bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effect of darodipine, a calcium antagonist, on rat cardiomyocytes against oxygen radical‐mediated injury
Authors: Matucci Rosanna , Francesca Ottaviani Maria , Barbieri Mario , Cerbai Elisabetta , Mugelli Alessandro
Published: January 2005

We used electrophysiological and electron spin resonance (e.s.r.) techniques to study the mechanism of the protective effect of darodipine on rat isolated cardiomyocytes exposed to an exogenous source of oxygen free radicals (OFR).

The L‐type calcium current (ICa,L), action potential and cell shortening were measured in patch‐clamped cells in the whole‐cell configuration. ICa,L blockade by darodipine was concentration‐dependent, peak current being reduced by 20% with 50 nM and by 58% with 100 nM darodipine. The lowest concentration of darodipine did not affect action potential or twitch profile.

Exposure to OFR‐generating solution (5 mM dihydroxyfumarate, DHF) caused the appearance of electrophysiological alterations and/or spontaneous activity in 73% of cells (n=26) within 5 min; action potential duration (APD) was prolonged (195±16 ms vs 140±6 ms in the control) and maximum diastolic potential (MDP) was reduced (−59.5±2.6 mV vs −69.8±0.8 mV in the control) (P<0.05, n=25).

A 2 min pretreatment with 50 nM darodipine significantly reduced the incidence of these arrhythmogenic events following a 5 min exposure to OFR (36% of cells, n=14; P<0.05 vs nonpretreated cells). Pretreatment with darodipine also prevented APD prolongation caused by OFR (137±12 ms after DHF vs 117±6 ms before DHF n=14, not significant) but not the decrease of MDP (−63.4±2.5 mV after DHF vs −70.9±1.0 mV before DHF, P<0.05).

The e.s.r. spectra obtained from the DHF‐DMPO solution in the absence of darodipine demonstrated the presence of two components corresponding to two DMPO adducts. The addition of darodipine (50–500 nM) led to a concentration‐dependent decrease in intensity of the signals, the intensity of the DMPO‐COO.− adduct being decreased more than that of the DMPO‐OH. adduct.

Our results demonstrate that darodipine dose‐depentently blocks ICa,L in rat isolated cardiomyocytes. Furthermore it exerts protective effects against free‐radical‐induced electrophysiological alterations independently of its calcium antagonistic properties; this effect is possibly due to trapping of specific radical species.
Recording of the saphenous vein compliance by an ultrasonic echo‐tracking device in the dog: effects of S 18149
Authors: Vayssettes‐Courchay Christine , Cordi Alex A. , Lacoste Jean‐Michel , Laubie Michel , Verbeuren Tony J.
Published: January 2005

Saphenous vein reactivity was recorded in the anaesthetized dog by use of an ultrasonic echo‐tracking device to measure the internal diameter of the vein and to calculate the venous compliance. This method was used to investigate the effects of a new partial α1/α2‐adrenoceptor agonist, S 18149, on the canine saphenous vein in vivo after intravenous (i.v.) or oral administration.

Venoconstrictions induced by i.v. or local administration of compounds were evaluated by continuous recording of the internal diameter of the saphenous vein with the echo‐tracking method. Venous compliance was calculated in two ways: (1) as the slope of the diameter‐pressure curve obtained by increasing the venous pressure with an inflatable cuff and (2) in veins in which pressure was higher than 12 mmHg, pulsatile variations in the venous diameter and venous pressure were detected and used to calculate the pulsatile compliance of the vein.

S 18149 administered i.v. at 0.5 μg kg−1 min−1 for 10 min induced a decrease in the saphenous vein diameter (−15±3%) and blood flow (−72±6%) associated with an increase in saphenous vein resistance; at the dose used, S 18149 did not modify venous pressure and caused only a weak increase in arterial pressure (+7±2 mmHg).

The pulsatile compliance of the saphenous vein averaged 8.65±1.37 mm2×100 mmHg−1 in control dogs and was significantly decreased to 5.13±0.68 mm2×100 mmHg−1 in the same animals after treatment with S 18149 at 100 μg kg−1 per os (n=10). The saphenous vein compliance calculated with the increased external pressure method averaged 24.90±1.49 μm mmHg−1 in control dogs and was significantly reduced in the same animals after treatment with S 18149 at 100 μg kg−1 per os to 9.06±3.42 μm mmHg−1 (n=5). When constrictions of the vein were induced with increasing doses of (−)‐phenylephrine, injected locally at 1, 3 or 6 μg min−1, only the responses obtained with the lower dose of (−)‐phenylephrine were increased in dogs treated with S 18149 100 μg kg−1 per os (−16±4% versus −4±3%, n=5).

These results show that the high resolution echo‐tracking device previously used for arterial compliance measurements, allows the detection of pulsatile changes in the canine saphenous vein and thus permits calculation of both the pulsatile and the static compliance of superficial veins in vivo. Using this technique, we have demonstrated that the novel α‐adrenoceptor agonist S 18149 constricts the canine saphenous vein in vivo and decreases the saphenous vein compliance after oral administration.
Acute non‐insulin‐like stimulation of rat muscle glucose metabolism by troglitazone in vitro
Authors: Fürnsinn C. , Neschen S. , Noe C. , Bisschop M. , Roden M. , Vogl C. , Schneider B. , Waldhäusl W.
Published: January 2005

The direct short‐term effects of troglitazone on parameters of glucose metabolism were investigated in rat soleus muscle strips.

In muscle strips from Sprague‐Dawley rats, troglitazone (3.25 μmol l−1) increased basal and insulin‐stimulated glucose transport by 24% and 41%, respectively (P<0.01 each).

In the presence of 5 nmol l−1 insulin, stimulation of glucose transport by 3.25 μmol l−1 troglitazone was accompanied by a 36% decrease in glycogen synthesis, while glycolysis was increased (112% increase in lactate production) suggesting a catabolic response of intracellular glucose handling.

