bluuebunny/crossref_metadata_2025 huggingface dataset:
Ionic basis for the regulation of spontaneous excitation in detrusor smooth muscle cells of the guinea‐pig urinary bladder
Authors: Hashitani Hikaru , Brading Alison F
Published: September 2003

The regulatory mechanisms of spontaneous excitation in detrusor smooth muscles of the guinea‐pig urinary bladder were investigated using intracellular microelectrode and muscle tension recording techniques.

Detrusor smooth muscle cells exhibited nifedipine‐sensitive spontaneous action potentials. Their frequency was highly sensitive to membrane polarization and was reduced by lowering the temperature. Lowering the temperature also reduced the frequency of spontaneous contractions and increased their amplitude.

Charybdotoxin (50 nM) and iberiotoxin (0.1 μM) increased the amplitude and duration of action potentials, and abolished after hyperpolarizations (AHPs). Both agents also increased the amplitude and duration of spontaneous contractions, and reduced their frequency. Apamin (0.1 μM) did not change the shape of action potentials but often converted individual action potentials into bursts. It also increased the amplitude and duration of spontaneous contractions, and reduced their frequency. 4‐aminopyrideine (4‐AP, 1 mM) increased the frequency of action potentials without affecting their shape, and increased the amplitude and frequency of spontaneous contractions.

Cyclopiazonic acid (CPA, 10 μM) and ryanodine (50 μM) increased the amplitude of action potentials, and suppressed AHPs. Both agents also increased the amplitude and duration of spontaneous contractions, and reduced their frequency. 1,2‐(Bis (2‐aminophenoxy) ethane‐N,N,N′, N′‐tetraacetic acid tetrakis (acetoxymethyl ester) (50 μM) dramatically increased the amplitude and duration of the action potential, and abolished AHPs.

Spontaneous action potentials in detrusor smooth muscles cells result from the opening of L‐type Ca2+ channels, and their frequency is regulated by voltage‐dependent mechanisms and by some metabolic process. Both the activation of large conductance Ca2+‐activated K+ (BK) channels and Ca2+‐mediated inactivation of the Ca2+ channels are involved in the repolarizing phase of action potentials. The Ca2+ influx through L‐type Ca2+ channels triggers calcium‐induced calcium release via ryanodine receptors and activates BK channels to generate AHPs. Both small conductance Ca2+‐activated K+ channels and voltage‐sensitive K+ channels may contribute to the resting membrane potential and regulate the frequency of action potentials. The regulatory mechanisms of action potentials are closely related to the regulation of spontaneous contractions.

British Journal of Pharmacology (2003) 140, 159–169. doi:10.1038/sj.bjp.0705320
GLP‐1 derivative liraglutide in rats with β‐cell deficiencies: influence of metabolic state on β‐cell mass dynamics
Authors: Sturis Jeppe , Gotfredsen Carsten F , Rømer John , Rolin Bidda , Ribel Ulla , Brand Christian L , Wilken Michael , Wassermann Karsten , Deacon Carolyn F , Carr Richard D , Knudsen Lotte Bjerre
Published: September 2003

Liraglutide is a long‐acting GLP‐1 derivative, designed for once daily administration in type II diabetic patients. To investigate the effects of liraglutide on glycemic control and β‐cell mass in rat models of β‐cell deficiencies, studies were performed in male Zucker diabetic fatty (ZDF) rats and in 60% pancreatectomized rats.

When liraglutide was dosed s.c. at 150 μg kg−1 b.i.d. for 6 weeks in ZDF rats 6–8 weeks of age at study start, diabetes development was markedly attenuated. Blood glucose was approximately 12 mM lower compared to vehicle (P<0.0002), and plasma insulin was 2–3‐fold higher during a normal 24‐h feeding period (P<0.001). Judged by pair feeding, approximately 53% of the antihyperglycemic effect observed on 24‐h glucose profiles was mediated by a reduction in food intake, which persisted throughout the study and averaged 16% (P<0.02).

Histological analyses revealed that β‐cell mass and proliferation were significantly lower in prediabetic animals still normoglycemic after 2 weeks treatment compared to vehicle‐treated animals that had begun to develop diabetes. When the treatment period was 6 weeks, the liraglutide‐treated animals were no longer completely normoglycemic and the β‐cell mass was significantly increased compared to overtly diabetic vehicle‐treated animals, while β‐cell proliferation was unaffected.

In the experiments with 60% pancreatectomized rats, 8 days treatment with liraglutide resulted in a significantly lower glucose excursion in response to oral glucose compared to vehicle treatment. Again, part of the antihyperglycemic effect was due to reduced food intake. No effect of liraglutide on β‐cell mass was observed in these virtually normoglycemic animals.

In conclusion, treatment with liraglutide has marked antihyperglycemic effects in rodent models of β‐cell deficiencies, and the in vivo effect of liraglutide on β‐cell mass may in part depend on the metabolic state of the animals.

British Journal of Pharmacology (2003) 140, 123–132. doi:10.1038/sj.bjp.0705397
Effect of selective PKC isoform activation and inhibition on TNF‐α‐induced injury and apoptosis in human intestinal epithelial cells
Authors: Chang Q , Tepperman B L
Published: September 2003

We have investigated the effects of specific PKC isoforms in TNF‐α mediated cellular damage using a human intestinal cell line (SCBN).

TNF‐α treatment induced a decrease in the extent of intestinal cellular viability as determined by a formazan‐based assay and an increase in the apoptotic index as assessed by immunohistology. These changes in cellular integrity were found to be related to the degradation of I‐κBα, mobilization of NF‐κB and release of mitochondrial cytochrome c.

TNF‐α treatment also induced the activation of selective PKC isoforms which were associated with the decrease in cellular viability and an increase of cellular apoptosis.

Nonselective PKC antagonists, such as GF109203X and Gö6976 as well as isoform‐selective PKC‐inhibiting peptides would reverse the cellular injury as well as reduce the degradation of I‐κBα and mitochondrial cytochrome c release. These effects were most highly correlated with changes in PKCδ and ɛ primarily.

Intestinal cellular injury could be induced by treating cells with agonists selective for PKCδ and ɛ mainly.

