bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that spontaneous contractile activity in the rat myometrium is not inhibited by NO‐mediated increases in tissue levels of cyclic GMP
Authors: Hennan James K. , Diamond Jack
Published: January 2005

There is conflicting evidence in the literature concerning the role of cyclic GMP in the regulation of myometrial contractility and the importance of hormonal status on the uterine response to cyclic GMP‐elevating agents. The objective of the present study was to investigate further the importance of cyclic GMP in the control of uterine contractility, by monitoring the effects of cyclic GMP‐elevating agents on spontaneous contractions and cyclic GMP levels in myometrial strips from pregnant rats and from ovariectomized rats under the influence of oestrogen and/or progesterone.

Sodium nitroprusside (SNP) 5 mM, atrial natriuretic peptide (ANP) 100 nM, L‐arginine 1 mM and 8‐bromo‐cyclic GMP 100 mM had no relaxant effect on the spontaneous contractions of myometria from pregnant rats or from ovariectomized rats under the influence of oestrogen or progesterone.

Tissue levels of cyclic GMP were significantly elevated by SNP in all treatment groups, including pregnant animals. For example, in ovariectomized, progesterone‐treated rats, SNP raised cyclic GMP levels approximately 8 fold from a basal level of 2.9±0.4 pmol mg−1 protein to 24.8±4.0 pmol mg−1 protein. ANP increased cyclic GMP levels approximately 2 fold in all treatment groups, except in the pregnant animals. L‐Arginine elevated cyclic GMP significantly only in ovariectomized, vehicle‐treated myometria.

The activity of cyclic GMP‐dependent protein kinase (PKG) was significantly increased (3 fold) in myometria exposed to SNP (5 mM). Thus, the inability of SNP to relax uterine preparations was not due to a failure of SNP‐elevated cyclic GMP to activate PKG.

The more potent NO donor, S‐nitroso‐N‐acetylpenicillamine (SNAP), at a concentration of 100 μM was able to inhibit spontaneous contractions significantly in myometrial preparations from both non‐ovariectomized and ovariectomized rats treated with oestrogen or progesterone.

Tissue levels of cyclic GMP were markedly increased by SNAP at concentrations of 10, 30 and 100 μM. At 100 μM, cyclic GMP levels increased from 1.9±0.2 pmol mg−1 protein to 74.0±18.0 pmol mg−1 protein. However, complete or partial blockade of SNAP‐induced increases in cyclic GMP levels by the soluble guanylyl cyclase inhibitor, ODQ (25 μM), had no effect on the relaxant response to SNAP. Thus, the relaxant effect of SNAP in this tissue appears to be mediated via a mechanism independent of cyclic GMP.

Taken as a whole, the results of the present study indicate that cyclic GMP does not play an important role in the control of contractility in the rat uterus.

British Journal of Pharmacology (1998) 123, 959–967; doi:10.1038/sj.bjp.0701678
Opposite effects of nitric oxide donors on DNA single strand breakage and cytotoxicity caused by tert‐butylhydroperoxide
Authors: Guidarelli Andrea , Sestili Piero , Cantoni Orazio
Published: January 2005

The effects of three different NO donors on tert‐butylhydroperoxide (tB‐OOH)‐induced DNA cleavage and toxicity were investigated in U937 cells.

Treatment with S‐nitroso‐N‐acetyl‐penicillamine (SNAP, 1–30 μm), while not in itself DNA‐damaging, potentiated the DNA strand scission induced by 200 μm tB‐OOH in a concentration‐dependent fashion. The enhancing effects of SNAP were observed with two different techniques for the assessment of DNA damage. Decomposed SNAP was inactive. S‐nitrosoglutathione (GSNO, 300 μm) and (Z)‐1‐[(2‐aminoethyl)‐N‐(2‐ammonioethyl) amino]diazen‐1‐ium‐1,2‐diolate (DETA‐NO, 1 mm) also increased DNA cleavage generated by tB‐OOH and these responses, as well as that mediated by SNAP, were prevented by the NO scavenger 2‐phenyl‐4,4,5,5‐tetramethylimidazolin‐1‐oxyl‐3‐oxide (PTIO).

SNAP neither inhibited catalase activity nor increased the formation of DNA lesions in cells exposed to H2O2. Furthermore, SNAP did not affect the rate of rejoining of the DNA single strand breaks generated by tB‐OOH.

Under the conditions utilized in the DNA damage experiments, treatment with tB‐OOH alone or associated with SNAP did not cause cell death. However, SNAP as well as GSNO markedly reduced the lethal response promoted by millimolar concentrations of tB‐OOH and these effects were abolished by PTIO. Decomposed SNAP was inactive.

It is concluded that low levels of NO donors, which probably release physiological concentrations of NO, enhance the accumulation of DNA single strand breaks in U937 cells exposed to tB‐OOH. This NO‐mediated effect appears to (a) not depend on inhibition of either DNA repair (which would increase the net accumulation of DNA lesions by preventing DNA single strand break removal) or catalase activity (which would also enhance the net accumulation of DNA lesions since H2O2 is one of the species mediating the tB‐OOH‐induced DNA cleavage) and (b) be caused by enforced formation of tB‐OOH‐derived DNA‐damaging species. In contrast to these results, similar concentrations of NO prevented cell death caused by millimolar concentrations of tB‐OOH. Hence, DNA single strand breakage generated by tB‐OOH in the absence or presence of NO does not represent a lethal event.

British Journal of Pharmacology (1998) 123, 1311–1316; doi:10.1038/sj.bjp.0701683
Mechanisms of relaxations of bovine isolated bronchioles by the nitric oxide donor, GEA 3175
Authors: Hernández Medardo , Elmedal Britt , Mulvany Michael J. , Simonsen Ulf
Published: January 2005

The present study was designed to investigate the effects and mechanisms of relaxation induced by the nitric oxide (NO) donor, GEA 3175 (a 3‐aryl‐substituted oxatriazole derivative) on bovine bronchioles (effective lumen diameter 200–800 μm) suspended in microvascular myographs for isometric tension recording.

