bluuebunny/crossref_metadata_2025 huggingface dataset:
Blockade of nitrergic transmission by hydroquinone, hydroxocobalamin and carboxy‐PTIO in bovine retractor penis: role of superoxide anion
Authors: Paisley Karen , Martin William
Published: July 2012

The effects of inhibiting endogenous Cu/Zn superoxide dismutase (SOD) with diethyldithiocarbamate (DETCA) were examined on the ability of hydroquinone, hydroxocobalamin and carboxy‐PTIO to block nitrergic relaxation in the bovine retractor penis (BRP) muscle.

Incubation of strips of BRP with DETCA (3 mM) for 2 h reduced SOD activity from 73.1 ± 15.7 to 8.2 ± 1.9 units mg‐1 protein.

Hydroquinone (10 μm‐ 1mM) produced weak inhibition of nitrergic (4 Hz, 10 s) relaxation in control strips of BRP, but powerful inhibition in strips treated with DETCA (3 mM, 2 h). Exogenous SOD (250 units ml‐1) produced a partial blockade of the ability of hydroquinone to inhibit nitrergic relaxation in DETCA‐treated strips.

In an assay of SOD‐inhibitable reduction of cytochrome C, hypoxanthine (0.1 mM)/xanthine oxidase (16 munits ml‐1) and pyrogallol (10 μm), led to the rapid generation of superoxide anion. Hydroquinone (10 μm) also led to the generation of the free radical, although the rate of generation was slower.

Two NO‐scavenging agents, hydroxocobalamin (0.1 μm‐1 mM) and carboxy‐PTIO (0.1‐1 mM), produced concentration‐dependent blockade of nitrergic relaxation of the BRP. The magnitude of the blockade induced by these agents was unaffected following treatment with DETCA or SOD.

The findings with hydroquinone support our previous proposal that endogenous Cu/Zn SOD plays a vital role in protecting nitrergic neurotransmission from inactivation by superoxide anion. Results with hydroxocobalamin and carboxy‐PTIO are consistent with the known ability of these agents to scavenge NO. The nitrergic neurotransmitter in the BRP thus appears to have the properties of NO.
Effect of a non‐sulphonylurea hypoglycaemic agent, KAD‐1229 on hormone secretion in the isolated perfused pancreas of the rat
Authors: Kinukawa Mayumi , Ohnota Hideki , Ajisawa Yukiyoshi
Published: July 2012

We examined the cooperative effect of a newly synthesized oral hypoglycaemic agent, KAD‐1229 with glucose on insulin, glucagon and somatostatin secretion in the isolated perfused pancreas of the rat.

KAD‐1229 stimulated concentration‐dependently the first phase of insulin secretion without the second phase in the presence of 2.8 mM glucose, while it stimulated both the first and the second phase of insulin release in the presence of 5.6 mM glucose. It was confirmed that the first phase of insulin release is depolarization‐induced release with no other additional signal transduction.

KAD‐1229 also enhanced insulin release evoked by 16.7 mM glucose, a concentration known to inhibit the ATP‐sensitive K+ current completely.

A low concentration (2.8 mM) of glucose stimulated somatostatin release transiently, while a higher concentration (16.7 mM) of glucose exerted a sustained stimulation. KAD‐1229 stimulated somatostatin secretion in a concentration‐dependent manner irrespective of glucose concentrations.

When glucagon release was stimulated with 2.8 mM glucose, KAD‐1229 inhibited this hypoglycaemia‐induced glucagon secretion.

When pancreata from rats pretreated with streptozotocin (STZ) 60 mg kg−1 were perfused, the basal secretion of glucagon was markedly elevated, and the glucagon response to the low glucose was abolished. Further, the insulin and somatostatin responses to KAD‐1229 were largely attenuated. KAD‐1229 showed transient enhancement followed by inhibition of the glucagon release from the STZ‐pretreated rat pancreas.

We conclude that KAD‐1229 stimulates insulin and somatostatin release, while it inhibits glucagon release following transient stimulation.
Involvement of secretory phospholipase A2 activity in the zymosan rat air pouch model of inflammation
Authors: Payá Miguel , Terencio M. Carmen , Ferrándiz M. Luisa , Alcaraz M. José
Published: July 2012

In the zymosan rat air pouch model of inflammation we have assessed the time dependence of phospholipase A2 (PLA2) accumulation in the inflammatory exudates as well as cell migration, myeloperoxidase activity, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels.

A significant increase in PLA2 activity was detected in 1,200 g supernatants of exudates 8 h after injection of zymosan into rat air pouch. This event coincided with peaks in cell accumulation (mainly neutrophils) and myeloperoxidase activity in exudates and was preceded by a rise in eicosanoid levels.

This enzyme (without further purification) behaved as a secretory type II PLA2 with an optimum pH at 7 − 8 units, lack of selectivity for arachidonate release and dependence on mM calcium concentrations for maximal activity.

The PLA2 inhibitors manoalide and scalaradial inhibited this enzyme activity in vitro in a concentration‐dependent manner. Scalaradial also inhibited zymosan stimulated myeloperoxidase release in vitro.

Injection of the marine PLA2 inhibitor scalaradial together with zymosan into the pouch at doses of 0.5, 1 and 5 μmol per pouch resulted in a dose‐dependent inhibition of PLA2 activity in exudates collected 8 h later. Myeloperoxidase levels and cell migration were also decreased, while eicosanoid levels were not modified.

Colchicine administration to rats prevented infiltration and decreased PLA2 levels in the 8 h zymosan‐injected air pouch.

These results indicate that during inflammatory response to zymosan in the rat air pouch a secretory PLA2 activity is released into the exudates. The source of this activity is mainly the neutrophil which migrates into the pouch.

Scalaradial exerts anti‐inflammatory effects in the zymosan air pouch.
An interaction of ondansetron and dexamethasone antagonizing cisplatin‐induced acute and delayed emesis in the ferret
Authors: Rudd J.A. , Naylor R.J.
Published: July 2012

Cisplatin, 5 mg kg−1, i.p., administered as a single treatment, induced an acute (day 1) and delayed (days 2 and 3) emetic response in the ferret that was used to investigate the potential anti‐emetic activity of ondansetron and dexamethasone and their interaction over a three day period.

