bluuebunny/crossref_metadata_2025 huggingface dataset:
Aggregation of pigment granules in single cultured Xenopus laevis melanophores by melatonin analogues
Authors: Sugden D.
Published: July 2012

Isolated melanophores were differentiated from aggregates of neural crest obtained from neurula stage Xenopus laevis embryos after 2 days in culture.

Condensation of pigment granules in these cells by melatonin (5‐methoxy N‐acetyltryptamine, aMT) and various novel analogues was monitored with an image analysis system to quantitate the area occupied by pigment in individual cells.

Melanophores exposed to vehicle (a maximum of 0.1% MeOH) showed little (< 5%) change in pigment area. aMT produced a dramatic condensation of pigment granules (EC50 = the concentration producing a half maximal condensation, 9 pm). The response was rapid, reached a maximum (∼80% decrease in pigmented area) by 10 min, and was reversible after removal of aMT from the culture medium.

Aggregation to aMT was blocked by treating melanophores with pertussis toxin (1 μg ml−1, 7h) indicating a role for a guanosine 5′ triphosphate (GTP)‐binding protein in transducing the aMT receptor signal.

Structure‐activity studies indicated that analogues of aMT lacking a side‐chain N‐acyl substituent (5‐methoxytryptamine, MT) or a group at the 5‐position of the indole ring (N‐acetyltryptamine, aT) were unable to induce pigment aggregation (EC50 > 10 μm).

Lengthening the side‐chain N‐acyl group (N‐propionyl, N‐butanoyl) was tolerated to some degree but eventually (N‐valeroyl and larger) activity diminished. Of the 5‐position analogues tested 5‐methoxy (aMT) was by far the most potent.

Halogen substitution in the 6‐position of the indole ring led to some loss of activity as did a 6‐OH substitution. The 6‐OCH3 compound was inactive.

These studies demonstrate the utility of this model in investigations of structure‐activity relationships at the aMT receptor and suggest that it may be a valuable system for determining the transduction mechanisms coupled to the aMT receptor.
Effects of PAF‐antagonists in mouse ear oedema induced by several inflammatory agents
Authors: Merlos M. , Gómez L.A. , Giral M. , Vericat M.L. , García‐Rafanell J. , Forn J.
Published: July 2012

Several platelet activating factor (PAF)‐antagonists of different chemical structures were tested in the arachidonic acid‐, tetradecanoylphorbol acetate‐, dithranol‐, and benzoic acid‐induced mouse ear oedema models.

Topical application of UR‐10324, UR‐11353, CV‐6209 and WEB‐2086 markedly inhibited ear oedema induced by the four irritants tested, mimicking the profile obtained with dexamethasone. YM‐461 was highly effective only in the dithranol‐induced ear oedema, while BN‐52021 failed to inhibit ear oedema in all models tested.

Leukocyte recruitment into the inflamed ears was prevented by PAF‐antagonists, as measured by myeloperoxidase activity in the supernatants of ear homogenates.

A relationship between PAF‐antagonist and anti‐inflammatory activities was found in some cases, but other mechanisms cannot be excluded to explain the topical anti‐inflammatory effect of these compounds.

Our results suggest that topical formulations containing PAF‐antagonists could be useful in the treatment of some inflammatory skin diseases and provide evidence on the involvement of PAF in these inflammatory processes.
Effects of vasoactive intestinal peptide, helodermin and galanin on responses of guinea‐pig lung parenchyma to histamine, acetylcholine and leukotriene D4
Authors: Conroy Dolores M. , Samhoun Marwa N. , Piper Priscilla J.
Published: July 2012

The effect of vasoactive intestinal peptide (VIP) was studied on the contractile response of guinea‐pig lung parenchymal strips (GPP) induced by bronchoconstrictor agonists, such as leukotriene D4 (LTD4), histamine and acetylcholine (ACh). This effect of VIP was compared with helodermin, a peptide that is structurally related to VIP, and galanin, another neuropeptide that is thought to co‐exist with VIP.

VIP (10 nm) induced a potent and reversible inhibition of the contractions of GPP induced by LTD4 (1–30 pmol) but did not affect those due to ACh (1–100 nmol) or histamine (1–30 nmol). A ten fold higher concentration of VIP (100 nm) did not further inhibit LTD4‐induced responses or reduce those induced by histamine or ACh.

Helodermin (10 nm) had a similar inhibitory effect on contractions of GPP induced by LTD4 (3–30 pmol) but did not affect contractions induced by histamine (1–10 nmol).

Indomethacin (2.8 μm) and salbutamol (10 nm) significantly reduced responses elicited by LTD4 and histamine but not those due to ACh. A ten fold higher concentration of salbutamol (100 nm) further inhibited the contractions due to LTD4 and histamine and at this concentration responses induced by ACh were inhibited.

VIP (10 nm) and helodermin (10 nm) significantly reduced the LTD4‐induced release of thromboxane A2 (TXA2), measured as TxB2 by radioimmunoassay, from GPP. The smaller release of TxA2 induced by histamine was not significantly reduced in the presence of VIP.

In comparative studies, galanin (10–100 nm) did not affect contractions of GPP induced by either LTD4, histamine or ACh. In contrast to VIP and helodermin, both at 0.1–3 nmol, which induced dose‐related relaxations of guinea‐pig trachea, galanin was inactive on this preparation in doses of up to 3 nmol.

In conclusion, our results show that contractions of GPP induced by LTD4 are more sensitive to inhibition by VIP and helodermin than are contractions due to histamine or ACh. This inhibition appears to be associated with the different contribution of released TxA2 to contractions evoked by the agonists. VIP and helodermin inhibit the cyclo‐oxygenase‐dependent component of the LTD4‐induced response, as in the case of indomethacin.
Endothelin‐1 inhibits pre‐stimulated tracheal submucosal gland secretion and epithelial albumin transport
Authors: Yurdakos E. , Webber S.E.
Published: July 2012

Endothelin‐1 potently contracts smooth muscle, including that in the airways. However, its effect on airway mucosal function has not so far been studied.

