bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential abilities of phorbol esters in inducing protein kinase C (PKC) down‐regulation in noradrenergic neurones
Authors: Kotsonis P , Funk L , Prountzos C , Iannazzo L , Majewski H
Published: January 2005

The ability of several phorbol ester protein kinase C (PKC) activators (phorbol 12, 13‐dibutyrate, PDB; phorbol 12, 13‐diacetate, PDA; and 12‐deoxyphorbol 13‐acetate, dPA) to down‐regulate PKC was studied by assessing their effects on electrical stimulation‐induced (S‐I) noradrenaline release from rat brain cortical slices and phosphorylation of the PKC neural substrate B‐50 in rat cortical synaptosomal membranes.

In cortical slices which were incubated for 20 h with vehicle, acute application of PDB, PDA and dPA (0.1 – 3.0 μM) enhanced the S‐I noradrenaline release in a concentration‐dependent manner to between 200 – 250% of control in each case. In slices incubated with PDB (1 μM for 20 h), subsequent acute application of PDB (0.1 – 3.0 μM) failed to enhance S‐I release, indicating PKC down‐regulation. However, in tissues incubated with PDA or dPA (3 μM) for 20 h, there was no reduction in the facilitatory effect of their respective phorbol esters or PDB (0.1 – 3.0 μM) when acutely applied, indicating that PKC was not down‐regulated. This was confirmed using Western blot analysis which showed that PDB (1 μM for 20 h) but not PDA (3 μM for 20 h) caused a significant reduction in PKCα.

Incubation with PDB for 20 h, followed by acute application of PDB (3 μM) failed to increase phosphorylation of B‐50 in synaptosomal membranes, indicating down‐regulation. In contrast, tissues incubated with PDA or dPA for 20 h, acute application of their respective phorbol ester (10 μM) or PDB (3 μM) induced a significant increase in B‐50 phosphorylation.

Acutely all three phorbol esters elevate noradrenaline release to about the same extent, yet PDA and dPA have lower affinities for PKC compared to PDB, suggesting unique neural effects for these agents. This inability to cause functional down‐regulation of PKC extends their unusual neural properties. Their neural potency and lack of down‐regulation may be related to their decreased lipophilicity compared to other phorbol esters.

We suggest that PKC down‐regulation appears to be related to binding affinity, where agents with high affinity, irreversibly insert PKC into artificial membrane lipid and generate Ca2+‐independent kinase activity which degrades and deplete PKC. We suggest that this mechanism may also underlie the ability of PDB to down‐regulate PKC in nerve terminals, in contrast to PDA and dPA.

British Journal of Pharmacology (2001) 132, 489–499; doi:10.1038/sj.bjp.0703813
Neural mechanisms underlying migrating motor complex formation in mouse isolated colon
Authors: Brierley Stuart M , Nichols Kim , Grasby Dallas J , Waterman Sally A
Published: January 2005

Little is known about the intrinsic enteric reflex pathways associated with migrating motor complex (MMC) formation. Acetylcholine (ACh) mediates the rapid component of the MMC, however a non‐cholinergic component also exists. The present study investigated the possible role of endogenous tachykinins (TKs) in the formation of colonic MMCs and the relative roles of excitatory and inhibitory pathways.

MMCs were recorded from the circular muscle at four sites (proximal, proximal‐mid, mid‐distal and distal) along the mouse colon using force transducers.

The tachykinin (NK1 and NK2) receptor antagonists SR‐140 333 (250 nM) and SR‐48 968 (250 nM) reduced the amplitude of MMCs at all recording sites, preferentially abolishing the long duration contraction. Residual MMCs were abolished by the subsequent addition of atropine (1 μM).

The neuronal nitric oxide synthase inhibitor, Nωnitro‐L‐arginine (L‐NOARG, 100 μM), increased MMC amplitude in the distal region, whilst reducing the amplitude in the proximal region. In preparations where MMCs did not migrate to the distal colon, addition of L‐NOARG resulted in the formation of MMCs. Subsequent addition of apamin (250 nM) or suramin (100 μM) further increased MMC amplitude in the distal region, whilst suramin increased MMC amplitude in the mid‐distal region. Apamin but not suramin reduced MMC amplitude in the proximal region. Subsequent addition of SR‐140 333 and SR‐48 968 reduced MMC amplitude at all sites. Residual MMCs were abolished by atropine (1 μM).

In conclusion, TKs, ACh, nitric oxide (NO) and ATP are involved in the neural mechanisms underlying the formation of MMCs in the mouse colon. Tachykinins mediate the long duration component of the MMC via NK1 and NK2 receptors. Inhibitory pathways may be involved in determining whether MMCs are formed.

British Journal of Pharmacology (2001) 132, 507–517; doi:10.1038/sj.bjp.0703814
Activity of adenosine diphosphates and triphosphates on a P2YT‐type receptor in brain capillary endothelial cells
Authors: Simon J , Vigne P , Eklund K M , Michel A D , Carruthers A M , Humphrey P P A , Frelin C , Barnard E A
Published: January 2005

A P2Y (nucleotide) receptor activity in a clonal population (B10) of rat brain capillary endothelial cells is coupled to inhibition of adenylyl cyclase and has functional similarities to the P2YT (previously designated ‘P2T’) receptor for ADP of blood platelets. However, the only P2Y receptor which was detectable in a previous study of B10 cells by mRNA analysis was the P2Y1 receptor, which elsewhere shows no transduction via cyclic nucleotides. We have sought here to clarify these issues.

The inhibition of forskolin‐stimulated adenylyl cyclase induced by purified nucleotides was measured on B10 cells. The EC50 value for 2‐methylthioADP (2‐MeSADP) was 2.2 nM and, surprisingly, 2‐MeSATP was an almost equally strong agonist (EC50=3.5 nM). ATP and 2‐ClATP were weak partial agonists (EC50=26 μM and 10 μM respectively) and under appropriate conditions could antagonise the activity on 2‐MeSADP.

A known selective antagonist of the platelet P2YT receptor, 2‐propylthioadenosine‐5′‐(β,γ)‐difluoromethylene) triphosphonate (AR‐C 66096), was a competitive antagonist of this B10 cell receptor, with pKB=7.6. That ligand is inactive at the P2Y1 receptor in the same cells. Conversely, the competitive P2Y1 receptor antagonists, the 3′, 5′‐ and 2′, 5′‐adenosine bis‐monophosphates, are, instead, weak agonists at the adenylyl cyclase‐inhibitory receptor.

