bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition by antioxidants of nitric oxide synthase expression in murine macrophages: role of nuclear factor kB and interferon regulatory factor 1
Authors: Hecker Markus , Preiß Christiane , Klemm Peter , Busse Rudi
Published: July 2012

In view of the potential deleterious effects of high amounts of nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS) in inflammation, the prevention of the expression of this enzyme represents an important therapeutic goal. In cytokine‐stimulated cells, activation of nuclear factor kB (NF‐kB) is crucial for the increase in iNOS gene expression. Since NF‐kB activation appears to involve a redox‐sensitive step, we have investigated whether three structurally unrelated antioxidants, 5,7‐dihydroxyflavone (chrysin), 3,4‐dichloroisocoumarin (DCI) and N‐acetyl 5‐hydroxytryptamine (N‐acetylserotonin, NAS), affect iNOS expression in cultured RAW 264.7 monocyte/macrophages stimulated with bacterial lipopolysaccharide (LPS, 140 ng ml−1) and interferon‐γ (IFNγ, 5 u ml−1).

During a 6 h incubation period neither LPS nor IFNγ alone exerted a significant effect but when combined, caused a prominent increase in nitrite formation, iNOS mRNA and protein abundance. Co‐incubation with chrysin (50 μm), DCI (50 μm) or NAS (1 mM) markedly attenuated this increase in iNOS gene expression.

DCI, but not chrysin or NAS, prevented the activation of NF‐kB in cells exposed to LPS plus IFNγ for 30 min. In contrast, all three antioxidants significantly blunted the DNA‐binding activity of interferon regulatory factor 1 (IRF‐1), which mediates the synergistic effect of IFNγ on iNOS gene expression in cells treated for 2 h with LPS plus IFNγ.

DCI thus appears to inhibit iNOS gene expression at the transcriptional level by preventing the activation of both NF‐kB and IRF‐1. The inhibitory effect of DCI on NF‐kB activation, however, does not seem to be related to its antioxidative properties, since DCI, unlike chrysin or NAS, is a potent serine protease inihbitor which stabilizes the inactive NF‐kB complex by protecting the inhibitory IkB‐α subunit from proteolytic degradation.

The virtually identical inhibitory effect of chrysin, DCI and NAS on the activation of IRF‐1 points to a redox‐sensitive step in the activation of this transcription factor, which in contrast to NF‐kB requires de novo protein synthesis.

Since iNOS gene expression in human cells and tissues usually requires the combination of several cytokines, antioxidants such as chrysin and NAS which do not interfere with the activation of NF‐kB may be of therapeutic value for selectively inhibiting the enhanced expression of this enzyme in inflammation.
Effects of cyclic GMP elevation on isoprenaline‐induced increase in cyclic AMP and relaxation in rat aortic smooth muscle: role of phosphodiesterase 3
Authors: Delpy Eric , Coste Hervé , de Gouville Anne‐Charlotte le Monnier
Published: July 2012

In rat aortic rings precontracted with phenylephrine, the β‐adrenoceptor agonist isoprenaline (10 nM to 30 μm) produces greater relaxant effects in preparations with endothelium than in endothelium‐denuded preparations. The aim of this study was to determine the mechanisms involved in this effect and in particular investigate the possibility of a synergistic action between adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) and guanosine 3′:5′‐cyclic monophosphate (cyclic GMP).

Isoprenaline‐induced relaxation of rat aortic rings precontracted with phenylephrine was greatly reduced by the nitric oxide (NO) synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 300 μm) or the soluble guanylate cyclase inhibitors methylene blue (10 μm) or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 10 μm) but unaffected by indomethacin (10 μm), a cyclo‐oxygenase inhibitor. Similarly, in intact rings, the concentration‐response curve of forskolin (10 nM to 1 μm) was shifted to the right upon endothelium removal or treatment with methylene blue.

In endothelium‐denuded rat aortic rings, isoprenaline‐induced relaxation was potentiated by the guanylate cyclase activators atrial natriuretic factor (ANF, 1 to 10 nM) and sodium nitroprusside (SNP, 1 to 10 nM), and to a greater extent in the presence of the cyclic GMP‐specific phosphodiesterase (PDE 5) inhibitor, 1,3dimethyl‐6‐(2‐propoxy‐5‐methane sulphonylamidophenyl) pyrazolo [3,4‐d] pyrimidin‐4‐(5H)‐one (DMPPO, 30 nM). Relaxation induced by isoprenaline was also potentiated by the cyclic GMP‐inhibited PDE (PDE 3) inhibitor cilostamide (100 nM).

Intracellular cyclic nucleotide levels were measured either in rat cultured aortic smooth muscle cells or in de‐endothelialized aortic rings. In both types of preparation, isoprenaline (5 nM and 10 μm) increased cyclic AMP levels and this effect was potentiated by cilostamide (10 μm), by rolipram, a cyclic AMP‐specific PDE (PDE 4) inhibitor (10 μm) and by cyclic GMP‐elevating agents (50 nM ANF or 30 nM SNP plus 100 nM DMPPO). In isoprenaline‐stimulated conditions, the increase in cyclic AMP induced by rolipram was further potentiated by cilostamide and by cyclic GMP‐elevating agents. Cilostamide and cyclic GMP‐elevating agents did not potentiate each other, suggesting a similar mechanism of action.

We conclude that in vascular smooth muscle (VSM) cells an increase in cyclic GMP levels may inhibit PDE 3 and, thereby, cyclic AMP catabolism. Under physiological conditions of constitutive NO release, and to a greater extent in the presence of the PDE 5 inhibitor DMPPO, cyclic GMP should act synergistically with adenylate cyclase activators to relax VSM.
The effect of 5‐HT and selective 5‐HT receptor agonists and antagonists on rat dorsal vagal preganglionic neurones in vitro
Authors: Albert Anthony P. , Spyer K. Michael , Brooks Penelope A.
Published: July 2012

Whole‐cell patch‐clamp recordings were made from 142 visually identified rat dorsal vagal preganglionic neurones (DVMs). Applications of 5‐hydroxytryptamine (5‐HT, 20 μm, 2 min) elicited a slow depolarization (8.2 ± 0.5 mV, n = 59) in 95% of the cells tested, accompanied by an increase in excitability. In (68%) of DVMs the depolarization was associated with an increase in apparent membrane resistance (Rm, 22.7 ± 2.2%). These depolarizations and increases in Rm (14.3 ± 2.6%, n = 8) were maintained in a medium which blocked synaptic transmission.