We have used the ferret whole trachea in vitro to examine the effect of endothelin‐1 on tracheal smooth muscle tone, transepithelial potential difference (p.d.), submucosal gland secretion (including lysozyme secretion from serous cells) and active epithelial albumin transport. In addition we have examined the effects of endothelin on submucosal gland secretion and albumin transport pre‐stimulated with the muscarinic agonist methacholine and the α‐adrenoceptor agonist phenylephrine. The effects of the Ca2+ channel blocker nifedipine on the responses to endothelin have also been assessed.

Endothelin (0.1–100 nm) produced concentration‐dependent increases in intraluminal tracheal pressure indicating smooth muscle contraction, and in the negativity of the transepithelial p.d. These effects were partially inhibited by nifedipine (10 μm).

Endothelin (0.01–100 nm) had no significant effect on baseline rates of mucus, lysozyme or albumin outputs, but produced concentration‐dependent reductions in maintained methacholine‐ and phenylephrine‐induced mucus, lysozyme and albumin outputs. In general endothelin was more potent against methacholine‐induced effects. All of the concentration‐response curves for endothelin were shallow and some appeared to be biphasic, suggesting the possibility of more than one mechanism of action of endothelin.

The effects of endothelin (at concentrations > 1 nm) on phenylephrine‐induced mucus volume, lysozyme and albumin outputs were significantly inhibited by nifedipine. Similarly the effect of endothelin (> 1 nm) on methacholine‐induced mucus volume and albumin outputs (but not lysozyme output) was attenuated by nifedipine. The effects of endothelin (at concentrations < 1 nm) on methacholine and phenylephrine‐induced responses were generally not affected by nifedipine.

Thus, endothelin contracts ferret tracheal smooth muscle and increases transepithelial p.d. at least in part by opening dihydropyridine‐sensitive Ca2+ channels. Endothelin does not directly stimulate submucosal gland secretion or epithelial albumin transport, but inhibits methacholine‐ and phenylephrine‐induced secretion and transport. The inhibitory effects produced by higher concentrations of endothelin may be mediated partially by activation of dihydropyridine‐sensitive Ca2+ channels, although the explanation for this is not clear. The mechanism of action of endothelin in attenuating stimulated secretion and epithelial transport at lower concentrations is unknown.;
Human big‐endothelin‐1 and endothelin‐1 release prostacyclin via the activation of ET1 receptors in the rat perfused lung
Authors: D'Orléans‐Juste Pedro , Télémaque Sabine , Claing Audrey , Ihara Masaki , Yano Mitsuo
Published: July 2012

Although ET1 and ET2 binding sites were found in rat lung membranes, a selective ET1 receptor antagonist, BQ‐123 (10 μm), did not displace [125I]‐endothelin‐1 ([125I]ET‐1) from ET2 sites, illustrating the selectivity of the angatonist for ET1 receptors. In rat perfused lungs, BQ‐123 (1 μm) markedly reduced the prostacyclin (PGI2) releasing properties of endothelin‐1 (ET‐1: 5 nm) and human big‐ET‐1 (100 nm) suggesting that both peptides induce the release of PGI2 via the selective activation of ET1 receptors.
Characterization of a novel aquaretic agent, OPC‐31260, as an orally effective, nonpeptide vasopressin V2 receptor antagonist
Authors: Yamamura Yoshitaka , Ogawa Hidenori , Yamashita Hiroshi , Chihara Tomihiko , Miyamoto Hisashi , Nakamura Shigeki , Onogawa Toshiyuki , Yamashita Tatsuya , Hosokawa Tetsumi , Mori Toyoki , Tominaga Michiaki , Yabuuchi Youichi
Published: July 2012

OPC‐31260, a benzazepine derivative, has been studied for its ability to antagonize the binding of arginine vasopressin (AVP) to receptors in rat liver (V1) and kidney (V2) plasma membranes, for antagonism of the antidiuretic action of AVP in alcohol‐anaesthetized rats and for diuretic action in conscious normal rats.

OPC‐31260 caused a competitive displacement of [3H]‐AVP binding to both V1 and V2 receptors with IC50 values of 1.2 ± 0.2 × 10−6 m and 1.4 ± 0.2 × 10−8 m, respectively.

OPC‐31260 at doses of 10 to 100 μg kg−1, i.v., inhibited the antidiuretic action of exogenously administered AVP in water‐loaded, alcohol‐anaesthetized rats in a dose‐dependent manner. OPC‐31260 did not exert an antidiuretic activity suggesting that it is not a partial V2 receptor agonist.

After oral administration at doses of 1 to 30 mg kg−1 in normal conscious rats, OPC‐31260 dose‐dependently increased urine flow and decreased urine osmolality. The diuretic action of OPC‐31260 was characterized as aquaresis, the mode of diuretic action being different from previously known diuretic agents such as furosemide, hydrochlorothiazide and spironolactone.

The results indicate that OPC‐31260 is a selective V2 receptor antagonist and behaves as an aquaretic agent. OPC‐31260 will be a useful tool in studying the physiological role of AVP and in the treatment of various conditions characterized by water retention.
Sodium‐dependence and ouabain‐sensitivity of the synthesis of dopamine in renal tissues of the rat
Authors: Soares‐da‐Silva P. , Fernandes M. Helena
Published: July 2012

The present study has examined the influence of sodium chloride (0–160 mm) and ouabain (100 and 500 μm), an inhibitor of the enzyme Na+–K+ ATPase, on the synthesis of dopamine in slices of rat renal cortex loaded with exogenous l‐dihydroxyphenylalanine (l‐DOPA). The deamination of newly‐formed dopamine into 3,4‐dihydroxyphenylacetic acid (DOPAC) was also examined. The assay of l‐DOPA, dopamine and DOPAC in kidney slices was performed by high performance liquid chromatography (h.p.l.c.). with electrochemical detection.