The inhibition of adenylyl cyclase by 2‐MeSADP was completely abolished by pertussis toxin.

In summary, these brain endothelial cells possess a P2YT‐type receptor in addition to the P2Y1 receptor. The two have similarities in agonist profiles but are clearly distinguishable by antagonists and by their second messenger activations. The possible relationships between the B10 and platelet P2YT receptors are discussed.

British Journal of Pharmacology (2001) 132, 173–182; doi:10.1038/sj.bjp.0703816
Activation of potassium conductance by ophiopogonin‐D in acutely dissociated rat paratracheal neurones
Authors: Ishibashi Hitoshi , Mochidome Takanobu , Okai Junpei , Ichiki Hiroyuki , Shimada Hideaki , Takahama Kazuo
Published: January 2005

The effect of ophiopogonin‐D (OP‐D), a steroidal glycoside and an active component of Bakumondo‐to, a Chinese herbal antitussive, on neurones acutely dissociated from paratracheal ganglia of 2‐week‐old Wistar rats was investigated using the nystatin‐perforated patch recording configuration.

Under current‐clamp conditions, OP‐D (10 μM) hyperpolarized the paratracheal neurones from a resting membrane potential of −65.7 to −73.5 mV.

At the concentration of 1 μM and above, OP‐D concentration‐dependently activated an outward current accompanied by an increase in the membrane conductance under voltage‐clamp conditions at a holding potential of −40 mV.

The reversal potential of the OP‐D‐induced current (IOP‐D) was −79.4 mV, which is close to the K+ equilibrium potential of −86.4 mV. The changes in the reversal potential for a 10 fold change in extracellular K+ concentration was 53.1 mV, indicating that the current was carried by K+.

The IOP‐D was blocked by an extracellular application of 1 mM Ba2+ by 59.0%, but other K+ channel blockers, including 4‐aminopyridine (3 mM), apamin (1 μM), charybdotoxin (0.3 μM), glibenclamide (1 μM), tolbutamide (0.3 mM) and tetraethylammonium (10 mM), did not inhibit the IOP‐D.

OP‐D also inhibited the ACh‐ and bradykinin‐induced depolarizing responses which were accompanied with firing of action potentials.

The results suggest that OP‐D may be of benefit in reducing the excitability of airway parasympathetic ganglion neurones and consequently cholinergic control of airway function and further, that the hyperpolarizing effect of OP‐D on paratracheal neurones via an activation of K+ channels might explain a part of mechanisms of the antitussive action of the agent.

British Journal of Pharmacology (2001) 132, 461–466; doi:10.1038/sj.bjp.0703818
Evidence for cocaine and methylecgonidine stimulation of M2 muscarinic receptors in cultured human embryonic lung cells
Authors: Yang Yinke , Ke Qingen , Cai Jingbo , Xiao Yong‐Fu , Morgan James P
Published: January 2005

Muscarinic cholinoceptor stimulation leads to an increase in guanylyl cyclase activity and to a decrease in adenylyl cyclase activity. This study examined the effects of cocaine and methylecgonidine (MEG) on muscarinic receptors by measurement of cyclic GMP and cyclic AMP content in cultured human embryonic lung (HEL299) cells which specifically express M2 muscarinic receptors.

A concentration‐dependent increase in cyclic GMP production was observed in HEL299 cells incubated with carbachol, cocaine, or MEG for 24 h. The increase in cyclic GMP content was 3.6 fold for 1 μM carbachol (P<0.01), 3.1 fold for 1 μM cocaine (P<0.01), and 7.8 fold for 1 μM MEG (P<0.001), respectively. This increase in cyclic GMP content was significantly attenuated or abolished by the muscarinic receptor antagonist atropine or the M2 blocker methoctramine.

In contrast, cocaine, MEG, and carbachol produced a significant inhibition of cyclic AMP production in HEL299 cells. Compared to the control, HEL299 cells treated with 1 μM cocaine decreased cyclic AMP production by 30%. MEG and carbachol at 1 μM decreased cyclic AMP production by 37 and 38%, respectively. Atropine or methoctramine at 1 or 10 μM significantly attenuated or abolished the cocaine‐induced decrease in cyclic AMP production. However, the antagonists alone had neither an effect on cyclic GMP nor cyclic AMP production. Pretreatment of HEL299 cells with pertussis toxin prevented the cocaine‐induced reduction of cyclic AMP production.

Western blot analysis showed that HEL299 cells specifically express M2 muscarinic receptors without detectable M1 and M3. Incubation of HEL299 cells with cocaine, carbachol, and atropine did not alter the expression of M2 protein levels. However, the inducible isoform of nitric oxide synthase (iNOS) was induced in the presence of cocaine or carbachol and this induction was significantly attenuated after addition of atropine or methoctramine.

The present data show that cocaine and MEG significantly affect cyclic GMP and cyclic AMP production in cultured HEL299 cells. Our results also show that these effects result from the drug‐induced stimulation of M2 muscarinic receptors accompanied with no alterations of receptor expression. However, the induction of iNOS by cocaine may result in the increase in cyclic GMP production.

British Journal of Pharmacology (2001) 132, 451–460; doi:10.1038/sj.bjp.0703819
In vivo evidence for KCa channel opening properties of acetazolamide in the human vasculature
Authors: Pickkers Peter , Hughes Alun D , Russel Frans G M , Thien Theo , Smits Paul
Published: January 2005

The selective carbonic anhydrase inhibitor acetazolamide is known to increase blood flow in several organs. Acetazolamide directly dilates isolated resistance arteries associated with activation of calcium‐activated potassium (KCa) channels. We examined both the presence and mechanism of the direct vascular action of acetazolamide in vivo in humans.