The response to 5‐HT was associated with a reversal potential (Erev) of −91 ± 1 mV at an extracellular K+ concentration ([K+]o) of 4.2 mM. This correlated well with the K+ equilibrium potential (EK = −89 mV).

The depolarizing effect of 5‐HT was attenuated by the 5‐HT2A/2C receptor antagonists, ketanserin (1 μm), LY 53,857 (1 μm) and the 5‐HT1A/2A receptor antagonist, spiperone (1 γM). The 5‐HT1A receptor antagonist, pindobind 5‐HT1A (5 μm), had no effect on the depolarizing response to 5‐HT.

The effect of 5‐HT was mimicked by the 5‐HT2A/2C receptor agonist, α‐methyl‐5‐HT (50 μm), the 5‐HT1 receptor agonist, 5‐carboxamidotryptamine (20 μm) and the putative 5‐HT4 agonist, 5‐methyoxytryptamine (50 μm). The selective 5‐HT4 receptor antagonist, GR113808, had no effect on the depolarizing effect of 5‐HT or 5‐MEOT on DVMs.

The 5‐HT3 antagonists, MDL 72222 (10 μm) and ICS‐205–930 (1 and 10 μm), partially reduced the effect of 5‐HT. The 5‐HT3 receptor agonist, 2‐methyl‐5‐HT (100–300 μm), excited a proportion of neurones tested (56%) by evoking a depolarizing and/or an increase in postsynaptic potentials (p.s.ps).

These results are consistent with direct, postsynaptic actions of 5‐HT on DVMs via 5‐HT2A receptors, being mediated, in part, by the reduction of K+ conductance.
The nitric oxide‐cyclic GMP pathway and synaptic plasticity in the rat superior cervical ganglion
Authors: Southam Eric , Charles Sarah L. , Garthwaite John
Published: July 2012

We have investigated the possibility that nitric oxide (NO) and soluble guanylyl cyclase, an enzyme that synthesizes guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) in response to NO, contributes to plasticity of synaptic transmission in the rat isolated superior cervical ganglion (SCG).

Exposure of ganglia to the NO donor, nitroprusside, caused a concentration‐dependent accumulation of cyclic GMP which was augmented in the presence of the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine. The compound, 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), a selective inhibitor of soluble guanylyl cyclase, completely blocked this cyclic GMP response.

As assessed by extracellular recording, nitroprusside (100 μm) and another NO donor, S‐nitrosoglutathione (30 μm) increased the efficacy of ganglionic synaptic transmission in response to electrical stimulation of the preganglionic nerve, an effect that was reversible and which could be replicated by the cyclic GMP analogue, 8‐bromo‐cyclic GMP. Ganglionic depolarizations resulting from stimulation of nicotinic receptors with carbachol were not increased by nitroprusside. The potentiating actions of the NO donors on synaptic transmission, but not that of 8‐bromo‐cyclic GMP, were inhibited by ODQ.

Brief tetanic stimulation of the preganglionic nerve resulted in a long‐term potentiation (LTP) of synaptic transmission that was unaffected by ODQ, either in the absence or presence of the NO synthase inhibitor, NG‐nitro‐L‐arginine (L‐NOARG, 100 μm). A lack of influence of L‐NOARG was confirmed in intracellular recordings of LTP of the excitatory postsynaptic potential. Furthermore, under conditions where tetanically‐induced LTP was saturated, nitroprusside was still able to potentiate synaptic transmission, as judged from extracellular recording.

We conclude that NO is capable of potentiating ganglionic neurotransmission and this effect is mediated through the stimulation of soluble guanylyl cyclase and the accumulation of cyclic GMP. However, this potentiation is distinct from LTP of nicotinic synaptic transmission, in which neither NO nor soluble guanylyl cyclase appear to participate.
Role of a copper (I)‐dependent enzyme in the anti‐platelet action of S‐nitrosoglutathione
Authors: Gordge M.P. , Hothersall J.S. , Neild G.H. , Dutra A.A. Noronha
Published: July 2012

S‐nitrosoglutathione (GSNO) is a potent and selective anti‐platelet agent, despite the fact that its spontaneous rate of release of nitric oxide (NO) is very slow. Our aim was to investigate the mechanism of the anti‐aggregatory action of GSNO.

The biological action of GSNO could be mediated by NO released from S‐nitrosocystylglycine, following enzymatic cleavage of GSNO by γ‐glutamyl transpeptidase. The anti‐aggregatory potency of GSNO was not, however, altered by treatment of target platelets with the γ‐glutamyl transpeptidase inhibitor acivicin (1 mM). γ‐Glutamyl transpeptidase is not, therefore, involved in mediating the action of GSNO.

The rate of breakdown of S‐nitrosoalbumin was increased from 0.19 ± 0.086 nmol min−1 to 1.52 ± 0.24 nmol min−1 (mean±s.e.mean) in the presence of cysteine (P < 0.05, n = 4). Inhibition of platelet aggregation by S‐nitrosoalbumin was also significantly increased by cysteine (P < 0.05, n = 4), suggesting that the biological activity of S‐nitrosoalbumin is mediated by exchange of NO from the protein carrier to form the unstable compound cysNO. Breakdown of GSNO showed a non‐significant acceleration in the presence of cysteine, from 0.56 ± 0.22 to 1.77 ± 0.27 nmol min−1 (mean±s.e.mean) (P = 0.064, n = 4), and its ability to inhibit platelet aggregation was not enhanced by cysteine. This indicates that the anti‐platelet action of GSNO is not dependent upon transnitrosation to form cysNO.

