bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation by calcineurin of 5‐HT3receptor function in NG108‐15 neuroblastoma x glioma cells
Authors: Boddeke H.W.G.M. , Meigel I. , Boeijinga P. , Arbuckle J. , Docherty R.J.
Published: July 2012

. We have investigated the mechanism of regulation of 5‐HT3receptor channel sensitivity in voltage‐clamped (−80 mV) NG108‐15 neuroblastoma cells.

. The 5‐HT‐induced inward current activated rapidly. The fast onset was followed by a biphasic decay which was characterized by two time constants, τ1(1.1 ± 0.21s) and τ2(8.9 ± 1.6s), respectively. Brief applications of 5‐HT, applied at 2 min intervals, induced a decrease in the amplitude of the 5‐HT3receptor‐mediated peak inward currents.

. Buffering of intracellular calcium with the calcium chelator BAPTA (10 mM) instead of EGTA (10 mM) attenuated the 5‐HT‐induced loss of responsiveness of 5‐HT3receptors. Omission of calcium from the extracellular medium yielded a similar attenuation of loss of responsiveness.

. Inclusion of the protein kinase inhibitor, staurosporine (1 μm) or of okadaic acid (1 μm), an inhibitor of protein phosphatases 1 and 2A, in the intracellular buffer solution did not affect 5‐HT3receptor sensitivity.

. Injection of cyclosporin A‐cyclophilin A complex (20 nM), which potently inhibits calcineurin, did not affect the time constants of the biphasic decay of the 5‐HT response τ1(1.4 ± 0.28s) and τ2(11.3 ± 1.7s). The complex, however, prevented the loss of 5‐HT3receptor responsiveness upon repeated application of 5‐HT. A similar, but weaker effect was observed after intracellular application of the autoinhibitory peptide domain of calcineurin (1 μm).

. The recovery of desensitized 5‐HT3receptors upon a second application of 5‐HT (1 μm) showed a half‐life time (τ1/2) of 2.6 ± 0.12 min in control cells which was reduced to 1.6 ± 0.09 min in cells treated with cyclosporin A‐cyclophilin A (20 nM) complex.

. We conclude that calcineurin does not affect the fast decay of the 5‐HT3receptor response but may be involved in a slower process which regulates channel activity.
Evidence for P‐glycoprotein‐modulated penetration of morphine‐6‐glucuronide into brain capillary endothelium
Authors: Huwyler Jörg , Drewe Jürgen , Klusemann Caterina , Fricker Gert
Published: July 2012

Morphine‐6‐glucuronide is one of the major metabolites of morphine. The potent analgesic action of this compound together with its potential lower apparent toxicity in man, when compared with morphine, indicated its clinical importance.

Primary cultures of porcine brain capillary endothelial cells were used to study brain penetration of morphine‐6‐glucuronide. Biochemical characterization of the cell cultures revealed a marked enrichment in enzymatic activity of alkaline phosphatase (56 fold) and angiotensin converting enzyme (230 fold) as compared to whole brain tissue. By immunostaining the presence of vimentin, factor VIII, the tight junction associated protein ZO‐1, and P‐glycoprotein was shown. Functional characterization revealed that the carrier system responsible for transport of neutral amino acids was intact.

Uptake and transport of morphine‐6‐glucuronide was marginal and in the range of the extracellular marker sucrose. However, uptake of morphine‐6‐glucuronide was enhanced significantly (P<0.0001) in presence of the inhibitors of P‐glycoprotein, verapamil or vincristine. The finding that morphine‐6‐glucuronide may serve as a substrate for P‐glycoprotein was confirmed in multidrug‐resistant P388 tumour cells.

We conclude that penetration of the blood‐brain barrier by morphine‐6‐glucuronide may depend on the expression of the product of the multidrug‐resistance (MDR) gene in brain capillary endothelial cells.
Effects of RP 73401, a novel, potent and selective phosphodiesterase type 4 inhibitor, on contractility of human, isolated bronchial muscle
Authors: Naline E. , Qian Y. , Advenier C. , Raeburn D. , Karlsson J.‐A.
Published: July 2012

The aim of this study was to investigate the smooth muscle relaxant effects of the novel, selective phosphodiesterase (PDE) type 4 inhibitor, RP 73401 in comparison with the classical PDE 4 inhibitor, rolipram, the non‐selective PDE inhibitor, theophylline and the β‐adrenoceptor agonist, isoprenaline on the human, isolated bronchus.

At resting tone, the rank order of potency (pD2) for the relaxants was RP 73401 ≥ rolipram ≥ isoprenaline > > theophylline. In terms of maximum relaxation produced (Emax) the PDE 4‐selective inhibitors were similar, but the maximal effects (70–75% of theophylline, 3 mM) were lower than that observed with isoprenaline (98% of theophylline, 3 mM) or theophylline itself (100%).

On the human isolated bronchus pre‐contracted with acetylcholine (ACh, 0.1 or 1.0 mM), the rank order of potency remained the same. The maximal responses to RP 73401 and rolipram were however markedly reduced (Emax 39.9–46.6%) compared with isoprenaline (Emax 79–85%).

In tissues pre‐contracted with ACh (0.1 mM), RP 73401 and rolipram (10−9‐10−7 m) significantly and concentration‐dependently increased tissue sensitivity to isoprenaline. RP 73401 and rolipram were similar in potency. Both selective PDE 4 inhibitors also significantly increased the maximal relaxant effects of isoprenaline. These effects were not observed with the PDE 3 inhibitor, siguazodan.

In terms of retention by tissues (an index of duration of action), the onset of action of RP 73401 (2.11 ± 0.53 min) and rolipram (1.70 ± 0.45 min) was significantly slower than that of isoprenaline (0.33 ± 0.06 min) or theophylline (1.17 ± 0.25 min). The retention of RP 73401 (89.0 ± 21.9 min) on the human isolated bronchial tissues after washing was however dramatically longer than that of rolipram (18.3 ± 4.5 min), theophylline (3.43 ± 0.58 min) or isoprenaline (2.81 ± 0.31 min).