Forearm vasodilator responses of 30 healthy volunteers to infusion of placebo and increasing doses of acetazolamide (1‐3‐10 mg min−1 dl−1) into the brachial artery were recorded by venous occlusion plethysmography, before and after local administration of L‐NMMA (0.2 mg min−1 dl−1, an inhibitor of NO‐synthase, n=6), indomethacin (5.0 μg min−1 dl−1, an inhibitor of prostaglandin synthesis, n=6), glibenclamide (10 μg min−1 dl−1, an inhibitor of KATP channels, n=6), tetraethylammonium (0.1 mg min−1 dl−1, an inhibitor of KCa channels, n=6) or placebo (NaCl 0.9%, n=6). Lower dosages of acetazolamide did not affect vascular tone (n=6).

Acetazolamide infusions increased forearm blood flow from 2.41±0.17 to 2.99±0.18, 4.09±0.26 and 6.77±0.49 ml min−1 dl−1 in the infused forearm (P<0.001), with no significant changes in the non‐infused forearm, blood pressure or heart rate. Acetazolamide‐induced vasodilation was not inhibited by L‐NMMA, indomethacin, or glibenclamide but was significantly attenuated by TEA (vasodilation: 23±6, 82±19, 241±38% versus 27±8, 44±22, 42±35%).

We conclude that acetazolamide exerts a direct vasodilator effect in vivo in humans mediated by vascular KCa channel activation. This makes acetazolamide the first drug known that specifically modulates this channel.

British Journal of Pharmacology (2001) 132, 443–450; doi:10.1038/sj.bjp.0703825
Separation of cannabinoid receptor affinity and efficacy in delta‐8‐tetrahydrocannabinol side‐chain analogues
Authors: Griffin Graeme , Williams Stephanie , Aung Mie Mie , Razdan Raj K , Martin Billy R , Abood Mary E
Published: January 2005

The activities of a number of side‐chain analogues of delta‐8‐tetrahydrocannabinol (Δ8‐THC) in rat cerebellar membrane preparations were tested.

The affinities of each compound for the CB1 receptor were compared by their respective abilities to displace [3H]‐SR141716A and their efficacies compared by stimulation of [35S]‐GTPγS binding.

It was found that the affinities varied from 0.19±0.03 nM for 3‐norpentyl‐3‐[6′‐cyano,1′,1′‐dimethyl]hexyl‐Δ8‐THC to 395±66.3 nM for 5′‐[N‐(4‐chlorophenyl)]‐1′,1′‐dimethyl‐carboxamido‐Δ8‐THC.

The efficacies of these compounds varied greatly, ranging from the very low efficacy exhibited to acetylenic compounds such as 1′‐heptyn‐Δ8‐THC and 4′‐octyn‐Δ8‐THC to higher efficacy compounds such as 5′‐(4‐cyanophenoxy)‐1′,1′‐dimethyl‐Δ8‐THC and 5′‐[N‐(4‐aminosulphonylphenyl)]‐1′,1′ dimethyl‐carboxamido Δ8‐THC. All agonist activities were antagonized by the CB1‐selective antagonist SR141716A.

It was found that a ligand's CB1 affinity and efficacy are differentially altered by modifications in the side‐chain. Decreasing the flexibility of the side‐chain reduced efficacy but largely did not alter affinity. Additionally, the positioning of electrostatic moieties, such as cyano groups, within the side‐chain also has contrasting effects on these two properties.

In summary, this report details the characterization of a number of novel Δ8‐THC analogues in rat cerebellar membranes. It provides the first detailed pharmacological analysis of how the inclusion of electrostatic moieties in the side‐chain and also how alteration of the side‐chain's flexibility may differentially affect a CB1 cannabinoid receptor ligand's affinity and efficacy.

British Journal of Pharmacology (2001) 132, 525–535; doi:10.1038/sj.bjp.0703827
β1‐Adrenoceptors compensate for β3‐adrenoceptors in ileum from β3‐adrenoceptor knock‐out mice
Authors: Hutchinson Dana S , Evans Bronwyn A , Summers Roger J
Published: January 2005

This study examines β1‐, β2‐ and β3‐adrenoceptor (AR)‐mediated responses, mRNA levels and radioligand binding in ileum from β3‐AR knock‐out (−/−) (KO) and wild type (+/+) (FVB) mice.

In KO and FVB mice, SR59230A (100 nM) (β3‐AR antagonist) antagonized responses to (−)‐isoprenaline in both KO and FVB mice. (−)‐Isoprenaline mediated relaxation of ileum was antagonized weakly by ICI118551 (100 nM) (β2‐AR antagonist). Responses to (−)‐isoprenaline were more strongly antagonized by CGP20712A (100 nM) (β1‐AR antagonist), propranolol (1 μM) (β1‐/β2‐AR antagonist), carvedilol (100 nM) (non‐specific β‐AR antagonist), and CGP12177A (100 nM) (β1‐/β2‐AR antagonist) in ileum from KO than in FVB mice.

Responses to CL316243 (β3‐AR agonist) in ileum from FVB mice were antagonized by SR59230A (100 nM) but not by propranolol (1 μM) or carvedilol (100 nM). CL316243 was ineffective in relaxing ileum from KO mice.

CGP12177A had no agonist actions in ileum from either KO or FVB mice.

β1‐AR mRNA levels were increased 3 fold in ileum from KO compared to FVB mice. This was associated with an increased maximum number of β1‐/β2‐AR binding sites (Bmax). β2‐AR mRNA levels were unaffected while no β3‐AR mRNA was detected in KO mice.

In mouse ileum, β3‐ARs and to a lesser extent β1‐ARs are the predominant adrenoceptor subtypes mediating relaxation in ileum from FVB mice. In KO mice β1‐ARs functionally compensate for the lack of β3‐ARs, and this is associated with increased β1‐AR mRNA and levels of binding.

British Journal of Pharmacology (2001) 132, 433–442; doi:10.1038/sj.bjp.0703828
Improved diabetic syndrome in C57BL/KsJ‐db/db mice by oral administration of the Na+‐glucose cotransporter inhibitor T‐1095
Authors: Arakawa Kenji , Ishihara Tomomi , Oku Akira , Nawano Masao , Ueta Kiichiro , Kitamura Kazuyuki , Matsumoto Mamoru , Saito Akira
Published: January 2005

The therapeutic effects of an orally active inhibitor of Na+‐glucose cotransporter (SGLT), T‐1095 (a derivative of phlorizin; 3‐(benzo[b]furan‐5‐yl)‐2′,6′‐dihydroxy‐4′‐methylpropiophenone 2′‐O‐(6‐O‐methoxycarbonyl‐β‐D‐glycopyranoside)) were examined in C57BL/KsJ‐db/db (db/db) mice, a genetic animal model of obese type 2 diabetes.

