bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of lipocortin‐1 in the inhibitory action of dexamethasone on eosinophil trafficking in cutaneous inflammatory reactions in the mouse
Authors: Teixeira Mauro M. , Das Anuk M. , Miotla Jadwiga M. , Perretti Mauro , Hellewell Paul G.
Published: January 2005

The ability of glucocorticosteroids to inhibit tissue eosinophilia may be an important feature of their anti‐inflammatory action in allergic diseases. Our previous work showed that an effect of dexamethasone on the release of eosinophils from the bone marrow could explain its inhibitory action on eosinophil accumulation in a mouse air‐pouch model. Thus, it was unclear from that study whether dexamethasone could interfere with the process of eosinophil trafficking. In the present study, therefore, we used a newly developed mouse model to evaluate the effects of systemic treatment with dexamethasone on the recruitment of 111In‐labelled blood eosinophils to sites of cutaneous inflammation in the mouse and whether lipocortin‐1 (LC‐1) was involved.

The i.d. injection of ovalbumin (OVA) in sensitized mice induced a dose‐dependent recruitment of 111In‐labelled blood eosinophils which peaked at 4 to 8 h after antigen challenge. Systemic treatment with dexamethasone (50 μg per mouse, 3 h after antigen) effectively inhibited 111In‐eosinophil recruitment in this reaction by 70 to 85%. Similarly, a 1 h pretreatment with dexamethasone significantly suppressed 111In‐eosinophil induced by platelet‐activating factor (PAF), leukotriene B4(LTB4) and the chemokine macrophage inflammatory protein‐1α (MIP‐1α) by 40 to 70%.

Two experimental approaches were used to evaluate the role of LC‐1: treatment with LC‐1 fragment Ac2‐26 and use of an anti‐LC‐1 antiserum. LC‐1 fragment Ac2 ‐26 (100 μg per mouse) failed to affect 111In‐eosinophil recruitment. Moreover, pretreatment of animals with an anti‐LC‐1 antiserum failed to reverse the inhibitory effects of dexamethasone on 111In‐eosinophil recruitment induced by MIP‐1α and by antigen in sensitized mice.

In contrast, the LC‐1 fragment significantly inhibited glycogen‐induced neutrophil recruitment into the peritoneal cavity of mice. Furthermore, the anti‐LC‐1 antiserum reversed the inhibitory effects of dexamethasone on the glycogen‐induced neutrophil recruitment.

Thus, our results suggest that dexamethasone can inhibit the recruitment of eosinophils in mouse skin independent of an action on the bone marrow. However, by use of two different approaches, we showed that LC‐1 does not play a role in mediating the inhibitory action of dexamethasone on eosinophil migration into cutaneous inflammatory reactions in the mouse. These data add further support to a LC‐1‐independent action of dexamethasone on eosinophils in vivo.

British Journal of Pharmacology (1998) 123, 538–544; doi:10.1038/sj.bjp.0701625
Regulation by tolbutamide and diazoxide of the electrical activity in mouse pancreaticβ‐cells recordedin vivo
Authors: Gomis Ana , Valdeolmillos Miguel
Published: January 2005

The glucose‐dependence of β‐cell electrical activity and the effects of tolbutamide and diazoxide were studied in anaesthetized mice.

In untreated animals there was a direct relationship between glycaemia and the burst pattern of electrical activity. Animals with high glucose concentration showed continuous electrical activity. The application of insulin led to a steady decrease in blood glucose concentration and a transition from continuous to oscillatory activity at 7.7±0.1 mMglucose (mean±s.d.) and a subsequent transition from oscillatory to silent at 4.7±0.6 mMglucose.

At physiological blood glucose concentrations the electrical activity was oscillatory. The injection of tolbutamide (1800 mg kg−1) transformed this oscillatory pattern into one of continuous electrical activity. The increased electrical activity was associated with a decrease in blood glucose concentration from 7.1±0.9 (control) to 5.5±1.0 mM(10 min after tolbutamide injection). The effects of tolbutamide are consistent with a direct blocking effect on the KATPchannel that leads to membrane depolarization.

The injection of diazoxide (6000 mg kg−1) hyperpolarized the cells and transformed the oscillatory pattern into a silent one. This is consistent with a direct stimulant effect by diazoxide on the KATPchannel. The use of tolbutamide or diazoxide correspondingly led to the lengthening or shortening of the active phase of electrical activity, respectively. This indicates thatin vivo, such activity can be modulated by the relative degree of activation or inhibition of the KATPchannel.

These results indicate that under physiological conditions, tolbutamide and diazoxide have direct and opposite effects on the electrical activity of pancreatic β‐cells, most likely through their action on KATPchannels. This is consistent with previous work carried out onin vitromodels and explains the drugs hypo‐ and hyperglycaemic effects.

British Journal of Pharmacology(1998)123, 443–448; doi:10.1038/sj.bjp.0701628
Epoxyeicosatrienoic acids, potassium channel blockers and endothelium‐dependent hyperpolarization in the guinea‐pig carotid artery
Authors: Chataigneau Thierry , Félétou Michel , Duhault Jacques , Vanhoutte Paul M.
Published: January 2005

Using intracellular microelectrodes, we investigated the effects of 17‐octadecynoic acid (17‐ODYA) on the endothelium‐dependent hyperpolarization induced by acetylcholine in the guinea‐pig isolated internal carotid artery with endothelium.

In the presence of Nω‐nitro‐L‐arginine (L‐NOARG, 100 μM) and indomethacin (5 μM) to inhibit nitric oxide synthase and cyclo‐oxygenase, acetylcholine (1 μM) evoked an endothelium‐dependent hyperpolarization which averaged −16.4 mV starting from a resting membrane potential of −56.8 mV. There was a negative correlation between the amplitude of the hyperpolarization and the absolute values of the resting membrane potential.

The acetylcholine‐induced endothelium‐dependent hyperpolarization was not altered by charybdotoxin (0.1 μM) or iberiotoxin (30 nM). It was partially but significantly reduced by apamin (0.5 μM) to −12.8±1.2 mV (n=10) or the combination of apamin plus iberiotoxin (−14.3±3.4 mV, n=4). However, the combination of charybdotoxin and apamin abolished the hyperpolarization and under these conditions, acetylcholine evoked a depolarization (+7.1±3.7 mV, n=8).

