bluuebunny/crossref_metadata_2025 huggingface dataset:
KW‐3902, a selective high affinity antagonist for adenosine A1 receptors
Authors: Nonakay Hiromi , Ichimura Michio , Takeda Masami , Kanda Tomoyuki , Shimada Junichi , Suzuki Fumio , Kase Hiroshi
Published: July 2012

We demonstrate that 8‐(noradamantan‐3‐yl)‐1,3‐dipropylxanthine (KW‐3902) is a very potent and selective adenosine A1 receptor antagonist, assessed by radioligand binding and cyclic AMP response in cells.

In rat forebrain adenosine A1 receptors labelled with [3H]‐cyclohexyladenosine (CHA), KW‐3902 had a Ki value of 0.19 nM, whereas it showed a Ki value of 170 nM in rat striatal A2A receptors labelled with [3H]‐2‐[p‐(2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS21680), indicating 890 fold A1 receptor selectivity versus the A2A receptor. KW‐3902 at 10 μm showed no effect on recombinant rat A3 receptors expressed on CHO cells.

Saturation studies with [3H]‐KW‐3902 revealed that it bound with high affinity (Kd = 77 pM) and limited capacity (Bmax = 470 fmol mg−1 of protein) to a single class of recognition sites. A high positive correlation was observed between the pharmacological profile of adenosine ligands inhibiting the binding of [3H]‐KW‐3902 and that of [3H]‐CHA.

KW‐3902 showed potent A1 antagonism against the inhibition of forskolin‐induced cyclic AMP accumulation in DDT1 MF‐2 cells by the Arselective agonist, cyclopentyladenosine with a dissociation constant (KB value) of 0.34 nM. KW‐3902 antagonized 5′‐N‐ethylcarboxamidoadenosine‐elicited cyclic AMP accumulation via A2B receptors with a KB value of 52 nM.

KW‐3902 exhibited marked species‐dependent differences in the binding affinities. The highest affinity was for the rat A1 receptor (Ki = 0.19 nM) and these values for guinea‐pig and dog A1 receptors were 1.3 and 10 nM, respectively.
P2‐purinoceptors mediating spasm of the isolated uterus of the non‐pregnant guinea‐pig
Authors: Piper Angela S. , Hollingsworth Michael
Published: July 2012

The isolated uterus of the non‐pregnant guinea‐pig has been suggested to contain P1‐, and possibly P2‐purinoceptors mediating spasm. The presence of P1‐purinoceptors has been confirmed and these receptors have been further characterized.

In the presence of the adenosine uptake inhibitor, S‐(4‐nitrobenzyl)‐6‐thioinosine (NBTI, 300 nM) and a pA100 concentration of the P1‐purinoceptor antagonist 8‐sulphophenyltheophylline (140 μm), the potency order of agonists as spasmogens was: 2 methylthio ATP»αβ methylene ATP = UTP = ATP» βγ methylene ATP. This order is not consistent with any single recognised P2‐purinoceptor subtype.

Indomethacin (1 μm) treatment abolished responses to 2 methylthio ATP, αβ methylene ATP and UTP, while spasm to ATP was significantly inhibited. When the endometrial and circular smooth muscle cell layers were removed, spasmogenic responses to ATP, 2 methylthio ATP, αβ methylene ATP and UTP were significantly reduced.

2 methylthio ATP was able to cause desensitization to itself, but not to UTP, indicating that these agonists act at different receptor sites.

The P2‐purinoceptor antagonist, suramin antagonized 2 methylthio ATP with a pA2 of 5.9±0.3. Suramin was also an antagonist of ATP and UTP. In the case of ATP, the antagonism was not dependent on suramin concentration, while for UTP the interaction appeared to be non‐equilibrium. Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS, 10 μm) had no effect on spasm to ATP, UTP or 2 methythio ATP.

In the presence of indomethacin, responses to ATP were unaffected by 8‐sulphophenyltheophylline (140 μm) or by suramin (100 μm), but PPADS (10 μm) antagonized ATP.

These results suggest that the isolated uterus of the non‐pregnant guinea‐pig contains a mixture of P2‐purinoceptors. P2U‐ (or UTP‐selective pyrimidinoceptors) and P2Y‐purinoceptors appear to be present, probably mainly located on the endometrial or circular smooth muscle layer. Activation of these receptors leads to spasm via increases in prostanoid generation. There appears also to be a third class of non‐P2X‐, non P2Y‐purinoceptor present, at which ATP is an agonist and PPADS is an antagonist, located on the longitudinal smooth muscle, activation of which causes spasm independent of changes in prostanoids.
Translocation of lipocortin (annexin) 1 to the membrane of U937 cells induced by phorbol ester, but not by dexamethasone
Authors: Kang Sin Ae , Cho Young Ju , Moon Hee‐Bom , Na Doe Sun
Published: July 2012

Induction of lipocortin 1 secretion by dexamethasone has been demonstrated, although the secretory mechanism is still unknown. We have studied the effects of 12‐tetradecanoyl phorbol 13‐acetate (TPA) and/or dexamethasone on the expression, translocation, and secretion of lipocortin 1 in U937 cells.

The expression of lipocortin 1 and its mRNA increased during TPA‐induced differentiation of U937 cells to a maximum of 1.9 fold and 8.2 fold, respectively, after 48 h. Both the protein and the mRNA levels decreased after 48 h.

TPA caused the translocation of lipocortin 1 from the cytosol to the membrane of U937 cells in a time‐dependent manner, as determined by Western blot analysis. The translocation was concurrent with the differentiation of the cells. After 48 h of TPA treatment, 82.6 ± 6.5% of lipocortin 1 was present in the membrane fraction compared to 41.6 ± 1.7% in untreated cells.

The amount of lipocortin 1 that was externally bound (associated) with the membrane increased to 3.2 fold as the cytosol to membrane translocation of lipocortin 1 increased.

Dexamethasone decreased the externally bound lipocortin 1, but had no effect on the cytosol to membrane translocation.

