bluuebunny/crossref_metadata_2025 huggingface dataset:
Extrahepatic metabolism of frusemide in anaesthetized rabbits
Authors: Vergés José , Héroux Lucie , Maurice Hélène , du Souich Patrick
Published: July 2012

Frusemide is removed from the body by biotransformation and renal secretion, but since frusemide metabolism is not altered in patients with hepatic cirrhosis, the role of the liver may be questioned. The aim of the study was to investigate which organs contribute to the first‐pass metabolism and systemic clearance of frusemide.

Groups of anaesthetized New Zealand rabbits were administered frusemide proximally (prox) and distally (dist) to different organs, and blood was sampled from the abdominal aorta. The area under frusemide plasma concentrations‐time curve (AUC0‐∞) was calculated and frusemide extraction by an organ was estimated from the ratio (AUCdist‐AUCprox)/AUCdist. The small intestine extracted 83% of the absorbed dose of frusemide but the first‐pass uptake by the liver and lungs was negligible.

To assess the contribution of the intestine and the kidneys to the systemic clearance of frusemide, it was injected into the jugular vein and blood was sampled proximal and distal to each organ. The kidneys extracted 24% of frusemide circulating in the renal arteries; on the other hand, the ability of the intestine to extract frusemide from the systemic circulation could not be detected.

The lungs did not metabolize frusemide in vitro; the rate of metabolism of frusemide in vitro by kidneys was similar to that estimated in the intestine, and both rates were faster (P<0.05) than that observed in the liver.

It is concluded that in rabbits, presystemic metabolism of frusemide is carried out by the intestine, and that systemic clearance of frusemide is mainly performed by the kidneys, although other organs, such as the intestine and the liver, must contribute to it.
PPADS: an antagonist at endothelial P2Y‐purinoceptors but not P2U‐purinoceptors
Authors: Brown Colin , Tanna Bhavna , Boarder Michael R.
Published: July 2012

Bovine aortic endothelial (BAE) cells contain two co‐existing receptors for extracellular ATP, the P2Y and P2U‐purinoceptors. Here we have determined whether the proposed P2x‐purinoceptor antagonist, pyridoxalphosphate‐6‐azophenyl‐2″, 4″‐disulphonic acid (PPADS) could distinguish between these two receptor subtypes.

Cells labelled with myo‐[2‐3H]‐inositol were stimulated with increasing concentrations of either the P2Y‐agonist, 2MeSATP, or the P2U‐agonist, UTP in the absence or presence of 30 μm PPADS. The accumulation of total [3H]‐inositol (poly)phosphates mediated by 2MeSATP was markedly attenuated by PPADS, whereas the response to UTP was not significantly affected.

Stimulation of BAE cells with increasing concentrations of ATP showed a reduced response in the presence of 10 μm PPADS, but this effect of the antagonist was not significant. By contrast, inhibition of the response to ADP was profound and highly significant.

These observations show that PPADS is not a selective P2X‐purinoceptor antagonist, but is able to distinguish between P2Y‐ and P2U‐purinoceptors in BAE cells, and indicate that this compound may provide a useful tool in the study of multiple subtypes of P2‐purinoceptors. Furthermore the results are consistent with the hypothesis that ATP interacts with both receptor subtypes, but that the action of ADP is primarily at the P2Y‐purinoceptor in these endothelial cells.
Evidence for arginine vasopressin as the primary activator of the HPA axis during adjuvant‐induced arthritis
Authors: Chowdrey H.S. , Larsen P.J. , Harbuz M.S. , Jessop D.S. , Aguilera G. , Eckland D.J.A. , Lightman S.L.
Published: July 2012

Adjuvant‐induced arthritis (AA) is an experimental inflammation of the joints that results in chronic activation of the hypothalamo‐pituitary‐adrenal (HPA) axis.

In this study the role of hypothalamic corticotrophin‐releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of the HPA axis in this condition both in Sprague‐Dawley (SD), and Piebald‐Viral‐Glaxo (PVG) rats has been further characterized.

The increase in AVP peptide content of portal blood (as early as day 11), just prior to the onset of arthritis is confirmed and further increases, peaking at day 16 are shown, coincident with the progression of inflammation in the PVG rats.

The increase in AVP is associated with a significant increase in the expression of AVP but not CRF mRNAs in the medial parvocellular division of the hypothalamic paraventricular nucleus (PVN) of arthritic SD rats.

In the presence of maximal inflammation of SD rats there was a significant decrease in the maximum binding of [125I]‐Tyr‐oCRF to anterior pituitary membranes, whereas AVP receptor concentration in anterior pituitary membranes from both PVG and SD rats showed a significant increase with respect to controls.

The basal adrenocorticotrophin (ACTH) secretion in vitro was similar in both control and arthritic SD rats but that from arthritic PVG rat pituitaries was significantly greater than the respective controls (436±91 v 167±23 pg/tube). The ACTH response of pituitaries of arthritic PVG rats to CRF or the combination of CRF and AVP was significantly higher compared with the controls, although the ACTH response of arthritic SD rat pituitaries was unchanged.

The results are consistent with the view that activation of the parvocellular vasopressin system has an important role in the adaptation of the HPA axis to experimentally‐induced chronic stress of arthritis.
Reduction by prostaglandin E1 or prostaglandin E0 of myocardial infarct size in the rabbit by activation of ATP‐sensitive potassium channels
Authors: Hide Emma J. , Ney Peter , Piper Julie , Thiemermann Christoph , Vane John R.
Published: July 2012

This study examined whether pretreatment of rabbits with infusions of prostaglandin El (PGE1) or prostaglandin E0 (PGE0) (which were terminated prior to the onset of ischaemia) reduce myocardial infarct size arising from coronary artery occlusion (60 min) and reperfusion (120 min). In addition, we investigated whether the observed cardioprotective effects of these two prostaglandins were due to the activation of ATP‐sensitive potassium (KATP) channels.

