bluuebunny/crossref_metadata_2025 huggingface dataset:
The KATP channel opener diazoxide protects cardiac myocytes during metabolic inhibition without causing mitochondrial depolarization or flavoprotein oxidation
Authors: Lawrence C L , Billups B , Rodrigo G C , Standen N B
Published: January 2005

The KATP channel opener diazoxide has been proposed to protect cardiac muscle against ischaemia by opening mitochondrial KATP channels to depolarize the mitochondrial membrane potential, ΔΨm. We have used the fluorescent dye TMRE to measure ΔΨm in adult rat freshly isolated cardiac myocytes exposed to diazoxide and metabolic inhibition.

Diazoxide, at concentrations that are highly cardioprotective (100 or 200 μM), caused no detectable increase in TMRE fluorescence (n=27 cells). However, subsequent application of the protonophore FCCP, which should collapse ΔΨm, led to large increases in TMRE fluorescence (>300%).

Metabolic inhibition (MI: 2 mM NaCN+1 mM iodoacetic acid (IAA) led to an immediate partial depolarization of ΔΨm, followed after a few minutes delay by complete depolarization which was correlated with rigor contracture. Removal of metabolic inhibition led to abrupt mitochondrial repolarization followed in many cells by hypercontracture, indicated by cell rounding and loss of striated appearance.

Prior application of diazoxide (100 μM) reduced the number of cells that hypercontracted after metabolic inhibition from 63.7±4.7% to 24.2±1.8% (P<0.0001). 5‐hydroxydeanoate (100 μM) reduced the protection of diazoxide (46.8±2.7% cells hypercontracted, P<0.0001 vs diazoxide alone).

Diazoxide caused no detectable change in flavoprotein autofluorescence (n=26 cells).

Our results suggest that mitochondrial depolarization and flavoprotein oxidation are not inevitable consequences of diazoxide application in intact cardiac myocytes, and that they are also not essential components of the mechanism by which it causes protection.

British Journal of Pharmacology (2001) 134, 535–542; doi:10.1038/sj.bjp.0704289
Cannabinoid CB1‐receptor mediated regulation of gastrointestinal motility in mice in a model of intestinal inflammation
Authors: Izzo Angelo A , Fezza Filomena , Capasso Raffaele , Bisogno Tiziana , Pinto Luisa , Iuvone Teresa , Esposito Giuseppe , Mascolo Nicola , Di Marzo Vincenzo , Capasso Francesco
Published: January 2005

We have studied the effect of cannabinoid agonists (CP 55,940 and cannabinol) on intestinal motility in a model of intestinal inflammation (induced by oral croton oil in mice) and measured cannabinoid receptor expression, endocannabinoids (anandamide and 2‐arachidonylglycerol) and anandamide amidohydrolase activity both in physiological and pathophysiological states.

CP 55,940 (0.03 – 10 nmol mouse−1) and cannabinol (10 – 3000 nmol mouse−1) were more active in delaying intestinal motility in croton oil‐treated mice than in control mice. These inhibitory effects were counteracted by the selective cannabinoid CB1 receptor antagonist SR141716A (16 nmol mouse−1). SR141716A (1 – 300 nmol mouse−1), administered alone, increased intestinal motility to the same extent in both control and croton oil‐treated mice

Croton oil‐induced intestinal inflammation was associated with an increased expression of CB1 receptor, an unprecedented example of up‐regulation of cannabinoid receptors during inflammation.

High levels of anandamide and 2‐arachidonylglycerol were detected in the small intestine, although no differences were observed between control and croton oil‐treated mice; by contrast anandamide amidohydrolase activity increased 2 fold in the inflamed small intestine.

It is concluded that inflammation of the gut increases the potency of cannabinoid agonists possibly by ‘up‐regulating’ CB1 receptor expression; in addition, endocannabinoids, whose turnover is increased in inflamed gut, might tonically inhibit intestinal motility.

British Journal of Pharmacology (2001) 134, 563–570; doi:10.1038/sj.bjp.0704293
Reduction of oxidative stress and AT1 receptor expression by the selective oestrogen receptor modulator idoxifene
Authors: Bäumer Anselm T , Wassmann Sven , Ahlbory Katja , Strehlow Kerstin , Müller Cornelius , Sauer Heinrich , Böhm Michael , Nickenig Georg
Published: January 2005

The beneficial vasoprotective effects of oestrogens are hampered by their side effects on secondary sexual organs. Selective oestrogen receptor modulators (SERM) such as idoxifene may exert beneficial vascular effects without influencing cancerogenesis in breast or uterus.

In order to investigate vascular effects of selective oestrogen receptor modulators, we examined the impact of idoxifene on production of reactive oxygen species as well as AT1 receptor expression in vascular smooth muscle cells (VSMC).

Idoxifene caused a concentration‐ and time‐dependent down‐regulation of AT1 receptor mRNA expression, as assessed by Northern analysis. The maximal effect was reached with 10 μmol l−1 idoxifene after a 4 h incubation period (33±7% of control levels). Western blots showed a similar down‐regulation of AT1 receptor protein to 36±11% of control levels.

Confocal laserscanning microscopy using the redox sensitive marker 2′,7′‐dichlorofluorescein (DCF) and measurement of NAD(P)H oxidase activity in cell homogenates revealed that idoxifene effectively blunted the angiotensin II‐induced production of reactive oxygen species.

In order to investigate the signal transduction involved in SERM‐induced modulation of AT1 receptor expression, VSMC were preincubation with PD98059, genistein, wortmannin, or Nω‐Nitro‐L‐arginine. The results suggested that idoxifene caused AT1 receptor down‐regulation through nitric oxide‐dependent pathways.

In conclusion, idoxifene reduces angiotensin II‐evoked oxidative stress in VSMC. This could in part be explained by idoxifene‐induced down‐regulation of AT1 receptor expression. These results demonstrate that the selective oestrogen receptor modulator idoxifene may exert beneficial vascular effects which could be useful for therapeutic regimen in postmenopausal women at risk for cardiovascular diseases.

