bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effects of the tricyclic antidepressant amoxapine on glycine uptake mediated by the recombinant GLYT1 and GLYT2 glycine transporters
Authors: Núñez Enrique , López‐Corcuera Beatriz , Vázquez Jesús , Giménez Cecilio , Aragón Carmen
Published: January 2005

We examined the effects of nine different tricyclic antidepressant drugs on the glycine uptake mediated by the glycine transporter 1b (GLYT1b) and glycine transporter 2a (GLYT2a) stably expressed in human embryonic kidney 293 cells. Desipramine, imipramine, clomipramine, nomifensine and mianserin had no effect on the activity of the glycine transporters. Doxepin, amitriptyline and nortriptyline inhibited the two transporter subtypes to a similar extent.

Amoxapine displayed a selective inhibition of GLYT2a behaving as a 10 fold more efficient inhibitor of this isoform than of GLYT1b.

Kinetic analysis of the initial rates of glycine uptake by GLYT2a as a function of either glycine, chloride or sodium concentration, in the absence and presence of amoxapine indicated that amoxapine behaved as a competitive inhibitor of both glycine and chloride and a mixed‐type inhibitor with respect to sodium.

A kinetic model was developed which explains adequately these data, and gives information about the order of binding of sodium and chloride ions to GLYT2a.

Our results may contribute to the development of the glycine transporter pharmacology. Additionally, the inhibition of the glycine uptake by GLYT2 is suggested to have some role in the sedative and psychomotor side effects of amoxapine.

British Journal of Pharmacology (2000) 129, 200–206; doi:10.1038/sj.bjp.0703049
Primary porcine enterocyte and hepatocyte cultures to study drug oxidation reactions
Authors: Bader A , Hansen T , Kirchner G , Allmeling C , Haverich A , Borlak J T
Published: January 2005

Primary porcine hepatocytes and enterocytes were isolated and cultured in Williams' E medium for up to 10 days to investigate potential organ differences in the metabolism of the immunosuppressive compound tacrolimus (FK 506) and of two investigational drugs (KC11346 and KC12291). Using LC‐MS (FK506) and HPLC‐FL (KC 11346/12291) a number of metabolites with identical mass and/or identical retention time could be detected.

In the case of tacrolimus hepatocytes and enterocytes produced the same spectrum of metabolites, e.g. bisdemethyl‐tacrolimus, demethyl‐tacrolimus, demethyl‐hydroxy‐tacrolimus and hydroxy‐tacrolimus, albeit at varying intensities.

Treatment of enterocyte cultures with dexamethasone increased the overall metabolite formation very significantly (up to 36 fold).

The metabolism of tacrolimus was also studied with preparations of insect cells, that express specifically high levels of individual human cytochrome P450 (CYP) isoenzymes. All metabolites could be generated with microsomal preparations specifically expressing CYP3A4, but hydroxy‐tacrolimus was exclusively produced by CYP3A5.

In the case of the investigational drugs KC 11346 and KC 12291 only three metabolites were formed by cultured enterocytes whereas hepatocytes produced 10 and 20 metabolites, respectively.

When assessed at the protein level CYP1A and CYP3A were expressed in cultures of porcine enterocytes for up to 10 days but porcine hepatocytes expressed additionally CYP2C9/10.

In conclusion, primary enterocytes and hepatocytes can be successfully cultured for several days while maintaining mono‐oxygenase activity and may therefore be used as a tool for studying intestinal and hepatic metabolism.

British Journal of Pharmacology (2000) 129, 331–342; doi:10.1038/sj.bjp.0703062
A comparison of the anti‐inflammatory and anti‐nociceptive activity of nitroaspirin and aspirin
Authors: Al‐Swayeh O A , Clifford R H , Del Soldato P , Moore P K
Published: January 2005

Nitroaspirin (2.5–50 mg kg−1, i.p. or 2.5–100 mg kg−1, p.o.) and aspirin (2.5–100 mg kg−1, i.p. or p.o.) exhibit anti‐inflammatory activity in the carrageenan‐induced hindpaw oedema model in the rat. When administered i.p., nitroaspirin was a more effective anti‐oedema agent than aspirin particularly in the ‘early’ phase (i.e. up to 60 min) of the response. The ED50 values for nitroaspirin and aspirin as inhibitors of the ‘late’ phase response (measured at 180 min) were 64.3 μmol kg−1 and >555 μmol kg−1, respectively. When administered p.o., neither nitroaspirin nor aspirin exhibited significant anti‐inflammatory activity in the ‘early’ phase and were of similar potency in the ‘late’ phase. Thus, at the highest dose used (100 mg kg−1, 360 min) orally administered nitroaspirin (aspirin in parenthesis) inhibited oedema formation by 46.9±1.6% (47.2±3.8%, both n=6, P<0.05).

Nitroaspirin and aspirin (25–200 mg kg−1, p.o.) caused dose‐related inhibition of the hyperalgesia to mechanical stimulation following intraplantar injection of carrageenan in the rat. ED50 values were 365 μmol kg−1 and 784 μmol kg−1, respectively. Neither drug influenced the threshold for mechanical stimulation in the contralateral (i.e. untreated) hindpaw.

Nitroaspirin and aspirin (2.5–100 mg kg−1, p.o.) caused dose‐related inhibition of acetic acid induced abdominal constrictions in the mouse (ED50 values of 154.7 μmol kg−1 and 242.8 μmol kg−1, respectively).

Nitroaspirin and aspirin (>200 mg kg−1, p.o.) reduced the ‘late’ phase (but not the ‘early’ phase) of the formalin‐induced hindpaw licking assay in the mouse. Similarly, nitroaspirin and aspirin (>50 mg kg−1, p.o.) prolonged tail withdrawal latency following application of a noxious heat stimulus in the mouse.