Ondansetron, 1 mg kg−1, i.p., administered three times per day in two experiments, antagonized significantly the retching and vomiting that occurred on days 1 and 2 by 60–76 and 73–84%. On the third day of treatment there was a trend for a 38% reduction in one experiment and a 74% reduction in the other.

There was a trend for dexamethasone, 1 mg kg−1, i.p., administered as a single daily injection for three days, to reduce by 37% the retching and vomiting response that occurred on day 1, the reduction of 77% on day 2 achieved significance and dexamethasone non‐significantly increased the retching and vomiting response by 46% on day 3. However, dexamethasone 1 mg kg−1 i.p. administered three times per day for three days significantly reduced the retching+vomiting reponse by 85, 97 and 86% on days 1, 2 and 3 respectively.

The combination of dexamethasone, 1 mg kg−1, i.p., as single daily injections with ondansetron, 1 mg kg−1, i.p., administered three times per day improved the control of the retching and vomiting response, significantly reducing the total numbers of retches and vomits by more than 70% over a three day period. The combination of dexamethasone (1.0 mg kg−1) and ondansetron (1.0 mg kg−1), both administered three times daily, abolished cisplatin‐induced emesis over the three day period.

The three times per day administration of ondansetron, 1 mg kg−1, i.p., plus dexamethasone, 1 mg kg−1, i.p., administered only on day 1 prevented day 1 emesis but did not modify the retching and vomiting that occurred on days 2 and 3.

The present results indicate that ondansetron and dexamethasone significantly reduce cisplatininduced emesis in the ferret during both the acute and delayed phase; drug/co‐treatment can exert an additive action to abolish cisplatin‐induced emesis. The ferret model may be useful to detect anti‐emetic drug action for treatment of chemotherapy‐induced acute and delayed emesis in man.
Improvement by dynorphin A (1–13) of galanin‐induced impairment of memory accompanied by blockade of reductions in acetylcholine release in rats
Authors: Hiramatsu M. , Mori H. , Murasawa H. , Kameyama T.
Published: July 2012

Human galanin (0.32 nmol per rat, i.c.v.), an endogenous neuropeptide, administered 30 min before acquisition or retention trials, significantly impaired the acquisition of learning and recall of memory in a step‐through type passive avoidance performance.

The role of dynorphin A (1–13) in learning and memory is controversial. Dynorphin A (1–13) (0.5 nmol per rat, i.c.v.) administered 5 min before galanin injection, completely antagonized these impairments.

Galanin significantly decreased acetylcholine release in the hippocampus 40 to 120 min after injection as determined by in vivo brain microdialysis. This peptide also decreased acetylcholine release, albeit to a lesser extent, from the frontal cortex.

Dynorphin A (1–13) (0.5 nmol per rat, i.c.v.) 5 min before galanin injection, completely blocked the decrease in extracellular acetylcholine concentration induced by galanin.

These antagonistic effects of dynorphin A (1–13) were abolished by treatment with nor‐binaltorphimine (5.44 nmol per rat, i.c.v.), a selective κ‐opioid receptor antagonist, 5 min before dynorphin A (1–13).

Dynorphin A (1–13) (0.5 nmol) itself had no effect on learning and memory and on the acetylcholine concentration in the hippocampus or the frontal cortex in normal rats.

These results suggest that the neuropeptide dynorphin A (1–13) ameliorates the galanin‐induced impairment of learning and memory accompanied by abolition of reductions in acetylcholine release via κ‐opioid receptors.
Role of protein kinase C in mesenteric pressor responses of rats with portal hypertension
Authors: Atucha Noemí M. , Ortíz M. Clara , Martínez Concepción , Quesada Tomás , García‐Estañ Joaquín
Published: July 2012

Hyporesponsiveness to vasoconstrictors is a characteristic abnormality of liver diseases of uncertain origin. In the present study, we have evaluated the involvement of protein kinase C (PKC) in the reduced pressor response to methoxamine (MTX) of a rat model of portal hypertension induced by partial portal vein ligation (PVL). Experiments were performed in the isolated and perfused mesentery.

The pressor response to MTX was reduced in PVL compared to that of control animals (Sham) and pretreatment with NG‐nitro‐L‐arginine (L‐NOARG, 10−4 m) or removal of the endothelium potentiated the response of both groups. However, only removal of the endothelium completely eliminated the reduced pressor response to MTX of the PVL vessels.

Pretreatment of the mesentric vessels with calphostin C (10−6 m), a PKC inhibitor, reduced the response to MTX of Sham to a level similar to that of untreated PVL vessels, but did not change that of PVL animals.

Mesenteric pressor responses to a PKC activator, phorbol 12,13‐dibutyrate (PDBu), were similar in vessels from both PVL and Sham rats and pretreatment with L‐NOARG or removal of the endothelium enhanced those responses while indomethacin (10−5 m) decreased them. In all cases, the responses to PDBU were similar in PVL vessels compared to Sham.

These results indicate that the reduced pressor response to MTX of the mesenteric vascular bed of PVL rats is due to an endothelial alteration, compatible with an enhanced production of nitric oxide. The lack of response to calphostin C in PVL vessels suggests an impairment in agonist‐induced PKC activation. Since direct activation of PKC induces a normal pressor response, it is concluded that the endothelial alteration interacts with the mechanism producing PKC activation, which results in a lower pressor response of the PVL mesenteric vaculature.
Effect of gadolinium on stretch‐induced changes in contraction and intracellularly recorded action‐ and afterpotentials of rat isolated atrium
Authors: Tavi P. , Laine M. , Weckström M.
Published: July 2012

Atrial arrhythmias, like atrial fibrillation and extrasystoles, are common in clinical situations when atrial pressure is increased. Although cardiac mechanoelectrical feedback has been under intensive study for many years, the mechanisms of stretch‐induced arrhythmias are not known in detail. This is partly due to methodological difficulties in recording intracellular voltage during stretch stimulation. In this study we investigated the effects of gadolinium (Gd3+), a blocker of stretch‐activated (SA) channels, on stretch‐induced changes in rat atrial action potentials and contraction force.