British Journal of Pharmacology (2001) 134, 579–584; doi:10.1038/sj.bjp.0704299
Glycosylation and the activation of proteinase‐activated receptor 2 (PAR2) by human mast cell tryptase
Authors: Compton Steven J , Renaux Bernard , Wijesuriya Suranga J , Hollenberg Morley D
Published: January 2005

Human mast cell tryptase appears to display considerable variation in activating proteinase‐activated receptor 2 (PAR2). We found tryptase to be an inefficient activator of wild‐type rat‐PAR2 (wt‐rPAR2) and therefore decided to explore the factors that may influence tryptase activation of PAR2.

Using a 20 mer peptide (P20) corresponding to the cleavage/activation sequence of wt‐rPAR2, tryptase was as efficient as trypsin in releasing the receptor‐activating sequence (SLIGRL…). However, in the presence of either human‐PAR2 or wt‐r PAR2 expressing cells, tryptase could only activate PAR2 by releasing SLIGRL from the P20 peptide, suggesting that PAR2 expressed on the cells was protected from tryptase activation.

Three approaches were employed to test the hypothesis that PAR2 receptor glycosylation restricts tryptase activation. (a) pretreatment of wt‐rPAR2 expressing cells or human embryonic kidney cells (HEK293) with vibrio cholerae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase‐mediated PAR2 activation. (b) Inhibiting receptor glycosylation in HEK293 cells with tunicamycin enabled tryptase‐mediated PAR2 activation. (c) Wt‐rPAR2 devoid of the N‐terminal glycosylation sequon (PAR2T25−), but not rPAR2 devoid of the glycosylation sequon located on extracellular loop‐2 (PAR2T224A), was selectively and substantially (>30 fold) more sensitive to tryptase compared with the wt‐rPAR2.

Immunocytochemistry using antisera that specifically recognized the N‐terminal precleavage sequence of PAR2 demonstrated that tryptase released the precleavage domain from PAR2T25− but not from wt‐rPAR2.

Heparin : tryptase molar ratios of greater than 2 : 1 abrogated tryptase activation of PAR2T25−.

Our results indicate that glycosylation of PAR2 and heparin‐inhibition of PAR2 activation by tryptase could provide novel mechanisms for regulating receptor activation by tryptase and possibly other proteases.

British Journal of Pharmacology (2001) 134, 705–718; doi:10.1038/sj.bjp.0704303
Properties of Ca2+ release induced by clofibric acid from the sarcoplasmic reticulum of mouse skeletal muscle fibres
Authors: Ikemoto Takaaki , Endo Makoto
Published: January 2005

To characterize the effect of clofibric acid (Clof) on the Ca2+ release mechanism in the sarcoplasmic reticulum (SR) of skeletal muscle, we analysed the properties of Clof‐induced Ca2+ release under various conditions using chemically skinned skeletal muscle fibres of the mouse.

Clof (>0.5 mM) released Ca2+ from the SR under Ca2+‐free conditions buffered with 10 mM EGTA (pCa >8).

Co‐application of ryanodine and Clof at pCa >8 but not ryanodine alone reduced the Ca2+ uptake capacity of the SR. Thus, Ca2+ release induced by Clof at pCa >8 must be a result of the activation of the ryanodine receptor (RyR).

At pCa >8, (i) Clof‐induced Ca2+ release was inhibited by adenosine monophosphate (AMP), (ii) the inhibitory effect of Mg2+ on the Clof‐induced Ca2+ release was saturated at about 1 mM, and (iii) Clof‐induced Ca2+ release was not inhibited by procaine (10 mM). These results indicate that Clof may activate the RyR‐Ca2+ release channels in a manner different from Ca2+‐induced Ca2+ release (CICR).

In addition to this unique mode of opening, Clof also enhanced the CICR mode of opening of RyR‐Ca2+ release channels.

Apart from CICR, a high concentration of Ca2+ might also enhance the unique mode of opening by Clof.

These results suggest that some features of Ca2+ release activated by Clof are similar to those of physiological Ca2+ release (PCR) in living muscle cells and raise the possibility that Clof may be useful in elucidating the mechanism of PCR in skeletal muscle.

British Journal of Pharmacology (2001) 134, 719–728; doi:10.1038/sj.bjp.0704306
Thrombin‐induced platelet endostatin release is blocked by a proteinase activated receptor‐4 (PAR4) antagonist
Authors: Ma Li , Hollenberg Morley D , Wallace John L
Published: January 2005

Endostatin is a potent endogenous inhibitor of angiogenesis that was recently shown to be stored in platelets and released in response to thrombin, but not ADP. In the present study, we have tested the hypothesis that thrombin‐induced endostatin release from rat platelets is mediated via proteinase‐activated receptor‐4 (PAR4). Immunoprecipitation and Western blotting confirmed that endostatin is contained within rat platelets. Aggregation and release of endostatin could be elicited by thrombin (0.5 – 1.0 U ml−1) or by specific PAR4 agonist (AYPGKF‐NH2; AY‐NH2; 15 – 50 μM). Significant release of endostatin could be induced by a dose of thrombin below that necessary for induction of aggregation. An adenosine diphosphate (ADP) scavenger, apyrase, inhibited the platelet aggregation induced by thrombin, but not the release of endostatin. In contrast, a selective PAR4 antagonist (trans‐cinnamoyl‐YPGKF‐NH2; tcY‐NH2) prevented endostatin release and aggregation induced by thrombin or by AY‐NH2. We conclude that thrombin‐induced endostatin release from rat platelets is PAR4‐mediated via an ADP‐independent mechanism that can occur independently of platelet aggregation.

British Journal of Pharmacology (2001) 134, 701–704; doi:10.1038/sj.bjp.0704312
Methylamine and benzylamine induced hypophagia in mice: Modulation by semicarbazide‐sensitive benzylamine oxidase inhibitors and aODN towards Kv1.1 channels
Authors: Pirisino R , Ghelardini C , Banchelli G , Galeotti N , Raimondi L
Published: January 2005

In starved mice, the anorectic activity of methylamine (MET) and benzylamine (BZ), both substrates of semicarbazide‐sensitive benzylamine oxidases (Bz‐SSAO), was compared with that of the potassium channel blocking agents charybdotoxin (ChTX), tetraethylammonium (TEA), gliquidone (GLI), ammonium chloride (NH4+) and of the anoressants amphetamine (AMPH) and nicotine (NIC). After i.c.v. administration, an approximate ranking order of potency was: ChTXAMPH>NIC=TEAGLIMET>BZ>NH4+.