In conclusion, this study has shown that TNF‐α treatment can induce the activation of PKCδ and ɛ in the human intestinal cell line, SCBN, and this response is closely associated with an increase in cellular damage and apoptosis. PKCδ and ɛ primarily mediate the release of mitochondrial cytochrome c and degradation of I‐κBα and hence mobilization of NF‐κB, which are responsible for the pathway leading to cell injury.

British Journal of Pharmacology (2003) 140, 41–52. doi:10.1038/sj.bjp.0705398
Evidence for the involvement of central 5‐HT7 receptors in the micturition reflex in anaesthetized female rats
Authors: Read Kate E , Sanger Gareth J , Ramage Andrew G
Published: September 2003

The effects of the selective 5‐HT7 receptor antagonists SB‐269970 (3‐300 μg kg−1; n=5–6) and SB‐656104 (30 μg kg−1; n=5) administered centrally (i.c.v.) were investigated on the ‘micturition reflex’ in the urethane anaesthetized female rat.

In cystometric recordings, SB‐269970 caused significant increases in volume of 58±15 and 138±33% and pressure of 140±46 and 149±60% thresholds at 10 and 30 μg kg−1. These changes were associated with significant decreases in distension‐induced bladder contraction of 62±14 and 60±11%, respectively. However, there was no change in residual volume. At the higher doses, SB‐269970 blocked the micturition reflex. SB‐656104 had similar effects to SB‐269970 but in addition significantly increased the residual volume.

SB‐269970 (10 μg kg−1; n=5) given i.v. had no effect on the micturition reflex.

SB‐269970 (30 μg kg−1; n=4) given intrathecally (i.t.) had no effect on micturition reflex, although the selective 5‐HT1A receptor antagonist WAY‐100635 given i.t. after SB‐269970 caused a significant increase in the volume threshold.

Using an isovolumetric method in which urethral changes were measured, SB‐269970 (30 μg kg−1; n=4; i.c.v.) failed to have any effect on these urethral‐evoked changes although they significantly reduced the amplitude of the bladder contraction.

These data demonstrate that 5‐HT7 receptors located supraspinally in the rat are involved in the control of micturition.

British Journal of Pharmacology (2003) 140, 53–60. doi:10.1038/sj.bjp.0705399
Partial agonist behaviour depends upon the level of nociceptin/orphanin FQ receptor expression: studies using the ecdysone‐inducible mammalian expression system
Authors: McDonald J , Barnes T A , Okawa H , Williams J , Calo' G , Rowbotham D J , Lambert D G
Published: September 2003

Partial agonism is primarily dependent upon receptor density and coupling efficiency. As these parameters are tissue/model dependent, intrinsic activity in different tissues can vary. We have utilised the ecdysone‐inducible expression system containing the human nociceptin/orphanin FQ (N/OFQ) peptide receptor (hNOP) expressed in Chinese hamster ovary cells (CHOINDhNOP) to examine the activity of a range of partial agonists in receptor binding, GTPγ35S binding and inhibition of adenylyl cyclase studies.

Incubation of CHOINDhNOP cells with ponasterone A (PON) induced hNOP expression ([leucyl‐3H]N/OFQ binding) of 24, 68, 191 and 1101 fmol mg−1 protein at 1, 2, 5 and 10 μM PON, respectively. At 191 fmol mg−1, protein hNOP pharmacology was identical to that reported for other traditional expression systems.

pEC50 values for GTPγ35S binding ranged from 7.23 to 7.72 (2–10 μM PON) for the partial agonist [Phe1ψ(CH2–NH)Gly2]N/OFQ(1–13)–NH2 ([F/G]N/OFQ(1–13)–NH2) and 8.12–8.60 (1–10 μM PON) for N/OFQ(1–13)–NH2 and Emax values (stimulation factor relative to basal) ranged from 1.51 to 3.21 (2–10 μM PON) for [F/G]N/OFQ(1–13)–NH2 and 1.28–6.95 (1–10 μM) for N/OFQ(1–13)–NH2. Intrinsic activity of [F/G]N/OFQ(1–13)–NH2 relative to N/OFQ(1–13)–NH2 was 0.3–0.5. [F/G]N/OFQ(1–13)–NH2 did not stimulate GTPγ35S binding at 1 μM PON, but competitively antagonised the effects of N/OFQ(1–13)–NH2 with a pKB=7.62.

pEC50 values for cAMP inhibition ranged from 8.26 to 8.32 (2–10 μM PON) for [F/G]N/OFQ(1–13)–NH2 and 9.42–10.35 for N/OFQ(1–13)–NH2 and Emax values (% inhibition) ranged from 19.6 to 83.2 for [F/G]N/OFQ(1–13)–NH2 and 40.9–86.0 for N/OFQ(1–13)–NH2. The intrinsic activity of [F/G]N/OFQ(1–13)–NH2 relative to N/OFQ(1–13)–NH2 was 0.48–0.97.

In the same cellular environment with receptor density as the only variable, we show that the profile of [F/G]N/OFQ(1–13)–NH2 can be manipulated to encompass full and partial agonism along with antagonism.

British Journal of Pharmacology (2003) 140, 61–70. doi:10.1038/sj.bjp.0705401
Comparative protection against rat intestinal reperfusion injury by a new inhibitor of sPLA2, COX‐1 and COX‐2 selective inhibitors, and an LTC4receptor antagonist
Authors: Arumugam Thiruma V , Arnold Naomi , Proctor Lavinia M , Newman Michelle , Reid Robert C , Hansford Karl A , Fairlie David P , Shiels Ian A , Taylor Stephen M
Published: September 2003

A new group IIa sPLA2inhibitor was compared with selective inhibitors of COX‐1, COX‐2 and an LTC4antagonist for effects on local and remote tissue injuries following ischaemia and reperfusion (I/R) of the small intestine in rats.

In an acute model of ischaemia (30 min) and reperfusion (150 min) injury in the absence of inhibitors, there was significant intestinal haemorrhage, oedema and mucosal damage, neutropenia, elevated serum levels of aspartate aminotransferase (AST) and hypotension.

Preischaemic treatment with the inhibitor of sPLA2(Group IIa), at 5 mg kg−1i.v. or 10 mg kg−1p.o. significantly inhibited I/R‐induced neutropenia, the elevation of serum levels of AST, intestinal oedema and hypotension.