By intracellular voltage recordings from rat isolated atria we studied the effects of Gd3+ (80 μm) on stretch‐induced changes in action potentials. The stretch was induced by increasing pressure inside the atrium (1 mmHg to 7 mmHg). An elastic electrode holder that moved along the atrial tissue was used in the recordings. Thus the mechanical artifacts were eliminated and the cell‐electrode contact was made more stable. To examine the influence of Gd3+ on atrial contraction we stretched the atria at different diastolic pressure levels (1 to 7 mmHg) with Gd3+ application of (80 μm) or diltiazem (5.0 μm). Contraction force was monitored by recording the pressure changes generated by the atrial contractions.

Our results show that: (1) atrial stretch induces delayed afterdepolarizations (DADs), increase in action potential amplitude and increase in relative conduction speed; (ii) Gd3+ blocks stretch‐induced DADs and action potential changes; (iii) Gd3+ inhibits pressure‐stimulated increase in the atrial contraction force, while similar inhibition is not observed with diltiazem, a blocker of L‐type calcium channels.

This study suggests that Gd3+ inhibits stretch‐induced changes in cell electrophysiology and contraction in the rat atrial cells and that the effects of gadolinium are due to rather specific block of stretch‐activated ion channels with only a small effect on voltage‐activated calcium channels.
Prevention by (±)‐8‐hydroxy‐2‐(di‐npropylamino)tetralin of both catalepsy and the rises in rat striatal dopamine metabolism caused by haloperidol
Authors: Andersen Heidi L. , Kilpatrick Ian C.
Published: July 2012

The influence of (±)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT) on haloperidol‐induced increases in the dopamine metabolites, 3,4‐dihydroxyphenylacetic acid (DOPAC) and 4‐hydroxy‐3‐methoxyphenylacetic acid (HVA), was measured in three microdissected brain regions of the rat following a quantitative assessment of catalepsy.

Haloperidol alone (2.66 μzmol kg−1, i.p.) caused a robust cataleptic response. Given 30 min after haloperidol, 8‐OH‐DPAT (76 or 760 nmol kg−1, s.c.) prevented catalepsy in 30% and 100% of rats, respectively.

Haloperidol significantly increased the DOPAC (by 2 to 4 fold) and HVA (by 3 to 7 fold) contents of the caudate‐putamen, nucleus accumbens and medial prefrontal cortex. Given alone, only the lower dose of 8‐OH‐DPAT caused a significant biochemical change, a doubling of cortical DOPAC.

In the cases where catalepsy was prevented by either dose of 8‐OH‐DPAT, the haloperidol‐induced increases in DOPAC and HVA were consistently lower in the caudate‐putamen. This pattern was true for the rise in cortical HVA but only in response to the lower dose of 8‐OH‐DPAT. In contrast, neither dose of 8‐OH‐DPAT was able to influence the haloperidol‐induced rises in cortical DOPAC. In the nucleus accumbens, 8‐OH‐DPAT did not affect the haloperidol‐induced increases in the dopamine metabolites, irrespective of the dose employed or the resulting behaviour. When catalepsy was not prevented, 8‐OH‐DPAT did not alter the neurochemical responses to haloperidol in any region.

These results suggest that part of the mechanism by which 8‐OH‐DPAT prevents haloperidol‐induced catalepsy is reflected by a reversal of the compensatory increase in meso‐striatal and/or meso‐cortical dopamine neuronal activity that normally accompanies postsynaptic dopamine receptor blockade with haloperidol.
Comparison of the uptake of [3H]‐gabapentin with the uptake of L‐[3H]‐leucine into rat brain synaptosomes
Authors: Thurlow Richard J. , Hill David R. , Woodruff G.N.
Published: July 2012

Gabapentin is a novel anticonvulsant with an unknown mechanism of action. Homogenate binding studies described elsewhere have suggested that [3H]‐gabapentin binds to a site in brain similar to the large neutral amino acid (LNAA) uptake site, termed system‐L.

This study describes an investigation into the uptake of [3H]‐gabapentin into a crude synaptosomal preparation from cerebral cortex of rat brain. Characterization studies showed that [3H]‐gabapentin is taken up into synaptosomes by a system that is similar to that responsible for the uptake of L‐[3H]‐leucine. This system is sodium‐independent, temperature‐sensitive and requires ATP for function.

Kinetic studies of [3H]‐gabapentin uptake produced a Michaelis constant (KM = 160 μm) similar to that observed for L‐[3H]‐leucine (KM = 110.3 μm). Vmax values were 837.1 pmol mg−1 protein min−1 and 2.192 nmol mg−1 protein min−1 respectively.

Gabapentin and L‐leucine mutually inhibit their uptake. Lineweaver‐Burke plots of these data demonstrate that inhibition occurs by a competitive mechanism. Further to this the Dixon transformation of the data illustrates that these two substrates share a common uptake site by the similarity between their calculated Ki and KM values (gabapentin inhibition of L‐[3H]‐leucine uptake: Ki = 160 μm; L‐leucine inhibition of [3H]‐gabapentin uptake: Ki = 262 μm).

Studies into the effect of gabapentin, the system‐L‐specific ligand 2‐(—)‐endoamino‐bicycloheptane‐2‐carboxylic acid (BCH), and the system‐A‐specific ligand α‐(methyl‐amino)‐isobutyric acid (MeAIB), on the initial rate of uptake of [3H]‐glycine, L‐[3H]‐glutamate, L‐[3H]‐glutamine, and L‐[3H]‐leucine were performed. At 100 μm, gabapentin significantly inhibited initial rate of uptake of [3H]‐glycine (29%), L‐[3H]‐glutamate (22%) and L‐[3H]‐leucine (40%).

