bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of the novel anti‐inflammatory compounds, N‐[2‐(cyclohexyloxy)‐4‐nitrophenyl] methanesulphonamide (NS‐398) and 5‐methanesulphonamido‐6‐(2, 4‐difluorothiophenyl)‐1‐indanone (L‐745, 337), on the cyclo‐oxygenase activity of human blood prostaglandin endoperoxide synthases
Authors: Panara Maria R. , Greco Anita , Santini Giovanna , Sciulli Maria G. , Rotondo Maria T. , Padovano Roberto , di Giamberardino Maria , Cipollone Francesco , Cuccurullo Franco , Patrono Carlo , Patrignani Paola
Published: July 2012

We have evaluated the selectivity of ketoprofen and two novel nonsteroidal anti‐inflammatory drugs, N‐[2‐(cyclohexyloxy)‐4‐nitrophenyl]methanesulphonamide (NS‐398) and 5‐methanesulphonamido‐6‐(2, 4‐difluorothiophenyl)‐1‐indanone (L‐745, 337), in inhibiting the cyclo‐oxygenase activity of prostaglandin endoperoxide synthase‐2 (PGHS‐2) vs PGHS‐1 in human blood monocytes and platelets, respectively.

Heparinized whole blood samples were drawn from healthy volunteers pretreated with aspirin, 300 mg 48 h before sampling, to suppress the activity of platelet PGHS‐1 and incubated at 37°C for 24 h with increasing concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 μg ml−1). Immunoreactive PGE2 levels were measured in plasma by a specific radioimmunoassay as an index of the cyclo‐oxygenase activity of LPS‐induced monocyte PGHS‐2.

The effects of the same inhibitors on platelet PGHS‐1 activity were assessed by allowing whole blood samples, drawn from the same subjects in aspirin‐free periods, to clot at 37°C for 1 h in the presence of the compounds and measuring immunoreactive thromboxane B2 (TXB2) levels in serum by a specific radioimmunoassay.

Under these experimental conditions, ketoprofen enantioselectively inhibited the cyclo‐oxygenase activity of both PGHS‐1 and PGHS‐2 with equal potency (IC50 ratio: approx. 0.5 for both enantiomers), while L‐745, 337 and NS‐398 achieved selective inhibition of monocyte PGHS‐2 (IC50 ratio: > 150). L‐745, 337 and NS‐398 did not affect LPS‐induced monocyte PGHS‐2 biosynthesis to any detectable extent.

We conclude that L‐745, 337 and NS‐398 are selective inhibitors of the cyclo‐oxygenase activity of human monocyte PGHS‐2. These compounds may provide adequate tools to test the contribution of this novel pathway of arachidonate metabolism to human inflammatory disease.
Characteristics of the NANC post‐stimulus (‘rebound’) contraction of the urinary bladder neck muscle in sheep
Authors: Thornbury K.D. , Donaghy K.M. , Peake Jennifer
Published: July 2012

Strips of muscle from sheep bladder neck were set up for tension recording and subjected to electrical field stimulation (EFS) to stimulate their intramural nerves.

In the presence of atropine (1 μm) and guanethidine (1 μm), the response to 1 Hz EFS was biphasic, characterized by a relaxation during the stimulus period, followed by a post‐stimulus contraction. A similar biphasic response was also seen following bolus application of nitric oxide (NO).

In the absence of atropine and guanethidine, the relaxations were masked by contractions during stimulation; however, the post‐stimulus contractions were unaffected. L‐NAME (100 μm) blocked the post‐stimulus contractions and L‐arginine (1 mM) restored them, suggesting that they were NO‐mediated.

M&B 22948, a phosphodiesterase inhibitor, prolonged the relaxations and abolished the post‐stimulus contractions. This suggests that rapid removal of cyclic GMP is required for post‐stimulus contraction to occur.

When the number of pulses in the stimulus train was kept constant, the size of the post‐stimulus contraction increased as the duration of the preceding period of stimulation increased. Maximal post‐stimulus contractions were obtained following stimulation for >40 s.

The L‐channel antagonist, nifedipine (1 μm) and verapamil (1 μm), had little effect on the amplitude of the post‐stimulus contractions. 7 In contrast, ryanodine (8 μm) reduced the post‐stimulus contractions by over 90%. Caffeine (20 mM) also abolished the post‐stimulus contractions and cyclopiazonic acid (CPA, 10 μm) reduced them by 76%. However, in the presence of CPA a slower post‐stimulus contraction developed. Nifedipine (1 μm) reduced this by 40%.

In conclusion, these results support a role for NO in the post‐stimulus contraction of the sheep bladder neck muscle. The post‐stimulus contraction depends more on release of intracellular Ca2+, than Ca2+ influx through L‐type channels.
Roles of endothelin receptors in the regional and systemic vascular responses to ET‐1 in the anaesthetized ganglion‐blocked rat: use of selective antagonists
Authors: Allcock Graham H. , Warner Timothy D. , Vane John R.
Published: July 2012

Endothelin‐1 (ET‐1) produces vasoconstriction, via activation of ETA and ETB receptors on vascular smooth muscle, and vasodilatation via ETB receptors on endothelial cells. Here we have used the ETA receptor‐selective antagonist, BQ‐123, the ETB receptor‐selective antagonist, BQ‐788 and the ETA/ETB receptor non‐selective antagonist, PD 145065, to study the role of these receptors in mediating the haemodynamic changes induced by an infusion of ET‐1 to the anaesthetized ganglion‐blocked rat.

Infusion of ET‐1 (10 pmol kg−1 min−1) increased the mean arterial pressure (MAP) by 57.5 ± 5.1 mmHg over 70 min. This pressor response was reduced by about 50% by coinfusion of BQ‐123 (10 nmol kg−1 min−1), but was unaffected by either BQ‐788 (10 nmol kg−1 min−1) or PD 145065 (10 nmol kg−1 min−1).