In the anaesthetized rabbit, infarct size (expressed as a percentage of the area at risk) after 60 min of coronary artery occlusion followed by 2 h of reperfusion was 59±4% (n= 10). PGE1 or PGE0 treatment (1.0 μg kg−1 min−1), administered as 1 h pretreatments (0.05 ml min−1, i.v.), significantly reduced infarct size to 44±6% (n = 6) or 42±1% (n = 6), respectively. PGE1 or PGE0 pretreatment resulted in a significant reduction in mean arterial blood pressure, which returned to baseline within 15 min of discontinuation of the infusion (i.e. prior to LAL ligation).

The reduction in infarct size afforded by PGE1 was abolished by pretreatment of rabbits with the KATP channel blockers, glibenclamide (60±4%; n = 8) or 5‐hydroxydecanoate (58±6%; n = 6). Similarly, glibenclamide also largely attenuated the reduction in infarct size afforded by PGE0 (52±3%; n = 8).

We propose that a 1 h pretreatment of PGE1 or PGE0 reduces infarct size by activating protein kinase C resulting in the opening of KATP channels.
Endothelin ETA receptor expression in human cerebrovascular smooth muscle cells
Authors: Yu Julie C.M. , Pickard John D. , Davenport Anthony P.
Published: July 2012

Endothelin (ET) has been implicated in cerebrovasospasm for example, following subarachnoid haemorrhage, and blocking the interaction of ET with its receptors on cerebral vessels, may be of therapeutic benefit. The aim of our study was to characterize endothelin receptor subtypes on medial smooth muscle cells of human cerebral vessels. Cultures of vascular smooth muscle cells were explanted from human cerebral resistance vessels and characterized as human brain smooth muscle cells (HBSMCs).

Over a 48 h incubation period, HBSMC cultures secreted comparable levels of immunoreactive (IR) big endothelin‐1 (big ET‐1) and IR endothelin (ET): 12.7± 10.3 and 8.3 + 5.6 pmol/106 cells, respectively (mean ± s.e.mean from three different individuals), into the culture medium.

Total RNA was extracted from cultures of human brain smooth muscle cells. Reverse‐transcriptase polymerase chain reaction (RT‐PCR) assays and subsequent product separation by agarose gel electrophoresis revealed single bands corresponding to the expected product sizes encoding cDNA for ETA (299 base pairs) and ETB (428 base pairs) (n = 3 different cultures).

Autoradiography demonstrated the presence of specific binding sites for [125I]‐ET‐1 which labels all ET receptors, and [125I]‐PD151242, an ETA subtype‐selective antagonist which exclusively labels ETA receptors, but no specific binding was detected using ETB subtype‐selective [125I]‐BQ3020 (n= 3 different cultures, in duplicate).

In saturation binding assays, [125I]‐ET‐1 bound with high affinity: KD = 0.8 ± 0.1 nm and Bmax = 690±108 fmol mg−1. A one‐site fit was preferred and Hill slopes were close to unity over the concentration range (10−12 to 10−8 m). [125I]‐PD151242 also bound with similar affinity: KD = 0.4 ± 0.1 nm and Bmax = 388 ± 68 fmol mg−l (mean ± s.e.mean, n = 3 different cultures). Again, a one‐site fit was preferred and Hill slopes were close to unity over the concentration range. Unlabelled PD151242 competed for the binding of [125I]‐ET‐I monophasically and analysis of the competition curves indicated that a one‐site fit was preferred over a two‐site model, implying that the cultures express mainly ETA receptors.

Although messenger RNA encoding both ETA and ETB receptors was detected, autoradiographical analysis, as well as binding studies indicate that human cultured brain smooth muscle cells express only ETA receptor protein. Antagonism of this subtype may be necessary to block the actions of ET‐1 in the human cerebral resistance vessels in the vasospasm observed subsequent to subarachnoid haemorrhage.
Endothelium‐dependent relaxations mediated by inducible B1 and constitutive B2 kinin receptors in the bovine isolated coronary artery
Authors: Drummond G.R. , Cocks T.M.
Published: July 2012

Rings of bovine left anterior descending coronary artery (LAD) were contracted with the thromboxane A2‐mimetic, U46619 (1–30 nM), to approximately 40% of their maximum contraction to 125 mM KCl Krebs solution (KPSSmax) for comparison of responses to the B1 and B2 kinin receptor agonists, des‐Arg9‐bradykinin (des‐Arg9‐BK) and bradykinin (BK), respectively. Relaxation responses were normalized as percentages of the initial U46619‐induced contraction level, while contractile responses were expressed as percentages of KPSSmax.

After 6 h of in vitro incubation in Krebs solution at 37°C, des‐Arg9‐BK (pEC50, 8.00 ±0.08; maximum response (Rmax), 93.9 ±1.9%) and BK (pEC50, 9.75 ±0.07; Rmax, 100.1 ±0.7%) caused endothelium‐dependent relaxations in precontracted rings of bovine LAD which were competitively and selectively antagonized by the B. receptor antagonist, des‐Arg9‐[Leu8]‐BK (pA2, 6.27 ±0.11) and the B2 receptor antagonist Hoe‐140 (pA2, 9.63 +0.14), respectively.

At 3 h of in vitro incubation, the sensitivity (pEC50, 7.45 ±0.10) and Rmax (84.6 ±3.3%) to des‐Arg9‐BK were significantly less than those obtained in the same tissues at 6 h (pEC50, 7.94 ± 0.06; Rmax, 91.4±2.5%), whereas endothelium‐dependent relaxations to BK and ACh were unaffected by incubation time.

Relaxation responses to des‐Arg9‐BK, but not BK, at both 3 h and 6 h were significantly attenuated by the protein synthesis inhibitors, cycloheximide (30 and 100 μm) and actinomycin D (2 μm).

At 6 h, the nitric oxide (NO) synthase inhibitor, NG‐nitro‐L‐arginine (L‐NOARG, 100 μm), caused a significant 2 fold decrease in pEC50 (9.58 ±0.03) but had no effect on Rmax for BK. For des‐Arg9‐BK, L‐NOARG (100 μm) caused a marked and significant decrease in both the pEC50 and Rmax and revealed contractions to low concentrations of des‐Arg9‐BK. In both cases, L‐NOARG inhibition was reversed in the presence of L‐arginine (10 mM).