17‐ODYA (10 μM) produced a significant hyperpolarization of the resting membrane potential which averaged −59.6 mV and a partial but significant inhibition of the acetylcholine‐induced endothelium‐dependent hyperpolarization (−10.9 mV).

Apamin did not modify the effects of 17‐ODYA but in the presence of charybdotoxin or iberiotoxin, 17‐ODYA no longer influenced the resting membrane potential or the acetylcholine‐induced hyperpolarization.

When compared to solvent (ethanol, 1% v/v), epoxyeicosatrienoic acids (EpETrEs) (5,6‐, 8,9‐, 11,12‐ and 14,15‐EpETrE, 3 μM) did not affect the cell membrane potential and did not relax the guinea‐pig isolated internal carotid artery.

These results indicate that, in the guinea‐pig internal carotid artery, the involvement of metabolites of arachidonic acid through the cytochrome P450 pathway in endothelium‐dependent hyperpolarization is unlikely. Furthermore, the hyperpolarization mediated by the endothelium‐derived hyperpolarizing factor (EDHF) is probably not due to the opening of BKCa channels.

British Journal of Pharmacology (1998) 123, 574–580; doi:10.1038/sj.bjp.0701629
The effect of chronic treatment with trandolapril on cyclic AMP‐ and cyclic GMP‐dependent relaxations in aortic segments of rats with chronic heart failure
Authors: Toyoshima Hiroko , Nasa Yoshihisa , Kohsaka Yumi , Isayama Yoko , Yamaguchi Fuminari , Sanbe Atsushi , Takeo Satoshi
Published: January 2005

Characteristics of cyclic GMP‐ and cyclic AMP‐mediated relaxation in aortic segments of rats with chronic heart failure (CHF) and the effects of chronic treatment with an angiotensin I converting enzyme (ACE) inhibitor, trandolapril, were examined 8 weeks after coronary artery ligation.

Cardiac output indices of coronary artery‐ligated and sham‐operated rats were 125±8 and 189±10 ml min−1 kg−1, respectively (P<0.05), indicating the development of CHF at this period.

The maximal relaxant response of aortic segments to 10 μM acetylcholine in rats with CHF and sham‐operated rats was 64.0±5.7 and 86.9±1.9%, respectively (P<0.05), whereas the relaxant response to sodium nitroprusside (SNP) remained unchanged. Tissue cyclic GMP content in rats with CHF was lower than that of sham‐operated rats.

In endothelium‐intact segments of rats with CHF, the maximal relaxant response to 10 μM isoprenaline (44.5±6.7%) was lower that sham‐operated rats (81.3±2.5%, P<0.05) and the concentration‐response curve for NKH477, a water‐soluble forskolin, was shifted to the right without a reduction in the maximal response. Isoprenaline‐induced relaxation of aortic segments was attenuated by NG‐nitro‐L‐arginine methyl ester (L‐NAME) in sham‐operated rats, but not in rats with CHF. Relaxation to 30 μM dibutyryl cyclic AMP in rats with CHF (26.8±2.7%) was lower than that in sham‐operated rats (63.4±11.8%, P<0.05).

Trandolapril (3 mg kg−1 day−1) was orally administered from the 2nd to 8th week after the operation. Aortic blood flow of rats with CHF (38.5±3.6 ml min−1) was lower than that of sham‐operated rats (55.0±3.0 ml min−1), and this reduction was reversed (54.1±3.4 ml min−1) by treatment with trandolapril. The diminished responsiveness described above was normalized in the trandolapril‐treated rat with CHF (i.e., the maximal relaxation to acetylcholine, 94.7±1.0%; that to isoprenaline, 80.5±2.8%; that to dibutyryl cyclic AMP, 54.7±6.2%). However, aortic segments of trandolapril‐treated rats with CHF, L‐NAME did not attenuate isoprenaline‐induced relaxation and the tissue cyclic GMP level was not fully restored, suggesting that the ability of the endothelium to produce NO was still partially damaged.

The results suggest that vasorelaxation in CHF, diminished mainly due to dysfunction in endothelial nitric oxide (NO) production and cyclic AMP‐mediated signal transduction, was partially restored by long‐term treatment with trandolapril. The mechanism underlying the restoration may be attributed in part to prevention of CHF‐induced endothelial dysfunction.

British Journal of Pharmacology (1998) 123, 344–352; doi:10.1038/sj.bjp.0701635
The relevance of resting tension to responsiveness and inherent tone of human bronchial smooth muscle
Authors: Watson N. , Magnussen H. , Rabe K F.
Published: January 2005

In the present study the effects of resting tension on isometric responses of human airway smooth muscle to contractile and relaxant stimuli were investigated. Also, its effects on inherent smooth muscle tone were examined, in an effort to determine a pre‐defined resting tension which can be considered optimal for in vitro studies.

Bronchial ring preparations (2–4 mm internal diameter) were suspended in tissue baths at a range of levels of resting tension (200–1600 mg). The responses to electrical field stimulation (EFS, 30 V, 0.2 ms, 8–30 Hz), carbachol (3 μM), isoprenaline (1 μM) and the 5‐lipoxygenase inhibitor, zileuton (10 μM) were investigated along with the ability of the preparations to recover stable resting tension after 60 min of washing post challenge.

EFS induced monophasic contractions at low stimulation frequency (8 Hz). However, with increasing frequency and tension (15 and 30 Hz; > 400 mg) contractile responses were accompanied by a relaxant component. The magnitude of contractile responses increased with increasing resting tension to a plateau at between 500 and 700 mg. Relaxant responses when present, increased in magnitude with increasing resting tension.

The level of resting tension did not significantly alter responses to carbachol, but tissues at tensions 1000 mg showed poor tension recovery after washing. Isoprenaline‐induced relaxations increased with increasing resting tension (P<0.05) and tension recovery after washing was complete. The 5‐lipoxygenase sensitive portion of the tension (33±4%) was not altered by the level of resting tension.