British Journal of Pharmacology (2000) 129, 343–350; doi:10.1038/sj.bjp.0703064
Differential regulation of mammalian brain‐specific proline transporter by calcium and calcium‐dependent protein kinases
Authors: Jayanthi Lankupalle D , Wilson Jennifer J , Montalvo Jocelyn , DeFelice Louis J
Published: January 2005

This study examined the role of [Ca2+]I and Ca2+‐dependent kinases in the modulation of high‐affinity, mammalian brain‐specific L‐proline transporter (PROT).

β‐PMA (phorbol 12‐myristate 13‐acetate), an activator of protein kinase C (PKC), inhibits PRO uptake, and bisindolylmalemide I (BIM), a potent PKC inhibitor, prevents β‐PMA inhibition. Down‐regulation of PKC by chronic treatment with β‐PMA enhances PROT function indicating PROT regulation by tonic activity of PKC.

Thapsigargin, which increases [Ca2+]I levels by inhibiting Ca2+‐ATPase, inhibits PROT and exhibits additive inhibition when co‐treated with β‐PMA. KN‐62, a Ca2+/calmodulin‐dependent kinase II (CaMK II) inhibitor, but not BIM (a PKC inhibitor) prevents the inhibition by thapsigargin. These data suggest that PKC and CaMK II modulate PROT and that thapsigargin mediates its effect via CaMK II.

Thapsigargin raises [Ca2+]I and increases PRO‐induced current on a second time scale, whereas the inhibitory effect of thapsigargin occurs only after 10 min of treatment. These data suggest that Ca2+ differentially regulate PROT: Ca2+ initially enhances PRO transport but eventually inhibits transport function through CaMK II pathway.

Ca2+‐induced stimulation exemplifies the acute regulation of a neurotransmitter transporter, which may play a critical role in the profile of neurotransmitters during synaptic transmission

British Journal of Pharmacology (2000) 129, 465–470; doi:10.1038/sj.bjp.0703071
Characterization of MPP+ secretion across human intestinal Caco‐2 cell monolayers: role of P‐glycoprotein and a novel Na+‐dependent organic cation transport mechanism
Authors: Bleasby Kelly , Chauhan Seema , Brown Colin D A
Published: January 2005

In the kidney, a number of transport proteins involved in the secretion of permanently charged organic cations have recently been cloned. To evaluate the possible similarities between intestine and kidney in the handling of organic cations we investigated the transport of 1‐methyl‐4‐phenylpyridinium (MPP+) across monolayers of intestinal Caco‐2 cells. MPP+ is a prototypic substrate of the cloned organic cation transporters hOCT1 and hOCT2.

In Caco‐2 cell monolayers, the basolateral to apical flux of MPP+ was significantly greater than the apical to basolateral flux, consistent with net secretion of MPP+.

Net secretion of MPP+ was abolished by addition of either 10 μM cyclosporin A or 100 μM verapamil to the apical membrane. In contrast, secretion of MPP+ was unaffected by addition of either TEA (2 mM) or decynium‐22 (2 μM) to either apical or basolateral membranes. These results suggest that MPP+ secretion is mediated primarily by P‐glycoprotein located at the apical membrane. We found no evidence of a role for hOCT1 or hOCT2 in the secretion of MPP+.

In addition to net secretion of MPP+, we found evidence of a Na+‐dependent MPP+ uptake mechanism at the apical membrane of Caco‐2 cells.

Na+‐dependent MPP+ uptake was sensitive to inhibition by the organic cations; decynium‐22 (2 μM), TEA (2 mM) and cimetidine (5 mM) but not by carnitine, guanidine or proline.

These results suggest that net secretion of MPP+ across the apical membrane of Caco‐2 cells is a function of the relative contributions of MPP+secretion mediated by P‐glycoprotein and MPP+ absorption mediated by a novel Na+‐dependent transport mechanism.

British Journal of Pharmacology (2000) 129, 619–625; doi:10.1038/sj.bjp.0703078
Prejunctional muscarinic inhibitory control of acetylcholine release in the human isolated detrusor: involvement of the M4 receptor subtype
Authors: D'Agostino Gianluigi , Bolognesi Maria L , Lucchelli Adele , Vicini Davide , Balestra Barbara , Spelta Valeria , Melchiorre Carlo , Tonini Marcello
Published: January 2005

Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response.

In detrusor strips preincubated with [3H]‐choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 μM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]‐ACh (76%) and [3H]‐choline (24%).

Electrically‐evoked [3H]‐ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 μM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM–1 μM), and enhanced by atropine (0.1–100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release.

The effects of various subtype‐preferring muscarinic receptor antagonists were evaluated on [3H]‐ACh release and muscle contraction. The rank potency (−log EC50) orders at pre‐ and postjunctional level were: atropine 4‐diphenyl‐acetoxy‐N‐piperidine (4‐DAMP)>mamba toxin 3 (MT‐3)>tripitramine>para‐fluorohexahydrosiladiphenidol (pF‐HHSiD)methoctraminepirenzepine>tripinamide, and atropine4‐DAMP>pF‐HHSiD>>pirenzepine=tripitramine>tripinamide>methoctramine>>MT‐3, respectively.

The comparison of pre‐ and post‐junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.

British Journal of Pharmacology (2000) 129, 493–500; doi:10.1038/sj.bjp.0703080
Differential tetraethylammonium sensitivity of KCNQ1–4 potassium channels
Authors: Hadley J K , Noda M , Selyanko A A , Wood I C , Abogadie F C , Brown D A
Published: January 2005

In Shaker‐group potassium channels the presence of a tyrosine residue, just downstream of the pore signature sequence GYG, determines sensitivity to tetraethylammonium (TEA). The KCNQ family of channels has a variety of amino acid residues in the equivalent position. We studied the effect of TEA on currents generated by KCNQ homomers and heteromers expressed in CHO cells. We used wild‐type KCNQ1–4 channels and heteromeric KCNQ2/3 channels incorporating either wild‐type KCNQ3 subunits or a mutated KCNQ3 in which tyrosine replaced threonine at position 323 (mutant T323Y). IC50 values were (mM): KCNQ1, 5.0; KCNQ2, 0.3; KCNQ3, >30; KCNQ4, 3.0; KCNQ2+KCNQ3, 3.8; and KCNQ2+KCNQ3(T323Y), 0.5. While the high TEA sensitivity of KCNQ2 may be conferred by a tyrosine residue lacking in the other channels, the intermediate TEA sensitivity of KCNQ1 and KCNQ4 implies that other residues are also important in determining TEA block of the KCNQ channels.

