bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization and localization of thromboxane A2 receptor in human and guinea‐pig nasal mucosa using radiolabelled (+)‐S‐145
Authors: Arimura A , Miwa M , Hasegawa H , Kishino J , Notoya M , Yasui K , Komori M , Iwata S 
Published: January 2005

TxA2 receptor (TP‐receptor) antagonists such as S‐1452 and Bay u 3405 have been shown to be effective in alleviating nasal blockage in patients with allergic rhinitis as well as guinea‐pig allergic rhinitis models. The present study was conducted to examine the existence and localization of the TP‐receptor in human and guinea‐pig nasal mucosa by in vitro receptor binding autoradiography using radiolabelled (+)‐S‐145, which is a potent and specific TP‐receptor antagonist with an extremely slow dissociation rate.
We ascertained the binding specificity of [3H]‐(+)‐S‐145 in human and guinea‐pig platelet membranes by comparing the ability of four TP‐receptor ligands of U‐46619, (+)‐S‐145, I‐(+)‐S‐145 and Bay u 3405 to displace the specific binding of [3H]‐(+)‐S‐145 and [3H]‐U‐46619. The rank order of potency (Ki) for the displacement was correlated highly with that determined from [3H]‐U‐46619 binding to the same preparations.
Quantitative autoradiography using a radioluminographic imaging plate system, in which the radioactivity of [3H]‐(+)‐S‐145 is expressed as photostimulated luminescence (PSL) per area (mm2), revealed that specific binding of [3H]‐(+)‐S‐145 to human and guinea‐pig nasal mucosa was saturable. Scatchard analysis showed about three fold higher affinity and two fold greater maximal binding to the nasal mucosa of humans than that of guinea‐pigs: the KD and Bmax values in human mucosa were 2.82±0.35 nM and 6.47±0.33 PSL mm−2 and those in guinea‐pig mucosa were 8.23±1.93 nM and 3.37±0.66 PSL mm−2, respectively.
Specific [3H]‐(+)‐S‐145 binding to cryostat sections of human and guinea‐pig nasal mucosa was displaced by another TP‐receptor antagonist, Bay u 3405, and a TP‐receptor agonist, U‐46619. The order of potency (Ki value: nM) was (+)‐S‐145 (2.5) > Bay u 3405 (15.4) >> U‐46619 (359.6) in human nasal mucosa and (+)‐S‐145 (22.8) > U‐46619 (49.8) & Bay u 3405 (62.1) in guinea‐pig nasal mucosa. These rank orders showed rather good correlation with those obtained for the respective platelet membranes.
Autoradiographs of human nasal mucosa demonstrated that specific [125I]‐(+)‐S‐145 binding sites mainly exist on the smooth muscle layers of venous sinusoids and arterioles in the lamina propria, with few or no binding sites in the epithelium and nasal gland.
We concluded that radiolabelled (+)‐S‐145 can be used as a TP‐receptor ligand for autoradiographic study, and that the TP‐receptor is exclusively located on smooth muscle layers of venous sinusoids and arterioles in the nasal mucosa. The potent vasoconstrictive activity of TxA2 may cause reduction of local blood flow followed by mucosal oedema probably through mechanisms of vascular injury such as ischaemia‐reperfusion.
AT2 antagonist‐sensitive potentiation of angiotensin II‐induced constriction by NO blockade and its dependence on endothelium and P450 eicosanoids in rat renal vasculature
Authors: Muller Catherine , Endlich Karlhans , Helwig Jean‐Jacques 
Published: January 2005

We showed earlier that NO inhibition caused a left‐shift and augmented Emax of the concentration‐response curve of AT1‐mediated (angiotensin II)‐induced vasoconstrictions (AII‐VC) in the rat kidney. The 0.01–0.1 nM AII‐VC unmasked by the potentiating effect of NO inhibition, were sensitive not only to AT1 (L158809), but also to AT2 receptor (PD123319) antagonists. We now demonstrate the role of endothelium and eicosanoids in the NO‐masked AT1/AT2‐mediated component of the AII‐VC in isolated indomethacin‐perfused kidneys of the rat.
L‐NAME increased 0.1 nM AII‐VC 7.2 fold. Pretreatment of the kidneys with factor VIII antibody/complement or with the detergent CHAPS to damage endothelium, decreased carbachol‐induced vasodilatation and blunted by 60 and 30% respectively, the enhancement of AII‐VC during NO inhibition.
L‐NAME also increased 3 μM noradrenaline (NA)‐induced vasoconstriction (NA‐VC) 8.1 fold. In contrast to AII‐VC, endothelium damage was without effect on the enhancement of NA‐VC by L‐NAME, suggesting a dominant role of endothelium‐derived NO in the enhancement of NA‐VC.
During NO inhibition, ETYA (2 μM; an inhibitor of all arachidonic acid derived pathways) and α‐naphtoflavone (10 μM; an inhibitor of the cytochrome P450 isozymes), decreased by 85% the 0.1 nM AII‐VC.
In conclusion, during NO inhibition, the AT1‐mediated constriction to low concentrations of AII, which is sensitive to AT2 antagonists, depends on intact endothelium, and can be blocked by inhibition of eicosanoid synthesis. The results suggest that the AII‐mediated vasoconstriction through AT1 receptors is potentiated in the absence of NO, by the release of eicosanoids from the endothelium through AT2 receptors.
Epigallocatechin suppression of proliferation of vascular smooth muscle cells: correlation with c‐jun and JNK
Authors: Lu Liang‐Huei , Lee Shoei‐Sheng , Huang Huei‐Chen 
Published: January 2005