At 6 h, removal of the endothelium abolished relaxation responses to des‐Arg9‐BK and BK, and for des‐Arg9‐BK, but not BK, unmasked concentration‐dependent contractions (pEC50, 7.57 ±0.09; Rmax, 83.4±9.1%). The sensitivity of contractions to des‐Arg9‐BK increased slightly from 3h (pEC50, 7.37 ±0.08) to 6 h (pEC50, 7.62 ±0.12) of in vitro incubation; however, there was a small but significant depression in the maximum response over this time (Rmax, 126.8 ±8.5% and 103.3 ±8.6% for 3 h and 6 h of incubation respectively).

In conclusion, the bovine LAD contains inducible B1 and constitutive B2 endothelial cell kinin receptors, both of which mediate endothelium‐dependent relaxation partly via the release of NO. B1 receptors were also present on the smooth muscle layer of the bovine LAD.
Enhancement of immobility in a forced swimming test by subacute or repeated treatment with phencyclidine: a new model of schizophrenia
Authors: Noda Y. , Yamada K. , Furukawa H. , Nabeshima T.
Published: July 2012

Immobility induced by forced swimming is well known as an animal model of depression. To develop an animal model for the negative symptoms of schizophrenia, in particular the depressive symptoms, the effect of phencyclidine (PCP) on immobility in the forced swimming test was investigated in mice, since PCP produces such negative symptoms in humans.

Repeated treatment with PCP (10 mg kg−1 day−1, s.c., once a day for 14 days) prolonged the immobility time in the forced swimming test 24 h after the final injection compared with saline treatment; the effect was not obtained by single or 5 treatments with PCP (10 mg kg−1, s.c.), or by repeated treatment with methamphetamine (0.5 and 1 mg kg−1 day−1, s.c., once a day for 14 days).

The enhancing effect of PCP (10 mg kg−1 day−1, s.c.) on the immobility persisted for at least 21 days after the withdrawal of the drug.

Haloperidol (0.3 and mg kg−1, p.o.), ritanserin (3 and 10 mg kg−1, p.o.), risperidone (0.1‐1 mg kg−, p.o.), and clozapine (3 and 10 mg kg−1, p.o.) failed to attenuate the immobility induced by the forced swimming in mice repeatedly treated with saline when the drugs were administered 1 h before the forced swimming test. However, ritanserin (30 mg kg−1) and clozapine (30 mg kg−1) did attenuate this immobility.

The enhancing effect of PCP on the immobility was attenuated by ritanserin (3 and 10 mg kg−1, p.o.), risperidone (0.3 mg kg−1, p.o.), and clozapine (3 and 10 mg kg−1, p.o.), whereas haloperidol (0.3 and 1 mg kg−1, p.o.) had no effect.

These results suggest that the enhancement of immobility in the forced swimming test brought about by repeated PCP treatment could be used as a model of the negative symptoms, particularly the depression, of schizophrenia. This effect of PCP appeared to be mediated, at least in part, via 5‐HT2A receptors.
Multiple pathways underlying endothelium‐dependent relaxation in the rabbit isolated femoral artery
Authors: Plane Frances , Pearson Timothy , Garland Christopher J.
Published: July 2012

In isolated segments of the rabbit femoral artery stimulated with noradrenaline, both acetylcholine (1nM‐10μM) and the calcium ionophore A23187 (1nM‐100 μM) evoked endothelium‐dependent smooth muscle relaxation and hyperpolarization while bradykinin (0.01–100 nM) had no effect.

The nitric oxide synthase inhibitors, NG‐nitro‐L‐arginine (L‐NOARG; 100 μM; 20 min) or NG‐nitro‐L‐arginine methyl ester (L‐NAME; 100 μM; 20 min) each abolished the hyperpolarization and the majority of the relaxation to acetylcholine (maximal response reduced from 96.8 ±2.3% to 2.0 ± 1.4%).

The potassium channel blocker, glibenclamide (10 μM; 10 min) also abolished the change in membrane potential to acetylcholine but did not modify the smooth muscle relaxation.

In contrast, neither L‐NAME nor glibenclamide modified the comparable responses of the femoral artery to A23187, which were also unaffected by the cyclo‐oxygenase inhibitor, indomethacin (10 μM).

In artery segments stimulated with potassium chloride (25 mM), the maximal change in tension and membrane potential evoked by A23187 (100 μM) was significantly reduced from 95.0 ±4.5% and 23.0±2.0mV to 69.0 ±10.1% and 12.0±1.5 mV, respectively. Under these conditions L‐NAME further reduced the relaxation but not the accompanying hyperpolarization to A23187.

Endothelium‐denuded arterial segments sandwiched with endothelium‐intact ‘donor’ segments gave qualitatively similar relaxant responses to those described above for acetylcholine and A23187.

Exogenous nitric oxide (0.5–10μM) stimulated a transient relaxation in precontracted artery segments, which at concentrations above 5 μM was accompanied by smooth muscle hyperpolarization (maximum 8.5 ± 3.2 mV; n = 4). The hyperpolarization but not the relaxation to nitric oxide was abolished by either glibenclamide or 25 mM potassium.

These data indicate that in the femoral artery, acetylcholine‐induced relaxation can be attributed solely to the release of nitric oxide from the endothelium, which then stimulates relaxation independently of a change in smooth muscle membrane potential. In contrast, both the relaxation and hyperpolarization evoked by A23187 appear to be mediated predominantly by nitric oxide‐independent pathways which appear to involve a diffusible factor released from the endothelium. The results suggest that this diffusible hyperpolarizing factor can be released from endothelial cells in the femoral artery by A23187 but not by acetylcholine.
Regulation of cytosolic calcium by collagen in single human platelets
Authors: Poole Alastair W. , Watson Stephen P.
Published: July 2012

There is controversy in the literature as to whether collagen is able to induce directly a rise in cytosolic calcium concentration ([Ca2+]i) in human platelets. We have addressed this question by observing the cytosolic calcium response of single fura‐2‐loaded human platelets settling onto a collagen‐coated surface using dynamic fluorescence ratio imaging.

Following a short lag phase after adherence to collagen fibres, platelets underwent a rapid rise in cytosolic calcium from basal values of 80 ± 13 nM (n = 24) to a peak of 475 ± 42 nM (n = 24) which was sustained for the remaining period of the experiment.