These results suggest that in human bronchial ring preparations of 2–4 mm internal diameter, resting tensions within the range 400–1000 mg could be considered optimal for isometric tension recordings of protocols involving both contraction and relaxation procedures.

British Journal of Pharmacology (1998) 123, 694–700; doi:10.1038/sj.bjp.0701637
Mediators and mechanisms of relaxation in rabbit urethral smooth muscle
Authors: Waldeck Kristian , Ny Lars , Persson Katarina , Andersson Karl‐Erik
Published: January 2005

Electrophysiological and mechanical experiments were performed to investigate whether the nitric oxide (NO)‐mediated relaxation of rabbit urethral smooth muscle is associated with a hyperpolarization of the membrane potential. In addition, a possible role for vasoactive intestinal peptide (VIP) and carbon monoxide (CO) as relaxant agents in rabbit urethra was investigated.

Immunohistochemical experiments were performed to characterize the NO‐synthase (NOS) and VIP innervation. Possible target cells for NO were studied by using antisera against cyclic GMP. The cyclic GMP‐immunoreactivity was investigated on tissues pretreated with 1 mM IBMX, 0.1 mM zaprinast and 1 mM sodium nitroprusside.

Intracellular recordings of the membrane potential in the circular smooth muscle layer revealed two types of spontaneous depolarizations, slow waves with a duration of 3–4 s and an amplitude of 30–40 mV, and faster (0.5–1 s), more irregular depolarizations with an amplitude of 5–15 mV. The resting membrane potential was 39±1 mV (n=12). Application of NO (30 μM), CO (30 μM) or VIP (1 μM) did not change the resting membrane potential.

Both NO (1–100 μM) and VIP (1 nM–1 μM) produced concentration‐dependent relaxations amounting to 87±4% and 97±2% (n=6), respectively. The relaxant effect of CO (1–30 μM) amounted to 27±4% (n=5) at the highest concentration used.

Immunohistochemical experiments revealed a rich supply of NOS‐immunoreactive nerve fibres in the smooth muscle layers. Numerous spinous cyclic GMP‐immunoreactive cells were found interspersed between the smooth muscle bundles, mainly localized in the outer layer. These cells had long processes forming a network surrounding the smooth muscle bundles. VIP‐immunoreactivity was sparse in comparison to NOS‐immunoreactive nerves.

The rich supply of NOS‐immunoreactive nerve fibres supports the view that NO is an important NANC‐mediator in the rabbit urethra. In contrast to several other tissues, the relaxant effect of NO in the rabbit urethra does not seem to be mediated by hyperpolarization. The network of cyclic GMP‐immunoreactive cells may constitute target cells for NO, but their function remains to be established.

British Journal of Pharmacology (1998) 123, 617–624; doi:10.1038/sj.bjp.0701645
Blockade of the development of morphine tolerance by U‐50,488, an AVP antagonist or MK‐801 in the rat hippocampal slice
Authors: Su Mei‐Tsu , Lin Wei‐Bin , Lue Wei‐Ming , Cheng Chen‐Yu , Tao Pao‐Luh
Published: January 2005

In this study, we investigated the effects of different drugs (a κ‐opioid receptor agonist U‐50,488, a vasopressin receptor antagonist dPTyr(Me)AVP or an N‐methyl‐D‐aspartate (NMDA) receptor antagonist MK‐801) on the development of morphine tolerance in rat hippocampal slices.

Hippocampal slices (450 μm) of Sprague‐Dawley rats (250–300 g) were used. Slices were continuously superfused with artificial CSF or drugs at 1 ml min−1. Nichrome wire electrodes were placed in the Schaffer‐collateral pathway and used to deliver biphasic 0.2 ms pulses of 5–30 V (0.033 Hz). A glass microelectrode was placed in the CA1 area to record population spikes.

When the slices were superfused with 10 μM morphine, the amplitude of population spikes increased 2–3 fold in 30–40 min. However, this effect of morphine decreased, i.e. tolerance developed after continuous superfusion of morphine for 2–6 h.

When either U‐50,488 (200 nM) or dPTyr(Me) AVP (500 pM) or MK‐801 (500 pM) was co‐superfused with morphine (10 μM), it significantly blocked the development of morphine tolerance. Nor‐BNI (a κ‐opioid receptor antagonist, 200 nM) significantly reversed the inhibitory effect of U‐50,488 but not those of dPTyr(Me)AVP or MK‐801 on the development of morphine tolerance.

These data indicate that κ‐opioid receptors, AVP receptors and NMDA receptors are all involved in the development of morphine tolerance. The suppression of κ‐opioid receptor activity after chronic morphine may occur before the activation of AVP receptors or NMDA receptors during the development of morphine tolerance.

British Journal of Pharmacology (1998) 123, 625–630; doi:10.1038/sj.bjp.0701646
Modulation of transcription factor NF‐κB by enantiomers of the nonsteroidal drug ibuprofen
Authors: Scheuren Nicole , Bang Holger , Münster Tino , Brune Kay , Pahl Andreas
Published: January 2005

The nonsteroidal drug ibuprofen exists as an R(−)‐ and S(+)‐enantiomer. Only the S(+)‐enantiomer is an effective cyclo‐oxygenase inhibitor, while the R(−)‐enantiomer is inactive in this respect. Thus the molecular mechanism by which R(−)‐ibuprofen exerts its anti‐inflammatory and antinociceptive effects remains unknown.

In this study the effects of the enantiomers of ibuprofen on modulation of transcription factors have been examined with electrophoretic mobility‐shift assay (EMSA), transient transfection experiments, confocal immunofluorescence and nuclear import experiments, to determine their selectivity and potency as inhibitors of the activation of transcription factor nuclear factor‐κB (NF‐κB).