After infusion of ET‐1 for 70 min the cardiac output had fallen from 102.6±11.3 to 55.7 ± 7.6 ml min−1 and the total peripheral resistance had increased from 3.24 ± 0.6 to 10.0±0.8 mmHg ml−1 min−1 (per 100g body weight). BQ‐123 decreased the magnitudes of these changes whereas BQ‐788 potentiated them. PD 145065 was without effect.

ET‐1 increased the vascular resistances of all the organs studied except the brain and stomach. These changes were attenuated by BQ‐123 in the kidneys, skin, adrenal glands and caecum and potentiated by BQ‐788 in the kidneys, small intestine, large intestine and mesentery. PD 145065 had little effect on the individual tissues.

Thus, BQ‐123, a selective ETA receptor antagonist, inhibits the pressor and vascular constrictor effects of ET‐1 more actively than PD 145065. As BQ‐788 potentiates some of the vasoconstrictor effects of ET‐1 and increases the effects of ET‐1 on total peripheral resistance, the predominant role of ETB receptors in the rat circulation is to limit the pressor effects of ET‐1.
Comparative behavioural profile of centrally administered tachykinin NK1, NK2 and NK3 receptor agonists in the guinea‐pig
Authors: Piot Odile , Betschart Josette , Grall Isabelle , Ravard Sophie , Garret Claude , Blanchard Jean‐Charles
Published: July 2012

The NK, tachykinin receptor agonists, septide, [Sar9, Met(O2)11]SP and [Pro9]SP produced locomotor hyperactivity (10–20 min) when injected intracerebroventricularly (i.c.v.) in the guinea‐pig. Producedlocomotor The most potent in eliciting this hyperactivity was septide (from 0.63 to 5 μg), compared to [Sar9, Met(O2)11]SP, which was active at 2.5 and 5 μg and [Pro9]SP which induced a non‐significant increase even at 10 μg.

Wet‐dog shakes were elicited by septide, [Sar9, Met(O2)11]SP and [Pro9]SP injected by the i.c.v. route in the guinea‐pig. [Sar9, Met(O2)11]SP, active from 0.16 to 2.5 μg was more potent than septide (active at 1.25 μg) and [Pro9]SP (active at 0.63 μg) in eliciting such behaviour. To a lesser extent, grooming was also observed after injection of these agonists.

The NK2 tachykinin receptor agonist, [Lys5, MeLeu9, Nle10]NKA(4–10), up to the dose of 10 μg i.c.v. had no effect in the guinea‐pig. It neither modified locomotor activity nor induced a characteristic behavioural response. At higher doses (20μg), some toxic effects were noted.

The NK3 tachykinin receptor agonist, senktide, contrasts with the NK1 receptor agonists in that it elicited only wet‐dog shakes, at doses ranging from 0.32 to 1.25 μg. It neither modified locomotor activity (1 pg) nor induced grooming (up to 5 μg) in the guinea‐pig.

To our knowledge, these results are the first demonstration that the guinea‐pig could be useful to differentiate tachykinin agonists on the basis of their behavioural profile, distinct from those obtained in mice and rats.
Selective inhibition by barbiturates of the synthesis of endothelium‐derived hyperpolarizing factor in the rabbit carotid artery
Authors: Lischke Volker , Busse Rudi , Hecker Markus
Published: July 2012

Several lines of evidence suggest that both volatile and intravenous anaesthetics may interfere with the synthesis and release of endothelium‐derived vasoactive factors. We have investigated the effects of three different barbiturates on the release of nitric oxide (NO) and endothelium‐derived hyperpolarizing factor (EDHF) in phenylephrine (1 μm)‐preconstricted, endothelium‐intact ring segments of the rabbit carotid artery. The segments were pretreated with the cyclo‐oxygenase inhibitor, diclofenac (1 μm), to prevent the formation of vasoactive prostanoids, such as prostacyclin (PGI2).

Acetylcholine (ACh) elicited a concentration‐dependent relaxation (EC50 0.15 μm) in control segments which was not significantly different from the relaxant responses of segments pretreated with methohexitone (0.03‐0.3 mM), phenobarbitone (0.1–0.3 mM) or thiopentone (0.1‐0.3 mM).

Inhibition of NO synthesis with NG–nitro‐L‐arginine (0.1 mM) significantly reduced the maximum relaxant response to ACh from 96 to 40%. This NO/PGI2‐independent relaxation appeared to be mediated by the release of EDHF, since it was strongly diminished in the presence of the K+ca inhibitors, tetrabutylammonium (1–3 mM) and charybdotoxin (10 nM), following preconstriction with potassium calcium (40 mM) or removal of the endothelium. Thiopentone or methohexitone markedly attenuated the EDHF‐mediated relaxant response to ACh, while phenobarbitone had no effect. The endothelium‐independent relaxation elicited by sodium nitroprusside (0.01 −10 μm), on the other hand, was only marginally affected by these anaesthetics.

The cytochrome P450 inhibitor, clotrimazole (3–100 μm), mimicked the inhibitory effect of thiopentone and methohexitone on the NO/PGI2‐independent relaxant response to ACh. Moreover the cytochrome P450‐catalyzed O‐dealkylation of 7‐ethoxycoumarin by rabbit liver microsomes was inhibited in the presence of thiopentone or methohexitone (0.3‐1 mM), while phenobarbitone was without effect.

These findings suggest that thiopentone and methohexitone selectively attenuate the EDHF‐mediated relaxant response to ACh in the rabbit carotid artery, presumably by interfering with its synthesis from arachidonic acid via the cytochrome P450 epoxygenase pathway.
NANC neurotransmission in lamina propria of the rabbit urethra: regulation by different subsets of calcium channels
Authors: Zygmunt Pia K.E. , Zygmunt Peter M. , Högestätt Edward D. , Andersson Karl‐Erik
Published: July 2012

Electrical field stimulation (EFS) of the rabbit urethral lamina propria elicited a frequency‐dependent non‐adrenergic, non‐cholinergic (NANC) relaxation, which was abolished by NΩ–nitro‐L‐arginine (l‐NOARG).