Platelets pretreated with the copper (I)‐specific chelator bathocuproine disulphonic acid (BCS), then resuspended in BCS‐free buffer, showed resistance to the inhibitory effect of GSNO. These findings suggest that BCS impedes the action of GSNO by binding to structures on the platelet, rather than by chelating free copper in solution.

Release of NO from GSNO was catalysed enzymatically by ultrasonicated platelet suspensions. This enzyme had an apparent Km for GSNO of 12.4 ± 2.64 μm and a Vmax of 0.21 ± 0.03 nmol min−1 per 108 platelets (mean±s.e.mean, n = 5). It was inhibited by BCS, but not by the iron chelator bathophenathroline disulphonic acid, nor by acivicin.

We conclude that the stable S‐nitrosothiol compound GSNO may exert its anti‐platelet action via enzymatic, rather than spontaneous release of NO. This is mediated by a copper‐dependent mechanism. The potency and platelet‐selectivity of GSNO may result from targeted NO release at the platelet surface.
Effects of γ‐aminobutyric acid (GABA) agonists and GABA uptake inhibitors on pharmacosensitive and pharmacoresistant epileptiform activity in vitro
Authors: Pfeiffer Michael , Draguhn Andreas , Meierkord Hartmut , Heinemann Uwe
Published: July 2012

Lowering of the extracellular Mg2+‐concentration induces various patterns of epileptiform activity in combined rat entorhinal cortex‐hippocampal brain slices. After a prolonged period of exposure to Mg2+‐free medium seizure‐like events in the entorhinal cortex change to a state of late recurrent discharges which cannot be blocked by clinically available antiepileptic drugs. This late epileptiform activity thus represents a useful model to test the effects of new anticonvulsant substances.

A mechanism possibly underlying the development of sustained seizure‐like activity is the loss of synaptically released γ‐aminobutyric acid (GABA). Drugs which increase the amount of GABA available in presynaptic endings might thus be useful in the treatment of these therapeutically complicated forms of epilepsy.

Therefore, we studied the effects of various substances increasing GABA‐mediated inhibition on early and late forms of epileptiform activity. GABA and the GABAA receptor agonist muscimol blocked both the pharmacosensitive discharges in the hippocampus and entorhinal cortex as well as the late recurrent discharges in the medial entorhinal cortex. The GABAB receptor agonist baclofen blocked the recurrent short discharges very potently, but did not consistently block seizure‐like events and late recurrent discharges in the entorhinal cortex.

GABA uptake blockers showed a differential potency to block the various discharge patterns. Whereas nipecotic acid and β‐alanine suppressed all forms of epileptiform activity albeit at high concentrations (1–5 mM), tiagabine was much more potent in blocking the hippocampal recurrent short discharges and the seizure‐like events in the medial entorhinal cortex, but could not block the late recurrent discharges.

Our data support the idea that prolonged neuronal overactivity might result in a loss of synaptically available GABA. Selective block of uptake into glia cells or substitution of the transmitter may therefore be an efficient strategy for the treatment of severe prolonged epileptic discharges whereas block of neuronal GABA uptake fails to counteract synchronized discharges in this situation.
Influence of levosimendan, pimobendan, and milrinone on the regional distribution of cardiac output in anaesthetized dogs
Authors: Pagel Paul S. , Hettrick Douglas A. , Warltier David C.
Published: July 2012

The distribution of cardiac output during administration of levosimendan, a new myofilament calcium sensitizer, is unknown. We examined and compared the effects of levosimendan, pimobendan, and milrinone on regional tissue perfusion by use of the radioactive microsphere technique in barbiturate‐anaesthetized dogs.

Haemodynamics and regional blood flow were determined before and during infusions of levosimendan (0.75, 1.5, and 3.0 μg kg−1 min−1), pimobendan (10, 20, and 40 μg kg−1 min−1), or milrinone (1.0, 2.0, and 4.0 μg kg−1 min−1).

All three drugs caused similar increases in heart rate, cardiac output, and left ventricular + dP/dt and decreases in end‐diastolic pressure and systemic vascular resistance. No changes in subendocardial, midmyocardial, and subepicardial blood flow occurred during administration of levosimendan. However, a redistribution of blood flow from subendocardium to subepicardium was observed. Pimobendan increased midmyocardial and subepicardial blood flow and reduced the endo/epi ratio to a greater degree than levosimendan. Milrinone did not affect myocardial perfusion.

Levosimendan increased blood flow to the renal medulla and decreased renal medullary and cortical vascular resistance. Levosimendan increased blood flow to the small intestine and liver and reduced vascular resistance in these organs. Pimobendan increased hepatic blood flow to a greater degree than levosimendan but did not alter small intestinal perfusion. All three drugs decreased splenic blood flow to similar degrees. Levosimendan and pimobendan reduced cerebral vascular resistance. Levosimendan and milrinone reduced skeletal muscle vascular resistance.

The results indicate that levosimendan, pimobendan, and milrinone cause subtlety different alterations in regional tissue perfusion while producing similar haemodynamic effects.
Depression of glutamatergic transmission by nociceptin in the neonatal rat hemisected spinal cord preparation in vitro
Authors: Faber E.S.L. , Chambers J.P. , Evans R.H. , Henderson G.
Published: July 2012

The present study explored the action of nociceptin, the putative endogenous ligand for the orphan opioid receptor (ORL1), on the rat hemisected spinal cord preparation. Electrical stimulation of a dorsal root evokes a glutamatergic population ventral root potential (DR‐VRP) in the corresponding ventral root. Low intensity stimulation evokes two A fibre‐mediated components; a compound action potential of motonuerones superimposed on a population e.p.s.p. (excitatory postsynaptic potential); at higher stimulus intensities sufficient to activate C fibres a more prolonged population e.p.s.p. is evoked. All three components were depressed by nociceptin in a concentration‐dependent manner with IC50 values (s.e.mean) of 119±2 nM (n = 4), 241±3 nM (n = 4) and 32±2 nM (n = 4), respectively. The depressant actions of nociceptin (30 nM and 300 nM) were not reversed by the opioid antagonist naloxone (1 μm). Nociceptin (100 nM and 300 nM) had no effect on the afferent volleys in the dorsal root. Nociceptin therefore appears to be acting as an inhibitory peptide at the spinal level through a naloxone‐insensitive opioid receptor.
Interleukin‐8 production by the human colon epithelial cell line HT‐29: modulation by interleukin‐13
Authors: Kolios George , Robertson Duncan A.F. , Jordan Nicola J. , Minty Adrian , Caput Daniel , Ferrara Pascal , Westwick John
Published: July 2012

We have determined which cytokines induce and modulate the production of the chemokine interleukin‐8 (IL‐8) by the human colonic epithelial cell line HT‐29.

Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin‐1α (IL‐1α), tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), interleukin‐13 (IL‐13), interleukin‐10 (IL‐10) or vehicle added alone or in combination. The production of IL‐8 was determined by enzyme‐linked immunosorbent assay (ELISA) and IL‐8 messenger RNA expression by Northern blot analysis.

The production of IL‐8 in unstimulated cells was undetectable by both ELISA and Northern blot analysis.

HT‐29 cells produced IL‐8 following stimulation with IL‐1α or TNF‐α in a time‐ and a concentration‐dependent manner, while IFN‐γ, IL‐10 and IL‐13 did not induce IL‐8 production by HT‐29 cells.

IL‐13 was found to up‐regulate significantly (P < 0.01) the IL‐1α but not the TNF‐α‐induced IL‐8 generation by HT‐29 cells. In contrast, IL‐10 had no effect on either IL‐1α or TNF‐α‐induced IL‐8 production.

Experiments using cycloheximide demonstrated that this synergistic effect of IL‐13 and IL‐1α on IL‐8 secretion was not through de novo protein synthesis. Using actinomycin‐D, we demonstrated that the IL‐13 up‐regulation was at the level of transcription rather than messenger RNA stability.

These findings suggest that colonic epithelial cells have a functional IL‐13 receptor, which is coupled to an up‐regulation of IL‐1α, but not TNF‐α induced IL‐8 generation.
Analogues of diverse structure are unable to differentiate native melatonin receptors in the chicken retina, sheep pars tuberalis and Xenopus melanophores
Authors: Pickering Hannah , Sword Susan , Vonhoff Stefan , Jones Rob , Sugden David
Published: July 2012

The pineal hormone melatonin exerts its biological effects through specific, high affinity G‐protein coupled receptors. Recently, three melatonin receptor subtypes (Mel1a, Mel1b and Mel1c) have been cloned. Neither the cloned subtypes, nor the native receptors have yet been compared in a detailed pharmacological analysis.

The present study examined the structure‐activity relationships of a series of 21 melatonin analogues, by comparing their potency on the pigment aggregation response in Xenopus laevis melanophores with their affinity in radioligand binding competition studies in chicken retina and sheep pars tuberalis (PT), two tissues in which melatonin is known to mediate a biological response.

All but four of the analogues were full melatonin receptor agonists producing a concentration‐related redistribution of pigment granules in cultured Xenopus melanophores. The remaining analogues produced little pigment aggregation at 10 μm.

Saturation studies with 2‐[125I]‐iodomelatonin identified a single binding site in the chicken retina and sheep PT membranes, with a KD of 36.6 ± 2.8 and 37.3 ± 4.3 pM, and a maximal number of binding sites (Bmax) of 16.6 ± 0.5, and 40.1 ± 1.7 fmol mg−1 protein, respectively.

Comparison of the potency/affinity of the analogues for the binding sites gave a highly significant correlation in each case, retina/melanophore, r = 0.97 (P < 0.001, n = 17), PT/melanophore, r = 0.97 (P < 0.001, n = 17) and PT/retina, r = 0.98 (P < 0.001, n = 21).

Despite their large range in affinity and structural diversity these melatonin agonists were unable to distinguish between melatonin receptors in the chicken retina, sheep pars tuberalis and Xenopus melanophores.
Pharmacological and functional characterization of bradykinin receptors in canine cultured tracheal epithelial cells
Authors: Luo Shue‐Fen , Tsai Chuen‐Tao , Wu Wen‐Bin , Pan Shiow‐Lin , Tsai Yih‐Jeng , Yang Chuen‐Mao
Published: July 2012

A direct [3H]‐bradykinin ([3H]‐BK) binding assay has been used to characterize the BK receptors in canine cultured tracheal epithelial cells (TECs). Based on receptor binding assay, TECs have specific, saturable, high‐affinity binding sites for [3H]‐BK.

The specific [3H]‐BK binding was time‐ and temperature‐dependent. Equilibrium of association of [3H]‐BK with the BK receptors was attained within 30 min at room temperature and 1 h at 4°C, respectively.

Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 1.5 ± 0.2 nM and a maximum receptor density (Bmax) of 53.2 ± 5.2 fmol mg−1 protein. The Hill coefficient for [3H]‐BK binding was 1.00 ± 0.02. The association (K1 and dissociation (K‐1) rate constants were (7.6 ± 1.1) × 106 M−1 min−1 and (9.2 ± 1.5) × 10 M−3 min−1, respectively. KD, calculated from the ratio of K‐1 and K1 was 1.2 ± 0.3 nM, a value close to that calculated from Scatchard plots of binding isotherms.

Neither a B1 receptor selective agonist (des‐Arg9‐BK, 0.1 nM‐10 μm) nor antagonist ([Leu8, des‐Arg9]‐BK, 0.1 nM‐10 μm) significantly inhibited [3H]‐BK binding to TECs, which excludes the presence of B1 receptors in canine TECs.