R(−)‐ibuprofen (IC50: 121.8 μM) as well as the S(+)‐enantiomer (IC50: 61.7 μM) inhibited the activation of NF‐κB in response to T‐cell stimulation. The effect of ibuprofen was specific because, at concentrations up to 10 mM, ibuprofen did not affect the heat shock transcription factor (HSF) and the activation of NF‐κB by prostaglandin E2 (PGE2). Very high concentrations of ibuprofen (20 mM) did not prevent NF‐κB binding to DNA in vitro. Immunofluorescence and nuclear import experiments indicate that the site of ibuprofen action appeared to be upstream of the dissociation of the NF‐κB‐IκB‐complex.

Our data raise the possibility that R(−)‐ibuprofen exerts some of its effects by inhibition of NF‐κB activation.

British Journal of Pharmacology (1998) 123, 645–652; doi:10.1038/sj.bjp.0701652
Mechanism of antidiuresis caused by bendroflumethiazide in conscious rats with diabetes insipidus
Authors: Grønbeck Lene , Marples David , Nielsen Søren , Christensen Sten
Published: January 2005

The mechanism underlying the antidiuretic effect of thiazide diuretics in diabetes insipidus (DI) is unknown. This study addressed two specific questions: is the reduction in urine flow rate (V) related to a decrease in the delivery of fluid from the pars recta of the proximal tubules (‘distal delivery’), and are there any changes in the expression and/or intracellular distribution of vasopressin stimulated water channels (AQP2) in the collecting ducts, during chronic thiazide‐induced antidiuresis?

Nine Brattleboro rats with vasopressin‐deficient DI were treated for 5 days with bendroflumethiazide (BFTZ), 9 mg kg−1 day−1 orally, and 9 Brattleboro rats were left untreated. BFTZ‐treated DI rats showed a fall in V from ∼200 to ∼75 ml day−1 and an increase in urine osmolality from ∼130 to ∼400 mosmol kg−1.

BFTZ‐induced antidiuresis was associated with a persistent loss of sodium, but not of potassium. After 5 days of treatment, clearance studies in conscious rats showed a tendency towards decreases in effective renal plasma flow (−7%), GFR (−12%) and lithium clearance (CLi; used as marker for distal delivery) (−25%), compared with untreated controls, but none of these changes were statistically significant. There was no apparent relationship between CLi and V in BFTZ‐treated or untreated DI rats.

BFTZ treatment did not change the expression of AQP2 in homogenates of cortex, outer or inner medulla from DI rats, or from normal Long Evans rats. Light and electron microscopic immunocyto‐chemistry revealed no changes in intracellular distribution of AQP2 in principal cells from inner medullary collecting ducts of BFTZ‐treated DI rats.

We concluded, (i) that although the antidiuretic effect of BFTZ in rats with DI is associated with a net loss of Na, the decrease in V shows no association with changes in distal delivery, as estimated by CLi. (ii) Antidiuretic treatment with BFTZ does not alter the expression of subcellular distribution of AQP2 water channels in the collecting ducts. The mechanism underlying the chronic antidiuresis caused by thiazide diuretics in DI remains elusive.

British Journal of Pharmacology (1998) 123, 737–745; doi:10.1038/sj.bjp.0701653
Functional and morphological damage of endothelium in rabbit ear artery following irradiation with cobalt60
Authors: Qi Fazhi , Sugihara Tsuneki , Hattori Yuichi , Yamamoto Yuhei , Kanno Morio , Abe Kazuhiro
Published: January 2005

The relaxant actions of acetylcholine and A23187 were examined in the rabbit central ear artery at different intervals following exposure to different doses of radiation with a cobalt60 unit. The artery was irradiated with a dose of 10 Gy, 20 Gy and 45 Gy. Radiation caused dose‐ and time‐dependent impairment of the endothelium‐dependent relaxations. The impaired endothelium‐dependent relaxations occurred as early as 1 week postirradiation and persisted throughout the experimental period (10 weeks).

The endothelium‐independent response to sodium nitroprusside was well preserved up to 6 weeks after irradiation. The contractile response to noradrenaline was unaltered by irradiation throughout the experimental period, but in contrast to control vessels, an increase in the sensitivity to noradrenaline in the presence of the nitric oxide synthase (NOS) inhibitor NG‐nitro‐L‐arginine was not observed in the irradiated vessels.

The impaired endothelium‐dependent relaxations in the irradiated vessels were not improved by pretreatment with the NOS substrate L‐arginine, the cyclo‐oxygenase inhibitor indomethacin or the free radical scavengers superoxide dismutase and catalase.

Scanning electron microscopy indicated morphologically intact endothelial cells within the first 4 weeks after irradiation.

Western blot analysis showed a significant decrease in the expression of endothelial NOS (eNOS) in the irradiated vessels.

These data indicate that endothelial cell function is specifically impaired in the irradiated vessels before morphological endothelial cell damage can be detected. This impairment may be related to diminished eNOS expression.

British Journal of Pharmacology (1998) 123, 653–660; doi:10.1038/sj.bjp.0701654
Influence of excitatory amino acids on basal and sensory stimuli‐induced release of 5‐HT in the locus coeruleus
Authors: Singewald Nicolas , Kaehler Stefan T. , Hemeida Ramadan , Philippu Athineos
Published: January 2005

The interactions between 5‐hydroxytryptaminergic neurones and excitatory amino acid utilizing neurones were studied in the locus coeruleus of conscious, freely moving rats. The locus coeruleus was superfused with artificial cerebrospinal fluid through a push‐pull cannula and 5‐hydroxytryptamine (5‐HT) was determined in the superfusate that was continuously collected in time periods of 10 min.

Superfusion of the locus coeruleus with the NMDA receptor antagonist AP5 (10 μM), kynurenic acid (1 mM), or the AMPA/kainate receptor antagonist DNQX (10 μM) reduced the 5‐HT release in the locus coeruleus.

Superfusion with the agonists NMDA (50 μM), kainic acid (50 μM) or AMPA (10 μM) enhanced the release rate of 5‐HT. AP5 (10 μM) blocked the stimulant effect of NMDA, while tetrodotoxin (1 μM) failed to influence the NMDA‐induced release of 5‐HT. In the presence of 10 μM DNQX, the releasing effect of 50 μM kainic acid was abolished.