Clorgyline (2.5 mg kg−1 i.p.) or deprenyl (10 mg kg−1 i.p.) potentiated the anorectic effect of i.c.v.‐administered BZ, NIC and AMPH. The effect of TEA was increased only by deprenyl, while MET, NH4+, ChTX and GLI were not affected by either of the inhibitors.

The Bz‐SSAO inhibitors α‐aminoguanidine (50 mg kg−1 i.p.), B24 (100 mg kg−1 i.p.) and MDL 72274 (2.5 mg kg−1 i.p.) potentiated the effect of i.p., but not of i.c.v.‐administered MET.

Antisense oligodeoxyribonucleotides (aODN) to Kv1.1 potassium channels abolished the effect of BZ and TEA, but was ineffective in reducing the activity of MET and other compounds.

These results suggest that MET is endowed with peculiar hypophagic effects at dosage levels that are not able to affect gross behaviour in mice. The effect of MET, differently from BZ, seems unrelated to an increase in the central release of monoaminergic mediators, as well as to a Kv1.1 blocking activity. Through a reduction of the endogenous breakdown of MET, Bz‐SSAO inhibitors enhance the central pharmacological activity of this amine.

British Journal of Pharmacology (2001) 134, 880–886; doi:10.1038/sj.bjp.0704316
Intracellular distribution of psychotropic drugs in the grey and white matter of the brain: the role of lysosomal trapping
Authors: Daniel Władysława A , Wójcikowski Jacek , Pałucha Agnieszka
Published: January 2005

Since the brain is not a homogenous organ (i.e. the phospholipid pattern and density of lysosomes may vary in its different regions), in the present study we examined the uptake of psychotropic drugs by vertically cut slices of whole brain, grey (cerebral cortex) and white (corpus callosum, internal capsule) matter of the brain and by neuronal and astroglial cell cultures.

Moreover, we assessed the contribution of lysosomal trapping to total drug uptake (total uptake=lysosomal trapping+phospholipid binding) by tissue slices or cells conducting experiments in the presence and absence of ‘lysosomal inhibitors’, i.e., the lysosomotropic compound ammonium chloride (20 mM) or the Na+/H+‐ionophore monensin (10 μM), which elevated the internal pH of lysosomes. The initial concentration of psychotropic drug in the incubation medium was 5 μM.

Both total uptake and lysosomal trapping of the antidepressants investigated (imipramine, amitriptyline, fluoxetine, sertraline) and neuroleptics (promazine, perazine, thioridazine) were higher in the grey matter and neurones than in the white matter and astrocytes, respectively. Lysosomal trapping of the psychotropics occurred mainly in neurones where thioridazine sertraline and perazine showed the highest degree of lysosomotropism.

Distribution interactions between antidepressants and neuroleptics took place in neuronesviamutual inhibition of lysosomal trapping of drugs.

A differential number of neuronal and glial cells in the brain may mask the lysosomal trapping and the distribution interactions of less potent lysosomotropic drugs in vertically cut brain slices.

A reduction (viaa distribution interaction) in the concentration of psychotropics in lysosomes (depot), which leads to an increase in their level in membranes and tissue fluids, may intensify the pharmacological action of the combined drugs.

British Journal of Pharmacology(2001)134, 807–814; doi:10.1038/sj.bjp.0704319
Effect of long‐term treatment with trandolapril on Hsp72 and Hsp73 induction of the failing heart following myocardial infarction
Authors: Tanonaka Kouichi , Toga Wakako , Yoshida Hiroyuki , Furuhama Ken‐ichi , Takeo Satoshi
Published: January 2005

The effect of long‐term treatment of rats with chronic heart failure (CHF) following acute myocardial infarction with trandolapril, an angiotensin I‐converting enzyme (ACE) inhibitor, on heat shock‐induced Hsp72 and Hsp73 production was examined.

Acute myocardial infarction was induced by coronary artery ligation (CAL). The animals with CAL showed symptoms of CHF at the 8th week after the operation. The hearts isolated from animals with CAL at the 2nd and 8th week after surgery were subjected to hyperthermia at 42°C for 15 min followed by 6‐h perfusion (hyperthermia/6‐h perfusion).

In the hearts isolated from the animals at the 2nd week, an approximate 20% decline in the rate pressure product (RPP) was seen after hyperthermia/6‐h perfusion, which was similar to that in non‐operated controls. In contrast, a significant reduction in the RPP after hyperthermia/6‐h perfusion was seen in the hearts of rats with CHF. These hearts did not increase Hsp72 and Hsp73 production after hyperthermia. The decline in RPP was associated with failure in the production of myocardial Hsp72 and Hsp73.

When rats with CAL were treated with 3 mg kg−1 day−1 trandolapril from the 2nd to 8th week after the operation, the decline in RPP of the failing heart after hyperthermia was similar to that of the sham‐operated rats. The induction of myocardial Hsp72 and Hsp73 production of the coronary artery‐ligated rats after hyperthermia was reversed by treatment with trandolapril.

These findings suggest that the preserved ability to induce Hsp72 and Hsp73 production in the heart with CAL by trandolapril treatment may be attributed to the increased tolerance against heat stress‐induced deterioration of myocardial contractile function.

British Journal of Pharmacology (2001) 134, 969–976; doi:10.1038/sj.bjp.0704323
GABA receptors modulate trigeminovascular nociceptive neurotransmission in the trigeminocervical complex
Authors: Storer R James , Akerman Simon , Goadsby Peter J
Published: January 2005

GABA (γ‐aminobutyric acid) receptors involved in craniovascular nociceptive pathways were characterised by in vivo microiontophoresis of GABA receptor agonists and antagonists onto neurones in the trigeminocervical complex of the cat.

