bluuebunny/crossref_metadata_2025 huggingface dataset:
The mechanism of the inhibitory effect of polyamines on the induction of nitric oxide synthase: role of aldehyde metabolites
Authors: Szabó Csaba , Southan Garry J. , Thiemermann Christoph , Vane John R.
Published: July 2012

We have recently found that in the presence, but not in the absence, of foetal calf serum, spermine inhibits the production of nitric oxide (NO) in cultured J774.2 macrophages stimulated with bacterial endotoxin (lipopolysaccharide; LPS) or with 7‐interferon (IFN), showing that polyamines may act as suppressants of NO‐mediated immune functions. Here, we have studied the mechanisms and the specificity of this inhibitory action.

Other polyamines, as well as spermine, inhibit the formation of NO in cultured J774.2 macrophages, with the order of potency being spermine > spermidine > > putrescine = cadaverine. This inhibition of NO formation is not due to any cytotoxic effect of these agents for they neither reduced mitochondrial respiration nor increased the release of lactate dehydrogenase into the supernatant.

Spermine is not a direct inhibitor of the activity of iNOS in induced J774.2 cells as measured by its lack of effect on the conversion of L‐arginine to L‐citrulline in homogenates. Neither spermine, nor its metabolites, interfere with the production of nitrite from NO or act as scavengers of NO. Thus, spermine is an inhibitor of the induction of iNOS.

Spermine inhibits nitrite formation in the presence of foetal, newborn or adult bovine serum, but not rat or human serum.

The effect of sper mine on nitrite production can be prevented by isoniazid, hydrazine or hydroxy‐lamine, inhibitors of spermine oxidase, as well as by phenylhydrazine, an aldehyde inhibitor. We have, therefore, tested the effects of spermine dialdehyde or malon dialdehyde on the induction of iNOS. Spermine dialdehyde (SDA, 10−5m) inhibits nitrite formation by IFN‐activated J774.2 cells in the absence of serum when given as a pretreatment but not when given 6 h after stimulation. In contrast, malon dialdehyde was ineffective. Thus, aldehyde metabolites of spermine, such as SDA, account for the inhibitory effect of polyamines on the induction of NOS in vitro.

The inhibitory effect of polyamines on iNOS induction appears to be fairly specific to iNOS, for spermine does not inhibit LPS‐induced production of prostaglandin F2α or tumour necrosis factor.
Contribution of intra‐ and extracellular Ca2+ to noradrenaline exocytosis induced by ouabain and monensin from guinea‐pig vas deferens
Authors: Katsuragi Takeshi , Ogawa Shusuke , Furukawa Tatsuo
Published: July 2012

Contributions of intra‐ and extracellular Ca2+ to noradrenaline (NA) release evoked by increasing intracellular Na+ concentrations (ouabain plus monensin) from adrenergic nerves of guinea‐pig vas deferens were evaluated under conditions eliminating carrier‐mediated NA release (with 100 μm cocaine).

Ouabain (100 μm) plus monensin (10 μm), unlike 100 mm KCl, produced a marked NA release which was unchanged by Ca2+‐removal.

In normal solution but not in Ca2+‐free solution, the release of NA evoked by ouabain plus monensin was reduced by adenosine, clonidine and neuropeptide Y, and by Ca2+‐channel blockers such as ω‐conotoxin GVIA and nifedipine. The release of NA was also decreased by cromakalim in a glibenclamide‐sensitive fashion.

In contrast, in the absence but not in the presence of Ca2+, the drug‐evoked NA release was inhibited by mitochondrial inhibitors (carbonylcyanide‐m‐chlorophenylhydrazone and olivomycin) and further by immobilizers of intracellular Ca2+ (TMB‐8 and BAPTA‐AM) and calmodulin antagonists (W‐7 and trifluoperazine).

These findings suggest that the release of NA evoked by elevation of [Na+1 from adrenergic nerves in the presence and absence of Ca2+ involves, in part, exocytotic processes which are triggered by depolarization‐induced Ca2+ influx and by utilization of Ca2+ from intracellular Ca2+ store sites such as mitochondria, respectively.
Evidence for P2‐purinoceptor‐mediated inhibition of noradrenaline release in rat brain cortex
Authors: von Kügelgen Ivar , Späth Leni , Starke Klaus
Published: July 2012

Some postganglionic sympathetic axons possess P2Y‐like P2‐purinoceptors which, when activated, decrease the release of noradrenaline. We examined the question of whether such receptors also occur at the noradrenergic axons in the rat brain cortex. Slices of the brain cortex were preincubated with [3H]‐noradrenaline, then superfused with medium containing desipramine (1 μm) and stimulated electrically, in most experiments by trains of 4 pulses/100 Hz.

The selective adenosine A1‐receptor agonist, N6‐cyclopentyl‐adenosine (CPA; 0.03‐3 μm) as well as the non‐subtype‐selective agonist 5′‐N‐ethylcarboxamido‐adenosine (NECA; 0.3‐3 μm) reduced the evoked overflow of tritium, whereas the adenosine A2a‐receptor agonist, 2‐p‐(2‐carbonylethyl)‐phenethylamino‐5′‐N‐ethylcarboxamido‐adenosine (CGS‐21680; 0.003–30 μm) and the adenosine A3‐receptor agonist N6‐2‐(4‐aminophenyl)ethyl‐adenosine (APNEA; 0.03‐3 μm) caused no change. Of the nucleotides tested, ATP (30–300 μm), adenosine‐5′‐0‐(3‐thiotriphosphate) (ATP7S; 30–300 μm), adenosine‐5′‐0‐(2‐thiodiphosphate) (ADPγS; 30–300 μm), P1, P4‐di(adenosine‐5−)‐tetraphosphate (Ap4A; 30–300 μm) and the preferential P2Y‐purinoceptor agonist, 2‐methylthio‐ATP (300 μm) decreased the evoked overflow of tritium. The P2X‐purinoceptor agonist, α,β‐methylene‐ATP (3–300 μm) caused no change.