Pain elicited by tail pinch, as well as noise‐induced stress, increased the release of 5‐HT. Superfusion of the locus coeruleus with 10 μM AP5 reduced the tail pinch‐induced 5‐HT release. AP5 (10 μM) did not affect the noise‐induced release of 5‐HT which was reduced, when the locus coeruleus was superfused simultaneously with this concentration of AP5 and 1 μM kynurenic acid. DNQX (10 mM) failed to influence the release of 5‐HT induced by tail pinch or noise.

The findings suggest that 5‐hydroxytryptaminergic neurones of the locus coeruleus are tonically modulated by excitatory amino acids via NMDA and AMPA/kainate receptors. The release of 5‐HT elicited by tail pinch and noise is mediated to a considerable extent through endogenous excitatory amino acids acting on NMDA receptors, while AMPA/kainate receptors are not involved in this process.

British Journal of Pharmacology (1998) 123, 746–752; doi:10.1038/sj.bjp.0701656
Different efficacy of specific agonists at 5‐HT3 receptor splice variants: the role of the extra six amino acid segment
Authors: Niemeyer M‐I. , Lummis S C R.
Published: January 2005

Whole cell voltage clamp electrophysiology and radioligand binding were used to examine the agonist characteristics of the two splice variants of the 5‐HT3 receptor which have been cloned from neuronal cell lines. Homo‐oligomeric 5‐HT3 receptors were examined in HEK 293 cells stably transfected with either long (5‐HT3‐L) or short (5‐HT3‐S) receptor subunit DNAs.

Functional homo‐oligomeric receptors were formed from both subunits, and responses to 5‐HT3 receptor agonists (5‐hydroxytryptamine (5‐HT), 2‐methyl 5‐HT and m‐chlorophenylbiguanide) were qualitatively similar.

Maximum currents (Rmax) elicited by the 5‐HT3 receptor agonists m‐chlorophenylbiguanide (mCPBG) and 2‐methyl‐5‐HT (2‐Me‐5‐HT), as compared to 5‐HT, differed in the two splice variants: Rmax mCPBG/Rmax 5‐HT values were 0.68±0.04 and 0.91±0.01 in 5‐HT3‐L and 5‐HT3‐S receptors, respectively. Comparable values for 2‐Me‐5‐HT were 0.30±0.02 and 0.23±0.02.

Radioligand binding data showed no difference in affinity of agonist or antagonist binding sites; thus the six amino acid deletion appears to cause differences in agonist efficacy.

The role of the 6 amino acid insertion was further investigated by use of site‐directed mutagenesis to create two mutant receptors, one where serine 286 was replaced with alanine, and the second where all 6 amino acids were replaced with alanines.

Examination of the mutant receptors when stably expressed in HEK 293 cells revealed agonist properties resembling long and not short 5‐HT3 receptors. Thus specific amino acids in this region are not responsible for the observed differences.

The data show intracellular structure can have significant effects on ligand‐gated ion channel function, and suggest that minor changes in structure may be responsible for differences in function observed when ligand‐gated ion channel proteins are modulated intracellularly.

British Journal of Pharmacology (1998) 123, 661–666; doi:10.1038/sj.bjp.0701657
P2U‐receptor mediated endothelium‐dependent but nitric oxide‐independent vascular relaxation
Authors: Malmsjö M. , Edvinsson L. , Erlinge D.
Published: January 2005

The dilator effect of extracellular adenosine triphosphate (ATP) has mainly been characterized as a direct effect on smooth muscle or as an endothelium‐dependent effect mediated by nitric oxide (NO) or prostaglandins. We tested the hypothesis that endothelium‐derived hyperpolarizing factor (EDHF) may also be involved. Dilator effects were studied in vitro by continuous recording of isomeric tension in cylindrical segments of rat blood vessels precontracted by noradrenaline (NA), in the presence of indomethacin (10 μM).

By screening different blood vessels in the rat we found that both acetylcholine (ACh) and ATP dilate mesenteric arteries with a resting tone of 1 mN by an endothelium‐dependent non‐NO mechanism. With an increased resting tone (4 mN) the dilatation was mediated by NO. Thus by varying the resting tension the different dilator mechanisms could be examined. However, in the carotid artery the dilatation was solely mediated by an endothelium‐dependent NO mechanism, even at different resting tones (1 and 4 mN).

The N‐nitro‐L‐arginine methyl ester (L‐NAME)‐resistant dilatation to ACh and ATP was further inhibited by the NO‐scavenger 2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (PTIO), indicating L‐NAME insensitive NO‐synthesis.

In carotid arteries and mesenteric arteries at high resting tones (4 mN) the ATP‐dilatation was totally inhibited by endothelium removal or L‐NAME (10−3 M). In mesenteric arteries at low resting tone (1 mN) the ATP, UTP (uridine‐triphosphate) and 2‐MeSATP (2methylthioATP)‐dilatation was totally inhibited by endothelium removal. However, L‐NAME in combination with indomethacin attenuated only 5% of the UTP dilatation, 70% of the ATP dilatation but all of the 2‐MeSATP‐dilatation. The inhibitors of Ca2+‐activated K+ channels charybdotoxin (0.5×10−7 M) together with apamin (10−6 M), and the cytochrome P450 inhibitor, SKF 525A (10−4 M), each in combination with indomethacin, L‐NAME and PTIO (0.5×10−3 M) totally abolished the remaining ATP and UTP‐dilatation. This indicates a dilatation mediated by an endothelium‐dependent non‐NO factor, probably EDHF.

Agonist potency (UTP>ATP≈#62;2‐MeSATP), indicates that the EDHF‐mediated dilatation was stimulated by a P2U‐receptor, possibly by a selective pyrimidine‐receptor. In contrast, a P2Y‐receptor stimulated NO‐mediated dilatation (2‐MeSATP=ATP>UTP).

In conclusion, the dilator effects of ATP and especially UTP can be mediated by an endothelium‐dependent non‐NO‐mediated mechanism, probably EDHF, mediated by a P2U‐receptor, possibly a selective pyrimidine‐receptor, while NO‐mediated dilatation is stimulated mainly by a P2Y1‐receptor. Furthermore, the EDHF‐dilatation is dependent on the resting tone of the blood vessel.