Whereas insulin retained its stimulant effect on [3H]‐2‐deoxy‐glucose transport in hypoxia‐stimulated muscle (by 44%; c.p.m. mg−1 h−1: 852±77 vs 1229±75, P<0.01), 3.25 μmol l−1 troglitazone failed to increase glucose transport under hypoxic conditions (789±40 vs 815±28, NS) suggesting that hypoxia and troglitazone address a similar, non‐insulin‐like mechanism.

No differences between troglitazone and hypoxia were identified in respective interactions with insulin.

Troglitazone acutely stimulated muscle glucose metabolism in a hypoxia/contraction‐like manner, but it remains to be elucidated whether this contributes to the long‐term antidiabetic and insulin enhancing potential in vivo or is to be regarded as an independent pharmacological effect.
Investigation of the inhibitory effect of NG‐nitro‐L‐arginine methyl ester on the antihypertensive effect of the angiotensin AT1 receptor antagonist, GR138950
Authors: Anderson Ian K. , Drew Geoffrey M.
Published: January 2005

The effect of systemic administration of the nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME) on the antihypertensive effects of the angiotensin AT1 receptor antagonist, GR138950, the angiotensin‐converting enzyme (ACE) inhibitor, enalapril, or hydralazine has been evaluated in unrestrained, conscious renal artery ligated hypertensive (RALH) rats. The effect of the phosphodiesterase type V inhibitor, zaprinast on the antihypertensive effect of GR138950 in RALH rats was also examined. The effect of GR138950 on blood pressure, and plasma and urine cyclic GMP levels was compared to that of zaprinast in conscious RALH rats.

GR138950, enalapril or hydralazine caused marked reductions in blood pressure associated with immediate tachycardia in conscious RALH rats. L‐NAME pretreatment attenuated the antihypertensive effects of GR138950 or enalapril but not that of hydralazine in conscious RALH rats. The initial tachycardia caused by GR138950 or enalapril but not hydralazine was attenuated by L‐NAME pretreatment. L‐NAME alone caused a transient (20 min) pressor response and a prolonged (6 h) bradycardia in conscious RALH rats.

Pretreatment with indomethacin did not affect the cardiovascular effect of GR138950 in conscious RALH rats. Indomethacin alone did not significantly change basal blood pressure or heart rate in RALH rats.

Zaprinast pretreatment did not affect the antihypertensive effect of GR138950 in conscious RALH rats but potentiated the depressor response to sodium nitroprusside. Zaprinast alone caused a small reduction in basal blood pressure but did not change basal heart rate in RALH rats.

The antihypertensive effect of GR138950 was not associated with an increase in plasma or urine cyclic GMP levels in conscious RALH rats, whereas zaprinast caused a small fall in blood pressure associated with increases in plasma and urine cyclic GMP.

The ability of L‐NAME to inhibit the antihypertensive action of GR138950 or enalapril suggests that these agents release nitric oxide (NO) and/or enhance the cardiovascular effects of NO as part of their mechanism of action. However, the inability of zaprinast to potentiate the antihypertensive effects of GR138950 and the finding that GR138950 did not increase urine and plasma cyclic GMP levels are not consistent with this view. Attenuation of the response to GR138950 or enalapril, but not hydralazine, suggests a selective interaction between L‐NAME and inhibitors of the renin‐angiotensin system, although the nature of this interaction is unknown.
Increased feeding in fatty Zucker rats by the thiazolidinedione BRL 49653 (rosiglitazone) and the possible involvement of leptin and hypothalamic neuropeptide Y
Authors: Wang Qiong , Dryden Simon , Frankish Helen M. , Bing Chen , Pickavance Lucy , Hopkins David , Buckingham Robin , Williams Gareth
Published: January 2005

The thiazolidinedione BRL 49653 (rosiglitazone) induces hyperphagia and weight gain in obese, insulin‐resistant fatty Zucker rats but not in lean insulin‐sensitive rats. We investigated whether these responses might involve neuropeptide Y (NPY), leptin and insulin.

BRL 49653 (1 mg kg−1 day−1, orally) was given for 7 or 20 days to fatty and lean Zucker and Wistar rats.

In lean rats of either strain, BRL 49653 had no effect on food intake, body weight, plasma insulin and corticosterone, NPY or NPY mRNA levels.

Fatty rats given BRL 49653 showed a 30% increase in food intake and accelerated body weight gain (both P<0.01) after 7 and 20 days, but without significant changes in regional hypothalamic NPY or NPY mRNA levels.

Plasma leptin levels were twice as high in untreated fatty Zucker rats as in lean rats (P<0.01), but were unaffected by BRL 49653 given for 20 days. However, BRL 49653 reduced insulin levels by 42% and increased corticosterone levels by 124% in fatty rats (both P<0.01).

Hyperphagia induced in fatty Zucker rats by BRL 49653 does not appear to be mediated by either a fall in circulating leptin levels or increased activity of hypothalamic NPYergic neurones. The fall in plasma insulin and/or rise in corticosterone levels during BRL 49653 treatment may be involved, consistent with the postulated role of these hormones in the control of food intake.
The effect of losigamone (AO‐33) on electrical activity and excitatory amino acid release in mouse cortical slices
Authors: Srinivasan Jayashri , Richens Alan , Davies John A.
Published: January 2005

Losigamone is a novel anticonvulsant the mechanism of action of which is not known. This study investigated the effect of losigamone on spontaneous, NMDA‐ and AMPA‐induced depolarizations in the cortical wedge preparation of the DBA/2 mouse (which are susceptible to sound‐induced seizures) and on endogenous amino acid release from BALB/c mouse cortical slices.

Cortical wedges exhibit spontaneous depolarizations in magnesium‐free medium and losigamone was effective in significantly reducing these spontaneous depolarizations at concentrations of 100 μM and above.

NMDA‐induced depolarizations were significantly reduced by losigamone at concentrations of 25 μM and above. Losigamone had no effect on AMPA‐induced depolarizations.

Veratridine (20 μM) and potassium (60 mM) were used to stimulate the release of amino acids from mouse cortex. Veratridine‐stimulated release of glutamate was significantly reduced by losigamone at concentrations of 100 μM and above, while potassium‐stimulated release was significantly reduced by losigamone at 200 μM.