Pretreatment with the COX‐2 inhibitor celebrex (10 mg kg−1i.v.) and the LTC4antagonist zafirlukast (1 mg kg−1i.v.) also showed marked improvement with I/R‐induced AST, oedema and neutropenia. Hypotension was only reduced by the LTC4antagonist. The COX‐1 inhibitor flunixin (1 mg kg−1i.v.) did not effect improvement in the markers of tissue injury.

Histological examination of rat I/R injury showed that all of the drugs offered some protection to the mucosal layer damage compared to no drug treatment. Given i.v., the sPLA2inhibitor was more effective than either the COX‐1 or COX‐2 inhibitors in preventing rat I/R injury.

These results indicate that a potent new inhibitor of sPLA2(group IIa) protects the rat small intestine from I/R injury after oral or intravenous administration. COX‐2 and LTC4inhibitors also showed some beneficial effects against intestinal I/R injury. Our study suggests that sPLA2(Group IIa) may have a pathogenic role in intestinal I/R in rats.

British Journal of Pharmacology(2003)140, 71–80. doi:10.1038/sj.bjp.0705402
Mechanisms of anion secretion in Calu‐3 human airway epithelial cells by 7,8‐benzoquinoline
Authors: Cuthbert A W , MacVinish L J
Published: September 2003

Cultured epithelial monolayers of Calu‐3 human airway cells were used to measure anion secretion in response to a number of phenanthrolines and benzoquinolines, using short‐circuit current measurements. Calu‐3 cells are derived from serous cells of submucosal glands of the airways and are a target for conditions in which muco‐ciliary clearance is compromised.

Compounds studied were 5,6‐benzoquinoline, 5‐chloro‐1,10‐phenanthroline, 7,8‐benzoquinoline, 5‐nitro‐1,10‐phenanthroline, benzo[c]cinnoline and 1,10‐phenanthroline, which gave EC50 values of 34, 48, 123, 235, 192 and 217 μM, respectively. Of these, 7,8‐benzoquinoline was chosen for further detailed study. Concentration–response relationships for all the compounds had Hill slopes greater than 1.

Permeabilisation of the apical surface of epithelia with nystatin in the presence of an apical to basolateral potassium ion gradient reduced the EC50 for 7,8‐benzoquinoline to 31 μM and altered the Hill slope to close to 1.

Using apically permeabilised epithelia it was shown that 7,8‐benzoquinoline activates an intermediate‐conductance calcium‐sensitive potassium channel (KCNN4) and a cAMP‐sensitive potassium channel (KCNQ1/KCNE3) in the basolateral epithelial membranes.

7,8‐Benzoquinoline was shown to increase chloride conductance of apical epithelial membranes, presumed to be by activation of the cystic fibrosis transmembrane conductance regulator.

7,8‐Benzoquinoline had a minor effect on cAMP accumulation in Calu‐3 cells, probably by inhibition of phosphodiesterase, which may contribute to its effect on CFTR‐ and cAMP‐sensitive potassium channels.

The usefulness of these novel actions in promoting secretion in airway submucosal glands is discussed.

British Journal of Pharmacology (2003) 140, 81–90. doi:10.1038/sj.bjp.0705403
Galanin and neuropeptide Y reduce cholinergic transmission in the heart of the anaesthetised mouse
Authors: Smith‐White Margaret A , Iismaa Tiina P , Potter Erica K
Published: September 2003

This study investigated the effects of galanin (GAL) on inhibition of cholinergic (vagal) activity in the mouse heart using control galanin knockout (GAL‐KO) and GAL‐1R receptor knockout (GAL‐1R‐KO) mice.

In pentobarbitone anaesthetised mice, supramaximal stimulation every 30 s of the vagus nerve innervating the heart, increased pulse interval (PI) by approximately 50 ms or decreased heart rate by approximately 100 beats min−1. This response was attenuated by intravenous administration of GAL (dose ranged from 0.8 to 13 nmol kg−1) in a dose‐dependent manner.

In GAL‐KO mice, the magnitude of inhibition of the increase in PI (ΔPI) following a bolus dose of GAL was not different from the ΔPI in control mice, and neuropeptide Y (NPY), previously shown to attenuate vagal inhibitory activity in mice, evoked a comparative inhibition of ΔPI in GAL‐KO mice.

In GAL‐1R‐KO mice, an intravenous, bolus injection of GAL had no inhibitory effect on vagal activity.

In control mice, stimulation of the sympathetic nerve at 25 V, 10 Hz for 2 min in the presence of propranolol evoked a long‐lasting attenuation of ΔPI. The inhibitory effect on ΔPI was reduced in the presence of the NPY Y2 antagonist, BIIE0246.

In GAL‐1R‐KO mice, stimulation of the sympathetic nerve in the presence of propranolol evoked an attenuation of ΔPI not significantly different from the response in control mice in the presence of BIIE0246. Following administration of BIIE0246 in GAL‐1R‐KO mice, the inhibition of ΔPI that followed stimulation of the sympathetic nerve was abolished.

These findings support the view that the nerve terminals of parasympathetic neurons in the mouse heart possess both GAL‐1R and NPY Y2 receptors which, when activated, reduce acetylcholine release.

British Journal of Pharmacology (2003) 140, 170–178. doi:10.1038/sj.bjp.0705404
Inhibition of leukotriene synthesis with MK‐886 prevents a rise in blood pressure and reduces noradrenaline‐evoked contraction in L‐NAME‐treated rats
Authors: Stanke‐Labesque Françoise , Hardy Gaëlle , Caron Françoise , Cracowski Jean‐Luc , Bessard Germain
Published: September 2003

Long‐term treatment of rats with Nω‐nitro‐L‐arginine methyl ester (L‐NAME) induces hypertension associated with inflammatory and vascular changes. Leukotrienes are proinflammatory vasoactive products that are suspected to be involved in the pathogenesis of hypertension. We investigated, in rats chronically treated with L‐NAME, the involvement of leukotrienes in the in vivo regulation of blood pressure and the in vitro contraction elicited by noradrenaline in isolated aorta.

Rats were randomly assigned to four groups and orally treated for 3 weeks with L‐NAME (1 mg ml−1), L‐NAME (1 mg ml−1) plus the leukotriene biosynthesis inhibitor MK‐886 (0.1 mg ml−1), MK‐886 (0.1 mg ml−1) alone or vehicle (Methocel, 0.1%). All the drugs were added to the drinking fluid.