Ω‐Conotoxin GVIA, a selective blocker of N‐type voltage‐operated calcium channels (VOCCs), and Ω‐conotoxin MVIIC (blocker of N‐ and Q‐type VOCCs) inhibited the NANC relaxation, and the inhibition was inversely related to the frequency of stimulation. Combined, the two toxins were more effective than co‐conotoxin GVIA alone.

The relaxation induced by the nitric oxide (NO) donor, 3‐morpholino‐sydnonimine (SIN‐1) was not affected by Ω‐conotoxin MVIIC.

Ω‐Agatoxin IVA (blocker of P‐type VOCCs) did not attenuate the NANC relaxation.

Reduction of the calcium concentration from 1.5 to 0.5 mM reduced the NANC relaxation at low but not at high frequencies of stimulation; the relaxation induced by SIN‐1 was not affected.

EFS (20Hz, 30 s) increased the cyclic GMP level 3 fold in normal Krebs solutions, but was unable to enhance significantly the cyclic GMP level after calcium omission. L‐NOARG reduced the cyclic GMP content in ‘calcium‐free’ medium, indicating an ongoing NO synthesis that was independent of extracellular calcium.

Caffeine, ryanodine and thapsigargin (inhibitors of sarcoplasmic calcium release), and CGP 37157 (inhibitor of mitochondrial sodium/calcium exchange) had no effect on the NANC relaxation.

It is suggested that nitrergic nerve activation in the rabbit urethral lamina propria is mediated in part by N‐type (co‐conotoxin GVIA‐sensitive) and in part by Q‐type (co‐conotoxin MVIIC‐sensitive) VOCCs. With high frequences of stimulation, another mechanism, possibly calcium‐independent, appears to become operational.
Modulation of the isoprenaline‐induced membrane hyperpolarization of mouse skeletal muscle cells
Authors: van Mil H.G.J. , Kerkhof C.J.M. , van Heukelom J. Siegenbeek
Published: July 2012

The hyperpolarization of the resting membrane potential, Vm, induced by isoprenaline in the lumbrical muscle fibres of the mouse, was investigated by use of intracellular microelectrodes.

In normal Krebs‐Henseleit solution (potassium concentration: K+o=5.7mM, ‘control’), Vm was −74.0 ± 0.2 mV; lowering K+o to 0.76 mM (′low K+o) resulted in either a hyperpolarization (Vm= −95.7 ±2.9 mV), or a depolarization (Vm= −52.0 ±0.3 mV).

Isoprenaline (≥200 nM) induced a hyperpolarization of Vm by ΔVm= − 5.6 ±0.4 mV in control solution.

When Vm hyperpolarized after switching to low K+o, the addition of isoprenaline resulted in increased hyperpolarization Vm: ΔVm=−16.3±3.2 mV to a final Vm=−110.1 ±3.4 mV. Adding iso‐prenaline when Vm depolarized in low K+o, leads to a hyperpolarization of either by − 11.6±0.5mV to −63.6 ±0.8 mV or by −51.7 ±2.7 mV to −106.9 ±3.9 mV.

Ouabain (0.1 to 1 mm) did not suppress the hyperpolarization by isoprenaline in 5.7 mM K+o (ΔVm=−6.7±0.4mV) or the hyperpolarization of the depolarized cells in low K+o (ΔVm=−9.7 Δ1.5mV).

The hyperpolarization is a logarithmically decreasing function of K+o in the range between 2 and 20 mM (12 mV/decade).

IBMX and 8Br‐cyclic AMP mimicked the response to isoprenaline whereas forskolin (FSK) induced in low K+o a hyperpolarization of −7.0 ±0.7 mV that could be augmented by addition of isoprenaline (ΔVm=−8.2±1.8mV).

In control and low K+o, Ba2+ (0.6 mm) inhibited the hyperpolarization induced by isoprenaline, IBMX or 8Br‐cyclic AMP. Other blockers of the potassium conductance such as TEA (5 mm) and apamin (0.4 μm) had no effect.

We conclude that in the lumbrical muscle of the mouse the isoprenaline‐induced hyperpolarization is primarily due to an increase in potassium permeability.
Behavioural interactions between 5‐hydroxytryptophan, neuroleptic agents and 5‐HT receptor antagonists in modifying rodent responding to aversive situations
Authors: Costall B. , Naylor R.J.
Published: July 2012

The ability of 5‐hydroxytryptophan, 5‐HT2 receptor antagonists and typical and atypical neuroleptic agents to modify behavioural responding to aversive situations was investigated in the mouse light/dark test and rat social interaction.

The administration of 5‐hydroxytryptophan inhibited rat social interaction and the exploratory behaviour of mice in the light/dark test.

The 5‐HT2 receptor antagonists, ketanserin, ritanserin, MDL11939, methysergide and RP62203, the neuroleptic agents, spiperone, haloperidol and benperidol, and the atypical neuroleptic agent, clozapine, when administered alone failed to modify mouse or rat behaviour. In contrast, when administered alone, sulpiride in rats and mice and thioridazine in rats disinhibited behaviour.

Methysergide, RP62203, ketanserin, ritanserin and MDL 11939 antagonized the inhibitory effects of 5‐hydroxytryptophan or reversed the inhibitory effects to one of disinhibition.

Low doses of spiperone (but not haloperidol or benperidol) also antagonized the inhibitory effects of 5‐hydroxytryptophan in the rat but not the mouse. Higher doses of the three neuroleptic agents caused locomotor depression in both rats and mice which obscured any specific changes in behavioural responding to the aversive situations.

The disinhibitory profile of sulpiride in both mice and rats and thioridazine in rats was evident during their interaction with 5‐hydroxytryptophan. Thioridazine in the mouse and clozapine in rats and mice also reversed the inhibitory effects of 5‐hydroxytryptophan to one of disinhibition.

