bluuebunny/crossref_metadata_2025 huggingface dataset:
Species specificity in the blood cholesterol‐lowering effect of YM‐16638
Authors: Goto Shoichiro , Shimokawa Teruhiko , Ugawa Toru , Hisamichi Nami , Masuyama Youichi , Iizumi Yuichi , Sato Noboru , Takenaka Toichi , Kodama Tatsuhiki
Published: July 2012

. The compound YM‐16638, [[5‐[[3‐(4‐acetyl‐3‐hydroxy‐2‐propylphenoxy)propyl]thio]‐1,3,4‐thiadiazol‐ 2‐yl]thio] acetic acid was developed in a series of in vitro and in vivo studies as a leukotriene D4 receptor antagonist.

. In a clinical trial as a leukotriene antagonist drug, this compound was found to have a potent serum cholesterol lowering effect in normolipidaemic healthy male volunteers.

. In the present study, we investigated the serum cholesterol lower effect of this compound in various species of experimental animals.

. Administration of YM‐16638 did not cause a significant decrease in serum total cholesterol (TC) in mice (up to 200 mg kg−1, body weight per day for 28 days), rats (200 mg kg−1 for 15 days) or rabbits (90 mg kg−1 for 18 days). In hamsters, administration of YM‐16638 orally or by peritoneal injection at 50 mg kg−1 or more daily for 7 days caused a significant decrease in serum TC and the rate of body weight gain. In monkeys, serum TC did not change in YM‐16638‐administered squirrel monkeys (50 mg kg−1 daily for 3 weeks), but a significant decrease in serum TC was observed in cynomolgus monkeys (33% decrease at 30 mg kg−1 for 4 weeks) and rhesus monkeys (27% decrease at 30 mg kg−1 for 3 weeks) without any serious decrease in body weight. These results were consistent with those in a phase I study in human subjects. In contrast, serum alanine aminotransferase (ALT) level decreased in all animals after YM‐16638 treatment.

. From these results, we conclude that YM‐16638 has a potent hypocholesterolaemic effect, but that this effect if species‐specific and is only recognized clearly in human subjects and old‐world monkeys.
Effect of 1‐aminocyclopropanecarboxylic acid on N‐methyl‐d‐aspartate‐stimulated [3H]‐noradrenaline release in rat hippocampal synaptosomes
Authors: Clos M.V. , Sanz A. Garcia , Trullas R. , Badia A.
Published: July 2012

The effect of 1‐aminocyclopropanecarboxylic acid (ACPC), a partial agonist at the glycine site of the N‐methyl‐d‐aspartate (NMDA) receptor complex that exhibits neuroprotective, anxiolytic and antidepressant‐like actions, was investigated in a functional assay for presynaptic NMDA receptors.

NMDA (100 μm) produced a 36% increase of tritium efflux above basal efflux in rat hippocampal synaptosomes preincubated with [3H]‐noradrenaline ([3H]‐NA), reflecting a release of tritiated noradrenaline. This effect was prevented by 10 μm 7‐chlorokynurenic acid, an antagonist of the glycine site of the NMDA receptor.

Glycine enhanced the effect of NMDA with Emax and EC50 values of 84 ± 11% and 1.82 ± 0.04 μm, respectively. ACPC potentiated the effect of NMDA on tritium overflow with a lower EC50 (43 ± 6 nM) and a lower maximal effect (Emax = 40 ± 9%) than glycine. Furthermore, ACPC (0.1 μm) shifted the EC50 of glycine from 1.82 μm to ≥ 3 mM.