The specific binding of [3H]‐BK to canine TECs was inhibited by the B2 receptor selective antagonists ([D‐Arg0, Hyp3, Thi5, D‐Tic7, Oic8]‐BK (Hoe 140, 0.1 nM‐10 μm) and [D‐Arg0, Hyp3, Thi5,8, D‐Phe7]‐BK, 0.1 nM‐10 μm) and agonists (BK and kallidin, 0.1 nM‐10 μm) with a best fit by a one‐binding site model. The order of potency for the inhibition of [3H]‐BK binding was kallidin = BK = Hoe 140 > [D‐Arg0, Hyp3, Thi5,8, D‐Phe7]‐BK.

BK and kallidin significantly induced concentration‐dependent accumulation of IPs with a half‐maximal response (EC50) at 17.6 ± 3.5 and 26.6 ± 5.3 nM, respectively, while the B1‐selective agonist, des‐Arg9‐BK did not stimulate IPs accumulation and the B1‐selective antagonist [Leu8, des‐Arg9‐BK did not inhibit BK‐induced IPs accumulation. Two B2‐selective antagonists, Hoe 140 and [D‐Arg0, Hyp3, Thi5,8, D‐Phe7]‐BK, inhibited BK‐stimulated IPs accumulation with apparent pKB values of 8.8 ± 0.3 and 7.0 ± 0.3, respectively.

It is concluded that the pharmacological characteristics of the BK receptors in canine cultured TECs are primarily of the B2 receptor subtype which might regulate the function of tracheal epithelium through the activation of this receptor subtype coupling to PI hydrolysis.
Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase
Authors: MacAllister Raymond J. , Parry Heather , Kimoto Masumi , Ogawa Tadashi , Russell Rachel J. , Hodson Harold , Whitley Guy St.J. , Vallance Patrick
Published: July 2012

Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes the endogenous nitric oxide synthase inhibitors NG‐monomethyl‐L‐arginine and NG, NG‐dimethy‐L‐arginine to citrulline, was identified by Western blotting in rat and human tissue homogenates.

S‐2‐amino‐4(3‐methylguanidino)butanoic acid (4124W) inhibited the metabolism of [14C]‐NG‐monomethyl‐L‐arginine to [14C]‐citrulline by rat liver homogenates (IC50 416 ± 66μm; n = 9), human cultured endothelial cells (IC50 250 ± 34 μm; n = 9)and isolated purified dimethylarginine dimethylaminohydrolase.

Addition of 4124W to culture medium increased the accumulation of endogenously‐generated NG, NG‐dimethy‐L‐arginine in the supernatant of human cultured endothelial cells from 3.1 ± 0.3 to 5 ± 0.7 μm (n=15; P < 0.005).

4124W (1 μm‐1 mM) had no direct effect on endothelial nitric oxide synthase activity but caused endothelium‐dependent contraction of rat aortic rings (1 mM 4124W increased tone by 81.5 ± 9.6% of that caused by phenylephrine 100 nM). This effect was reversed by L‐arginine (100 μm). 4124W reversed endothelium‐dependent relaxation of human saphenous vein (19.2 ± 6.7% reversal of bradykinin‐induced relaxation at 1 mM 4124W).

These data suggest that inhibition of dimethylarginine dimethylaminohydrolase increases the intracellular concentration of NG, NG‐dimethyl‐L‐arginine sufficiently to inhibit nitric oxide synthesis. Inhibiting the activity of DDAH may provide an alternative mechanism for inhibition of nitric oxide synthases and changes in the activity of DDAH could contribute to pathophysiological alterations in NO generation.
Analysis of relaxation and repolarization mechanisms of nicorandil in rat mesenteric artery
Authors: Fujiwara Toshimasa , Angus Jarnes A.
Published: July 2012

The mechanism by which nicorandil causes relaxation of rat isolated small mesenteric arteries mounted on a Mulvany myograph was investigated by use of a combination of putatively mechanism‐specific antagonists.

In arteries precontracted by the thromboxane‐mimetic, U46619, the EC50 for cromakalim and levcromakalim‐induced relaxation curves were raised by 36 and 17 fold by glibenclamide (3 μm) while the EC50 for nicorandil‐induced relaxation was unaffected by either glibenclamide or methylene blue (10 μm). A combination of these antagonists raised the EC50 for nicorandil by 8 fold.

In U46619‐contracted arteries, nifedipine (100 nM) did not affect the cromakalim relaxation curve but it raised the EC50 for nicorandil by 5 fold. The combination of methylene blue, glibenclamide and nifedipine further inhibited the maximum relaxation to nicorandil.

In separate experiments, membrane potential (Em) and force responses were measured simultaneously. In arteries depolarized and contracted by U46619 both nicorandil and cromakalim repolarized (δEm, 35mV) and relaxed (100%) the vessels. Glibenclamide (3μm) did not alter the relaxation‐concentration curve to nicorandil but reduced the maximum repolarization to A10.8 mV. In contrast the Em and relaxation‐response curves to cromakalim were shifted to the right by glibenclamide by 30–100 fold.

In unstimulated arteries, nicorandil (but not cromakalim) ‐induced hyperpolarization was significantly antagonized by methylene blue (10 μm) (6.6 fold rightward shift) or nifedipine (100 nM) (2.6 fold). In depolarized arteries (U46619), nifedipine but not methylene blue inhibited the nicorandil‐induced hyperpolarization.

In arteries precontracted to 50% tissue maximum by either KCl or U46619, nifedipine (100 nM) relaxed the artery but failed to repolarize the Em. Presumably voltage‐operated calcium channels (VOCC) were blocked preventing contraction but the artery remained depolarized, presumably through non VOCC mechanisms.

These data suggest that nicorandil may relax small arteries through 3 parallel pathways, (i) NO‐donor mediated stimulation of guanylate cyclase and increase in cyclic GMP, (ii) K+ATP channel opening, and (iii) nifedipine‐sensitive VOCC inhibition. Em data suggest that nicorandil‐induced repolarization is caused principally through opening K+ATP channels. Blockade of this hyperpolarization by glibenclamide is not sufficient to alter the relaxation, indicating dissociation of nicorandil‐induced changes in membrane potential and relaxation.