These data indicate that RP 73401 is a potent and long‐acting relaxant of human bronchial muscle in vitro. RP 73401 is more potent than the classical PDE 4‐selective inhibitor rolipram and the non‐selective PDE inhibitor theophylline and is retained in bronchial tissue for a much longer period of time.
Effect of intrarenally infused parathyroid hormone‐related protein on renal blood flow and glomerular filtration rate in the anaesthetized rat
Authors: Massfelder Thierry , Parekh Niranjan , Endlich Karlhans , Saussine Christian , Steinhausen Michael , Helwig Jean‐Jacques
Published: July 2012

Parathyroid hormone‐related protein (PTHrP) is expressed in the kidney and acts on vascular PTH/PTHrP receptors to vasodilate the isolated kidney and to stimulate renin release. However, effects of PTHrP on renal blood flow (RBF) and glomerular filtration rate (GFR) in vivo have not been assessed in the absence of its cardiac, peripheral and central effects. We investigated the renal effects of PTH and PTHrP infused into the left renal artery of anaesthetized rats.

Intrarenal infusions, adjusted to generate increasing concentrations of human PTHrP(1–34) and rat PTH(1–34) in renal plasma (2 × 10−11 to 6 × 10−9 m) produced a comparable dose‐dependent increase in RBF. The rise was 4% at the lowest and 34% at the highest concentrations of peptides. Up to a concentration of 2 × 10−9 m, mean arterial pressure (MAP) and heart rate were not affected, but at 6 × 10−9 m, intrarenally infused peptides reached the peripheral circulation, and caused a fall in MAP within a few minutes. While MAP returned to basal value after the last peptide infusion, RBF remained more than 10% above control for at least 30 min.

Two competitive PTH/PTHrP receptor antagonists, [Nle8,18, Tyr34]‐bPTH(3–34)amide and [Leu11, D‐Trp12]‐hPTHrP(7–34)amide (2 × 10−8 m) were devoid of agonist activity, but markedly antagonized the dose‐dependent increase in RBF elicited by PTHrP.

GFR and urine flow were measured in left PTHrP‐infused experimental kidney and right control kidney. Renal PTHrP concentration of 10−10 m elevated left RBF by 10%, and GFR by 20% without significantly increasing filtration fraction, and increased urine flow by 57%. In the right control kidney GFR and diuresis did not change.

The results indicate that PTHrP has similar renal haemodynamic effects as PTH and increases RBF, GFR and diuresis in anaesthetized rats.
Impaired activation of adenylyl cyclase in lung of the Basenjigreyhound model of airway hyperresponsiveness: decreased numbers of high affinity β‐adrenoceptors
Authors: Emala C.W. , Aryana A. , Hirshman C.A.
Published: July 2012

To evaluate mechanisms involved in the impaired β‐adrenoceptor stimulation of adenylyl cyclase in tissues from the Basenji‐greyhound (BG) dog model of airway hyperresponsiveness, we compared agonist and antagonist binding affinity of β‐adrenoceptors, β‐adrenoceptor subtypes, percentage of β‐adrenoceptors sequestered, and coupling of the β‐adrenoceptor to Gsα in lung membranes from BG and control mongrel dogs. We found that lung membranes from the BG dog had higher total numbers of β‐adrenoceptors with a greater percentage of receptors of the β subtype as compared to mongrel lung membranes.

Agonist and antagonist binding affinity and the percentage of β‐adrenoceptors sequestered were not different in BG and mongrel dog lung membranes. However, the percentage of β‐adrenoceptors in the high affinity state for agonist was decreased in BG lung membranes suggesting an uncoupling of the receptor from Gsα.

Impaired coupling between the β‐adrenoceptor and G protein documented by the decreased numbers of β‐adrenoceptors in the high affinity state in BG lung membranes, is a plausible explanation for the reduced stimulation of adenylyl cyclase and the resultant reduction in airway smooth muscle relaxation in this model.
Tachykininergic synaptic transmission in the coeliac ganglion of the guinea‐pig
Authors: Zhao F.‐Y. , Saito K. , Yoshioka K. , Guo J.‐Z. , Murakoshi T. , Konishi S. , Otsuka M.
Published: July 2012

The responses of coeliac ganglion neurones of the guinea‐pig to electrical stimulation of the mesenteric nerves and applications of tachykinin receptor agonists were investigated by use of intracellular recording techniques.

Ganglion neurones were classified into three groups based on firing patterns in response to a depolarizing current pulse: phasic (38% of the population), tonic (39%) and atypical (23%). In the majority of phasic neurones (91%) a long after‐hyperpolarization (LAH) lasting 5–8 s followed action potentials induced by a train of depolarizing current pulses. In contrast, LAH was rarely observed in tonic neurones (5%).

In most of tonic neurones (90%) slow excitatory post‐synaptic potentials (e.p.s.ps) lasting 3–10 min were evoked by repetitive electrical stimulation of the mesenteric nerves. Prolonged depolarizations were also evoked in most tonic neurones by applications of substance P (SP), neurokinin A (NKA) or senktide, a tachykinin NK3 receptor agonist.

In most of phasic neurones (73%), mesenteric nerve stimulation did not induce an obvious depolarization but induced a prolonged inhibition of LAH lasting 3–10 min. Bath‐applied tachykinin receptor agonists similarly induced an inhibition of LAH without causing depolarization in most of the phasic neurones.

GR71251 (5 μm), a tachykinin NK1 receptor antagonist, partially depressed the nerve‐evoked slow e.p.s.ps in tonic neurones and the nerve‐evoked LAH inhibition in phasic neurones.