The A1‐selective antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX; 10 nm) attenuated the effects of the nucleosides CPA (apparent pKB value 9.8) and NECA as well as of the nucleotides ATP (apparent pKB 9.3), ATP7S (apparent pKB 9.2) and ADPβS (apparent pKB 8.7). CGS‐21680 and APNEA were ineffective also in the presence of DPCPX. The A2‐selective antagonist 1,3‐dipropyl‐8‐(3,4‐dimethoxystyryl)‐7‐methylxanthine (KF‐17837) reduced the effects of CPA, NECA and ATPγS only when given at a concentration of 300 nm but not at 10 nm.

The P2‐purinoceptor antagonists, suramin (300 μm), reactive blue 2 (30 μm) and cibacron blue 3GA (30 μm) did not change the effect of CPA. Suramin and cibacron blue 3GA shifted the concentration‐response curve of ATPγS to the right (apparent pKB values 3.7 and 5.0, respectively). Reactive blue 2 also attenuated the effect of ATPγS, and cibacron blue 3GA attenuated the effect of ATP, but in these cases the agonist concentration‐response curves were not shifted to the right. There was no antagonistic effect of suramin against ATP and ADPβS.

The results indicate that rat cerebrocortical noradrenergic axons possess, in addition to the known adenosine A1‐receptor, a separate purinoceptor for nucleotides (P2) which, in contrast to the A1‐receptor, is blocked by suramin, reactive blue 2 and cibacron blue 3GA. Nucleotides such as ATP and ATPγS activate both receptors. Inconsistencies in antagonist effects against nucleotides are probably due to this activation of two receptors. The presynaptic P2‐purinoceptor is P2Y‐like, as it is in the peripheral sympathetic nervous system.
Effects of dihydropyridine calcium antagonists on rat midbrain dopaminergic neurones
Authors: Mercuri Nicola B. , Bond Antonello , Calabresi Paolo , Stratta Francesca , Stefani Alessandro , Bernardi Giorgio
Published: July 2012

The effects of the dihydropyridine calcium channel antagonists, nifedipine and nimodipine (300 nm‐30 μm) were tested in vitro on intracellularly recorded dopaminergic neurones in the rat ventral mesencephalon.

Bath applied nifedipine and nimodipine inhibited in a concentration‐dependent manner the spontaneous firing discharge of the action potentials, whereas, the dihydropyridine calcium channel agonist, Bay K 8644 increased the firing rate.

Pacemaker oscillations and bursts of action potentials were produced by loading the cells with caesium. Nifedipine and nimodipine reduced the rate and the duration of the caesium‐induced membrane oscillations and decreased the number of action potentials in a burst. During the blockade of potassium currents the dopaminergic neurones often developed a prolonged (100–800 ms) afterdepolarization that was also inhibited by dihydropyridines.

The spontaneous discharge of calcium spikes was also inhibited by both dihydropyridine calcium antagonists. The apparent input resistance and the level of membrane potential were not affected by the dihydropyridine calcium antagonists.

If the action potential duration was less than 150 ms the shape of the spike was not clearly influenced by both calcium antagonists. However, when the duration of the action potential was longer than 150–200 ms due to the intracellular injection of caesium ions plus the extracellular application of tetraethylammonium (10–50 μm), both nifedipine and nimodipine reversibly shortened the plateau potential.

It is suggested that nifedipine and nimodipine depress the rhythmic and bursting activity of the dopaminergic cells and shorten the calcium action potential by blocking dihydropyridine‐sensitive high‐threshold calcium currents.
Characterization of P2‐purinoceptors in the smooth muscle of the rat tail artery: a comparison between contractile and electrophysiological responses
Authors: Evans R.J. , Kennedy C.
Published: July 2012

The electrophysiological actions of the P2‐purinoceptor agonists, adenosine 5′‐triphosphate (ATP), 2‐methylthioATP (2‐meSATP), α,β‐methyleneATP (α,β‐meATP) and uridine 5′‐triphosphate (UTP) were studied under concentration and voltage‐clamp conditions in acutely dissociated rat tail artery smooth muscle cells. For comparison, their actions as vasoconstrictors were studied in intact ring preparations.

Rapid application of ATP (100 nm‐1 μm) via a U‐tube supervision system activated concentration‐dependent inward currents with a latency to onset of less than 3 ms. The inward current decayed by more than 95% during a 2 s application of 300 nm and 1 μm ATP.

2‐meSATP (100nM‐1 μm) and α,β‐meATP (100 nm‐1 μm) also evoked transient inward currents. The agonist order of potency was ATP = 2‐meSATP ≥ α,β‐meATP. UTP (300nM‐1μm) did not produce a change in the holding current.

A second application of ATP (300 nm and 1 μm) 10 min after the first, evoked currents which were one third of the initial amplitude. This decline was dependent upon activation of the P2‐purinoceptor. Similar results were seen with 2‐meSATP and α,β‐meATP (both 300 nm and 1 μm). Cross‐desensitization was seen between ATP and 2‐meSATP or α,β‐meATP.

Inward currents evoked by ATP, 2‐meSATP and α,β‐meATP (all 1 μm) were abolished by the P2‐purinoceptor antagonist suramin (100 μm).

αβ‐meATP (100 nm‐30 μm), 2‐meSATP (3 μm‐100 μm), ATP (3 μm‐ 1 μm) and UTP (3 μm‐ 1 μm) produced concentration‐dependent contractions of rat tail artery rings. When measured at a level equal to 50% of the maximum response to noradrenaline, the rank order of agonist potency was α,β,‐meATP > > 2‐meSATP >UTP >ATP.

This study shows that the rank order of agonist potency at the P2X‐purinoceptor which mediates contractions of the rat isolated tail artery is very different from the potency order for evoking the inward current which initiates the contractions. It is concluded that this difference may be due to the relative absence of breakdown of some of the agonists in the single cell system compared with artery rings.
Inhibition by SK&F 96365 of Ca2+ current, IL‐2 production and activation in T lymphocytes
Authors: Chung S. Clare , McDonald Thomas V. , Gardner Phyllis
Published: July 2012

By use of whole cell patch‐clamp and Indo‐1 fluorescence studies of the Jurkat T leukaemic cell line, we show that the new organic antagonist of receptor‐mediated Ca2+ entry, SK&F 96365, inhibits the T cell Ca2+ current in a dose‐dependent fashion, with an IC50 of 12 μm.