These results highlight the ‘chameleon’ actions of nicorandil where there is no apparent association of Em repolarization with relaxation, in contrast to the parallel responses for cromakalim.
A possible structural determinant of selectivity of boldine and derivatives for the α1A‐adrenoceptor subtype
Authors: Madrero Y. , Elorriaga M. , Martinez S. , Noguera M.A. , Cassels B.K. , D'Ocon P. , Ivorra M.D.
Published: July 2012

The selectivity of action of boldine and the related aporphine alkaloids, predicentrine (9‐O‐methylboldine) and glaucine (2,9‐O‐dimethylboldine) on α1‐adrenoceptor subtypes was studied by examining [3H]‐prazosin competition binding in rat cerebral cortex. WB 4101 and benoxathian were used as selective α1A‐adrenoceptor antagonists.

In the competition experiments [3H]‐prazosin (0.2 nM) binding was inhibited by WB 4101 and benoxathian. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components (pKi = 9.92 and 8.29 for WB 4101, 9.35 and 7.94 for benoxathian). The two antagonists recognized approximately 37% of the sites with high affinity from among the total [3H]‐prazosin specific binding sites.

Boldine, predicentrine and glaucine also competed for [3H]‐prazosin (0.2 nM) binding with shallow and biphasic curves recognizing 30–40% of the sites with high affinity. Drug affinities (pKi) at the high and low affinity sites were, 8.31 and 6.50, respectively, for boldine, 8.13 and 6.39 for predicentrine, and 7.12 and 5.92 for glaucine. The relative order of selectivity for α1A‐adrenoceptors was boldine (70 fold α1A‐selective) = predicentrine (60 fold, α1A‐selective) > glaucine (15 fold, α1A‐selective).

Pretreatment of rat cerebral cortex membranes with chloroethylclonidine (CEC, 10 μm) for 30 min at 37°C followed by thorough washing out reduced specific [3H]‐prazosin binding by approximately 70%. The CEC‐insensitive [3H]‐prazosin binding was inhibited by boldine monophasically (Hill slope = 0.93) with a single pKi value (7.76).

These results suggest that whereas the aporphine structure shared by these alkaloids is responsible for their selectivity of action for the a α1A‐adrenoceptor subtype in rat cerebral cortex, defined functional groups, namely the 2‐hydroxy function, induces a significant increase in α1A‐subtype selectivity and affinity.
A cyclic GMP‐dependent housekeeping Cl− channel in rabbit gastric parietal cells activated by a vasodilator ecabapide
Authors: Sakai Hideki , Ikari Akira , Kumano Eiichi , Takeguchi Noriaki
Published: July 2012

The membrane potential of rabbit gastric parietal cells is dominated by a Cl− channel with a sub‐picosiemens single channel conductance in the basolateral membrane. The effects of 3‐[[[2‐(3,4‐dimethoxyphenyl)ethyl]carbamoyl]methyl]amino‐N‐methylbenzamide (DQ‐2511: ecabapide), a vasodilator, on the opening of this Cl− channel, the cyclic GMP content and the intracellular free Ca2+ concentration ([Ca2+]i) of parietal cells were investigated by whole‐cell patch‐clamp technique, enzyme immunoassay and Fura 2‐fluorescence measurement.

Ecabapide stimulated the opening of the Cl− channel as determined by the reversal potential. This stimulation was concentration‐dependent, and its EC50 value was 0.2 μm. Both the basal and ecabapide‐induced openings of the channel were inhibited by 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB, 500 μm), a Cl− channel blocker. Another Cl− channel blocker, niflumic acid (500 μm) was much less effective.

The power spectra of the currents before and after the addition of ecabapide (10 μm) were analysed. Both spectra contained only one Lorentzian (1/f2) component.

6‐Anilino‐5,8‐quinolinedione (LY83583; 5 μm), which prevents activation of soluble guanylate cyclase, significantly inhibited both the basal and ecabapide (10 μm)‐induced openings of the Cl− channel.

Ecabapide (0.01 −100 μm) concentration‐dependently elevated the cyclic GMP content in the parietal cell‐rich suspension. The EC50 value was 0.2 μm.

In single Fura 2‐loaded parietal cells, ecabapide (10–100 μm) did not increase [Ca2+]i.

These results indicate that ecabapide stimulates an intracellular production of cyclic GMP in the parietal cell without increasing [Ca2+]i, and leads to an activation of the housekeeping Cl− channel.
Inhibition of ecto‐ATPase by PPADS, suramin and reactive blue in endothelial cells, C6 glioma cells and RAW 264.7 macrophages
Authors: Chen Bin C. , Lee Chii‐M. , Lin Wan‐W.
Published: July 2012

Previous studies have shown that bovine pulmonary artery endothelium (CPAE) has P2Y and P2U purinoceptors, rat C6 glioma cells have P2u purinoceptors and mouse RAW 264.7 cells have pyrimidinoceptors, all of which are coupled to phosphoinositide‐specific phospholipase C (PI‐PLC). The dual actions of PPADS, suramin and reactive blue as antagonists of receptor subtypes and ecto‐ATPase inhibitors were studied in these three cell types.

In CPAE, suramin, at 3–100 μm, competitively inhibited the PI responses induced by 2MeSATP and UTP, with pA2 values of 5.5 ± 0.3 and 4.4 ± 0.4, respectively. Reactive blue, at 1–3 μm, produced shifts to the right of the 2MeSATP and UTP curves, but no further right shift at 10 μm. PPADS, at 10 μm, caused a 3 fold right shift of the 2MeSATP curve, but no further shift at concentrations up to 100 μm. In contrast, a dose‐dependent shift to the left of the UTP curve and a weak inhibition of the ATP response were seen with PPADS.

In RAW 264.7 cells, suramin and reactive blue, but not PPADS, competitively inhibited the UTP response, with pA2 values of 4.8 ± 0.5 and 5.8 ± 0.7, respectively.

In C6 glioma cells, although suramin and reactive blue inhibited the ATP response, a potentiation effect on ATP and UTP responses was seen with PPADS.