Capsaicin (0.1–5 μm) induced a prolonged depolarization in tonic neurones and an inhibition of LAH in phasic neurones.

A mixture of peptidase inhibitors potentiated the depolarization and the LAH inhibition evoked by nerve stimulation, SP and NKA, but not those evoked by senktide.

It is concluded that tonic neurones respond to repetitive mesenteric nerve stimulation preferentially with slow e.p.s.ps and that phasic neurones respond preferentially with LAH inhibition. The present study further suggests that SP and NKA, released from axon collaterals of primary afferent neurones, produce slow e.p.s.ps in tonic neurones and the LAH inhibition in phasic neurones via NK1 receptors.
Potentiation of chloride responses to glycine by three 5‐HT3 antagonists in rat spinal neurones
Authors: Chesnoy‐Marchais D.
Published: July 2012

Modulations of Cl− responses to glycine by 5‐hydroxytryptamine ligands were studied in cultured spinal neurones, by the whole‐cell recording technique.

Three 5‐HT3 antagonists were found to potentiate reversibly responses to low concentrations of glycine. Potentiations were induced by micromolar concentrations of LY‐278,584 (1–10 μm) and by concentrations of MDL‐72222 or ICS‐205,930 between 10 nM and 1 μm.

Potentiations were observed over the whole voltage range without any change in the reversal potential of the glycine responses and without affecting the resting conductance.

The degree of potentiation was variable among cells. It increased with the concentration of the modulator, but only up to 100 nM for MDL‐72222 and ICS‐205,930.

The potentiation appeared to result from an increase in the affinity for glycine of glycine receptors.

Neither the blockade of glycine uptake by Na+ removal, nor the excision of membrane patches prevented the potentiation.

At high concentrations (10 μm), both MDL‐72222 and ICS‐205,930 had, in contrast, a blocking effect on glycine responses.

Potentiation by LY‐278,584 and a dose‐dependent modulation by MDL‐72222 were also observed for taurine responses.

The effects on glycine responses of various ligands of 5‐HT3 receptors (including agonists) are discussed. The ability of LY‐278,584, MDL‐72222 and ICS‐205,930 to potentiate glycine responses appears to be independent of their known 5‐HT3 receptor antagonist properties. It would be interesting to look for chemically related drugs that would be specific potentiators of glycine responses.
Facilitation of imipramine efflux from the brain by systemic specific antibodies
Authors: Ragusi Corinne , Boschi Gabrielle , Rips Richard , Scherrmann Jean‐Michel
Published: July 2012

This study investigated the capacity of circulating anti‐tricyclic antidepressant (TCA) IgG to increase the efflux of imipramine (Imip) from the rat brain.

A tracer amount of [3H]‐Imip (40 pmol) was injected into the cerebral lateral ventricle and its efflux was determined in control rats and in rats given anti‐TCA antibody. The monoclonal anti‐TCA IgG1 was injected i.v. 48 h before Imip at 4 IgG:Imip molar ratios (10, 100, 1000 and 10000). The [3H]‐Imip in arterial and venous plasma was measured for up to 60 min, and in the brain and peripheral organs (heart, liver, lung, kidney) 5 and 60 min after Imip injection.

The arterial plasma concentration of Imip in control rats was significantly higher (26.7 ± 2.1 pM) than the venous one (17.7 ± 2.0 pM) at 5 min, indicating that Imip released from brain becomes distributed in peripheral tissues. These concentrations were not significantly different at 60 min suggesting that Imip was, at this time, redistributing from extravascular tissues to the blood. In rats given anti‐TCA IgG, any Imip leaving the brain was immediately bound by the circulating antibody at 5 min. This greatly reduced the Imip in the heart (63.9%) and lung (61.3%) at the highest IgG:Imip ratio. The brain Imip was markedly lower at 60 min (31.5% with an IgG:Imip ratio of 1000 and 57.5% at a ratio of 10000). The two lowest IgG:Imip ratios had less effect on the plasma Imip because of the relative low affinity of the anti‐TCA IgG (3.8 × 107 M−1).

These data indicate that the anti‐TCA IgG facilitated the efflux of Imip from the brain, even though these antibodies cannot cross the blood‐brain barrier. This may be an efficient system for increasing drug organ clearance, as more than half the Imip in the brain was actively removed by the antibody in 1 h.
Further evidence for the presence of cannabinoid CB1 receptors in guinea‐pig small intestine
Authors: Pertwee Roger G. , Fernando Susanthi R. , Nash Joanne E. , Coutts Angela A.
Published: July 2012

CP 50,556, CP 55,940, nabilone, CP 56,667, Δ9‐tetrahydrocannabinol (THC) and cannabinol each inhibited electrically‐evoked contractions of the myenteric plexus‐longitudinal muscle preparation of guinea‐pig small intestine in a concentration‐related manner. The IC50 values of these cannabinoids, respectively 3.45, 3.46, 30.61, 162.94, 214.63, and 3913.5 nM, correlate well with previously obtained potency values for displacement of [3H]‐CP 55,940 from cannabinoid binding sites.

Electrically‐evoked contractions of the myenteric plexus‐longitudinal muscle preparation were also inhibited by AM 630 (6‐iodo‐pravadoline) and by WIN 55,212‐2 (IC50 = 1923.0 and 5.54 nM, respectively). The present finding that AM 630 is an agonist, contrasts with a previous observation that it behaves as a cannabinoid receptor antagonist in the mouse isolated vas deferens.

SR141716A produced dose‐related parallel rightward shifts in the log concentration‐response curves of CP 55,940, WIN 55,212‐2, THC and AM 630 for inhibition of electrically‐evoked contractions of the myenteric plexus‐longitudinal muscle preparation. SR141716A (1 μm) did not reverse the inhibitory effects of normorphine and clonidine on electrically‐evoked contractions or potentiate the contractile response to acetylcholine.