The higher renal SGLT activity in db/db mice than normoglycaemic C57BL/KsJ‐db/+m (db/+m) mice may support the rationale for using an SGLT inhibitor in the treatment regimen for type 2 diabetes. Both T‐1095 and its metabolite, T‐1095A, which had approximately 10 times more potency, effectively inhibited renal SGLT activity of these mice in vitro.

Single oral administration of T‐1095 (10, 30, 100 mg kg−1, p.o.) to db/db mice caused a dose‐dependent reduction in blood glucose levels and a concomitant increase in glucose excretion into urine. In contrast, T‐1095 only slightly affected blood glucose levels in db/+m mice.

Chronic administration of T‐1095 (0.1% w w−1 pellet chow, for 12 weeks) decreased blood glucose and haemoglobin A1C levels, and improved glucose intolerance in db/db mice. The age‐related decrease in plasma insulin levels was markedly inhibited and there was a 2.5 fold increase of insulin content in the pancreas of T‐1095‐treated db/db mice. Food consumption was not changed, while impaired body weight gain was ameliorated by T‐1095 treatment.

Both the development of albuminuria and the expansion of glomerular mesangial area in db/db mice were significantly suppressed by chronic T‐1095 treatment, indicating the prevention of the progression of diabetic nephropathy.

These results demonstrate that the SGLT inhibitor T‐1095 is able to improve the metabolic abnormalities and inhibit the development of diabetic complications in db/db mice. Thus, T‐1095 can be used for therapy of type 2 diabetic patients.

British Journal of Pharmacology (2001) 132, 578–586; doi:10.1038/sj.bjp.0703829
A N‐terminal PTHrP peptide fragment void of a PTH/PTHrP‐receptor binding domain activates cardiac ETA receptors
Authors: Schlüter Klaus‐Dieter , Katzer Christian , Piper Hans Michael
Published: January 2005

Adult ventricular cardiomyocytes show an unusual structure‐function relationship for cyclic AMP‐dependent effects of PTHrP. We investigated whether PTHrP(1 – 16), void of biological activity on classical PTHrP target cells, is able to mimic the positive contractile effect of PTHrP(1 – 34), a fully biological agonist on cardiomyocytes.

Adult ventricular cardiomyocytes were paced at a constant frequency of 0.5 Hz and cell contraction was monitored using a cell‐edge‐detection system. Twitch amplitudes, expressed as per cent cell shortening of the diastolic cell length, and rate constants for maximal contraction and relaxation velocity were analysed.

PTHrP(1 – 16) (1 μmol l−1) mimicked the contractile effects of PTHrP(1 – 34) (1 μmol l−1). It increased the twitch amplitude from 5.33±0.72 to 8.95±1.10 (% dl l−1) without changing the kinetic of contraction.

PTH(1 – 34) (10 μmol l−1) affected the positive contractile effect of PTHrP(1 – 34), but not that of PTHrP(1 – 16).

RpcAMPS (10 μmol l−1) inhibited the positive contractile effect of PTHrP(1 – 34), but not that of PTHrP(1 – 16).

The positive contractile effect of PTHrP(1 – 16) was antagonized by the ETA receptor antagonist BQ123.

Sarafotoxin 6b and PTHrP(1 – 16), but not PTHrP(1 – 34), replaced 3H‐BQ123 from cardiac binding sites.

We conclude that N‐terminal PTHrP peptides void of a PTH/PTHrP‐receptor binding domain are able to bind to, and activate cardiac ETA receptors.

British Journal of Pharmacology (2001) 132, 427–432; doi:10.1038/sj.bjp.0703830
Evidence for expression and function of phosphodiesterase type 5 (PDE‐V) in rat resistance arteries
Authors: Sampson L J , Hinton J M , Garland C J
Published: January 2005

Evidence is provided for expression and a functional role for phosphodiesterase type V (PDE‐V) in the rat isolated small mesenteric artery. The reverse transcription polymerase chain reaction (RT – PCR) demonstrated mRNA for PDE‐V, while Western blotting and immunocytochemical studies showed corresponding protein expression. Smooth muscle relaxation to the nitric oxide donor, diethylamine NONOate (DEA NONOate; 1 nM – 10 μM; pEC50=6.7±0.3) was potentiated significantly by the specific inhibitor of PDE‐V, 4‐[[3,4‐(methylenedioxy)benzyl]amino]‐6‐chloroquinazoline (MBCQ; 1 μM; pEC50=10.5±0.04). These data show that PDE‐V is expressed in both the smooth muscle and endothelial cells of a resistance artery, and the enzyme can significantly influence nitric oxide‐evoked vasorelaxation.

British Journal of Pharmacology (2001) 132, 13–17; doi:10.1038/sj.bjp.0703831
Differential modulation by GTPγS of agonist and inverse agonist binding to h5‐HT1Areceptors revealed by [3H]‐WAY100,635
Authors: Newman‐Tancredi Adrian , Verrièle Laurence , Millan Mark J
Published: January 2005

The interaction of serotonergic ligands at human (h) 5‐HT1Areceptors expressed in Chinese hamster ovary cells was examined with the selective ‘neutral’ 5‐HT1Aantagonist [3H]‐WAY100,635. Its binding was saturable (KD=0.056 nM) with a Bmax(3.65 pmol mg−1) significantly higher than that of two other selective 5‐HT1Aradioligands: the partial agonist, [3H]‐S15535 (2.77 pmol mg−1) and the agonist, [3H]‐8‐OH‐DPAT (2.02 pmol mg−1).

The influence of GTPγS (100 μM) on the binding affinity of 15 serotonergic agonists, partial agonists, antagonists and inverse agonists was investigated in competition binding experiments with [3H]‐WAY100,635.

Agonists, including 5‐HT, 8‐OH‐DPAT and buspirone, displayed biphasic isotherms which shifted to the right in the presence of GTPγS. In contrast, isotherms of theinverseagonists, methiothepin, (+)butaclamol and spiperone, were shifted to the left in the presence of GTPγS. Unlabelled WAY100,635 was the only ligand that was unaffected by GTPγS, consistent with ‘neutral’ antagonist properties.