The mechanisms of the antiproliferative effect of epigallocatechin, one of the catechin derivatives found in green tea, in vascular smooth muscle cells were studied. The proliferative response was determined from the uptake of tritiated thymidine.
In the concentration range of 10−6 to 10−4 m, catechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin, epigallocatechin gallate, concentration‐dependently inhibited the proliferative response stimulated by serum in rabbit cultured vascular smooth muscle cells. Catechin and epicatechin were less effective in inhibiting the serum‐stimulated smooth muscle cell proliferation, indicating that the galloyl group may be important for full inhibitory activity.
Epigallocatechin (EGC) inhibited the proliferative responses in different cells including rat aortic smooth muscle cells (A7r5 cells), rabbit cultured aortic smooth muscle cells, human coronary artery smooth muscle cells, and human CEM lymphocytes in a concentration‐dependent manner. The possible mechanisms of the antiproliferative effect of EGC were further studied in A7r5 cells.
The membranous protein tyrosine kinase activity stimulated by serum in A7r5 cells was significantly reduced by 10−5 m EGC. In contrast, the cytosolic protein kinase C activity stimulated by phorbol ester was unaffected by directly incubating with EGC (10−6−10−4 m).
We also performed Western blot analysis using the anti‐phosphotyrosine monoclonal antibody PY‐20. EGC (10−5 m) reduced the levels of tyrosine phosphorylated proteins with different molecular weights, indicating that EGC may inhibit the protein tyrosine kinase activity or stimulate the protein phosphatase activity.
Reverse transcription‐polymerase chain reaction analysis of c‐fos, c‐jun and c‐myc mRNA levels demonstrated that c‐jun mRNA level after serum‐stimulation was significantly reduced by 10−5 m EGC. However, the reduction of c‐fos and c‐myc mRNA levels by 10−5 m EGC did not achieve significance.
Western blot analysis using the antibody against JNK (c‐jun N‐terminal kinase) and ERK (extracellular signal‐regulated kinase) demonstrated that the level of phosphorylated JNK1, but not phosphorylated ERK1 and ERK2, was reduced by 10−5 m EGC. Direct measurement of kinase activity by immune complex kinase assay confirmed that JNK1 activity was inhibited by EGC treatment. These results demonstrate that EGC preferentially reduced the activation of JNK/SAPK (stress‐activated protein kinase) signal transduction pathway.
It is suggested that the antiproliferative effect of epigallocatechin on vascular smooth muscle cells may partly be mediated through inhibition of protein tyrosine kinase activity, reducing c‐jun mRNA expression and inhibiting JNK1 activation. Tea catechins may be useful as a template for the development of drugs to prevent the pathological changes of atherosclerosis and post‐angioplasty restenosis.

British Journal of Pharmacology (1998) 124, 1227–1237; doi:10.1038/sj.bjp.0701912
Mutational analysis of the potential phosphorylation sites for protein kinase C on the CCKA receptor
Authors: Smeets R L L. , Fouraux M A. , Pouwels W , van Emst‐de Vries S E. , Ronken E , De Pont J J H H M. , Willems P H G M. 
Published: January 2005