These results show that ACPC can reduce the potentiation by glycine of NMDA‐evoked [3H]‐NA release and hence, may act as an antagonist at the glycine site of presynaptic hippocampal NMDA receptors when the concentration of glycine is high.
Characterization of nitric oxide synthase activity in sheep urinary tract: functional implications
Authors: García‐Pascual A. , Costa G. , Labadia A. , Persson K. , Triguero D.
Published: July 2012

To define further the role of nitric oxide (NO) in urinary tract function, we have measured the presence of nitric oxide synthase (NOS) activity, and its relationship with functional NO‐mediated responses to electrical field stimulation (EFS) in the urethra, the detrusor and the ureter from sheep. NOS activity was assayed by the conversion of L‐[14C]‐arginine to L‐[14C]‐citrulline. Endogenous production of citrulline was confirmed by thin layer chromatography.

NOS enzymatic activity was detected in the cytosolic fraction from tissue homogenates with the following regional distribution (pmol citrulline mg−1 protein min−1): urethra (33 ± 3.3), detrusor (13.1 ± 1.1) and ureter (1.5 ± 0.2). No activity was detected in the particulate fraction of any region.

NOS activity was dependent on Ca2+‐calmodulin and required exogenously added NADPH and tetrahydrobyopterin (BH4) for maximal activity. Exclusion of calmodulin from the incubation mixture did not modify NOS activity, but it was significantly reduced in the presence of the calmodulin antagonist, calmidazolium, suggesting the presence of enough endogenous calmodulin to sustain the observed NOS activity.

NOS activity was inhibited to a greater extent by NG‐nitro‐l‐arginine (l‐NOARG) and its methyl ester (l‐NAME) than by NG‐monomethyl‐l‐arginine (l‐NMMA), while 7‐nitroindazole (7‐NI) was a weak inhibitor and L‐cannavine had no effect.

Citrulline formation could be inhibited by superoxide dismutase in an oxyhaemoglobin‐sensitive manner, suggesting feedback inhibition of NOS by NO.

EFS induced prominent NO‐mediated relaxations in the urethra while minor or no responses were observed in the detrusor and the ureter, respectively. Urethral relaxations to EFS were inhibited by NOS inhibitors with the rank order of potency: L‐NOARG = L‐NAME > 7‐NI > L‐NMMA.

In conclusion, we have demonstrated the presence of NO‐synthesizing enzymatic activity in the sheep urinary tract which shows similar characteristics to the constitutive NOS isoform found in brain. We suggest that the enzymatic activity measured in the urethral muscle layer may account for the NO‐mediated urethral relaxation during micturition whereas regulation of detrusor and ureteral motor function by NOS containing nerves is less likely.
Antiproliferative and c‐myc mRNA suppressive effects of tranilast on newborn human vascular smooth muscle cells in culture
Authors: Miyazawa Keiji , Hamano Shuichiro , Ujiie Arao
Published: July 2012

Newborn human vascular smooth muscle cells (VSMCs) proliferated faster and were more sensitive to platelet‐derived growth factor‐BB (PDGF‐BB) than those from adults. In this study, we investigated mechanism of the inhibitory effect of tranilast on PDGF‐BB‐induced proliferation of VSMCs from newborns.

Tranilast (30–300 μm) concentration‐dependently inhibited the VSMC proliferation in randomly growing cultures stimulated with PDGF‐BB.

Tranilast (30–300 μm) concentration‐dependently inhibited the [3H]‐thymidine incorporation into DNA in VSMCs that had been synchronized by 48 h serum depletion and then stimulated by addition of PDGF‐BB. However, tranilast had little influence on unscheduled DNA synthesis in quiescent cells or on RNA and protein synthesis, unlike aphidicolin, actimomycin D, and cycloheximide.

In synchronized VSMC cultures, tranilast still inhibited the PDGF‐BB‐induced DNA synthesis even when added 18 h after stimulation of the quiescent cells. The mode of the antiproliferative action of tranilast was different from that of NiCl2, genistein, or staurosporin. In addition, flow cytometry of synchronized VSMCs treated with tranilast revealed a blockade of PDGF‐inducible cell‐cycle progression at the G1/S checkpoint.