In summary, we present evidence that the atypical neuroleptic agents, thioridazine and clozapine, with their known affinity for the 5‐HT2 receptors, can mimic the actions of reference 5‐HT2 receptor antagonists to antagonize the inhibitory effects of 5‐hydroxytryptophan in rodent models of anxiety. The results are intepreted in terms of drug action on different 5‐HT2 and other 5‐HT receptor subtypes. In addition, thioridazine and sulpiride have disinhibitory effects in their own right which remain to be explained.
Discrimination by benextramine between the NPY‐Y1 receptor subtypes present in rabbit isolated vas deferens and saphenous vein
Authors: Palea S. , Corsi M. , Rimland J.M. , Trist D.G.
Published: July 2012

In order to characterize the neuropeptide Y (NPY) Y1 receptors known to be present in rabbit isolated vas deferens and saphenous vein, the pharmacological activity of the selective NPY Y1 receptor agonists, [Leu31Pro34] NPY and various other peptide agonists, together with the putative NPY antagonist, benextramine, were compared in the two tissues.

In rabbit isolated saphenous vein, cumulative dose‐response curves to various NPY agonists were obtained. All the peptides tested caused contractions which developed quite slowly. The rank order of potency obtained was: PYY>NPY>[Leu31, Pro34] NPY = NPY2–36>hPP>>NPY13–36 = NPYl8‐36. Incubation with benextramine (BXT) at 100 μM for 30min irreversibly abolished the contractile response to [Leu31, Pro34] NPY but was ineffective against NPY18‐3‐induced contractions.

Cumulative dose‐response curves to [Leu31, Pro34] NPY were performed in the same preparation before and after incubation with 100 μM BXT for 20 min in order to inactivate NPY Y1 receptors. The pKA (‐logATA) estimation for [Leu31, Pro34] NPY was 7.60 ±0.30 using the operational model and 7.20 ± 0.33 using the null method; the difference between the two methods was not statistically significant (P = 0.36).

Prostatic segments of rabbit vas deferens were electrically stimulated with single pulses. Immediately after stabilization of the contractile response, a cumulative dose‐response curve to various NPY agonists was obtained in each tissue. The rank order of potency for twitch inhibition was: PYY>[Leu3l, Pro34] NPY ≥ NPY ≥ hPP>NPY2–36gGt;NPY13–36≫NPY18–36 which indicates the presence of a prejunctional NPY Y1 receptor. BXT at 100 μM incubated for 10 or 60 min did not antagonize the response to [Leu3l, Pro34] NPY.

We conclude that rabbit isolated saphenous vein contains a population of post‐junctional NPY Y1 receptors irreversibly blocked by BXT, as well as a population of post‐junctional NPY Y2 receptors, which are insensitive to BXT. In contrast, the rabbit isolated vas deferens express a prejunctional NPY Y1 receptor subtype which is not blocked by BXT. Tetramine disulphides such as BXT could be useful tools in classifying NPY receptors.
Hyperpolarization by GABAB receptor agonists in mid‐brain periaqueductal gray neurones in vitro
Authors: Chieng B. , Christie M.J.
Published: July 2012

The effects of GABAB receptor stimulation on membrane properties of rat periaqueductal gray neurones were studied by use of intracellular recordings from single neurones in superfused brain shoes. Intracellular staining with biocytin was used to characterize the anatomical location of each impaled neurone.

The GABAB receptor agonist, baclofen, directly hyperpolarized or produced an outward current (single electrode voltage‐clamp) in all 66 neurones tested. Baclofen‐induced hyperpolarizations were concentration‐dependent with an EC50 of approximately 0.6 μM and maximum hyperpolarization with 10 μM baclofen. Hyperpolarizations persisted in the presence of tetrodotoxin (1 μM, n = 2).

2‐OH‐saclofen, a selective GABAB receptor antagonist, competitively antagonized baclofen‐induced hyperpolarizations (n = 4) with equihbrium dissociation constants estimated in two neurones to be 6 and 23 μM. Naloxone (1 μM) did not prevent hyperpolarizations induced by baclofen (n = 34).

Hyperpolarizations induced by baclofen were associated with an increased inwardly rectifying potassium conductance. Ba2+ superfusion (5 to 10 mM) blocked this conductance increase (n=4). Elevation of extracellular potassium concentration (from 2.5 to 6.5 mM) shifted the reversal potential in agreement with predictions of the Nerast equation.

Hyperpolarizations produced by baclofen (10 μM) desensitized (>5% inhibition of the maximum response) in 7/22 neurones during continuous superfusion for 5 min. Strong desensitization (>25% inhibition of the maximum response) was observed in only 2/22 neurones in the ventrolateral periaqueductal gray. In contrast 6/9 neurones in the laterodorsal tegmental nucleus displayed strong desensitization.

These studies demonstrate that baclofen acting on GABAB receptors increases potassium conductance in all lateral and ventrolateral periaqueductal gray neurones. The neurones hyperpolarized by baclofen are likely to be involved in the well‐established antinociceptive actions of baclofen in the ventrolateral periaqueductal gray, but might also be involved in other functions because many of them lie outside the main ‘antinociceptive’ zone of this region. The cellular mechanisms underlying baclofen‐induced antinociception presumably differ from the postulated antinociceptive action of opioids, thought to be mediated via disinhibition of periaqueductal gray neurones which project to the ventromedial medulla.
α1A‐Adrenoceptor‐mediated contractile responses of the human vas deferens
Authors: Furukawa K. , Rosario D.J. , Smith D.J. , Chappie C.R. , Uchiyama T. , Chess‐Williams R.
Published: July 2012

The predominant α‐adrenoceptor mediating contractions of the human vas deferens has been characterised in vitro by use of subtype selective antagonists.

Responses of human epididymal vas deferens were obtained to phenylephrine in the presence of amine uptake inhibitors and propranolol. The effects of the α1‐adrenoceptor antagonists, 5‐methylurapidil, oxymetazoline, WB4101, prazosin and chloroethylclonidine were examined and also the L‐type calcium channel blocker, nifedipine.

5‐Methylurapidil, WB4101, oxymetazoli and prazosin acted as competitive antagonists of the responses to phenylephrine, yielding pA2 values of 8.8, 9.2, 7.7 and 8.8 respectively. All four antagonists produced Schild plots with slopes similar to unity and maximum responses to phenylephrine were not altered in the presence of any of the antagonists.