The mean arterial blood pressure (MABP) increased significantly in L‐NAME‐treated rats (173.3±9.4 mmHg (n=25)) vs Methocel‐treated rats (110.7±4.8 mmHg (n=11), P<0.001). Chronic treatment with MK‐886 prevented this rise in MABP.

Aortic rings with or without endothelium were suspended in organ baths for recording isometric changes in response to noradrenaline. Pretreatment with either MK‐886 (10 μM), the CysLT1 receptor antagonist MK571 (1 μM) or the dual CysLT1/CysLT2 receptor antagonist BAY‐u9773 (0.1 μM) reduced (P<0.05) noradrenaline‐induced contractions in intact aortic rings from L‐NAME‐treated rats only.

Noradrenaline (0.3 μM) induced a two‐fold increase in cysteinyl leukotriene (CysLT) release (measured by enzyme immunoassay) in intact aortic rings from L‐NAME‐treated rats only.

These data suggested (1) a role for the 5‐lipoxygenase pathway in the regulation of blood pressure in L‐NAME‐treated rats and (2) the involvement of endothelial CysLTs in noradrenaline‐induced contraction in aorta from L‐NAME‐treated rats.

British Journal of Pharmacology (2003) 140, 186–194. doi:10.1038/sj.bjp.0705405
Dexamethasone improves vascular hyporeactivity induced by LPS in vivo by modulating ATP‐sensitive potassium channels activity
Authors: D'Emmanuele Di Villa Bianca R , Lippolis L , Autore G , Popolo A , Marzocco S , Sorrentino L , Pinto A , Sorrentino R
Published: September 2003

Septic shock represents an important risk factor for patients critically ill. This pathology has been largely demonstrated to be a result of a myriad of events. Glucocorticoids represent the main pharmacological therapy used in this pathology.

Previously we showed that ATP‐sensitive potassium (KATP) channels are involved in delayed vascular hyporeactivity in rats (24 h after Escherichia coli lipopolysaccharide (LPS) injection). In LPS‐treated rats, we observed a significant hyporeactivity to phenylephrine (PE) that was reverted by glybenclamide (GLB), and a significant increase in cromakalim (CRK)‐induced hypotension.

We evaluated the effect of dexamethasone (DEX 8 mg kg−1 i.p.) whether on hyporeactivity to PE or on hyperreactivity to CRK administration, in vivo, in a model of LPS (8 × 106 U kg−1 i.p.)‐induced endotoxemia in urethane‐anaesthetised rats.

DEX treatment significantly reduced, in a time‐dependent manner, the increased hypotensive effect induced by CRK in LPS‐treated rats. This effect was significantly (P<0.05) reverted by the glucocorticoid receptor antagonist RU38486 (6.6 mg kg−1 i.p.).

GLB‐induced hypertension (40 mg kg−1 i.p.), in LPS‐treated rats, was significantly inhibited by DEX if administered at the same time of LPS.

Simultaneous administration of DEX and LPS to rats completely abolished the hyporeactivity to PE observed after 24 h from LPS injection.

In conclusion, our results suggest that the beneficial effect of DEX in endotoxemia could be ascribed, at least in part, to its ability to interfere with KATP channel activation induced by LPS. This interaction may explain the improvement of vascular reactivity to PE, mediated by DEX, in LPS‐treated rats, highlighting a new pharmacological activity to the well‐known anti‐inflammatory properties of glucocorticoids.

British Journal of Pharmacology (2003) 140, 91–96. doi:10.1038/sj.bjp.0705406
Role of L‐citrulline transport in nitric oxide synthesis in rat aortic smooth muscle cells activated with LPS and interferon‐γ
Authors: Wileman Samantha M , Mann Giovanni E , Pearson Jeremy D , Baydoun Anwar R
Published: September 2003

L‐citrulline, a coproduct of nitric oxide synthase (NOS)‐catalysed metabolism of L‐arginine to nitric oxide (NO), is an important intermediate of the urea cycle and a precursor for L‐arginine biosynthesis in vascular cells.

In the present study, we have examined the characteristics of L‐citrulline transport, regulation by lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) and the ability of L‐citrulline to sustain NO synthesis in rat cultured aortic smooth muscle cells.

L‐citrulline transport was saturable with an apparent Km=1.6±0.2 mM and Vmax=5.9±0.6 pmol μg−1 protein min−1. Transport was pH‐insensitive, partially Na+‐dependent and markedly inhibited by substrates selective for amino‐acid transport systems L and N but not by L‐arginine or substrates for systems A, ASC, xc‐ or XAG. Moreover, transport was not altered in cells treated with LPS (100 μg ml−1) and IFN‐γ (50 U ml−1) for 0–24 h.

Unlike L‐arginine, L‐citrulline could not sustain maximal NO production in cells expressing iNOS.

Our findings provide the first evidence in vascular smooth muscle cells that L‐citrulline transport is mediated via a low‐affinity carrier with characteristics resembling systems L and N. Moreover, in L‐arginine‐deprived rat aortic smooth muscle cells, L‐citrulline cannot sustain maximal NO release via iNOS.

British Journal of Pharmacology (2003) 140, 179–185. doi:10.1038/sj.bjp.0705407
Capacitative calcium entry as a pulmonary specific vasoconstrictor mechanism in small muscular arteries of the rat
Authors: Snetkov Vladimir A , Aaronson Philip I , Ward Jeremy P T , Knock Gregory A , Robertson Tom P
Published: September 2003

The effect of induction of capacitative Ca2+ entry (CCE) upon tone in small (i.d. 200–500 μm) intrapulmonary (IPA), mesenteric (MA), renal (RA), femoral (FA), and coronary arteries (CA) of the rat was examined.

Following incubation of IPA with 100 nM thapsigargin (Thg) in Ca2+‐free physiological salt solution (PSS), a sustained contraction was observed upon reintroduction of 1.8 mM Ca2+, which was unaffected by either diltiazem (10 μM) or the reverse mode Na+/Ca2+ antiport inhibitor KB‐R7943 (10 μM). An identical protocol failed to elicit contraction in MA, RA, or CA, while a small transient contraction was sometimes observed in FA.