In segments of bovine bronchioles contracted to 5‐hydroxytryptamine, GEA 3175 (10−8–10−4 M) induced concentration‐dependent reproducible relaxations. These relaxations were slow in onset compared to other NO‐donors such as 3‐morpholinosydonimine‐hydrochloride (SIN‐1) and S‐nitroso‐N‐acetylpenicillamine (SNAP).

In 5‐hydroxytryptamine‐contracted preparations the order of relaxant potency (pD2) was: salbutamol (7.80)>GEA 3175 (6.18)>SIN‐1 (4.90)>SNAP (3.55). In segments contracted to acetylcholine, the relaxant responses were reduced and GEA 3175 relaxed the bronchioles with pD2=4.41±0.12 and relaxations of 66±10% (n=4), while SNAP and salbutamol caused relaxations of 19±6% (n=4) and 27±6% (n=8) at the highest concentration used, respectively.

Oxyhaemoglobin (10−5 M), the scavenger of nitric oxide, caused rightward shifts of the concentration‐relaxation curves to GEA 3175 and NO. 1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one (ODQ, 3×10−6 M) and LY 83583 (10−6 M), the inhibitors of soluble guanylate cyclase, also reduced the relaxations induced by GEA 3175 and nitric oxide. However, ODQ did not affect salbutamol‐evoked relaxation in the bovine small bronchioles.

GEA 3175‐induced relaxations were reduced in potassium‐rich (60 mmol l−1 K+) solution. Glibenclamide (10−6 M) markedly inhibited the relaxations induced by the opener of ATP‐sensitive K+ channels, levcromakalim (3×10−8–10−5 M), but it did not modify the relaxations induced by GEA 3175 or salbutamol. Apamin (5×10−7 M), a blocker of the small Ca2+‐activated K+‐channels did not affect the relaxations to GEA 3175. In contrast, blockers of large Ca2+‐activated K+‐channels, charybdotoxin (3×10−8–10−7 M) and iberiotoxin (10−8 M), did inhibit the relaxations to GEA 3175. The combination of apamin and charybdotoxin did not induce an additional inhibitory effect on the relaxations to GEA 3175 compared to charybdotoxin alone.

In preparations where a concentration‐response curve to GEA 3175 or NO was first obtained in the presence of LY 83583, incubation with charybdotoxin (10−7 M) did produce an additional inhibitory effect of the relaxations. However, in the presence of ODQ (3×10−6 M), iberiotoxin (10−8 M) did not produce additional reduction of the NO‐ or GEA 3175‐induced relaxations.

The present results suggest that the slow‐releasing NO‐donor GEA 3175 is more potent than the traditional NO donors in inducing relaxations of bovine bronchioles. GEA 3175, as for exogenously added NO, elicits relaxations through a cyclic GMP‐dependent mechanism followed by opening of large conductance Ca2+‐activated K+‐channels.

British Journal of Pharmacology (1998) 123, 895–905; doi:10.1038/sj.bjp.0701684
Interaction of N‐benzoyl‐D‐phenylalanine and related compounds with the sulphonylurea receptor of β‐cells
Authors: Schwanstecher Christina , Meyer Miriam , Schwanstecher Mathias , Panten Uwe
Published: January 2005

The structure activity relationships for the insulin secretagogues N‐benzoyl‐D‐phenylalanine (NBDP) and related compounds were examined at the sulphonylurea receptor level by use of cultured HIT‐T15 and mouse pancreatic β‐cells. The affinities of these compounds for the sulphonylurea receptor were compared with their potencies for KATP‐channel inhibition. In addition, the effects of cytosolic nucleotides on KATP‐channel inhibition by NBDP were investigated.

NBDP displayed a dissociation constant for binding to the sulphonylurea receptor (KD value) of 11 μM and half‐maximally effective concentrations of KATP‐channel inhibition (EC50 values) between 2 and 4 μM (in the absence of cytosolic nucleotides or presence of 0.1 mM GDP or 1 mM ADP).

In the absence of cytosolic nucleotides or presence of GDP (0.1 mM) maximally effective concentrations of NBDP (0.1–1 mM) reduced KATP‐channel activity to 47% and 44% of control, respectively. In the presence of ADP (1 mM), KATP‐channel activity was completely suppressed by 0.1 mM NBDP.

The L‐isomer of N‐benzoyl‐phenylalanine displayed a 20 fold lower affinity and an 80 fold lower potency than the D‐isomer.

Introduction of a p‐nitro substituent in the D‐phenylalanine moiety of NBDP did not decrease lipophilicity but lowered affinity and potency by more than 30 fold.

Introduction of a p‐amino substituent in the D‐phenylalanine moiety of NBDP (N‐benzoyl‐p‐amino‐D‐phenylalanine, NBADP) reduced lipophilicity and lowered affinity and potency by about 10 fold. This loss of affinity and potency was compensated for by formation of the phenylpropionic acid derivative of NBADP. A similar difference in affinity was observed for the sulphonylurea carbutamide and its phenylpropionic acid derivative.

Replacing the benzene ring in the D‐phenylalanine moiety of NBDP by a cyclohexyl ring increased lipophilicity, and the KD and EC50 values were slightly lower than for NBDP. Exchange of both benzene rings in NBDP by cyclohexyl rings further increased lipophilicity without altering affinity and potency.

This study shows that N‐acylphenylalanines interact with the sulphonylurea receptor of pancreatic β‐cells in a stereospecific manner. Their potency depends on lipophilic but not aromatic properties of their benzene rings. As observed for sulphonylureas, interaction of N‐acylphenylalanines with the sulphonylurea receptor does not induce complete inhibition of KATP‐channel activity in the absence of inhibitory cytosolic nucleotides.