This offers a model system with which the function and the secretion mechanism of lipocortin 1 can be studied. Our data is consistent with the hypothesis that the secretory mechanism is through an unknown pathway, involving the translocation of lipocortin 1 from the cytosol to the internal membranes, and then, its secretion to the external membrane.
Effect of lacidipine on fatty and proliferative lesions induced in hypercholesterolaemic rabbits
Authors: Soma M.R. , Donetti E. , Seregni R. , Barberi L. , Fumagalli R. , Paoletti R. , Catapano A.L.
Published: July 2012

The in vivo antiatherogenic activity of the calcium antagonist, lacidipine, was investigated in two different types of atherosclerotic lesions (proliferative and fatty lesions) induced in rabbits.

The proliferative lesion was obtained by positioning a hollow silastic collar around one carotid artery, while aortic fatty lesions were induced by cholesterol feeding. Cholesterol (1%) and lacidipine (1,3, and 10 mg kg−1) were given daily mixed with standard diet for 8 weeks to White New Zealand rabbits. The intimal hyperplasia (proliferative lesion) was induced 6 weeks after dietary and drug treatment started.

The neointimal formation was determined by measuring cross sectional thickness of intimal (I) and medial (M) tissue of fixed arteries. In untreated animals (n=5), 14 days after collar positioning an intimal hyperplasia was clearly detectable: the arteries with no collar (sham) showed an I/M tissue ratio of 0.03±0.02, whereas in the carotid with collar the ratio was 0.62±0.12. In lacidipine‐treated animals a significant and dose‐dependent effect on proliferative lesions at all three doses tested, was observed. I/M ratios were 0.47±0.02, 0.40±0.09, 0.32±0.02 for doses 1, 3, and 10 mg kg−1 day−1, respectively (P<0.05).

The fatty lesion extent was significantly reduced by lacidipine at the 10 mg kg−1 day−1 dose, although a trend was also observed with lower dosage.

These results suggest a direct antiatherosclerotic effect of lacidipine, independent of modulation of risk factors such as hypercholesterolaemia and/or hypertension. Furthermore, the proliferative lesions are apparently more sensitive to lacidipine than are lipid‐rich lesions.
Paracrine renal endothelin system in rats with liver cirrhosis
Authors: Hocher Berthold , Zart Rüdiger , Diekmann Fritz , Rohmeiss Peter , Distler Armin , Neumayer Hans H. , Bauer Christian , Gross Peter
Published: July 2012

Liver cirrhosis was induced in rats by CCl4 administration. We analysed the expression of endothelin receptor subtypes in the renal cortex and medulla using Scatchard analysis and receptor autoradiography, and measured plasma as well as renal‐tissue endothelin‐1 concentrations using a specific radioimmunoassay. Furthermore, we analysed the effects of the non‐selective (A/B) endothelin receptor antagonist, bosentan (6 and 100 mg kg−1 day−1) on mean arterial blood pressure, water and sodium excretion and glomerular filtration rate.

Our study revealed an overexpression of the endothelin B receptor (ETB) in the renal medulla of rats with liver cirrhosis (Cir: 2775 ± 299 fmol mg−1; Con:1695 ± 255 fmol mg−1; n = 8; means ± s.d., P < 0.01), whereas the density of ETB in the cortex and the endothelin A receptor (ETA) in the cortex and medulla were similar in both cirrhotic and control rats. Receptor autoradiography showed that the upregulation of medullary ETB in cirrhotic rats was due to an upregulation of ETB in the inner medullary collecting duct cells.

The tissue endothelin‐1 concentrations were increased in the renal medulla of cirrhotic rats (Cir:271 ± 68 pg g−1wet wt.; Con: 153 ± 36 pg g−1 wet wt., n = 8; means ± s.d., P < 0.01).

The glomerular filtration rate was slightly decreased in cirrhotic rats but not altered after bosentan treatment in either cirrhotic or control rats. Bosentan decreased sodium excretion to a similar extent in both cirrhotic and control rats, whereas water excretion was significantly reduced by both dosages of bosentan in cirrhotic rats only (Cir + vehicle: 12.5 ± 0.62 ml day−1, Cir + 6 mg kg−1 day−1 bosentan: 8.6 ± 1.0 ml day−1; Cir + 100 mg kg−1 day−1 bosentan:7.4 ± 0.6 ml day−1; n = 10; means ± s.e.mean).

We therefore suggest that the upregulation of the medullary ETB in cirrhotic rats is involved in the regulation of water excretion in rats with CCl4‐induced liver cirrhosis.
Rhythmic relaxations of active tension in the rabbit large arteries induced by a combination of cyclopiazonic acid and Bay K 8644
Authors: Omote Masayuki , Mizusawa Hideo
Published: July 2012

We previously demonstrated that cyclopiazonic acid (CPA), an inhibitor of Ca2+‐ATPase in the sarcoplasmic reticulum, induced rhythmic relaxations of active tension in the endothelium‐denuded small arteries of the mesentery and the ear of the rabbit, but that this agent failed to induce rhythmic responses in the endothelium‐denuded rabbit femoral artery.

In the present study, an attempt was made to induce rhythmic relaxations of active tension in the endothelium‐denuded rabbit femoral artery and the thoracic aorta, both of which were suspended in organ chambers for isometric tension recordings, by using CPA plus Bay K 8644, an L‐type Ca2+ channel agonist, to induce an excessive increase in cytosolic Ca2+.

CPA or Bay K 8644 alone failed to produce rhythmic relaxations in the femoral artery that had been contracted with phenylephrine. In contrast, rhythmic responses were induced by the sequential treatment of the femoral artery with CPA and Bay K 8644.

The rhythmic relaxations of active tension in the femoral artery induced by CPA plus Bay K 8644 were inhibited by charybdotoxin and by iberiotoxin, both of which are antagonists of the Ca2+‐activated K+ channel, but not by glibenclamide, a blocker of the ATP‐sensitive K+ channel.

The endothelium‐denuded rabbit aorta also exhibited rhythmic responses by the sequential addition of CPA and Bay K 8644. These responses were sensitive to charybdotoxin.