The magnitude of affinity changes induced by GTPγS for 13 ligands was highly correlated (r=0.98) with their efficacy (positive and negative) previously determined by [35S]GTPγS binding.

In contrast, the napthylpiperazine derivative and high efficacy agonist, S14506, displayed only a modest GTPγS shift, in accordance with previous indications of ‘atypical’ binding properties of this ligand. A further full agonist, S14671, which is chemically closely‐related to S14506, also displayed a minimal GTPγS shift, underpinning this observation.

In conclusion, [3H]‐WAY100,635 constitutes a useful neutral antagonist radioligand for the characterization of drug actions at h5‐HT1Areceptors. GTPγS‐induced affinity changes of agonist and inverse agonist competition isotherms generally correlate well with ligand efficacy, with the notable exception of two chemically‐similar agents, S14506 and S14671, which are efficacious agonists, yet relatively insensitive to h5‐HT1Areceptor/G‐protein coupling changes.

British Journal of Pharmacology(2001)132, 518–524; doi:10.1038/sj.bjp.0703832
Contractile actions of proteinase‐activated receptor‐derived polypeptides in guinea‐pig gastric and lung parenchymal strips: evidence for distinct receptor systems
Authors: Saifeddine M , Al‐Ani B , Sandhu S , Wijesuriya S J , Hollenberg M D
Published: January 2005

We have measured the contractile activities and relative potencies (EC50s) of six thrombin PAR1 receptor‐derived receptor‐activating peptides (PAR‐APs): AparafluroFRChaCit‐y‐NH2 (Cit‐NH2); SFLLRNP(P7); SFLLRNP‐NH2 (P7‐NH2); SFLLR (P5); SFLLR‐NH2 (P5‐NH2); TFLLR‐NH2 (TF‐NH2) and a PAR2 receptor activating peptide [SLIGRL‐NH2 (SL‐NH2)] (a) in a guinea‐pig lung peripheral parenchymal strip preparation and (b) in a gastric longitudinal smooth muscle preparation.

The relative potencies of the PAR‐APs in the lung preparation (Cit‐NH2≅TF‐NH2≅P5‐NH2>P7≅P5≅P7‐NH2; SL‐NH2 not active) differed appreciably from their relative potencies in the gastric preparation: Cit‐NH2≅TF‐NH2≅P7‐NH2≅P5‐NH2>P7≅percnt;SL‐NH2.

The contractile actions of the PAR1‐selective peptide, TF‐NH2 in the gastric preparation were entirely dependent on extracellular calcium and were blocked by tyrosine kinase inhibitors (genistein, tyrphostin 47/AG213, PP1) and by the cyclooxygenase inhibitor, indomethacin, whereas in the lung preparation, the PAR1‐mediated contractile response was only partially dependent on extracellular calcium and was refractory to the actions of either tyrosine kinase inhibitors or indomethacin.

Partial sequencing of the PAR cDNAs detected by RT – PCR both in whole lung and in the peripheral parenchymal strip bioassay tissue demonstrated the presence of both PAR1 and PAR2 mRNA; the expression of PAR2 was detected by immunohistochemistry.

The data point to the presence of distinct receptor systems for the PAR1‐APs in guinea‐pig lung parenchymal and gastric smooth muscle and indicate that PAR2 does not regulate contractile activity in peripheral parenchymal guinea‐pig lung tissue

British Journal of Pharmacology (2001) 132, 556–566; doi:10.1038/sj.bjp.0703839
Pharmacological manipulation of the inflammatory cascade by the superoxide dismutase mimetic, M40403
Authors: Salvemini Daniela , Mazzon Emanuela , Dugo Laura , Riley Dennis P , Serraino Ivana , Caputi Achille P , Cuzzocrea Salvatore
Published: January 2005

M40403 is a low molecular weight, synthetic manganese containing superoxide dismutase mimetic (SODm) that removes superoxide anions (.O2−) without interfering with other reactive species known to be involved in inflammatory responses (e.g. nitric oxide, NO and peroxynitrite, ONOO‐).

As such, M40403 represents an important pharmacological tool to dissect the roles of .O2− in acute and chronic inflammation. For this purpose, the pharmacological profile of M40403 was evaluated in carrageenan‐induced pleurisy.

Injection of carrageenan into the pleural cavity of rats elicited an acute inflammatory response characterized by: fluid accumulation in the pleural cavity which contained a large number of neutrophils (PMNs) as well as an infiltration of PMNs in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx), prostaglandin E2 (PGE2), tumour necrosis factor α, (TNFα), interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and interleukin‐10 (IL‐10).

All parameters of inflammation were attenuated by M40403 except for NOx, PGE2 and IL‐10 which remained unaltered. Furthermore, carrageenan induced an upregulation of the adhesion molecules ICAM‐1 and P‐selectin, as well as nitrotyrosine and poly (ADP‐ribose) synthetase (PARS) as determined by immunohistochemical analysis of lung tissues.

The degree of staining for the ICAM‐1, P‐selectin, nitrotyrosine and PARS was reduced by M40403.

These results clearly indicate that .O2− plays a critical role in the development of the inflammatory response by altering key components of the inflammatory cascade. Therefore, synthetic enzymes of SOD such as M40403, offers a novel therapeutic approach for the management of various inflammatory diseases where these radicals have been postulated to play a role.

British Journal of Pharmacology (2001) 132, 815–827; doi:10.1038/sj.bjp.0703841
Effect of chronic morphine treatment on α2‐adrenoceptor mediated autoinhibition of transmitter release from sympathetic varicosities of the mouse vas deferens
Authors: Karunanithi Shanker , Lavidis Nickolas A
Published: January 2005

The effect of chronic morphine treatment (CMT) on sympathetic innervation of the mouse vas deferens and on α2‐adrenoceptor mediated autoinhibition has been examined using intracellular recording of excitatory junction potentials (EJPs) and histochemistry.