Extracellular recordings were made from neurones in the trigeminocervical complex activated by supramaximal electrical stimulation of superior sagittal sinus, which were subsequently stimulated with L‐glutamate.

Cell firing evoked by microiontophoretic application of L‐glutamate (n=30) was reversibly inhibited by GABA in every cell tested (n=19), the GABAA agonist muscimol (n=10) in all cells tested, or both where tested, but not by iontophoresis of either sodium or chloride ions at comparable ejection currents. Inhibited cells received wide dynamic range (WDR) or nociceptive specific input from cutaneous receptive fields on the face or forepaws.

The inhibition of trigeminal neurones by GABA or muscimol could be antagonized by the GABAA antagonist N‐methylbicuculline, 1(S),9(R) in all but two cells tested (n=16), but not by the GABAB antagonist 2‐hydroxysaclofen (n=11).

R(−)‐baclofen, a GABAB agonist, inhibited the firing of three out of seven cells activated by L‐glutamate. Where tested, this inhibition could be antagonized by 2‐hydroxysaclofen. These baclofen‐inhibited cells were characterized as having low threshold mechanoreceptor/WDR input.

GABA thus appears to modulate nociceptive input to the trigeminocervical complex mainly through GABAA receptors. GABAA receptors may therefore provide a target for the development of new therapeutic agents for primary headache disorders.

British Journal of Pharmacology (2001) 134, 896–904; doi:10.1038/sj.bjp.0704325
NCX4016 (NO‐Aspirin) has multiple inhibitory effects in LPS‐stimulated human monocytes
Authors: Minuz Pietro , Degan Maurizio , Gaino Stefania , Meneguzzi Alessandra , Zuliani Valeria , Santonastaso Clara Lechi , Soldato Piero Del , Lechi Alessandro
Published: January 2005

NCX4016 (2 acetoxy‐benzoate 2‐(2‐nitroxymethyl)‐phenyl ester, NicOx S.A., France) is an anti‐thrombotic agent, chemically related to acetylsalicylic acid (ASA) and able to release NO.

We tested the effects of NCX4016 and ASA on the release of the thromboxane (TX) A2 metabolite TXB2, tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), expression and activity of tissue factor (TF) in stimulated, adherent human monocytes.

Both ASA and NCX4016 1 – 1000 μmol l−1 dose‐dependently reduced TXB2 concentration, measured by RIA in the supernatant of 10 μg ml−1 LPS‐stimulated cells. NCX4016 activity was comparable to that of equimolar ASA when incubation lasted 6 h (NCX4016 30 μmol l−1: −86.0±10.1%, NCX4016 300 μmol l−1: −92.2±9.0%, ASA 30 μmol l−1: −92.3±7.5%, ASA 300 μmol l−1: −97.3±1.0%, n=6, M±s.d.). Most of the activity of NCX4016 up to 100 μmol l−1 was prevented by 10 μmol l−1 ODQ, inhibitor of cyclic GMP.

NCX4016 100 – 300 μmol l−1 reduced TNF‐α (NCX4016 300 μmol l−1=−77.2±19.9%, n=6) and IL‐6 (NCX4016 300 μmol l−1: −61.9±15.2%, n=6) in LPS stimulated monocytes while ASA had no significant effects.

TF activity (NCX4016 300 μmol l−1: 53.7±39.9%, n=4) and immunoreactive TF (NCX4016 300 μmol l−1: −93.9±7.9%, n=7), measured in the supernatant of stimulated cells, were also dose‐dependently inhibited by NCX4016 but not by ASA.

The present results indicate that NCX4016 inhibits TXA2 generation as well as cytokine release and TF in human monocytes partly via NO‐dependent mechanisms. NCX4016 may have a favourable profile of activities in the clinical setting of athero‐thrombosis.

British Journal of Pharmacology (2001) 134, 905–911; doi:10.1038/sj.bjp.0704326
Phenanthrolines – a new class of CFTR chloride channel openers
Authors: Duszyk Marek , MacVinish Lesley , Cuthbert Alan W
Published: January 2005

A number of phenanthrolines and benzoquinolines were examined for their ability to activate epithelial chloride secretion by measuring short circuit current (SCC) using the mouse colon epithelium. 1,10 phenanthroline stimulated electrogenic chloride secretion with an EC50 of 612±10 μM and a Hill slope of 4.9±0.3. A similar pharmacology was demonstrated by both 1,7 and 4,7 phenanthrolines, 7,8 benzoquinoline and phenanthridine.

Evidence that the increase in SCC caused by 1,10 phenanthroline was due to chloride secretion is based upon (a) inhibition of the current by furosemide, (b) failure of cystic fibrosis (CF) colons to respond and (c) an associated net flux of 36Cl−.

1,10 Phenanthroline affected neither the generation of cyclic AMP or the concentration of intracellular Ca2+ in colonic epithelial cells.

1,10 phenanthroline affected the chloride conductance of the apical membrane, as shown by an increase in chloride current in ‘apical membrane only’ preparations in the presence of an apical to basolateral chloride gradient. The increase in chloride current was inhibited by 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB) and was not present in CF colons.

Additionally, 1,10 phenanthroline activated basolateral K+ channels, both Ca2+‐ and cyclic AMP‐sensitive channels, as shown by inhibitor studies with charybdotoxin (ChTX) and XE991, and after the apical membrane was permeabilized with nystatin.

The phenanthrolines and benzoquinolines described here, with dual actions affecting CFTR and basolateral K+ channels, may constitute useful lead compounds for adjunct therapy in CF.

British Journal of Pharmacology (2001) 134, 853–864; doi:10.1038/sj.bjp.0704328
Renoprotective effects of carvedilol in hypertensive‐stroke prone rats may involve inhibition of TGFβ expression
Authors: Wong Victoria Y , Laping Nicholas J , Nelson Allen H , Contino Lisa C , Olson Barbara A , Gygielko Eugene , Campbell Wallace G , Barone Frank , Brooks David P
Published: January 2005

The effect of carvedilol on renal function, structure and expression of TGFβ and the matrix proteins fibronectin, collagen I and collagen III, was evaluated in spontaneously hypertensive stroke‐prone (SHR‐SP) rats fed a high fat, high salt diet.