British Journal of Pharmacology (1998) 123, 1023–1030; doi:10.1038/sj.bjp.0701686
Induction of nitric oxide synthase by protein synthesis inhibition in aortic smooth muscle cells
Authors: Marczin Nándor , Go Carolyn Y. , Papapetropoulos Andreas , Catravas John D.
Published: January 2005

The role of de novo protein synthesis in inducible NO synthase (iNOS) activation was investigated in vitro by evaluating the effects of protein synthesis inhibitors cycloheximide (CH) and anisomycin (ANI) on iNOS activity, protein and mRNA levels in rat aortic smooth muscle cells (RASMC).

As determined by cyclic GMP accumulation, substrate (L‐arginine)‐ and inhibitor (NG‐monomethyl‐L‐arginine, NMMA)‐sensitive iNOS activity was significantly elevated in CH‐ or ANI‐treated RASMC after 24 h.

Lipopolysaccharide (LPS) produced a time‐dependent increase in cyclic GMP levels with maximal stimulation at 6 h and a decline to near baseline at 24 h. CH attenuated LPS‐induced cyclic GMP accumulation at 3 and 6 h. However, cyclic GMP levels were superinduced at later times by CH. The concentration‐dependence of cyclic GMP stimulation by cycloheximide was biphasic both in the absence and presence of LPS, with maximal stimulation at 10 μM and inhibition at higher concentrations.

Increased iNOS activity by CH was associated with elevated levels of immunoreactive iNOS protein as judged by Western blotting in LPS‐ and CH‐treated cells.

CH‐induced iNOS activity and superinduction of iNOS by CH in cells treated with LPS were both significantly inhibited by actinomycin D, a transcription inhibitor.

RT‐PCR revealed elevated iNOS mRNA levels after 12 h of exposure to CH. The combination of LPS and CH caused a significant increase in iNOS gene expression relative to LPS‐ or CH stimulation alone.

These results show that partial protein synthesis inhibition by CH alone upregulates iNOS mRNA and superinduces iNOS mRNA in cytokine‐treated RASMC, which is translated to the functional enzyme generating biologically active NO. Thus iNOS activation in these cells not only requires new protein synthesis but it also appears to be negatively regulated by newly synthesized proteins.

British Journal of Pharmacology (1998) 123, 1000–1008; doi:10.1038/sj.bjp.0701687
Phosphodiesterase profile of human B lymphocytes from normal and atopic donors and the effects of PDE inhibition on B cell proliferation
Authors: Gantner Florian , Götz Christine , Gekeler Volker , Schudt Christian , Wendel Albrecht , Hatzelmann Armin
Published: January 2005

CD19+ B lymphocytes were purified from the peripheral blood of normal and atopic subjects to analyse and compare the phosphodiesterase (PDE) activity profile, PDE mRNA expression and the importance of PDE activity for the regulation of B cell function.

The majority of cyclic AMP hydrolyzing activity of human B cells was cytosolic PDE4, followed by cytosolic PDE7‐like activity; marginal PDE3 activity was found only in the particulate B cell fraction. PDE1, PDE2 and PDE5 activities were not detected.

By cDNA‐PCR analysis mRNA of the PDE4 subtypes A, B (splice variant PDE4B2) and D were detected. In addition, a weak signal for PDE3A was found.

No differences in PDE activities or mRNA expression of PDE subtypes were found in B cells from either normal or atopic subjects.

Stimulation of B lymphocytes with the polyclonal stimulus lipopolysaccharide (LPS) induced a proliferative response in a time‐ and concentration‐dependent manner, which was increased in the presence of interleukin‐4 (IL‐4). PDE4 inhibitors (rolipram, piclamilast) led to an increase in the cellular cyclic AMP concentration and to an augmentation of proliferation, whereas a PDE3 inhibitor (motapizone) was ineffective, which is in accordance with the PDE profile found. The proliferation enhancing effect of the PDE4 inhibitors was partly mimicked by the cyclic AMP analogues dibutyryl (db) cyclic AMP and 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole‐3′,5′‐cyclic monophosphorothioate, Sp‐isomer (dcl‐cBIMPS), respectively. However, at concentrations exceeding 100 μM db‐cyclic AMP suppressed B lymphocyte proliferation, probably as a result of cytotoxicity. Prostaglandin E2 (PGE2, 1 μM) and forskolin (10 μM) did not affect B cell proliferation, even when given in combination with rolipram.

Inhibition of protein kinase A (PKA) by differentially acting selective inhibitors (KT 5720, Rp‐8‐Br‐cyclic AMPS) decreased the proliferative response of control cells and reversed the proliferation enhancing effects of rolipram.

Importantly, PDE4 activity in LPS/IL‐4‐activated B lymphocytes decreased by about 50% compared to unstimulated control values.

We conclude that an increase in cyclic AMP, mediated by down‐regulation of PDE4 activity, is involved in the stimulation of B cell proliferation in response to LPS/IL‐4. B cell proliferation in response to a mitogenic stimulus can be further enhanced by pharmacological elevation of cyclic AMP.

British Journal of Pharmacology (1998) 123, 1031–1038; doi:10.1038/sj.bjp.0701688
Possible mechanism of the negative inotropic effect of α1‐adrenoceptor agonists in rat isolated left atria after exposure to free radicals
Authors: Peters Stephan L M. , Batink Harry D. , Michel Martin C. , Pfaffendorf Martin , van Zwieten Pieter A.
Published: January 2005

This study was designed to investigate the mechanism(s) of the negative inotropic effects of α1‐adrenoceptor agonists observed in rat isolated left atria after exposure to free radicals.

Ouabain and calphostin C were used in contraction experiments to block the sodium pump and protein kinase C. Methoxamine‐induced phospholipase C and Na+/K+ ATPase activities were measured.

Methoxamine (300 μM) increased contractile force by 1.6±0.2 mN in control atria but decreased contractile force in electrolysis‐treated atria by 2.0±0.1 mN (P<0.05), as determined 10 min after methoxamine addition. In contrast, the positive inotropic effects of endothelin‐1 (30 nM) and isoprenaline (10 μM) were reduced from 2.6±0.3 to 1.3±0.1 mN and from 2.6±0.3 to 1.7±0.2 mN, respectively, by electrolysis treatment (P<0.05), but not converted into a negative inotropic action.