These findings indicate that, like small arteries, the large femoral and aortic arteries of the rabbit are also capable of displaying rhythmic relaxations of active tension; these relaxations may be in part attributed to the activation of the Ca2+‐activated K+ channel as a result of the Ca2+ overload caused by CPA and Bay K 8644.
Rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity: potent block by the selective m4 ligand muscarinic toxin 3 (MT3)
Authors: Olianas Maria C. , Adem Abdu , Karlsson Evert , Onali Pierluigi
Published: July 2012

In rat striatal membranes, muscarinic toxin 3 (MT3), a selective ligand of the cloned m4 receptor subtype, antagonized the acetylcholine (ACh) inhibition of forskolin‐and dopamine D1 receptor‐stimulated adenylyl cyclase activities with pA2 values of 8.09 and 8.15, respectively.

In radioligand binding experiments, MT3 increased the Ki but did not change the Bmax value of [3H]‐ N‐methylscopolamine (3H]‐NMS) binding to rat striatal muscarinic receptors. The toxin displaced the major portion of the [3H]‐NMS binding sites with a Ki of 8.0 nM.

In rat myocardium, MT3 antagonized the ACh inhibition of adenylyl cyclase with a Ki value of 860 nM.

In rat cerebral cortical membranes prelabelled with [3H]‐myo‐inositol, MT3 counteracted the methacholine stimulation of [3H]‐inositol phosphates formation with a Ki value of 113 nM.

The present study shows that MT3 is a potent antagonist of the striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity. This finding provides strong evidence for the classification of these receptors as pharmacologically equivalent to the m4 gene product (M4). On the other hand, the weaker potencies of MT3 in antagonizing the muscarinic responses in cerebral cortex and in the heart are consistent with the reported lower affinities of the toxin for the cloned ml and m2 receptor subtypes, respectively.
Effects of the non‐peptide, non‐selective endothelin antagonist, bosentan, on regional haemodynamic responses to NG‐monomethyl‐L‐arginine in conscious rats
Authors: Gardiner S.M. , Kemp P.A. , March J.E. , Bennett T.
Published: July 2012

Male, Long Evans rats (350–450 g) were chronically instrumented to allow monitoring of mean arterial blood pressure, heart rate and changes in renal, mesenteric and hindquarters haemodynamics. In the first experiment, animals (n = 8) were given a bolus i.v. injection of the nitric oxide synthase inhibitor, NG‐monomethyl‐L‐arginine hydrochloride (L‐NMMA; 30 mg kg−1) on four consecutive days. Fifteen min prior to L‐NMMA administration on the fourth day, the endothelin, ETA‐, ETB‐receptor antagonist, bosentan, was injected (30 mg kg−1, i.v.). Relative to the response on the third day, bosentan caused 33±4%, 24±3%, 14±3%, and 18±5% inhibition of the pressor, and renal, mesenteric and hindquarters vasoconstrictor effects of L‐NMMA, respectively.

In the second experiment, bosentan was given 15 min before L‐NMMA in a group of rats (n = 6) which had not received L‐NMMA previously. Relative to the responses to L‐NMMA on the first day in the previous experiment, bosentan caused a 30%, 24%, 18% and 27% inhibition of the pressor, and renal, mesenteric and hindquarters vasoconstrictor effects of L‐NMMA, respectively.

The results indicate a significant contribution from endothelin to the haemodynamic effects of L‐NMMA in conscious rats. However, since our previous studies have shown the renin‐angiotensin and sympathoadrenal systems are not involved, it is likely that the major component of the cardiovascular response to L‐NMMA in conscious rats is due to loss of vasodilator action of endothelial nitric oxide.
Inhibition of glycolysis and enhanced mechanical function of working rat hearts as a result of adenosine A1 receptor stimulation during reperfusion following ischaemia
Authors: Finegan Barry A. , Lopaschuk Gary D. , Gandhi Manoj , Clanachan Alexander S.
Published: July 2012

This study examined effects of adenosine and selective adenosine A1 and A2 receptor agonists on glucose metabolism in rat isolated working hearts perfused under aerobic conditions and during reperfusion after 35 min of global no‐flow ischaemia.

Hearts were perfused with a modified Krebs‐Henseleit buffer containing 1.25 mM Ca2+, 11 mM glucose, 1.2 mM palmitate and insulin (100 μu ml−1), and paced at 280 beats min−1. Rates of glycolysis and glucose oxidation were measured from the quantitative production of 3H2O and 14CO2, respectively, from [5‐3H/U‐14C]‐glucose.

Under aerobic conditions, adenosine (100 μm) and the adenosine A1 receptor agonist, N6‐cyclohexyladenosine (CHA, 0.05 μm), inhibited glycolysis but had no effect on either glucose oxidation or mechanical function (as assessed by heart rate systolic pressure product). The improved coupling of glycolysis to glucose oxidation reduced the calculated rate of proton production from glucose metabolism. The adenosine A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX 0.3 μm) did not alter glycolysis or glucose oxidation per se but completely antagonized the adenosine‐ and CHA‐induced inhibition of glycolysis and proton production.

During aerobic reperfusion following ischaemia, CHA (0.05 μm) again inhibited glycolysis and proton production from glucose metabolism and had no effect on glucose oxidation. CHA also significantly enhanced the recovery of mechanical function. In contrast, the selective adenosine A2a receptor agonist, CGS‐21680 (1.0 μm), exerted no metabolic or mechanical effects. Similar profiles of action were seen if these agonists were present during ischaemia and throughout reperfusion or when they were present only during reperfusion.

DPCPX (0.3 μm), added at reperfusion, antagonized the CHA‐induced improvement in mechanical function. It also significantly depressed the recovery of mechanical function per se during reperfusion. Both the metabolic and mechanical effects of adenosine (100 μm) were antagonized by the nonselective A1/A2 antagonist, 8‐sulphophenyltheophylline (100 μm).