The effect of this protocol on the intracellular Ca2+ concentration ([Ca2+]i) was assessed using Fura PE3‐loaded IPA, MA, and FA. Reintroduction of Ca2+ into the bath solution following Thg treatment in Ca2+‐free PSS caused a large, rapid, and sustained increase in [Ca2+]i in all the three types of artery.

100 nM Thg induced a slowly developing noisy inward current in smooth muscle cells (SMC) isolated from IPA, which was due to an increase in the activity of single channels with a conductance of ∼30 pS. The current had a reversal potential near 0 mV in normal PSS, and persisted when Ca2+‐dependent K+ and Cl− currents were blocked; it was greatly inhibited by 1 μM La3+, 1 μM Gd3+, and the IP3 receptor antagonist 2‐APB (75 μM), and by replacement of extracellular cations by NMDG+.

In conclusion, depletion of intracellular Ca2+ stores with Thg caused capacitative Ca2+ entry in rat small muscular IPA, MA, and FA. However, a corresponding contraction was observed only in IPA. CCE in IPA was associated with the development of a small La3+‐ and Gd3+‐sensitive current, and an increased Mn2+ quench of Fura PE‐3 fluorescence. These results suggest that although CCE occurs in a number of types of small arteries, its coupling to contraction appears to be of particular importance in pulmonary arteries.

British Journal of Pharmacology (2003) 140, 97–106. doi:10.1038/sj.bjp.0705408
17β‐Estradiol regulates the expression of endothelin receptor type B in the heart
Authors: Nuedling Simone , Eickels Martin van , Alléra Axel , Doevendans Pieter , Meyer Rainer , Vetter Hans , Grohé Christian
Published: September 2003

Little is known about the interaction of 17β‐estradiol (E2) and the vasoactive endothelin system in the heart. Endothelin signaling is activated in a failing heart and may contribute to myocardial dysfunction and remodeling. Therefore, we investigated the regulation of proteins of the endothelin system (ppET‐1, ECE and ETA‐R and ETB‐R) in the hearts of female spontaneously hypertensive rats (SHR) with respect to E2.

Relative expression levels of the respective cardiac mRNA obtained from sham‐operated, ovariectomized and ovariectomized E2‐substituted SHR were quantified by real‐time PCR. Ovariectomy led to a significant upregulation of the ETB‐R mRNA (2.6±0.8‐fold) in the left ventricular myocardium, which was not attendant with an alteration of ETA‐R, ECE and ppET‐1 mRNA expression.

An upregulation of the relative expression level of ETB‐R protein due to ovariectomy was also demonstrated by radioligand binding assay.

Upregulation of both ETB‐R mRNA and ETB‐R protein expression was completely inhibited by E2 replacement.

To confirm these results in in vitro experiments, we quantified the mRNA of ET‐R subtypes from isolated cardiomyocytes in the presence and absence of E2 (10−8 M, 24 h). Our data showed a markedly downregulated level of ETB‐R mRNA in cardiomyocytes stimulated with E2. ETB‐R downregulation was not attendant with the alteration of ETA‐R, ECE and ppET‐1 mRNA expression.

Taken together, these data demonstrate that estrogen regulates the expression of ETB‐R in rat ventricular myocardium in vivo and in vitro. These observations may help to understand gender‐based differences found in cardiovascular disease.

British Journal of Pharmacology (2003) 140, 195–201. doi:10.1038/sj.bjp.0705409
Aminoguanidine prevents age‐related aortic stiffening in Fisher 344 rats: aortic impedance analysis
Authors: Chang Kuo‐Chu , Hsu Kwan‐Lih , Peng Ying‐I , Lee Fong‐Chu , Tseng Yung‐Zu
Published: September 2003

We determined the effects of long‐term treatment with aminoguanidine (AG), an inhibitor of advanced glycation end products, on the mechanical properties of the arterial system in aged Fisher 344 rats, using the aortic impedance analysis.

Normotensive rats were treated from 18 to 24 months with AG (1 g/l−1 in drinking water) and compared with a control group. Pulsatile aortic pressure and flow signals were measured and then subjected to Fourier transformation for the analysis of aortic input impedance. Wave transit time was determined using the impulse response function of the filtered aortic input impedance spectra.

With no alteration in body weight, rats treated with AG had decreased heart weight compared with the aged untreated controls.

AG did not affect arterial blood pressure; however, the age‐related increase in total peripheral resistance was prevented by AG.

AG retarded the age‐related decline in aortic distensibility, as evidenced by a reduction of 25.2% in aortic characteristic impedance and an increase of 28.1% in wave transit time.

Meanwhile, the increase in wave reflection factor in aging rats was reduced by 32.3% by AG. Both the increased wave transit time and the decreased wave reflection factor suggest that AG may prevent the age‐related augmentation in systolic loading condition for the left ventricle coupled to the arterial system.

We conclude that long‐term treatment with AG may impart significant protection against aortic stiffening and cardiac hypertrophy in aged Fisher 344 rats.

British Journal of Pharmacology (2003) 140, 107–114. doi:10.1038/sj.bjp.0705410
2′, 3′‐O‐(2,4,6,Trinitrophenyl)‐ATP and A‐317491 are competitive antagonists at a slowly desensitizing chimeric human P2X3 receptor
Authors: Neelands Torben R , Burgard Edward C , Uchic Marie E , McDonald Heath A , Niforatos Wende , Faltynek Connie R , Lynch Kevin J , Jarvis Michael F
Published: September 2003

Rapid desensitization of ligand‐gated ion channel receptors can alter the apparent activity of receptor modulators, as well as make detection of fast‐channel activation difficult. Investigation of the antagonist pharmacology of ATP‐sensitive homomeric P2X3 receptors is limited by agonist‐evoked fast‐desensitization kinetics.

In the present studies, chimeric receptors were created using the coding sequence for the N‐terminus and the first transmembrane domain of either the nondesensitizing human P2X2a or fast‐desensitizing P2X3 receptor joined to the sequence encoding the extracellular loop, second transmembrane domain, and C‐terminus of the other receptor (designated P2X2–3 and P2X3–2, respectively). These clones were stably transfected into 1321N1 astrocytoma cells for biophysical and pharmacological experiments using both electrophysiological and calcium‐imaging methods.