The tyrphostin protein tyrosine kinase inhibitor, ST271, reduced substantially the proportion of platelets which exhibited a rise in [Ca2+]i on adherence to collagen and transformed the response in remaining cells to one of oscillations.

In contrast, and as a control for collagen, laminin‐coated surfaces induced adherence of human platelets without elevating intracellular [Ca2+]; the cells however remained responsive to ADP.

We conclude that collagen directly induces a rise in cytosolic calcium in single human platelets through a tyrosine kinase‐mediated pathway.
Drug‐induced defaecation in rats: role of central 5‐HT1A receptors
Authors: Croci Tiziano , Landi Marco , Bianchetti Alberto , Manara Luciano
Published: July 2012

We investigated the acute effects of 5‐hydroxytryptamine (5‐HT), and of the 5‐HT1A receptor agonists, 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), buspirone and SR 57746A, on rat faecal pellet output and water content.

5‐HT, 8‐OH‐DPAT, buspirone and SR 57746A, a new selective 5‐HT1A receptor agonist, displaced [3H]‐8‐OH‐DPAT from specific binding sites in rat hippocampus membranes (Ki, nM: 1.8, 1.2, 15, 3.1 respectively) and stimulated rat defaecation dose‐dependently. SR 57746A and buspirone induced 1 g dry weight of faeces at 1.3 and 6.1 mgkg−1, p.o. (ADO respectively. 8‐OH‐DPAT and 5‐HT stimulated defaecation after s.c. injection (ADl5 0.07 and 7.5mgkg−1, respectively). All these agents increased faecal water content.

The putative 5‐HTlA receptor antagonist, pindolol, injected s.c. or i.c.v., significantly reduced the defaecation induced by systemically administered 8‐OH‐DPAT, buspirone or SR 57746A, but not 5‐HT.

Pretreatment with p‐chlorophenylalanine (i.p.) or 5,7‐dihydroxytryptamine (i.c.v.), according to protocols designed to cause either generalized or CNS‐limited 5‐HT depletion respectively, also reduced the defaecation induced by buspirone or SR 57746A.

No specific 5‐HT1A binding sites could be labelled by incubating rat colon membranes with [3H]‐8‐OH‐DPAT, and in vitro preparations of rat colon segments showed no response to 8‐OH‐DPAT or SR 57746A up to 5 μM.

After eight days' repeated daily treatment, complete tolerance developed to the stimulant effects of SR 57746A and buspirone on faecal water content, but not on faecal pellet output. This suggests that faecal mass excretion and water exchange through the gut wall are affected by independent mechanisms.

The present findings support the involvement of central 5‐HT1A receptors in intestinal propulsion and regulation of luminal fluid content, presumably accounting for the drug‐induced defaecation in rats.
Deoxyribose analogues of N6‐cyclopentyladenosine (CPA): partial agonists at the adenosine A1 receptor in vivo
Authors: Mathôt R.A.A. , Van der Wenden E.M. , Soudijn W. , IJzerman A.P. , Danhof M.
Published: July 2012

The purpose of the present study was to quantify the cardiovascular effects of the 2′‐, 3′‐ and 5′‐deoxyribose analogues of the selective adenosine A1 receptor agonist, N6‐cyclopentyladenosine (CPA) in vivo. The blood concentration‐effect relationships of the compounds were assessed in individual rats and correlated to their receptor binding characteristics.

The pharmacokinetics and pharmacodynamics of the compounds were determined after a single intravenous infusion of 0.80 mg kg−1 (2.5 μmol kg−1) of 5′dCPA, 1.2 mg kg−1 (3.8 μmol kg−1) of 3′dCPA or 20 mg kg−1 (63 μmol kg−1) of 2′ dCPA. The heart rate (HR) and mean arterial blood pressure (MAP) were monitored continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentration.

The relationship between blood concentrations and the reductions in both heart rate and blood pressure were described according to the sigmoidal Emax model. For the bradycardiac effect, the potencies based on free drug concentrations (EC50, u) of 5′dCPA, 3′dCPA and 2′dCPA in blood were 5.9 ± 1.7, 18 ± 4 and 260 ± 70 ng ml−1 (19 ± 6, 56 ± 11 and 830 ± 210 nM), respectively, and correlated well with the adenosine A1 receptor affinity in vitro. The Emax value of 2′dCPA was significantly less than those of the other compounds, suggesting that this compound may be regarded as a partial agonist when compared to the other analogues. The rank order of the maximal reduction in heart rate of the compounds corresponded well with the order of the GTP‐shifts, as determined in vitro.

It is concluded that deoxyribose derivatives of CPA may be partial agonists for the adenosine A1 receptor and may serve as tools for further investigation of adenosine receptor partial agonism in vivo.
Mucosa‐dependent muscarinic liberation of prostaglandins from rat isolated trachea
Authors: Brunn Gemot , Wessler Ignaz , Racke Kurt
Published: July 2012

The present study examined whether cholinoceptor stimulation modulates the release of arachidonic acid‐derived mediators from rat isolated tracheae.

Tracheae were preincubated with [3H]‐arachidonic acid and the outflow of 3H‐compounds was determined. Acetylcholine and the muscarinic agonist, carbachol but not nicotine, increased the rate of tritium outflow maximally by about 30%. The M3 receptor‐preferring antagonist p‐fluoro‐hexahydrosiladiphenidol was more effective than pirenzepine and methoctramine in antagonizing the effect of acetylcholine.

High performance liquid chromatography analysis (methanol gradient) of the released 3H‐compounds showed that one peak, co‐eluting with [14C]‐prostaglandin E2 ([14C]‐PGE2) and [3H]‐PGD2, was enhanced almost 10 fold following muscarinic receptor activation, whereas the outflow of [3H]‐arachidonic acid remained unaffected.

Using an acetonitril gradient separation it was shown that [3H]‐PGE2, [3H]‐PGD2 and [3H]‐PGF2α are released spontaneously, but [3H]‐PGE2 represented the major fraction of 3H‐prostaglandins. Acetylcholine enhanced the release of all three 3H‐prostaglandins, but the effect on PGE2 was most pronounced and most consistent.