NMDA antagonism and inhibition of excitatory amino acid release may contribute to the anticonvulsant effect of losigamone.
Modulation of dopamine release from rat striatum by protein kinase C: interaction with presynaptic D2‐dopamine‐autoreceptors
Authors: Iannazzo L. , Sathananthan S. , Majewski H.
Published: January 2005

Interactions between dopamine receptors and protein kinase C (PKC) have been proposed from biochemical studies. The aim of the present study was to investigate the hypothesis that there is an interaction between protein kinase C and inhibitory D2‐dopamine receptors in the modulation of stimulation‐induced (S‐I) dopamine release from rat striatal slices incubated with [3H]‐dopamine. Dopamine release can be modulated by protein kinase C and inhibitory presynaptic D2 receptors since phorbol dibutyrate (PDB) and (−)‐sulpiride, respectively, elevated S‐I dopamine release.

The protein kinase C inhibitors polymyxin B (21 μm) and chelerythrine (3 μm) had no effect on stimulation‐induced (S‐I) dopamine release. However, when presynaptic dopamine D2 receptors were blocked by sulpiride (1 μm), an inhibitory effect of both PKC inhibitors on S‐I dopamine release was revealed. Thus, sulpiride unmasks an endogenous PKC effect on dopamine release which suggests that presynaptic D2 receptors normally suppress endogenous PKC activity. This is supported by results in striatal slices which were pretreated with PDB to down‐regulate PKC. In this case the facilitatory effect of sulpiride was completely abolished.

The inhibitory effect of the dopamine D2/D3 agonist quinpirole on S‐I dopamine release was partially attenuated by PKC down‐regulation. Since the effect of sulpiride was completely abolished under the same conditions, this suggests that exogenous agonists may target a PKC‐dependent as well as a PKC‐independent pathway. The inhibitory effect of apomorphine was not affected by either polymyxin B or PKC down‐regulation, suggesting that it operated exclusively through a PKC‐independent mechanism.

These results suggest that there are at least two pathways involved in the inhibition of dopamine release through dopamine receptors. One pathway involves dopamine receptor suppression of protein kinase C activity, perhaps through inhibition of phospholipase C activity and this is preferentially utilized by neuronally‐released dopamine. The other pathway which seems to be utilized by exogenous agonists does not involve PKC.
Age‐related changes in purinergic and adrenergic components of sympathetic neurotransmission in guinea‐pig seminal vesicles
Authors: Pinna Christian , Rubino Annalisa , Burnstock Geoffrey
Published: January 2005

Purinergic and adrenergic components of sympathetic neurotransmission and contractile responses to exogenous α,β‐methylene ATP and noradrenaline have been investigated in the seminal vesicles of 1‐day (new‐born), 2‐weeks (young), 12‐weeks (adult) and 2‐years old (aged) guinea‐pigs.

In seminal vesicles of new‐born guinea‐pigs electrical field stimulation (EFS; 80 V, 0.5 ms for 30 s, 2–32 Hz) evoked tonic frequency‐related contractions. In 2‐weeks old guinea‐pigs the tonic contraction masked an initial phasic component of the neurogenic responses, whereas in 12‐weeks and 2‐years old guinea‐pigs, neurogenic responses were biphasic, a phasic response being followed by a tonic contraction. In all experimental groups, prazosin (10−6 M) blocked the tonic contraction while desensitization of P2X receptors by α,β‐methylene ATP (10−4 M) abolished the phasic responses.

The phasic purinergic component of the neurogenic response was significantly higher in 12‐weeks and 2‐years old animals, compared with 2‐weeks old guinea‐pigs. At 32 Hz phasic contractions were (mN mg−1 tissue): 0.047±0.012, 0.018±0.040 and 0.147±0.026 in 2‐weeks, 12‐weeks and 2‐years old guinea‐pigs, respectively. In contrast, the tonic adrenergic component of the neurogenic contraction significantly declined at 12‐weeks and 2‐years compared with 2‐weeks old guinea‐pigs.

Contractile responses (mN mg−1 tissue) to the highest concentration of α,β‐methylene ATP tested were significantly higher in 2‐weeks (0.248±0.022) than in 1‐day old animals (0.113±0.012) and decreased in 12‐weeks (0.163±0.016) and 2‐years old guinea‐pigs (0.200±0.008). The pD2 values for the purinoceptor agonist were also significantly lower in adult (4.74±0.20) and aged guinea‐pigs (5.22±0.08) compared with 2‐weeks old animals (5.91±0.27). Conversely, responses to the highest concentration of noradrenaline gradually decreased with age, without significant changes in the pD2 values. Contractile responses to KCl (240 mM) did not differ significantly between the experimental groups.

These results demonstrate age‐related changes in purinergic and adrenergic components of sympathetic neurotransmission in the guinea‐pig seminal vesicles. The purinergic component is absent in new‐born animals and it appears fully developed in adult and old guinea‐pigs, while the adrenergic component decreases with age. Pre‐ and postjunctional mechanisms contributing to the age‐related changes of sympathetic neurotransmission are discussed.
Neuropeptide Y (NPY) and peptide YY (PYY) effects in the epididymis of the guinea‐pig: evidence of a pre‐junctional PYY‐selective receptor
Authors: Haynes John M. , Hill Stephen J. , Selbie Lisa A.
Published: January 2005

The effects of peptide YY (PYY), neuropeptide Y (NPY) and structurally related peptides upon field stimulation‐induced and phenylephrine‐mediated contractile responses in the cauda epididymis of the guinea‐pig were investigated.

Preparations of cauda epididymis responded to field stimulation with contractions which were completely attenuated by both the neurotoxin, tetrodotoxin (500 nM), and also by the α‐adrenoceptor antagonist, phentolamine (3 μM). PYY (n=7) and the truncated peptide analogue PYY(3–36) (n=5) inhibited field stimulation‐induced contractions (pIC50+s.e.mean: 8.9±0.2 and 9.4±0.2, respectively). Pancreatic polypeptide (PP, up to 1 μM, n=6), NPY (up to 100 nM, n=6) and the NPY analogues [Leu31,Pro34]NPY (n=6) and NPY (13–36) (both up to 1 μM, n=5) had no significant effect.