Gabapentin is taken up into synaptosomes by a system similar to system‐L, responsible for the uptake of large neutral amino acids. Gabapentin will also inhibit the uptake of certain excitatory amino acids in this synaptosomal preparation. The implications of these findings for the mechanism of action for gabapentin are unclear. The data presented here may suggest an intracellular site for mechanism of action for this compound. Similarly changes in levels of amino acid pools may be involved in the mechanism of gabapentin's anticonvulsant action.
The effect of dopamine D1 receptor stimulation on the up‐regulation of histamine H3‐receptors following destruction of the ascending dopaminergic neurones
Authors: Ryu Jong Hoon , Yanai Kazuhiko , Zhao Xiao‐Lan , Watanabe Takehiko
Published: July 2012

The binding of [3H]‐(R)α‐methylhistamine and [3H]‐Nα‐methylhistamine to histamine H3‐receptors, [3H]‐SCH23390 to dopamine D1‐receptors, and [3H]‐YM09151‐2 to dopamine D2‐receptors was investigated by quantitative receptor autoradiography in the rat brain following 6‐hydroxydopamine injection into the substantia nigra.

The levels of [3H]‐(R)α‐methylhistamine binding sites in the denervated striatum and substantia nigra were significantly higher than those in the contralateral side from 1 week to 12 weeks after nigral lesions. The H3‐receptor binding was maximal at 3 weeks after nigral lesions and maintained until 12 weeks.

The increased number of histamine H3‐receptors was decreased to the level of the contralateral side by chronic treatment with a selective dopamine D1 agonist, SKF38393, but not modified by a selective dopamine D2 agonist, quinpirole.

Dopamine D1‐ and D2‐receptors in the striatum were similarly up‐regulated after unilateral nigral lesion. On the other hand, the number of dopamine D2‐receptors in the substantia nigra was markedly decreased after administration of 6‐hydroxydopamine.

The treatment with (S)α‐fluoromethylhistidine increased the H3‐receptor binding in both the ipsilateral and contralateral sides. As a result, the magnitude of the ratio of the H3‐receptor binding between ipsilateral and contralateral sides was partially attenuated by treatment with (S)‐α‐fluoromethylhistidine.

These results strongly suggest that the expression of histamine H3‐receptors in the striatum and substantia nigra is influenced through D1‐receptors by tonic nigrostriatal dopaminergic inputs.
Effects of hydroxyethylrutosides on hypoxia‐induced activation of human endothelial cells in vitro
Authors: Janssens Dominique , Michiels Carine , Arnould Thierry , Remacle José
Published: July 2012

A clinically available mixture of hydroxyethylrutosides (HR) was examined as inhibitors of endothelial cell activation by hypoxia in vitro. Thus, the effects of HR on ATP depletion, phospholipase A2 activation and neutrophil adherence were investigated in hypoxia‐activated human umbilical vein endothelial cells in primary cell culture.

Our results show that HR inhibited two important steps of the activation of endothelial cells by hypoxia: the decrease in ATP content, which is the starting point of the process, and the activation of phospholipase A2 one enzyme responsible for the release of inflammatory mediators. This inhibition was dose‐dependent with 70 to 90% inhibition at 500 μg ml−1 of HR.

In addition, hypoxia‐activated endothelial cells increased their adhesiveness for neutrophils. This process could also be prevented in a dose‐dependent manner if endothelial cells were incubated in the presence of HR. This inhibition was confirmed by a morphological study.

In conclusion, the results of this study suggest that a possible explanation for the improvement in venous insufficiency by HR observed clinically could be their ability to inhibit the activation of endothelial cells during blood stasis.
Regulation of cytosolic calcium in skeletal muscle cells of the mdx mouse under conditions of stress
Authors: Leijendekker Wim J. , Passaquin Anne‐Catherine , Metzinger Laurent , Rüegg Urs T.
Published: July 2012

In Duchenne muscular dystrophy (DMD) dysregulation of cytosolic calcium appears to be involved in the degeneration of skeletal muscle fibres. Therefore, we have studied the regulation of the free cytosolic calcium concentration ([Ca2+]c) under specific stress conditions in cultured myotubes isolated from the hind limbs of wild‐type (C57BL10) and dystrophin‐deficient mutant mdx mice. [Ca2+]c in the myotubes was estimated by the use of the Ca2+‐sensitive fluorescent dye, fura‐2.

Resting [Ca2+]c was similar in mdx and normal myotubes (35 ± 9 nM and 38 ± nM, respectively). However, when mdx myotubes were exposed to a high extracellular calcium concentration ([Ca2+]e) of 40 mM, the [Ca2+]c was elevated to 84 ± 29 nM, compared to 49 ± 7 nM in normal myotubes.

Lowering the osmolarity of the superfusion solution from 300 mOsm to 100 mOsm resulted also in a rise in [Ca2+]c which was about two times higher for mdx (243 ± 65 nM) than for C57BL10 (135 ± 37 nM). Replacing extracellular Ca2+ by EGTA (0.2 mM) prevented the rise in [Ca2+]c in both mdx and normal myotubes when exposed to the low osmolarity solution.

Gadolinium ion (50 μm), an inhibitor of Ca2+ entry, antagonized the rise in [Ca2+]c of myotubes superfused with 40 mM [Ca2+]c by 20–40% for both mdx and C57BL10 cells, but did not significantly reduce the rise in [Ca2+]c when the cells were exposed to the hypo‐osmotic buffer (100 mOsm).

Incubation of the cell culture for 3–5 days from the onset of induction of myotube formation with the membrane permeable protease inhibitor, calpeptin (50 μm) abolished the rise in [Ca2+]c in mdx myotubes upon exposure to hypo‐osmotic shock.

Treatment of the cell culture for 3–5 days with α‐methylprednisolone (PDN, 10 μm) attenuated the rise in [Ca2+]c following hypo‐osmotic stress for both normal and mdx myotubes by about 50%.

The results described here suggest an increased permeability of mdx myotubes to Ca2+ under specific stress conditions. The ameliorating effect of PDN on [Ca2+]c could explain, at least partly, the beneficial effect of this drug on DMD patients.
PPADS and suramin as antagonists at cloned P2Y‐ and P2U‐purinoceptors
Authors: Charlton Steven J. , Brown Colin A. , Weisman Gary A. , Turner John T. , Erb Laurie , Boarder Michael R.
Published: July 2012

The effect of suramin and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) on the stimulation of phospholipase C in 1321N1 cells transfected with the human P2U‐purinoceptor (h‐P2U‐1321N1 cells) or with the turkey P2Y‐purinoceptor (t‐P2Y‐1321N1 cells) was investigated. 2‐Methylthioadenosine triphosphate (2MeSATP) was used as the agonist at t‐P2Y‐1321N1 cells and uridine triphosphate (UTP) at h‐P2U‐1321N1 cells.