After removal of the mucosa the muscarinic effect of acetylcholine on total tritium and on that of the 3H‐prostaglandins ([3H]‐PGE2/PGD2 peak) was abolished.

Acetylcholine also enhanced the outflow of radioimmunologically determined PGE2 in a mucosa‐dependent manner.

After inhibition of cyclo‐oxygenase by 3 μM indomethacin, the outflow of 3H‐prostaglandins was reduced to almost undetectable levels and acetylcholine evoked a marked release of [3H]‐arachidonic acid. The phospholipase A2 inhibitor, quinacrine (up to 100 μm) also blocked the effect of acetylcholine on the outflow of 3H‐prostaglandins, but this was not followed by a compensatory increase in the outflow of [3H]‐arachidonic acid.

In conclusion, activation of muscarinic receptors which have characteristics of the M3 subtype can evoke release of prostaglandins from the airway mucosa.
Characterization of the pharmacology and regional distribution of (S)‐[3H]‐5‐fluorowillardiine binding in rat brain
Authors: Hawkins L.M. , Beaver K.M. , Jane D.E. , Taylor P.M. , Sunter D.C. , Roberts P.J.
Published: July 2012

This study examined the binding of the new radioligand (S)‐[3H]‐5‐fluorowillardiine to rat brain synaptic membranes. Specific binding represented greater than 80% of the total binding and was increased by 10% in the presence of 100 mM potassium thiocyanate (KSCN).

In the absence of KSCN, (S)‐[3H]‐5‐fluorowillardiine identified two binding sites with KD1 = 22.5nM, Bmax1= 1.4 pmol mg‐1 protein and KD2=1.5 μM, Bmax2= 10.8 pmol mg−1 protein. In the presence of 100 mM KSCN the affinities of both the binding sites were increased, yielding values of KD1 = 6.9nM and KD2 = 0.4 μm KSCN was without effect on the Bmax values.

(S)‐[3H]‐5‐fluorowillardiine binding was displaced by non‐NMDA receptor ligands with the rank order of potency: 2,3‐dihydroxy‐6‐nitro‐7‐sulphamoyl‐benzo(F)quinoxaline (NBQX) > domoate > (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) ≅ L‐glutamate > 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) > kainate >> (R)‐5‐fluorowillardiine. In contrast, both N‐methyl‐D‐aspartate (NMDA) and the metabotropic glutamate receptor agonist, (lS,3R)‐l‐aminocyclopentane‐l,3‐dicarboxylic acid (ACPD) were inactive.

By use of quantitative autoradiography the regional distribution of (S)‐[3H]‐5‐fluorowillardiine binding in rat brain was assessed. The highest levels of binding were in the dentate gyrus and the CA1 region of the hippocampus. Lower levels of binding were detected in the cerebral cortex, olfactory system, lateral septum, caudate putamen and nucleus accumbens.

We conclude that the pharmacological profile and regional distribution of (S)‐[3H]‐5‐fluorowillardiine binding is consistent with its specific interaction with AMPA receptors. The advantages of high percentage specific binding and recognition of the two binding sites, in the absence of KSCN, suggest that (S)‐[3H]‐5‐fluorowillardiine may be the ligand of choice for the future study of AMPA receptors.
Mechanism of endothelium‐dependent relaxation induced by substance P in the coronary artery of the pig
Authors: Kuroiwa Mari , Aoki Hiroki , Kobayashi Sei , Nishimura Junji , Kanaide Hideo
Published: July 2012

Using front‐surface fluorometry of fura‐2‐loaded porcine coronary arterial strips with the endothelium intact, we investigated the mechanisms of vasorelaxation induced by substance P (SP). Fura‐2 fluorescence signals which indicated the cytosolic Ca2+‐concentration ([Ca2+]i), were observed to arise exclusively from the smooth muscle cells in these strips.

During the contractions induced by U46619 (100 nM), a thromboxane A2 analogue, an SP‐induced endothelium‐dependent, biphasic vasorelaxation was observed, which consisted of an initial rapid relaxation phase followed by a sustained phase, with a transient decrease in [Ca2+]i. Pretreatment with indomethacin (Ind) had no effect on the SP‐induced relaxation; however, pretreatment with NG‐nitro‐L‐arginine (l‐NOARG) partially, but significantly inhibited the decrease in both the [Ca2+]i and tension induced by SP; in addition, the sustained phase of SP‐induced relaxation was almost completely abolished. Thus, part of the relaxation was considered to be mediated by L‐NOARG‐sensitive relaxing factor (endothelium‐derived relaxing factor: EDRF).

During the 40 mM K+‐depolarization‐induced contraction which may eliminate the effects of endothelium‐derived hyperpolarizing factor (EDHF), the vasorelaxation induced by SP was completely inhibited by L‐NOARG.

During the vasorelaxation induced by SP, the [Ca2+]itension relationships shifted to the right of the contractions induced by either U46619 or high K+‐depolarization.

Using front‐surface fluorometry of fura‐2 loaded porcine aortic valvular strips, we examined the effects of SP on [Ca2+]i in endothelial cells in situ. SP induced a rapid increase in [Ca2+]i of endothelial cells in situ followed by a small sustained phase in normal PSS (5.9 mM K+). The increase in extracellular K+ had no apparent effect on the SP‐induced [Ca2+]i elevation of endothelial cells.

We thus conclude that: (1) SP‐induced vasorelaxation is mediated by an L‐NOARG‐sensitive factor (EDRF) and an L‐NOARG‐resistant factor; and (2) the first, rapid, phase of the relaxation is mediated by both factors while the sustained phase seems to be mediated mainly by EDRF. The underlying mechanisms of L‐NOARG‐resistant relaxation have yet to be elucidated, but EDHF appears to be a potentially contributing factor.
Determination of KA values by controlled receptor expression in Xenopus oocytes
Authors: Murakoshi Hideyuki , Nunoki Kazuo , Ishii Kuniaki , Taira Norio
Published: July 2012

In the present study we estimated the KA value of endothelin‐1 (ET‐1) for ETA‐receptors by a new method in which the level of expression of ETA‐receptors in Xenopus oocytes was altered in a controlled way.