Doses of naloxone and yohimbine that reversed the inhibitory effects of normorphine or clonidine on electrically‐evoked contractions of the myenteric plexus‐longitudinal muscle preparation did not affect the inhibitory response to WIN 55,212‐2.

Electrically‐evoked release of acetylcholine from strips of myenteric plexus‐longitudinal muscle was decreased by 200 nM CP 55,940 and this inhibitory effect was almost completely reversed by 1 μm SR141716A. Acetylcholine‐induced contractions were not affected by 200 nM CP 55,940.

These results support the hypothesis that guinea‐pig small intestine contains prejunctional cannabinoid CB1 receptors through which cannabinoids act to inhibit electrically‐evoked contractions by reducing release of the contractile transmitter, acetylcholine.

THC was found to be more susceptible to antagonism by SR141716A than CP 55,940 or AM 630, raising the possibility that guinea‐pig small intestine contains more than one type of cannabinoid receptor.

At concentrations of 10 nM and above, SR141716A produced small but significant increases in the amplitude of electrically‐evoked contractions of the myenteric plexus‐longitudinal muscle preparation suggesting that this tissue may release an endogenous cannabinoid receptor agonist or that some cannabinoid receptors in this tissue are precoupled and that SR141716A can reduce the number of receptors in this state.
The inhibitory effects of iberiotoxin and 4‐aminopyridine on the relaxation induced by β1‐ and β2‐adrenoceptor activation in rat aortic rings
Authors: Satake N. , Shibata M. , Shibata S.
Published: July 2012

In rat aortic rings contracted by phenylephrine, the relaxation induced by isoprenaline was partly inhibited by iberiotoxin, (ibTX), tetraethylammonium, 4‐aminopyridine (4‐AP) and 1,9‐dideoxyforskolin, but not by glibenclamide.

In the presence of 4‐AP, 1,9‐dideoxyforskolin failed to inhibit further the relaxant response to isoprenaline. Cromakalim‐induced relaxation was inhibited by glibenclamide.

In the absence of endothelium, ibTX and 4‐AP still inhibited the relaxant response to isoprenaline.

The inhibitory effect of ibTX on the relaxant response to isoprenaline was eliminated by pretreatment with ICI‐118,551, a β2‐adrenoceptor antagonist, but not by atenolol, a β1‐adrenoceptor antagonist.

The inhibitory effect of 4‐AP on the relaxation induced by isoprenaline was abolished by atenolol, but not by ICI‐118, 551.

The inhibitory effect of ibTX on the isoprenaline‐induced relaxation in the presence of atenolol was completely abolished by MDL 12,330A, an adenylate cyclase inhibitor. Further, the inhibitory effect of 4‐AP on the isoprenaline‐induced relaxation in the presence of ICI‐118,551 was markedly reduced by MDL 12,330A.

The relaxation induced by dibutyryl cyclic AMP was partly inhibited by 4‐AP but not by ibTX. However, in the presence of KT5720, an inhibitor of cyclic AMP‐dependent protein kinase, ibTX failed to inhibit further the relaxation induced by isoprenaline.

These results suggest that, in rat aortic rings, Kca channels are involved in the relaxation induced by isoprenaline. In addition, Kca channels are mainly activated by β2‐adrenoceptors through cyclic AMP‐dependent pathways. Further, the inhibition of isoprenaline‐relaxation by 4‐AP may be related to the activation of β1‐adrenoceptors and cyclic AMP formation.
Changes in superoxide dismutase mRNA expression by streptozotocin‐induced diabetes
Authors: Kamata Katsuo , Kobayashi Tsuneo
Published: July 2012

Experiments were designed to investigate the involvement of superoxide anions in the attenuated endothelium‐dependent relaxation of the rat aorta from streptozotocin (STZ)‐induced diabetic rats.

The endothelium‐dependent relaxation responses to acetylcholine (ACh, 10−7 m) in helical strips of the aorta precontracted with noradrenaline (NA, 5 × 10−8∼3 × 10−7 m) were significantly decreased in STZ‐induced diabetic rats. The recovery phase of the relaxation after single administration of ACh in the STZ‐induced diabetic rats was more rapid than those in control vessels.

Preincubation of aortic strips with superoxide dismutase (SOD, 60 u ml−1) normalized the recovery phase of the relaxation of diabetic aorta after single administration of ACh, whereas catalase (150 u ml−1) or indomethacin (10−5 m) had no effects on the relaxation.

SOD (180 u ml−1) caused relaxation in NA precontracted aortic strips and the degree of the SOD‐induced relaxation was significantly greater in diabetic aorta as compared with age‐matched control vessels.

When the changes in mRNA expressions of Mn‐SOD or Cu‐Zn‐SOD were observed, Mn‐SOD mRNA expression was markedly decreased, and Cu‐Zn‐SOD was slightly decreased in diabetic aorta.

These results suggest that the rapid destruction of NO by superoxide anions may occur in the STZ‐induced diabetic rats, and this may be due to a decrease in mRNA expression of Mn‐SOD or Cu‐Zn‐SOD.
The role of ketoconazole in the QTc interval prolonging effects of H1‐antihistamines in a guinea‐pig model of arrhythmogenicity
Authors: Gras J. , Llenas J. , Palacios J.M. , Roberts D.J.
Published: July 2012

We have carried out experiments to re‐evaluate the influence of ketoconazole (400 mg kg−1, p.o.) on the effects of ebastine, terfenadine and loratadine on the QTc interval in conscious guinea‐pigs. Following a previously described protocol of oral drug administration, but using telemetric recording of the ECG, we have found that the prolongation of the QTc interval attributed to ebastine and terfenadine is in fact entirely due to ketoconazole, and that neither terfenadine, ebastine nor loratadine produce any additional effects on subsequent administration.
PK 11195 blockade of benzodiazepine‐induced inhibition of forskolin‐stimulated adenylate cyclase activity in the striatum
Authors: Tenn Catherine C. , Neu John M. , Niles Lennard P.
Published: July 2012

The effects of benzodiazepine receptor antagonists on the inhibition of forskolin‐stimulated adenylate cyclase (AC) activity by various benzodiazepine (BZ) and indoleamine agonists in the rat striatum were investigated.