These data demonstrate that inhibition of glycolysis and improved recovery of mechanical function during reperfusion of rat isolated hearts are mediated by an adenosine A1 receptor mechanism. Improved coupling of glycolysis and glucose oxidation during reperfusion may contribute to the enhanced recovery of mechanical function by decreasing proton production from glucose metabolism and the potential for intracellular Ca2+ accumulation, which if not corrected leads to mechanical dysfunction of the post‐ischaemic myocardium.
Localization and activity of haem oxygenase and functional effects of carbon monoxide in the feline lower oesophageal sphincter
Authors: Ny Lars , Alm Per , Ekström Peter , Larsson Bengt , Grundemar Lars , Andersson Karl‐Erik
Published: July 2012

In the feline lower oesophageal sphincter (LOS), the distribution of the carbon monoxide (CO) producing enzymes haem oxygenase (HO)−‐1 and −2 was studied by immunohistochemistry and confocal microscopy, the HO activity was measured and the possible role for CO as a mediator of relaxation was investigated.

HO‐2 immunoreactivity was abundant in nerve cell bodies of the submucosal and myenteric plexus. Approximately 50% of the HO‐2‐containing myenteric cell bodies were also nitric oxide synthase‐ and vasoactive intestinal peptide (VIP)‐immunoreactive. In addition, HO‐2 immunoreactivity was seen in nerve fibres, in non‐neuronal cells dispersed in the smooth muscle and in arterial endothelium. HO‐1 immunoreactivity was confined to non‐neuronal cells in the smooth muscle, similar to those positive for HO‐2.

Activity of HO, measured as CO production, was observed in LOS homogenates at a rate of 1.00±0.05 nmol mg−1 protein h−1. This production was inhibited by the HO inhibitor, zinc protoporphyrin‐IX (ZnPP).

In isolated circular smooth muscle strips of LOS, developing spontaneous tone, exogenously administered CO evoked a concentration‐dependent relaxation reaching a maximum of 93±3%. This relaxation was accompanied by an increase in cyclic GMP, but not cyclic AMP levels. The relaxant response was attenuated by methylene blue, but unaffected by tetrodotoxin. Repeated exposure to CO resulted in a progressive reduction of the relaxant response.

ZnPP caused a rightward‐shift of the concentration‐response curves for the relaxant responses to VIP, peptide histidine isoleucine, and pituitary adenylate cyclase activating peptide 27.

ZnPP and tin protoporphyrin‐IX (another inhibitor of HO) did not affect nonadrenergic, noncholinergic relaxations induced by electrical field stimulation. Nor did ZnPP affect relaxations induced by 3‐morpholino‐sydnonimine or forskolin.

The present findings, showing localization of HO immunoreactivity to both neuronal and non‐neuronal cells of the feline LOS, ability of LOS to produce CO and a relaxant effect of CO in circular LOS muscle, suggest a role for CO as a peripheral messenger.
Potent antagonism by BIM‐23056 at the human recombinant somatostatin sst5 receptor
Authors: Wilkinson Graeme F. , Thurlow Richard J. , Sellers Lynda A. , Coote Jim E. , Feniuk Wasyl , Humphrey Patrick P.A.
Published: July 2012

We have investigated the effects of somatostatin (SRIF) and the linear octapeptide BIM‐23056 on changes in intracellular calcium ion concentration ([Ca2+]i) and on the formation of inositol‐1, 4, 5‐trisphosphate (Ins(1, 4, 5)P3) in CHO‐K***1 cells transfected with the human recombinant SRIF sst5 receptor. SRIF elicited concentration‐dependent increases in [Ca2+]i, with a pEC50 of 7.02±0.06, while BIM‐23056 (1 × 10−7 m) behaved not as an agonist but as a potent, surmountable antagonist of these increases in [Ca2+]i. The SRIF concentration‐effect curve for increases in [Ca2+]i was shifted rightward producing an estimated pKB for the antagonist of 8.0. BIM‐23056 (1 × 10−7 m) also significantly attenuated Ins(1, 4, 5)P3 increases due to SRIF, but had no effect on either basal or uridine 5′‐triphosphate (UTP) (1 × 10−4 m) stimulated increases in the levels of [Ca2+]i or Ins(1, 4, 5)P3.
Inhibition of neuromuscular transmission in the myenteric plexus of guinea‐pig ileum by ω‐conotoxins GVIA, MVIIA, MVIIC and SVIB
Authors: Hong S.J. , Roan Yow‐Fen , Chang C.C.
Published: July 2012

The effects of a number of Ca2+ channel blockers on the transmural electrical stimulation or receptor agonist‐elicited contractile responses of guinea‐pig ileum were compared.

ω‐Conotoxins (MVIIA, GVIA, SVIB and MVIIC), but not ω‐agatoxin IVA, completely blocked the twitch responses evoked by low frequency (0.1 Hz) transmural stimulation without inhibition of the contractures evoked by exogenous acetylcholine. The concentration‐inhibition curves were shifted by changes of external Ca2+.

The tetanic contractures produced by a high frequency (30 Hz) train of stimulation were inhibited by ω‐conotoxins by only 25–30%, except for ω‐conotoxin MVIIC, which produced about 55% inhibition, all significantly less than that produced by atropine (about 70%) or tetrodotoxin (about 85%). Combinations of ω‐conotoxins did not produce additive inhibitory effects.

The four ω‐conotoxins as well as atropine produced similar partial inhibition (53–62%) of the contractures evoked by dimethylphenylpiperazinium, while tetrodotoxin inhibited the contracture completely.

Nifedipine and Ni2+ depressed the nerve stimulation‐evoked twitch response and tetanic contracture as well as acetylcholine contracture.