The accumulation of newly‐formed dopamine and DOPAC in kidney slices loaded with l‐DOPA (50 and 100 μm) was found to be dependent on the concentration of NaCl in the medium. A similar picture could be observed for DOPAC. The fractional rate of accumulation (k; mm NaCl−1) was at 50 and 100 μm l‐DOPA, respectively, 0.00305 ± 0.00036 and 0.00328 ± 0.00029 for dopamine and 0.00672 ± 0.00072 and 0.00641 ±0.00069 for DOPAC. The sodium‐dependent formation of dopamine was completely abolished when the experiments were performed in the absence of oxygen.

In experiments performed in the presence of 120 mm NaCl, but not in conditions of low sodium (20 mm NaCl in the medium), ouabain (100 and 500 μm) was found to inhibit the accumulation of newly‐formed dopamine and DOPAC (14–57% reduction; P < 0.05); this effect was more marked at 50 and 100 μm l‐DOPA. When the experiments were performed in the absence of oxygen the renal production of dopamine and DOPAC became markedly decreased (respectively, 40% and 77% reduction; P < 0.05) and ouabain (100 and 500 μm) was no longer able to reduce the accumulation of both newly‐formed dopamine and DOPAC.

The formation of dopamine and its deamination to DOPAC in kidney homogenates closely depended on the concentration of l‐DOPA added to the medium; ouabain (100, 500 and 1000 μm) was found to affect neither the formation of dopamine nor its deamination to DOPAC when homogenates were used instead of kidney slices.

In conclusion, the results presented here show that the formation of dopamine in kidney slices loaded with l‐DOPA is sodium‐dependent and ouabain‐sensitive suggesting the involvement of a co‐transport system of sodium and DOPA into the tubular epithelial cell.
The effects of acute and repeated nicotine treatment on nucleus accumbens dopamine and locomotor activity
Authors: Benwell Maureen E.M. , Balfour David J.K.
Published: July 2012

The effects of acute and subchronic nicotine and (+)‐amphetamine on the extracellular levels of dopamine and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in nucleus accumbens (NAc) have been studied in conscious, freely‐moving rats by use of in vivo microdialysis.

In rats which had been habituated to the test apparatus for approximately 80 min, the acute subcutaneous (s.c.) administration of nicotine (0.1 or 0.4 mg kg−1) caused a dose‐dependent increase (P < 0.01) in spontaneous activity and evoked significant increases (P < 0.05) in the extracellular levels of DOPAC and HVA.

Measurements made 24 h after the last injection of nicotine showed that pretreatment with the higher doses tested (0.4 mg kg−1) resulted in increased basal levels of dopamine (P < 0.01) and decreased basal levels of DOPAC (P < 0.05) in the NAc dialysates.

Pretreatment with nicotine (0.1 or 0.4 mg kg−1 daily for 5 days) enhanced the effects of the drug on spontaneous locomotor activity and enhanced the effects of the drug on extracellular levels of dopamine to the extent that the response became significant (P < 0.05).

If a dopamine uptake inhibitor, nomifensine, was added to the Ringer solution used to dialyse the probe, the s.c. administration of both acute and subchronic nicotine (0.4 mg kg−1) resulted in significant increases (P < 0.05) in the dopamine concentration in the dialysate. Under these conditions, pretreatment with nicotine prior to the test day prolonged (P < 0.05) the dopamine response to a challenge dose of nicotine.

Subcutaneous injections of (+)‐amphetamine (0.2 or 0.5 mg kg−1) evoked dose‐dependent increases in both spontaneous activity and the concentration of dopamine in NAc dialysates. These responses were unaffected by 5 days pretreatment with the drug.

The results of this study support the conclusion that the enhanced locomotor response to nicotine observed in animals pretreated with the drug prior to the test day is associated with potentiation of its effects on dopamine secretion in the NAc.
Changes in adenosine sensitivity in the hippocampus of rats with streptozotocin‐induced diabetes
Authors: Morrison P.D , Mackinnon M.W.B. , Bartrup J.T. , Skett P.G. , Stone T.W.
Published: July 2012

Hippocampal slices have been used to assess the sensitivity of the CNS to adenosine and γ‐aminobutyric acid (GABA) in diabetes. The effects of adenosine, 2‐chloroadenosine, GABA, muscimol and baclofen were studied on orthodromic synaptic potentials recorded in the CA1 region of slices taken from normal rats or animals made diabetic by the injection of streptozotocin.

In diabetic animals the sensitivity to adenosine was increased 4 fold compared with normal rats. The potency of 2‐chloroadenosine was unchanged.

The nucleoside transport inhibitor, hydroxynitrobenzylthioinosine (HNBTI), increased the potency of adenosine in slices from normal rats but not in slices from diabetic rats.

No change was observed in the potency of GABA or muscimol, although a small but significant decrease was detected in the EC50 value for baclofen.

Treatment of diabetic animals with insulin restored the potency of adenosine to control levels.

It is concluded that the diabetic state is accompanied by substantial changes of adenosine sensitivity due to the loss of nucleoside uptake processes. Secondary neurochemical changes following from this in human diabetic patients may contribute to the reported behavioural changes.
Effect of metformin on glucose metabolism in the splanchnic bed
Authors: Bailey C.J. , Wilcock Carol , Day Caroline
Published: July 2012

Use of the antihyperglycaemic agent, metformin, is often associated with a small rise in circulating lactate. This study investigates the source of the lactate and examines the effect of metformin on glucose metabolism by the intestine and liver of rats.

Changes in plasma glucose and lactate were measured in the inferior vena cava (IVC), hepatic portal vein (HPV), hepatic vein (HV) and aorta (A) after intrajejunal administration of metformin (50 and 250 mg kg−1) without and with glucose (2 g kg−1).

Metformin 250 mg kg−1 reduced the hyperglycaemic response to a glucose challenge, associated with a greater reduction of glucose concentrations in the HPV (average decrease of 33% at 60 and 120 min) than at other sites.

Both doses of metformin increased lactate concentrations in the glucose‐loaded state: the highest concentration (2.5 fold increase) was recorded in the HPV 60 min after administration of 250 mg kg−1 metformin, with a high lactate concentration persisting in the HV at 120 min. Metformin 250 mg kg−1 also increased lactate concentrations in the basal state, with highest concentrations (2 fold increase) in the HPV.