The NPY Y1 receptor antagonist BIBP3226 ((R)‐N2‐(diphenylacetyl)‐N[(4‐hydroxyphenyl)‐methyl]‐argininamide) at 750 nM (n=6) and 7.5 μM (n=6) did not affect the PYY‐mediated inhibition of field stimulation‐induced contractions (pIC50 8.9±0.3 and 9.0±0.3, respectively). In the presence of BIBP3226 (7.5 μM), NPY (n=6) inhibited field stimulation‐induced contractions (pIC50 8.0±0.2).

NPY, PYY and PYY(3–36) inhibited [3H]‐noradrenaline release from preparations of epididymis (pIC50 values 7.9±0.7, 9.6±0.8 and 10.0±0.9, respectively, all n=6). The agonists PP and [Leu31,Pro34]PYY (both up to 100 nM) were without significant effect (both n=6).

In preparations of cauda epididymis, stimulated with threshold concentrations of the α1‐adrenoceptor agonist, phenylephrine (1 μM), both NPY (n=6) and PYY (n=7) elicited concentration‐dependent increases in contractile force (with pEC50 values of 8.9±0.2 and 8.6±0.1, respectively). The effects of both NPY (n=6) and PYY (n=6) were antagonized by preincubation with BIBP3226 (75 nM; apparent pKB±s.e. values 8.3±1.0 and 8.2±0.6, respectively). The peptide analogues NPY(13–36) (n=5), PYY (3–36) (n=7) and [Leu31,Pro34]NPY (n=5) did not significantly augment responses to threshold concentrations of phenylephrine.

These results are consistent with the proposal that distinct NPY receptors mediate the (prejunctional) inhibition of field stimulation‐induced contractions and the (postjunctional) potentiation of responses to phenylephrine in the cauda epididymis of the guinea‐pig. The rank order of agonist potency (NPYPYY≫NPY(13–36), [Leu31,Pro34]NPY and PYY(3–36) and the high potency of BIBP3226 indicate that the postjunctional receptor may be Y1‐like. The rank orders of agonist potency in inhibiting field stimulation‐induced contractile responses and [3H]‐noradrenaline release (PYY(3–36)PYY> NPY≫PP, NPY(13–36), [Leu31,Pro34]NPY and PYY(3–36)PYY>NPY≫;PP,[Leu31,Pro34]PYY, respectively) are consistent with the action of these peptides at a PYY‐preferring receptor subtype, which may be distinct from the presently characterized NPY receptor subtypes.
Synthesis of kininogen and degradation of bradykinin by PC12 cells
Authors: Dendorfer A. , Wellhöner P. , Braun A. , Roscher A. A. , Dominiak P.
Published: January 2005

In this study, the abilities of PC12 cells to synthesize and degrade kinins were investigated. Kinin formation was assessed as kinin and kininogen content of cells and supernatants in serum‐free incubations by use of a bradykinin‐specific radioimmunoassay. Expression of kininogen mRNA was demonstrated by reverse‐transcriptase PCR. Kinin degradation pathways of intact PC12 cells were characterized by identification of the kinin fragments generated from tritiated bradykinin either in the absence or presence of the angiotensin I‐converting enzyme inhibitor ramiprilat.

Kinin immunoreactivity in the supernatant of PC12 cell cultures accumulated in a time‐dependent fashion during incubations in serum‐free media. This effect was solely due to de novo synthesis and release of kininogen (35 pg bradykinin  h−1 mg−1 protein) since it could be suppressed by cycloheximide. Continuous synthesis of kininogen was a specific property of PC12 cells, as it was not observed in cultured macro‐ or microvascular endothelial cells. PC12 cells contained only minor amounts of stored kininogen. The rate of kininogen synthesis was not affected by ramiprilat, bacterial lipopolysaccharide, nerve growth factor or dexamethasone, but was stimulated 1.4 fold when cells were pretreated for 1 day with 1 μm desoxycorticosterone.

By use of cDNA probes specific for kininogen subtype mRNAs, expression of low‐molecular‐weight kininogen and T‐kininogen in PC12 cells was confirmed. Expression of high molecular weight kininogen mRNA was also shown, though only at the lowest limit of detection of the assay.

Degradation of tritiated bradykinin by PC12 cells occurred with a half‐life of 48 min resulting in the main fragments [1–7]‐ and [1–5]‐bradykinin. The degradation rate of bradykinin decreased to 15% in the presence of ramiprilat (250 nm). Apart from angiotensin I‐converting enzyme direct cleavage of bradykinin to [1–7]‐ and [1–5]‐bradykinin still occurred under this condition as a result of additional kininase activities.

Along with previous findings of B2‐receptor‐mediated catecholamine release, these results now confirm the hypothesis that a cellular kinin system is expressed in PC12 cells. The presence of such a system may reflect a role of kinins as local neuromodulatory mediators in the peripheral sympathetic system.
Prostanoids synthesized by cyclo‐oxygenase isoforms in rat spinal cord and their contribution to the development of neuronal hyperexcitability
Authors: Willingale H. L. , Gardiner N. J. , McLymont N. , Giblett S. , Grubb B. D.
Published: January 2005

The responses of wide dynamic range spinal dorsal horn neurones to noxious mechanical stimulation of the ankle or knee joint were tested before and after spinal administration of the non‐selective cyclo‐oxygenase (COX) inhibitors, indomethacin and meclofenamic acid. Neither of these drugs altered the responses of these neurones to noxious mechanical stimulation.

Wind‐up of a spinal nociceptive reflex evoked by electrical stimulation of the sural nerve at C‐fibre strength was dose‐dependently inhibited by intravenous administration of indomethacin, a non‐selective COX inhibitor, and SC58125, a selective COX‐2 inhibitor. Intrathecal administration of indomethacin also reduced the wind‐up of this nociceptive reflex.