Many G protein‐coupled receptors contain potential phosphorylation sites for protein kinase C (PKC), the exact role of which is poorly understood. In the present study, a mutant cholecystokininA (CCKA) receptor was generated in which the four consensus sites for PKC action were changed in an alanine. Both the wild‐type (CCKAWT) and mutant (CCKAMT) receptor were stably expressed in Chinese hamster ovary (CHO) cells.
Binding of [3H]‐cholecystokinin‐(26‐33)‐peptide amide (CCK‐8) to membranes prepared from CHO‐CCKAWT cells and CHO‐CCKAMT cells revealed no difference in binding affinity (Kd values of 0.72 nM and 0.86 nM CCK‐8, respectively).
The dose‐response curves for CCK‐8‐induced cyclic AMP accumulation and inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) formation were shifted to the left in CHO‐CCKAMT cells. This leftward shift was mimicked by the potent inhibitor of protein kinase activity, staurosporine. However, the effect of staurosporine was restricted to CHO‐CCKAWT cells. This demonstrates that attenuation of CCK‐8‐induced activation of adenylyl cyclase and phospholipase C‐β involves a staurosporine‐sensitive kinase, which acts directly at the potential sites of PKC action on the CCKA receptor in CCK‐8‐stimulated CHO‐CCKAWT cells.
The potent PKC activator, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), evoked a rightward shift of the dose‐response curve for CCK‐8‐induced cyclic AMP accumulation in CHO‐CCKAWT cells but not CHO‐CCKAMT cells. This is in agreement with the idea that PKC acts directly at the CCKA receptor to attenuate adenylyl cyclase activation.
In contrast, TPA evoked a rightward shift of the dose‐response curve for CCK‐8‐induced Ins(1,4,5)P3 formation in both cell lines. This demonstrates that high‐level PKC activation inhibits CCK‐8‐induced Ins(1,4,5)P3 formation also at a post‐receptor site.
TPA inhibition of agonist‐induced Ca2+ mobilization was only partly reversed in CHO‐CCKAMT cells. TPA also inhibited Ca2+ mobilization in response to the G protein activator, Mas‐7. These findings are in agreement with the idea that partial reversal of agonist‐induced Ca2+ mobilization is due to the presence of an additional site of PKC inhibition downstream of the receptor and that the mutant receptor itself is not inhibited by the action of PKC.
The data presented demonstrate that the predicted sites for PKC action on the CCKA receptor are the only sites involved in TPA‐induced uncoupling of the receptor from its G proteins. In addition, the present study unveils a post‐receptor site of PKC action, the physiological relevance of which may be that it provides a means for the cell to inhibit phospholipase C‐β activation by receptors that are not phosphorylated by PKC.
Modulation of GABAA receptors and inhibitory synaptic currents by the endogenous CNS sleep regulator cis‐9,10‐octadecenoamide (cOA)
Authors: Lees George , Edwards Michelle D. , Hassoni Abdul A. , Robin Ganellin C , Galanakis Dimitrious 
Published: January 2005

Cis‐9,10‐octadecenoamide (cOA) accumulates in the CSF of sleep‐deprived cats and may represent a novel signalling molecule. Synthetic cOA has been shown to induce physiological sleep when injected into laboratory rats. Here we assess the cellular mode of action of cOA in vitro.
In all rat cultured cortical neurones (pyramidal cells) examined, the synthetic brain lipid (3.2–64 μM) enhanced the responses to subsaturating GABA concentrations (up to circa 2×) in a concentration‐dependent manner (EC50, circa 15 μM).
(20 μM) cOA significantly enhanced the affinity of exogenous GABA for its receptor without changing the Hill slope or the maximal response. These effects were not voltage‐dependent or secondary to shifts in ECl.
In the absence of GABA, cOA directly evoked small inhibitory currents in a subpopulation (<7%) of sensitive cells.
20 μM cOA reversibly enhanced the duration of spontaneous inhibitory post synaptic currents (circa 2 fold) without significantly altering their amplitude.
At 32–64 μM, cOA reversibly reduced the incidence and amplitude of both inhibitory post synaptic currents (i.p.s.cs) and excitatory post synaptic currents (e.p.s.cs) in the cultured neuronal circuits in common with other depressant drugs acting at the GABAA receptor.
32 μM Oleic acid did not modulate exogenous GABA currents or synaptic activity suggesting that cOAs actions are mediated through a specific receptor.
A specific, protein‐dependent interaction with GABAA receptors was confirmed in Xenopus oocytes. Recombinant human receptors were modulated by 10 μM cOA (and diazepam) only when a γ2 subunit was co‐expressed with α1β2: the cOA response was not sensitive to the specific benzodiazepine antagonist flumazenil (1 μM).
cOA may represent an endogenous ligand for allosteric modulatory sites on isoforms of GABAA receptors which are crucial for the regulation of arousal and have recently been implicated in the circadian control of physiological sleep.
Tandospirone‐induced K+ current in acutely dissociated rat dorsal raphe neurones
Authors: Jin Young‐Ho , Akaike Norio 
Published: January 2005