A biphasic inhibition of forskolin‐stimulated AC activity by the peripheral‐type agonist, Ro5‐4864, and a multiphasic inhibition by the non‐selective BZ, diazepam, was observed. One phase of AC inhibition is consistent with a Gi‐coupled receptor‐mediated action, whereas the other phases appear to involve a direct effect on the enzyme itself.

While the central‐type antagonist, flumazenil, had no effect on the ability of Ro5‐4864 to inhibit AC activity, the peripheral‐type receptor ligand, PK 11195, abolished the first phase of inhibition.

PK 11195 and pertussis toxin were found to block the inhibitory effect of various BZs and the indoleamines, melatonin and 2‐iodomelatonin, on induced AC activity.

Saturation binding studies, conducted at 30°C with [3H]‐diazepam revealed a single binding site in the rat striatum (KD = 19.3 ± 0.80 nM) which significantly decreased in affinity in the presence of GTP (KD = 30.5 ± 2.6 nM; P < 0.05). No significant change in Bmax was observed.

These findings indicate the presence of Gi‐coupled BZ receptors in the rat striatum. Thus, suppression of cyclic AMP production may contribute to the diverse neuropharmacological effects of BZs, melatonin and related drugs.
Evidence of differential effects produced by ethanol on specific phospholipid biosynthetic pathways in rat hepatocytes
Authors: Carrasco María P. , Sanchez‐Amate María C. , Marco Carmen , Segovia Josefa L.
Published: July 2012

The aim of the present study was to investigate the effects of ethanol in vitro on the phospholipid biosynthetic pathways in hepatocytes isolated from the rat. We have used [methyl‐14C]‐choline, [1‐3H]‐ethanolamine and L‐[3‐3H]‐serine as exogenous precursors of the corresponding phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS).

Incubation of hepatocytes in the presence of ethanol significantly alters the incorporation of radiolabel from [14C]‐choline and [3H]‐ethanolamine into the metabolic intermediates and the final products of the CDP‐choline and CDP‐ethanolamine pathways. Radioactivity in the metabolic intermediates of both pathways was significantly decreased and the amount of label in PE was reduced whilst that of PC was not modified.

In the presence of 4‐methylpyrazole, an inhibitor of alcohol dehydrogenase (ADH) activity, ethanol produces a reduction in the label of choline phosphate, ethanolamine phosphate and a significant decrease in the amount of PC and PE radiolabel.

On the other hand, ethanol increases the incorporation of serine into phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, although this effect is observed only in the absence of 4‐methylpyrazole, indicating that this alteration is produced by some metabolite generated as a consequence of hepatic alcohol metabolism.

Ethanol also interferes with the methylation of phosphatidylethanolamine produced via the CDP‐ethanolamine pathway but it does not alter phosphatidylethanolamine methylation when this phospholipid is produced by mitochondrial phosphatidylserine decarboxylation, suggesting the existence of different intramembrane pools of phosphatidylethanolamine, which may exhibit different sensitivity to alcohol.

Our results indicate that ethanol exerts two different effects on phospholipid metabolism in hepatocytes: a stimulatory effect on the incorporation of exogenous substrates into different phospholipids probably related to an alteration in the availability of lipogenic substrates as a consequence of ethanol metabolism, and another inhibitory effect produced by ethanol per se, which can be observed only when ethanol metabolism is inhibited by the presence of a specific inhibitor of alcohol dehydrogenase activity.
The stimulation of capsaicin‐sensitive neurones in a vanilloid receptor‐mediated fashion by pungent terpenoids possessing an unsaturated 1,4‐dialdehyde moiety
Authors: Szallasi A. , Jonassohn M. , Ács G. , Bíró T. , Ács P. , Blumberg P.M. , Sterner O.
Published: July 2012

The irritant fungal terpenoid isovelleral caused protective eye‐wiping movements in the rat upon intraocular instillation and showed cross‐tachyphylaxis with capsaicin, the pungent principle in hot pepper.

Isovelleral induced a dose‐dependent calcium uptake by rat dorsal root ganglion neurones cultured in vitro with an EC50 of 95 nM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine.

Isovelleral inhibited specific binding of [3H]‐resiniferatoxin (RTX), an ultrapotent capsaicin analogue, to rat trigeminal ganglion or spinal cord preparations with an IC50 of 5.2 μm; in experiments in which the concentration of [3H]‐RTX was varied, isovelleral changed both the apparent affinity (from 16 pM to 37 pM) and the co‐operativity index (from 2.1 to 1.5), but not the Bmax.

The affinity of isovelleral for inducing calcium uptake or inhibiting RTX binding was in very good agreement with the threshold dose (2.2. nmol) at which it provoked pungency on the human tongue.

For a series of 14 terpenoids with an unsaturated 1,4‐dialdehyde, a good correlation was found between pungency on the human tongue and affinity for vanilloid receptors on the rat spinal cord.