These observations suggest that, in the myenteric plexus, a subset of N‐type Ca2+ channel dominates under low frequency stimulation, while high frequency stimulation may recruit additional channels and non‐cholinergic pathways.
The relaxant 5‐HT receptor in the dog coronary artery smooth muscle: pharmacological resemblance to the cloned 5‐ht7 receptor subtype
Authors: Terrón José A.
Published: July 2012

The relaxant effect of 5‐hydroxytryptamine (5‐HT) in the dog isolated coronary artery deprived of endothelium is mediated by a receptor unrelated to the 5‐HT1, 5‐HT2, 5‐HT3 or 5‐HT4 types. Based upon the pharmacological characteristics of this relaxant 5‐HT receptor and those reported for the new members of the 5‐HT receptor family, the present study explored the possibility that the relaxant 5‐HT receptor referred to above, corresponds to the cloned 5‐ht7 subtype. Thus, the relaxing and/or blocking effects of several 5‐HT receptor drugs as well as some typical and atypical antipsychotic drugs with high affinity for the cloned 5‐ht7 receptor in precontracted ring segments were analyzed.

5‐HT, 5‐carboxamidotryptamine (5‐CT) and 5‐methoxytryptamine, but not 8‐OH‐DPAT or sumatriptan, produced concentration‐dependent relaxations in endothelium‐denuded canine coronary artery rings precontracted with prostaglandin F2α (2 μm). Clozapine (1 μm) produced in some cases a small relaxing effect and antagonized 5‐HT‐ and 5‐CT‐induced relaxation suggesting a partial agonist effect. In the presence of the 5‐HT1D receptor antagonist, GR127935 (100 nM), the rank order of agonist potency was 5‐CT > 5‐HT > clozapine ≥ 5‐methoxytryptamine. 8‐OH‐DPAT and sumatriptan remained inactive as agonists.

In GR 127935‐treated preparations, methiothepin (3 nM) and mianserin (1 μm), as well as the antipsychotics, clozapine (1 μm), pimozide (300 nM), risperidone (3 nM) and spiperone (1 μm), failed to induce a significant relaxation in prostaglandin F2α‐precontracted vessels, but produced significant rightward displacements of the concentration‐response curves to 5‐HT and 5‐CT without significantly reducing the Emax. In a final set of experiments with 5‐CT, metergoline (100 nM) and mesulergine (300 nM) behaved as competitive antagonists. In contrast, lisuride (3 nM) noncompetitively antagonized 5‐CT‐induced relaxation. The estimated affinity (apparent pKB values) of the above antagonist drugs for the relaxant 5‐HT receptor significantly correlated with their reported affinity at the cloned 5‐ht7 receptor.

Taken together, the above pharmacological data may suggest that the relaxant 5‐HT receptor in the smooth muscle of the canine coronary artery is similar to the cloned 5‐ht7 receptor subtype.
Cyclic AMP‐dependent regulation of K+ transport in the rat distal colon
Authors: Diener M. , Hug F. , Strabel D. , Scharrer E.
Published: July 2012

The effect of agonists of the cyclic AMP pathway and of 293B, a chromanole‐derived K+ channel blocker, on K+ transport in the rat distal colon was studied by measuring unidirectional fluxes, uptake, and efflux of Rb+ in mucosa‐submucosa preparations and by patch‐clamp of crypt epithelia from isolated crypts.

293B concentration‐dependently inhibited basal and forskolin‐stimulated short‐circuit current. In isolated crypts 293B blocked a basal K+ conductance but had no effect on cyclic AMP‐evoked depolarization induced by the opening of apical Cl− channels. When the effect of cyclic AMP on Cl− conductance was prevented by substituting Cl− with gluconate, an inhibition of total cellular K+ conductance by forskolin and a membrane‐permeable cyclic AMP analogue was unmasked.

Unidirectional ion flux measurements revealed that 293B suppressed the increase in JRbsm induced by forskolin. This, together with the inhibition of cyclic AMP‐induced anion secretion indicates that the drug blocks K+ channels, presumably both in the apical and the basolateral membrane. Forskolin caused not only inhibition of K+ absorption, but also stimulation of K+ secretion. The inhibition was diminished, but not blocked, in the presence of inhibitors of the apical H+‐K+‐ATPase, vanadate and ouabain. Forskolin stimulated serosal, bumetanide‐sensitive Rb+ uptake, whereas mucosal, ouabain/vanadate‐sensitive uptake remained unaffected.

Efflux experiments revealed that forskolin caused a redistribution of cellular K+ efflux reducing the ratio of basolateral versus apical Rb+ efflux.

These results suggest that intracellular cyclic AMP exerts its effects on K+ transport by several mechanisms: an increase in the driving force for K+ efflux due to the depolarization induced by opening of Cl− channels, a stimulation of the basolateral uptake of K+ via the Na+‐K+‐Cl−‐cotransporter, and a decrease of the ratio of basolateral versus apical K+ conductance leading to an enhanced efflux of K+ into the lumen and a reduced K+ efflux to the serosal compartment.
Contribution of calcium‐activated potassium channels to the vasodilator effect of bradykinin in the isolated, perfused kidney of the rat
Authors: Rapacon M. , Mieyal P. , McGiff J.C. , Fulton D. , Quilley J.
Published: July 2012

NO‐ and prostaglandin‐independent, endothelium‐dependent vasodilator responses to bradykinin are attributed to release of a hyperpolarizing factor. Therefore, the contribution of K+ channels to the renal vasodilator effect of bradykinin was examined in rat perfused kidneys that were preconstricted with phenylephrine and treated with NG‐nitro‐L‐arginine (L‐NOARG) and indomethacin to inhibit NO and prostaglandin synthesis.

The non‐specific K+ channel inhibitors, TEA and TBA reduced vasodilator responses to bradykinin and cromakalim but not those to nitroprusside.

Glibenclamide, an inhibitor of ATP‐sensitive K+ channels, blocked the vasodilator response to cromakalim without affecting responses to bradykinin.

Charybdotoxin, a selective inhibitor of Ca2+‐activated K+ channels, greatly attenuated vasodilator responses to bradykinin without affecting those to cromakalim or nitroprusside.

Iberiotoxin and leiurotoxin, inhibitors of large and small conductance Ca2+‐activated K+ channels, respectively, were without effect on vasodilator responses to bradykinin, cromakalim or nitroprusside.