Kvl.2 (a delayed rectifier type K channel) cRNA at the fixed concentration of 0.2 μg μl−1 was mixed with ETA‐receptor cRNA at various concentration ratios (10−3‐3). Oocytes were examined 2–4 days after the injection of the cRNA mixtures.

In these oocytes, ET‐1 suppressed the amplitude of Kvl.2 current in a dose‐dependent manner in the range of 0.1–100 nM; the maximum inhibition produced by ET‐1 was larger and the EC50 value for the inhibition by ET‐1 was smaller as the mixture ratio was increased. Double‐reciprocal plots of equiactive concentrations of ET‐1 in 1/1‐ and 1/30‐injected oocytes yielded a KA for ET‐1 of 7.4 nM. The number of ETA‐receptors in 1/30‐injected oocytes was 13% of that in 1/1‐injected oocytes, whereas the inhibition of the current in 1/30‐injected oocytes was about 60% of that in 1/1‐injected oocytes. This suggests the presence of spare receptors of ETA in the latter.

A saturation binding experiment estaimated a KD value of 0.1 nM for ET‐1 at ETA‐receptors and the number of ETA‐receptors in 1/30‐injected oocytes was 23% of that in 1/1‐injected ones. This value was not significantly different from that estimated by the above new method. However, there was a discrepancy between KA and 1KD, which could be due to factors unique to the expression system employed in the present study.
Mastoparan‐induced phosphatidylcholine hydrolysis by phospholipase D activation in human astrocytoma cells
Authors: Mizuno Kouichi , Nakahata Norimichi , Ohizumi Yasushi
Published: July 2012

The effect of mastoparan on phosphatidylcholine hydrolysis was examined in 1321N1 human astrocytoma cells. Mastoparan (3–30 μm) caused an accumulation of diacylglycerol (DG) and phosphatidic acid (PA) accompanied by choline release in a concentration‐ and time‐dependent manner.

In the presence of 2% n‐butanol, mastoparan (3–100 μM) induced phosphatidylbutanol (PBut) accumulation in a concentration‐ and time‐dependent manner, suggesting that mastoparan activates phospholipase D (PLD). Propranolol (30–300 μM), a phosphatidate phosphohydrolase inhibitor, inhibited DG accumulation induced by mastoparan, supporting this idea.

Depletion of extracellular free calcium ion did not alter the effect of mastoparan on PLD activity.

A protein kinase C (PKC) inhibitor, calphostin C (1 μm), did not inhibit mastoparan‐induced PLD activation but the ability of mastoparan to stimulate phospholipase D activity was decreased in the PKC down regulated cells.

PLD activity stimulated by mastoparan was not prevented by pretreatment of the cells with pertussis toxin (PT) or C3 ADP‐ribosyltransferase. Furthermore, guanine nucleotides did not affect PLD activity stimulation by mastoparan in membrane preparations.

Mastoparan stimulated PLD in several cell lines such as RBL‐2H3, RBL‐1, HL‐60, P388, endothelial cells, as well as 1321N1 human astrocytoma cells.

These results suggest that mastoparan induces phosphatidylcholine (PC) hydrolysis by activation of PLD, not by activation of phosphatidylcholine‐specific phospholipase C (PC‐PLC); mastoparan‐induced PLD activation is not mediated by G proteins.
Pharmacological characterization of behavioural responses to SK&F 83959 in relation to ‘D1‐like’ dopamine receptors not linked to adenylyl cyclase
Authors: Deveney Aaron M. , Waddington John L.
Published: July 2012

Behavioural responses to the new benzazepine derivative, SK&F 83959, a compound that both fails to stimulate adenylyl cyclase and inhibits the stimulation of adenylyl cyclase induced by dopamine, were characterized in detail.

In rat striatal membrane preparations, radioligand binding studies with [3H]‐SCH 23390 and [3H]‐spiperone indicated SK&F 83959 had a high affinity and > 250 fold selectivity for D1 over D2 receptors.

Using a rapid time‐sampling behavioural check list technique, SK&F 83959 (0.01‐1.25 mg kg−1) induced grooming in the manner of all known D1 receptor agonists, together with some vacuous chewing, which declined at higher doses with the emergence of directed chewing and rearing as an adjunct to prominent sniffing; no stereotyped behaviour was evident.

Grooming to SK&F 83959 (0.05 mg kg−1) was blocked by the selective D1 receptor antagonists, SCH 23390 (0.01‐1.0 mg kg−1) and BW 737C (0.04–5.0 mg kg−1) and was attenuated by the selective D2 receptor antagonist, YM 09151‐2 (0.005‐0.5 mg kg−1); vacuous chewing to SK&F 83959 was not influenced by either SCH 23390 or BW 737C and was enhanced by YM 09151‐2.

The paradoxical induction of typical D1 receptor agonist‐induced grooming by SK&F 83959, an agent satisfying criteria for a D1 receptor antagonist as classically defined, together with its blockade by typical D1 antagonists, strongly suggests mediation via a ‘D1‐like’ site that appears to respond similarly to agents independent of whether they exert agonist or antagonist actions at the classical adenylyl cyclase‐coupled D1 receptor. This direct functional evidence for a ‘D1‐like’ site that is not linked to adenylyl cyclase readily complements neurochemical data suggesting the existence of a cyclase‐independent ‘D1‐like’ receptor that may be coupled to phosphoinositide hydrolysis.
Evidence that 5‐HT1D receptors mediate inhibition of sympathetic ganglionic transmission in anaesthetized cats
Authors: Jones James F.X. , Martin Graeme R. , Ramage Andrew G.
Published: July 2012