Northern blotting showed that tranilast (30–300 μm) concentration‐dependently suppressed constitutive c‐myc mRNA expression even when added 18 h after PDGF‐BB‐stimulation of quiescent VSMCs. Tranilast still had an inhibitory effect on the induction of c‐myc mRNA when de novo protein synthesis was inhibited by cycloheximide and did not shorten the degradation of c‐myc mRNA at the post‐transcriptional level, demonstrating that tranilast directly inhibited c‐myc mRNA expression at the transcriptional level.

These results suggest that the inhibitory effect of tranilast on PDGF‐BB‐induced proliferation is due to S‐phase blockade and may be, at least in part, involved in the direct suppression of c‐myc gene expression. Tranilast did not cause cell toxicity and may therefore hold promising potential for the prevention of vascular proliferative diseases.
Reversal by naloxone of the spinal antinociceptive actions of a systemically‐administered NSAID
Authors: Herrero Juan F. , Headley P. Max
Published: July 2012

Possible interactions between non‐steroidal anti‐inflammatory drugs (NSAIDs) and endogenous opioids were examined in electrophysiological experiments in α‐chloralose anaesthetized spinalized rats without or with carrageenan‐induced acute inflammation of one hindpaw. Spinal reflex responses, monitored as single motor unit discharges, were elicited by noxious pinch and electrical stimuli.

The μ‐opioid agonist, fentanyl, was an effective depressant of reflexes under all conditions (ED50 6–14 μg kg−1, i.v.). In rats without peripheral inflammation the NSAID, flunixin, a niflumic acid derivative, had only a small effect that was not dose‐dependent. However, in animals with unilateral inflammation, flunixin reduced spinal reflexes evoked both by noxious pinch stimuli (that activate peripheral nociceptors; ID50 4 mg kg−1, i.v.) and by electrical stimuli (that bypass nociceptor endings; ID50 6.5–11 mg kg−1, i.v.), indicating that it has a central site of action at doses comparable to those used clinically.

The opioid antagonist, naloxone (1 mg kg−1, i.v.), reversed all actions of fentanyl. It did not reverse the small effects that flunixin had in rats without inflammation, showing that the NSAID is not a direct opioid agonist. In rats with carrageenan‐induced inflammation of the hindpaw, however, naloxone fully reversed or prevented the antinociception by flunixin, but not that by the α2‐adrenoceptor agonist, medetomidine.

We conclude that under conditions of peripheral inflammation and the resultant central changes, the NSAID, flunixin, has antinociceptive actions that are mediated by endogenous opioids acting within the spinal cord.
Tyrphostin inhibition of ATP‐stimulated DNA synthesis, cell proliferation and Fos‐protein expression in vascular smooth muscle cells
Authors: Erlinge David , Heilig Markus , Edvinsson Lars
Published: July 2012

We and others have shown that extracellular ATP (adenosine triphosphate), released from sympathetic nerves and platelets, stimulates growth of vascular smooth muscle cells (SMC). To study the importance of tyrosine kinases for ATP‐mediated proliferation in vascular smooth muscle cells we used tyrphostins, a recently developed group of highly specific inhibitors of tyrosine kinases.

ATP induced a powerful concentration‐dependent increase in DNA synthesis measured by [3H]‐thymidine incorporation in rat aorta SMC (RASMC) and an increase in total cell number after 72 h of incubation as measured by an enzymatic cell proliferation assay. Tyrphostin 25 (10−5 m) had no effect per se on basal DNA synthesis but reduced ATP‐stimulated DNA synthesis and increase in cell number in a dose‐dependent manner. Higher concentrations of ATP could not reverse the inhibitory effect of tyrphostin 25. The potency of several (six) other tyrphostins was also examined and found to be slightly greater than tyrphostin 25 with equal efficacy.

When RASMC were incubated with 10−5 m ATP for 2 h, nearly all of the cells (87±5%) were intensely stained with an antibody to the Fos protein while in the controls only 1±2% of the cells were weakly stained. Tyrphostin 25 greatly reduced the Fos‐protein staining (14±2%).