The ecto‐ATPase inhibitory activity of these three receptor antagonists were determined. All three inhibited ecto‐ATPase present in CPAE, C6 and RAW 264.7 cells, with IC50 values of 4, 4.8 and 4.7 for PPADS, 4, 4.4 and > > 4 for suramin, and 4.5, 4.7 and 4.7 for reactive blue.

This study indicates that PPADS, suramin and reactive blue are ecto‐ATPase inhibitors. This property, combined with their antagonistic selectivity for receptor subtypes, can result in inhibition of, potentiation of, or lack of effect on agonist‐mediated PI responses. Reactive blue is a more potent antagonist than suramin on P2Y, P2U and pyrimidinoceptors, and PPADS is a weak antagonist for P2Y receptors.
Functional, endogenously expressed 5‐hydroxytryptamine 5‐ht7 receptors in human vascular smooth muscle cells
Authors: Schoeffter P. , Ullmer C. , Bobirnac I. , Gabbiani G. , Lübbert H.
Published: July 2012

Human uterine artery smooth muscle cells in culture were shown to express constitutively both 5‐ht7 receptor mRNA and 5‐ht7‐like receptors functionally linked to cyclic AMP formation. 5‐Carboxamidotryptamine (5‐CT) and 5‐HT enhanced forskolin‐stimulated cyclic AMP accumulation in these cells, with pEC50 values of 7.12 and 6.25, sumatriptan being very weakly active. Both methiothepin (0.1 μm) and clozapine (1 μm), but not the 5‐HT4‐receptor antagonist, SDZ 205–557 (10 μm) antagonized the effects of 5‐CT. In reverse transcriptase‐polymerase chain reaction analysis, the mRNA for 5‐ht7, but not for 5‐HT4 or 5‐ht6 receptors was found to be strongly expressed in the same cells. These findings represent a further step toward the recognition of 5‐ht7 receptors as real, functional receptors.
Early Exercise of Put Options on Stocks
Authors: BARRACLOUGH KATHRYN , WHALEY ROBERT E.
Published: July 2012

U.S. exchange‐traded stock options are exercisable before expiration. While put options should frequently be exercised early to earn interest, they are not. In this paper, we derive an early exercise decision rule and then examine actual exercise behavior during the period January 1996 through September 2008. We find that more than 3.96 million puts that should have been exercised early remain unexercised, representing over 3.7% of all outstanding puts. We also find that failure to exercise cost put option holders $1.9 billion in forgone interest income and that this interest is systematically captured by market makers and proprietary firms.
Nurse‐Physician Relationships in Ambulatory Oncology Settings
Authors: Friese Christopher R. , Manojlovich Milisa
Published: July 2012

Purpose: The purpose of this study was to explore nurses’ perceptions of nurse‐physician relationships in ambulatory oncology settings, which are linked to patient safety.

Design: This cross‐sectional, descriptive study analyzed survey data collected in 2010 from oncology nurses employed in ambulatory settings. The sampling frame was the nurse licensure database in one state in the Southeastern United States. Nurses completed the Practice Environment Scale of the Nursing Work Index (PES‐NWI), reported on the quality of care in their setting, and commented on factors that promoted or inhibited high‐quality care delivery.

Methods: Data analysis used three study variables: empirically derived values from the PES‐NWI, a scale of nurse‐reported quality of care in their setting, and open‐text comments about features in their workplace that promoted or hindered high‐quality care. After categorizing open‐text comments, analysis of variance was used to evaluate differences in PES‐NWI subscales by comment category. Chi‐square test statistics were calculated to examine differences in overall practice environment and quality of care by comment category.

Results: Nurses reported their relationships with physicians as generally favorable. Qualitative findings suggest two themes that influence how nurses characterize their working relationships with physicians: (a) physician behaviors and (b) structural factors. Both PES‐NWI scores and quality of care were rated significantly higher by nurses who wrote favorably about physicians.

Conclusions: Favorable nurse‐physician relationships in ambulatory settings may reflect positive workplaces and promote high‐quality care.

Clinical Relevance: Consistent with findings from inpatient units, nurse‐physician relationships are important to the quality of ambulatory oncology care. Systematic measurement and attention to reported deficits in these relationships may promote higher quality care.

Journal of Nursing Scholarship, 2012; 00:0, 1–8. ©2012 Sigma Theta Tau International.
Controllable Synthesis of Layered Zircons by Low‐Temperature Hydrothermal Method
Authors: Lei Binglong , Peng Cheng , Wu Jianqing
Published: July 2012

Series of intensive investigations into porous, morphologically distinctive and well‐defined zircons have been conducted. Single‐to‐multiple layered zircons were controllably synthesized for the first time by modulating parameters of hydrothermal duration, pH, and concentration under low‐temperature hydrothermal conditions. The layered zircons possess high specific surface area and micro‐to‐meso pore structure, which make them suitable candidates for catalyst supports applied at high temperature. The larger d‐spacing and the shift of the crystal plane (200) of these hydrothermal zircons are interpreted mainly for the structural incorporation of fluorine ions and OH groups.
Secular trends in impact factor of neonatology publications over a 10‐year period
Authors: Marom Ronella , Mimouni Francis B. , Cohen Shlomi , Lubetzky Ronit , Mandel Dror
Published: July 2012

Aim:  To test the hypotheses that published randomized clinical trials (RCTs) in neonatology with negative results (NR) are more likely to be published in journals with lower impact factor (IF) than those with positive results (PR); that there is an increase in the number of yearly published RCTs; that studies with large sample sizes are likely to be published in journals with higher IF.

Methods:  We used all English‐written RCTs registered in MEDLINE between 1/1/2001–31/12/2010 in the field of neonatology. Each RCT was classified as having a PR or NR. IF of each journal was determined for the year of publication.

Results:  We identified 329 RCTs. Yearly number of RCTs varied between 19 and 46, with no significant consistent linear increase over the years. There was no significant change over the years in average IF or in average patient size. IF and sample size of the studies were not significantly higher in studies with PR than in studies with NR.