In anaesthetized cats, 5‐carboxamidotryptamine (5‐CT) or 5‐hydroxytryptamine (5‐HT) (0.3–300 μg kg−1, i.v.) inhibited the postganglionic compound action potential evoked by preganglionic electrical stimulation (0.5 Hz) with a similar potency in the stellate and splanchnic ganglia. In the 5‐HT experiments transmission thorough the inferior mesenteric ganglia was also recorded. The maximal inhibitory effect of 5‐HT was greater on the stellate and splanchnic ganglia (60 ± 4 and 52 ± 5%) than on the inferior mesenteric (15 ± 2%). The effects of 5‐HT were unaffected by pretreatment with antagonists (1 mg kg−1;i.v.) for 5‐HT2 (BW501C67), 5‐HT1A (WAY‐100635) and 5‐HT3 receptors (ondansetron). However, responses to both 5‐HT and 5‐CT were attenuated significantly by GR127935 (1 mg kg−1) except the responses to 5‐HT at the inferior mesenteric ganglia. These results are consistent with the involvement of 5‐HT1D receptors mediating inhibition of sympathetic ganglionic transmission in vivo.
Relationship between agonist binding, phosphorylation and immunoprecipitation of the m3‐muscarinic receptor, and second messenger responses
Authors: Tobin A.B. , Willars G.B. , Burford N.T. , Nahorski S.R.
Published: July 2012

Phosphoinositidase C‐linked m3‐muscarinic receptors expressed in Chinese hamster ovary cells (CHO‐m3 cells) are phosphorylated on serine following agonist stimulation

m3‐Muscarinic receptor phosphorylation is concentration‐dependent requiring a carbachol concentration of 13.2 μm for half maximal stimulation

The phosphorylation concentration‐response curve lies to the left of the curve for carbachol binding to muscarinic receptors (KD=100μm) in membranes from CHO‐m3 cells. In contrast, receptor phosphorylation closely correlates with receptor‐mediated phosphoinositidase C activation (EC50 for inositol 1,4,5 trisphosphate accumulation during the peak and plateau phases were 7.14 μm and 5.92 μm respectively) but not with rapid agonist‐mediated calcium elevation (EC50 = 0.32 μm) measured in fura‐2‐AM loaded cells

These data suggest a dissociation of receptor phosphorylation from agonist occupation. Such an apparent ‘receptor reserve’ for m3‐muscarinic receptor phosphorylation may be indicative of a mechanism that is dependent on a small amplification of the receptor signal, though probably dissociated from the calcium signal.
[3H]‐RS‐45041‐190: a selective high‐affinity radioligand for I2 imidazoline receptors
Authors: MacKinnon Alison C. , Redfern William S. , Brown Christine M.
Published: July 2012

RS‐45041‐190 (4‐chloro‐2‐(imidazolin‐2‐yl)isoindoline) is an I2 imidazoline receptor ligand with the highest affinity and selectivity so far described; [3H]‐RS‐45041‐190 has a tritium atom attached to the 7‐position on the isoindoline ring

[3H]‐RS‐45041‐190 binding to rat kidney membranes was saturable (Bmax = 223.1 ± 18.4 fmol mg−1 protein) and of high affinity (Kd = 2.71 ±0.59 nM). Kinetic studies revealed that the binding was rapid and reversible, with [3H]‐RS‐45041‐190 interacting with two sites or two affinity states

Competition studies showed that 60–70% of [3H]‐RS‐45041‐190 binding (1 nM) was specifically to imidazoline binding sites of the I2 subtype, characterized by high affinity for idazoxan (pIC50 7.85 ±0.03) and cirazoline (pIC50 8.16 ±0.05). The remaining 30–40% was displaced specifically by the monoamine oxidase A inhibitors, clorgyline and pargyline

α1 and α2‐adrenoceptor, Ii imidazoline, histamine, 5‐hydroxytryptamine or dopamine receptor ligands had low affinity suggesting that [3H]‐RS‐45041‐190 did not label receptors of these classes

In autoradiography studies, [3H]‐RS‐45041‐190 labelled discrete regions of rat brain corresponding to the distribution of I2 subtypes, notably the subfornical organ, arcuate nucleus, interpeduncular nucleus, medial habenular nucleus and lateral mammillary nucleus, and additional sites in the locus coeruleus, dorsal raphe and dorsomedial hypothalamic nucleus

[3H]‐RS‐45041‐190 therefore labels I2 receptors with high affinity, and an additional site which has high affinity for some monoamine oxidase inhibitors.
Evidence that activation of 5‐HT2 receptors in the forebrain of anaesthetized cats causes sympathoexcitation
Authors: Anderson Ian K. , Martin Graeme R. , Ramage Andrew G.
Published: July 2012

The aim of the present experiments was to determine whether the effects of lateral ventricular application of 5‐HT on cardiovascular and respiratory variables in anaesthetized cats are mediated by forebrain 5‐HT2 receptors. This was carried out by determining whether the effects of 5‐HT are blocked by the 5‐HT2 antagonist, cinanserin and if they are mimicked by the selective 5‐HT2 agonist, 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI)

Cats were anaesthetized with a mixture of oc‐chloralose and pentobarbitone sodium, neuromuscularly blocked and artificially ventilated. The following cardiovascular and respiratory variables were recorded: renal, splanchnic and cardiac sympathetic nerve activities, phrenic nerve activity, heart rate, arterial blood pressure, femoral arterial conductance and tracheal pressure. All drugs were administered via the lateral ventricle and the action of these agonists was restricted to forebrain sites by a cannula placed in the Aqueduct of Sylvius

Cumulative doses of 5‐HT (10–160 nmol kg−1) and DOI (80–320 nmol kg−1) injected into the lateral ventricle caused significant increases in blood pressure, heart rate, cardiac and splanchnic sympathetic nerve activity and a decrease in femoral arterial conductance. DOI and 5‐HT caused a greater increase in cardiac compared with splanchnic nerve activity and failed to change renal nerve activity

‐HT but not DOI significantly increased the magnitude and the number of phrenic bursts as well as significantly increasing tracheal pressure. The effects of 5‐HT also differed from DOI in that 5‐HT evoked maximal pressor and near maximal sympathoexcitatory effects after the first dose, whereas the pressor and sympathoexcitatory effects of DOI were graded over the complete dose‐range

The 5‐HT2 antagonist, cinanserin (265 nmol kg−1, i.c.v.) caused significant falls in blood pressure, heart rate and cardiac nerve activity and an increase in femoral arterial conductance. Splanchnic and renal sympathetic nerve activity, phrenic nerve activity and tracheal pressure were unaffected by cinanserin. After pretreatment with cinanserin all cardiovascular and respiratory effects of 5‐HT were significantly attenuated