Carvedilol treatment for 11 to 18 weeks did not alter systolic blood pressure in SHR‐SP rats, however, it resulted in a significant reduction in heart rate.

Carvedilol treatment reduced renal fibrosis and total, active and chronic renal damage to levels approaching those of WKY rats on a normal diet.

Urinary protein excretion was higher in SHR‐SP rats (51±10 mg day−1) than WKY rats (18±2 mg day−1) and this was further increased when SHR‐SP rats were fed a high fat, high salt diet (251±120 mg day−1). Treatment with carvedilol resulted in significantly lower urinary protein excretion (37±15 mg day−1).

The expression of TGFβ mRNA was significantly higher in SHR‐SP rats compared to WKY rats and a further increase was observed when rats were fed a high fat, high salt diet. Renal TGFβ expression was significantly reduced by treatment with carvedilol. The expression of fibronectin and collagen I and collagen III mRNA showed a pattern similar to that observed with TGFβ mRNA expression. Collagen I mRNA expression followed a pattern similar to renal fibrosis.

These data indicate that carvedilol can provide significant renal protection in the absence of any antihypertensive activity and that the mechanisms involved in this action may include reduced expression of profibrotic factors such as TGFβ.

British Journal of Pharmacology (2001) 134, 977–984; doi:10.1038/sj.bjp.0704329
A rapid and transient synthesis of nitric oxide (NO) by a constitutively expressed type II NO synthase in the guinea‐pig suprachiasmatic nucleus
Authors: Starkey Sarah J , Grant Andrea L , Hagan Russell M
Published: January 2005

We have measured extracellular NO/NO2− concentrations in guinea‐pig suprachiasmatic nucleus (SCN) brain slices using fast cyclic voltammetry. A rapid and transient signal equivalent to 2.2±0.2 μM NO/NO2− (mean±s.e.mean, n=13) was detected at 1.26 V, the peak oxidation potential for NO, following local electrical stimulation (five pulses of 0.1 ms duration at 100 Hz, delivered every 5 min).

The NO/NO2− signal was inhibited by the non‐selective nitric oxide synthase (NOS) inhibitors L‐NAME, L‐NMMA and the highly selective type II NOS (iNOS) inhibitor 1400 W (Garvey et al., 1997) in a concentration‐dependent manner. IC50 values were 229 μM (65 – 801, n=3, geomean and 95% confidence intervals (C.I.)), 452 nM (88 – 2310, n=5), and 14.2 μM (3.6 – 54.4, n=5), with maximum inhibitions of 82.8±6.7, 46.0±8.1, and 90.6±3.6%, respectively.

Exposure of the slices to the protein synthesis inhibitor cyclohexamide or the inhibitor of type II NOS induction dexamethasone immediately following slice cutting, and for a subsequent 4 – 5 h, did not inhibit the NO/NO2− signal.

The evoked NO/NO2− signal was not reduced following 6 h perfusion in Ca2+‐free media, consistent with a Ca2+‐independent type II NOS activity.

PCR for type II NOS revealed the presence of this isotype in the SCN, even immediately following removal of the brain.

These studies provide the first evidence to suggest a functional, constitutively‐active type II NOS within the brain of normal, healthy adult animals, and add type II NOS to the multiple isotypes of NO synthase playing a role within the mammalian SCN.

British Journal of Pharmacology (2001) 134, 1084–1092; doi:10.1038/sj.bjp.0704330
Dominant role of an endothelium‐derived hyperpolarizing factor (EDHF)‐like vasodilator in the ciliary vascular bed of the bovine isolated perfused eye
Authors: McNeish Alister J , Wilson William S , Martin William
Published: January 2005

The roles of the endothelium‐derived nitric oxide, prostacyclin and endothelium‐derived hyperpolarizing factor (EDHF) in mediating vasodilator responses to acetylcholine and bradykinin were assessed in the ciliary vascular bed of the bovine isolated perfused eye preparation.

Vasodilatation to acetylcholine or bradykinin was unaffected by the nitric oxide synthase inhibitor, L‐NAME (100 μM), or the cyclo‐oxygenase inhibitor, flurbiprofen (30 μM), but was virtually abolished following treatment with a high concentration of KCl (30 mM), or by damaging the endothelium with the detergent, CHAPS (0.3%, 2 min).

Acetylcholine‐induced vasodilatation was unaffected by glibenclamide (10 μM), an inhibitor of ATP‐sensitive K+ channels (K+ATP), but was significantly attenuated by TEA (10 mM), a non‐selective inhibitor of K+ channels.

The small conductance calcium‐sensitive K+ channel (SK+Ca) inhibitor, apamin (100 nM), and the large conductance calcium‐sensitive K+ channel (BK+Ca) inhibitor, iberiotoxin (50 nM), had no significant effect on acetylcholine‐induced vasodilatation. In contrast, the intermediate (IK+Ca)/large conductance calcium‐sensitive K+ channel inhibitor, charybdotoxin (50 nM), powerfully blocked these vasodilator responses, and uncovered a vasoconstrictor response.

The combination of apamin (100 nM) with a sub‐threshold concentration of charybdotoxin (10 nM) significantly attenuated acetylcholine‐induced vasodilatation, but the combination of apamin (100 nM) with iberiotoxin (50 nM) had no effect.

In conclusion, blockade by a high concentration of KCl, by charybdotoxin, or by the combination of apamin with a sub‐threshold concentration of charybdotoxin, strongly suggests that vasodilatation in the bovine isolated perfused eye is mediated by an EDHF.