Suramin caused a parallel shift to the right of the concentration‐response curves for 2MeSATP in the t‐P2Y‐1321N1 cells, yielding a Schild plot with a slope of 1.16 ± 0.08 and a pA2 value of 5.77 ± 0.11.

Suramin also caused a shift to the right of concentration‐response curves for UTP in the h‐P2U‐1321N1 cells, and on Schild plots gave a slope different from unity (1.57 ± 0.19) and an apparent pA2 value of 4.32 ± 0.13. Suramin was therefore a less potent antagonist at the P2U‐purinoceptor than the P2Y‐purinoceptor.

In the presence of the ectonucleotidase inhibitor, ARL 67156 (6‐N,N‐diethyl‐β,γ‐dibromomethylene‐D‐ATP) there was no significant difference in the EC50 or shapes of curves with either cell type, and no difference in pA2 values for suramin.

PPADS caused an increase in the EC50 for 2MeSATP in the t‐P2Y‐1321N1 cells. The Schild plot had a slope different from unity (0.55 ± 0.15) and an X‐intercept corresponding to an apparent pA2 of 5.98 ± 0.65.

PPADS up to 30 μm had no effect on the concentration‐response curve for UTP with the h‐P2U‐1321N1 cells.

In conclusion, suramin and PPADS show clear differences in their action at the 2 receptor types, in each case being substantially more effective as an antagonist at the P2Y‐purinoceptor than at the P2U‐purinoceptor. Ectonucleotidase breakdown had little influence on the nature of the responses at the two receptor types, or in their differential sensitivity to suramin.
Downregulation of nitrovasodilator‐induced cyclic GMP accumulation in cells exposed to endotoxin or interleukin‐1β
Authors: Papapetropoulos Andreas , Abou‐Mohamed Gamal , Marczin Nandor , Murad Ferid , Caldwell R. William , Catravas John D.
Published: July 2012

Induction of nitric oxide synthase (iNOS) results in overproduction of nitric oxide (NO), which may be a principal cause of the massive vasodilatation and hypotension observed in septic shock. Since NO‐induced vasorelaxation is mediated via the soluble isoform of guanylate cyclase (sGC), the regulation of sGC activity during shock is of obvious importance, but yet poorly understood. The aim of the present study was to investigate the activation of sGC by sodium nitroprusside (SNP) before and after exposure of rat aortic smooth muscle cells to endotoxin (LPS) or interleukin‐1β (IL‐1β).

Exposure of rat aortic smooth muscle cells to SNP (10 μm) elicited up to 200 fold increases in cyclic GMP. This effect was attenuated by 30–70% in IL‐1β‐ or LPS‐pretreated cells, in a pretreatment time‐and IL‐1β‐ or LPS‐concentration‐dependent manner. When, however, cells were exposed to IL‐1β or LPS and then stimulated with the particulate guanylate cyclase activator, atriopeptin II, no reduction in cyclic GMP accumulation was observed.

Pretreatment of rats with LPS (5 mg kg−1, i.v.) for 6 h led to a decrease in aortic ring SNP‐induced cyclic GMP accumulation.

The IL‐1β‐induced reduction in SNP‐stimulated cyclic GMP accumulation in cultured cells was dependent on NO production, as arginine depletion abolished the downregulation of cyclic GMP accumulation in response to SNP.

Reverse‐transcriptase‐polymerase chain reaction analysis revealed that the ratio of steady state mRNA for the α1 subunit of sGC to glyceraldehyde phosphate dehydrogenase was decreased in LPS‐ or IL‐1β‐treated cells, as compared to vehicle‐treated cells.

Protein levels of the α1 sGC subunit remained unaltered upon exposure to LPS or IL‐1β, suggesting that the early decreased cyclic GMP accumulation in IL‐1β‐ or LPS‐pretreated cells was probably due to reduced sGC activation. Thus, the observed decreased responsiveness of sGC to NO stimulation following cytokine or LPS challenge may represent an important homeostatic mechanism to offset the extensive vasodilatation seen in sepsis.
Role of intracellular Ca2+ in the K channel opener action of CGRP in the guinea‐pig ureter
Authors: Maggi Carlo Alberto , Giuliani Sandro , Santicioli Paolo , Brading Alison F.
Published: July 2012

The aim of this study was to assess the role of sarcoplasmic reticulum (SR) calcium (Ca2+) in the smooth muscle relaxant and hyperpolarizing actions of calcitonin gene‐related peptide (CGRP) in the guinea‐pig ureter.

CGRP (0.1 μm) rapidly and transiently reduced myogenic phasic contractions (twitches) produced by electrical field stimulation (EFS). Approximately 70% of the response to CGRP was antagonized by glibenclamide (1 μm).

Cyclopiazonic acid (CPA, 10 μm), ryanodine (100 μm) and thapsigargin (1 μm) reduced only the glibenclamide‐sensitive component of the response to CGRP (0.1 μm) but did not modify the mechanoinhibitory effect of cromakalim (3 μm). A low concentration of CPA (1 μm), assumed to produce a limited impairment of Ca2+ uptake from the stores, prolonged the duration of the inhibitory response to CGRP. Pre‐exposure to caffeine (5 mM) inhibited the suppression of twitches by CGRP or cromakalim.

When the frequency of EFS was increased, the suppression of twitches by CGRP was reduced. Under these conditions, CPA (1 μm) again prolonged the duration of the inhibitory response to CGRP.

CGRP (0.1 μm) and cromakalim (3 μm) markedly depressed the phasic component of contractions to 80 mM KCl. CPA (10 μm) antagonized the inhibitory effect of CGRP but not that of cromakalim. Inhibition of the tonic contraction to 80 mM KCl by CGRP was insensitive to CPA.