These results implicate K+ channels, specifically Ca2+‐activated K+ channels of intermediate conductance, in the renal vasodilator effect of bradykinin and, thereby, support a role for a hyperpolarizing factor.
Potentiation of purinergic transmission by angiotensin in prostatic rat vas deferens
Authors: Sum Chi‐Shing , Wan David C.C. , Cheung Wing‐Tai
Published: July 2012

Angiotensin II (AII) elicited only a minute, if any, direct contractile response in smooth muscle cells of prostatic rat vas deferens, but it potentiated contractile responses to field stimulation.

Angiotensin‐potentiated contractile response to field stimulation was concentration‐dependent, and the order of potency was AII > AIII ≅ AI. The EC50 of AII was 8.11 ± 2.79 nM.

AII did not modify the contractile response of exogenous noradrenaline (NA) on non‐stimulated prostatic vas deferens. Furthermore, the concentration‐response curve for AII‐potentiated contractile responses to field stimulation in reserpine‐treated rats did not significantly differ from the control group.

Desensitization of purinoceptors with 30 μm α, β‐methylene‐ATP almost completely abolished the potentiation of the contractile response to field stimulation by AII.

The response to AII in the prostatic rat vas deferens was blocked by the AT1 selective antagonist losartan, but not by the AT2 selective antagonist CGP 42112. Losartan acted as a competitive antagonist with a pA2 value of 8.75.

In conclusion, AII potentiated purinergic transmission in the prostatic rat vas deferens via the AT1 receptor.
Pharmacological characterization of metabotropic glutamate receptors potentiating NMDA responses in mouse cortical wedge preparations
Authors: Mannaioni Guido , Carlà Vincenzo , Moroni Flavio
Published: July 2012

Mouse cortical wedge preparations were used in order to study the effects of metabotropic glutamate receptor (mGluR) agonists and antagonists on the depolarization induced by N‐methyl‐D‐aspartate (NMDA) or by (S)‐α‐amino‐4‐bromo‐3‐hydroxy‐5‐isoxazolepropionic acid (AMPA).

(1S, 3R)‐1‐aminocyclopentane‐1, 3‐dicarboxylic acid (1S, 3R‐ACPD) (30–300 μm) significantly potentiated the depolarizations induced by NMDA, leaving unchanged those mediated by AMPA. This potentiation developed slowly and lasted for up to 60 min provided that the slices were continuously perfused with the mGluR agonist.

Concentration‐response curves to NMDA in the absence and in the presence of 1S, 3R‐ACPD (100 μm) indicated that the potentiation was due to increased affinity of the NMDA receptor complex for its agonist. The maximal responses to NMDA were not potentiated.

Selective agonists of group 1 mGluR such as quisqualate (Quis) (30 μm) or (RS)‐3, 5‐dihydroxyphenylglycine (DHPG) (300 μm) did not potentiate NMDA responses. Similarly, selective agonists of group 2 mGluRs, such as (2S, 3S, 4S)‐α‐carboxycyclopropyl‐glycine (L‐CCG‐I) (3–30 μm), and of group 3, such as L‐2‐amino‐4‐phosphonobutyric acid (L‐AP4) (100 μm) were inactive in our test. A number of other putative mGluR agents having partial agonist activity on mGluRs in brain slices and in expression systems, such as 1R, 3S‐ACPD (500 μm), DL‐2‐amino‐3‐phosphonopropionic acid (DL‐AP3) (300 μm) and (S)‐4‐carboxy‐3‐hydroxyphenylglycine (S‐4C3HPG; 500 μm), when placed in the experimental protocol we used, did not change NMDA responses.

Available mGluR antagonists, such as DL‐AP3 (1 mM), (+)‐α‐methyl‐4‐carboxyphenylglycine (MCPG) (500 μm), S‐4‐carboxyphenylglycine (4CPG; 500 μm) and S‐4‐carboxy‐3‐hydroxyphenylglycine (S‐4C3HPG; 500 μm), did not reduce 1S, 3R‐ACPD potentiation of NMDA responses.

It is concluded that the potentiation of NMDA currents induced by the mGluR agonist 1S, 3R‐ACPD, in mouse cortical wedges, has a pharmacological profile which is different from that of the three mGluR groups so far described in expression systems.
The contribution of nitric oxide to endotoxin‐induced ocular inflammation: interaction with sensory nerve fibres
Authors: Wang Zun‐Yi , Alm Per , Håkanson Rolf
Published: July 2012

The actions of nitric oxide (NO) have been investigated in an endotoxin‐evoked ocular inflammatory model in the rabbit, with particular emphasis on the relationship between NO, sensory nerves (C‐fibres) and the C‐fibre neuropeptides, calcitonin gene‐related peptide (CGRP) and pituitary adenylate cyclase activating peptide (PACAP).

Endotoxin, injected intravitreally, evoked inflammatory responses, i.e. conjunctival hyperaemia, miosis and protein extravasation, reflected by the aqueous flare response (AFR). In control rabbits, the maximum AFR was 66.5 ± 9.5 (arbitrary units). Pretreatment with the NO synthase (NOS) inhibitor, NG‐nitro‐l‐arginine (L‐NAME, 200 mg kg−1) given by intravenous injection, inhibited the endotoxin‐evoked responses; the AFR was 16.5 ± 1.9 (n = 8, P < 0.001) and the conjunctival hyperaemia was abolished.

Endotoxin‐evoked ocular inflammation is associated with the release of CGRP and PACAP from C‐fibres. In the eyes challenged with endotoxin, the concentrations of PACAP‐27, −38 and CGRP in the aqueous humour were 58.2 ± 10.9, 54.4 ± 12.4 and 5526 ± 519 (pmoll−1), respectively. L‐NAME inhibited the release of PACAP‐27, −38 and CGRP; the concentrations were 14.3 ± 2.5, 13.5 ± 2.5 and 510 ± 67 (pmoll−1), respectively (n = 8, P < 0.01 or 0.001).

Intravitreal injection of 0.3 nmol CGRP induced conjunctival hyperaemia and AFR; the maximum AFR was 140.2 ± 11.4. L‐NAME suppressed the response induced by CGRP; the AFR was 23.4 ± 5.5 (n = 8, P < 0.001). L‐NAME abolished the conjunctival hyperaemia induced by PACAP‐27 and −38 (0.3 nmol) and reduced the AFR.