The results suggest that isovelleral‐like compounds produce their irritant effect by interacting with vanilloid receptors on capsaicin‐sensitive sensory neurones. Since these pungent diterpenes are structurally distinct from the known classes of vanilloids, these data provide new insights into structure‐activity relations and may afford new opportunities for the development of drugs targeting capsaicin‐sensitive pathways.
Attenuation by prolonged nitric oxide synthase inhibition of the enhancement of fibrinolysis caused by environmental stress in the rat
Authors: Kawabata Atsufumi , Hata Taeko
Published: July 2012

Nitric oxide (NO) suppresses platelet aggregation and plasminogen activator inhibitor (PAI) release from platelets, playing physiological and/or pathological roles in the haemostatic system. We investigated the effect of NG‐nitro‐L‐arginine methyl ester (L‐NAME), an NO synthase inhibitor, on the disseminated intravascular coagulation (DIC)‐like phenomena in rats under environmental stress, induced by prolonged fluctuation in air temperature, known as SART (specific alternation of rhythm in temperature) stress.

Exposure of rats to SART stress for 7 days caused mild DIC‐like symptoms such as thrombocytopenia, hypofibrinogenemia, decreased factor VIII: coagulant activity and shortened euglobulin clot lysis time (ECLT). The enhanced fibrinolysis was accompanied by a marked decrease in the activity of plasma PAI.

L‐NAME, but not its D‐enantiomer, when administered orally at 0.3–10 mg kg−1, twice a day for 7‐day exposure to stress, inhibited the stress‐induced decrease in fibrinogen levels in a dose‐dependent manner, whereas it failed to alter platelet count, factor VIII:coagulant activity and plasma protein levels in stressed rats. All these parameters in unstressed rats were resistant to L‐NAME at 10 mg kg−1.

Repeated treatment with 10 mg kg−1 of L‐NAME blocked the shortening of ECLT and the decrease in PAI activity following stress exposure, although it was without effect in unstressed rats.

The inhibitory effects of L‐NAME at 10 mg kg−1 on the stress‐induced alterations in fibrinogen levels and in ECLT were significantly reduced by coadministered L‐arginine at 1000 mg kg−1.

These findings demonstrate that repeated administration of L‐NAME attenuates the enhanced fibrinolysis, without aggravating thrombocytopenia, in SART‐stressed rats. Endogenous NO appears to contribute to the stress‐induced development of fibrinolysis by suppressing plasma PAI activity, most probably as a result of inhibition of the PAI release from platelets.
Interaction of biphenylimidazole and imidazoleacrylic acid nonpeptide antagonists with valine 108 in TM III of the AT1 angiotensin receptor
Authors: Nirula Vaneet , Zheng Wei , Sothinathan Renuka , Sandberg Kathryn
Published: July 2012

Interspecies amino acid exchange, based on pharmacological differences between mammalian AT1 and amphibian xAT angiotensin II receptors, previously demonstrated that Val108 in transmembrane III (ValIII:08) is a critical structural requirement for binding the biphenylimidazole, losartan. Here, we investigated a series of biphenylimidazole and imidazoleacrylic acid nonpeptides to determine the general role of Val108 in nonpeptide recognition. Substitution of Val108 in the rAT1b receptor with He, the corresponding residue in xATa, significantly reduced ligand affinities from both nonpeptide classes (Fmut values (mutant IC50/rAT1bIC50): losartan > L‐162,389 > L‐162,313 > L‐162,017 = L‐163,491 > SB‐203,220 > SK&F‐108,566). While distinct molecular requirements exist for biphenylimidazole and imidazoleacrylic acid binding, these results suggest that Val108 is a common structural determinant of nonpeptide recognition on the AT1 receptor.
Tachykinin inhibition of acid‐induced gastric hyperaemia in the rat
Authors: Heinemann Akos , Jocic Milana , Herzeg Gemot , Holzer Peter
Published: July 2012

Primary afferent neurones releasing the vasodilator, calcitonin gene‐related peptide, mediate the gastric hyperaemic response to acid back‐diffusion. The tachykinins neurokinin A (NKA) and substance P (SP) are located in the same neurones and are co‐released with calcitonin gene‐related peptide. In this study we investigated the effect and possible role of tachykinins in the acid‐evoked gastric vasodilatation in urethane‐anaesthetized rats.

Gastric acid back‐diffusion, induced by perfusing the stomach with 15% ethanol in the presence of 0.05 m HCl, increased gastric mucosal blood flow by 60–90%, as determined by the hydrogen clearance technique. NKA and SP (0.14‐3.78 nmol min−1 kg−1, infused intra‐aortically) inhibited the gastric mucosal hyperaemic response to acid back‐diffusion in a dose‐dependent manner, an effect that was accompanied by aggravation of ethanol/acid‐induced macroscopic haemorrhagic lesions.

The inhibitory effect of NKA (1.26 nmol min−1 kg−1) on the acid‐induced gastric mucosal vasodilatation was prevented by the tachykinin NK2 receptor antagonist, MEN 10,627 (200 nmol kg−1) but left unaltered by the NK1 receptor antagonist, SR 140,333 (300 nmol kg−1) and the mast‐cell stabilizer, ketotifen (4.6 μmol kg−1).

Under basal conditions, with 0.05 m HCl being perfused through the stomach, NKA (1.26 nmol min−1 kg−1) reduced gastric mucosal blood flow by about 25%, an effect that was abolished by SR 140,333 but not MEN 10,627 or ketotifen.

SR 140,333, MEN 10,627 or ketotifen had no significant effect on basal gastric mucosal blood flow nor did they modify the gastric mucosal hyperaemic reaction to acid back‐diffusion.

The effect of NKA (1.26 nmol min−1 kg−1) in causing vasoconstriction and inhibiting the vasodilator response to acid back‐diffusion was also seen when blood flow in the left gastric artery was measured with the ultrasonic transit time shift technique.

Arginine vasopressin (AVP, 0.1 nmol min−1 kg−1) induced gastric mucosal vasoconstriction under basal conditions but was unable to inhibit the dilator response to acid back‐diffusion.