In an inositol phosphate assay we observed that the stimulation of phospholipase C by methoxamine was attenuated by electrolysis when the (electrolyzed) medium from the organ bath was used, but the phospholipase C responses were restored by the use of fresh medium. However, fresh medium did not counteract the negative inotropic effect of methoxamine. Accordingly, the negative inotropic effect of methoxamine is not directly related to the impaired phospholipase C responses seen in atria subjected to electrolysis.

Ouabain (10 μM) and the protein kinase C inhibitor calphostin C (50 nM), completely prevented the negative inotropic effect of 300 μM methoxamine in electrolysis‐treated atria.

Measurement of the Na+/K+ ATPase activity, revealed that in control atria, α1‐adrenoceptor stimulation with 300 μM methoxamine, decreased the Na+/K+ ATPase activity by 14.4±7.7%. In contrast, methoxamine increased the Na+/K+ ATPase activity by 48.8±8.9% (P<0.05) in electrolysis‐treated atria. Interestingly, this increase in Na+/K+ ATPase activity was completely counteracted by calphostin C (1.4±0.1% over basal).

These results indicate that the negative inotropic effects of α1‐adrenoceptor agonists, observed in rat isolated left atria exposed to free radicals, are likely to be caused by protein kinase C‐mediated phosphorylation and subsequent activation of the Na+/K+ ATPase.

British Journal of Pharmacology (1998) 123, 952–958; doi:10.1038/sj.bjp.0701689
S‐nitrosothiols and the nitrergic neurotransmitter in the rat gastric fundus: effect of antioxidants and metal chelation
Authors: De Man Joris G. , De Winter Benedicte Y. , Moreels Tom G. , Herman Arnold G. , Pelckmans Paul A.
Published: January 2005

The effects of the antioxidants ascorbic acid and α‐tocopherol and of the metal chelator ethylenediaminetetraacetic acid (EDTA) were studied on relaxations in response to S‐nitrosothiols, authentic nitric oxide (NO) and nitrergic non‐adrenergic non‐cholinergic stimulation of the rat gastric fundus.

The S‐nitrosothiols S‐nitrosocysteine (1–100 nM), S‐nitrosoglutathione (0.01–3 μM) and S‐nitroso‐N‐acetylpenicillamine (0.01–3 μM) induced concentration‐dependent relaxations of the rat gastric fundus muscle strips, which were precontracted with prostaglandin F2α. The relaxations to all S‐nitrosothiols were concentration‐dependently enhanced by the antioxidants ascorbic acid (0.1–3 μM) and α‐tocopherol (3–30 μM) and inhibited by the metal chelator EDTA (26 μM).

Ascorbic acid and α‐tocopherol alone did not induce a relaxation of the precontracted rat gastric fundus muscle strip. However, when ascorbic acid (1 μM) or α‐tocopherol (1 μM) were injected in the organ bath 1 minute after S‐nitrosoglutathione (0.1 μM) or after S‐nitroso‐N‐acetylpenicillamine (0.1 μM), they induced an immediate, sharp and transient relaxation. This relaxation was inhibited by the superoxide generator pyrogallol (2 μM). Such a relaxation to ascorbic acid or α‐tocopherol was not observed in the presence of S‐nitrosocysteine (10 nM).

Electrical field stimulation (0.5–4 Hz) of the precontracted rat gastric fundus strips induced frequency‐dependent nitrergic relaxations which were mimicked by authentic NO (3–300 nM) and by acidified sodium nitrite NaNO2 (0.3–10 μM). Ascorbic acid (0.3–3 μM), α‐tocopherol (3–30 μM) or EDTA (26 μM) did not affect the relaxations to nitrergic stimulation, NO or NaNO2.

In summary, relaxations to S‐nitrosothiols in the rat gastric fundus are enhanced by the antioxidants ascorbic acid and α‐tocopherol and inhibited by the metal chelator EDTA. However, relaxations to nitrergic stimulation of the rat gastric fundus or those to authentic NO were not affected by the antioxidants or by the metal chelator. These results indicate that antioxidants and metal chelators have a different effect on the biological activity of S‐nitrosothiols and on that of the nitrergic neurotransmitter. Therefore, our results suggest that S‐nitrosothiols do not act as intermediate compounds in nitrergic neurotransmission in the rat gastric fundus.

British Journal of Pharmacology (1998) 123, 1039–1046; doi:10.1038/sj.bjp.0701692
Effects of bradykinin on signal transduction, cell proliferation, and cytokine, prostaglandin E2 and collagenase‐1 release from human corneal epithelial cells
Authors: Wiernas T K. , Davis T L. , Griffin B W. , Sharif N A.
Published: January 2005

We recently demonstrated the presence of phospholipase C‐coupled bradykinin (BK) B2‐receptors in human primary and SV40 virus‐immortalized corneal epithelial (CEPI) cells.

The aims of the present studies were to demonstrate the specific binding of [3H]‐BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca2+‐mobilization ([Ca2+]i), cell proliferation (via [3H]‐thymidine incorporation), and the release of various cytokines, collagenase‐1 (matrix metalloproteinase‐1) and prostaglandin E2 (PGE2).

Specific [3H]‐BK binding comprised 83±2% of the total binding, and was of high affinity (Kd=1.66±0.52 nM, n=5), saturable (Bmax=640±154 fmol g−1 wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK‐related peptides: Hoe‐140 (D‐Arg‐[Hyp3,Thi5,D‐Tic7,Oic8]BK; icatibant): Ki=0.17±0.07 nM; BK: Ki=1.0±0.11 nM; [Tyr8]‐BK: Ki=12.9±2.3 nM; [des‐Arg9]‐BK: Ki>9,200 nM (all n=3–5)).