The inflammatory cells that infiltrated the uvea, cornea and aqueous humour in large numbers in response to intravitreal injection of endotoxin were found to express inducible NOS. L‐NAME prevented the appearance of such cells.

Our findings suggest that NO plays an important role in the endotoxin‐evoked ocular inflammation in the rabbit: NO activates C‐fibres causing release of C‐fibre neuropeptides into the aqueous humour. In addition, NO mediates some of the ocular effects of CGRP and PACAP, since L‐NAME suppressed the AFR induced by these peptides.
Prevention of vascular nitroglycerin tolerance by inhibition of protein kinase C
Authors: Zierhut Wolfgang , Ball Howard A.
Published: July 2012

We investigated whether in vivo inhibition of protein kinase C (PKC) can prevent the development of vascular tolerance and restore the sensitivity of isolated vessels to nitroglycerin (NTG). Tolerance was induced in male Wistar rats by a constant i.v. infusion of NTG 1 mg kg−1 h−1, a dose which did not alter blood pressure. After 72 h, the aorta was removed and the sensitivity of aortic rings to NTG tested. Chronic NTG infusion resulted in a 5.5 fold decrease in NTG‐sensitivity as compared with controls (vehicle), indicating the development of vascular tolerance. The simultaneous in vivo administration of the specific PKC inhibitor N‐benzoyl‐staurosporine (30 mg kg−1 day−1) prevented this decrease in NTG sensitivity. These results suggest a role for PKC activation in the development of vascular NTG tolerance.
Clonidine‐induced increase in osmolar clearance and free water clearance via activation of two distinct α2‐adrenoceptor sites
Authors: Intengan H.D. , Smyth D.D.
Published: July 2012

Clonidine, an α2‐adrenoceptor agonist, will increase urine flow rate in the anaesthetized rat by increasing both free water and osmolar clearance. In the present study, we investigated whether these effects of clonidine were mediated at two sites which could be distinguished pharmacologically in uninephrectomized male Sprague‐Dawley rats.

Clonidine (1.0 nmol kg−1 min−1) infused into the renal artery increased osmolar and free water clearance. Following pretreatment with prazosin (0.15 mg kg−1, i.v.), an antagonist with reported selectivity for the α2b‐adrenoceptor subtype, the increase in free water but not osmolar clearance was decreased. Pretreatment with the opioid receptor antagonist, naltrexone (3.0 mg kg−1, i.v.) attenuated the increase in osmolar but not free water clearance. This disparate antagonism of clonidine by prazosin and naltrexone was consistent with two distinct sites.

We submit the hypothesis that the α2a‐ and α2b‐adrenoceptor subtypes mediated the clonidine‐induced osmolar and free water clearance. The blockade in free water clearance by prazosin indicated a possible role of the α2b‐adrenoceptor subtype whereas the α2a‐adrenoceptor subtype was considered as the site mediating the clonidine‐induced increase in osmolar clearance. UK‐14,304 (1.0 nmol kg−1 min−1), a mixed α2‐adrenoceptor/imidazoline receptor agonist with selectivity for the α2a‐subtype, increased only osmolar clearance. This increase was blocked by naltrexone but not prazosin pretreatment. The imidazoline receptor was not involved, as naltrexone failed to alter the moxonidine (3.0 nmol kg−1‐min−1) induced increase in osmolar clearance. These data suggested to us that the α2a‐/α2b‐subtype hypothesis should be investigated more closely in future studies.

These findings indicate that the increase in osmolar and free water clearance following clonidine can be distinguished pharmacologically indicating that two sites were involved. Furthermore, we propose the hypothesis that the α2a‐adrenoceptor subtype mediated osmolar clearance whereas the α2b‐subtype mediated free water clearance. The prazosin‐sensitive increase in free water clearance following clonidine suggested a possible role for the α2b‐subtype. The naltrexone‐sensitive increase in osmolar clearance following clonidine and UK‐14,304 (but not moxonidine) suggested a possible role of the α2a‐subtype. Clearly, this postulate requires further study.
Protective action of hydroxyethyl rutosides on singlet oxygen challenged cardiomyocytes
Authors: Olbrich Hans‐Georg , Grabisch Peter , Großmann Alexander , Rinne Thorsten , Klepzig Harald , Mutschler Ernst
Published: July 2012

The effect of a standardized mixture of β‐hydroxyethyl rutosides against oxidative damage in singlet oxygen‐challenged isolated cardiac myocytes from adult rats was investigated. The morphology of the myocytes was evaluated as an indicator for cell viability (elongated, rod shaped cells vs. hypercontracted, rounded cells). The determination of the production of thiobarbituric acid reactive substances served as an indicator for lipid peroxidation.

Exposure to singlet oxygen which was generated by photo‐excitation of rose bengal (10−7 m) reduced the number of rod shaped (vital) cardiomyocytes by 78.5 ± 2.5% and increased the production of thiobarbituric acid reactive substances by 1180 ± 150% in comparison to incubation with control buffer.

Coincubation of the cells with β‐hydroxyethyl rutosides (concentration range: 6.7 pg ml−1 to 670 μg ml−1) increased the number of rod shaped cardiomyocytes after exposure to singlet oxygen in a dose‐dependent bell‐shaped manner. A significant protective effect was observed at β‐hydroxyethyl rutosides concentrations ranging from 0.67 ng ml−1 to 67 ng ml−1.

In spite of their protective action, β‐hydroxyethyl rutosides did not reduce the accumulation of thiobarbituric acid reactive substances, used as an indicator for lipid peroxidation.

The data suggest that β‐hydroxyethyl rutosides exert a protective action against oxygen radical‐induced damage of cardiac myocytes at very low concentrations without interfering with lipid peroxidation.
Effects of cytochrome P450 inhibitors on potassium currents and mechanical activity in rat portal vein
Authors: Edwards Gillian , Zygmunt Peter M. , Högestätt Edward D. , Weston Arthur H.
Published: July 2012

The effects of the cytochrome P450 inhibitors, proadifen, clotrimazole and 17‐octadecynoic acid (17‐ODYA) on K‐currents in freshly‐isolated single cells derived from rat portal vein and on mechanical activity in whole veins were studied.