The effects of tandospirone (TDS) on dissociated rat dorsal raphe neurones were investigated using the patch‐clamp method.
Under current‐clamp conditions, TDS hyperpolarized the cell membrane, resulting in the reduction of firing rates.
Under voltage‐clamp conditions, TDS induced an inward rectifying K+ current in a concentration‐dependent manner.
The TDS‐induced K+ currents (ITDS) were mimicked by 8‐OH‐DPAT, a 5‐HT1A agonist. The ITDS was blocked by spiperone, a 5‐HT1A receptor antagonist, in a concentration‐dependent manner.
N‐Ethylmaleimide, an agent which uncouples between the receptor and the G‐protein, irreversibly blocked the ITDS.
In neurones perfused intracellularly with a pipette‐solution containing GTP using the conventional whole‐cell patch recording, the ITDS showed a gradual rundown. When the neurones were perfused with GTPγS, TDS activated the inwardly rectifying K+ current in an irreversible manner.
In the inside‐out patch recording mode, TDS‐activated single K+ channel currents (iTDS) which also showed an inward rectification. When the GDP in cytosolic side was completely replaced with GTP, the open probability of iTDS significantly increased.
These results indicate that the activation of 5‐HT1A receptors by TDS directly opens the inward rectifying K+ channels via a G‐protein mediated process.
Agonist action at D2(long) dopamine receptors: ligand binding and functional assays
Authors: Gardner B , Strange P G. 
Published: January 2005

The activities of a range of agonists at D2(long) dopamine receptors expressed in CHO cells have been determined in ligand binding and in a functional assay, the stimulation of [35S]‐GTPγS binding.
For several agonists (apomorphine, dopamine, pergolide, quinpirole, NPA, ropinirole, talipexole) binding in the absence of added guanine nucleotides was best described in terms of interaction at higher and lower affinity states, whereas for other agonists (bromocriptine, DHEC, lisuride, 3‐PPP) a one binding site model was a good description of the data. In the presence of GTP (100 μM) all agonist binding data were best described by a one site model.
All of the agonists tested increased [35S]‐GTPγS binding above the basal level and the maximal effects and potencies of the agonists in this test were different. There was no clear relation betwen the ability of an agonist to stabilize the formation of the ternary complex of agonist/receptor/G‐protein and the maximal activity of the agonist or the amplification factor (ratio of dissociation constant for binding to receptor to EC50 in functional assay).
A comparison was made between the profiles of the D2(short) and D2(long) receptor isoforms in these assays.
Functional coupling of a recombinant Human 5‐HT5A receptor to G‐proteins in HEK‐293 cells
Authors: Hurley P. T. , McMahon R. A. , Fanning P. , O'Boyle K. M. , Rogers M. , Martin F. 
Published: January 2005

We have cloned, expressed and pharmacologically characterized the Human 5‐HT5A receptor.
We have shown that ligand activation of the Human 5‐HT5A receptor results in functional coupling to G‐proteins in HEK‐293 cells.
Stimulation of the receptor with 5‐CT (5‐carboxamidotryptamine) resulted in a dose‐dependent increase in the % [35S]‐GTPγS binding over the basal level. This is the first study to describe such G‐protein activation for the Human 5‐HT5A receptor in any cell.
A dose‐dependent inhibition of cyclic AMP accumulation was observed in the recombinant Human 5‐HT5A receptor cell line, suggesting a functional coupling to a Gαi, G‐protein in the HEK‐293 cell line.
A ligand‐stimulated reduction in the detectable level of the catalytic domain of protein kinase A (PKA) in nuclear extracts isolated from Human 5‐HT5A expressing cells was observed. This observation was consistent with the reduction in the level of cyclic AMP accumulation, in response to receptor activation.

British Journal of Pharmacology (1998) 124, 1238–1244; doi:10.1038/sj.bjp.0701928
Activation of a nonspecific cation current in rat cultured retinal pigment epithelial cells: involvement of a Gαi subunit protein and the mitogen‐activated protein kinase signalling pathway
Authors: Ryan Jennifer S. , Kelly Melanie E. M. 
Published: January 2005