British Journal of Pharmacology (2000) 129, 413–415; doi:10.1038/sj.bjp.0703086
Comparison of novel cannabinoid partial agonists and SR141716A in the guinea‐pig small intestine
Authors: Coutts Angela A , Brewster Nicola , Ingram Tracey , Razdan Raj K , Pertwee Roger G
Published: January 2005

The controversial nature of the CB1 receptor antagonist, SR141716A, in the guinea‐pig small intestine was investigated by comparing it with four analogues of Δ8‐tetrahydrocannabinol (Δ8‐THC): O‐1184, O‐1238, O‐584 and O‐1315.

These compounds (10–1000 nM) inhibited the electrically‐evoked contractions with a rank order of potency of O‐1238>O‐1184>O‐584>O‐1315. Log concentration‐response curves for O‐1238, O‐1184 and O‐1315 were significantly shifted to the right by SR141716A and the maxima were significantly less than that of the CB1 agonist, WIN55212‐2, an indication of partial agonism.

Partial saturation of the triple bond in O‐1184 to a cis double bond (O‐1238) increased its potency as an agonist (pEC50 from 6.42 to 7.63) and as an antagonist of WIN55212‐2, (pKB, from 8.36 to 9.49). Substitution of the terminal azide group by an ethyl group (O‐584) or removal of the phenolic hydroxyl group (O‐1315) had no significant effect on the agonist or antagonist potency. None of these analogues increased the twitch response in a manner resembling that of SR141716A.

O‐1184 (10 and 100 nM) shifted the log concentration‐response curve of WIN55212‐2 for inhibition of the twitch responses to the right with pKB values of 8.29 and 8.38, respectively.

We conclude that these Δ8‐THC analogues behave as partial agonists rather than silent antagonists at CB1 binding sites in this tissue. There was no evidence of antagonism of endocannabinoids thus supporting the hypothesis that, in this tissue, SR141716A is an inverse agonist of constitutively active CB1 receptors.

British Journal of Pharmacology (2000) 129, 645–652; doi:10.1038/sj.bjp.0703094
M‐type K+ currents in rat cultured thoracolumbar sympathetic neurones and their role in uracil nucleotide‐evoked noradrenaline release
Authors: Nörenberg W , Von Kügelgen I , Meyer A , Illes P , Starke K
Published: January 2005

Cultured sympathetic neurones are depolarized and release noradrenaline in response to extracellular ATP, UDP and UTP. We examined the possibility that, in neurones cultured from rat thoracolumbar sympathetic ganglia, inhibition of the M‐type potassium current might underlie the effects of UDP and UTP.

Reverse transcriptase‐polymerase chain reaction indicated that the cultured cells contained mRNA for P2Y2‐, P2Y4‐ and P2Y6‐receptors as well as for the KCNQ2‐ and KCNQ3‐subunits which have been suggested to assemble into M‐channels.

In cultures of neurones taken from newborn as well as from 10 day‐old rats, oxotremorine, the M‐channel blocker Ba2+ and UDP all released previously stored [3H]‐noradrenaline.

The neurones possessed M‐currents, the kinetic properties of which were similar in neurones from newborn and 9–12 day‐old rats.

UDP, UTP and ATP had no effect on M‐currents in neurones prepared from newborn rats. Oxotremorine and Ba2+ substantially inhibited the current.

ATP also had no effect on the M‐current in neurones prepared from 9–12 day‐old rats. Oxotremorine and Ba2+ again caused marked inhibition. In contrast to cultures from newborn animals, UDP and UTP attenuated the M‐current in neurones from 9–12 day‐old rats; however, the maximal inhibition was less than 30%.

The results indicate that inhibition of the M‐current is not involved in uracil nucleotide‐induced transmitter release from rat cultured sympathetic neurones during early development. M‐current inhibition may contribute to release at later stages, but only to a minor extent. The mechanism leading to noradrenaline release by UDP and UTP remains unknown.

British Journal of Pharmacology (2000) 129, 709–723; doi:10.1038/sj.bjp.0703096
Effects of local delivery of trapidil on neointima formation in a rabbit angioplasty model
Authors: Zacharowski Kai , Schneidmüller Dorien , Ibe Waltraut , Großer Tilo , Buerke Michael , Meyer Jürgen , Darius Harald
Published: January 2005

Smooth muscle cell (SMC) proliferation can result in luminal reduction of a vessel following balloon angioplasty. This study was designed (i) to determine if local administration of trapidil (triazolopyrimidine) into a vessel wall reduces neointima formation, and (ii) to explore the mechanism involved in the subsequent reduction in cell proliferation.

Following balloon angioplasty in 40 anaesthetized New Zealand White rabbits, trapidil (50–200 mg) or its vehicle (saline) was injected into the dilated vessel wall of the right femoral artery. Experimental groups and time of investigation: (I) vehicle (2 weeks, n=3), (II) trapidil‐100 mg (2 weeks, n=3), (III) vehicle (3 weeks, n=8), (IV) trapidil‐50 mg (3 weeks, n=5); (V) trapidil‐100 mg (3 weeks, n=9) or (V) trapidil‐200 mg (3 weeks, n=7).

After 2 weeks, there was a significant reduction of intimal hyperplasia (expressed as intima to media area ratio) in the trapidil group compared with vehicle (0.44±0.04 vs 0.93±0.04, *P<0.05) and also a significant reduction in cell proliferation (% ratio of BrdU‐positive cells to total cell number: vehicle 14±2% vs trapidil 6±1%, *P<0.05).