British Journal of Pharmacology (1998) 123, 719–729; doi:10.1038/sj.bjp.0701660
Metabotropic glutamate receptors depress glutamate‐mediated synaptic input to rat midbrain dopamine neurones in vitro
Authors: Wigmore Mark A. , Lacey Michael G.
Published: January 2005

Glutamate (AMPA receptor‐mediated) excitatory postsynaptic potentials (e.p.s.ps.), evoked by electrical stimulation rostral to the recording site, were examined by intracellular microelectrode recording from dopamine neurones in parasagittal slices of rat ventral midbrain.

The e.p.s.p. was depressed by the group III metabotropic glutamate (mGlu) receptor agonist L‐2‐amino‐4‐phosphonobutyric acid (L‐AP4; 0.01–30 μM) by up to 60% with an EC50 of 0.82 μM. The depression induced by L‐AP4 (3 μM) was reversed by the group III preferring mGlu receptor antagonist, α‐methyl‐4‐phosphonophenylglycine (MPPG; 250 μM).

The group I and II mGlu agonist, 1S,3R‐aminocyclopentanedicarboxylic acid (ACPD; 3–30 μM) also depressed the e.p.s.p. in a concentration‐dependent manner. The effect of ACPD (10 μM) was reversed by (+)‐α‐methyl‐4‐carboxyphenylglycine (MCPG; 1 mM; 4 cells). This effect of ACPD was also partially antagonized (by 50.3±15.7%, 4 cells) by MPPG (250 μM).

The selective agonist at group I mGlu receptors, dihydroxyphenylglycine (DHPG; 100 μM), decreased e.p.s.p. amplitude by 27.1±8.2% (7 cells), as did the group II mGlu receptor‐selective agonist (1S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycyclopropyl)glycine (DCG‐IV; 1 μM) by 26.7±4.3% (5 cells).

DHPG (10–100 μM) caused a depolarization of the recorded cell, as did ACPD (3–30 μM), whereas no such postsynaptic effect of either L‐AP4 or DCG‐IV was observed.

These results provide evidence for the presence of presynaptic inhibitory metabotropic glutamate autoreceptors from the mGlu receptor groups II and III on descending glutamatergic inputs to midbrain dopamine neurones. Group I mGlu receptors mediate a postsynaptic depolarization, and can also depress glutamatergic transmission, but may not necessarily be localized presynaptically. These sites represent novel drug targets for treatment of schizophrenia and movement disorders of basal ganglia origin.

British Journal of Pharmacology (1998) 123, 667–674; doi:10.1038/sj.bjp.0701662
The mechanism of action of cantharidin in smooth muscle
Authors: Knapp Jörg , Bokník Peter , Huke Sabine , Lüss Hartmut , Müller Frank U. , Müller Thorsten , Nacke Peter , Schmitz Wilhelm , Vahlensieck Ute , Neumann Joachim
Published: January 2005

The aim of this study was to investigate the mechanism(s) of the vasoconstrictor effect of cantharidin in bovine preparations.

Catalytic subunits of protein phosphatase type 1 (PP 1) and type 2A (PP 2A) were immunologically identified in coronary arteries, isolated smooth muscle cells and ventricular myocardium.

The mRNAs coding for catalytic subunits of PP 1α, PP 1β and PP 2Aα were identified by hybridization with specific cDNA‐probes in total RNA from coronary arteries, isolated smooth muscle cells and ventricles.

The activities of catalytic subunits of PP 1 and PP 2A separated by column chromatography from coronary arteries, isolated smooth muscle cells and ventricles were inhibited by cantharidin in a concentration‐dependent manner.

Cantharidin increased the phosphorylation state of smooth muscle proteins including the regulatory light chains of myosin in 32P‐labelled intact smooth muscle cells in a concentration‐dependent manner.

Cantharidin did not affect cytosolic calcium concentrations in aortic smooth muscle cells.

It is suggested that cantharidin contracts smooth muscle preparations by increasing the phosphorylation state of regulatory proteins due to inhibition of phosphatase activities. Thus, cantharidin might be a useful tool to study the function of phosphatases in smooth muscle.

British Journal of Pharmacology (1998) 123, 911–919; doi:10.1038/sj.bjp.0701668
Reversion of muscarinic autoreceptor agonist‐induced acetylcholine decrease and learning impairment by dynorphin A (1–13), an endogenous κ‐opioid receptor agonist
Authors: Hiramatsu Masayuki , Murasawa Hiroyasu , Mori Hiromasa , Kameyama Tsutomu
Published: January 2005

We investigated whether carbachol, a muscarinic receptor agonist, induces learning and memory impairment, and if so, dynorphin A (1–13), an endogenous κ‐opioid receptor agonist, ameliorates the impairment of learning and memory induced by carbachol, by use of a step‐through type passive avoidance task.

Carbachol induced a dose‐related dual response. Carbachol (1.66 pmol per rat) administered directly into the hippocampus significantly shortened the step‐through latency, while lower (0.166 pmol per rat) and higher (16.6 pmol per rat) doses of carbachol did not induce learning or memory impairment.

Dynorphin A (1–13) (0.5 nmol per rat, i.c.v.) administered 5 min after carbachol injection significantly reversed carbachol‐induced impairment of learning and memory.

Perfusion with carbachol (3×10−4 M) significantly decreased acetylcholine release in the hippocampus during perfusion as determined by in vivo brain microdialysis. This decrease in acetylcholine release was suppressed by co‐perfusion with a low dose of atropine (10−7 M).

Dynorphin A (1–13) (0.5 nmol per rat, i.c.v.) immediately before carbachol perfusion completely blocked this decrease in extracellular acetylcholine concentration induced by carbachol.

These antagonistic effects of dynorphin A (1–13) were abolished by treatment with nor‐binaltorphimine (5.44 nmol per rat, i.c.v.), a selective κ‐opioid receptor antagonist, 5 min before dynorphin A (1–13) treatment.