In sucrose gap experiments, a 5 min exposure to CGRP (0.1 μm) or cromakalim (3 μm) produced a sustained membrane hyperpolarization. Caffeine (5 mM) produced a glibenclamide‐sensitive transient hyperpolarization followed by a sustained depolarization. When tested in a Ca2+‐free medium the hyperpolarization produced by CGRP, cromakalim or caffeine was reduced. In normal Krebs, pre‐exposure to CPA (10 μm, 60 min) only abolished the hyperpolarization induced by CGRP. In contrast, 5 min after a caffeine challenge (5 mM) the hyperpolarizations induced by CGRP or cromakalim were reduced. The CGRP‐induced hyperpolarization was insensitive to apamin (0.1 μm) or charybdotoxin (0.1 μm).

We conclude that the K channel‐opening action of CGRP in the guinea‐pig ureter requires the mobilization of intracellular Ca2+ from a caffeine‐ and CPA‐sensitive store, leading to transient activation of glibenclamide‐sensitive K channels. The K channel‐opening action of caffeine appears to involve Ca2+ mobilization from a store which is insensitive to depletion by CPA.
Pharmacological analysis of dopamine stimulation of [35S]‐GTPγS binding via human D2short and D2long dopamine receptors expressed in recombinant cells
Authors: Gardner B. , Hall D.A. , Strange P.G.
Published: July 2012

The activation of G‐proteins by agonist‐occupied D2 or D3 dopamine receptors in membranes from recombinant cells expressing the cloned receptors has been analysed by a [35S]‐guanosine 5′‐[γ‐thio] triphosphate ([35S]‐GTPγS) binding assay.

The rate of [35S]‐GTPγS binding was increased by dopamine in a dose‐dependent manner in membranes from CHO cells stably expressing either the D2short or D2long dopamine receptor.

The dopamine‐induced stimulation of [35S]‐GTPγS binding could be inhibited by a range of antagonists. Affinities for antagonists derived from the inhibition of the dopamine stimulation of [35S]‐GTPγS binding correlated very well with affinities derived from radioligand binding studies.

When the maximum [35S]‐GTPγS binding responses stimulated by dopamine acting at different receptor subtypes were compared, there was a tendency for the stimulation via the D2short receptor to be greater than via the D2long receptor and for the stimulation via the D3 dopamine receptor to be less than for either D2 receptor. These differences in maximal response were also seen when the inhibitory effects of dopamine on adenylyl cyclase via the three receptor subtypes were compared.

The stimulation of [35S]‐GTPγS binding by dopamine in membranes from recombinant cells therefore provides an excellent system for studying the molecular nature of agonism and the receptor/G‐protein interactions for these receptors.
Inhibition of a store‐operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908
Authors: Encabo A. , Romanin C. , Birke F.W. , Kukovetz W.R. , Groschner Klaus
Published: July 2012

The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells.

LOE 908 inhibited store‐operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 μm and 4 μm, respectively.

LOE 908 did not affect thapsigargin‐ or ionomycin‐induced Ca2+ release from intracellular stores up to concentrations of 3 μm.

LOE 908 reversibly suppressed thapsigargin‐ as well as ionomycin‐induced whole‐cell membrane currents.

The LOE 908‐sensitive membrane conductance corresponded to a cation permeability of 5.5 and 6.9 fold selectivity for Ca2+ over K+ in the presence of thapsigargin and ionomycin, respectively.

Our results suggest that the isoquinoline, LOE 908 is a novel, potent inhibitor of the store‐operated (capacitive) Ca2+ entry pathway in endothelial cells.
The effect of the PKC inhibitor GF109203X on the release of Ca2+ from internal stores and Ca2+ entry in DDT1 MF‐2 cells
Authors: Sipma Henk , van der Zee Lucie , van den Akker Jan , den Hertog Adriaan , Nelemans Adriaan
Published: July 2012

The effects of the specific protein kinase C (PKC) inhibitor, GF109203X, were measured on the cytoplasmic Ca2+ concentration ([Ca2+]i), and on histamine H1 receptor‐ and thapsigargin‐mediated increases in [Ca2+]i in DDT1 MF‐2 smooth muscle cells.

After pretreatment of cells with GF109203X (5 μm, 45 min), the histamine (100 μm)‐induced initial rise in [Ca2+]i, representing Ca2+ mobilization from internal stores, was inhibited (by 59 ± 7%). The slowly declining phase of the histamine induced Ca2+ response, reflecting Ca2+ entry, was enhanced (83 ± 26%) in the presence of the PKC inhibitor.

The histamine induced release of Ca2+ from internal stores, measured after blocking Ca2+ entry with LaCl3 was inhibited by GF109203X in a concentration‐dependent manner (IC50:3.1 ± 1.1 μm).

Histamine‐induced formation of inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) was not changed in the presence of GF109203X.

The PKC activating phorbol ester, phorbol 12‐myristate 13‐acetate (PMA, 1 μm), strongly reduced histamine‐induced Ins(1,4,5)P3 formation (58 ± 16%). This effect was reversed by GF109203X (5 μm). Furthermore, PMA diminished histamine evoked Ca2+ release (50 ± 6%) and blocked Ca2+ entry completely.

The rise in [Ca2+]i caused by blocking endoplasmic reticulum Ca2+‐ATPase with thapsigargin (1 μm), was strongly reduced (57 ± 3%) after pretreatment of cells with GF109203X. Downregulation of PKC by long‐term pretreatment of cells with PMA (1 μm, 48 h) did not abolish this effect of GF109203X (48 ± 3% inhibition).

In permeabilized DDT1 MF‐2 cells preloaded with 45Ca2+ in the presence of GF109203X, the amount of 45Ca2+ released by Ins(1,4,5)P3 (10 μm) was markedly reduced (42 ± 9%). GF109203X did not release Ca2+ itself and did not impair Ins(1,4,5)P3 receptor function.

Uptake of 45Ca2+ by intact cells, representing Ca2+ entry, was enhanced by GF109203X (65 ± 11%), by histamine (24 ± 6%) and also by thapsigargin (121 ± 10%). The GF109203X‐ and the thapsigargin‐induced uptake of 45Ca2+ were not additive.