Western blot analysis of proteins extracted from normal rat spinal cord revealed the presence of both cyclo‐oxygenase (COX)‐1 and COX‐2 proteins.

Immunocytochemistry of sections of normal rat spinal cord with specific COX‐1 antiserum revealed little specific COX‐1‐like immunoreactivity in the grey matter. With the same antiserum, intense COX‐1‐like immunoreactivity was observed in the cytoplasm, nuclear membrane and axonal processes of small to medium sized (<1000 μm2) dorsal root ganglion (DRG) cell bodies.

Immunocytochemistry of sections of normal rat spinal cord incubated with specific COX‐2 antiserum showed intense COX‐2‐like immunoreactivity (COX‐2‐li) in the superficial dorsal horn of the spinal cord (laminae I and II) and around the central canal (lamina X). COX‐2‐li was also observed in some neurones in deep dorsal horn and in individual motor neurones in ventral horn. COX‐2‐li was not observed in the cell bodies of DRG.

Superfusion of the lumbar spinal cord of normal rats with artificial CSF and subsequent radioimmunoassay revealed the presence of prostaglandin D2 (PGD2)<PGE2, but not PGI2 (determined by measurement of the stable metabolite, 6‐keto‐PGF1α) or PGF2α.

These data suggest that eicosanoids synthesized by an active COX pathway in the spinal cord of normal animals may contribute to nociceptive processing, but only when the spinal cord neurones are rendered hyperexcitable following C‐fibre stimulation. Selective inhibition of one or both of the COX isoforms in normal animals may represent a novel target for spinal analgesia.
Potency and kinetics of nitric oxide‐mediated vascular smooth muscle relaxation determined with flash photolysis of ruthenium nitrosyl chlorides
Authors: Carter T. D. , Bettache N. , Ogden D.
Published: January 2005

Flash photolysis of thermally stable, photolabile ‘caged’ precursors permits rapid and precise changes of ligand concentration at their site of action. This approach was used to determine the concentration‐dependence and time course of NO‐mediated relaxation of aortic smooth muscle, by use of two photolabile NO donors, trichloronitrosylruthenium (Ru(NO)Cl3) and dipotassium pentachloronitrosylruthenate (K2Ru(NO)Cl5). At concentrations up to 500 μM, both compounds were non‐toxic before photolysis, and produced non‐toxic by‐products on photolysis. Photolytic release of NO produced relaxations of intact and endothelium‐denuded aortic rings precontracted with noradrenaline (0.1–0.5 μM), with an EC50 for NO‐mediated relaxations of 10.5 nM and 13 nM, respectively. NO‐mediated relaxations were reversibly blocked by 1 μM oxyhaemoglobin. The time course of NO‐mediated relaxation comprised a delay of 3–7 s, followed by a sigmoidal decline in tension with peak rates that were strongly dependent on NO concentration.

British Journal of Pharmacology (1997) 122, 971–973; doi:10.1038/sj.bjp.0701549
Role of potassium channels in the nitrergic nerve stimulation‐induced vasodilatation in the guinea‐pig isolated basilar artery
Authors: Jiang Fan , Li Chun Guang , Rand Michael J
Published: January 2005

We studied the effects of various K+ channel blockers on the vasodilator responses of guinea‐pig isolated basilar arteries to nitrergic nerve stimulation, the nitric oxide (NO) donor sodium nitroprusside (SNP), and the membrane permeable guanosine‐3′, 5′‐cyclic monophosphate (cyclic GMP) analogue 8‐bromo‐cyclic GMP (8‐Br‐cyclic GMP).

In endothelium‐denuded preparations which were contracted with prostaglandin F2α (1 μM), electrical field stimulation (EFS, 10 Hz for 30 s) produced a vasodilatation which was totally blocked by the nitric oxide synthase (NOS) inhibitor NG‐nitro‐L‐arginine methyl ester L‐NAME; 100 μM) (n=3) and by the selective NO‐sensitive guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one (ODQ; 1 μM) (n=4). The vasodilator response to SNP (100 nM) was not reduced by L‐NAME but was abolished by ODQ (1 μM) (n=4).

EFS‐elicited vasodilatation was partly but significantly reduced by the non‐selective K+ channel blockers tetraethylammonium (TEA, 1 and 3 mM) and 4‐aminopyridine (4‐AP, 3 mM), and by the large‐conductance calcium‐activated K+ channel (KCa channel) blockers charybdotoxin (ChTX, 150 nM) and iberiotoxin (IbTX, 30 and 100 nM). In contrast, the ATP‐sensitive K+ channel (KATP channel) blocker glibenclamide (1–10 μM) and the small‐conductance KCa channel blocker apamin (100–500 nM) did not affect EFS‐induced vasodilatation.

The vasodilator response elicited by SNP (10–100 nM) was significantly reduced by TEA (3 mM) and ChTX (150 nM) but not by apamin (500 nM) or glibenclamide (1 μM). The vasodilatation elicited by 8‐Br‐cyclic GMP (100 μM) was also reduced by TEA (3 mM) and ChTX (150 nM).

The results indicate that the vasodilatations induced by nitrergic nerve stimulation and the NO donor SNP in endothelium‐denuded guinea‐pig basilar artery depend on the formation of intracellular cyclic GMP. The increased cyclic GMP level activates large‐conductance KCa channels which partly mediate the vasodilator response. Neither KATP channels nor apamin‐sensitive small‐conductance KCa channels are involved in nitrergic transmitter‐mediated vasodilatation.

British Journal of Pharmacology (1998) 123, 106–112; doi:10.1038/sj.bjp.0701552
Effects of oral administration of different doses of purified micronized flavonoid fraction on microvascular reactivity after ischaemia/reperfusion in the hamster cheek pouch
Authors: Bouskela E. , Cyrino F. Z. G. A. , Lerond L.
Published: January 2005

The effects of a purified micronized flavonoid fraction (S5682) on mean internal diameter and blood flow of arterioles and venules, as well as the functional capillary density (FCD) were evaluated in the hamster cheek pouch microvasculature before and after 90 min of total ischaemia.