BK potently stimulated PI turnover (EC50=2.3±0.3 nM; n=7) and [Ca2+]i mobilization (EC50=8–20 nM) in CEPI cells and both responses were inhibited in a concentration‐dependent manner by 100 nM–10 μM Hoe‐140, a selective B2‐receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, U73122 (1‐(6‐((17β‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl)amino)hexyl)‐1H‐pyrrole‐2,5‐dione) (IC50=3.0±1.6 μM). BK‐induced [Ca2+]i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine.

BK (0.1 nM–10 μM) significantly (P<0.05–0.001) stimulated [3H]‐thymidine incorporation into CEPI cellular DNA. However, while interleukin‐1α (IL‐1α; 10 ng ml−1) potently stimulated the release of IL‐6, IL‐8 and granulocyte macrophage colony‐stimulating factor from CEPI cells, BK (0.1 nM–10 μM) was without effect.

Whilst phorbol‐12‐myristate‐13‐acetate (PMA; 3 μg ml−1) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP‐1 and PGE2 from CEPI cells, BK (0.1 nM–10 μM) was without any significant effect under these conditions.

In conclusion, these data indicate that the CEPI cells express high‐affinity [3H]‐BK binding sites representing B2‐subtype BK receptors coupled to PI turnover and [Ca2+]i mobilization which appear to stimulate [3H]‐thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE2, various cytokines and MMP‐1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.

British Journal of Pharmacology (1998) 123, 1127–1137; doi:10.1038/sj.bjp.0701700
Myogenic nitric oxide synthase activity in canine lower oesophageal sphincter: morphological and functional evidence
Authors: Salapatek Anne Marie F. , Wang Yu‐Fang , Mao Yu‐Kang , Lam Annette , Daniel Edwin E.
Published: January 2005

Studies on canine lower oesophageal sphincter (LOS) evaluated the existence and function of a myogenic, nitric oxide synthase (NOS) by use of immunocytochemistry for NOS isozymes, NADPH‐d histochemistry, [3H]‐L‐arginine to [3H]‐L‐citrulline transformation. In addition, functional studies in the muscle bath were performed.

Smooth muscle bundles or freshly isolated smooth muscle cells of LOS were NADPH‐d reactive but did not recognize some antibodies against neural, endothelial or inducible NOS. NADPH‐d reactivity and immunoreactivity to a neural NOS antibody were colocalized in LOS enteric nerves. Muscle plasma membrane‐enriched fractions from fresh and cultured LOS cells converted [3H]‐L‐arginine to [3H]‐L‐citrulline; activity was mostly Ca2+/calmodulin‐dependent.

N‐Nitro‐L‐arginine (L‐NOARG) persistently increased tone (blocked by L‐arginine) in muscle strips despite blockade of nerve function. Nifedipine prevented or abolished L‐NOARG‐induced, but not carbachol‐induced, contraction showing that tone increase by L‐NOARG required functional L‐Ca channels.

Membrane‐bound, myogenic NOS in canine LOS may release NO continuously when Ca2+ entry through L‐Ca channels occurs under physiological conditions and thereby modulate tone in LOS.

British Journal of Pharmacology (1998) 123, 1055–1064; doi:10.1038/sj.bjp.0701701
Induction of nitric oxide synthase in vivo and cell injury in rat duodenal epithelium by a water soluble extract of Helicobacter pylori
Authors: Lamarque Dominique , Kiss József , Tankovic Jacques , François Flejou Jean , Delchier Jean‐Charles , Whittle Brendan J R.
Published: January 2005

Helicobacter pylori (Hp) infection, which involves the gastric antrum and duodenal mucosa, may be involved in peptic ulceration by stimulating the local release of cytoxic or pro‐inflammatory factors.

Nitric oxide (NO) is known to be cytotoxic at high concentration. The aim of the present study was therefore to investigate the ability of a water soluble extract of Hp to induce NO synthase in duodenal mucosa and epithelial cells following its administration in vivo in rats and determine its association with cell damage.

Administration of Hp water extract (4 ml kg−1) led to the expression of the calcium‐independent inducible nitric oxide synthase (iNOS) after 4 h in the duodenum, determined as [14C]‐arginine conversion to citrulline.

This iNOS activity was not reduced by pretreatment with anti‐neutrophil serum (0.4 ml kg−1, i.p., 3 h before challenge). However, dexamethasone pretreatment (1 mg kg−1, i.v., 2 h before the extract), or administration of the NO synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME, 5 mg kg−1, i.v., 2.5 h after the extract) reduced this activity.

Furthermore, iNOS was expressed in duodenal isolated epithelial cells 4 h after the i.v. challenge with the extract, at a time when the cellular viability was also reduced, as assessed by trypan blue exclusion.

Dexamethasone pretreatment, administration of L‐NAME, or pretreatment with polymyxin B (1 mg kg−1, i.v.) which binds endotoxin, reduced both the iNOS activity and epithelial cell damage.

The induction of NO synthase by the Hp extract thus results in duodenal epithelial cell injury and such actions could play a role in pathogenesis of peptic ulcer disease.

British Journal of Pharmacology (1998) 123, 1073–1078; doi:10.1038/sj.bjp.0701706
Pharmacological detection of AMPA receptor heterogeneity by use of two allosteric potentiators in rat hippocampal cultures
Authors: Sekiguchi Masayuki , Takeo Jiro , Harada Takayuki , Morimoto Takako , Kudo Yoshihisa , Yamashita Shinya , Kohsaka Shinichi , Wada Keiji
Published: January 2005

In order to examine whether a recently developed allosteric potentiator for AMPA receptors, 4‐[2‐(phenylsulphonylamino)ethylthio]‐2,6‐difluoro‐phenoxyacetamide (PEPA), can be utilized as an indicator of AMPA receptor heterogeneity, the action of PEPA upon the increase of intracellular free calcium ion concentration ([Ca2+]i) elicited by AMPA was investigated in rat hippocampal cultures, and the action was compared with that of cyclothiazide, a well characterized allosteric modulator of AMPA receptors.

PEPA dose‐dependently potentiated AMPA‐induced increase of [Ca2+]i. In 90% (72 out of 80) of the cells in which cyclothiazide acts, PEPA potentiated the increased [Ca2+]i induced by AMPA with pronounced cell‐to‐cell variation in rat hippocampal cultures.