These data suggest that GF109203X reduces the filling‐state of intracellular Ins(1,4,5)P3 sensitive Ca2+ stores by inhibiting the Ca2+ uptake into these stores, thereby promoting store‐dependent (capacitive) Ca2+ entry.
Interaction of the islet nitric oxide system with L‐arginine‐induced secretion of insulin and glucagon in mice
Authors: Åkesson Björn , Mosén Henrik , Panagiotidis Georgios , Lundquist Ingmar
Published: July 2012

Several recent in vitro studies have suggested that production of nitric oxide (NO) from the islet NO system may have an important regulatory influence on the secretion of insulin and glucagon. In the present paper we have investigated, mainly with an in vivo approach, the influence and specificity of the NO synthase (NOS) blocker NG‐nitro‐L‐arginine methyl ester (L‐NAME) on L‐arginine‐induced secretion of insulin and glucagon.

In freely fed mice, L‐NAME pretreatment (1.2 mmol kg−1) influenced the dynamics of insulin and glucagon release following an equimolar dose of L‐arginine, the specific substrate for NOS activity, in that the NOS inhibitor enhanced the insulin response but suppressed the glucagon responses. This was reflected in a large decrease in the plasma glucose levels of the L‐NAME pretreated animals.

L‐NAME pretreatment did not influence the insulin and glucagon secretory responses to the L‐arginine‐enantiomer D‐arginine, which cannot serve as a substrate for NOS activity.

Replacing L‐NAME pretreatment by pretreatment with D‐arginine or L‐arginine itself, which both carry the same cationic charge and are devoid of NOS inhibitory properties, did not mimic the effects of L‐NAME on L‐arginine‐induced hormone release.

Fasting the animals for 24 h totally abolished the L‐NAME‐induced potentiation of L‐arginine stimulated insulin release suggesting that the sensitivity of the β‐cell secretory machinery to NO‐production is greatly changed in the fasting state. However, the L‐NAME‐induced suppression of L‐arginine stimulated glucagon release was unaffected by starvation.

In isolated islets from freely fed mice, L‐arginine (5 mM) stimulated insulin release was greatly enhanced and glucagon release markedly suppressed by the presence of the NOS inhibitor L‐NAME in the incubation medium. These effects were abolished in isolated islets taken from 24 h fasted mice.

Our present results, which showed that the NOS inhibitor L‐NAME markedly enhances insulin release but suppresses glucagon release induced by L‐arginine in the intact animal, give strong support to our previous hypothesis that the islet NO system is a negative modulator of insulin secretion and a positive modulator of glucagon secretion. Additionally, we observed that the importance of the β‐cell NO‐production for secretory mechanisms, as evaluated by the effect of L‐NAME on L‐arginine‐induced insulin release, was greatly changed after starvation, an effect less prominent with regard to glucagon release.
Stereoselective recognition of the enantiomers of phenglutarimide and of six related compounds by four muscarinic receptor subtypes
Authors: Waelbroeck M. , Lazareno S. , Pfaff O. , Friebe T. , Tastenoy M. , Mutschler E. , Lambrecht G.
Published: July 2012

We have compared the binding properties of the enantiomers of phenglutarimide (1) and of six related compounds to M1 receptors in NB‐OK‐1 cells, M2 receptors in rat heart, M3 receptors in rat pancreas and the M4 receptors of rat striatum, with their functional (antimuscarinic) properties in rabbit vas deferens (Mj/M4‐like), guinea‐pig atria (M2) and guinea‐pig ileum (M3) receptors. The binding properties of the enantiomers of three of the compounds were also measured on cloned human ml‐m4 receptors expressed by CHO cells, using [3H]‐N‐methylscopolamine ([3H]‐NMS) as radioligand.

The high affinity enantiomers behaved as competitive antagonists in binding and pharmacological studies. (S)‐phenglutarimide (pKiM1 = 9.0/9.3) and (R)‐thienglutarimide (pKiM1 = 8.6/9.2) recognized selectively the native M1 > M4 > M3 > M2 receptors in tissues as well as the respective cloned receptors.

The pA2 values at the inhibitory heteroreceptors in the rabbit vas deferens, and at the guinea‐pig atria and ileum for the seven more potent enantiomers were compatible with the previous classification of these receptors as M1/M4‐like, M2 and M3, respectively.

Replacement of the phenyl by a thienyl ring or of the diethylamino by a piperidino group in the phenglutarimide molecule did not affect markedly the potencies of the high affinity enantiomer. In contrast, replacement of the phenyl by a cyclohexyl ring decreased 20 fold the active enantiomers potency. Methylation of the piperidine‐2,6‐dione nitrogen also reduced markedly the eutomers' affinities, more on the M1 than on the other subtypes.

The selectivity profiles (recognition of four receptor subtypes) of six of the seven less active enantiomers were different from the corresponding more active enantiomers selectivity profiles, suggesting that the preparations used in this study were pure. However, we cannot not exclude the hypothesis that the batch of (S)‐thienglutarimide used in this study was contaminated by less than 0.02% of the eutomer.

In contrast with the eutomer binding site, replacement of the phenyl ring by a thienyl or cyclohexyl ring did not affect binding of the low affinity enantiomers to the muscarinic receptor or the [3H]‐NMS‐receptor complex. The replacement of the diethylamino group by a piperidine ring, and N‐methylation of the piperidine‐2,6 dione moiety increased slightly these enantiomers' potencies.

The muscarinic receptors were extremely stereoselective, and had up to 20 000 fold lower affinity for the less active enantiomers. However, the stereochemical requirements of the muscarinic receptor subtypes were different for the enantiomers of compounds 1–7, being most stringent at M1 receptors.

The weaker enantiomers behaved as competitive antagonists in pharmacological studies, at least in the concentration‐range investigated.
Influence of authentic nitric oxide on basal cytosolic [Ca2+] and Ca2+ release from internal stores in human platelets
Authors: Sang Kim‐Hanh Le Quan , Lantoine Frédérique , Devynck Marie‐Aude
Published: July 2012

Nitric oxide (NO) donors inhibit platelet function and Ca2+ mobilization evoked by different agonists. This led us to investigate the direct effects of authentic NO on basal cytosolic Ca2+ concentration ([Ca2+]i) and on Ca2+ mobilization induced by thrombin or by two inhibitors of intracellular Ca2+‐ATPases, thapsigargin and 2,5‐di‐(t‐butyl)‐l,4‐benzohydroquinone (t‐BuBHQ).

