentration‐dependent fashion, with maximal inhibitions of 83% at 10 μM pranlukast and 78% at 10 μM zafirlukast, and IC50 values of 0.3 μM for pranlukast and 0.6 μM for zafirlukast. Combined mucosal and serosal administration of the antagonists (5 μM each) gave degrees of inhibition of mucosal‐serosal 10 μM LTD4‐evoked 35SO4 output similar to those of the drugs given mucosally. Pranlukast (0.5 μM) caused a parallel rightward shift of the LTD4 concentration‐response curve with a pKB of 7. Pranlukast did not inhibit ATP‐induced 35SO4 output.

Ovalbumin (10–500 μg ml−1) challenge of tracheae from guinea‐pigs actively sensitized with ovalbumin caused a concentration‐related increase in 35SO4 output with a maximal increase of 20 fold above vehicle controls at 200 μg ml−1. The combination of the antihistamines pyrilamine and cimetidine (0.1 mM each) did not inhibit ovalbumin‐induced 35SO4 output in sensitized guinea‐pigs. Neither mucosal (10 μM or 100 μM) nor mucosal‐serosal (100 μM) histamine had any significant effect on 35SO4 output.

Pranlukast or zafirlukast (5 μM each) significantly suppressed ovalbumin‐induced secretion in tracheae from sensitized guinea‐pigs by 70% and 65%, respectively.

We conclude that LTD4 or ovalbumin challenge of sensitized animals provokes mucus secretion from guinea‐pig trachea in vitro and this effect is inhibited by the CysLT1 receptor antagonists pranlukast and zafirlukast. These antagonists may be beneficial in the treatment of allergic airway diseases in which mucus hypersecretion is a clinical symptom, for example asthma and allergic rhinitis.
Effects of steroid treatment on activation of nuclear factor κB in patients with inflammatory bowel disease
Authors: Ardite E , Panés J , Miranda M , Salas A , Elizalde J I , Sans M , Arce Y , Bordas J M , Fernández‐Checa J C , Piqué J M
Published: January 2005

Nuclear factor κB (NFκB) is a transcription factor that controls several genes important for immunity and inflammation. The aim of this study was to assess if activation of NFκB plays a role in the pathogenesis of inflammatory bowel disease (IBD), and whether steroid treatment affects NFκB activation. Activation of NFκB was analysed in colon biopsy samples of 13 patients with active IBD (8 Crohn's colitis, 5 ulcerative colitis) by electrophoretic mobility‐shift assays, under basal conditions and 3 weeks after treatment with 0.75 mg kg−1 day−1 prednisolone. The presence of interleukin‐8 mRNA in biopsies was assessed by RT–PCR. A specific NFκB band was present in all nuclear extracts from inflamed mucosa, whereas the band was barely detectable in uninflamed colonic mucosa. NFκB bands were super‐shifted by antibodies against p50 subunit, whereas antibodies against p65, p52, c‐Rel, or Rel B did not modify the mobility of the band. Increased interleukin‐8 mRNA was detected at the same sites of NFκB activation. Steroid‐induced healing of colonic inflammation was associated with disappearance of NFκB from nuclear extracts. These results support the notion that NFκB plays an important role in the pathogenesis of IBD, and that blockade of NFκB activation is one of the mechanisms by which steroids suppress the inflammatory cascade in IBD.