Chimeric P2X2–3 and P2X3–2 receptors were inwardly rectifying and agonist responses showed desensitization properties similar to the wild‐type human P2X2a and P2X3 receptors, respectively.

The P2X2–3 chimera displayed an agonist pharmacological profile similar to the P2X3 wild‐type receptor being activated by low concentrations of both ATP and α,β‐meATP. In contrast, the P2X3–2 chimera had markedly reduced sensitivity to both agonists.

The P2X3 receptor antagonists TNP‐ATP and A‐317491 were shown to be potent, competitive antagonists of the P2X2–3 chimera (Ki=2.2 and 52.1 nM, respectively), supporting the hypothesis that rapid receptor desensitization can mask the competitive antagonism of wild‐type homomeric P2X3 receptors.

British Journal of Pharmacology (2003) 140, 202–210. doi:10.1038/sj.bjp.0705411
Role of cannabinoid CB1 receptors and tumor necrosis factor‐α in the gut and systemic anti‐inflammatory activity of SR 141716 (Rimonabant) in rodents
Authors: Croci Tiziano , Landi Marco , Galzin Anne‐Marie , Marini Pietro
Published: September 2003

We investigated the effect of the cannabinoid CB1 receptor antagonist, SR 141716, on indomethacin‐induced small intestine inflammation and Escherichia coli lipopolysaccharide (LPS)‐induced plasma TNF‐α (TNF) release in comparison to the cannabinoid CB2 receptor antagonist, SR 144528, in rodents.

In rats, indomethacin induced significant ulcer formation in the small intestine; this was accompanied by an increase in tissue TNF levels and myeloperoxidase (MPO) activity. SR 141716 prevented the ulcers and the rise in TNF levels (ID50 3.3, 0.4 mg kg−1, respectively) and MPO activity. SR 144528 prevented intestinal ulcers only.

The effect of SR 141716 against indomethacin‐induced ulcers and increase of plasma TNF levels after LPS was also studied in wild‐type and CB1 receptor knockout mice. Indomethacin induced intestinal ulcers in mice, but not tissue TNF production and MPO activity. SR 141716 reduced the ulcers to a similar extent in wild‐type and CB1 receptor knockout mice. In rats and wild‐type mice, but not in CB1 receptor knockout mice, SR 141716 inhibited the LPS‐induced increase in plasma TNF levels.

These findings provide evidence that the indomethacin model of intestinal lesions differs in rat and mouse and support the existence of several mechanisms for the antiulcer activity of SR141716, the most important involving the inhibition of TNF production. The potent anti‐inflammatory activity of SR141716 in rodents indicated its potential therapeutic interest in chronic immune‐inflammatory diseases.

British Journal of Pharmacology (2003) 140, 115–122. doi:10.1038/sj.bjp.0705412
Dexamethasone enhances interaction of endogenous Annexin 1 with L‐selectin and triggers shedding of L‐selectin in the monocytic cell line U‐937
Authors: De Coupade Catherine , Solito Egle , Levine Jon D
Published: September 2003

L‐selectin, constitutively expressed by leukocytes, is involved in the initial binding of leukocytes to activated endothelium. Anti‐inflammatory drugs like glucocorticoids can induce shedding of L‐selectin, but the mechanism is still unknown. Annexin 1, a protein whose synthesis and externalization/secretion are induced during the inflammatory response, has been proposed as a mediator of the anti‐inflammatory actions of glucocorticoids.

The monocytic cell line U‐937 strongly expresses Annexin 1 after 24 h of phorbol 12‐myristate 13‐acetate (PMA, 1 nM) treatment and externalizes/releases the protein after additional 16 h of dexamethasone (1 μM) treatment.

This study investigated the possible regulation of cell surface L‐selectin shedding by endogenous Annexin 1, and its role in glucocorticoid‐induced L‐selectin shedding in the U‐937 cell line.

PMA‐ and dexamethasone treatment‐induced L‐selectin shedding was potentially mediated by Annexin 1, since neutralizing antibodies against Annexin 1 reduced dexamethasone‐ and Annexin 1‐induced shedding.

Immunoprecipitation and binding assays provided support for the suggestion that this effect could be mediated by an interaction between externalized Annexin 1 and L‐selectin. Such interaction involved the N‐terminal domain of Annexin 1 and was calcium‐dependent. Confocal microscopy studies demonstrated increased colocalization of Annexin 1 and L‐selectin on the cell surface.

Overall, our study provides new insights into the potential role of endogenous ANXA1 as a mediator of dexamethasone‐induced L‐selectin shedding, which may contribute to the anti‐inflammatory activity of glucocorticoids.

British Journal of Pharmacology (2003) 140, 133–145. doi:10.1038/sj.bjp.0705413
Relationship between peroxisome proliferator‐activated receptors (PPARα and PPARγ) and endothelium‐dependent relaxation in streptozotocin‐induced diabetic rats
Authors: Kanie Noriyasu , Matsumoto Takayuki , Kobayashi Tsuneo , Kamata Katsuo
Published: September 2003

The aim of the present study was to investigate the causal relationship between peroxisome proliferator‐activated receptor (PPAR) and endothelium‐dependent relaxation in streptozotocin (STZ)‐induced diabetic rats.

Acetylcholine (ACh)‐induced endothelium‐dependent relaxation was significantly weaker in diabetic rats than in age‐matched controls. The decreased relaxation in diabetes was improved by the chronic administration of bezafibrate (30 mg kg−1, p.o., 4 weeks).

The expressions of the mRNAs for PPARα and PPARγ were significantly decreased in STZ‐induced diabetic rats (compared with the controls) and this decrease was restored partially, but not completely, by the chronic administration of bezafibrate.

Superoxide dismutase activity in the aorta was not significantly different between diabetic rats and bezafibrate‐treated diabetic rats.

The expression of the mRNA for the p22phox subunit of NAD(P)H oxidase was significantly higher in diabetics than in controls, but it was lower in bezafibrate‐treated diabetic rats than in nontreated diabetic rats. Although the expression of the mRNA for prepro ET‐1 (ppET‐1) was markedly increased in diabetic rats (compared with controls), this increase was prevented to a significant extent by the chronic administration of bezafibrate.