Male hamsters were treated for ten days, twice a day, with oral doses of S5682 (5, 20, 80 and 160 mg kg−1 day−1) or placebo (10% lactose solution). The cheek pouch preparation was placed under an intravital microscope coupled to a closed circuit TV system. Local ischaemia was obtained by a cuff mounted around the neck of the everted pouch where it leaves the mouth of the hamster.

Measurements were performed before ischaemia, at the onset of reperfusion and 10, 20, 30, 45 and 60 min thereafter. Diameters were measured by means of an image shearing device. Red blood cell (RBC) velocity was analysed by use of the dual‐slit photometric technique. Blood flow was calculated from diameters and RBC velocities. FCD, defined as the number of capillaries with flowing blood per field of observation, was also assessed.

During reperfusion, placebo‐treated animals showed a significant vasodilatation, a decrease in blood flow and FCD and S5682‐treated animals showed a clear trend, dose‐dependent, towards maintaining these parameters closer to the value found before ischaemia.

In conclusion, our results indicate that S5682 improves the microvascular reactivity and FCD after ischaemia/reperfusion. These data suggest that S5682 could function as an antioxidant, which may explain its beneficial therapeutic effect in chronic venous insufficiency where oxidative stress is involved in the pathological mechanism.
Heterogeneity of α2‐adrenoceptors in human and rat myometrium and differential expression during pregnancy
Authors: Bouet‐Alard Rachel , Mhaouty‐Kodja Sakina , Limon‐Boulez Isabelle , Coudouel Noelline , Maltier Jean Paul , Legrand Chantal
Published: January 2005

The aim of this study was first, to characterize α2‐adrenoceptor subtypes in human and rat pregnant myometrium and second, to investigate the possibility of a differential expression of the putative subtypes according to the stage of pregnancy.

In both species, specific [3H]‐rauwolscine binding was inhibited by five different compounds with an order of affinity characteristic of the one described for α2‐adrenoceptors (yohimbineclonidine>noradrenaline>phenylephrine>propranolol). Binding affinities (pKi) for the compounds tested were, in human and rat, respectively: 7.63 and 8.93 for yohimbine, 6.91 and 8.71 for clonidine, 6.23 and 6.09 for noradrenaline, 5.37 and 5.73 for phenylephrine, 4.64 and 4.72 for propranolol.

By use of non‐linear iterative curve fitting procedures and by fitting the data to a two‐site model, analysis of [3H]‐rauwolscine inhibition binding curves performed in the presence of oxymetazoline (α2A‐selective), ARC239, prazosin or chlorpromazine (α2B‐ and α2C‐selective) indicated that pregnant human and rat myometrium contain at least two pharmacologically distinct α2‐adrenoceptor subtypes (α2A, α2B and/or α2C). RNA blot analysis with probes specific for each cloned human and rat α2‐adrenoceptor subtype demonstrated that α2A‐ and α2B‐subtypes were present in both species but α2C seems to be expressed only in human tissues.

In the pregnant rat myometrium, subtype selective compounds competition curves revealed a predominant expression of α2A‐adrenoceptors at mid‐pregnancy whereas, at term, α2A‐ and α2B‐subtypes density reached approximately the same level (α2A : α2B ratio=73 : 27 at mid‐pregnancy and=43 : 57 at term). In addition, quantification of α2A‐ and α2B‐transcripts by densitometry, following data normalization with an oligo(dT)12–18 probe, showed a pattern of expression comparable to the one characterized by pharmacological studies.

In conclusion, these data demonstrate heterogeneity of α2‐adrenoceptors in pregnant human and rat myometria and an alteration of the α2A‐/α2B‐subtypes expression pattern during rat pregnancy. Such observations lead us to suggest a multiple role for α2‐adrenoceptors in regulating specific functions of myometrium throughout the time course of pregnancy.
Prolonged effect of a novel S‐nitrosated glyco‐amino acid in endothelium‐denuded rat femoral arteries: potential as a slow release nitric oxide donor drug
Authors: Megson I. L. , Greig I. R. , Gray G. A. , Webb D. J. , Butler A. R.
Published: January 2005

The vasodilator properties of a novel S‐nitrosated glyco‐amino acid (RIG200) were investigated in isolated rat femoral arteries and compared with those of the parent S‐nitrosothiol compound, S‐nitroso‐N‐acetylpenicillamine (SNAP).

Spectrophotometric analysis revealed that 2.5 mm solutions of RIG200 decomposed more slowly (half‐life (t1/2)=216.2±26.7 min) than SNAP (t1/2=37.2±13.8 min) in Krebs buffer at 24°C. Furthermore, the rate of decomposition of SNAP, but not of RIG200, was significantly reduced by the Cu(I) chelator, neocuproine. We concluded that the relative stability of RIG200 is due, at least in part, to its resistance to trace Cu(I)‐catalyzed decomposition. Nitric oxide (NO) generation from SNAP and RIG200 was confirmed by use of an NO electrode.

Experiments to investigate the vasodilator effects of RIG200 were carried out on isolated femoral arteries taken from adult male Wistar rats (400–550 g). Lengths of artery (7–8 mm long) were cannulated, dissected free and perfused at constant flow rate (0.6 ml min−1) with Krebs buffer. Vessels were precontracted with phenylephrine (10.2±0.3 μm) and developed pressures of 91.8 ± 4 mmHg, detected upstream by a differential pressure transducer.

Concentration‐dependent vasodilator responses to bolus injections of SNAP or RIG200 (10 μl; 10−8–10−3 m) made into the perfusate of endothelium‐intact vessels were transient, recovering the pre‐injection pressure in <20 min.

Responses to equivalent bolus injections of SNAP in endothelium‐denuded vessels were also transient but those in response to concentrations of RIG200 >10−5 m were sustained. Responses to 10−3 m RIG200 were sustained for periods >4 h. Sustained vasodilatation was reversed by the NO scavenger, ferrohaemoglobin (10 μm) but was unaffected by the NO synthase inhibitor, Nω‐nitro‐L‐arginine methyl ester (200 μm), indicating involvement of NO from a source other than NO synthase.