When cells were stepped from −90 mV to a series of test potentials (from −80 to +50 mV), a delayed rectifier current (IK(V)) and an A‐type current (IK(A)) could be identified. Proadifen (10 μm), clotrimazole (30 μm) and 17‐ODYA (5 μm) each inhibited IK(A) but had little effect on IK(A).

When cells were held at −10 mV to inactivate the time‐dependent K‐currents, IK(V) and IK(A), levcromakalim (3 μm) induced a time‐independent outward K‐current (IK(ATP)) which was totally inhibited by clotrimazole (30 μm) and almost fully inhibited by proadifen (10 μm). 17‐ODYA (5 μm) had no effect on IK(ATP) and exerted only a minor inhibitory action on this current at 20 μm.

17‐ODYA (5 μm) potentiated current flow through the large conductance, Ca‐sensitive K‐channel (BKCa). In contrast, proadifen (10 μm) had no effect on IBK(Ca) whereas clotrimazole (30 μm) exerted a small but significant inhibitory action.

Proadifen (10 μm) and clotrimazole (30 μm) each inhibited the magnitude but increased the frequency of spontaneous contractions in whole portal veins. 17‐ODYA (5 μm) had no effect on spontaneous contractions but these were inhibited when the concentration of 17‐ODYA was increased to 50 μm.

The spasmolytic effect of levcromakalim on spontaneous contractions was antagonized by proadifen (10–30 μm) in a concentration‐dependent manner but 17‐ODYA (up to 50 μm) was without effect.

These results in portal vein show that cytochrome P450 inhibitors exert profound effects on a variety of K‐channel subtypes. This suggests that enzymes dependent on this cofactor may be important regulators of K‐channel activity in smooth muscle. The relevance of these findings for the identification of the pathway involved in the synthesis of the endothelium‐derived hyperpolarizing factor is discussed.
Inhibition by P1075 and pinacidil of a calcium‐independent chloride conductance in conditionally‐immortal renal glomerular mesangial cells
Authors: Barber Robert D. , Henderson Robert M.
Published: July 2012

Depolarization of mesangial cells has been shown to occur following an outward movement of chloride ions from the cell. We have shown previously that mesangial cells from the H‐2Kb‐tsA58 transgenic mouse possess a significant whole‐cell chloride conductance and consequently are a suitable preparation for the study of potential chloride channel inhibitors.

The effects on the whole‐cell chloride conductance of the chloride channel inhibitor, 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) and the potassium channel openers, (KCOs) P1075 and pinacidil were investigated in mesangial cells from the H‐2Kb‐tsA58 transgenic mouse cultured in permissive conditions (at 33dGC in the presence of 50 u ml−1 murine γ‐interferon).

In symmetrical solutions of 140 mM tetramethylammonium chloride (TMAC1) the whole‐cell chloride conductance was 1.08 ± 0.05 nS (n = 63) and this could be reversibly inhibited by 5 × 10−5 m NPPB.

Both P1075 and pinacidil inhibited the whole‐cell chloride conductance. This inhibition was not reversible after drug washout and was demonstrated only when drugs were applied to the extracellular surface of the cells. Very low concentrations of the drugs were found to reduce the chloride conductance after 16 h incubation but under no circumstances studied was the conductance totally inhibited, leaving a mean residual current of 0.33 ± 0.03 nS (n = 12).

The effects of different peptide calcium concentrations on the magnitude of the residual current in the presence of the drugs were investigated. The residual current was reduced with 10−8 m calcium in the pipette and increased with 10−3 m pipette calcium. Therefore, these data suggest that P1075 and pinacidil selectively inhibit a calcium‐independent chloride conductance in mesangial cells from the H‐2Kb‐tsA58 transgenic mouse.
Discrimination between neuropeptide Y and peptide YY in the rat tail artery by the neuropeptide Y1‐selective antagonist, BIBP 3226
Authors: Gicquiaux Hervé , Tschöpl Martin , Doods Henri N. , Bucher Bernard
Published: July 2012

The ability of the novel, nonpeptide, neuropeptide Y (NPY) Y1selective antagonist, BIBP 3226 {(R)‐N2‐(diphenylacetyl)‐N‐[(4‐hydroxyphenyl)methyl]‐D‐arginine amide}, to antagonize the increase in perfusion pressure induced by NPY and peptide Y (PYY) was tested in the perfused rat tail artery, a postjunctional Y1‐receptor bioassay, precontracted by 1 μm phenylephrine.

NPY and PYY produced a concentration‐dependent enhancement of the vasoconstrictor response evoked by 1 μm phenylephrine. Although NPY and PYY are roughly equipotent, the maximal contractile response elicited by PYY was about twice that elicited by NPY.

Increasing concentrations of BIBP 3226 caused a parallel and rightward shift in the NPY concentration‐response curve without depressing the maximal response. The contractile effect of NPY was potently inhibited in a competitive manner. The pA2 value for BIBP 3226 was 7.01 ± 0.08, a value equivalent to that observed in the rabbit saphenous vein. Although increasing concentrations of BIBP 3226 shifted the concentration‐response curve of PYY to the right without any significant decrease in the maximal vasoconstrictor response, the antagonism appeared non‐competitive as the slope of the Schild plot was significantly different from unity (0.58 ± 0.04).

In conclusion, these data confirm that BIBP 3226 is a potent and selective nonpeptide Y1 receptor antagonist. Moreover, they show that complex interactions occur between BIBP 3226 and postjunctional receptors activated by PYY. We postulate that BIBP 3226 might discriminate between the effects of NPY and PYY at the postjunctional level in the rat tail artery. It may be that distinct receptors for NPY and PYY exist; these may or may not allosterically interact with each other. Another working hypothesis would be that there is a single receptor complex with allosterically interacting binding sites for the two peptides.