Cytosolic Ca2+ concentration was evaluated with Fura‐2, in the absence of Ca2+ influx. Addition of 5 μm NO increased by 48% the basal cytosolic [Ca2+] of resting human platelets whereas a lower concentration (0.1 μm) did not induce significant modifications. This NO‐induced Ca2+ increase was inversely correlated with the basal level of cytosolic [Ca2+].

NO pretreatment for 30 to 120 s decreased by 42 to 57% the transient [Ca2+]i peak evoked by 0.10 u ml−1 thrombin and strongly attenuated the initial rate of [Ca2+]i rise induced by 1 μm thapsigargin or by 20 μm t‐BuBHQ. The two components of the thapsigargin response, the Ca2+ release due to inhibition of Ca2+ pumps and the thromboxane A2‐dependent self‐amplification mechanism, were inhibited by NO. The observation that a subsequent stimulation was still capable of eliciting Ca2+ release suggests the presence of NO‐insensitive Ca2+ stores.

These findings indicate that nitric oxide can modulate basal cytosolic [Ca2+] in unstimulated human platelets and decrease the Ca2+ mobilization from NO‐sensitive internal stores evoked by stimulation of receptors or by Ca2+‐ATPase inhibitors. This underlines the important role of nitric oxide in the modulation of platelet Ca2+ handling.
Endothelin‐1‐induced contraction in isolated aortae from normotensive and DOCA‐salt hypertensive rats: effect of magnesium
Authors: Laurant Pascal , Berthelot Alain
Published: July 2012

The contractile responses to endothelin‐1 and the effect on these of various magnesium concentrations, were studied in isolated aortic rings from normotensive Sprague‐Dawley rats and deoxycorticosterone acetate‐salt (DOCA‐salt) hypertensive rats.

Contractions induced by endothelin‐1 were smaller in endothelium‐denuded aortae from DOCA‐salt hypertensive rats than in those from normotensive rats. The absence of calcium in the medium attenuated endothelin‐1‐induced contractions of aortae from both normotensive and DOCA‐salt rats, but the contraction was greater in aortae from DOCA‐salt hypertensive rats. Ryanodine (which inhibits the release of intracellular calcium) inhibited endothelin‐1‐induced contractions in aortae from DOCA‐salt hypertensive rats to a greater extent than in aortae from normotensive rats.

A high extracellular magnesium concentration (4.8 mM) attenuated endothelin‐1‐induced contractions in tissues from DOCA‐salt hypertensive rats but not in tissues from normotensive rats. In the absence of calcium, a high concentration of magnesium attenuated endothelin‐1‐induced contraction in aortae from both normotensive and hypertensive rats. In the presence of ryanodine, a high concentration of magnesium did not modify the contraction in preparations from either strain.

Absence of magnesium attenuated endothelin‐1‐induced contractions in aortae from both normotensive and DOCA‐salt hypertensive rats. In the absence of calcium, removal of magnesium totally inhibited endothelin‐1‐induced contraction in tissues from normotensive rats but had no effect in those from hypertensive rats. In the presence of ryanodine, the lack of magnesium inhibited endothelin‐1‐induced contractions in aortae from DOCA‐salt hypertensive rats but increased the sensitivity to endothelin‐1 of aortae from normotensive rats.

The presence of endothelium did not modify the effect of high magnesium on endothelin‐1‐induced contractions in aortae from normotensive and DOCA‐salt hypertensive rats. Conversely, the attenuating effect of magnesium removal on endothelin‐1‐induced contractions did not occur when endothelium was present.

In conclusion, endothelin‐1‐induced contraction was blunted in aortae from DOCA‐salt hypertensive rats. The blunted response was related to altered calcium utilization during contraction. Changes in extracellular magnesium concentration differentially alter endothelin‐1‐induced contraction in aortae from normotensive and hypertensive rats, possibly by interfering with calcium utilization during contraction. Magnesium may be required for the contractile response to endothelin‐1 and increasing magnesium may limit the vascular effects of endothelin‐1 in blood vessels from DOCA‐salt hypertensive rats.
Differential regulation of 5‐hydroxytryptamine release by GABAA and GABAB receptors in midbrain raphe nuclei and forebrain of rats
Authors: Tao Rui , Ma Zhiyuan , Auerbach Sidney B.
Published: July 2012

Extracellular 5‐hydroxytryptamine (5‐HT) was determined in dorsal raphe nucleus (DRN), median raphe nucleus (MRN) and nucleus accumbens by use of microdialysis in unanaesthetized rats.

Infusion of the γ‐aminobutyric acid (GABA)A receptor agonist muscimol into DRN and MRN resulted in decreased 5‐HT in DRN and MRN, respectively. Muscimol infusion into nucleus accumbens had no effect on 5‐HT.

Infusion of the GABAA receptor antgonist bicuculline into DRN resulted in increased DRN and nucleus accumbens 5‐HT. Bicuculline infusion into MRN had no effect on 5‐HT. This suggests that endogenous GABA had a tonic, GABAA receptor‐mediated inhibitory effect on 5‐HT in DRN, but not in MRN.

Infusion of the GABAB receptor agonist baclofen into DRN produced a decrease in DRN 5‐HT. Baclofen infusion into nucleus accumbens resulted in decreased nucleus accumbens 5‐HT. This suggests that GABAB receptors are present in the area of cell bodies and terminals of 5‐hydroxytryptaminergic neurones.

Infusion of the GABAB receptor antagonists phaclofen and 2‐hydroxysaclofen had no effect on midbrain raphe and forebrain 5‐HT. This suggests that GABAB receptors did not contribute to tonic inhibition of 5‐HT release.

In conclusion, 5‐HT release is physiologically regulated by distinct subtypes of GABA receptors in presynaptic and postsynaptic sites.