These data show that NKA has two fundamentally different effects on the gastric circulation. Firstly, NKA reduces gastric blood flow by activation of NK1 receptors. Secondly, NKA inhibits the gastric hyperaemic response to acid back‐diffusion through an NK2 receptor‐mediated mechanism. These two tachykinin effects appear to take place independently of each other since they are mediated by different receptors. This concept is further supported by the inability of AVP to mimic tachykinin inhibition of the gastric vasodilator response to acid back‐diffusion.
Actions of picrodendrin antagonists on dieldrin‐sensitive and ‐resistant Drosophila GABA receptors
Authors: Hosie Alastair M. , Ozoe Yoshihisa , Koike Kazuo , Ohmoto Taichi , Nikaido Tamotsu , Sattelle David B.
Published: July 2012

A series of terpenoid compounds, recently isolated from Picrodendron baccatum, share a picrotoxane skeleton with picrotoxinin, an antagonist of ionotropic GABA receptors. Referred to as picrodendrins, they inhibit the binding of [35S]‐tert‐butylbicyclophosphorothionate (TBPS) to rat GABAA receptors. Hitherto, their effects on GABA receptors have not been investigated electrophysiologically. Under two‐electrode voltage‐clamp, the actions of picrodendrins and related terpenoids have been assayed on homo‐oligomeric GABA receptors formed by the expression of a Drosophila GABA receptor subunit (RDLac) in Xenopus oocytes.

All the terpenoids tested, dose‐dependently antagonized currents induced by 30 μm (EC50) GABA.

Tutin and its analogues (dihydrotutin and isohyenanchin) differ in the structure of their axial C4 substituents. Of these compounds, tutin, which bears an isopropenyl group at this carbon atom, was the most potent antagonist of RDLac homo‐oligomers, whereas isohyenanchin, which bears a hydroxyisopropyl group, was the least potent antagonist tested.

Picrodendrins differ mainly in the structure of their C9 substituents. The IC50s of picrodendrins ranged from 17 ± 1.3 nM (picrodendrin‐Q) to 1006 ± 1.3 nM (picrodendrin‐O). As such, the most potent picrodendrins (Q, A and B) were approximately equipotent with picrotoxinin as antagonists of RDLac homo‐oligomers.

Certain picrodendrin compounds effected a use‐dependent blockade of RDLac homo‐oligomers. Such a biphasic block was not observed with tutin analogues.

Picrotoxin‐resistant RDLacA3025 homo‐oligomers, which have a single amino acid substitution (A302S) in the 2nd transmembrane region, were markedly less sensitive to picrodendrin‐O than the wild‐type, dieldrin‐sensitive, homo‐oligomers.

The relative potency of tutin analogues demonstrates that the structure‐activity relationship of the C4 substituent of picrotoxane‐based compounds is conserved in vertebrates and insects. However, the relative order of potency of picrodendrins on RDLac homo‐oligomers is distinctly different from that observed in previous radioligand binding studies performed on vertebrate GABAA receptors. As picrodendrin compounds differ in the structure of their C9 substituents, these data suggest that the optimal convulsant pharmacophores of vertebrate GABAA receptors and RDLac homo‐oligomers differ with respect to this substituent.
Agonist pharmacology of two Drosophila GABA receptor splice variants
Authors: Hosie Alastair M. , Sattelle David B.
Published: July 2012

The Drosophila melanogaster γ‐aminobutyric acid (GABA) receptor subunits, RDLac and DRC 17‐1‐2, form functional homo‐oligomeric receptors when heterologously expressed in Xenopus laevis oocytes. The subunits differ in only 17 amino acids, principally in regions of the N‐terminal domain which determine agonist pharmacology in vertebrate ionotropic neurotransmitter receptors. A range of conformationally restricted GABA analogues were tested on the two homo‐oligomers and their agonist pharmacology compared with that of insect and vertebrate iontropic GABA receptors.

The actions of GABA, isoguvacine and isonipecotic acid on RDLac and DRC 17‐1‐2 homo‐oligomers were compared, by use of two‐electrode voltage‐clamp. All three compounds were full agonists of both receptors, but were 4–6 fold less potent agonists of DRC 17‐1‐2 homo‐oligomers than of RDLac. However, the relative potencies of these agonists on each receptor were very similar.

A more complete agonist profile was established for RDLac homo‐oligomers. The most potent agonists of these receptors were GABA, muscimol and trans‐aminocrotonic acid (TACA), which were approximately equipotent. RDLac homo‐oligomers were fully activated by a range of GABA analogues, with the order of potency: GABA>ZAPA ((Z)‐3‐[(aminoiminomethyl)thio]prop‐2‐enoic acid) > isoguvacine > imidazole‐4‐acetic acid ≥ isonipecotic acid ≥ cis‐aminocrotonic acid (CACA) > β‐alanine. 3‐Aminopropane sulphonic acid (3‐APS), a partial agonist of RDLac homo‐oligomers, was the weakest agonist tested and 100 fold less potent than GABA.

SR95531, an antagonist of vertebrate GABAA receptors, competitively inhibited the GABA responses of RDLac homo‐oligomers, which have previously been found to be insensitive to bicuculline. However, its potency (IC50 500 μm was much reduced when compared to GABAA receptors.

The agonist pharmacology of Drosophila RDLac homo‐oligomers exhibits aspects of the characteristic pharmacology of certain native insect GABA receptors which distinguish them from vertebrate GABA receptors. The high potency and efficacy of isoguvacine and ZAPA distinguishes RDLac homo‐oligomers from bicuculline‐insensitive vertebrate GABAC receptors, while the low potency of SR95531 and 3‐APS distinguishes them from GABAA receptors. The differences in the potency of agonists on RDLac and DRC 17‐1‐2 homo‐oligomers observed in the present study may assist in identification of further molecular determinants of GABA receptor function.
Characterization of endothelin receptors in the human umbilical artery and vein
Authors: Bogoni G. , Rizzi A. , Calo G. , Campobasso C. , D'Orleans‐Juste P. , Regoli D.
Published: July 2012

The aim of the present study was to characterize pharmacologically endothelin receptors that are present in human umbilical vessels.