We suggest that a possible explanation for the prolonged effect of RIG200 is retention of the compound by the vascular wall, facilitated by endothelial denudation. Slow decomposition of RIG200 in situ would release sufficient NO to maintain a ‘vasodilator tone’ which persists for more than 4 h. Selective retention by damaged vessels could have important therapeutic implications with regard to targeted delivery of NO, restoring protection to areas deprived of endogenous NO, whilst avoiding unwanted hypotension.
Pharmacological evidence for the existence of multiple P2 receptors in the circular muscle of guinea‐pig colon
Authors: Zagorodnyuk Vladimir , Maggi Carlo Alberto
Published: January 2005

By using the sucrose gap technique, we have investigated the effect of the metabolically stable P2Y receptor agonist, adenosine 5′‐O‐2‐thiodiphosphate (ADPβS), on the membrane potential and tension in the circular muscle of the guinea‐pig proximal colon. All experiments were performed in the presence of atropine (1 μM), guanethidine (3 μM), indomethacin (3 μM), nifedipine (1 μM), L‐nitroarginine (L‐NOARG, 100 μM) and of the tachykinin NK1 and NK2 receptor antagonists, SR 140333 (0.1 μM) and GR 94800 (0.1 μM), respectively.

ADPβS (100 μM for 15 s) evoked a tetrodotoxin‐ (1 μM) resistant hyperpolarization and contraction of the smooth muscle. In the presence of apamin (0.1 μM), the ADPβS‐induced hyperpolarization was converted to depolarization and the contraction was potentiated while tetraethylammonium (TEA, 10 mM) did not affect significantly the response to ADPβS. The combined application of apamin and TEA reproduced the effect observed with apamin alone.

Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acids (PPADS, 30 μM) slightly but significantly increased the ADPβS‐induced hyperpolarization, while the contraction evoked by ADPβS was reduced by about 80%. Suramin (100 μM) did not affect the ADPβS‐induced hyperpolarization but totally blocked the ADPβS‐induced contraction. In the presence of suramin (100 μM), a small relaxation of the circular muscle was observed upon application of ADPβS.

The contraction and hyperpolarization evoked by ADPβS were abolished in Ca2+‐free Krebs solution. The blocker of sarcoplasmic reticulum Ca2+ pump, cyclopiazonic acid (10 μM) reduced contraction and hyperpolarization induced by ADPβS by about 60 and 50%, respectively.

A comparison of our present and previous findings enables to conclude that at least 3 types of P2 receptors are present on the smooth muscle of the guinea‐pig colon, as follows: (1) inhibitory P2 receptors, producing an apamin‐sensitive hyperpolarization, which are activated by α,β‐methylene ATP (α,β‐meATP) and by endogenously released purines, sensitive to suramin and PPADS; (2) inhibitory P2 receptors, producing an apamin‐sensitive hyperpolarization, which are activated by ADPβS and are resistant to suramin and PPADS; (3) excitatory P2 receptors, producing contraction, which are activated by ADPβS and are sensitive to suramin and PPADS. The data also support the idea of the existence of a restricted pool of specialized junctional P2 receptors producing the apamin‐sensitive NANC inhibitory junction potential in response to endogenous ligand(s).

British Journal of Pharmacology (1998) 123, 122–128; doi:10.1038/sj.bjp.0701558
Internally applied endotoxin and the activation of BK channels in cerebral artery smooth muscle via a nitric oxide‐like pathway
Authors: Hoang L M , Mathers D A
Published: January 2005

In this study the role of nitric oxide synthase (NOS) in the acute activation of large conductance, Ca2+‐activated K+ channels (BK channels) by internally applied E. coli lipopolysaccharide (LPS, endotoxin) was examined in vascular smooth muscle cells.

Cerebrovascular smooth muscle cells (CVSMCs) were enzymatically dispersed from the middle, posterior communicating and posterior cerebral arteries of adult Wistar rats and maintained at 4°C for 2–4 days before recording with standard patch‐clamp techniques.

Acute application of LPS (100 μg ml−1) to inside‐out patches of CVSMC membrane isolated in a cell‐free environment rapidly and reversibly increased the open probability, Po of BK channels in these patches by 3.3±0.30 fold.

Acute application of the nitric oxide (NO) donor sodium nitroprusside (SNP, 100 μM) to inside‐out patches of CVSMC membrane, studied in the presence of intact cells, also reversibly increased Po, by some 1.8±0.2 fold over control.

Kinetic analysis showed that both LPS and SNP increased Po by accelerating the rate of BK channel reopening, rather than by retarding the closure of open channels.

Neither LPS nor SNP altered the reversal potential or conductance of BK channels.

The NOS substrate L‐arginine (1 μM) potentiated the acute activation of BK channels by LPS, while the synthetic enantiomer D‐arginine (1 μM) inhibited the action of LPS on BK channels.

The acute activation of BK channels by LPS was suppressed by pre‐incubation of cells with Nω‐nitro‐L‐arginine (50 μM) or Nω‐nitro‐L‐arginine methyl ester (1  mM), two competitive antagonists of nitric oxide synthases. Nω‐nitro‐D‐arginine (50 μM), a poor inhibitor of NOS in in vitro assays, had no effect on BK channel activation by LPS.

These results indicate that excised, inside‐out patches of CVSMC membrane exhibit a NOS‐like activity which is acutely activated when LPS is present at the cytoplasmic membrane surface. Possible relationships between this novel mechanism and the properties of known isoforms of nitric oxide synthase are discussed.

British Journal of Pharmacology (1998) 123, 5–12; doi:10.1038/sj.bjp.0701570
Evidence for the involvement of P‐glycoprotein on the extrusion of taken up L‐DOPA in cyclosporine A treated LLC‐PK1 cells
Authors: Soares‐da‐Silva P , Serrão M P , Vieira‐Coelho M A , Pestana M
Published: January 2005

The present work has examined the effects of short‐ (30 min) and long‐term (14 h) exposure to cyclosporine A (CsA) on the uptake of L‐DOPA, its decarboxylation to dopamine and the cellular extrusion of taken up L‐DOPA and of newly‐formed amine in monolayers of LLC‐PK1 cells.