In chronically saline treated (CST) preparations, morphine (1 μM) and the α2‐adrenoceptor agonist (clonidine, 1 μM) decreased the mean amplitude of EJPs evoked with 0.03 Hz stimulation by 81±8% (n=16) and 92±6% (n=7) respectively. In CMT preparations, morphine (1 μM) and clonidine (1 μM) decreased mean EJP amplitude by 68±8% (n=7) and 79±8% (n=7) respectively.

When stimulating the sympathetic axons at 0.03 Hz, the mean EJP amplitude recorded from smooth muscles acutely withdrawn from CMT was four times greater than for CST smooth muscles (40.7±3.8 mV, n=7 compared with 9.9±0.3 mV, n=7).

Part of the increase in mean EJP amplitude following CMT was produced by a 31% increase in the density of sympathetic axons and varicosities innervating the smooth muscle.

Results from the present study indicate that the effectiveness of α2‐adrenoceptor mediated autoinhibition is only slightly reduced in CMT preparations. Most of the cross tolerance which develops between morphine, clonidine and α2‐adrenoceptor mediated autoinhibition occurs as a consequence of increased efficacy of neuromuscular transmission which is produced by an increase in the probability of transmitter release and an increase in the density of sympathetic innervation.

British Journal of Pharmacology (2001) 132, 403–410; doi:10.1038/sj.bjp.0703842
Stereoselective effects of the enantiomers of a new local anaesthetic, IQB‐9302, on a human cardiac potassium channel (Kv1.5)
Authors: González T , Longobardo M , Caballero R , Delpón E , Sinisterra J V , Tamargo J , Valenzuela C
Published: January 2005

The N‐substituent of IQB‐9302 has the same number of carbons as bupivacaine, but it exhibits a different spatial localization (n‐butyl vs cyclopropylmethyl). Thus, the study of the effects of IQB‐9302 enantiomers on hKv1.5 channels will lead to a better knowledge of the determinants of stereoselective block.

The effects of the IQB‐9302 enantiomers were studied on hKv1.5 channels stably expressed in Ltk− cells using the whole‐cell configuration of the patch‐clamp technique. Drug molecular modelling was performed using Hyperchem software.

Block induced by IQB‐9302 was stereoselective with the R(+) enantiomer being 3.2‐fold more potent than the S(−) one (KD of 17.8±0.5 μM vs 58.6±4.0 μM).

S(−)‐ and R(+)IQB‐9302 induced‐block was time‐ and voltage‐dependent consistent with an electrical distance from the cytoplasmic side of 0.173±0.022 (n=12) and 0.181±0.018 (n=10), respectively.

Potency of block of pipecoloxylidide local anaesthetics was linearly related to the length between the cationic tertiary amine and the end of the substituent.

Molecular modelling shows that only when S(−) and R(+) enantiomers are superimposed by their aromatic ring, their N‐substituents are in opposite directions, which can explain the stereospecific block induced by bupivacaine and IQB‐9302 with hKv1.5 channels.

These results suggest that: (a) IQB‐9302 enantiomers block the open state of hKv1.5 channels, and (b) the length of the N‐substituent in these local anaesthetics and not its volume determines the potency and degree of their stereoselective hKv1.5 channel block.

British Journal of Pharmacology (2001) 132, 385–392; doi:10.1038/sj.bjp.0703844
Molecular and pharmacological evidence for modulation of kinin B1 receptor expression by endogenous glucocorticoids hormones in rats
Authors: Cabrini Daniela A , Campos Maria M , Tratsk Karla S , Merino Vanessa F , Silva José A , Souza Glória E P , Avellar Maria C W , Pesquero João B , Calixto João B
Published: January 2005

The effect of endogenous glucocorticoid hormones on the expression of rat B1 receptors was examined by means of molecular and pharmacological functional approaches.

Rats were adrenalectomized (ADX), and 7 days after this procedure the intradermal injection of B1 receptor agonist des‐Arg9‐BK produced a significant increase in the paw volume, while only a weak effect was observed in sham‐operated animals. A similar increase in the contractile responses mediated by B1 agonist des‐Arg9‐BK was also observed in the rat portal vein in vitro.

Chemical ADX performed with mitotane (a drug that reduces corticosteroid synthesis) produced essentially the same up‐regulation of B1 receptors as that observed in ADX rats.

The modulation of B1 receptor expression was evaluated by ribonuclease protection assay, employing mRNA obtained from the lungs and paw of ADX rats.

Additionally, both paw oedema and contraction of portal vein mediated by B1 agonist des‐Arg9‐BK in ADX rats, were markedly inhibited by treatment with dexamethasone, or COX‐2 inhibitor meloxican, or with the NF‐κB inhibitor PDTC. Interestingly, the same degree of inhibition was achieved when the animals were treated with a combination of submaximal doses of dexamethasone and PDTC.

The involvement of NF‐κB pathway was further confirmed by mobility shift assay using nuclear extracts from lung, paw and heart of ADX rats. It was also confirmed that the treatment of ADX rats with dexamethasone, PDTC or dexamethasone plus PDTC completely inhibit NF‐κB activation caused by absence of endogenous glucucorticoid.

Together, the results of the present study provide, for the first time, molecular and pharmacological evidence showing that B1 kinin receptor expression can be regulated through endogenous glucocorticoids by a mechanism dependent on NF‐κB pathway. Clinical significance of the present findings stem from evidence showing the importance of B1 kinin receptors in the mediation of inflammatory and pain related responses.

British Journal of Pharmacology (2001) 132, 567–577; doi:10.1038/sj.bjp.0703846
Mediator involvement in antigen‐induced bronchospasm and microvascular leakage in the airways of ovalbumin sensitized Brown Norway rats
Authors: Hele Dave J , Birrell Mark A , Webber Stephen E , Foster Martyn L , Belvisi Maria G
Published: January 2005

To determine which mediators are involved in antigen‐induced bronchospasm and microvascular leakage in the airways of ovalbumin sensitised Brown Norway rats we investigated the effect of a histamine H1 receptor antagonist, mepyramine, a 5‐HT receptor antagonist, methysergide, and a cys‐leukotriene‐1 receptor antagonist, montelukast.

Ovalbumin at 1 mg kg−1 i.v. caused a significant increase in microvascular leakage in the airways and at 3 mg kg−1 i.v. caused a significant increase in airways resistance.