These results suggest that downregulations of PPARα and PPARγ may lead to an increased expression of ppET‐1 mRNA in diabetic states and this increment may trigger endothelial dysfunction.

British Journal of Pharmacology (2003) 140, 23–32. doi:10.1038/sj.bjp.0705414
Tyr‐c[D‐Orn‐Tyr(Bzl)‐Pro‐Gly]: a novel antiproliferative acting somatostatin receptor agonist with μ‐opioid receptor‐sensitizing properties
Authors: Stirnweiss Jörg , Hartrodt Bianka , Greksch Gisela , Stürzebecher Uta , Böhmer Frank‐D , Neubert Klaus , Liebmann Claus
Published: September 2003

Here, we introduce a β‐casomorphin‐5‐derived cyclic pentapeptide, cCD‐2 (Tyr‐cyclo[D‐Orn‐Tyr(Bzl)‐Pro‐Gly]), which inhibits the cell growth of a variety of human cancer cell lines.

This opioid‐derived peptide possesses only low affinity for μ‐receptors, but enhances the agonist binding to μ‐receptors in vitro and potentiates the analgesic effect of morphin in vivo. The molecular mechanism of μ‐receptor sensitization by cCD‐2 is not yet known.

The antiproliferative effect of cCD‐2 is independent of μ‐, δ‐, and κ‐receptors.

Using SH‐SY5Y cells as model, we can demonstrate that cCD‐2 specifically binds to somatostatin receptors and stimulates the activity of protein tyrosine phosphatases, which are early downstream targets of SST receptors.

In SH‐SY5Y cells, cCD‐2 specifically increases the activity of the cytosolic PTP SHP‐2, stimulates the activity of mitogen‐activated protein kinase (MAPK), and elevates the expression of the cyclin‐dependent kinase inhibitor p21 (WAF1/Cip1), suggesting the involvement of SSTR1 receptor subtype in cCD‐2 action in this cell type.

In COS‐7 cells, for comparison, we found a stimulation of SHP‐2 as well as SHP‐1 in response to cCD‐2. The activation of SHP‐1, which is attributed to the SSTR2 receptor and negatively regulates the EGF receptor, corresponds with the ability of cCD‐2 to inhibit the EGF‐induced MAPK activation in COS‐7 cells.

Our results show that in SH‐SY5Y cells cCD‐2 inhibits cell growth via the SSTR1 receptor‐signalling pathway but may, in other cells, also use other SSTR subtypes and their signalling mechanisms.

cCD‐2 represents a novel type of opioid‐derived antiproliferative SST receptor agonist, which possesses low μ‐receptor affinity but may induce μ‐receptor sensitization and is structurally different from the hitherto known SST receptor agonists.

British Journal of Pharmacology (2003) 140, 13–22. doi:10.1038/sj.bjp.0705416
Different distribution of morphine and morphine‐6β‐glucuronide after intracerebroventricular injection in rats
Authors: Okura Takashi , Saito Masanori , Nakanishi Misato , Komiyama Noriyuki , Fujii Aki , Yamada Shizuo , Kimura Ryohei
Published: September 2003

We investigated the distribution of morphine and morphine‐6β‐glucuronide (M6G) in the brain and spinal cord after intracerebroventricular (i.c.v.) injection of each drug in rats.

The cerebrospinal fluid (CSF) concentration of M6G was 5–37 times greater than that of morphine 10, 60 and 120 min after the i.c.v. injection. The apparent elimination clearance of M6G from the CSF was 10 times lower than that of morphine.

The intrathecal CSF concentration of M6G measured by the microdialysis method was 29–79 times greater than that of morphine, and M6G was rapidly distributed into the intrathecal space after the i.c.v. injection.

M6G was detected in the cerebrum, brainstem, cerebellum and spinal cord at concentrations 2–21 times higher than morphine after the i.c.v. injection of each drug. The distribution volume of M6G in rat brain slices was three times lower than that of morphine, and close to the extracellular fluid space in the brain regions corresponding to the vicinity of the opioid receptors.

These brain distribution characteristics of M6G, namely, low clearance from the central nervous system, localization in the extracellular fluid and rapid distribution into the intrathecal space, may contribute to the potent analgesic effect of M6G after i.c.v. injection.

British Journal of Pharmacology (2003) 140, 211–217. doi:10.1038/sj.bjp.0705418
The ‘state’ of β‐adrenoceptors
Authors: Molenaar Peter
Published: September 2003

British Journal of Pharmacology (2003) 140, 1–2. doi:10.1038/sj.bjp.0705420
Atypical β‐adrenoceptors, different from β3‐adrenoceptors and probably from the low‐affinity state of β1‐adrenoceptors, relax the rat isolated mesenteric artery
Authors: Kozłowska Hanna , Szymska Urszula , Schlicker Eberhard , Malinowska Barbara
Published: September 2003

We examined whether β3‐ and/or atypical β‐adrenoceptors relax the rat isolated mesenteric artery.

Mesenteric arteries precontracted with phenylephrine were relaxed by β‐agonists with the following potencies (pD2): nonselective agonist isoprenaline (6.00)>nonconventional partial agonist cyanopindolol (5.45)>β2‐agonist fenoterol (4.98)>nonconventional partial agonist CGP 12177 (4.19)>β3‐agonist ZD 2079 (3.72). The β3‐agonist CL 316243 1 mM relaxed the vessel only marginally.

The concentration–response curves (CRCs) for cyanopindolol, CGP 12177 and ZD 2079 were not affected by the nonselective β‐antagonist propranolol 0.3 μM, the β2‐antagonist ICI 118551 1 μM and by CL 316243 60 μM, but shifted to the right by bupranolol (pA2 5.3–5.7), CGP 20712 (5.4) and SR 59230A (6.5–6.7) (the latter three drugs block atypical and/or β3‐adrenoceptors at high concentrations).

The CRC for isoprenaline was shifted to the right by propranolol (pA2 7.0) but, in the presence of propranolol 0.3 μM, not affected by SR 59230A 1 μM. The CRC for fenoterol was shifted to the right by propranolol (pA2 6.9) and ICI 118551 (6.8).

Removal of endothelium diminished the vasorelaxant effects of cyanopindolol, CGP 12177 and ZD 2079.