Tamsulosin (1 nM) caused rightward shifts of phenylephrine concentration‐response curves yielding an apparent pKB value of 10.0. However, maximum responses were also reduced by 51% with this concentration of antagonist.

Incubation of tissues with chloroethylclonidine (100 μM for 40 min) failed to alter responses significantly but the presence of nifedipine (1 μM) reduced maximum responses to phenylephrine by 32%.

The high affinity of 5‐methylurapidil, oxymetazoline and WB4101, together with the failure of chloroethylclonidine to antagonize responses, indicate that the predominant α‐adrenoceptor mediating contraction of the human vas deferens has the characteristics previously described for the pharmacologically‐defined α1A‐adrenoceptor. The data are also consistent with those described for the cloned α1C‐adrenoceptor subtype thereby supporting the hypothesis that the two receptors are identical. The human vas deferens therefore represents a readily accessible preparation for functional studies of the human α1A‐adrenoceptor.
Selectivity of the imidazoline α‐adrenoceptor agonists (oxymetazoline and cirazoline) for human cloned α1‐adrenoceptor subtypes
Authors: Hone Kuniko , Obika Kenji , Foglar Rudolf , Tsujimoto Gozoh
Published: July 2012

To investigate the structure‐activity relationships of α‐adrenoceptor agonists for the α1‐adrenoceptor subtypes*, we have compared the imidazoline class of compounds, oxymetazoline and cirazoline, with the phenethylamine, noradrenaline, in their affinities and also in their intrinsic activities in Chinese hamster ovary (CHO) cells stably expressing the cloned human α1‐adrenoceptor subtypes (α1a‐, α1b‐, and α1d‐subtypes)

Radioligand binding studies with [125I]‐HEAT showed that cirazoline and oxymetazoline had higher affinities at α1a‐subtype than at α1b‐ and (α1d‐subtypes, while noradrenaline had higher affinity at the α1d‐subtype than at α1a‐ and α1b‐subtypes.

In functional studies, cirazoline caused transients of cytosolic Ca2+ concentrations ([Ca2+] response) in a concentration‐dependent manner and developed a maximal response similar to that to noradrenaline in CHO cells expressing the α1a‐subtype, while it acted as a partial agonist at α1b‐ and α1d‐adrenoceptors. Oxymetazoline, on the other hand, was a weak agonist at α1a‐adrenoceptors, and has no intrinsic activity at the other subtypes.

Using the phenoxybenzamine inactivation method, the relationships between receptor occupancy and noradrenaline‐induced [Ca2+]i response for α1a‐ and α1d‐subtypes were found to be linear, whereas it was moderately hyperbolic for the α1b‐subtype, indicating the absence of receptor reserves in CHO cells expressing α1a‐ and α1d‐subtypes while there exists a small receptor reserve for CHO cells expressing the α1b‐subtype.

In summary, our data obtained in cells exclusively expressing a single receptor subtype support the idea that the relative role of agonist affinity and intrinsic activity may vary depending on the subtype of α1‐adrenoceptor.
Actions of ADP, but not ATP, on cytosolic free Ca2+ in single rat hepatocytes mimicked by 2‐methylthioATP
Authors: Dixon C. Jane , Cobbold Peter H. , Green Anne K.
Published: July 2012

Aequorin‐injected, single rat hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway, including ADP and ATP. We have previously described differences in the [Ca2+]i responses of aequorin‐injected hepatocytes to ADP and ATP.

The effects of the phosphorothioate analogue of ATP, 2‐methylthioATP (2‐meSATP), have been examined on single rat hepatocytes. This analogue is believed to be the most potent agonist at the P2Y1 subclass of purinoceptor.

The [Ca2+]i transients induced by 2‐meSATP were indistinguishable from those induced by ADP, and in contrast to those induced by ATP.

At high concentrations, 2‐meSATP and ADP both induced transients at high frequency. In contrast, hepatocytes responded to high concentrations of ATP with an initial rapid rise in [Ca2+]i, followed by a slowly decaying fall.

The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both 2‐meSATP‐ and ADP‐induced [Ca2+]i transients; the peak height and frequency of transients were enhanced. ATP‐induced transients, however, underwent either an increase in duration or conversion into a sustained rise in [Ca2+]i.

ATP‐induced transients were specifically potentiated by the co‐addition of α,β‐methyleneATP, whereas 2‐meSATP‐ and ADP‐induced transients were unaffected by this treatment.

We conclude that 2‐meSATP acts at the same receptor as ADP on rat hepatocytes, and that this is distinct from the receptor(s) mediating the effects of ATP.
Study of NO and VIP as non‐adrenergic non‐cholinergic neurotransmitters in the pig gastric fundus
Authors: Lefebvre R.A. , Smits G.J.M. , Timmermans J.‐P.
Published: July 2012

The contribution of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) to non‐adrenergic non‐cholinergic (NANC) relaxations in the pig gastric fundus was investigated.

Circular and longitudinal muscle strips were mounted for isotonic registration in the presence of atropine and guanethidine; tone was raised with 5‐hydroxytryptamine. Electrical field stimulation with 10 s trains at 5 min intervals induced short‐lasting, frequency‐dependent relaxations while continuous stimulation with cumulative increase of the stimulation frequency induced sustained frequency‐dependent relaxations in both types of strips; the electrically induced responses were abolished by tetrodotoxin.

The short‐lasting as well as the sustained electrically induced NANC relaxations were significantly reduced by NG‐nitro‐L‐arginme methyl ester (l‐NAME). Pretreatment with L‐arginine but not D‐arginine, prevented the inhibitory effect of L‐NAME except for sustained relaxations in the longitudinal muscle strips.

Sodium nitroprusside, forskolin, zaprinast and 3‐isobutyl‐l‐methylxanthine induced concentration‐dependent relaxations. Exogenous NO mimicked the short‐lasting electrically induced relaxations, while endogenous VIP evoked sustained relaxations. The responses to exogenous NO and VIP were not influenced by tetrodotoxin and L‐NAME.