ATP induced a concentration‐dependent increase in 45Ca2+‐influx and formation of inositol phosphates (IPtotal) in RASMC. These effects were not inhibited by tyrphostin 25.

Tyrphostin 25 did not alter ATP‐induced contraction in ring segments of rat aorta.

In conclusion, tyrphostin 25 inhibited ATP‐induced DNA synthesis, cell proliferation and Fosprotein expression, but not ATP‐induced 45Ca2+‐influx, inositolphosphate‐production or vasoconstriction. This indicates that the mitogenic effect of ATP on vascular smooth muscle cells is dependent on tyrosine kinases in contrast to the contractile effect of ATP in blood vessels.
Regulation of histamine H1 receptor coupling by dexamethasone in human cultured airway smooth muscle
Authors: Hardy Elaine , Farahani Mosavar , Hall Ian P.
Published: July 2012

The regulation of histamine‐induced [3H]‐inositol phosphate and intracellular calcium responses in human cultured airway smooth muscle cells was studied.

Histamine induced concentration‐dependent [3H]‐inositol phosphate formation (EC50 4 μm). This response was inhibited by a range of selective H1 receptor antagonists but not by the H2‐selective antagonist, tiotidone or the H3 receptor‐selective antagonist, thioperamide, indicating that an H1 receptor is involved in this response in human cultured airway smooth muscle cells.

Preincubation of human cultured airway smooth muscle cells with concentrations of dexamethasone > 10 nM for 22 h produced concentration‐dependent inhibition of histamine‐induced inositol phosphate formation. The maximum inhibition observed was 45% of the response in control cells. The inhibitory effect of dexamethasone was itself reversed by prior exposure to the glucocorticoid receptor antagonist, RU38486 (10 μm). Preincubation for 22 h with 1 μm dexamethasone produced inhibition of the inositol phosphate response to histamine at all concentrations of histamine inducing significant inositol phosphate formation in these cells. In contrast, the response to the G protein activator, NaF (0.1–20 mM) was unaltered by preincubation with dexamethasone.

Preincubation of human airway smooth muscle cells with 1 μm dexamethasone for time periods of < 6 h failed to inhibit histamine‐induced inositol phosphate formation in human airway smooth muscle cells.

Histamine also induced concentration‐dependent elevation of intracellular calcium levels in Fura 2‐loaded human airway smooth muscle cells. This response was inhibited by preincubation with 1 μm dexamethasone.

We conclude that signal transduction through the H1 receptor in human airway smooth muscle is subject to regulation by dexamethasone and that this may in part account for the protective effect of dexamethasone against spasmogen‐induced contractile responses in the airways.
Inhibition of tachykinin release from peripheral endings of sensory nerves by nociceptin, a novel opioid peptide
Authors: Giuliani Sandro , Maggi Carlo Alberto
Published: July 2012

The novel heptadecapeptide opioid, nociceptin, produced a concentration‐dependent (EC50 28 nM) suppression of the inotropic response of the guinea‐pig isolated renal pelvis to electrical stimulation, a response mediated by release of tachykinins from sensory nerves. Nociceptin did not affect the response to neurokinin A, indicating a prejunctional site of action on tachykininergic nerves. The effect of nociceptin was unchanged in the presence of the μ, δ and ***k opioid receptor antagonists, naloxone, naltrindole and nor‐binaltorphimine.
Electrophysiological mechanisms for antiarrhythmic efficacy and positive inotropy of liriodenine, a natural aporphine alkaloid from Fissistigma glaucescens
Authors: Chang Gwo‐Jyh , Wu Mei‐Hwan , Wu Yang‐Chang , Su Ming‐Jai
Published: July 2012

. The antiarrhythmic potential and electromechanical effects of liriodenine, an aporphine alkaloid isolated from the plant, Fissistigma glaucescens, were examined.