Two hours after intrajejunal administration of metformin, 50 mg kg−1, rings of tissue from the small intestine showed an average 22% decrease in glucose oxidation ([14C]‐glucose conversion to 14CO2) and a 10% increase in lactate production. Since glucose metabolism in the gut is predominantly anaerobic, metformin caused an overall 9.5% increase of intestinal glucose utilization.

Metformin, 10−6 and 10−4 mol l−1, did not significantly alter glucose oxidation or lactate production by isolated hepatocytes, but a very high concentration of metformin (10−2 mol l−1) increased lactate production by 60%.

The results support the view that metformin increased intestinal glucose utilization and lactate production by the intestine. Under basal conditions there was net extraction of lactate by the liver but not after an enteral glucose load.
l‐Arginine dilates rat pial arterioles by nitric oxide‐dependent mechanisms and increases blood flow during focal cerebral ischaemia
Authors: Morikawa Eiharu , Rosenblatt Sami , Moskowitz Michael A.
Published: July 2012

l‐Arginine (≥ 30 mg kg−1, i.v.), but not d‐arginine (300 mg kg−1) administered 5 min after unilateral common carotid/middle cerebral artery occlusion increased regional cerebral blood flow (rCBF) within the dorsolateral ischaemic cortex in spontaneously hypertensive rats. l‐Arginine (300 mg kg−1) increased rCBF from 22 ± 2.7 to 33 ± 4% of baseline as measured by laser‐Doppler flowmetry. This increase may explain the ability of l‐arginine to reduce infarct size following focal cerebral ischaemia, as reported previously. The mechanism appears to be mediated by nitric oxide since topical l‐NAME (1 μm), a nitric oxide synthase inhibitor, decreased pial arteriole calibre from 115 ± 2.2 to 106 ± 0.9% of baseline following l‐arginine infusion (300 mg kg−1).
Discrimination between ETA‐ and ETB‐receptor‐mediated effects of endothelin‐1 and [Ala1,3,11,15]endothelin‐1 by BQ‐123 in the anaesthetized rat
Authors: Bigaud Marc , Pelton John T.
Published: July 2012

The influence of BQ‐123 (a selective ETA‐receptor antagonist) on the haemodynamic response elicited by endothelin‐1 (ET‐1) and [Ala1,3,11,15]ET‐1 (a selective ETB‐receptor agonist) was studied in anaesthetized rats instrumented with ultrasonic Doppler flow probes on the carotid, coeliac, mesenteric, renal and iliac arteries.

BQ‐123 alone (1.6 μmol kg−1, i.v.) induced a decrease in femoral mean arterial pressure (AP), accompanied by a systemic vasodilatation. The response was maximal after 3 min and then returned slowly to baseline. None of these effects was observed after a 0.016 μmol kg−1 dose of BQ‐123.

ET‐1 (1 nmol kg−1, i.v.) induced a biphasic response characterized by a transient initial decrease in AP accompanied by regional vasodilatation (mainly in the carotid and iliac beds) and by immediate mesenteric and renal vasoconstrictions. This was followed, within 1 min, by a marked and prolonged increase in AP accompanied by systemic vasoconstriction. Pretreatment with BQ‐123 (1.6 μmol kg−1, i.v., 8 min before ET‐1) increased and prolonged the vasodilator effect of ET‐1 (mainly in the carotid, coeliac, mesenteric and iliac beds) and reduced its systemic vasoconstrictor effects with marked regional differences (the coeliac, mesenteric and renal beds being poorly affected).

[Ala1,3,11,15]ET‐1 (3 nmol kg−1, i.v.) induced an initial and marked decrease in AP accompanied by regional vasodilatation (mainly in the carotid, coeliac and iliac beds) and by mesenteric and renal vasoconstrictions. This was followed, within 5 min, by a small increase in AP and systemic vasoconstriction. All these effects were dose‐dependent. Pretreatment with BQ‐123 (1.6 μmol kg−1; 8 min before ET‐1) did not modify the early effect of [Ala1,3,11,15]ET‐1, but abolished its secondary vasoconstrictor effect except in the mesenteric bed.

This study demonstrates that pretreatment with BQ‐123 not only reduced a large part of the sustained vasoconstrictor activity of ET‐1, suggesting the involvement of ETA‐receptors, but also enhanced the early vasodilator activity of ET‐1 revealing a functional antagonism between the two effects. The vasodilator effect of [Ala1,3,11,15]ET‐1 was not affected by BQ‐123 and ET‐1 induced a similar vasodilatation, that was potentiated by BQ‐123, suggesting the involvement of ETB‐receptors in this vasodilator response. Marked regional differences were however observed which might be partly related to different levels of functional antagonism between ETB‐ and ETA‐mediated effects, but differences in receptor types, or subtypes, cannot be excluded, mainly in the mesenteric and renals beds.
Modulation of barrier function of bovine aortic and pulmonary artery endothelial cells: dissociation from cytosolic calcium content
Authors: Buchan Kevin W. , Martin William
Published: July 2012

Barrier function and cytosolic free calcium content [Ca2+]i was measured in monolayers of bovine pulmonary artery endothelial cells (BPAEC) and bovine aortic endothelial cells (BAEC).

Thrombin (1 u ml−1) increased albumin transfer across monolayers of BPAEC but not BAEC, yet induced biphasic increases in [Ca2+]i in both endothelial cell types, consisting of a rapid, initial phasic component which decayed to a lower, more sustained plateau phase.

4β‐Phorbol 12‐myristate 13‐acetate (PMA; 0.3–3000 nm) increased albumin transfer across monolayers of BPAEC and BAEC, but had no effect on basal levels of [Ca2+]i in either endothelial cell type.

Treatment of BPAEC and BAEC with forskolin (30 μm), an activator of adenylate cyclase, had no effect on resting transfer of albumin, but inhibited that stimulated by PMA (600 nm). It also inhibited the thrombin (1 u ml−1)‐induced increase in albumin transfer across monolayers of BPAEC, but enhanced the plateau phase of the associated increase in [Ca2+]i.