It is concluded that in the cat, as DOI and 5‐HT have similar effects on the cardiovascular variables recorded and as the effects of 5‐HT are blocked by cinanserin, 5‐HT can act on 5‐HT2 receptors located in the forebrain to cause differential sympathoexcitation and a rise in arterial blood pressure. Further, the sympathoexcitatory effects mediated by 5‐HT2 receptors located in the forebrain differ from those located in the hindbrain in that they mediate increases in cardiac nerve activity and heart rate and also have no effect on renal nerve activity.
Activation of phospholipase C in SH‐SY5Y neuroblastoma cells by potassium‐induced calcium entry
Authors: Smart D. , Wandless A. , Lambert D.G.
Published: July 2012

We used SH‐SY5Y human neuroblastoma cells to investigate whether depolarization with high K+ could stimulate inositol (1,4,5)trisphosphate (Ins(1,4,5)P3) formation and, if so, the mechanism involved

Ins(1,4,5)P3 was measured by a specific radioreceptor mass assay, whilst [Ca2+]i was measured fluorimetrically with the Ca2+ indicator dye, Fura‐2

Depolarization with K+ caused a time‐ and dose‐dependent increase in [Ca2+]j (peak at 27 s, EC50 of 50.0±9.0 mM) and Ins(1,4,5)P3 formation (peak at 30 s, EC50 of 47.4±1.1 mM)

Both the K+‐induced Ins(1,4,5)P3 formation and increase in [Ca2+]i were inhibited dose‐dependently by the L‐type voltage‐sensitive Ca2+ channel closer(R +)‐BayK8644, with IC50 values of 53.4 nM and 87.9 nM respectively.

These data show a close temporal and dose‐response relationship between Ca2+ entry via L‐type voltage‐sensitive Ca2+ channels and Ins(1,4,5)P3 formation following depolarization with K+, indicating that Ca2+ influx can activate phospholipase C in SH‐SY5Y cells
Modulation of calcium currents by G‐proteins and adenosine receptors in myenteric neurones cultured from adult guinea‐pig small intestine
Authors: Baidan Lessia V. , Zholos Alexander V. , Wood Jackie D.
Published: July 2012

Whole‐cell patch clamp methods were used to analyse voltage‐dependent calcium currents in cultured myenteric neurones enzymatically isolated from adult guinea‐pig small intestine

Activation of G‐proteins by intracellular administration of GTP‐γ‐S (100–200 μm in pipette) decreased the amplitude of high voltage activated Ca2+ current (ICa) by more than 50%. Residual ICa was activated more slowly and was non‐inactivating during 500 ms test pulses when GTP‐γ‐S was included in the pipette solution

Inclusion of 500 μm GDP‐β‐S in the patch pipettes increased the amplitude of ICa by over 30% without altering the voltage‐dependency

Extracellular application of 2‐chloroadenosine suppressed ICa dose‐dependently by reducing both transient and sustained components of the current

Pretreatment of the neurones with cholera toxin or forskolin did not alter the actions of GTP‐γ‐S or GDP‐β‐S or 2‐chloroadenosine

The results suggest that high threshold calcium channels in myenteric neurones are influenced by G‐proteins and that the inhibitory action of 2‐chloroadenosine on ICa involves G‐protein coupling of the adenosine receptors to the Ca2+ channel.
Expression of β3‐adrenoceptor mRNA in rat brain
Authors: Summers R.J. , Papaioannou M. , Harris S. , Evans B.A.
Published: July 2012

The reverse transcription/polymerase chain reaction was used to demonstrate β3‐adrenoceptor mRNA in rat brain regions. Levels were highest in hippocampus, cerebral cortex and striatum and lower in hypothalamus, brainstem and cerebellum.
SPECIAL REPORT Stimulation of cyclic AMP formation in the circular smooth muscle of human colon by activation of 5‐HT4‐like receptors
Authors: McLean Peter G. , Coupar Ian M.
Published: July 2012

5‐HT stimulated cyclic AMP generation in human colonic circular smooth muscle in a concentration‐dependent fashion (EC50 = 229.1 nM). DAU 6236 also increased cyclic AMP formation and was a partial agonist relative to 5‐HT. GR 113808 inhibited the cyclic AMP formation induced by 5‐HT with a ‐log Ki value of 9.1 and an apparent pA2 value of 9.2. Ondansetron and methysergide failed to inhibit cyclic AMP formation induced by 5‐HT. These results indicate that the 5‐HT4 receptors of human colonic circular muscle mediate relaxation and inhibition of spontaneous contractions via formation of cyclic AMP.
Role of brainstem adenosine A1 receptors in the cardiovascular response to hypothalamic defence area stimulation in the anaesthetized rat
Authors: St. Lambert J.H. , Dashwood M.R. , Spyer K.M.
Published: July 2012

The role of centrally located adenosine A1 receptors in the cardiovascular changes associated with the hypothalamic defence response has been investigated by in vitro autoradiography and the intraventricular application of an A1 receptor antagonist.

8‐Cyclopentyl‐1,3‐dipropylxanthine (DPCPX), a highly selective adenosine A1 antagonist and its vehicle, ethanol, were administered directly into the posterior portion of the fourth ventricle of α‐chloralose anaesthetized, paralysed and artificially ventilated rats.

DPCPX (0.01 to 0.3 mg kg−1) caused a dose‐dependent decrease in the magnitude of the evoked pressor response (from −13 to −23 mmHg) elicited on hypothalamic defence area stimulation at a dose 10 fold lower than that required to produce an equivalent effect following systemic administration whilst ethanol, the vehicle, had no effect.

In vitro autoradiography revealed a heterogeneous distribution of adenosine A1 binding sites in the lower brainstem of rats. Image analysis showed the ventrolateral medulla to have the highest density of A1 receptors. Intermediate levels of binding were seen in caudal regions of the nucleus tractus solitarii and the hypoglossal nucleus.

These data imply that a proportion of the cardiovascular response to hypothalamic defence area stimulation are produced by the activation of adenosine A1 receptors localized close to the surface of, or adjacent to, the fourth ventricle in the immediate vicinity of the injection site.