Whole‐cell patch‐clamp recording techniques were used to investigate the G protein subtype and related signalling molecules involved in activation of a nonspecific cation (NSC) current in rat cultured retinal pigment epithelial (RPE) cells.
Under control conditions, in 130 mm NaCl with K+ aspartate in the pipette, cytosolic dialysis with guanosine‐5′‐O‐(3‐triphosphate) (GTPγS, 0.1 mm) activated a large non‐inactivating NSC current in 80% of the cells recorded from.
Loading RPE cells with antibodies (10 μg‐ml−1) against the α subunit of all PTX‐sensitive G proteins (Gαi/o/t/z) reduced NSC current activation to 11%, while loading RPE cells with antibodies directed specifically against the α subunits of the Gi subclass (Gαi‐3) completely abolished current activation. In RPE cells loaded with anti‐Gαs activation of the NSC current was unaffected.
Investigation of the potential downstream mediators in the Gαi NSC channel pathway revealed that activation of the cation conductance was unaffected by treatment of RPE cells with the selective protein kinase C inhibitor GF 109203X (3 μm) or the selective CaM kinase II inhibitor KN‐93 (50 μm). However, NSC current activation was delayed and the current amplitude reduced in the presence of the nonselective kinase inhibitor H‐7 (100 μm) or the selective inhibitor of MAPKK (MEK) activation, PD 98059 (50 μm).
In the absence of GTPγS, the NSC current was not activated by superfusion of the cells with the cyclic GMP kinase activator dibutyryl‐cyclic GMP or with the adenylate cyclase activator forskolin.
These results support the involvement of a G protein of the Gαi subclass in the activation of a NSC current in rat RPE cells, and suggest a potential modulatory role for MAP kinase‐dependent phosphorylation in current regulation.

British Journal of Pharmacology (1998) 124, 1115–1122; doi:10.1038/sj.bjp.0701936
Effects of U‐50,488H withdrawal on catecholaminergic neurones of the rat ventricle
Authors: Milanés M. V. , Laorden M. L. 
Published: January 2005

In the present study the changes in noradrenaline (NA) and dopamine (DA) content and turnover during naloxone‐induced withdrawal were analysed in the right ventricle of rats chronically treated with the κ‐agonist U‐50,488H.
Rats were rendered tolerant by administration of U‐50,488H twice a day for 4 days. On the day of death the animals were injected with saline or naloxone (3 mg kg−1, s.c.) to precipitate a withdrawal syndrome.
After naloxone administration to U‐50,488H‐ treated rats we found neither behaviour signs of physical dependence nor changes in the tissue content of noradrenaline (NA). However, naloxone induced a decrease in both cardiac normetanephrine (NM) levels and NA turnover.
Similarly, naloxone enhanced the dopamine content and decreased the 3,4‐dihydroxyphenylacetic acid (DOPAC) concentration and dopamine turnover.
Importantly and in contrast to μ‐agonists, the present results demonstrate that U‐50,488H withdrawal produced a decrease in the NA and dopamine turnover, without behavioural signs of physical dependence.

British Journal of Pharmacology (1998) 124, 1060–1064; doi:10.1038/sj.bjp.0701939
Spatial heterogeneity in the mechanisms contributing to acetylcholine‐induced dilatation in the rabbit isolated ear
Authors: Berman Rodney S. , Griffith Tudor M. 
Published: January 2005

Using an X‐ray microangiographic technique in rabbit isolated perfused ears preconstricted with 5‐HT (300 nm) and histamine (300 nm), we investigated the combined actions of Nω‐nitro‐l‐arginine methyl ester (l‐NAME) and indomethacin on acetylcholine‐induced depressor responses.
Under control conditions, acetylcholine (10 nm–30 μm) induced a concentration‐dependent reversal of the pressor response, reaching a maximum of 66.0±13.6% (n = 6). In the presence of l‐NAME (300 μm) and indomethacin (10 μm), this depressor action was reduced, reaching a maximum of 38.6±5.9% (n = 6).
The control response was associated with substantial vasodilatation in the central ear artery (G0), a smaller dilatory action on first generation branch arteries (G1) and no effect on second generation branch arteries (G2). In the presence of l‐NAME and indomethacin, vasodilatation occurred in G2 with no effect in G0 or G1.
Two calcium‐activated K+ channels blockers, charybdotoxin (ChTX; 10 nm) and penitrem A (100 nm), further inhibited, but did not abolish, the l‐NAME‐ and indomethacin‐resistant response to acetylcholine (10 nm–300 μm). Both agents abolished the vasodilatory action of acetylcholine in G2.
In conclusion, l‐NAME and indomethacin induced a shift in acetylcholine‐induced vasodilatation from G0 and G1 to G2. This is consistent with the suggestion that nitric oxide dominates in larger vessels whilst other mechanisms dominate in smaller vessels. The l‐NAME‐ and indomethacin‐resistant component was inhibited by ChTX and penitrem A, suggesting it is mediated, at least in part, by activation of KCa channels and could therefore involve a hyperpolarising mediator such as endothelium‐derived hyperpolarising factor.

British Journal of Pharmacology (1998) 124, 1245–1253; doi:10.1038/sj.bjp.0701945