British Journal of Pharmacology (2001) 134, 912–920; doi:10.1038/sj.bjp.0704332
Differential effects of serotonin reuptake inhibitors on erectile responses, NO‐production, and neuronal NO synthase expression in rat corpus cavernosum tissue
Authors: Angulo Javier , Peiró Concepción , Sanchez‐Ferrer Carlos F , Gabancho Sonia , Cuevas Pedro , Gupta Sandeep , Tejada Iñigo Sáenz de
Published: January 2005

Increased incidence of impotence is associated with some selective serotonin‐reuptake‐inhibitors (SSRIs), but the pathophysiological mechanism is unknown. Paroxetine and citalopram are extensively used SSRIs, but only paroxetine has been shown to inhibit nitric oxide synthase (NOS) activity. NO is a key mediator of penile erection. Thus, the aim of this study was to determine the effects of paroxetine and citalopram on erectile function and NO production, in a rat model. Application of cavernosal nerve electrical stimulation produced frequency‐related intracavernosal pressure (ICP) increases, which were inhibited by the NOS inhibitor, NG‐nitro‐L‐arginine (0.3 mg kg−1). Acute or chronic (2 weeks) paroxetine‐treatment (10 mg kg−1) reduced ICP‐responses, while citalopram did not. Paroxetine, but not citalopram, significantly reduced nitrite+nitrate plasma levels by 61.4% and inhibited penile neuronal NOS (nNOS) protein expression by 31.2% after chronic treatment. The results show that paroxetine inhibits erectile responses in rats. We propose that this effect is due to reduced NO production and nNOS expression.

British Journal of Pharmacology (2001) 134, 1190–1194; doi:10.1038/sj.bjp.0704351
P2 receptor‐types involved in astrogliosis in vivo
Authors: Franke Heike , Krügel Ute , Schmidt Renate , Grosche Jens , Reichenbach Andreas , Illes Peter
Published: January 2005

In the nucleus accumbens (NAc) of rats, the involvement of P2X and P2Y receptors in the generation of astrogliosis in vivo, was investigated by local application of their respective ligands. The agonists used had selectivities for P2X1,3 (α,β‐methylene adenosine 5′‐triphosphate; α,β‐meATP), P2Y1,12 (adenosine 5′‐O‐(2‐thiodiphosphate; ADP‐β‐S) and P2Y2,4,6 receptors (uridine 5′‐O‐(3‐thiotriphosphate; UTP‐γ‐S). Pyridoxalphosphate‐6‐azophenyl‐2,4‐disulphonic acid (PPADS) was used as a non‐selective antagonist. The astroglial reaction was studied by means of immunocytochemical double‐labelling with antibodies to glial fibrillary acidic protein (GFAP) and 5‐bromo‐2′‐deoxyuridine (BrdU).

The agonist‐induced changes in comparison to the artificial cerebrospinal fluid (aCSF)‐treated control side reveal a strong mitogenic potency of ADP‐β‐S and α,β‐meATP, whereas UTP‐γ‐S was ineffective. The P2 receptor antagonist PPADS decreased the injury‐induced proliferation when given alone and in addition inhibited all agonist effects.

The observed morphogenic changes included hypertrophy of astrocytes, elongation of astrocytic processes and up‐regulation of GFAP. A significant increase of both GFAP‐immunoreactivity (IR) and GFA‐protein content (by using Western blotting) was found after microinfusion of α,β‐meATP or ADP‐β‐S. In contrast, UTP‐γ‐S failed to increase the GFAP‐IR. The morphogenic effects were also inhibited by pre‐treatment with PPADS.

A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3 and P2Y1 receptors on the GFAP‐labelled astrocytes.

In conclusion, the data suggest that P2Y (P2Y1 or P2Y12) receptor subtypes are involved in the generation of astrogliosis in the NAc of rats, with a possible minor contribution of P2X receptor subtypes.

British Journal of Pharmacology (2001) 134, 1180–1189; doi:10.1038/sj.bjp.0704353
Characterization of the prostanoid receptor types involved in mediating calcitonin gene‐related peptide release from cultured rat trigeminal neurones
Authors: Jenkins David W , Feniuk Wasyl , Humphrey Patrick P A
Published: January 2005

Prostaglandins and the vasodilator neuropeptide, calcitonin‐gene related peptide (CGRP), have both been implicated in the pathogenesis of migraine headache. We have used primary cultures of adult rat trigeminal neurones to examine the effects of prostanoids on CGRP release in vitro.

CGRP release was stimulated by prostaglandin E2 (PGE2) and the IP receptor agonist, carbaprostacyclin (cPGI2). These responses were extracellular calcium‐dependent, and the PGE2‐induced CGRP release was unaltered by inhibition of nitric oxide synthase (NOS), ATP receptor blockade, or the addition of adenosine deaminase.

Increases in CGRP levels were also observed in response to prostaglandin D2 (PGD2), and the EP2 receptor selective agonist, butaprost. No increases in CGRP release were observed in response to prostaglandin F2α (PGF2α) or the TP receptor selective agonist, U46619, or the EP3 receptor selective agonist, GR63799X.

The selective DP receptor antagonist, BWA868C, antagonized the PGD2‐, but not PGE2‐ or cPGI2‐induced release. Furthermore, the EP1 selective antagonist, ZM325802, failed to antagonize the PGE2‐induced CGRP release from these cells.

These data indicate that activation of DP, EP and IP receptors can each cause CGRP release from trigeminal neurones, and suggest that the predominant EP receptor subtype involved may be the EP2 receptor. Together with evidence that the cyclo‐oxygenase inhibitor, aspirin, particularly when administered intravenously is effective in treating acute migraine, these findings further suggest a role for prostaglandins in migraine pathophysiology.

British Journal of Pharmacology (2001) 134, 1296–1302; doi:10.1038/sj.bjp.0704357
Relationship between endogenous colony stimulating factors and apoptosis in human colon cancer cells: role of cyclo‐oxygenase inhibitors
Authors: Calatayud Sara , Warner Timothy D , Breese Emma J , Mitchell Jane A
Published: January 2005

Nonsteroidal anti‐inflammatory drug (NSAID) usage is associated with gastrointestinal inflammatory damage and aggravation of gut inflammatory conditions. NSAIDs also exert a preventive effect against colon cancer that seems to be due to increased colon cell apoptosis. NSAIDs have been shown to modulate the release of colony stimulating factors (CSFs) in some cells. In the present study we analysed the effect of these drugs on secretion of CSFs and apoptosis in human colon epithelial cells (HT‐29).