The ratio of the potentiation by PEPA to the potentiation by cyclothiazide (P/C ratio) also varied with cells between 0 and 2.15. It was found that the cultured hippocampal cells consisted of multiple populations with different P/C ratios. Among them two populations exhibited characteristic P/C ratios; low (0 to 0.15; 27 out of 80 cells, 34%) and high (2.00; 1 out of 80 cells, 1%) P/C ratios. The P/C ratios of the other populations were between 0.25 and 1.20, and these cells constituted 65% (52 out of 80 cells) of the cells tested.

Reverse transcriptase‐polymerase chain reaction analysis suggested that GluR2‐flip, GluR1‐flip, GluR2‐flop, and GluR1‐flop were abundantly expressed (in this rank order) in the cultures used.

In Xenopus oocytes expressing GluR1, GluR3, or these subunits plus GluR2, the potentiation of AMPA response by PEPA and by cyclothiazide varied with subunit and splice‐variant combinations, and the P/C ratio was between 0.19 and 2.20. Oocytes with low P/C ratios (0.19 to 0.50) and low sensitivity to PEPA potentiation (1.9 fold to 6.41 fold) were those expressing flip variants predominantly, and oocytes with high P/C ratios (1.8 to 2.2) were those expressing flop variants predominantly. Oocytes with intermediate P/C ratios (0.51 to 1.20) were those expressing various combinations of flip and flop variants, and it was impossible to specify the relative abundance of flip and flop variants in these cells. Therefore, the P/C ratio can be used to infer subunit/splice variant expression only when the ratio is low or high.

These results suggest that the potentiation by PEPA alone reveals cell‐to‐cell heterogeneity of AMPA receptors, but a comparison of the actions of PEPA and cyclothiazide further facilitates the detection of the heterogeneity.

British Journal of Pharmacology (1998) 123, 1294–1303; doi:10.1038/sj.bjp.0701707
Influence of potassium channel modulators on cognitive processes in mice
Authors: Ghelardini Carla , Galeotti Nicoletta , Bartolini Alessandro
Published: January 2005

The effect of i.c.v. administration of different potassium channel openers (minoxidil, pinacidil, cromakalim) and potassium channel blockers (tetraethylammonium, apamin, charybdotoxin, gliquidone, glibenclamide) on memory processes was evaluated in the mouse passive avoidance test.

The administration of minoxidil (10 μg per mouse i.c.v.), pinacidil (5–25 μg per mouse i.c.v.) and cromakalim (10–25 μg per mouse i.c.v.) immediately after the training session produced an amnesic effect.

Tetraethylammonium (TEA; 1–5 μg per mouse i.c.v.), apamin (10 ng per mouse i.c.v.), charybdotoxin (1 μg per mouse i.c.v.), gliquidone (3 μg per mouse i.c.v.) and glibenclamide (1 μg per mouse i.c.v.), administered 20 min before the training session, prevented the potassium channel opener‐induced amnesia.

At the highest effective doses, none of the drugs impaired motor coordination, as revealed by the rota rod test, or modified spontaneous motility and inspection activity, as revealed by the hole board test.

These results suggest that the modulation of potassium channels plays an important role in the regulation of memory processes. On this basis, the potassium channel blockers could be useful in the treatment of cognitive deficits.

British Journal of Pharmacology (1998) 123, 1079–1084; doi:10.1038/sj.bjp.0701709
Protein kinase C‐mediated inhibition of transmembrane signalling through CCKA and CCKB receptors
Authors: Smeets R L L. , Fouraux M A. , van Emst‐de Vries S E. , De Pont J J H H M. , Willems P H G M.
Published: January 2005

The rat CCKA and CCKB receptors were stably expressed in Chinese hamster ovary (CHO‐09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon.

Spectrofluorophotometry of Fura‐2‐loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin‐(26‐33)‐peptide amide (CCK‐8‐S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCKA cells, measured as an increase in Fura‐2 fluorescence emission ratio, 1000 fold more potently than its non‐sulphated form (CCK‐8‐NS) (EC50 values of 0.19 nM and 0.18 μM, respectively). By contrast, CCK‐8‐S and CCK‐8‐NS were equally potent in CCKB cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCKA receptor agonist JMV‐180 increased [Ca2+]i only in CCKA cells. Likewise, pentagastrin increased [Ca2+]i only in CCKB cells. Finally, CCK‐8‐S‐induced Ca2+ signalling through the CCKA receptor was most potently inhibited by the CCKA receptor antagonist L364,718, whereas the CCKB receptor antagonist L365,260 was more potent in CCKB cells.

Receptor‐mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3‐isobutyl‐1‐methylxanthine. CCK‐8‐S and, to a lesser extent, CCK‐8‐NS, but not JMV‐180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCKA cells. By contrast, none of these agonists increased cyclicAMP in CCKB cells.

Short‐term (3 min) pretreatment with the PKC activator 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) evoked a rightward shift of the dose‐response curve for the Ca2+ mobilizing effect of CCK‐8‐S in both cell lines. In addition, short‐term TPA pretreatment markedly reduced CCK‐8‐S‐induced cyclicAMP accumulation in CCKA cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF‐109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4‐α‐phorbol 12‐myristate 13‐acetate.

During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK‐8‐S‐induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down‐regulation of PKC‐α, suggesting the involvement of this PKC isotype in the inhibitory action of TPA.

This study demonstrates that following expression in CHO cells (i) both CCKA and CCKB receptors are coupled to Ca2+ mobilization, (ii) only CCKA receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.

British Journal of Pharmacology (1998) 123, 1189–1197; doi:10.1038/sj.bjp.0701713
Effect of 17β‐oestradiol on cytokine‐induced nitric oxide production in rat isolated aorta
Authors: Kauser Katalin , Sonnenberg Dagmar , Diel Patrick , Rubanyi Gabor M.
Published: January 2005

Studies were performed on isolated aortic rings without endothelium to investigate the effect of 17β‐oestradiol on cytokine‐induced nitric oxide production by the inducible nitric oxide synthase (iNOS).