After 3 weeks, there was a dose‐dependent reduction of intimal hyperplasia in the trapidil groups compared with vehicle (trapidil 50 mg 1.14±0.04; trapidil 100 mg 0.91±0.09*; trapidil 200 mg 0.77±0.09* vs vehicle 1.67±0.23, *P<0.05).

Thus, the local administration of trapidil to the rabbit femoral artery reduces the neointima formation, which occurs 2 or 3 weeks after balloon angioplasty via a mechanism, which is dependent on inhibition of cell proliferation.

British Journal of Pharmacology (2000) 129, 566–572; doi:10.1038/sj.bjp.0703098
Evidence that rat hepatocytes co‐express functional P2Y1and P2Y2receptors
Authors: Dixon C Jane , Woods Niall M , Webb Tania E , Green Anne K
Published: January 2005

Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase‐polymerase chain reaction (RT–PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y1, P2Y2, P2Y4and P2Y6).

The effects of UTP have been examined on single aequorin‐injected rat hepatocytes. The [Ca2+]itransients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP‐ and ATP‐induced [Ca2+]itransients.

UDP, an agonist at the P2Y6receptor, failed to induce transients in hepatocytes, indicating that functional P2Y6receptors coupled to increased [Ca2+]iare not expressed.

The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y2rather than P2Y4receptors.

Co‐application of 30 μMpyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP‐ and UTP‐induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients.

In conclusion the data presented here are consistent with the co‐expression of P2Y1and P2Y2receptors by rat hepatocytes.

British Journal of Pharmacology(2000)129, 764–770; doi:10.1038/sj.bjp.0703103
The group II metabotropic glutamate receptor agonist, DCG‐IV, alleviates akinesia following intranigral or intraventricular administration in the reserpine‐treated rat
Authors: Dawson Lee , Chadha Anita , Megalou Maria , Duty Susan
Published: January 2005

This study examined whether activation of group II metabotropic glutamate (mGlu) receptors in the substantia nigra pars reticulata (SNr) could reverse akinesia in a rodent model of Parkinson's disease (PD).

Male Sprague Dawley rats, stereotaxically cannulated above either the SNr or third ventricle, were rendered akinetic by injection of reserpine (5 mg kg−1 s.c.). Eighteen hours later, the rotational behaviour induced by unilateral injection of the group II mGlu receptor agonist, (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine (DCG‐IV), was examined.

Following intranigral injection, DCG‐IV (0.125–0.75 nmol in 0.1 μl) produced a dose‐dependent increase in net contraversive rotations (n=6–8 animals per dose), reaching a maximum of 395±51 rotations 60 min−1 after 0.75 nmol. The effects of DCG‐IV (0.5 nmol) were inhibited by 63.0±9.0% following 30 min pre‐treatment with the group II mGlu receptor antagonist, (2S)‐α‐ethylglutamic acid (EGLU; 100 nmol in 0.2 μl; n=6).

Following intraventricular injection, DCG‐IV (0.125–1.5 nmol in 2 μl) produced a dose‐dependent increase in bilateral locomotor activity (n=6–7 animals per dose), reaching a maximum of 180±21 locomotor units 30 min−1 after 0.5 nmol. Pre‐treatment with EGLU (200 nmol in 2 μl) inhibited the effects of DCG‐IV (0.5 nmol) by 68.2±12.3% (n=5).

These data show that activation of group II mGlu receptors in the SNr provides relief of akinesia in the reserpinized rat model of PD. The reversal seen following intraventricular administration supports the likely therapeutic benefit of systemically‐active group II mGlu receptor agonists in PD.

British Journal of Pharmacology (2000) 129, 541–546; doi:10.1038/sj.bjp.0703105
Pharmacological profile of BIBN4096BS, the first selective small molecule CGRP antagonist
Authors: Doods Henri , Hallermayer Gerhard , Wu Dongmei , Entzeroth Michael , Rudolf Klaus , Engel Wolfhard , Eberlein Wolfgang
Published: January 2005

Calcitonin gene‐related peptide (CGRP) is one of the most potent endogenous vasodilators known. This peptide is increased during migraine attacks and has been implicated in the pathogenesis of migraine headache. Here we report on the first small molecule selective CGRP antagonist: BIBN4096BS. In vitro, this compound is extremely potent at primate CGRP receptors exhibiting an affinity (Ki) for human CGRP receptors of 14.4±6.3 (n=4) pM. In an in vivo model, BIBN4096BS in doses between 1 and 30 μg kg−1 (i.v.) inhibited the effects of CGRP, released by stimulation of the trigeminal ganglion, on facial blood flow in marmoset monkeys. It is concluded that BIBN4096BS is a potent and selective CGRP antagonist.

British Journal of Pharmacology (2000) 129, 420–423; doi:10.1038/sj.bjp.0703110
The pharmacology of hSK1 Ca2+‐activated K+ channels expressed in mammalian cell lines
Authors: Shah M , Haylett D G
Published: January 2005

The pharmacology of hSK1, a small conductance calcium‐activated potassium channel, was studied in mammalian cell lines (HEK293 and COS‐7). In these cell types, hSK1 forms an apamin‐sensitive channel with an IC50 for apamin of 8 nM in HEK293 cells and 12 nM in COS‐7 cells. The currents in HEK293 cells were also sensitive to tubocurarine (IC50=23 μM), dequalinium (IC50=0.4 μM), and the novel dequalinium analogue, UCL1848 (IC50=1 nM). These results are very different from the pharmacology of hSK1 channels expressed in Xenopus oocytes and suggest the properties of the channel may depend on the expression system. Our findings also raise questions about the role of SK1 channels in generating the apamin‐insensitive slow afterhyperpolarization observed in central neurones.