HT‐29 cells secreted bioactive levels of GM‐CSF, G‐CSF and M‐CSF when stimulated with IL‐1ß and TNF‐α, and diclofenac (10−7 – 10−4 M), indomethacin (10−7 – 10−4 M) and sodium salicylate (10−5 – 10−2 M) induced concentration‐dependent increases in GM‐CSF secretion.

Reduced secretion of G‐CSF and M‐CSF and increased cell apoptosis were observed with the highest concentrations of these non‐selective NSAIDs.

No changes in any CSF release or HT‐29 cell apoptosis were detected in the presence of the COX‐2 selective inhibitor DFP (10−7 – 10−4 M).

Neither the exogenous addition of CSFs nor the blockade of secreted CSFs modified apoptosis in HT‐29 cells stimulated with cytokines and/or NSAIDs.

These results suggest that colon epithelial cells can contribute to local inflammatory responses by releasing CSFs and thus extend the life span of local leukocytes. Modulation of CSF levels by non‐selective NSAIDs may be involved in the pro‐inflammatory effects of these agents in the gut.

British Journal of Pharmacology (2001) 134, 1237–1244; doi:10.1038/sj.bjp.0704358
Effects of cannabinoids on adrenaline release from adrenal medullary cells
Authors: Niederhoffer Nathalie , Hansen Henrik H , Fernandez‐Ruiz Javier J , Szabo Bela
Published: January 2005

The objective of the present study was to analyse the peripheral effects of cannabinoids on adrenaline release from adrenal chromaffin cells.

In pithed rabbits with electrically stimulated sympathetic outflow, intravenous injection of the cannabinoid receptor agonists WIN55212‐2 and CP55940 (5, 50 and 500 μg kg−1) markedly lowered the plasma adrenaline concentration. The effect of WIN55212‐2 was attenuated by the selective CB1 cannabinoid receptor antagonist SR141716A (500 μg kg−1). WIN55212‐3 (same doses as WIN55212‐2), the enantiomer of WIN55212‐2 lacking affinity for cannabinoid receptors, had no effect on the plasma adrenaline concentration.

In rabbit isolated adrenal glands, the release of adrenaline elicited by electrical stimulation was measured by fast cyclic voltammetry. Electrically‐evoked adrenaline release was inhibited by WIN55212‐2 (0.3, 1, 3 and 10 μM) and this effect was antagonized by SR141716A (1 μM). The non‐cholinergic component of adrenaline release observed after blockade of nicotinic (by hexamethonium 100 μM) and muscarinic (by atropine 0.5 μM) acetylcholine receptors was not depressed by WIN55212‐2. WIN55212‐3 (10 μM) had no effect on adrenaline release.

No detectable specific CB1 receptor binding and mRNA expression were found in rabbit adrenal glands with autoradiography and in situ hybridization.

The results show that cannabinoids inhibit adrenaline secretion in rabbit isolated adrenal glands; the likely mechanism is a presynaptic CB1 receptor‐mediated inhibition of acetylcholine release from preganglionic sympathetic neurons. The inhibition of adrenaline secretion in adrenal glands most probably accounts for the decrease in the plasma adrenaline concentration observed after cannabinoid administration in pithed rabbits.

British Journal of Pharmacology (2001) 134, 1319–1327; doi:10.1038/sj.bjp.0704359
Antidepressants enhance glucocorticoid receptor function in vitro by modulating the membrane steroid transporters
Authors: Pariante Carmine M , Makoff Andrew , Lovestone Simon , Feroli Susan , Heyden Alexandra , Miller Andrew H , Kerwin Robert W
Published: January 2005

Previous data demonstrate that the tricyclic antidepressant, desipramine, induces glucocorticoid receptor (GR) translocation from the cytoplasm to the nucleus in L929 cells and increases dexamethasone‐induced GR‐mediated gene transcription in L929 cells stably transfected with the mouse mammary tumour virus‐chloramphenicol acetyltransferase (MMTV‐CAT) reporter gene (LMCAT cells) (Pariante et al., 1997).

To extend these findings, the present study has investigated the effects of 24 h coincubation of LMCAT cells with dexamethasone and amitriptyline, clomipramine, paroxetine, citalopram or fluoxetine.

All antidepressants, except fluoxetine, enhanced GR‐mediated gene transcription, with clomipramine having the greatest effect (10 fold increase). Twenty‐four hours coincubation of cells with desipramine, clomipramine or paroxetine, also enhanced GR function in the presence of cortisol, but not of corticosterone.

It is proposed that these effects are due to the antidepressants inhibiting the L929 membrane steroid transporter, which actively extrudes dexamethasone and cortisol from the cell, but not corticosterone. This is further confirmed by the fact that clomipramine failed to enhance GR‐mediated gene transcription in the presence of dexamethasone when the membrane steroid transporter was blocked by verapamil.

The membrane steroid transporters that regulate access of glucocorticoids to the brain in vivo, like the multiple drug resistance p‐glycoprotein, could be a fundamental target for antidepressant action.

British Journal of Pharmacology (2001) 134, 1335–1343; doi:10.1038/sj.bjp.0704368
Enhancement by histamine of vascular endothelial growth factor production in granulation tissue via H2 receptors
Authors: Ghosh Ajoy Kumar , Hirasawa Noriyasu , Ohuchi Kazuo
Published: January 2005

Roles of histamine in the production of vascular endothelial growth factor (VEGF) in the carrageenin‐induced granulation tissue in rats were analysed in vitro and in vivo.

Incubation of the minced granulation tissue in the presence of histamine (1 and 10 μM) increased the content of VEGF protein in the conditioned medium in a time‐ and concentration‐dependent manner. The levels of VEGF mRNA in the minced granulation tissue were also increased by histamine in a concentration‐dependent manner.

The increase in the content of VEGF protein in the conditioned medium by histamine (10 μM) was suppressed by the H2 receptor antagonist cimetidine (IC50 0.37 μM), but not by the H1 receptor antagonist pyrilamine maleate, the H3 receptor antagonist thioperamide or the cyclo‐oxygenase inhibitor indomethacin.