α‐Chymotrypsin abolished the responses to exogenous VIP but only moderately reduced NANC relaxations induced by continuous electrical stimulation. Zaprinast potentiated the relaxant responses to sodium nitroprusside and increased the duration of the NANC relaxations induced by electrical stimulation with 10 s trains in circular muscle strips but not in longitudinal muscle strips.

The cyclic GMP and cyclic AMP response to electrical stimulation, NO and VIP was measured in circular muscle strips. Short‐lasting as well as sustained electrical field stimulation induced an approximately 1.5 fold increase in cyclic GMP content, while NO induced nearly a 40 fold increase. An increase in cyclic AMP content was obtained only with sustained electrical field stimulation.

Immunocytochemistry for NO synthase (NOS) revealed immunoreactive neuronal cell bodies in the submucous and myenteric plexuses and nerve fibres in both the circular and longitudinal muscle layer; double‐labelling for NOS and VIP showed that VIP coexists in a major part of the intrinsic neurones. NADPH diaphorase‐histochemistry showed the same pattern of nitrergic neurones and nerves as NOS‐immunocytochemistry.

It is concluded that a cyclic GMP‐ and a cyclic AMP‐dependent pathway for relaxation is present in both the circular and longitudinal muscle layer of the pig gastric fundus. NO appears to contribute to short‐lasting as well as sustained NANC relaxations. A peptide, possibly VIP, may be involved during sustained stimulation at lower frequencies of stimulation.
Reflex circulatory collapse following intrapulmonary entrapment of activated platelets: mediation via 5‐HT3 receptor stimulation
Authors: Leañios O.L. , Hong E. , Amezcua J.L.
Published: July 2012

The role of 5‐HT2 and 5‐HT3 receptors in the mediation of direct and reflex vascular responses to intrapulmonary platelet activation was investigated.

Anaesthetized rabbits were challenged intravenously with an emulsion of autologous bone marrow that produced a sharp increase in pulmonary blood pressure, a fall in systemic blood pressure, platelet consumption and death.

Platelet depletion before the challenge nearly abolished all the cardiovascular effects and prevented death. Bilateral vagotomy prevented the fall in systemic blood pressure and death but did not prevent the increase in pulmonary pressure. The intravenous administration of the 5‐HT3 antagonist, MDL‐72222, resembled the protection afforded by vagotomy whereas the 5‐HT2 antagonist, ketanserin, only reduced the increase in pulmonary pressure without affecting the systemic response or mortality.

The effects of intravenous 5‐HT and of electrical stimulation of the cephalic ends of the cut vagi nerves were also explored. 5‐HT injection increased the pulmonary vascular pressure but its effects on systemic blood pressure were variable. These responses were modified by the 5‐HT antagonists in a manner that resembles their effects on bone marrow embolism. Afferent vagal stimulation produced a fall in systemic blood pressure that was not prevented by MDL‐72222.

This study indicates that a centrally mediated reduction of peripheral vascular tone is the cause of the potentially lethal circulatory collapse that follows the intrapulmonary entrapment of activated platelets. This reflex is initiated by the action of 5‐HT on 5‐HT3 receptors in the lung.
Locomotor activation and dopamine release produced by nicotine and isoarecolone in rats
Authors: Whiteaker P. , Garcha H.S. , Wonnacott S. , Stolerman I.P.
Published: July 2012

Isoarecolone was approximately 250 times less potent than nicotine as an inhibitor of [3H]‐nicotine binding to rat brain membranes. Isoarecolone failed to inhibit the binding of the nicotinic ligand [125I]‐α‐bungarotoxin or of the muscarinic ligand [3H]‐QNB.

Nicotine (0.01–30 μm) evoked the release of [3H]‐dopamine from striatal and frontal cortex synaptosomes, with EC50 values of approximately 0.5 μM in each case. This release was largely mecamylamine‐sensitive.

Isoarecolone (1–200 μm) evoked predominantly mecamylamine‐sensitive dopamine release from both striatal and cortical synaptosomes, with a potency at least 20 times less than that of nicotine. The maximum effect of isoarecolone was less than that of nicotine, particularly in the frontal cortex preparation.

In control rats treated chronically with saline, neither nicotine nor isoarecolone had clear effects on locomotor activity at the doses tested. Chronic treatment with nicotine clearly sensitized rats to the locomotor activating effect of nicotine; there was only weak cross‐sensitization to isoarecolone. The maximum effect of isoarecolone was seen at a dose about 40 times larger than that of nicotine.

The low potency and efficacy of isoarecolone in facilitating sensitized locomotor activity resembled its lower potency and efficacy, compared with nicotine, in evoking dopamine release in vitro. The agonist profile of the nicotinic receptor population mediating dopamine release may determine the pharmacological characteristics of consequent locomotor behaviour.
Enhancing effect of staurosporine on NO production in rat peritoneal macrophages via a protein kinase C‐independent mechanism
Authors: Sowa Grzegorz , Przewłocki Ryszard
Published: July 2012

Staurosporine (3–100 nM), frequently used as a protein kinase C (PKC) inhibitor, increased accumulation of nitrite in the culture medium of rat peritoneal macrophages up to 6 times above the control level. Moreover, when used in combination with the stable analogue of cyclic AMP, dibutyryl‐cyclic AMP (db cyclic AMP; 0.1 mM), and/or a cytokine, tumour necrosis factor‐α (TNFα; 100 u ml−1), staurosporine synergistically potentiated, up to 30 times, nitrite accumulation. On the other hand, the other PKC inhibitors, calphostin C and H‐7 (10 nM‐10 μm) were not effective under the same conditions. The staurosporine‐induced nitrite accumulation, in both the presence and the absence of TNFα and/or db cyclic AMP was effectively inhibited by the protein synthesis inhibitor, cycloheximide, or by the nitric oxide (NO) synthesis inhibitor, NG‐monomethyl‐L‐arginine (L‐NMMA). Thus our data suggest that staurosporine may enhance NO production in macrophages via intracellular mechanisms unrelated to the PKC inhibition.
Characterization of the thromboxane (TP‐) receptor subtype involved in proliferation in cultured vascular smooth muscle cells of rat
Authors: Ko Feng‐Nien , Yu Sheu‐Meei , Kang Ya‐Fei , Teng Che‐Ming
Published: July 2012