SK&F 96365 also inhibits [3H]‐thymidine incorporation and interleukin‐2 (IL‐2) synthesis in peripheral blood lymphocytes.

SK&F 96365 has no effect on Ca2+ stores release or K+ channels.

This is the first account of an organic inhibitor of the T cell Ca2+ current. The ability of SK&F 96365 to inhibit IL‐2 synthesis and cell proliferation suggests that a new class of related Ca2+ channel blockers can be developed as immunosuppressive agents.
Presence of neuropeptide Y in the rat seminal vesicle and its effects on noradrenaline‐ and nerve‐induced contractions
Authors: Iravani M.M. , Zar M.A.
Published: July 2012

Immunohistochemical and functional studies have been performed to localize and determine the effects of neuropeptide Y (NPY) in the rat seminal vesicle.

An abundant presence of NPY‐immunoreactive nerves, mainly concentrated in the smooth muscle layer of the seminal vesicle was found. Chronic 6‐hydroxydopamine treatment (four doses of 50 mg kg−1 i.p. on days 1, 2, 4 and 6; rats killed one week after the last injection) led to a large reduction but not abolition of the NPY‐immunoreactivity.

NPY (1‐250nM) did not affect the resting tone of the seminal vesicle.

The seminal vesicle was contracted by electrical field stimulation (EFS) and by exposure to 5 μm noradrenaline (NA). These contractions were abolished by phentolamine (1 μm). Tetrodotoxin (0.5 μm) abolished EFS‐evoked contractions but did not affect NA‐evoked contractions.

Seminal vesicles, from animals chronically‐treated with reserpine (5 mg kg−1 i.p. on days 1 and 2; rats killed on day 3), were contracted by NA but not by EFS.

NPY (0.25–250 nm), concentration‐dependently, inhibited EFS‐evoked contractions by up to 70% maximum inhibition. Contractions evoked by EFS with short trains of pulses were inhibited by NPY to a greater degree than those with longer trains.

NPY had no significant effect on NA‐evoked contractions.

These data provide strong evidence that the motor transmission in rat seminal vesicle is predominantly if not exclusively, adrenergic. It is further concluded that a rich NPY‐containing innervation is present in the smooth muscle layer of rat seminal vesicle. The primary effect of NPY is modulation of adrenergic motor transmission by a prejunctional inhibition of NA release.
Location of the sulphonylurea receptor at the cytoplasmic face of the β‐cell membrane
Authors: Schwanstecher Mathias , Schwanstecher Christina , Dickel Corinna , Chudziak Franz , Moshiri Ali , Panten Uwe
Published: July 2012

In insulin‐secreting cells the location of the sulphonylurea receptor was examined by use of a sulphonylurea derivative representing the glibenclamide molecule devoid of its cyclohexyl moiety (compound III) and a benzenesulphonic acid derivative representing the glibenclamide molecule devoid of its cyclohexylurea moiety (compound IV). At pH7.4 compound IV is only present in charged form.

Lipid solubility declined in the order tolbutamide > compound III > compound IV.

The dissociation constant (KD) for binding of compound IV to the sulphonylurea receptor in HIT‐cells (pancreatic β‐cell line) was similar to the KD value for tolbutamide and fourfold higher than the KD value for compound III.

In mouse pancreatic β‐cells, drug concentrations inhibiting adenosine 5′‐triphosphate‐sensitive K+ channels (KATP‐channels) half‐maximally (EC50) were determined by use of the patch‐clamp technique. When the drugs were applied to the extracellular side of outside‐out or the intracellular side of inside‐out membrane patches, the ratio of extracellular to intracellular EC50 values was 281 for compound IV, 25.5 for compound III and 1.2 for tolbutamide.

In mouse pancreatic β‐cells, measurement of KATP‐channel activity in cell‐attached patches and recording of insulin release displayed much higher EC50 values for compound IV than inside‐out patch experiments. A corresponding, but less pronounced difference in EC50 values was observed for compound III, whereas the EC50 values for tolbutamide did not differ significantly.

It is concluded that the sulphonylurea receptor is located at the cytoplasmic face of the β‐cell plasma membrane. Receptor activation is induced by the anionic forms of sulphonylureas and their analogues.
Regulation of nicotinic receptors in rat brain following quasi‐irreversible nicotinic blockade by chlorisondamine and chronic treatment with nicotine
Authors: El‐Bizri H. , Clarke P.B.S.
Published: July 2012

Chronic administration of nicotinic agonists in vivo increases the density of brain nicotinic binding sites. It has been proposed that this up‐regulation results from agonist‐induced functional blockade of nicotinic receptors. This hypothesis was tested by examining post mortem [3H]‐nicotine and [125I]‐α‐bungarotoxin ([125I]‐αBTX) binding following treatment in vivo with the quasi‐irreversible and insurmountable CNS nicotinic blocker chlorisondamine, given either alone or in combination with chronic nicotine administration.

In rats that had not received chlorisondamine pretreatment, chronic nicotine administration (0.6 mg kg−1 s.c, twice daily for 12 days) increased [3H]‐nicotine binding density (Bmax) in forebrain tissue sections by 19%, with no change in the apparent dissociation constant (KD). Chlorisondamine (10 mg kg−1, s.c), given once prior to the chronic treatment phase, neither increased [3H]‐nicotine binding by itself, nor altered the extent of nicotine‐induced up‐regulation. Nevertheless, chlorisondamine pretreatment resulted in a persistent blockade of CNS nicotinic receptors, as demonstrated by complete block of acute locomotor responses to nicotine.

In a second experiment, [3H]‐nicotine and [125I]‐αBTX binding was measured in tissue homogenates prepared from several brain regions. In the absence of chlorisondamine pretreatment, chronic nicotine administration (1 mg kg−1 s.c, twice daily for 12 days) increased the Bmax of [3H]‐nicotine binding in the cerebral cortex (by 34%), striatum (by 28%), midbrain (by 16%) and hippocampus (by 36%); KD was unchanged. As before, this up‐regulation was neither mimicked nor blocked by chlorisondamine pretreatment (10 mg kg−1, s.c, given twice), despite persistent blockade of acute locomotor responses to nicotine. Chronic nicotine treatment also increased the Bmax (but not KD) of [125I]‐αBTX binding in cerebral cortex (by 35%), hippocampus (by 46%) and midbrain (by 35%). Chlorisondamine altered neither Bmax nor KD when given alone, but significantly attenuated the nicotine‐induced up‐regulation of toxin binding sites in midbrain, with a similar trend in the other two regions.