The histamine‐induced increase in the content of VEGF protein in the conditioned medium was inhibited by the cyclic AMP antagonist Rp‐cAMP (IC50 6.8 μM), and the protein kinase A inhibitor H‐89 (IC50 12.5 μM), but not by the protein kinase C inhibitors Ro 31‐8425 and calphostin C or the tyrosine kinase inhibitor genistein.

Simultaneous injection of cimetidine (400 μg) and indomethacin (100 μg) into the air pouch of rats additively reduced the carrageenin‐induced increase in VEGF protein levels and angiogenesis in the granulation tissue as assessed by using carmine dye.

These findings indicate that histamine has an activity to induce VEGF production in the granulation tissue via the H2 receptor‐cyclic AMP‐protein kinase A pathway and augments angiogenesis in the granulation tissue.

British Journal of Pharmacology (2001) 134, 1419–1428; doi:10.1038/sj.bjp.0704372
Characterization of the receptors involved in the 5‐HT‐induced excitation of canine antral longitudinal muscle
Authors: Prins N H , Akkermans L M A , Lefebvre R A , Schuurkes J A J
Published: January 2005

We aimed to characterize the 5‐HT receptors involved in the 5‐HT‐induced effect on electrically induced contractions of dog antrum longitudinal muscle in vitro.

In the presence of L‐NOARG (0.1 mM), electrical field stimulation (EFS) induced atropine‐ and tetrodotoxin‐sensitive contractions. Tetrodotoxin or atropine left any agonist tested ineffective. These EFS‐induced contractions were on average enhanced by 5‐HT (0.3 μM), however, pronounced variation in the response to 5‐HT was observed. There were non‐significant trends of the selective 5‐HT3 receptor antagonist granisetron (1 μM), and methysergide (1 μM; preventing interactions of 5‐HT with 5‐HT1, 5‐HT2, 5‐ht5, 5‐HT6 and 5‐HT7 receptors) to increase the response to 5‐HT. The selective 5‐HT4 receptor antagonist GR 113808 (0.1 μM) displayed a non‐significant trend to inhibit the 5‐HT‐induced increase.

Combination experiments with methysergide (1 μM), granisetron (1 μM) and GR 113808 (0.1 μM) revealed that the 5‐HT (0.3 μM)‐induced response consisted of (1) an excitatory component blocked by GR 113808, (2) excitatory and inhibitory components both blocked by methysergide.

The selective 5‐HT4 receptor agonist prucalopride (0.3 μM) increased EFS‐induced contractions, an effect prevented by GR 113808 (0.1 μM).

The increase of EFS‐induced contractions by the preferential 5‐HT2 receptor agonist α‐Me‐5‐HT (0.3 μM) was antagonized by 5‐HT2B receptor antagonists.

The 5‐HT1/5‐HT7 receptor agonist 5‐carboxamidotryptamine (5‐CT; 0.3 μM) inhibited EFS‐induced contractions. This was prevented by methysergide (1 μM), the 5‐HT7 receptor antagonist mesulergine (0.3 μM) and the selective 5‐HT7 receptor antagonist SB‐269970 (0.3 μM).

In the presence of GR 113808 (0.1 μM), α‐Me‐5‐HT (1 μM) increased EFS‐induced contractions. The 5‐HT (0.3 μM)‐induced inhibition of the stimulation by α‐Me‐5‐HT was prevented by SB‐269970 (0.3 μM).

In conclusion, dog antral longitudinal muscle is endowed with (1) excitatory neuronal 5‐HT4 receptors and 5‐HT2B receptors and (2) inhibitory smooth muscle 5‐HT7 receptors.

British Journal of Pharmacology (2001) 134, 1351–1359; doi:10.1038/sj.bjp.0704376
Parathyroid hormone‐related protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets
Authors: Clemens Thomas L , Cormier Sarah , Eichinger Anne , Endlich Karlhans , Fiaschi‐Taesch Nathalie , Fischer Evelyne , Friedman Peter A , Karaplis Andrew C , Massfelder Thierry , Rossert Jérôme , Schlüter Klaus‐Dieter , Silve Caroline , Stewart Andrew F , Takane Karen , Helwig Jean‐Jacques
Published: January 2005

The cloning of the so‐called ‘parathyroid hormone‐related protein’ (PTHrP) in 1987 was the result of a long quest for the factor which, by mimicking the actions of PTH in bone and kidney, is responsible for the hypercalcemic paraneoplastic syndrome, humoral calcemia of malignancy. PTHrP is distinct from PTH in a number of ways. First, PTHrP is the product of a separate gene. Second, with the exception of a short N‐terminal region, the structure of PTHrP is not closely related to that of PTH. Third, in contrast to PTH, PTHrP is a paracrine factor expressed throughout the body. Finally, most of the functions of PTHrP have nothing in common with those of PTH. PTHrP is a poly‐hormone which comprises a family of distinct peptide hormones arising from post‐translational endoproteolytic cleavage of the initial PTHrP translation products. Mature N‐terminal, mid‐region and C‐terminal secretory forms of PTHrP are thus generated, each of them having their own physiologic functions and probably their own receptors. The type 1 PTHrP receptor, binding both PTH(1‐34) and PTHrP(1‐36), is the only cloned receptor so far. PTHrP is a PTH‐like calciotropic hormone, a myorelaxant, a growth factor and a developmental regulatory molecule. The present review reports recent aspects of PTHrP pharmacology and physiology, including: (a) the identification of new peptides and receptors of the PTH/PTHrP system; (b) the recently discovered nuclear functions of PTHrP and the role of PTHrP as an intracrine regulator of cell growth and cell death; (c) the physiological and developmental actions of PTHrP in the cardiovascular and the renal glomerulo‐vascular systems; (d) the role of PTHrP as a regulator of pancreatic beta cell growth and functions, and, (e) the interactions of PTHrP and calcium‐sensing receptors for the control of the growth of placental trophoblasts. These new advances have contributed to a better understanding of the pathophysiological role of PTHrP, and will help to identify its therapeutic potential in a number of diseases.

British Journal of Pharmacology (2001) 134, 1113–1136; doi:10.1038/sj.bjp.0704378