Treatment of the isolated aortic rings with interleukin‐1β (IL‐1β, 20 μ ml−1) led to the expression of iNOS mRNA and protein, as well as significant nitrite accumulation in the incubation media and suppression of phenylephrine (1 nM–10 μM)‐evoked contraction.

Cycloheximide (1 μM), a protein synthesis inhibitor, prevented iNOS protein expression, nitrite accumulation and the suppression of contractility by IL‐1β on the isolated aortic rings. 17β‐oestradiol (1 nM–10 μM) and the partial oestrogen receptor agonist 4‐OH‐tamoxifen (1 nM–10 μM) produced concentration‐dependent inhibition of IL‐1β‐induced nitrite accumulation and restored vasoconstrictor responsiveness to phenylephrine, similar to the iNOS inhibitor aminoguanidine (100 μM).

Semiquantitative PCR demonstrated decreased iNOS mRNA in the IL‐1β‐induced and 17β‐oestradiol‐treated rings. Western blot analysis of rat aorta homogenates revealed that 17β‐oestradiol treatment resulted in a reduction in IL‐1ß‐induced iNOS protein level.

Incubation with tumour necrosis factor α (TNFα, 1 ng ml−1) resulted in significant nitrite accumulation in the incubation media and suppression of the smooth muscle contractile response to phenylephrine, similar to IL‐1β. The effects of TNFα were also inhibited by co‐incubation of the rings with 17β‐oestradiol and 4‐OH‐tamoxifen (1 μM).

The anti‐transforming growth factor‐β1 (TGF‐β1) antibody, which inhibited TGF‐β1‐induced suppression of nitrite production from IL‐1β‐treated vascular rings, did not affect the inhibitory action of 17β‐oestradiol, suggesting that the effect of oestrogen on iNOS inhibition was not mediated by TGF‐β1.

These results show that the ovarian sex steroid, 17β‐oestradiol is a modulator of cytokine‐induced iNOS activity in rat vascular smooth muscle and its mechanism of action involves decrease of iNOS mRNA and protein.

British Journal of Pharmacology (1998) 123, 1089–1096; doi:10.1038/sj.bjp.0701715
Differential effects of cyclosporine A after acute antigen challenge in sensitized catsin vivoandex vivo
Authors: Mitchell Richard W. , Cozzi Phillip , Maurice Ndukwu I , Spaethe Stephen , Leff Alan R. , Padrid Philip A.
Published: January 2005

We determined the effect of cyclosporine A (CsA) treatment on mast cell degranulation and lung resistance (RL)in vivo, and tracheal smooth muscle (TSM) contractionex vivoafter antigen challenge in sensitized cats. We also determined the direct effects of addition of CsA to the tissue bath on antigen‐induced responses of TSMin vitro.

Cats (n=10) were sensitized by i.m. injection ofAscaris suumantigen (AA); 5 cats (CsA+) received CsA twice daily for 2 weeks before acute antigen challenge in doses sufficient to suppress interleukin‐2 secretion from feline peripheral blood mononuclear cellsex vivo.

Lung resistance increased comparably within 10 min of exposure to AA (P<0.03). Histamine content in bronchoalveolar lavage fluid from both groups increased comparably within 30 min of antigen challenge, from undetectable levels to 542±74 pg ml−1post AA for CsA+ and from 74±19 pg ml−1at baseline, to 970±180 pg ml−1post AA CsA− (P<0.05;P=NS vs CsA+).

In excised TSM, active tension elicited by exposure to AAin vitrowas 107±38% KCl in the CsA+ group vs 144±56% KCl in the CsA− group (P=NS). However, contraction of TSM (n=4) harvested from both groups was abolished or greatly diminished after AA challenge when tissues were pre‐incubated with 1 μMCsAin vitro(8±8% KCl,P<0.05 vs CsA+ and CsA−). This was associated with inhibited release of 5‐hydroxytryptamine into the organ bath fluid of tissues treated with CsAin vitroonly.

We demonstrated that CsA treatmentin vivodoes not inhibit the early phase asthmatic response or mast cell degranulation following antigen challenge in sensitized cats. Additionally, the effects of CsA on mast cell functionex vivodo not reflect lack of effects of CsA on mast cell functionin vivoin this animal model of atopic asthma.

British Journal of Pharmacology(1998)123, 1198–1204; doi:10.1038/sj.bjp.0701716
Prostaglandin E2 suppression of acetylcholine release from parasympathetic nerves innervating guinea‐pig trachea by interacting with prostanoid receptors of the EP3‐subtype
Authors: Spicuzza Lucia , Giembycz Mark A. , Barnes Peter J. , Belvisi Maria G.
Published: January 2005

We have demonstrated recently that exogenous prostaglandin E2 (PGE2) inhibits electrical field stimulation (EFS)‐induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea‐pig trachea. In the present study, we have attempted to characterize the pre‐junctional prostanoid receptor(s) responsible for the inhibitory action of PGE2 and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea‐pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS‐evoked [3H]‐ACh release.

In epithelium‐denuded tracheal strips pretreated with indomethacin (10 μM), PGE2 (0.1 nM–1 μM) inhibited EFS‐evoked [3H]‐ACh release in a concentration‐dependent manner with an EC50 and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP‐receptor agonist, PGF2α and the stable thromboxane mimetic, U46619 (each at 1 μM), also inhibited [3H]‐ACh release by 48%, 41% and 35%, respectively. PGD2 (1 μM) had no significant effect on [3H]‐ACh release.

The selective TP‐receptor antagonist, ICI 192,605 (0.1 μM), completely reversed the inhibition of cholinergic neurotransmission induced by U‐46619, but had no significant effect on similar responses effected by PGE2 and PGF2α.