The finding that chronic receptor blockade neither mimicked nor blocked the agonist‐induced up‐regulation of [3H]‐nicotine binding sites suggests that up‐regulation of these receptors is not determined by their functional status. In contrast, it appears that chronic nicotine‐induced up‐regulation of [125I]‐αBTX binding sites may result from receptor activation.
Effect of cytoplasmic pH on Ca2+‐stimulated eicosanoid biosynthesis in human platelets
Authors: Edwards Jacqueline S. , Ritter James M.
Published: July 2012

We have investigated the effect of cytoplasmic pH (pH;) on the relationship between platelet cytoplasmic Ca2+ concentration ([Ca2+]1) and eicosanoid biosynthesis. Stirred gel‐filtered human platelets loaded with fluorescent indicators of Ca2+ and H+ were suspended in balanced salt solutions at 37°C. [Ca2+]i was controlled by calcium ionophore (ionomycin). Increased [Ca2+]i was associated with increased production of thromboxane A2 (TXA2) as determined by radioimmunoassay of its stable hydrolysis product TXB2, and of 12‐hydroxy eicosatetraenoic acid (12‐HETE) measured by high performance liquid chromatography.

Varying pHi with a K+/H+ ionophore (nigericin) in platelets suspended in K+ rich solutions of pH 6.8, 7.4 or 7.8 with subsequent resuspension in solution of pH 7.4 containing albumin (1 g 1−1) and Ca2+ (mM) resulted in pH1 of 6.72 ± 0.05, 7.31 ± 0.02 and 7.71 ± 0.04 (mean ± s.e.mean, n = 5). Ionomycin (1.2 μm) increased [Ca2+1 by 97.1 ± 17.6, 191.9 ± 48.7 and 322.8 ± 55.7 nm at the different values of pH1 respectively; TXB2 production was 0.7 ± 0.2, 2.1 ± 0.4 and 10.7 ± 3.3 ngjig−1 protein, and 12‐HETE production was 150.9 ± 68.2, 184.4 ± 77.9 and 302.3 ± 62.8 ng μg−1 protein.

Ammonium chloride (50 μm) caused a small reduction in pHo while increasing pHi from 7.32 ± 0.04 to 7.89 ± 0.05 and increasing ionomycin (1.2μm)‐induced [Ca2+]4 responses from 94.1 ± 67.3 to 721.6 ± 288.3 nm. TXB2 production increased from 3.1 ± 2.1 to 17.3 ± 8.2 and 12‐HETE production increased from 100.5 ± 26.7 to 203.2 ± 36.4 ngμg−1 protein. Responses of [Ca2+]i and TXB2 production to epoxymethano prostaglandin H2 (U46619, an endoperoxide‐thromboxane receptor agonist) increased significantly in the presence of NH4Cl.

Alterations of pH1 (such as may occur under pathological conditions) influence [Ca2+]i responses and eicosanoid synthesis in human platelets.
The role of bradykinin B1 receptors in the maintenance of intra‐articular plasma extravasation in chronic antigen‐induced arthritis
Authors: Cruwys S.C. , Garrett N.E. , Perkins M.N. , Blake D.R. , Kidd B.L.
Published: July 2012

The role of bradykinin B1 and B2 receptors in bradykinin‐ and des‐Arg9‐bradykinin‐induced plasma extravasation in normal and inflamed rat knee joints was investigated by use of an antigen‐induced model of chronic arthritis. A modification of an Evans blue extraction technique allowed the unstimulated (basal) plasma extravasation to be assessed in this model. The contributions of bradykinin B1 and B2 receptors towards basal synovial plasma extravasation were determined.

In normal knees, intra‐articular injection of bradykinin (BK) induced plasma extravasation in a potent, dose‐dependent manner with a threshold of 0.01 nmol and an ED50 of 0.1 nmol. In day 5 arthritic knees, basal plasma extravasation was substantially enhanced. Lower doses of BK had no demonstrable effect and increases above basal extravasation were first observed at 0.1 nmol. Thereafter the dose‐response mirrored the response in normal knees and the maximal response was unaltered.

The B1 agonist, des‐Arg9‐BK, induced slight but significant plasma extravasation in normal knees but was less potent than bradykinin. This response was inhibited by the Bi receptor antagonist, des‐Arg9, [Leu8]‐BK. Lower doses of des‐Arg9‐BK bradykinin did not significantly increase basal extravasation in day 5 arthritic knees but, in contrast to BK, the maximal response was significantly enhanced.

The B2 antagonist, Hoe 140, inhibited BK‐induced plasma extravasation in normal joints over a dose‐range of 0.1‐1.0 nmol but was relatively inactive in day 5 inflamed knees. The B1 receptor antagonist, des‐Arg9, [Leu8]‐BK, was relatively inactive in normal joints but showed increased potency against BK‐induced plasma extravasation in day 5 arthritic joints.

Hoe 140 and des‐Arg9, [Leu8]‐BK both inhibited basal extravasation in arthritic joints on days 1 and 5 post‐challenge in a dose‐dependent fashion. Whilst Hoe 140 was the more potent inhibitor on day 1, it was less potent than des‐Arg9, [Leu8]‐BK on day 5.

Although the majority of responses to BK in normal tissue are mediated via B2 receptors, a small population of B1 receptors may exist in normal joint tissues. The data presented in this study suggest an evolving role for Bx receptors in the mediation of plasma extravasation in inflamed joint tissues. A role for BK antagonists in the treatment of arthritis is also suggested.
Pharmacological profile of the ATP‐mediated increase in L‐type calcium current amplitude and activation of a non‐specific cationic current in rat ventricular cells
Authors: Scamps Frédérique , Vassort Guy
Published: July 2012

The pharmacological profile of the ATP‐induced increase in ICa amplitude and of ATP activation of a non‐specific cationic current, IATP, was investigated in rat ventricular cells.