, Jen‐Ai Rd., 1st Section, Taipei, Taiwan

The effects of the thromboxane A2 (TxA2)‐mimetic, U‐46619, on the proliferation of vascular smooth muscle cells (VSMCs) were examined in a clonal smooth muscle cell line, A10, which was derived from foetal rat aorta

[3H]‐U‐46619 bound to A10 cells of passages 18–20 (pl8‐20) with two classes of sites. The high affinity site showed a Bmax of 3.0 ±1.8 fmol mg−1 protein with a KD value 1.0 ±0.1 nM, while the low affinity site showed a Bmaxx of 43.0 ±6.0 fmol mg protein and KD value of 129.0 ±7.9 nM. However, [3H]‐U‐46619 bound to A10 cells from passages 28–30 (p28‐30) at a single class of site with a Bmax 111.0 ±9.0 fmol mg−1 protein and a Ku value of 175.4 ±22.0 nM

Cinnamophilin and SQ29548 inhibited specific [3H]‐U‐46619 binding to pl8‐20 A10 cells in a concentration‐dependent manner with Ki values of 390.0 ±3.2 and 4.6 ±1.0 nM, respectively at a high affinity site, and 2.6 ±0.2 μm and 310.0 ±6.4 nM, respectively at the low affinity site

U‐46619 produced isometric contractions of rat aorta in a concentration‐dependent manner with an EC50 7.0 ±1.2 nM. Cinnamophilin and SQ29548 antagonized U‐46619‐induced aortic contractions with pA2 values 6.3 ±0.1 and 8.2 ±0.2, respectively

U‐46619 increased [3H]‐thymidine incorporation into DNA of pi8‐20 and p28‐30 A10 cells in a concentration‐dependent manner with EC50 values 362.7 ±27.0 and 302.5 ±20.1 nM, respectively. The U‐46619‐induced increase of [3H]‐thymidine incorporation into DNA of p28‐30 A10 cells was potentiated by PDGF (1 ng ml−1) and FCS (1%) and was inhibited by cinnamophilin (10 μm) and SQ29548 (1 μm) with estimated pKB values 5.4 ±1.2 and 6.3 ±0.9, respectively

Cell cycle analysis revealed that U‐46619‐increased cell cycle progression was primarily due to a rapid transition from the DNA synthetic (S) to the G2/mitotic (M) phase. Moreover, U‐46619 also increased protein synthesis and cell numbers in VSMC. All these effects of U‐46619 were inhibited by cinnamophilin and SQ29548

U‐46619 caused phosphoinositide breakdown and increased the intracellular Ca2+ concentration in VSMC, effects which were blocked by cinnamophilin and SQ29548

These data indicate there are two U‐46619 binding sites in A10 VSMC. The high affinity site is correlated to U‐46619‐induced vasoconstriction while the low affinity site is correlated to U‐46619‐mediated VSMC proliferation. These data also reveal that U‐46619 stimulates the cell cycle progression in VSMC primarily through a rapid transition from S to G2/M. Since cinnamophilin inhibits TP‐receptor‐mediated VSMC proliferation, it may thus hold promising potential for the prevention of atherosclerosis or vascular diseases.
Effects of nicorandil on the recovery of reflex potentials after spinal cord ischaemia in cats
Authors: Suzuki Toshio , Sekikawa Toshihiko , Nemoto Tetsuharu , Moriya Hideshige , Nakaya Haruaki
Published: July 2012

The pathophysiological significance of ATP‐sensitive K+ (KATP) channels in the central nervous system is not fully understood. In this study the effects of nicorandil (a hybrid vasodilator having a dual mechanism of action as a K+ channel opener and a nitrate) on the recovery of the spinal cord reflex potentials after spinal cord ischaemia were examined and compared with those of pinacidil and nitroprusside in anaesthetized spinal cats

Spinal cord ischaemia was produced by occlusion of the thoracic aorta and the bilateral internal mammary arteries for 10 min. Regional blood flow in the spinal cord was continuously measured with a laser‐Doppler flow meter. The monosynaptic (MSR) and polysynaptic reflex (PSR) potentials, elicited by electrical stimulation of the tibial nerve, were recorded from the lumbo‐sacral ventral root. The recovery process of spinal reflex potentials was reproducible when the occlusion was repeated twice at an interval of 120 min

Pretreatment with nicorandil (30–100 μg kg−1) accelerated the recovery of PSR potentials after spinal cord ischaemia. Such an accelerating effect on the recovery of PSR potentials was also shared by pinacidil (100 μg kg−1), another K+ channel opener. In addition, the accelerating effect of nicorandil (100 μg kg−1) on the recovery of PSR potentials was abolished by co‐administration of glibenclamide (3 mg kg−1), a sulphonylurea KATP channel blocker. Nitroprusside (8 μg kg−1 min−1) retarded rather than improved the recovery of PSR potentials after spinal cord ischaemia. AH of these drugs failed to improve the spinal cord blood flow during ischaemia and reperfusion

These results suggest that nicorandil promotes the recovery of polysynaptic reflex potentials after spinal cord ischaemia by opening the KATP channels of neurones rather than by increasing local blood flow. K+ channel openers may exert a salutary effect on the functional recovery of the ischaemic spinal cord.
Characterization of the effect of SR48692 on inositol monophosphate, cyclic GMP and cyclic AMP responses linked to neurotensin receptor activation in neuronal and non‐neuronal cells
Authors: Oury‐Donat F. , Thurneyssen O. , Gonalons N. , Forgez P. , Gully D. , Fur G. Le , Soubrie P.
Published: July 2012

Neurotensin stimulated inositol monophosphate (IP1) formation in both human colonic carcinoma HT29 cells and in mouse neuroblastoma N1E115 cells with EC50 values of 3.5 ± 0.5 nM (n = 4) and 0.46±0.02 nM (n = 3), respectively. Neurotensin also stimulated cyclic GMP production with an EC50 of 0.47±1.2 nM and inhibited cyclic AMP accumulation induced by forskolin (0.5 μm) with an IC50 of 1.33 + 1.5 nM (n = 3) on the N1E115 cell line