Histamine (1 mg kg−1 i.v.), 5‐HT (0.1 mg kg−1 i.v.) and leukotriene D4 (LTD4, 50 μg kg−1 i.v.) caused a significant increase in microvascular leakage in the airways.

Mepyramine (1 mg kg−1 i.v.), methysergide (0.1 mg kg−1 i.v.), or montelukast (30 mg kg−1 i.v.) inhibited histamine, 5‐HT or LTD4 ‐induced microvascular leakage respectively.

Methysergide (0.1 mg kg−1 i.v.) reduced ovalbumin‐induced microvascular leakage in the trachea and at 0.3 mg kg−1 i.v. inhibited bronchospasm (38 and 58%, respectively). Montelukast (30 mg kg−1 p.o.) reduced ovalbumin‐induced microvascular leakage in airway tissue to basal levels (78%) and inhibited ovalbumin‐induced bronchospasm (50%). Mepyramine (3 mg kg−1 i.v.) had no effect on ovalbumin‐induced leakage or bronchospasm.

A combination of all three compounds (mepyramine, methysergide and montelukast) reduced ovalbumin‐induced microvascular leakage in airway tissue to basal levels (70 – 78%) and almost completely inhibited bronchospasm (92%).

Antigen‐induced bronchospasm appears to equally involve the activation of 5‐HT and cys‐leukotriene‐1 receptors whereas ovalbumin‐induced microvascular leakage appears to be predominantly mediated by cys‐leukotriene‐1 receptors.

British Journal of Pharmacology (2001) 132, 481–488; doi:10.1038/sj.bjp.0703847
Regulation of 5‐HT1A receptor‐stimulated [35S]‐GTPγS binding as measured by quantitative autoradiography following chronic agonist administration
Authors: Hensler Julie G , Durgam Hymavathi
Published: January 2005

Because changes 5‐HT1A receptor number do not occur following repeated agonist treatment, we hypothesized that the basis for 5‐HT1A receptor desensitization involves changes in receptor‐G protein coupling. We measured the effect of repeated agonist administration on 5‐HT1A receptor‐stimulated [35S]‐GTPγS binding in forebrain areas, (i.e. anterior cingulate cortex, lateral septum, hippocampus, entorhinal cortex), and serotonergic cell body areas, the dorsal and median raphe nuclei.

Following treatment of rats with (±)8‐OH‐DPAT (1 mg kg−1, s.c.) for 7 or 14 days, 5‐HT1A receptor‐stimulated [35S]‐GTPγS binding was significantly attenuated in both the dorsal and median raphe nuclei.

5‐HT1A receptor‐stimulated [35S]‐GTPγS binding was significantly attenuated in the CA1 region of the hippocampus after 7, but not 14 days of 8‐OH‐DPAT administration. 5‐HT1A receptor‐stimulated [35S]‐GTPγS binding was not altered in other forebrain areas examined.

The binding of [3H]‐MPPF to 5‐HT1A receptor sites was not altered in any brain region examined following repeated agonist administration, suggesting that the observed changes in (±)8‐OH‐DPAT‐stimulated [35S]‐GTPγS binding were not due to changes in 5‐HT1A receptor number.

Our data indicate that in serotonergic cell body areas the regulation of presynaptic 5‐HT1A receptor function following repeated agonist administration occurs at the level of receptor‐G protein interaction. In forebrain areas, however, the regulation of postsynaptic 5‐HT1A receptor sensitivity appears not to be at the level of receptor‐G protein coupling.

British Journal of Pharmacology (2001) 132, 605–611; doi:10.1038/sj.bjp.0703855
α4 integrin‐dependent eosinophil recruitment in allergic but not non‐allergic inflammation
Authors: Teixeira Mauro M , Robinson Martyn K , Shock Anthony , Hellewell Paul G
Published: January 2005

Although anti‐α4 integrin mAbs reduce eosinophil accumulation in several models of allergic inflammation, it is not clear whether this occurs via a direct action to block eosinophil α4 integrins or indirectly on another cell type. The role of α4 integrins on the accumulation of 111In‐labelled eosinophils in allergic and non‐allergic inflammation in guinea‐pig skin was therefore investigated.

Intradermal injection of antigen in sensitized skin sites induced accumulation of 111In‐eosinophils that was reduced up to 70% by two anti‐α4 integrin mAbs. In contrast, accumulation of 111In‐eosinophils to intradermal chemoattractants was unaffected by the same mAbs.

Accumulation of 111In‐eosinophils in allergic and non‐allergic conditions was partly inhibited by a low dose of an anti‐β2 integrin mAb. In combination with anti‐α4 integrin mAb, responses were not further reduced suggesting that these adhesion pathways are not additive or synergic.

Pretreating skin sites with antiserum or contaminating LPS did not reveal an α4 integrin dependent pathway for chemoattractant‐induced 111In‐eosinophil accumulation. These data suggest that α4 integrins are involved in the response to antigen in sensitized skin sites.

Pretreating 111In‐eosinophil with α4 integrin mAb blocked their adhesion to fibronectin in vitro but did not inhibit their accumulation in allergic inflammation suggesting that the blocking effect in vivo was eosinophil independent.

These data support the concept that targeting α4 integrins on cells other than eosinophils could control eosinophil accumulation and have therapeutic potential in allergic diseases such as asthma and atopic dermatitis.

British Journal of Pharmacology (2001) 132, 596–604; doi:10.1038/sj.bjp.0703857
Assessment of CFTR chloride channel openers in intact normal and cystic fibrosis murine epithelia
Authors: Cuthbert Alan W
Published: January 2005

A method is described for the detection of CFTR chloride channel openers (ClCOs) and blockers. Murine colonic epithelia were used throughout, but the method is applicable to other epithelia and biopsy material.

The principle was to render the epithelial basolateral membranes electrically transparent so that the apical membrane alone could be voltage clamped. This was achieved by potassium depolarization on the basolateral side. Imposition of an apical to basolateral chloride gradient allowed the effects of ClCOs on an outward chloride current and on apical membrane conductance to be measured.

1‐ethyl‐2‐benzimidazolone (EBIO), forskolin, chlorzoxazone, and genistein all showed ClCO activity. In cystic fibrosis (CF) epithelia, either from CF null or CFΔF508 mice, EBIO showed only a minor effect, indicating that CFTR was the target in wild type tissues.