Treatment of BPAEC and BAEC with either atriopeptin II (100 nm), an activator of particulate guanylate cyclase, or 8 bromo cyclic GMP (30 μm) had no effect on resting or PMA (600 nm)‐stimulated transfer of albumin. Both agents did, however, inhibit the thrombin (1 u ml−1)‐induced increase in albumin transfer across monolayers of BPAEC, but had no effect on the associated increase in [Ca2+]i.

These data suggest a dissociation between the ability of agents that increase or decrease albumin transfer and their effects on [Ca2+]i. Consequently, activation of protein kinase C may be the major stimulus for trans‐endothelial transfer of macromolecular solutes. Endothelial barrier function is enhanced by elevation of either cyclic AMP or cyclic GMP content. Cyclic AMP appears to act by inhibiting the actions of protein kinase C, while cyclic GMP may act to inhibit a key step proximal to activation of this enzyme.
Enhanced responsiveness of ovalbumin‐sensitized guinea‐pig alveolar macrophages to tachykinins
Authors: Brunelleschi Sandra , Parenti Astrid , Ceni Elisabetta , Giotti Alberto , Fantozzi Roberto
Published: July 2012

We have evaluated the ability of substance P (SP), neurokinin A (NKA) and the selective NK2 receptor agonist [β‐Ala8]‐NKA(4–10) to induce superoxide anion (O2−) production and prostanoid (prostaglandin E2, thromboxane B2) release from alveolar macrophages (AMs) isolated from control or actively sensitized guinea‐pigs.

The dose‐response curves for NKA and SP were shifted to the left (three orders and one order of magnitude, respectively) in AMs isolated from sensitized animals, with no variation in maximal effects.

By evaluating the effects of [β‐Ala8]‐NKA(4–10), we observed that not only was the concentration‐response curve shifted to the left in both the functional parameters examined, but also maximal effects were significantly enhanced in AMs isolated from sensitized guinea‐pigs.

This varied responsiveness seems to be specific for tachykinins, as it was not reproduced by another AM stimulant, the bacterial peptide N‐formylmethionyl‐leucyl‐phenylalanine (fMLP).

Only small amounts of β‐glucuronidase were released following tachykinin or ovalbumin stimulation both in control and sensitized AMs.

These results indicate that AMs isolated from sensitized guinea‐pigs show an increased responsiveness to NK2 receptor stimulation and further stress the role played by AMs in allergic lung diseases.
Differential sensitivities of the prostacyclin and nitric oxide biosynthetic pathways to cytosolic calcium in bovine aortic endothelial cells
Authors: Parsaee Heydar , McEwan Jean R. , Joseph Sunil , MacDermot John
Published: July 2012

Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (PGI2; Kact = 24.1 nm) and endothelium‐derived relaxing factor (EDRF, NO; Kact = 0.7 nm) in a concentration‐dependent manner when exposed to bradykinin.

The bradykinin‐dependent release of PGI2 (but not EDRF) was inhibited by 1 μm isoprenaline or 5 μm forskolin, and the inhibitory effect of isoprenaline could be reversed by the β2‐adrenoceptor antagonist, ICI 118551. In contrast, isoprenaline had no capacity to inhibit PGI2 release stimulated by exogenous arachidonic acid.

Exposure of cells to bradykinin increased the cytosolic concentration of Ca2+ ions ([Ca2+]i; Kact = 4.8 nm), and the effect was inhibited by both 1 μm isoprenaline and 5 μm forskolin.

In similar experiments, exposure of cells to ionomycin also increased [Ca2+]i and the values of [Ca2+]i were calibrated in terms of the ionomycin concentration. In subsequent experiments involving exposure of endothelial cells to selected concentrations of ionomycin, it was possible to show that the biosynthesis of NO was triggered at ionomycin concentrations about one tenth of that required for PGI2 biosynthesis and that these corresponded to a [Ca2+]i threshold of 350 nm for PGI2 release while that for EDRF release was less than 200 nm.

These differences in Ca2+ ion sensitivity explain the selective inhibition of bradykinin‐stimulated PGI2 biosynthesis (to the exclusion of NO biosynthesis) by isoprenaline or forskolin, both of which attenuate bradykinin‐dependent increases in [Ca2+]i.
Blockade by antiarrhythmic drugs of glibenclamide‐sensitive K+ channels in Xenopus oocytes
Authors: Sakuta Hidenari , Okamoto Koichi , Watanabe Yasuhiro
Published: July 2012

The outward K+ current induced by KRN2391 (K+ channel opener) in Xenopus oocytes is blocked by glibenclamide. We have investigated the effects of various classes (I‐IV) of antiarrhythmic drugs on this KRN2391‐induced response.

All class I antiarrhythmic drugs (Na+ channel blockers) tested concentration‐dependently suppressed KRN2391‐induced responses with the rank order of potency (IC50 in μm), disopyramide (17.8) > aprindine (29.5) > propafenone (63.1) > ajmaline (145) > quinidine (151). Flecainide, SUN1165, lignocaine, mexiletine and procainamide were much less potent (IC50, 450– > 1000 μm) than quinidine.

The class II antiarrhythmic drugs (β‐blockers), timolol, (−)‐ and (±)‐ propranolol, and (+)‐propranolol (a non‐β‐blocker) inhibited KRN2391‐induced K+ currents in a concentration‐dependent manner with values for IC50 (μm) of 79, 131, 151 and 129, respectively, whilst butoxamine, oxprenolol, alprenolol, pindolol, nadolol, metoprolol and acebutolol were either weak (IC50, 300 μm — 600 μm) or virtually inactive (IC50, > 1000 μm).

The class III antiarrhythmic drugs, amiodarone and (+)‐sotalol scarcely affected KRN2391 responses.

All class IV drugs (Ca2+ antagonists) tested suppressed KRN2391‐induced responses in a concentration‐dependent manner with an IC50 of 6.3 μm for bepridil, 38 μm for prenylamine, 85 μm for verapamil and 135 μm for diltiazem.