The EC50 values for ICa increase and IATP activation were 0.36 μm and 0.76 μm respectively. Suramin (10 μm) and cibacron blue (1 μm) competitively antagonized both effects of ATP.

The rank order of efficacy and potency of ATP analogues in increasing ICa amplitude was 2‐methylthio‐ATP ≅ ATP ≅ ATPγS. The derivatives α,β‐methylene‐ATP, β,γ‐methylene‐ATP and β,γ‐imido‐ATP up to 500 μm had no significant effects.

The rank order of efficacy of ATP analogues in activating a non‐specific cationic current, IATP, was 2‐methylthio‐ATP > ATP > > ATPγS. The rank order of potency was 2‐methylthio‐ATP ≅ ATP. The EC50 of ATPγS could not be determined owing to its very low efficacy.

The ATP analogues α,β‐methylene‐ATP, β,γ‐methylene‐ATP and β,γ‐imido‐ATP at 500 μm did not activate IATP but acted as antagonists of activation of IATP by ATP.

The results suggest that the increase in ICa amplitude induced by external ATP is due to activation of P2Y‐purinoceptors.

The mechanism of IATP activation remains to be determined before the receptor subtype involved can be deduced.
Vasoconstrictor and vasodilator responses to various agonists in the rat perfused mesenteric arterial bed: selective inhibition by PPADS of contractions mediated via P2x‐purinoceptors
Authors: Windscheif Ursula , Ralevic Vera , Bäumert Hans G. , Mutschler Ernst , Lambrecht Günter , Burnstock Geoffrey
Published: July 2012

The effect of pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) on vasoconstrictor and/or vasodilator responses to various agonists and electrical field stimulation was investigated in the rat mesenteric arterial bed at basal tone and at tone raised by methoxamine (15–50 μm).

At basal tone, nucleotides produced vasoconstriction with the following rank order of potency: α,β‐methylene ATP > > 2‐methylthio ATP ≥ ATP = UTP. PPADS (0.3–10 μm) concentration‐dependently antagonized α,β‐methylene ATP‐, 2‐methylthio ATP‐ and ATP‐induced responses. UTP‐, noradrenaline‐ and nerve‐mediated (4–32 Hz) increases in perfusion pressure remained unaffected by 10 μm PPADS.

In raised tone preparations, nucleotides produced vasodilations, their rank order of potency being 2‐methylthio ATP > ATP > UTP. Responses to 2‐methylthio ATP were slightly antagonized, whereas ATP‐ and UTP‐induced responses remained unaffected by 10μm PPADS. In addition, acetylcholine‐and adenosine‐elicited relaxations were not influenced by 10 μm PPADS.

The present results confirm the previously described selective P2x antagonism by PPADS, this compound being ineffective at muscarinic M3‐ and adenosine P1‐receptors as well as at α1‐adrenoceptors. There was some inhibition of P2y‐purinoceptors but at a much higher concentration than required for inhibition of P2x‐purinoceptors.

In addition, this study provides evidence for the ineffectiveness of PPADS at both vasoconstriction‐and vasodilatation‐mediating P2u‐purinoceptors.
Haemodynamic actions of a novel sino‐atrial node function modulator, ZENECA ZD7288, in the anaesthetized dog: a comparison with zatebradine, atenolol and nitrendipine
Authors: Rouse W. , Johnson I.R.
Published: July 2012

ZENECA ZD7288 (4‐(N‐ethyl‐N‐phenylamino)‐1,2‐dimethyl‐6‐(methylamino) pyrimidinium chloride, formerly ICI D7288) is a novel sino‐atrial node function modulator which selectively slows sinus node rate. Its effects on haemodynamic function have been studied in pentobarbitone anaesthetized dogs, in comparison with zatebradine, atenolol and nitrendipine.

ZD7288 lowered heart rate in the dose‐range 0.02 to 1.0 mg kg−1 i.v. from 152 to 77 beats min−1. Myocardial contractile function (measured as both dPLV/dtmax and right ventricular free wall developed force) decreased along with rate. Stroke volume increased as rate decreased. Cardiac output decreased at doses in excess of 0.2 mg kg−1, i.v.

These haemodynamic changes were reversed when heart rate reduction was reversed by atrial pacing and are, therefore, considered to be indirect consequences of heart rate changes induced by ZD7288.

The effects of zatebradine paralleled those of ZD7288 (heart rate reduced from 149 to 60.5 beats min−1 over the dose‐range 0.02 to 1.0 mg kg−1, i.v.), except that dPLV/dtmax did not decrease with heart rate and increased during arial pacing.

Neither ZD7288 nor zatebradine had significant effects on atrio‐ventricular conduction at intrinsic heart rates, but both significantly and dose‐dependently prolonged the atrio‐ventricular conduction interval during atrial pacing at 180 beats min−1.

The observed effects of atenolol were commensurate with removal of p‐sympathetic cardiac drive. Atrial pacing was found not to restore the pre‐atenolol haemodynamic state completely.

Nitrendipine up to 0.2 mg kg−1 i.v. induced changes indicative of direct vasodilatation accompanied by reflex compensation, followed by cardiac depression at higher doses. Atrial pacing failed to compensate for the effects of vasodilatation, but caused atrio‐ventricular conduction block at doses above O.S mg kg−1, i.v.

The data show ZD7288 has marked heart rate slowing properties and that accompanying haemodynamic changes appear to be secondary to the rate changes, being reversed by atrial pacing even in the continued presence of the drug. Heart rate slowing without depression of contractile function should prove to be of benefit in the treatment of myocardial ischaemia, particularly in the presence of myocardial dysfunction.
Pharmacological reactivity of human epicardial coronary arteries: phasic and tonic responses to vasoconstrictor agents differentiated by nifedipine
Authors: Stork Andrew P. , Cocks Thomas M.
Published: July 2012

Human epicardial coronary artery rings, freshly obtained from cardiac transplantation patients, commonly exhibited phasic contractile activity in vitro. This activity occurred either spontaneously or in response to vasoconstrictor stimulation.

Nifedipine pretreatment (1 nm‐0.1 μm) reduced both types of phasic contractions in a concentration‐dependent manner. At 0.1 μm nifedipine, spontaneous contractions were completely abolished, as were phasic contractions induced by U46619, endothelin‐1 or 5‐hydroxytryptamine (5‐HT).