Endothelin‐1 (ET‐1) and endothelin‐2 (ET‐2) are potent stimulants of both the human umbilical artery (pEC50 7.9 and 7.5) and vein (pEC50 8.1 and 8.0). Endothelin‐3 (ET‐3) is inactive on the artery but contracts the vein (pEC50 7.6). IRL1620 is inactive in both vessels. The order of potency of agonists is suggestive of a typical ETA receptor in the artery (ET‐1 =ET‐2 > > ET‐3) and a mixture of ETA and ETB receptors in the vein (ET‐1 = ET‐2 ≥ ET‐3).

The selective ETA receptor antagonist, BQ123, competitively inhibits the effect of ET‐1 in the human umbilical artery (pA2 6.9), while in the vein, only a mixture of BQ123 and BQ788 (a selective ETB antagonist) weakly displaces to the right the cumulative concentration‐response curve to ET‐1. Contractions induced by ET‐3 in the vein are inhibited by BQ788 (pA2 7.6), but not by BQ123.

Inhibition of Ca2+ channels by nifedipine (0.1 μm) is accompanied by a significant decrease of the maximal response to ET‐1 by 40% in the artery and by 30% in the vein. The response of the vein to ET‐3 is almost abolished by nifedipine.

The results indicate that: (i) endothelins contract the human isolated umbilical artery via stimulation of an ETA receptor type; (ii) the contraction induced by ET‐1 in the vein is mediated by both ETA and ETB receptors, while ET‐3 stimulates the ETB receptor; (iii) the contribution of Ca2+ channels to the contraction mediated by the ETB receptor appears to be more important than to that mediated by the ETA receptor.
The involvement of sensory neuropeptides in toluene diisocyanate‐induced tracheal hyperreactivity in the mouse airways
Authors: Scheerens Heleen , Buckley Theresa L. , Muis Thea , Van Loveren Henk , Nijkamp Frans P.
Published: July 2012

Recently, we developed a murine model to investigate toluene diisocyanate (TDI)‐induced occupational asthma. After skin‐sensitization and intranasal challenge with TDI (1%) mice exhibited tracheal hyperreactivity 24 h after the challenge.

The aim of the present study was to investigate the possible role for sensory neuropeptides in the development of this tracheal hyperreactivity.

First, we demonstrated that direct application of TDI in vitro induced the release of tachykinins from the sensory nerves in the mouse isolated trachea. Second, capsaicin pretreatment, resulting in the depletion of sensory neuropeptides, completely abolished the TDI‐induced tracheal hyperreactivity 24 h after the challenge. Third, the selective neurokinin1 (NK1)‐receptor antagonist RP 67580 (0.2 μmol kg−1) also inhibited tracheal hyperreactivity when it was administered before the challenge. However, administration of RP 67580 during the sensitization phase did not result in a suppression of the TDI‐induced tracheal hyperreactivity 24 after the challenge.

When TDI‐sensitized mice were topically challenged with TDI a marked ear swelling response was observed. The cutaneous response after TDI application was not affected by capsaicin pretreatment or RP 67580 administration.

These results clearly show that sensory neuropeptides, particularly tachykinins, are essential for the development of TDI‐induced tracheal hyperreactivity during the effector phase. The differences between the airways and skin with respect to the sensory neuropeptides is intriguing and could suggest a local action for the tachykinins in the airways.
Investigation on colour, fastness properties and HPLC‐DAD analysis of silk fibres dyed withRubia tinctoriumL. andQuercus ithaburensisDecaisne
Authors: Deveoglu Ozan , Sahinbaskan Burcu Y , Torgan Emine , Karadag Recep
Published: July 2012

Silk fabric samples were dyed according to the various procedures using madder (Rubia tinctoriumL.) and walloon oak (Quercus ithaburensisDecaisne) extracts. The colour coordinates,K/S, as well as wash, light, rub and perspiration fastness values were determined. A reversed‐phase high‐performance liquid chromatography with diode‐array detection was utilised for the identification of the components of dyes present in the dyed fabrics and in the plant extracts.
Levels of macrophage migration inhibitory factor and glucocorticoids in chronic wound patients and their potential interactions with impaired wound endothelial progenitor cell migration
Authors: Grieb Gerrit , Simons David , Eckert Lena , Hemmrich Melanie , Steffens Guy , Bernhagen Jürgen , Pallua Norbert
Published: July 2012

Macrophage migration inhibitory factor (MIF), a structurally and functionally unique pleiotropic mediator in inflammation and immune processes, was identified decades ago. There is now strong evidence that MIF promotes revascularization and is involved in wound healing processes. However, its exact role in wound healing is still a matter of debate. A cohort of 33 patients was recruited, including 14 patients with acute and 19 patients with chronic wounds. Both serum and wound fluid samples were collected from each patient, and MIF and cortisol concentrations were determined. To functionally underscore MIF's potential role in wound revascularization, a chemotaxis assay was adapted to test whether and to what extent serum samples and wound fluids of each group promote the chemotactic migration of endothelial progenitor cells (EPCs). MIF serum levels were significantly higher in chronic wound patients than in acute wound patients. Wound exudates of chronic wounds, however, contained a significantly lower concentration of MIF. In chronic wound patients, EPC migration might be delayed, as suggested by in vitro chemotaxis experiments. Despite the overall descriptive nature of this study, we conclude that MIF is correlated with occurrence of chronic wound. The increased MIF levels in the serum of chronic wound patients might be due to MIF's systemic effect of its proinflammatory activities, while its locally decreased levels in chronic wound exudates might be responsible for impaired recruitment of EPCs. Additional prospective data and detailed in vivo models are needed for a more comprehensive understanding of the role of MIF in chronic wound healing.