In conclusion, antiarrhythmic drugs of classes I, II and IV potently blocked glibenclamide‐sensitive K+ channels in Xenopus oocytes.
Involvement of cyclic GMP in non‐adrenergic, non‐cholinergic inhibitory neurotransmission in dog proximal colon
Authors: Ward Sean M. , Dalziel Hugh H. , Bradley Michael E. , Buxton Iain L.O. , Keef Kathleen , Westfall David P. , Sanders Kenton M.
Published: July 2012

Nitric oxide (NO) may serve as a non‐adrenergic, non‐cholinergic (NANC) neurotransmitter released from enteric inhibitory nerves in the gastrointestinal tract. We tested whether guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) may serve as a second messenger in transducing the NO signal into inhibitory junction potentials (i.j.ps) and relaxation in the canine proximal colon.

The membrane permeable analogue of cyclic GMP, 8‐bromo cyclic GMP (8‐Br‐cyclic GMP) mimicked the effects of NO by hyperpolarizing cells near the myenteric border of the circular muscle layer and shortening slow waves in cells near the submucosal surface of the circular muscle layer. 8‐Br‐cGMP also inhibited spontaneous phasic contractions.

The specific cyclic GMP phosphodiesterase inhibitor, M&B 22948, hyperpolarized cells near the myenteric border and prolonged the duration of i.j.ps. M&B 22948 also inhibited phasic contractile activity.

Methylene blue failed to reduce significantly the amplitude and duration of i.j.ps and had variable effects on contractions.

Cyclic GMP levels were assayed in unstimulated muscles and in muscles exposed to exogenous NO and electrical field stimulation. Both stimuli hyperpolarized membrane potential, inhibited contractions, and elevated cyclic GMP levels.

Treatment of muscles with l‐NG‐nitroarginine methyl ester (l‐NAME) increased spontaneous contractile activity and lowered cyclic GMP levels. The inhibitory effect of M&B 22948 on contractions was greatly reduced after muscles were treated with l‐NAME.

These data support the concept that the effects of NANC nerve stimulation and NO (which may be one of the enteric inhibitory transmitters) may be mediated by cyclic GMP.
Inositol 1,4,5‐trisphosphate generation and calcium mobilisation via activation of an atypical P2 receptor in the neuronal cell line, N1E‐115
Authors: Iredale Philip A. , Martin Keith F. , Alexander Stephen P.H. , Hill Stephen J. , Kendall David A.
Published: July 2012

Alterations in the levels of intracellular calcium ([Ca2+]i) and d‐myo‐inositol‐1,4,5‐trisphosphate (InsP3) were measured in the murine neuroblastoma cell line clone, N1E‐115, by use of the calcium‐sensitive dye, fura‐2 and a radioreceptor assay, respectively.

Exposure of the cells to ATP (100 μm) elicited rapid and transient increases in [Ca2+]i and InsP3, with both responses reaching a maximum between 10– 20 s after agonist addition.

Investigation of concentration‐response data by use of various analogues of ATP suggests the presence of an extracellular receptor which fails to fit into the current classification of purinoceptors.

Cross‐desensitization experiments suggest that the same receptor can also be activated by the structurally different pyrimidine base, UTP.

Application of the tumour‐promoting agent, β‐phorbol‐12,13 dibutyrate (PDBu) caused a reduction in the increases in both [Ca2+]i and InsP3, suggesting a role for protein kinase C in feedback inhibition of purinoceptor responses in this cell line.

In summary, we present the first evidence for the existence of an atypical purinoceptor on a cell line of CNS origin. This receptor is linked to stimulation of phosphoinositide turnover and subsequent mobilisation of intracellular calcium.
Attenuation of vasoconstriction by endogenous nitric oxide in rat caudal artery
Authors: Vo Phuong A. , Reid Julianne J. , Rand Michael J.
Published: July 2012

The effects of NG‐nitro‐l‐arginine (l‐NNA), NG‐nitro‐l‐arginine methyl ester (l‐NAME), haemoglobin and methylene blue have been examined on vascular reactivity in the rat isolated caudal artery. The effects of l‐NNA and sodium nitroprusside were also investigated on the stimulation‐induced (S‐I) efflux of noradrenaline in the rat caudal artery.

l‐NNA (10 μm) and l‐NAME (10 μm) significantly attenuated the vasodilator responses to acetylcholine (1 nm − 1 μm), but had no effect on vasodilator responses to papaverine (1–100 μm).

Vasoconstrictor responses to sympathetic nerve stimulation (3 Hz, 10 s), noradrenaline (0.01–1 μm), methoxamine (1–10 μm), 5‐hydroxytryptamine (0.01–0.3 μm), phenylephrine (0.1–10 μm), endothelin‐1 (10 nm) and KCl (40 mm) were significantly enhanced by 10 μm l‐NNA. l‐NAME (10 μm) caused a significant enhancement of vasoconstrictor responses to noradrenaline and sympathetic nerve stimulation in endothelium‐intact, but not in endothelium‐denuded tissues.

Haemoglobin and methylene blue (both 10 μm) enhanced the vasoconstrictor responses to sympathetic nerve stimulation and noradrenaline. The enhancements were absent in endothelium‐denuded arterial segments.

In endothelium‐denuded arterial segments precontracted with phenylephrine, the vasodilator responses to the nitric oxide donor, sodium nitroprusside (0.1–300 nm), were decreased by increasing the level of precontraction.

l‐NNA (10 μm) had no effect on the S‐I efflux of radioactivity from arteries in which transmitter stores had been labelled with [3H]‐noradrenaline.

These results suggest that endothelial nitric oxide attenuates vasoconstrictor responses in the rat caudal artery through activation of soluble guanylate cyclase to decrease smooth muscle contractility. Therefore, the findings provide evidence that nitric oxide acts as a functional antagonist to oppose vasoconstriction.
Role of protein kinase C in the regulation of histamine and bradykinin stimulated inositol polyphosphate turnover in adrenal chromaffin cells
Authors: Boarder Michael R. , Challiss R.A. John
Published: July 2012

The possibility that bradykinin‐ or histamine‐stimulated inositol polyphosphate accumulation may be regulated by protein kinase C (PKC) in bovine adrenal chromaffin cells has been addressed.