For U46619 (0.1–100 nm), the largest phasic contractions (amplitude peak to trough) occurred over the mid‐range of concentrations used (1–10 nm). At higher concentrations (30–100 nm), phasic activity was reduced as the response reached a maximum. Estimated pEC50 values for the upper phasic and lower phasic curves were significantly different (8.71 ± 0.13 versus 7.90 ± 0.11; P < 0.05; n = 10). In the presence of nifedipine (0.1 μm), the purely tonic contraction curve to U46619 was similar to the lower phasic curve in the absence of nifedipine (pEC50 = 8.14 ± 0.06, n = 10). Similar results were obtained for endothelin‐1 (0.1 −100 nm).

Responses to 5‐HT (1 nm‐3 μm) were more variable. The largest phasic contractions were spread unevenly throughout the concentration‐response curve. In the presence of nifedipine (0.1 μm), the curve to 5‐HT was significantly depressed in range but not sensitivity (pEC50) when compared with the phasic curves.

In conclusion, activation of dihydropyridine‐sensitive voltage‐operated Ca2+ channels mediated the phasic contractions commonly observed in human epicardial coronary arteries. These contractions amplified the contractile responses to low concentrations of vasoconstrictors. Inhibition of phasic activity by the Ca2+ channel antagonist, nifedipine, allowed the tonic vasoconstrictor profile of human isolated coronary artery to be determined which is important information for the accurate quantitative assessment of vasodilator responses in this tissue in vitro.
Effects of tenoxicam and aspirin on the metabolism of proteoglycans and hyaluronan in normal and osteoarthritic human articular cartilage
Authors: Manicourt Daniel Henri , Egeren Anne Druetz‐Van , Haazen Ludo , de Deuxchaisnes Charles Nagant
Published: July 2012

As nonsteroidal antiinflammatory drugs may impair the ability of the chondrocyte to repair its damaged extracellular matrix, we explored the changes in the metabolism of newly synthesized proteoglycan (PG) and hyaluronan (HA) molecules produced by tenoxicam and aspirin in human normal cartilage explants and in osteoarthritic (OA) cartilage from age‐matched donors.

Explants were sampled from the medial femoral condyle and were classified by use of Mankin's histological‐histochemical grading system. Cartilage specimens were normal in 10 subjects, exhibited moderate OA (MOA) in 10 and had severe OA (SOA) in 10.

Cartilage explants were pulsed with [3H]‐glucosamine and chased in the absence and in the presence of either aspirin (190 μg ml1) or tenoxicam (4–16 μg ml−1). After papain digestion, the labelled chondroitin sulphate ([3H]‐PGs) and HA([3H]‐HA) molecules present in the tissue and media were purified by anion‐exchange chromatography.

In normal cartilage as well as in explants with MOA and SOA aspirin reduced more strongly PG and HA synthesis than the loss of [3H]‐HA and [3H]‐PGs.

In normal cartilage, tenoxicam did not affect PG metabolism whereas it reduced HA synthesis in a dose‐dependent manner and did not change or even increased the net loss of [3H]‐HA. In contrast, in OA cartilage, tenoxicam produced a stronger reduction in the loss of [3H]‐PGs than in PG synthesis and this decrease occurred at lower concentrations in cartilage with SOA (4–8 μg ml−1) than in cartilage with MOA (8–16 μg ml−1). In cartilage with MOA, the metabolic balance of HA was unaffected by tenoxicam whereas in cartilage with SOA, the drug decreased the loss of [3H]‐HA and concomitantly did not change or even increased HA synthesis.

The data obtained in short‐term in vitro cultures indicate that aspirin may produce OA‐like changes in normal cartilage and is likely to worsen the disease process in OA tissue. On the other hand, although tenoxicam may reduce the HA content of normal cartilage, and, in so doing, may produce OA‐like lesions, this drug should not per se accelerate joint failure in OA.
Prevention of thrombosis and rethrombosis and enhancement of the thrombolytic actions of recombinant tissue‐type plasminogen activator in the canine heart by DMP728, a glycoprotein IIb/IIIa antagonist
Authors: Lucchessi Benedict R. , Rote William E. , Driscoll Edward M. , Mu Dun‐Xue
Published: July 2012

We studied DMP728, a non‐peptide glycoprotein (GP) IIb/IIIa receptor antagonist, for prevention of coronary artery thrombosis or rethrombosis in a chronic canine model subjected to arterial injury.

In protocol I, DMP728 (1.0 mg kg−1, i.v., n = 8) or saline (n = 8) was administered and a 150 μA anodal current was applied to the intimal surface of the left circumflex coronary artery (LCX). Dogs were monitored for 6 h and again on each of 5 subsequent days.

Ex vivo platelet aggregation was inhibited but returned to baseline 1 day after drug administration. Thrombus weight was reduced (saline, 20.7 ± 5.0 mg; DMP728 1.7 ± 0.4mg; P < 0.05), as was infarct size [saline, 27.5 ± 4.3; DMP728, 1.6 ± 0.7 (per cent left ventricle); P < 0.05]. All control animals died by day 3, while all but one of the treated dogs survived the entire protocol (P < 0.05).

In protocol II, an LCX thrombus was induced and thrombolytic therapy was initiated 30 min later. DMP728 (1.0 mg kg−1, i.v., n = 8) or saline (n = 8) was administered 5 min after recombinant tissue‐type plasminogen activator infusion had begun. The incidence of reocclusion was reduced by DMP728 (saline, 4/8; DMP728, 1/8). One day after thrombolysis, 7/8 DMP728‐treated animals were alive compared with 1/8 in the control group (P = 0.01).

DMP728 inhibited ex vivo platelet aggregation, prevented primary and secondary occlusive thrombus formation, reduced thrombus weight and infarct size and increased survival in a chronic canine model of coronary artery thrombus formation. DMP728 is an effective anti‐platelet intervention when used as the singular adjunctive agent in association with thrombolytic therapy.
Regional effects of amphetamine, cocaine, nomifensine and GBR 12909 on the dynamics of dopamine release and metabolism in the rat brain
Authors: Karoum Tarouk , Chrapusta Stanislaw J. , Brinjak Raymond , Hitri Ana , Wyatt Richard J.
Published: July 2012

The effects of single‐dose regimens of amphetamine, cocaine, nomifensine and GBR 12909 on the dynamics of dopamine (DA) release and metabolism were evaluated in the frontal cortex, hypothalamus, nucleus accumbens and striatum. The regimens selected are known to produce substantial behavioural effects.