British Journal of Pharmacology (2000) 129, 627–630; doi:10.1038/sj.bjp.0703111
Role for endogenous endothelin in the regulation of plasma volume and albumin escape during endotoxin shock in conscious rats
Authors: Filep János G
Published: January 2005

To explore the role of endogenous endothelin (ET) in the regulation of vascular functions, we studied the effects endothelin receptor blockade on blood pressure, plasma volume and albumin escape during endotoxin shock in conscious, chronically catheterized rats. Red blood cell volume and plasma volume were determined by using chromium‐51‐tagged erythrocytes and iodine‐125‐labelled albumin, respectively.

Intravenous injection of lipopolysaccharide (LPS, 10 mg kg−1) resulted in hypotension, haemoconcentration, and increased total‐body albumin escape, which is reflected by a 30% reduction in plasma volume. Plasma ET‐1 concentrations increased 2.1 fold and 5.4 fold at 30 and 120 min post‐LPS, respectively.

LPS‐induced losses in plasma volume and albumin escape were significantly attenuated by pretreatment of animals with the dual ETA/ETB receptor antagonist bosentan (17.4 μmol kg−1, i.v. 15 min prior to LPS) or the ETA receptor antagonist FR 139317 (3.8 μmol kg−1, i.v.) during both the immediate and delayed phases of endotoxin shock. The inhibitory actions of bosentan and FR 139317 were similar. Both antagonists augmented the hypotensive action of LPS.

Administration of bosentan or FR 139317 70 min after injection of LPS also attenuated further losses in plasma volume and increases in total body and organ albumin escape rates with the exception of the lung and kidney.

These results indicate a role for endogenous endothelin in mediating losses in plasma volume and albumin escape elicited by LPS predominantly through activation of ETA receptors, and suggest that by attenuating these events, ETA or dual ETA/ETB receptor blockers may be useful agents in the therapy of septic shock.

British Journal of Pharmacology (2000) 129, 975–983; doi:10.1038/sj.bjp.0703114
Voltage‐dependent inhibition of the muscarinic cationic current in guinea‐pig ileal cells by SK&F 96365
Authors: Zholos A V , Tsytsyura Ya D , Philyppov I B , Shuba M F , Bolton T B
Published: January 2005

The effects of SK&F 96365 on cationic current evoked either by activating muscarinic receptors with carbachol or by intracellularly applied GTPγS (in the absence of carbachol) were studied using patch‐clamp recording techniques in single guinea‐pig ileal smooth muscle cells.

SK&F 96365 reversibly inhibited the muscarinic receptor cationic current in a concentration‐, time‐ and voltage‐dependent manner producing concomitant alteration of the steady‐state I‐V relationship shape which could be explained by assuming that increasing membrane positivity increased the affinity of the blocker. The inhibition was similar for both carbachol‐ and GTPγS‐evoked currents suggesting that the cationic channel rather than the muscarinic receptor was the primary site of the SK&F 96365 action.

Increased membrane positivity induced additional rapid inhibition of the cationic current by SK&F 96365 which was more slowly relieved during membrane repolarization. Both the inhibition and disinhibition time course could be well fitted by a single exponential function with the time constants decreasing with increasing positivity for the inhibition (e‐fold per about 12 mV) and approximately linearly decreasing with increasing negativity for the disinhibition.

At a constant SK&F 96365 concentration, the degree of cationic current inhibition was a sigmoidal function of the membrane potential with a potential of half‐maximal increase positive to about +30 mV and a slope factor of about −13 mV.

Increasing the duration of voltage steps at −80 or at 80 mV, increased the percentage inhibition; the degree of inhibition was almost identical at both potentials providing evidence that the same cationic channel was responsible for the cationic current both at negative and at positive potentials.

It is concluded that the distinctive and unique mode of SK&F 96365 action on the muscarinic receptor cationic channel is a valuable tool in future molecular biology studies of this channel.

British Journal of Pharmacology (2000) 129, 695–702; doi:10.1038/sj.bjp.0703115
Inhibition of the hypothalamic‐pituitary‐adrenal axis in food‐deprived rats by a CCK‐A receptor antagonist
Authors: Ruiz‐Gayo Mariano , Garrido Margarita M , Fuentes José A
Published: January 2005

The circadian activity of the hypothalamic‐pituitary‐adrenal (HPA) axis is regulated by caloric flow in rats. During the dark cycle, it has been shown that, in fasted rats, the time‐course profile of plasma concentrations of adrenocorticotropin (ACTH) and corticosterone parallels the profile of food intake in ad libitum fed animals.

Cholecystokinin (CCK) is involved in regulating food intake in rodents. CCK‐8 reduces food intake by acting on CCK‐A receptors subtype.

This work aims at establishing an eventual relationship between the modulatory role of CCK on food intake and its effect on HPA axis activity during fasting.

We studied the effect of CCK‐A and CCK‐B receptor antagonists on food intake during the first period of the dark cycle. Under these conditions we observed that the CCK‐A receptor antagonist, SR‐27897 (0.3 mg kg−1), but not the CCK‐B receptor antagonist, L‐365260 (1 mg kg−1), increases food‐intake.

In a second series of experiments we observed that the increase of both ACTH and corticosterone plasma level elicited by fasting, was prevented by SR‐27897, but not by L‐365260.

These results indicate that CCK‐A receptor blockade during fasting prevents the activation of the HPA axis.

British Journal of Pharmacology (2000) 129, 839–842; doi:10.1038/sj.bjp.0703117
Pharmacological characterization of small‐conductance Ca2+‐activated K+ channels stably expressed in HEK 293 cells
Authors: Strøbæk Dorte , Jørgensen Tino D , Christophersen Palle , Ahring Philip K , Olesen Søren‐Peter
Published: January 2005

Three genes encode the small‐conductance Ca2+‐activated K+ channels (SK channels). We have stably expressed hSK1 and rSK2 in HEK 293 cells and addressed the pharmacology of these subtypes using whole‐cell patch clamp recordings.