Initial experiments confirmed that the phorbol ester 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA) dramatically inhibited agonist‐stimulated [3H]‐inositol phosphate accumulations in [3H]‐inositol prelabelled cells. In contrast, the PKC inhibitor, Ro 31–8220, did not affect this response.

Histamine (100 μm) or bradykinin (100 nm) evoked rapid increases in inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5‐tetrakisphosphate (Ins(1,3,4,5)P4) mass accumulations (maximal accumulations within 10 s and 30 s, respectively) which declined towards basal values over a 10 min incubation period. TPA (1 μm) significantly attenuated the peak Ins(1,4,5)P3 response to bradykinin and histamine by 30% and 70% respectively. In contrast, TPA did not significantly affect agonist‐stimulated Ins(1,3,4,5)P4 responses.

Ro 31–8220 (10 μm) significantly enhanced the maximal Ins(1,4,5)P3 accumulations elicited by both bradykinin and histamine.

The results indicate that the initial Ins(1,4,5)P3 response to either bradykinin or histamine in bovine adrenal chromaffin cells can be attenuated by PKC activation by phorbol ester and enhanced by PKC inhibition by Ro 31–8220. In contrast, agonist‐stimulated Ins(1,3,4,5)P4 accumulation does not appear to be affected by these manipulations of PKC activity. Possible bases for differential modulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are discussed.
5‐Methylurapidil may discriminate between α1‐adrenoceptors with a high affinity for WB4101 in rat lung
Authors: Hiramatsu Yoshinori , Muraoka Ryusuke , Kigoshi Shigeru , Muramatsu Ikunobu
Published: July 2012

The α1‐adrenoceptors of rat lung with a high affinity for [3H]‐prazosin were subdivided into two populations (high and low affinity sites) by WB4101 and 5‐methylurapidil but the proportions were different between both drugs. After pretreatment with chlorethylclonidine, WB4101 recognized only high affinity sites, while 5‐methylurapidil still detected high and low affinity sites. These results indicate that α1‐adrenoceptors with a high affinity for WB4101 are not homogeneous in the rat lung, suggesting the possible existence of a new α1‐adrenoceptor subtype in addition to α1A and α1B subtypes.
Non‐competitive inhibition of GABAA responses by a new class of quinolones and non‐steroidal anti‐inflammatories in dissociated frog sensory neurones
Authors: Yakushiji Takashi , Shirasaki Tetsuya , Akaike Norio
Published: July 2012

The interaction of a new class of quinolone antimicrobials (new quinolones) and non‐steroidal anti‐inflammatory agents (NSAIDs) with the GABAA receptor‐Cl− channel complex was investigated in frog sensory neurones by use of the internal perfusion and ‘concentration clamp’ techniques.

The new quinolones and the NSAIDs (both 10−6−10−5 m) had little effect on the GABA‐induced chloride current (ICl) when applied separately. At a concentration of 10−4 m the new quinolones, and to a lesser degree the NSAIDs, produced some suppression of the GABA response.

The co‐administration of new quinolones and some NSAIDs (10−6−10−14 m) resulted in a marked suppression of the GABA response. The size of this inhibition was dependent on the concentration of either the new quinolone or the NSAID tested. The inhibitory potency of new quinolones in combination with 4‐biphenylacetic acid (BPAA) was in rank order norfloxacin (NFLX) ≫ enoxacin (ENX) > ciprofloxancin (CPFX) ≫ ofloxacin (OFLX), and that of NSAIDs in combination with ENX was BPAA ≫ indomethacin = ketoprofen > naproxen > ibuprofen > pranoprofen. Diclofenac, piroxicam and acetaminophen did not affect GABA responses in the presence of ENX.

In the presence of ENX or BPAA, there was a small shift to the right of the concentration‐response curve for GABA without any effect on the maximum response. However, the co‐administration of these drugs suppressed the maximum of the GABA concentration‐response curve, indicating a non‐competitive inhibition, for which no voltage‐dependency was observed.

Simultaneous administration of ENX and BPAA also suppressed pentobarbitone (PB)‐gated ICl. On the other hand, both PB and phenobarbitone reversed the inhibition of GABA‐induced ICl by co‐administration of ENX and BPAA.

The effect on GABAA responses of co‐administration of new quinolones and NSAIDs was not via an interaction with benzodiazepine receptors coupled to the GABAA receptor, since this effect was not reversed by Ro15–1788 or diazepam.

It is concluded that the co‐administration of new quinolones and some of the NSAIDs inhibit GABAergic transmission, and could result in convulsions.
Effect of milacemide on extracellular and tissue concentrations of dopamine and 5‐hydroxytryptamine in rat frontal cortex
Authors: Semba Jun'ichi , Doheny Mary , Patsalos Philip N. , Sarna Gurcharan , Curzon Gerald
Published: July 2012

Milacemide is a glycine prodrug which is both an inhibitor and a substrate for monoamine oxidase‐type B (MAO‐B) and also an inhibitor of MAO‐type A (MAO‐A). Its effects on dopamine and 5‐hydroxytryptamine (5‐HT) metabolism in rat frontal cortex tissue and dialysate were evaluated.

Dialysate dopamine concentrations increased linearly and dose‐dependently after milacemide administration (100, 200, 400 mg kg−1, i.p.), peaking at 1 h. A concomitant dose‐dependent decrease in dialysate 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) concentrations was observed but these changes were smaller (27% and 40% respectively) than the change in dopamine (125% after 400 mg kg−1 milacemide).

Dialysate 5‐HT was significantly increased only at 1.5 h after giving milacemide 400 mg kg−1. Dialysate 5‐hydroxyindoleacetic acid (5‐HIAA) concentration was not affected.

Milacemide (400 mg kg−1) at 1.5 h post‐administration significantly increased frontal cortex tissue concentrations of dopamine and 5‐HT; the percentage increase in dopamine being about four times that of 5‐HT. Metabolite concentrations, including 5‐HIAA, decreased. Changes in tissue and dialysate dopamine, DOPAC and HVA were approximately proportionate to each other.