3‐Methoxytyramine (3MT) and 3,4‐dihydroxyphenylacetic acid (DOPAC) rates of formation were used to assess DA metabolism by catechol‐O‐methyltransferase and monoamine oxidase respectively. The rate of formation of 3MT was used as an index of synaptic DA. The ratio and sum, respectively, of 3MT and DOPAC rates of formation were used to assess DA reuptake inhibition and turnover.

The effects of amphetamine on 3MT production and DOPAC steady‐state levels were similar in all regions, suggesting similar pharmacodynamic actions. Amphetamine increased 3MT formation and steady‐state levels, and reduced DOPAC steady‐state levels. DOPAC formation was significantly reduced only in the nucleus accumbens and striatum. Total DA turnover remained unchanged except in the nucleus accumbens. Apparently, the amphetamine‐induced increase in DA release occurred at the expense of intraneuronal DA metabolism and did not require stimulation of synthesis.

Nomifensine elevated 3MT formation in all regions. A similar effect was produced by cocaine except in the nucleus accumbens. GBR 12909 elevated 3MT production only in the hypothalamus, the striatum and the nucleus accumbens.

Cocaine selectively reduced DOPAC formation in the frontal cortex. Nomifensine increased and reduced, respectively, DOPAC formation in striatum and hypothalamus. GBR 12909 elevated DOPAC formation in all regions except the cortex, where pargyline did not reduce DOPAC levels in GBR 12909‐treated rats.

Ratios and sum of 3MT and DOPAC rates of formation also exhibited wide regional variations for each drug. In contrast to the other drugs, the ratio was not increased after GBR 12909. Apparently, the DA uptake properties of this drug are poorly related to its in vivo effects on the ratio of 3MT production to that of DOPAC, which should increase when DA reuptake is inhibited.

Total DA turnover was increased by GBR 12909 in the hypothalamus, nucleus accumbens and striatum, while cocaine and nomifensine increased it only in the nucleus accumbens and striatum respectively.

It is concluded that:

3MT and DOPAC rates of formation provide better indices of DA release and metabolism than do their steady‐state concentrations.

Some effects of DA uptake blockers on DA transmission, especially those of nomifensine and cocaine, may be attributed to increased DA release.

Patterns of regional effects of psychostimulants on the dynamics of DA release and metabolism may be better biochemical correlates of stimulant‐induced behaviours than would changes in any single region.
Pharmacological analysis of ecto‐ATPase inhibition: evidence for combined enzyme inhibition and receptor antagonism in P2X‐purinoceptor ligands
Authors: Crack B.E. , Beukers M.W. , McKechnie K.C.W. , IJzerman A.P. , Leff P.
Published: July 2012

Previous studies have shown that suramin and FPL 66301 are competitive antagonists at the P2X‐purinoceptor in the rabbit ear artery. Those studies employed α,β‐methylene ATP, a poorly hydrolysable ATP analogue, as the agonist. In this study these compounds have been tested using ATP as the agonist.

Suramin, in the concentration range 30–1000 μm, potentiated the contractile effects of ATP, producing a 3‐fold leftward shift of the ATP E/[A] curves. FPL 66301, in the concentration range 100–1000 μm, produced a significant but small (3‐fold) rightward shift of the ATP curves. These results are in marked contrast with previous studies using α,β‐methylene ATP in which 30‐fold rightward shifts were achieved using the same concentration ranges of suramin and FPL 66301.

Suramin and FPL 66301 were tested as ecto‐ATPase inhibitors in a human blood cell assay. Suramin inhibited the enzyme with a pIC50 of 4.3, FPL 66301 with a pIC50 of 3.3.

The pharmacological data were analysed using a theoretical model describing the action of a compound with dual enzyme inhibitory and receptor antagonistic properties on the effects of an agonist susceptible to enzymatic degradation. The model was found to fit the data well using the known pKBestimates for suramin and FPL 66301 and similar relative (but not absolute) pK1 estimates to those obtained for the compounds in the enzyme assay.

From this analysis it was concluded that the limited shifts of ATP E/[A] curves produced by suramin and FPL 66301 were the result of ‘self‐cancellation’ of the potentiating (enzyme inhibitory) and rightward‐shifting (receptor antagonistic) properties.

The analysis also indicated that the presence of ecto‐ATPase activity in the rabbit ear artery preparation has a marked effect on the apparent potency of ATP. The experimental p[A50] was 3.4, whereas the ‘true’ value, that is the value which would be obtained in the absence of ecto‐ATPase activity, was 6.0, some 400‐fold higher.

Two conclusions are drawn from this study. Firstly, caution must be exercised in the use of suramin and FPL 66301 as tools for receptor classification. Absence of overt antagonism by these compounds when metabolically unstable agonists are used could lead to erroneous claims for receptor subtypes. Secondly, the agonist potency order currently used to designate P2X‐ purinoceptors may require modification.
Possible roles of protein kinases in neutrophil chemotactic factor production by leucocytes in allergic inflammation in rats
Authors: Tanabe Jun‐Ichi , Watanabe Masako , Kondoh Sachiko , Mue Suetsugu , Ohuchi Kazuo
Published: July 2012

In an air pouch‐type allergic inflammation model in rats, leucocytes that had infiltrated into the pouch fluid collected 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils when they were incubated in the medium.

To clarify the mechanism of activation of the infiltrated leucocytes in producing these factors, the effects of protein kinase inhibitors on neutrophil chemotactic factor production were examined.