The bee venom peptide apamin blocked hSK1 as well as rSK2 with IC50 values of 3.3 nM and 83 pM, respectively.

The pharmacological separation between the subtypes was even more prominent when applying the scorpion peptide blocker scyllatoxin, which blocked hSK1 with an IC50 value of 80 nM and rSK2 at 287 pM.

The potent small molecule blockers showed little differentiation between the channel subtypes. The bis‐quinolinium cyclophane UCL 1684 blocked hSK1 with an IC50 value of 762 pM and rSK2 at 364 pM. The antiseptic compound dequalinium chloride blocked hSK1 and rSK2 with IC50 values of 444 nM and 162 nM, respectively.

The nicotinic acetylcholine receptor antagonist d‐tubocurarine was found to block hSK1 and rSK2 with IC50 values of 27 μM and 17 μM when measured at +80 mV. The inhibition by d‐tubocurarine was voltage‐dependent with increasing affinities at more hyperpolarized potentials.

The GABAA receptor antagonist bicuculline methiodide also blocked hSK1 and rSK2 in a voltage‐dependent manner with IC50 values of 15 and 25 μM when measured at +80 mV.

In conclusion, the pharmacological separation between SK channel subtypes expressed in mammalian cells is too small to support the notion that the apamin‐insensitive afterhyperpolarization of neurones is mediated by hSK1.

British Journal of Pharmacology (2000) 129, 991–999; doi:10.1038/sj.bjp.0703120
Bromoenol lactone enhances the permeabilization of rat submandibular acinar cells by P2X7 agonists
Authors: Chaïb N , Kabré E , Alzola E , Pochet S , Dehaye J P
Published: January 2005

The permeabilizing effect of P2X7 agonists was tested in rat submandibular acinar cells using the uptake of ethidium bromide as an index.

The uptake of ethidium bromide by acini incubated at 37°C in the presence of 1 mM ATP increased with time and reached after 5 min about 10% of maximal uptake measured in the presence of digitonin.

The response to ATP was dose‐dependent (half‐maximal concentration around 40 μM) and it was decreased when the temperature was lowered to 25°C.

Benzoyl‐ATP reproduced the response to ATP (half‐maximal concentration around 10 μM). UTP or 2‐methylthioATP had no effect.

The permeabilization in response to ATP was blocked by oxidized ATP and by magnesium and inhibited by Coomassie blue.

ATP increased the activity of a calcium‐insensitive phospholipase A2 (iPLA2).

Bromoenol lactone (BEL) inhibited the iPLA2 stimulated by ATP but potentiated the uptake of ethidium bromide in response to the purinergic agonist.

From these results it is concluded that the activation of P2X7 receptors permeabilizes rat submandibular acinar cells. The pore‐forming activity of the receptor might be negatively regulated by the concomitant activation of the iPLA2 by the receptor.

British Journal of Pharmacology (2000) 129, 703–708; doi:10.1038/sj.bjp.0703124
Decreased aortic glutathione levels may contribute to impaired nitric oxide‐induced relaxation in hypercholesterolaemia
Authors: Adachi Takeshi , Cohen Richard A
Published: January 2005

The aim of this study was to determine if the decrease in aortic total glutathione (GSH) levels in hypercholesterolaemia is related to the impairment of relaxation to acetylcholine (ACh) and exogenous nitric oxide (NO).

Isometric tension and vascular GSH levels were measured in thoracic aortic rings from rabbits fed for 12 weeks with 0.5% cholesterol diet. Hypercholesterolaemia decreased aortic GSH levels and impaired relaxation to ACh and NO.

To determine if GSH depletion impaired the response to NO, normal rabbit thoracic aorta was incubated with 1,3‐bis [2‐chloroethyl]‐1‐nitrosourea (BCNU; 0.2 mmol L−1), a GSH reductase inhibitor, or diazine‐dicarboxylic acid bis [N, N dimethylamide] (diamide; 1 mmol L−1), a thiol oxidizing agent. BCNU or diamide decreased aortic GSH levels and impaired ACh and NO‐induced relaxation. The effects of diamide on GSH levels and relaxation were partially prevented by co‐incubation with GSH ester (GSE; 2 mmol L−1).

Increasing GSH with GSE significantly enhanced NO‐induced relaxation in aorta from both hypercholesterolaemic and normal rabbits, however relaxation of hypercholesterolaemic rabbit aorta was not restored to normal.

These data suggest that other factors, perhaps related to the long‐term decrease in GSH levels, are responsible for reduced NO bioactivity in hypercholesterolaemia.

British Journal of Pharmacology (2000) 129, 1014–1020; doi:10.1038/sj.bjp.0703127
Exposure to glibenclamide increases rat beta cells sensitivity to glucose
Authors: Patanè Giovanni , Piro Salvatore , Anello Marcello , Rabuazzo Agata Maria , Vigneri Riccardo , Purrello Francesco
Published: January 2005

An increased sensitivity to glucose was observed in islets pre‐exposed for 1 h to glibenclamide (0.1 μmol 1−1), but not to tolbutamide (100 μmol l−1), as indicated by a shift to the left of the dose‐response curve (EC50 at 5.8±0.3 mmol l−1 glucose vs 10.6±0.8 in control islets; n=11, P<0.005).

According to this secretory pattern also glucose utilization at 2.5 and 5.0 mmol l−1 glucose was higher in islets exposed to glibenclamide.

Since binding to mitochondria results in an increased enzyme activity, we measured hexokinase (HK) and glucokinase (GK) activity both in a cytosolic and in a mitochondrion‐enriched fractions. Cytosolic hexokinase activity was similar in islets exposed to glibenclamide and in control islets but mitochondrial hexokinase activity was significantly increased after exposure to glibenclamide (124±7 vs 51±9 nmol μg prot−1 90 min−1, P<0.01), with no change in the enzyme protein content. In contrast, glucokinase activity in the two groups of islets was similar.