Conclusion:  The number of RCTs per year in the field of neonatology has stabilized in the past 10 years, and RCTs with positive or negative results are published in journals of similar IF.
Managing HIV/AIDS: Yunnan's Government-driven, Multi-sector Partnership Model
Authors: Wang Mei-Ling
Published: July 2012

This article seeks to unravel the management model used by China's Yunnan Province in its fight against HIV/AIDS. This government-driven, multi-sector partnership model has led to some capacity building and stabilization of HIV transmission among certain at-risk populations. The organizational strategies employed in this model might be useful for resource-poor countries in containing the spread of HIV/AIDS.
Roles of heterotypic CCN2/CTGF–CCN3/NOV and homotypic CCN2–CCN2 interactions in expression of the differentiated phenotype of chondrocytes
Authors: Hoshijima Mitsuhiro , Hattori Takako , Aoyama Eriko , Nishida Takashi , Yamashiro Takashi , Takigawa Masaharu
Published: July 2012

To identify proteins that regulate CCN2 activity, we carried out GAL4‐based yeast two‐hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS‐2/8. CCN2/CTGF and CCN3/NOV polypeptides were picked up as CCN2‐binding proteins, and CCN2–CCN2 and CCN2–CCN3 binding domains were identified. Direct binding between CCN2 and CCN3 was confirmed by coimmunoprecipitation in vitro and in vivo and surface plasmon resonance, and the calculated dissociation constants (Kd) were 1.17 × 10−9 m for CCN2 and CCN2, and 1.95 × 10−9 m for CCN2 and CCN3. Ectopically overexpressed green fluorescent protein–CCN2 and Halo–CCN3 in COS7 cells colocalized, as determined by direct fluorescence analysis. We present evidence that CCN2–CCN3 interactions modulated CCN2 activity such as enhancement of ACAN and col2a1 expression. Curiously, CCN2 enhanced, whereas CCN3 inhibited, the expression of aggrecan and col2a1 mRNA in HCS‐2/8 cells, and combined treatment with CCN2 and CCN3 abolished the inhibitory effect of CCN3. These effects were neutralized with an antibody against the von Willebrand factor type C domain of CCN2 (11H3). This antibody diminished the binding between CCN2 and CCN2, but enhanced that between CCN3 and CCN2. Our results suggest that CCN2 could form homotypic and heterotypic dimers with CCN2 and CCN3, respectively. Strengthening the binding between CCN2 and CCN3 with the 11H3 antibody had an enhancing effect on aggrecan expression in chondrocytes, suggesting that CCN2 had an antagonizing effect by binding to CCN3.
Integrated Reporting, Stakeholder Engagement, and Balanced Investing at American Electric Power
Authors: Parrot Katherine W. , Tierney Brian X.
Published: July 2012

The policies and practices of American Electric Power (AEP) encompass a number of paradoxes in the domain of sustainability. AEP is a large electric power provider with a predominantly coal‐fired power generation portfolio, which puts the company squarely in the center of national debates about global climate change and national air quality. At the same time, AEP is also a leader on several social and environmental fronts: integrated reporting, stakeholder engagement, technology innovation, and policy solutions for climate change.

In this article, the authors describe AEP's rationale for providing leadership in these areas and then explore how the company tries to balance stakeholder interests and financial, environmental, and social concerns in its capital investment decisions. Using these examples, the authors expand on and discuss the limitations of Michael Jensen's theory of “enlightened value maximization.”
How Does Sustainability Disclosure Drive Behavior Change?
Authors: Topping Nigel
Published: July 2012

In 2011, as part of the Carbon Disclosure Project (CDP), over 3,700 companies disclosed information about their energy use, emissions, risks, opportunities, and strategy to their institutional investors and customers. In this article, the Chief Innovation Officer of CDP discusses five ways that such disclosure is expected to lead to changes in corporate behavior.

The first two are “internal” mechanisms that tend to encourage constructive change more or less directly as a result of going through the process of disclosure. One is known as “WGMGM,” which is shorthand for the often‐cited principle that “what gets measured gets managed.” The second is the tendency of disclosure to bring about valuable changes in strategic thinking.

The next two are mechanisms that work to effect change through “external” channels. One relies on the use of data to enable investors, regulators, and other corporate stakeholders to draw comparisons among companies and industries in assessing the value implications of climate change disclosure. The second involves competitor benchmarking and the growing use of data in investment research.

Fifth and last, recent evidence suggests that a growing number of investors are willing to raise the bar in terms of the expectations they place on companies to act responsibly now in the face of current and expected future changes.

Each of these five change processes are discussed while citing evidence drawn from CDP's long experience of managing the disclosure process and working with both investors and companies to convert data into action.
Performance Measurement for Nonprofits
Authors: Glassman David M. , Spahn Kathy
Published: July 2012

The capital market for financing charitable work is far from efficient, and could be improved dramatically. Donors and the nonprofit agencies they fund do not share a standard set of measures and procedures to evaluate performance. Lacking measures of impact, donors—a group that includes government agencies, foundations, corporate CSR programs, and individuals—and charity “watchdogs” often focus on available information that typically has one main focus: the overhead expenses reported by nonprofits in public disclosures.

The problem, however, is that the use of overhead expense as a proxy for operational efficiency and effectiveness has many shortcomings. There is no shared understanding of what expenses constitute overhead; overhead spending says nothing about the quality of a nonprofit's programs; and an excessive focus on this single dimension can cause management to underinvest in infrastructure.

Management of nonprofits should be encouraged to communicate the results of their key programs—in particular, their impact on the communities they serve. For example, for at least their largest programs, nonprofits could describe the key performance indicators established at outset of their programs, and how they are currently performing against these benchmarks. A substantive management discussion that includes an explanation for any underperformance, and the corrective actions being taken, would help other agencies avoid similar pitfalls while providing potential donors with valuable information that improves not only their decision‐making, but the “efficiency” of the entire market for charitable giving.