Fenoterol and cyanopindolol also relaxed (endothelium‐intact) mesenteric arteries precontracted with serotonin. The relaxant effect of cyanopindolol was antagonized by bupranolol to about the same degree as in phenylephrine‐contracted vessels.

In conclusion, β2‐ and atypical β‐adrenoceptors (but not β3‐adrenoceptors) relax the rat mesenteric artery. The atypical β‐adrenoceptor, which is partially located endothelially, may differ from the low‐affinity state of the β1‐adrenoceptor.

British Journal of Pharmacology (2003) 140, 3–12. doi:10.1038/sj.bjp.0705421
Pharmacological comparison of the alternatively spliced short and long CCK2 receptors
Authors: Morton M F , Harper E A , Tavares I A , Shankley N P
Published: September 2003

The alternatively spliced, short and long cholecystokinin receptors (CCK2S and CCK2L) were expressed in NIH3T3 cells, and compared using radioligand‐binding assays with identical buffer and incubation conditions.

As judged by a saturation analysis, the selective CCK2‐receptor antagonist radioligand [3H]‐JB93182 did not discriminate between the CCK2S or CCK2L receptors.

A global analysis of competition studies, using a range of structurally diverse, CCK‐receptor selective ligands, provided further evidence that these receptor subtypes were pharmacologically indistinguishable. However, when analysed individually a number of small, yet significant differences were observed with some of the compounds.

These data are consistent with previous study that suggested a possible pharmacological difference between these isoforms, at least in terms of the CCK2‐receptor antagonist, L‐365,260. However, it would appear that the pharmacological profile of these compounds is not consistent with their affinity at the putative G1/G2 receptors previously described by Harper et al.

British Journal of Pharmacology (2003) 140, 218–224. doi:10.1038/sj.bjp.0705423
EDHF and endothelial potassiun channels: IKCa and SKCa
Authors: Félétou M , Vanhoutte Paul M , Weston Arthur H , Edwards Gillian
Published: September 2003

British Journal of Pharmacology (2003) 140, 225. doi:10.1038/sj.bjp.0705425
Endothelial calcium‐activated K+‐channels and EDHF signalling
Authors: Köhler Ralf , Eichler Ines
Published: September 2003

British Journal of Pharmacology (2003) 140, 226. doi:10.1038/sj.bjp.0705426
Evidence that central 5‐HT2A and 5‐HT2B/C receptors regulate 5‐HT cell firing in the dorsal raphe nucleus of the anaesthetised rat
Authors: Boothman L J , Allers K A , Rasmussen K , Sharp T
Published: September 2003

British Journal of Pharmacology (2003) 140, 227–228. doi:10.1038/sj.bjp.0705444
Purification of capsular polysaccharide from Streptococcus pneumoniae serotype 23F by a procedure suitable for scale‐up
Authors: Gonçalves Viviane Maimoni M. , Takagi Mickie , Lima Rodrigo B. , Massaldi Hugo , Giordano Roberto C. , Tanizaki Martha M.
Published: September 2003

Streptococcus pneumoniae is a pathogenic encapsulated bacterium, which causes pneumonia, bacteraemia and meningitis. Capsular polysaccharide conjugated to a carrier protein has been widely used as a vaccine antigen. Serotype 23F is one of the prevalent worldwide pneumococci. A simple and efficient method for capsular polysaccharide serotype 23F purification that can easily be scaled‐up was developed. This method consisted of using culture broth obtained by tangential microfiltration through a 0.22 μm membrane, broth microfiltrate concentration by tangential ultrafiltration in a 30 kDa spiral membrane, fractional ethanol precipitation (28–60%), nuclease and proteinase treatment, and concentration/diafiltration in a 30 kDa cassette membrane. The final polysaccharide recovery was 89%. The final protein and nucleotide contamination was 1.5% (w/w) and 0.3% (w/w) respectively. The final pure polysaccharide meets the requirements of the World Health Organization and residual proteinase was not found in the final product.
Continuous production of a peptidic fraction containing the intermediate opioid peptide LVV‐haemorphin‐7 (LVVh‐7) by peptic hydrolysis of bovine haemoglobin in a continuous membrane reactor
Authors: Kapel Romain , Froidevaux Renato , Nedjar‐Arroume Naima , Fertin‐Bazus Anne , Dhulster Pascal , Guillochon Didier
Published: September 2003

Peptic hydrolysis of native bovine haemoglobin at pH 3 yields the LVV‐haemorphin‐7 (Leu‐Val‐Val‐Tyr‐Pro‐Trp‐Thr‐Gln‐Arg‐Phe; LVVh‐7) opioid peptide corresponding to the residues‐31–40 fragment of the β‐chain of haemoglobin. This peptide is intermediate in the course of batch hydrolysis and is rapidly degraded. Indeed, it shows an optimum at 3% degree of hydrolysis (i.e. 2 min of reaction time). The hydrolysis was carried out in a continuous membrane reactor with a space time (ratio of the flux to the reactor volume) set to 2 min (corresponding to optimum LVVh‐7 production). This process allows the continuous production of a constant fraction of intermediate peptides containing LVVh‐7 for 48 min.
Biosynthesis of rifamycin SV by Amycolatopsis mediterranei MTCC17 in solid cultures
Authors: Krishna P. S. Murali , Venkateswarlu G. , Pandey Ashok , Rao Linga Venkateswar
Published: September 2003

Studies were performed on the production of rifamycin SV, an ansamycin compound, extensively used for curing tuberculosis, leprosy and several other mycobacterial infections, using a strain of Amycolatopsis mediterranei MTCC17 in solid cultures. Wheat bran was employed as a solid substrate. The culture produced 4 g of rifamycin SV/kg of substrate. Pre‐treatment of the substrate with dilute HCl was found to increase the yield of rifamycin SV by 300% (from 4 to 12 g·kg of substrate−1). Various process parameters were tested to establish the best conditions for the maximum production of the compound and a initial moisture level of 80%, inoculum size of 40%, initial substrate pH of 7.0, incubation temperature of 26 °C and a 7 day fermentation period were found to be optimal. Different solvents were used for the extraction of rifamycin SV from the fermented matter and methanol was found to be most suitable. Under optimized conditions, the yield of rifamycin SV further increased from 12 to 32 g·kg of substrate−1, showing an 8‐fold increase from the initial value.