When in islets < exposed to glibenclamide hexokinase binding to mitochondria was inhibited by the addition of 20 nmol l−1 dicyclohexylcarbodiimide (DCC), no increase of glucose sensitivity was observed (EC50 10.9±1.3 mmol l−1 glucose, n=3, similar to that of control islets).

These data indicate that a 1 h exposure to glibenclamide causes the beta cell to become more sensitive to glucose. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity, in particular the mitochondrial‐bound, more active, form. This mechanism may contribute to the hypoglycemic action of this drug.

British Journal of Pharmacology (2000) 129, 887–892; doi:10.1038/sj.bjp.0703131
Electrophysiological effects of protopine in cardiac myocytes: inhibition of multiple cation channel currents
Authors: Song Long‐Sheng , Ren Guo‐Jun , Chen Zhao‐Luan , Chen Zhi‐He , Zhou Zhao‐Nian , Cheng Heping
Published: January 2005

Protopine (Pro) from Corydalis tubers has been shown to have multiple actions on cardiovascular system, including anti‐arrhythmic, anti‐hypertensive and negative inotropic effects. Although it was thought that Pro exerts its actions through blocking Ca2+ currents, the electrophysiological profile of Pro is unclear. The aim of this study is to elucidate the ionic mechanisms of Pro effects in the heart.

In single isolated ventricular myocytes from guinea‐pig, extracellular application of Pro markedly and reversibly abbreviates action potential duration, and decreases the rate of upstroke (dV/dt)max, amplitude and overshoot of action potential in a dose‐dependent manner. Additionally, it produces a slight, but significant hyperpolarization of the resting membrane potential.

Pro at 25, 50 and 100 μM reduces L‐type Ca2+ current (ICa,L) amplitude to 89.1, 61.9 and 45.8% of control, respectively, and significantly slows the decay kinetics of ICa,L at higher concentration. The steady state inactivation of ICa,L is shifted negatively by 5.9–7.0 mV (at 50–100 μM Pro), whereas the voltage‐dependent activation of ICa,L remains unchanged. In contrast, Pro at 100 μM has no evident effects on T‐type Ca2+ current (ICa,T).

In the presence of Pro, both the inward rectifier (IK1) and delayed rectifier (IK) potassium currents are variably inhibited, depending on Pro concentrations.

Sodium current (INa), recorded in low [Na+]o (40 mM) solution, is more potently suppressed by Pro. At 25 μM, Pro significantly attenuated INa at most of the test voltages (−60∼+40 mV, with a 53% reduction at −30 mV.

Thus, Pro is not a selective Ca2+ channel antagonist. Rather, it acts as a promiscuous inhibitor of cation channel currents including ICa,L, IK, IK1 as well as INa. These findings may provide some mechanistic explanations for the therapeutic actions of Pro in the heart.

British Journal of Pharmacology (2000) 129, 893–900; doi:10.1038/sj.bjp.0703132
P2Y purinoceptor subtypes recruit different Mek activators in astrocytes
Authors: Lenz Guido , Gottfried Carmem , Luo Zhijun , Avruch Joseph , Rodnight Richard , Nie Wie‐Jia , Kang Yuan , Neary Joseph T
Published: January 2005

Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P2Y receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P2Y receptors expressed in astrocytes have not been defined and it is not known whether all P2Y receptor subtypes are coupled to Erk by identical or distinct signalling pathways.

We found that the P2Y receptor agonists ATP, ADP, UTP and 2‐methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf‐1, the first kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf‐1 was not stimulated by ATP. Furthermore, ATP did not activate B‐Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3.

Reverse transcriptase‐polymerase chain reaction (RT–PCR) studies using primer pairs for cloned rat P2Y receptors revealed that rat cortical astrocytes express P2Y1, a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P2Y2 and P2Y4, subtypes in rats for which ATP and UTP are equipotent. Transcripts for P2Y6, a pyrimidine‐preferring receptor, were not detected.

ATP did not increase cyclic AMP levels, suggesting that P2Y11, an ATP‐preferringz receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes.

These signal transduction and RT–PCR experiments reveal differences in the activation of cRaf‐1 by P2Y receptor agonists that are inconsistent with properties of the P2Y1, P2Y2 and P2Y4 receptors shown to be expressed in astrocytes, i.e. ATP≠UTP; ATP≠2MeSATP, ADP. This suggests that the properties of the native P2Y receptors coupled to the Erk cascade differ from the recombinant P2Y receptors or that astrocytes express novel purine‐preferring and pyrimidine‐preferring receptors coupled to the ERK cascade.

British Journal of Pharmacology (2000) 129, 927–936; doi:10.1038/sj.bjp.0703138
Postsynaptic 5‐hydroxytryptamine1A receptor activation increases in vivo dopamine release in rat prefrontal cortex
Authors: Sakaue M , Somboonthum P , Nishihara B , Koyama Y , Hashimoto H , Baba A , Matsuda T
Published: January 2005

5‐Hydroxytryptamine (5‐HT) plays a role in the regulation of 3,4‐dihydroxyphenylethylamine (dopamine) neurons in the brain, but the precise mechanism of regulation by 5‐HT1A receptors of dopamine release has not been defined. The present study describes the effect of 5‐{3‐[[(2S)‐1,4‐benzodioxan‐2ylmethyl]amino]propoxy}‐1,3‐benzodioxole HCl (MKC‐242), a highly potent and selective 5‐HT1A receptor agonist, on dopamine release in the prefrontal cortex using microdialysis in the freely moving rat.

Subcutaneous injection of MKC‐242 (0.3–1.0 mg kg−1) increased extracellular levels of dopamine in the prefrontal cortex.

The effect of MKC‐242 in the prefrontal cortex was antagonized by pretreatment with the selective 5‐HT1A receptor antagonist, N‐[2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐(2‐pyridinyl)cyclohexanecarboxamide (WAY100635; 1 mg kg−1, i.p.).