The competitive antagonism by the non‐peptide neurotensin receptor antagonist, SR48692 of neurotensin‐induced IP1 formation revealed pA2 values of 8.7 ±0.2 (n = 3) for HT29 and 10.1 ±0.2 (n = 3) for N1E115 cells. SR48692 also antagonized the cyclic GMP and cyclic AMP responses induced by neurotensin in the N1E115 cell line with pA2 values of 10.7±0.7 (n = 3) and 9.8±0.3 (n = 3), respectively

In CHO cells transfected with the rat neurotensin receptor, neurotensin stimulated IP1 and cyclic AMP formation with EC50 values of 3.0±0.5 nM (n = 3) and 72.2 ±20.7 nM (n = 3), respectively. Both effects were antagonized by SR48692, giving pA2 values of 8.4 ±0.1 (n = 3) for IP1 and 7.2 ±0.4 (n = 3) for cyclic AMP responses

Radioligand binding experiments, performed with [125I]‐neurotensin (0.2 nM), yielded IC50 values of 15.3 nM (n = 2) and 20.4 nM (n = 2) for SR48692 versus neurotensin receptor binding sites labelled in HT29 and N1E115 cells, respectively

In conclusion, SR48692 appears to be a potent, species‐independent antagonist of the signal transduction events triggered by neurotensin receptor activation in both neuronal and non‐neuronal cell systems.
Diadenosine tetraphosphate‐induced inhibition of ATP‐sensitive K+ channels in patches excised from ventricular myocytes
Authors: Jovanovic Aleksandar , Terzic Andre
Published: July 2012

Diadenosine 5′,5′‐P1, P4‐tetraphosphate (AP4A) has been termed ‘alarmone’ due to its role in intracellular signaling during metabolic stress. It is not known whether Ap4A could modulate ATP‐sensitive K+ (KATP) channels, a family of channels regulated by the metabolic status of a cell. We applied the single‐channel patch‐clamp technique to measure the effect of Ap4A on KATP channels. When applied to the intracellular side of patches, excised from guinea‐pig ventricular myocytes, Ap4A inhibited KATP channel activity, in a reversible and concentration‐dependent (half‐maximal concentration ∼ 17 μm) manner. We conclude that Ap4A, a naturally occurring diadenosine polyphosphate, is actually an inhibitor of the myocardial KATP channel.
In vitro and in vivo profile of SB 206553, a potent 5‐HT2C/5‐HT2B receptor antagonist with anxiolytic‐like properties
Authors: Kennett G.A. , Wood M.D. , Bright F. , Cilia J. , Piper D.C. , Gager T. , Thomas D. , Baxter G.S. , Forbes I.T. , Ham P. , Blackburn T.P.
Published: July 2012

SB 206553 (5‐methyl‐1‐(3‐pyridylcarbamoyl)‐1,2,3,5‐tetrahydropyrrolo[2,3‐f]indole) displays a high affinity (pK1 7.9) for the cloned human 5‐HT2C receptor expressed in HEK 293 cells and the 5‐HT2B receptor (pA2 8.9) as measured in the rat stomach fundus preparation. SB 206553 has low affinity for cloned human 5‐HT2A receptors expressed in HEK 293 cells (pK1 5.8) and (pK1 <6) for a wide variety of other neurotransmitter receptors.

SB 206553 appears to be a surmountable antagonist of 5‐HT‐stimulated phosphoinositide hydrolysis in HEK 293 cells expressing the human 5‐HT2C receptor (pKB 9.0).

The compound potently (ID50 5.5 mg kg−1, p.o., 0.27 mg kg−1, i.v.) inhibited the hypolocomotor response to m‐chlorophenylpiperazine (mCPP), a putative model of 5‐HT2C/5‐HT2B receptor function in vivo.

At similar doses (2–20 mg kg−1, p.o.) SB 206553 increased total interaction scores in a rat social interaction test and increased punished responding in a rat Geller‐Seifter conflict test. These effects are consistent with the possession of anxiolytic properties.

SB 206553 also increased suppressed responding in a marmoset conflict model of anxiety at somewhat higher doses (15 and 20 mg kg−1, p.o.) but also reduced unsuppressed responding.

These results suggest that SB 206553 is a potent mixed 5‐HT2C/5‐HT2B receptor antagonist with selectivity over the 5‐HT2A and all other sites studied and possesses anxiolytic‐like properties.
Cocaine‐induced microvascular vasoconstriction but differential systemic haemodynamic responses in Yucatan versus Yorkshire varieties of swine
Authors: Miao Lin , Núñez Boris D. , Susulic Vojken , Wheeler Suzanne , Carrozza Joseph P. , Ross James N. , Morgan James P.
Published: July 2012

Systemic and coronary haemodynamics were measured in 6 Yorkshire swine and 6 Yucatan miniature swine under isoflurane anaesthesia to investigate the influence of cocaine following its intravenous administration at 1, 3 and 7 mg kg−1.

Cocaine in Yorkshire swine decreased mean arterial pressure and rate pressure product (systolic pressure × heart rate), suggesting a cardiac depressant effect, whereas cocaine in Yucatan miniature swine increased these parameters, consistent with a hyperadrenergic state.

Cocaine in both Yorkshire swine and Yucatan miniature swine decreased coronary blood flow and coronary flow reserve, and increased coronary vascular resistance.

A modest generalized epicardial coronary artery constriction was observed by angiography, without evidence of focal spasm.

Our results confirm a marked vasoconstrictor effect of cocaine on the coronary arterial circulation, predominantly distal to the epicardial coronary arteries, but also indicate important differences in the systemic cardiovascular responses to the drug between two closely related strains of animals within the same species. Due to the similarities between the swine and human coronary arterial vasculature, we suggest that vasoconstriction in the coronary microcirculation may produce cardiac toxicity in man.