In the presence of benserazide (50 μM), L‐DOPA was rapidly accumulated in LLC‐PK1 cells (cultured in collagen‐treated plastic) attaining equilibrium at 30 min of incubation. Non‐linear analysis of the saturation curves revealed a Km of 113±16 μM and a Vmax of 5581±297 pmol mg−1 protein 6 min−1.

In the absence of benserazide, LLC‐PK1 cells incubated with increasing concentrations of L‐DOPA (10 to 500 μM) for 6 min accumulate newly‐formed dopamine by a saturable process with apparent Km and Vmax values of 31±6 μM and 1793±91 pmol mg−1 protein 6 min−1, respectively. The fractional outflow of newly‐formed dopamine was found to be 20%. Up to 200 μM of intracellular newly‐formed dopamine, the outward transfer of the amine was found to be a non‐saturable process.

Short‐term exposure to CsA (0.3, 1.0 and 3.0 μg ml−1) was found not to change the intracellular concentrations of newly‐formed dopamine, but increased the levels of dopamine in the incubation medium (143% to 224% increase) and the total amount of dopamine formed (31% to 59% increase). Long‐term exposure to CsA (0.03 to 3.0 μg ml−1) reduced the total amount of dopamine (15% to 39% reduction) and the intracellular levels of the amine (11% to 56% reduction), without changing dopamine levels in the incubation medium. Both short‐ and long‐term exposure to CsA resulted in a concentration‐dependent increase in the fractional outflow of newly‐formed dopamine.

Short‐term exposure to CsA (3.0 μg ml−1) reduced the apical extrusion of intracellular L‐DOPA by 15% (P<0.05), whereas long‐term exposure to CsA reverted this effect and decreased its intracellular availability (15% reduction; P<0.05).

Detection of P‐glycoprotein activity was carried out by measuring verapamil‐ or UIC2‐sensitive rhodamine 123 accumulation. Both UIC2 (3 μg ml−1) and verapamil (25 μM) significantly increased the accumulation of rhodamine 123 in LLC‐PK1 cells. A 30 min exposure to CsA was found not to affect the accumulation of rhodamine 123 in the presence of verapamil (25 μM), whereas a 14 h exposure to CsA was found to reduce the accumulation of rhodamine 123.

In conclusion, the increase and the reduction in the formation of dopamine after short‐ and long‐term exposure to CsA, respectively, correlate with the effects of the immunosuppressant on the apical cell extrusion of taken up L‐DOPA, suggesting the involvement of P‐glycoprotein. The effects of CsA on the fractional outflow of newly‐formed dopamine appear to be mediated by a different mechanism.

British Journal of Pharmacology (1998) 123, 13–22; doi:10.1038/sj.bjp.0701572
Role of nitric oxide synthase inhibition in the acute hypertensive response to intracerebroventricular cadmium
Authors: Demontis Maria Piera , Varoni Maria Vittoria , Volpe Anna Rita , Emanueli Costanza , Madeddu Paolo
Published: January 2005

In the rat, intracerebroventricular (i.c.v.) injection of cadmium, a pollutant with long biological half‐life, causes a sustained increase in blood pressure at doses that are ineffective by peripheral route. Since cadmium inhibits calcium‐calmodulin constitutive nitric oxide (NO) synthase in cytosolic preparations of rat brain, this mechanism may be responsible for the acute pressor action of this heavy metal.

To test this possibility, we evaluated the effect of i.c.v. injection of 88 nmol cadmium in normotensive unanaesthetized Wistar rats, which were i.c.v. pre‐treated with: (1) saline (control), (2) L‐arginine (L‐Arg), to increase the availability of substrate for NO biosynthesis, (3) D‐arginine (D‐Arg), (4) 3‐[4‐morpholinyl]‐sydnonimine‐hydrochloride (SIN‐1), an NO donor, or (5) CaCl2, a cofactor of brain calcium‐calmodulin‐dependent cNOSI. In additional experiments, the levels of L‐citrulline (the stable equimolar product derived from enzymatic cleavage of L‐Arg by NO synthase) were determined in the brain of vehicle‐ or cadmium‐treated rats.

The pressor response to cadmium reached its nadir at 5 min (43±4 mmHg) and lasted over 20 min in controls. L‐Citrulline/protein content was reduced from 35 up to 50% in the cerebral cortex, pons, hippocampus, striatus, hypothalamus (P<0.01) of cadmium‐treated rats compared with controls. Central injection of NG nitro‐L‐arginine‐methylester (L‐NAME) also reduced the levels of L‐citrulline in the brain.

Both the magnitude and duration of the response were attenuated by 1.21 and 2.42 μmol SIN‐1 (32±3 and 15±4 mmHg, P<0.05), or 1 μmol CaCl2 (6±4 mmHg, P<0.05). Selectivity of action exerted by SIN‐1 was confirmed by the use of another NO donor, S‐nitroso‐N‐acetyl‐penicillamine (SNAP). Both L‐Arg and D‐Arg caused a mild but significant attenuation in the main phase of the pressor response evoked by cadmium. However, only L‐Arg reduced the magnitude of the delayed, pressor response. Despite their similarity in ability to attenuate the cadmium‐induced pressure effect, L‐Arg and its isomer exerted differential biochemical changes in brain L‐citrulline, as L‐Arg normalized cadmium‐induced reduction in L‐citrulline levels, whereas i.c.v. D‐Arg did not.

We conclude that the pressor effect of i.c.v. cadmium is due, at least in part, to reduced NO formation, consequent to inhibition of brain NO synthase. Accumulation of cadmium in the central nervous system could interfere with central mechanisms (including NO synthase) implicated in the regulation of cardiovascular function.

British Journal of Pharmacology (1998) 123, 129–135; doi:10.1038/sj.bjp.0701573