5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB) was shown to block CFTR chloride channels. The blockade was pH and voltage‐dependent and indicated that while the charged form was the active moiety, movement into the cell depended on the unionized drug. It is concluded NPPB blocks CFTR from the cytosolic side and that the agent preferentially blocks at potentials opposing the inflow of chloride ions. No significant blockade was seen with either N‐phenylanthranilic acid (DPC) or with glibenclamide, under standard conditions.

The method described can be used to examine compounds reported to increase the trafficking of ΔF508 CFTR to the membrane or those capable of opening ΔF508 CFTR chloride channels and to differentiate between them. Further, the method distinguishes between chloride channel openers and those acting indirectly to increase the flux through CFTR chloride channels by indirect means, for example, hyperpolarization.

British Journal of Pharmacology (2001) 132, 659–668; doi:10.1038/sj.bjp.0703859
Transcriptional and translational regulation of calpain in the rat heart after myocardial infarction – effects of AT1 and AT2 receptor antagonists and ACE inhibitor
Authors: Sandmann Steffen , Yu Minghuan , Unger Thomas
Published: January 2005

Recent studies demonstrated that the cardiac calpain system is activated during ischaemic events and is involved in cardiomyocyte injury. The aim of this study was to investigate the contribution of AT1 and AT2 receptors in the regulation of calpain‐mediated myocardial damage following myocardial infarction (MI).

Infarcted animals were treated either with placebo, the ACE inhibitor ramipril (1 mg kg−1 d−1), the AT1 receptor antagonist valsartan (10 mg kg−1 d−1) or the AT2 receptor antagonist PD 123319 (30 mg kg−1 d−1). Treatment was started 7 days prior to surgery. On day 1, 3, 7 and 14 after MI, gene expression and protein levels of calpain I, II and calpastatin were determined in left ventricular free wall (LVFW) and interventricular septum (IS). At day 3 and 14 post MI, morphological investigations were performed.

Calpain I mRNA expression and protein levels were increased in IS 14 days post MI, whereas mRNA expression and protein levels of calpain II were maximally increased in LVFW 3 days post MI. Ramipril and valsartan decreased mRNA and protein up‐regulation of calpain I and II, and reduced infarct size and interstitial fibrosis. PD 123319 did not affect calpain I or II up‐regulation in the infarcted myocardium, but decreased interstitial fibrosis. Calpastatin expression and translation were not affected by AT receptor antagonists or ACE inhibitor.

Our data demonstrate a distinct, temporary‐spatial up‐regulation of calpain I and II following MI confer with the hypothesis of calpain I being involved in cardiac remodelling in the late and calpain II contributing to cardiac tissue damage in the early phase of MI. The up‐regulation of calpain I and II is partly mediated via the AT1 receptor and can be reduced by ACE inhibitors and AT1 receptor antagonists.

British Journal of Pharmacology (2001) 132, 767–777; doi:10.1038/sj.bjp.0703860
Characterization of KCNQ5/Q3 potassium channels expressed in mammalian cells
Authors: Wickenden Alan D , Zou Anruo , Wagoner P Kay , Jegla Tim
Published: January 2005

Heteromeric KCNQ5/Q3 channels were stably expressed in Chinese Hamster ovary cells and characterized using the whole cell voltage‐clamp technique. KCNQ5/Q3 channels were activated by the novel anticonvulsant, retigabine (EC50 1.4 μM) by a mechanism that involved drug‐induced, leftward shifts in the voltage‐dependence of channel activation (−31.8 mV by 30 μM retigabine). KCNQ5/Q3 channels were inhibited by linopirdine (IC50 7.7 μM) and barium (IC50 0.46 mM), at concentrations similar to those required to inhibit native M‐currents. These findings identify KCNQ5/Q3 channels as a molecular target for retigabine and raise the possibility that activation of KCNQ5/Q3 channels may be responsible for some of the anti‐convulsant activity of this agent. Furthermore, the sensitivity of KCNQ5/Q3 channels to linopirdine supports the possibility that potassium channels comprised of KCNQ5 and KCNQ3 may make a contribution to native M‐currents.

British Journal of Pharmacology (2001) 132, 381–384; doi:10.1038/sj.bjp.0703861
The sulphonylurea glibenclamide inhibits multidrug resistance protein (MRP1) activity in human lung cancer cells
Authors: Payen Léa , Delugin Laurence , Courtois Arnaud , Trinquart Yolande , Guillouzo André , Fardel Olivier
Published: January 2005

Glibenclamide, a sulphonylurea widely used for the treatment of non‐insulin‐dependent diabetes mellitus, has been shown to inhibit the activities of various ATP‐binding cassette (ABC) transporters. In the present study, its effects towards multidrug resistance protein 1 (MRP1), an ABC efflux pump conferring multidrug resistance and handling organic anions, were investigated.

Intracellular accumulation of calcein, an anionic dye substrate for MRP1, was strongly increased by glibenclamide in a dose‐dependent manner in MRP1‐overexpressing lung tumour GLC4/Sb30 cells through inhibition of MRP1‐related calcein efflux. By contrast, glibenclamide did not alter calcein levels in parental control GLC4 cells. Another sulphonylurea, tolbutamide, was however without effect on calcein accumulation in both GLC4/Sb30 and GLC4 cells.

Glibenclamide used at 12.5 μM was, moreover, found to strongly enhance the sensitivity of GLC4/Sb30 cells towards vincristine, an anticancer drug handled by MRP1.

Efflux of carboxy‐2′,7′‐dichlorofluorescein, an anionic dye handled by the ABC transporter MRP2 sharing numerous substrates with MRP1 and expressed at high levels in liver, was also strongly inhibited by glibenclamide in isolated rat hepatocytes.

In summary, glibenclamide reversed MRP1‐mediated drug resistance likely through inhibiting MRP1 activity and blocked organic anion efflux from MRP2‐expressing hepatocytes. Such effects associated with the known inhibitory properties of glibenclamide towards various others ABC proteins suggest that this sulphonylurea is a general inhibitor of ABC transporters.

British Journal of Pharmacology (2001) 132, 778–784; doi:10.1038/sj.bjp.0703863