A number of EP‐receptor agonists mimicked the ability of PGE2 to inhibit [3H]‐ACh release with a rank order of potency: GR63799X (EP3‐selective)>PGE2>M&B 28,767 (EP3 selective)>17‐phenyl‐ω‐trinor PGE2 (EP1‐selective). The EP2‐selective agonist, AH 13205 (1 μM), did not affect EFS‐induced [3H]‐ACh release.

AH6809 (10 μM), at a concentration 10 to 100 times greater than its pA2 at DP‐, EP1‐ and EP2‐receptors, failed to reverse the inhibitory effect of PGE2 or 17‐phenyl‐ω‐trinor PGE2 on [3H]‐ACh release.

These results suggest that PGE2 inhibits [3H]‐ACh release from parasympathetic nerves supplying guinea‐pig trachea via an interaction with prejunctional prostanoid receptors of the EP3‐receptor subtype. Evidence for inhibitory prejunctional TP‐ and, possibly, IP‐receptors was also obtained although these receptors may play only a minor role in suppressing [3H]‐ACh release when compared to receptors of the EP3‐subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea‐pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.

British Journal of Pharmacology (1998) 123, 1246–1252; doi:10.1038/sj.bjp.0701720
Role of serine esterases in mast cell activation
Authors: Emadi Khiav B , Pearce F L.
Published: January 2005

A variety of chymotryptic substrates and inhibitors prevented the release of histamine and prostaglandin D2 from rat peritoneal mast cells stimulated with anti‐IgE but not the calcium ionophore A23187 or a variety of polyamines.

The activity of the compounds was strikingly increased in cells reversibly permeabilized with ATP, indicating the importance of their effective incorporation into the cytosol.

The compounds produced a comparable inhibition of immunological, but not pharmacological, histamine release from human mast cells and basophils.

Treatment of rat mast cells with anti‐IgE led to a marked increase in the total chymotryptic activity expressed by the cells.

Immunological, but not pharmacological, stimulation of permeabilized rat mast cells loaded with a fluorescent chymotryptic substrate led to a pronounced and rapid increase in fluorescence, indicating activation of the enzyme and hydrolysis of the substrate. These changes were attenuated by chymotryptic inhibitors.

In total, these data provide compelling evidence for the direct involvement of a serine protease in IgE‐mediated histamine release from mast cells.

British Journal of Pharmacology (1998) 123, 1267–1273; doi:10.1038/sj.bjp.0701724
Acetylcholine sensitivity of biphasic Ca2+ mobilization induced by nicotinic receptor activation at the mouse skeletal muscle endplate
Authors: Dezaki Katsuya , Kimura Ikuko
Published: January 2005

Acetylcholine (ACh) was locally applied onto the endplate region in a mouse phrenic nerve‐diaphragm muscle preparation to measure intracellular free calcium ([Ca2+]i) entry through nicotinic ACh receptors (AChRs) by use of Ca2+‐aequorin luminescence.

ACh (0.1–3 mm, 20 μl) elicited biphasic elevation of [Ca2+]i (fast and slow Ca2+ mobilization) in muscle cells. The peak amplitude of the slow Ca2+ mobilization (not accompanied by twitch tension) was concentration‐dependently increased by ACh, whereas that of the fast component (accompanied by twitch tension) reached a maximum response at a lower concentration (0.1 mm) of applied ACh.

A pulse of nicotinic agonists, (−)‐nicotine (10 mm) and 1,1‐dimethyl‐4‐phenyl‐piperazinium (10 mm), but not a muscarinic agonist pilocarpine (10 mm), also elicited a biphasic Ca2+ signal.

Even though ACh release from motor nerve endings was blocked by botulinum toxin (5 μg, bolus i.p. before isolation of the tissue), the generation of both a fast and slow Ca2+ component caused by ACh application was observed.

These results strongly suggest that ACh locally applied onto the endplate region of skeletal muscle induces a slow Ca2+ signal reflecting Ca2+ entry through a postsynaptic nicotinic AChR, which has a low sensitivity to transmitter ACh.

British Journal of Pharmacology (1998) 123, 1418–1424; doi:10.1038/sj.bjp.0701725
Characterization of KATP channels in intact mammalian skeletal muscle fibres
Authors: Barrett‐Jolley Richard , McPherson Grant A.
Published: January 2005

The aim of this study was to characterize the KATP channel of intact rat skeletal muscle (rat flexor digitorum brevis muscle). Changes in membrane currents were recorded with two‐electrode voltage‐clamp of whole fibres.

The KATP channel openers, levcromakalim and pinacidil (10–400 μM), caused a concentration‐dependent increase in whole‐cell chord conductance (up to approximately 1.5 mScm−2). The activated current had a weak inwardly rectifying current‐voltage relation, a reversal potential near EK and nanomolar sensitivity to glibenclamide – characteristic of a KATP channel current. Concentration‐effect analysis revealed that levcromakalim and pinacidil were not particularly potent (EC50 ∼186 μM, ∼30 μM, respectively), but diazoxide was completely inactive.

The ability of both classical KATP channel inhibitors (glibenclamide, tolbutamide, glipizide and 5‐hydroxydecanoic acid) and a number of structurally related glibenclamide analogues to antagonize the levcromakalim‐induced current was determined. Glibenclamide was the most potent compound with an IC50 of approximately 5 nM. However, the non‐sulphonylurea (but cardioactive) compound 5‐hydroxydecanoic acid was inactive in this preparation.

Regression analysis showed that the glibenclamide analogues used have a similar rank order of potency to that observed previously in vascular smooth muscle and cerebral tissue. However, two compounds (glipizide and DK13) were found to have unexpectedly low potency in skeletal muscle.

These experiments revealed KATP channels of skeletal muscle to be at least 10× more sensitive to glibenclamide than previously found; this may be because of the requirement for an intact intracellular environment for the full effect of sulphonylureas to be realised. Pharmacologically, KATP channels of mammalian skeletal muscle appear to resemble most closely KATP channels of cardiac myocytes.

British Journal of Pharmacology (1998) 123, 1103–1110; doi:10.1038/sj.bjp.0701727