These results suggest that the neuropeptide dynorphin A (1–13) ameliorates the carbachol‐induced impairment of learning and memory, accompanied by attenuation of the reductions in acetylcholine release which may be associated with dysfunction of presynaptic cholinergic neurones via κ‐opioid receptors.

British Journal of Pharmacology (1998) 123, 920–926; doi:10.1038/sj.bjp.0701671
Selective cyclo‐oxygenase‐2 inhibitors and their influence on the protective effect of a mild irritant in the rat stomach
Authors: Gretzer Britta , Ehrlich Karlheinz , Maricic Nenad , Lambrecht Nils , Respondek Michael , Peskar Brigitta M.
Published: January 2005

The effects of the non‐selective cyclo‐oxygenase (COX) inhibitor indomethacin and the selective COX‐2 inhibitors, N‐[2‐(cyclohexyloxy)‐4‐nitrophenyl] methanesulphonamide (NS‐398), 5‐methanesulphonamido‐6‐(2,4‐difluorothio‐phenyl)‐1‐indanone (L‐745,337) and 5,5‐dimethyl‐3‐(3‐fluorophenyl)‐4‐(4‐methylsulphonyl) phenyl‐2(5H)‐furanone (DFU), on the protection induced by the mild irritant 20% ethanol were investigated in the rat stomach.

Instillation of 20% ethanol (1 ml, p.o.) effectively protected against gastric mucosal injury induced by subsequent instillation of 70% or 96% ethanol (1 ml, p.o.).

Oral administration of indomethacin (1.25–20 mg kg−1) dose‐dependently counteracted the protective effect of 20% ethanol (ID50: 3.5 mg kg−1).

Likewise, NS‐398 (0.1–1 mg kg−1), L‐745,337 (0.2–2 mg kg−1) and DFU (0.02–0.2 mg kg−1) inhibited the protective effect of 20% ethanol in a dose‐dependent manner with ID50 values of 0.3 mg kg−1, 0.4 mg kg−1 and 0.06 mg kg−1, respectively.

Inhibition of mild irritant‐induced protection was also found when NS‐398 (1 mg kg−1) was administered s.c. or when 96% ethanol was used to damage the mucosa.

Pretreatment with 16,16‐dimethyl‐prostaglandin (PG)E2 at 4 ng kg−1, a dose that did not protect against ethanol (70%)‐induced mucosal damage when given alone, completely reversed the effect of the selective COX‐2 inhibitors on the mild irritant‐induced protection.

Pretreatment with dexamethasone (3 mg kg−1, 24 and 2 h before instillation of 20% ethanol) did not affect the protective activity of the mild irritant, indicating that enzyme induction is not involved.

Indomethacin (20 mg kg−1, p.o.) did not prevent the protection conferred by sodium salicylate (100 mg kg−1), dimercaprol (30 μg kg−1), iodoacetamide (50 mg kg−1) and lithium (20 mg kg−1). Likewise, the protective effect of these agents was not counteracted by NS‐398 (1 mg kg−1, p.o.).

Whereas indomethacin (20 mg kg−1, p.o.) near‐maximally inhibited gastric mucosal formation of PGE2, 6‐keto‐PGF1α and thromboxane (TX) B2 as well as platelet TXB2 release, the selective COX‐2 inhibitors were ineffective.

The findings show that selective COX‐2 inhibitors, although lacking in ulcerogenic activity, prevent the protection conferred by a mild irritant. Prostaglandis generated by a constitutive COX‐2 could thus contribute to physiological functions involved in gastric homeostasis, although at present a non‐COX‐2‐related mechanism underlying the effect of the selective COX‐2 inhibitors tested on mild irritant‐induced protection cannot be completely excluded.

British Journal of Pharmacology (1998) 123, 927–935; doi:10.1038/sj.bjp.0701673
Emodin‐induced muscle contraction of mouse diaphragm and the involvement of Ca2+ influx and Ca2+ release from sarcoplasmic reticulum
Authors: Cheng Y W. , Kang J J.
Published: January 2005

The effects on skeletal muscle of emodin, an anthraquinone, were studied in the mouse isolated diaphragm and sarcoplasmic reticulum (SR) membrane vesicles.

Emodin dose‐dependently caused muscle contracture, simultaneously depressing twitch amplitude. Neither tubocurarine nor tetrodotoxin blocked the contraction suggesting that it was caused myogenically.

The contraction induced by emodin persisted in a Ca2+ free medium with a slight reduction in the maximal force of contraction. The contraction induced by emodin in the Ca2+ free medium was completely blocked when the internal Ca2+ pool of the muscle was depleted by ryanodine. These data suggest that the contraction caused by emodin is due to the release of Ca2+ from the intracellular ryanodine‐sensitive pool.

In contrast to the effect seen in the Ca2+ free medium, emodin induced a small but consisted contraction in the ryanodine‐treated muscle in Krebs medium. The contraction was blocked in the presence of dithiothreitol and was partially blocked by nifedipine, suggesting that oxidation of a sulphhydryl group on the external site of dihydropyridine receptor is involved.

Emodin dose‐dependently increased Ca2+ release from actively loaded SR vesicles and this effect was blocked by ruthenium red, a specific Ca2+ release channel blocker, and the thiol reducing agent, DTT, suggesting that emodin induced Ca2+ release through oxidation of the critical SH of the ryanodine receptor.

[3H]‐ryanodine binding was dose‐dependently potentiated by emodin in a biphasic manner. The degree of potentiation of ryanodine binding by emodin increased dose‐dependently at concentrations up to 10 μM but decreased at higher concentrations of 10–100 μM.

These data suggest that muscle contraction induced by emodin is due to Ca2+ release from the SR of skeletal muscle, as a result of oxidation of the ryanodine receptor and influx of extracellular Ca2+ through voltage‐dependent Ca2+ channels of the plasma membrane.

British Journal of Pharmacology (1998) 123, 815–820; doi:10.1038/sj.bjp.0701677
Inhibitory effect of peptides derived from the N‐terminus of lipocortin 1 on arachidonic acid release and proliferation in the A549 cell line: identification of E‐Q‐E‐Y‐V as a crucial component
Authors: Croxtall J D. , Choudhury Q , Flower R J.
Published: January 2005

The ability of the glucocorticoid‐induced protein lipocortin 1 (LC1) to inhibit arachidonic acid release and cell proliferation in A549 cells may be mimicked by a sequence taken from the N‐terminal, LC113–25 (FIENEEQEYVQTV). We have now synthesized and tested for biological activity a library of 25 smaller peptides derived from this sequence.