When the infiltrated leucocytes were incubated for 4h in medium containing the non‐selective protein kinase inhibitor K‐252a (1–100 ng ml−1, 2.14–214 nm), the tyrosine kinase inhibitor genistein (1–50 μg ml−1, 3.7–185 μm), and the more selective protein kinase C inhibitor H‐7 (5–100 μg ml−1, 13.7–274 μm); neutrophil chemotactic activity in the conditioned medium was decreased in a concentration‐dependent manner, but the adenosine 3′:5′‐cyclic monophosphate (cAMP)‐dependent protein kinase inhibitor H‐89 (1–1000 ng ml−1, 2.24–2240 nm) showed no effect.

Isoelectric focusing of the conditioned medium revealed that the leucocytes produced two neutrophil chemotactic factors, leucocyte‐derived neutrophil chemotactic factor (LDNCF) 1 and LDNCF‐2. Treatment of the leucocytes with K‐252a, genistein, and H‐7, but not H‐89, inhibited production of both LDNCF‐1 and LDNCF‐2.

These results suggest that activation of tyrosine kinase and protein kinase C., but not cAMP‐dependent protein kinase, is responsible for the production of LDNCF‐1 and LDNCF‐2.

The steroidal anti‐inflammatory drug dexamethasone and the protein synthesis inhibitor cyclohex‐imide inhibited neutrophil chemotactic factor production in a concentration‐dependent manner. Time‐course experiments showed that the inhibitory effect by dexamethasone was apparent even 30min after the incubation.

Mechanism for inhibiting the production of LDNCF‐1 and LDNCF‐2 by dexamethasone is also discussed.
A1 adenosine receptor inhibition of cyclic AMP formation and radioligand binding in the guinea‐pig cerebral cortex
Authors: Alexander Stephen P.H. , Curtis Alistair R. , Kendall David A. , Hill Stephen J.
Published: July 2012

A1 adenosine receptors were investigated by radioligand binding and functional studies in slices and particulate preparations from guinea‐pig cerebral cortex.

Binding of the adenosine receptor antagonist radioligand, 8‐cyclopentyl‐[3H]‐1,3‐dipropylxanthine (DPCPX) to guinea‐pig cerebral cortical membranes exhibited high density (1410 ± 241 fmol mg−1 protein) and high affinity (Kd 3.8 ± 0.3 nm).

[3H]‐DPCPX binding to guinea‐pig cerebral cortical membranes was displaced in a monophasic manner by adenosine receptor antagonists with the rank order of affinity (Ki values, nm): DPCPX (6) < xanthine amine congener (XAC, 153) < PD 115,199 (308).

Agonist displacement of [3H]‐DPCPX binding was biphasic and exhibited the following rank order at the low affinity site (Ki values): 2‐chloro‐N6‐cyclopentyl‐adenosine (CCPA, 513 nm) = N6‐R‐phenyl‐isopropyladenosine (R‐PIA, 526 nm) = M6‐cyclopentyladenosine (CPA, 532 nm) < 2‐chloroadenosine (2CA, 3.2 μm) = 5′‐N‐ethylcarboxamidoadenosine (NECA, 4.6 μm) < N6‐S‐phenylisopropyladenosine (S‐PIA, 19.9 μm).

In cerebral cortical slices, [3H]‐DPCPX binding was displaced by antagonists and agonists in an apparently monophasic manner with the rank order of affinity (Ki values, nm): DPCPX (14) < XAC (45) < R‐PIA (266) < PD 115,199 (666) < S‐PIA (21000).

Cyclic AMP accumulation stimulated by 30 μm forskolin in guinea‐pig cerebral cortical slices was inhibited by R‐PIA, CCPA and CPA up to 1 μm in a concentration‐dependent fashion with IQo values of 14, 18, and 22 nm, respectively. All three analogues inhibited the forskolin response to a similar extent (82–93% inhibition). NECA, S‐PIA and 2CA failed to inhibit the forskolin response, but rather enhanced the accumulation of cyclic AMP at concentrations of 100 nm or greater, presumably through activation of A2b adenosine receptors coupled to stimulation of cyclic AMP accumulation in guinea‐pig cerebral cortical slices.

The inhibition of forskolin‐stimulated cyclic AMP accumulation by CPA was antagonized with the rank order of affinity (Ki values, nm): DPCPX (6) < XAC (52) < PD 115,199 (505).

Xanthine‐based antagonists inhibited the adenosine receptor augmentation of histamine‐induced phosphoinositide turnover in guinea‐pig cerebral cortical slices with the rank order of affinity (Ki, nm): DPCPX (12) = XAC (17) < PD 155,199 (640).

In summary, we observe a good correlation between antagonist affinity at A1 receptors defined by radioligand binding, inhibition of cyclic AMP generation or augmentation of histamine‐evoked phosphoinositide turnover in guinea‐pig cerebral cortex.
Effects of β2‐adrenoceptor agonists on anti‐IgE‐induced contraction and smooth muscle reactivity in human airways
Authors: Gorenne Isabelle , Labat Carlos , Norel Xavier , De Montpreville Vincent , Guillet Marie‐Christine , Cavero Icilio , Brink Charles
Published: July 2012

The β2‐adrenoceptor agonists, salbutamol, salmeterol and RP 58802 relaxed basal tone of human isolated bronchial smooth muscle. Salmeterol‐ and RP 58802‐induced relaxations persisted for more than 4h when the medium was constantly renewed after treatment.

Salbutamol, salmeterol and RP 58802 reversed histamine‐induced contractions in human airways (pD2 values: 6.15 ± 0.21, 6.00 ± 0.19 and 6.56 ± 0.12, respectively).

Anti‐IgE‐induced contractions were significantly inhibited immediately after pretreatment of preparations with β2‐adrenoceptor agonists (10 μm). However, when tissues were treated with β2‐agonists and then washed for a period of 4 h, salmeterol was the only agonist which significantly inhibited the anti‐IgE response.

Histamine response curves were shifted to the right immediately after pretreatment of tissues with the β2‐adrenoceptor agonists (10 μm; 20 min), but maximal contractions were not affected. After a 4 h washing period, the histamine curves were not significantly different from controls. Concentration‐effect curves to acetylcholine (ACh) or leukotriene C4 (LTC4) were not significantly modified after β2‐agonist pretreatment.

These results suggest that β2‐adrenoceptor agonists may prevent anti‐IgE‐induced contraction by inhibition of mediator release rather than alterations of those mechanisms involved in airway smooth muscle contraction.