Local application of WAY100635 (10 μM) via a microdialysis probe antagonized the effect of systemic MKC‐242 in an increasing dopamine release, and locally infused 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (10 μM) increased dopamine release in the prefrontal cortex.

MKC‐242 increased cortical dopamine release in the rats pretreated with 5,7‐dihydroxytryptamine (150 μg, i.c.v.) that caused an almost complete reduction in cortical 5‐HT content.

The effect of MKC‐242 to increase dopamine release was also observed in the hippocampus, but not in the striatum or nucleus accumbens.

Fluoxetine, a selective serotonin reuptake inhibitor, increased dopamine release in the prefrontal cortex, but not in the nucleus accumbens, while buspirone, a 5‐HT1A receptor agonist, increased dopamine release in both brain regions.

The present results indicate that activation of postsynaptic 5‐HT1A receptors increases dopamine release in a brain region‐specific manner.

British Journal of Pharmacology (2000) 129, 1028–1034; doi:10.1038/sj.bjp.0703139
The mechanism of bradykinin‐induced endothelium‐dependent contraction and relaxation in the porcine interlobar renal artery
Authors: Ihara Eikichi , Hirano Katsuya , Derkach Dmitry N , Nishimura Junji , Nawata Hajime , Kanaide Hideo
Published: January 2005

The mechanism of endothelium‐dependent regulation of vascular tone of bradykinin was investigated by simultaneously monitoring the changes in the cytosolic Ca2+ concentration and the force of smooth muscle in fura‐2‐loaded strips of the porcine renal artery with endothelium.

During phenylephrine‐induced sustained contraction, bradykinin (>3×10−9 M) caused endothelium‐dependent triphasic changes in the force of the strips, composed of an initial relaxation, a subsequent transient contraction and a late sustained relaxation.

At low concentrations (10−10–10−9 M), bradykinin caused an endothelium‐dependent biphasic relaxation with no contraction.

A thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor antagonist (10−5 M ONO‐3708) completely inhibited, while a TXA2 synthase inhibitor (10−5 M OKY‐046) only partially inhibited, the transient contraction induced by bradykinin.

Under conditions where the bradykinin‐induced contraction was inhibited by ONO‐3708 during the phenylephrine‐induced contraction, bradykinin induced only a transient relaxation in the presence of NΩ‐nitro‐L‐arginine methyl ester (L‐NAME). This transient relaxation was inhibited when the precontraction was initiated by phenylephrine plus 40 mM extracellular K+. The removal of L‐NAME from this condition caused a partial reappearance of the initial relaxation and a complete reappearance of the sustained relaxation.

In conclusion, bradykinin caused the endothelium‐dependent triphasic regulation of vascular tone in the porcine renal artery. The concentrations of bradykinin required to induce a contraction was higher than that required to induce relaxation. Both TXA2 and PGH2 were involved in the bradykinin‐induced contraction. The initial relaxation was mediated by nitric oxide and hyperpolarizing factors while the sustained relaxation depended on nitric oxide.

British Journal of Pharmacology (2000) 129, 943–952; doi:10.1038/sj.bjp.0703141
Harmane produces hypotension following microinjection into the RVLM: possible role of I1‐imidazoline receptors
Authors: Musgrave I F , Badoer E
Published: January 2005

The β‐carboline, harmane (0.1–1.0 nmol) produces dose dependent hypotension when microinjected unilaterally into the rostral ventrolateral medulla (RVLM) of the anaesthetized rat. The potency of harmane on blood pressure is similar to that of the imidazoline, clonidine. The hypotensive effects of both clonidine and harmane are reversed by microinjection of the relatively I1‐receptor selective antagonist efaroxan (20 nmol). These results are consistent with harmane acting at an I1‐receptor in the RVLM. This is the first report of an endogenous ligand for I1‐receptors that has central effects on blood pressure.

British Journal of Pharmacology (2000) 129, 1057–1059; doi:10.1038/sj.bjp.0703142
Potassium‐ and acetylcholine‐induced vasorelaxation in mice lacking endothelial nitric oxide synthase
Authors: Ding Hong , Kubes Paul , Triggle Chris
Published: January 2005

The contribution of an endothelium‐derived hyperpolarizing factor (EDHF) was investigated in saphenous and mesenteric arteries from endothelial nitric oxide synthase (eNOS) (−/−) and (+/+) mice.

Acetylcholine‐induced endothelium‐dependent relaxation of saphenous arteries of eNOS(−/−) was resistant to Nω‐nitro‐L‐arginine (L‐NNA) and indomethacin, as well as the guanylyl cyclase inhibitor, 1H‐(1,2,4)oxadiazolo(4,3‐a) quinoxalin‐1‐one(ODQ).

Potassium (K+) induced a dose‐dependent vasorelaxation which was endothelium‐independent and unaffected by either L‐NNA or indomethacin in both saphenous and mesenteric arteries from eNOS(−/−) or (+/+) mice.

Thirty μM barium (Ba2+) and 10 μM ouabain partially blocked potassium‐induced, but had no effect on acetylcholine‐induced vasorelaxation in saphenous arteries.

Acetylcholine‐induced relaxation was blocked by a combination of charybdotoxin (ChTX) and apamin which had no effect on K+‐induced relaxation, however, iberiotoxin (IbTX) was ineffective against either acetylcholine‐ or K+‐induced relaxation.

Thirty μM Ba2+ partially blocked both K+‐ and acetylcholine‐induced relaxation of mesenteric arteries, and K+, but not acetylcholine‐induced relaxation was totally blocked by the combination of Ba2+ and ouabain.

These data indicate that acetylcholine‐induced relaxation cannot be mimicked by elevating extracellular K+ in saphenous arteries from either eNOS(−/−) or (+/+) mice, but K+ may contribute to EDHF‐mediated relaxation of mesenteric arteries.

British Journal of Pharmacology (2000) 129, 1194–1200; doi:10.1038/sj.bjp.0703144