The results are explicable in terms of an inhibition by milacemide of MAO‐A.
Neuropeptide Y (NPY) receptors in HEL cells: comparison of binding and functional parameters for full and partial agonists and a non‐peptide antagonist
Authors: Feth Friedhelm , Rascher Wolfgang , Michel Martin C.
Published: July 2012

We have compared the binding and Ca2+ mobilizing properties of various full agonists, partial agonists and a non‐peptide antagonist at the neuropeptide Y (NPY) receptor of human erythroleukemia (HEL) cells.

[125I]‐NPY binding to intact HEL cells was rapid, saturable, of high affinity and with a specificity typical for the Y1‐like subtype: NPY, peptide YY (PYY) and [Pro34]‐NPY competed for [125I]‐NPY binding with high affinity whereas NPY13–36 and NPY18–36 had only low affinity.

NPY, PYY and [Pro34]‐NPY potently increased intracellular Ca2+ in HEL cells and had equal efficacy. NPY13–36, vasoactive intestinal peptide (VIP) and pancreatic polypeptide (PP) increased intracellular Ca2+ only poorly.

Whereas VIP and PP did not significantly affect NPY‐stimulated Ca2+ mobilization, NPY13–36 inhibited NPY‐stimulated Ca2+ increases and shifted the NPY concentration‐response curve to the right without altering its maximal effect.

The agonist (pEC50) potencies of the various peptides corresponded well with the affinities of these compounds in the binding assay (pKi), whereas the antagonist potencies (pKb) of the peptide partial agonists and the pA2 value of the non‐peptide NPY antagonist (He 90481), calculated from functional data, were lower than the respective affinities determined in the binding studies.

A plot of the fractional Ca2+ response vs the fractional receptor occupancy did not reveal any nonlinear receptor‐effector coupling for NPY or [Pro34]‐NPY; a small receptor reserve might exist for PYY.

We conclude that the binding and functional properties of HEL cell NPY receptors are very similar. NPY, PYY and [Pro34]NPY are full agonists at these receptors, whereas NPY13–36 is a partial agonist.
The role of platelet‐activating factor and peptidoleukotrienes in the vascular changes of rat passive anaphylaxis
Authors: Fernández‐Gallardo Sagrario , Gijón Miguel Angel , García Carolina , Furió Vicente , Liu Fu‐Tong , Crespo Mariano Sánchez
Published: July 2012

The role of platelet‐activating factor (PAF) and peptidoleukotrienes as putative mediators of some of the vascular changes triggered by antigen was investigated in rats passively sensitized with monoclonal anti‐DNP (2,4‐dinitrophenyl) IgE.

Lethal anaphylaxis with respiratory distress, systemic hypotension, detachment of the intestinal mucosa, leukopenia and extravasation of protein‐rich plasma was observed after antigen challenge of rats sensitized with partially purified monoclonal IgE at concentrations of 15 mg protein kg−1.

Analysis of the peritoneal fluid obtained after i.v. challenge with DNP‐BSA (bovine serum albumin) showed the presence of significant amounts of PAF (101 ± 8 pg/rat), whereas this mediator was undetectable in control animals. Leukotriene D4 was the predominant peptidoleukotriene that could be recovered after antigen challenge, and showed an extremely high concentration (92 ± 15 ng/rat) as compared to PAF levels.

Extravasation of protein‐rich plasma was observed shortly after challenge and reached a maximum at 30 min. Treatment of animals with i.v. PCA 4248 (1–2 mg kg−1) and WEB 2086 (1 mg kg−1), two chemically unrelated compounds which are antagonists of the PAF‐receptor, produced a significant reduction of the extravasation of protein‐rich plasma.

The same degree of protection could be afforded by MK‐886, an inhibitor of leukotriene biosynthesis. Combined treatment with WEB 2086 and MK‐886 provided greater inhibition of protein‐rich plasma extravasation than either compound alone. PCA 4248 was also found to inhibit in a dose‐dependent manner the systemic hypotension observed upon DNP‐BSA challenge.

These data indicate that the lipid mediators PAF and peptidoleukotrienes are major effectors of the vascular disturbances observed in rat passive IgE‐mediated anaphylaxis.
Changes in plasma catecholamine and neuropeptide Y levels after sympathetic activation in dogs
Authors: Poncet Marie‐France , Damase‐Michel Christine , Tavernier Genevieve , Tran Marie‐Antoinette , Berlan Michel , Montastruc Jean‐Louis , Montastruc Paul
Published: July 2012

Plasma levels of noradrenaline (NA) and neuropeptide Y (NPY) were evaluated in two experimental models associated with an increase in sympathetic tone: conscious dogs which were subject to either sinoaortic denervation or acute administration of the α2‐adrenoceptor antagonist yohimbine.

Dogs that had undergone sinoaortic denervation exhibited a two fold increase in plasma NA without any change in NPY levels.

Yohimbine (0.05 mg kg−1 i.v. as a bolus) produced similar effects. A higher dose of yohimbine (0.5 mg kg−1 i.v.) increased both plasma NA (7 fold) and NPY (6.5 fold) levels.

The present results indicate that changes in plasma catecholamines and NPY are not always concomitant. They suggest that the simultaneous release of NA and NPY is only observed under in vivo conditions for a marked increase in sympathetic tone.
Endogenous noradrenaline release from guinea‐pig isolated trachea is inhibited by activation of M2 receptors
Authors: Racké Kurt , Hey Claudia , Wessler Ignaz
Published: July 2012

Overflow of endogenous noradrenaline (NA) from guinea‐pig isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses). The muscarinic receptor agonist oxotremorine inhibited the evoked overflow of NA in a concentration‐dependent manner (EC50 84 nm). Methoctramine, pirenzepine and p‐fluoro‐hexahydrosiladiphenidol (each 1 μm) shifted the concentration‐response curves of oxotremorine to the right with apparent pA2 values of 7.60, 6.74 and 6.18, respectively. It is concluded that sympathetic nerve terminals in the guinea‐pig trachea are endowed with inhibitory muscarinic M2 receptors.