Peptides were tested in two assays: A549 cells were prelabelled with tritiated arachidonic acid and thapsigargin (50 nM) and EGF (10 nM) used to stimulate the release of this fatty acid. Cell proliferation was determined by counting cell numbers following 3 day incubation with these peptides, or controls.

Many of the peptides were highly insoluble but could be more readily dissolved in aqueous solution in the presence of commercial liposomes or phosphatidyl serine (5 μM). Since neither of these agents alone had any effect on arachidonic acid release or cell proliferation, all peptides were tested in the presence of 5 μM phosphatidyl serine. Under these conditions LC113–25 was active in both assay systems with an IC40 of 40.7 and 57.0 μM respectively.

Deletion of amino acids from the C‐terminus of the peptide progressively diminished (2–3 fold) the molar potency of LC113–25 in both assays: after the removal of Val22 biological activity was virtually undetectable or very weak (<30% of LC113–25).

Removal of amino acids from the N‐terminus also lead to a progressive reduction (3–5 fold) in the molar potency of the peptides and biological activity became undetectable, or very weak, after the removal of Glu18.

All active peptides contained the core sequence EQEYV(Glu‐Gln‐Glu‐Tyr‐Val) which seems to represent a crucial component of the pharmacophore, although this sequence on its own was inactive and the shortest peptide with significant activity was LC118–25 (EQEYVQTV).

Methoxylation of Tyr21 abolished the ability of LC1 18–25 to inhibit cell proliferation and arachidonic acid release. A cyclized version of LC118–25 was also tested and found to be inactive.

LC118–25 (178 μM) inhibits cPLA2 activation in A549 cells as judged by a band‐shift assay, whereas equimolar concentrations of an inactive peptide LC119–25 were without effect in this assay system.

Several possible mechanisms whereby these peptides act are discussed in the light of LC1 biology and of the effect of glucocorticoids on cell function.

British Journal of Pharmacology (1998) 123, 975–983; doi:10.1038/sj.bjp.0701679
The effect of prolonged treatment with imipramine on the biosynthesis and functional characteristics of D2 dopamine receptors in the rat caudate putamen
Authors: Dziedzicka‐Wasylewska Marta , Rogoż Renata
Published: January 2005

The present study shows the effects of imipramine in a single dose (10 mg kg−1, p.o.) or following repeated (14 days, twice a day) treatment on the level of mRNA coding for D2 dopamine receptors in the rat caudate putamen (CP). Repeated administration of imipramine resulted in the increase of the level of mRNA coding for D2 dopamine receptors.

Radioligand binding studies with the D2 receptor agonist, [3H]‐N‐0437, indicated, that following imipramine administration, the affinity of the agonist for the D2 dopamine receptor significantly increased, though without any alterations in the Bmax.

Pharmacological manipulations (by use of forskolin, GppNHp and quinpirole) of the cyclic AMP generating system, ex vivo following administration of imipramine indicated that an up‐regulation of factors inhibiting cyclic GMP formation takes place.

Most probably it is the D2 dopamine receptor which undergoes functional up‐regulation, resulting from the enhancement of its biosynthesis.

British Journal of Pharmacology (1998) 123, 833–838; doi:10.1038/sj.bjp.0701681
Role of K+ channel opening and stimulation of cyclic GMP in the vasorelaxant effects of nicorandil in isolated piglet pulmonary and mesenteric arteries: relative efficacy and interactions between both pathways
Authors: Pérez‐Vizcaíno Francisco , Cogolludo Angel L. , Villamor Eduardo , Tamargo Juan
Published: January 2005

The effects of the K+ channel opener levcromakalim, the guanylate cyclase stimulant nitroprusside and the dual drug nicorandil (K+ channel opener and guanylate cyclase stimulant) were analysed in piglet isolated endothelium‐denuded pulmonary (PA) and mesenteric (MA) arteries stimulated by noradrenaline (NA) or by the thromboxane A2 mimetic U46619.

Nicorandil, levcromakalim and verapamil were less potent in PA than in MA, the efficacy of levcromakalim was also reduced in PA. The effects of nicorandil and levcromakalim were similar in arteries pre‐contracted by NA and U46619, whereas verapamil was more potent in arteries pre‐contracted by NA. Nitroprusside was equipotent in MA pre‐contracted by either NA or U46619 and in PA pre‐contracted by NA whereas in PA pre‐contracted by U46619, nitroprusside showed lower potency and efficacy.

The relaxant effects of levcromakalim and nitroprusside were inhibited by 10−5 M glibenclamide and 10−6 M ODQ, respectively. Nicorandil‐induced relaxation was inhibited by ODQ in all experimental conditions, whereas glibenclamide had inhibitory effects in PA and MA pre‐contracted by U46619, had no effect in PA pre‐contracted by NA and in MA pre‐contracted by NA it was only inhibitory in the presence of ODQ.

No apparent interactions were found between nitroprusside and levcromakalim as indicated by the lack of effects of pretreatment with one of them (producing 20–35% relaxation) on the potency of the relaxant response to the other. However, in PA pre‐contracted by U46619, where nitroprusside or levcromakalim induced only partial relaxation, the combination of both mechanisms (either by combining nitroprusside plus levcromakalim or by nicorandil) was able to induce full vasodilatation.

In conclusion, K+ channel opening and guanylate cyclase stimulation are independent pathways that induce additive vasorelaxation in piglet PA and MA. The mechanism of action of nicorandil is dependent on the artery and on the nature of the agonist employed to precontract the artery. The relative efficacy of K+ channel opening vs guanylate cyclase stimulation may partially explain the preferential contribution of each mechanism to the relaxant effects of nicorandil.

British Journal of Pharmacology (1998) 123, 847–854; doi:10.1038/sj.bjp.0701693