. In the Langendorff perfused (with constant pressure) rat heart, at a concentration of 0.3 to 3 μm, liriodenine was able to convert a polymorphic ventricular tachyrhythmia induced by the ischaemia‐reperfusion (EC50 = 0.3 μm).

. In isolated atrial and ventricular muscle, liriodenine increased the contractile force and slowed the spontaneous beating of the right atrium.

. The liriodenine‐induced positive inotropy was markedly attenuated by a transient outward K+ channel blocker, 4‐aminopyridine (4‐AP) but was not significantly affected by prazosin, propranolol, verapamil or carbachol.

. In rat isolated ventricular myocytes, liriodenine prolonged action potential duration and decreased the maximal upstroke velocity of phase 0 depolarization (V̇max) and resting membrane potential in a concentration‐dependent manner. The action potential amplitude was not significantly changed.

. Whole‐cell voltage clamp study revealed that liriodenine blocked the Na+ channel (INa) concentration‐dependently (IC50 = 0.7 μm) and caused a leftward shift of its steady‐state inactivation curve. However, its recovery rate from the inactivated state was not affected. The L‐type Ca2+ currents (ICa) were also decreased, but to a lesser degree (IC50 = 2.5 μm, maximal inhibition = 35%).

. Liriodenine inhibited the 4‐AP‐sensitive transient outward current (Ito) (IC50 = 2.8 μm) and moderately accelerated its rate of decay. The block of Ito was not associated with changes in the voltage‐dependence of the steady‐state inactivation curve or in the process of recovery from inactivation of the current. Liriodenine also reduced the amplitude of a slowly inactivating, steady‐state outward current (Iss) (IC50 = 1.9 μm). These effects were consistent with its prolonging effect on action potential duration. The inwardly rectifying background K+ current (IK***1), was also decreased but to a less degree.

. Compared to quinidine, liriodenine exerted a stronger degree of block on INa, comparable degree of block on IK1, and lesser extent of block on ICa and Ito.

. It is concluded that, through inhibition of Na+ and the Ito channel, liriodenine can suppress ventricular arrhythmias induced by myocardial ischaemia reperfusion. The positive inotropic effect can be explained by inhibition of the Ito channel and the subsequent prolongation of action potential duration. These results provide a satisfactory therapeutic potential for the treatment of cardiac arrhythmias.
Differential effects of angiostatic steroids and dexamethasone on angiogenesis and cytokine levels in rat sponge implants
Authors: Hori Yozo , Hu De‐En , Yasui Kiyoshi , Smither Rachel L. , Gresham G. Austin , Fan Tai‐Ping D.
Published: July 2012

. Subcutaneous implantation of sterile polyether sponges elicited a reproducible neovascular response in rats, as determined by blood flow measurement with a 133Xe clearance technique and confirmed histologically. This model was used to monitor the levels of two cytokines during angiogenesis and to compare the activities of angiostatic steroids and anti‐inflammatory steroids.

. Initial experiments followed the neovascular development over a 20‐day period. Daily local injection of hydrocortisone caused a dose‐dependent (0.5, 5 and 50 μg per sponge) inhibition of the basal sponge‐induced angiogenesis. However, daily systemic treatment of hydrocortisone (2, 10 and 50 mg kg−1, s.c.) was less effective at inhibiting angiogenesis, and this inhibition was not sustained by day 20 after sponge implantation.

. To investigate the involvement of cytokines during the course of angiogenesis, we measured the endogenous levels of tumour necrosis factor‐α (TNF‐α) and interleukin 6 (IL‐6) in sponge implants. Levels of IL‐6 and TNF‐α peaked at day 7 and day 11 after implantation, respectively. These cytokine levels subsided through the completion of angiogenesis by day 20.

. Subsequent experiments were carried out over a 14‐day period. Among the three angiostatic steroids tested, U‐24067 (6α‐fluoro‐17,21‐dihydroxy‐16α‐methylpregna‐4,9(11)‐diene‐3,20‐dione‐21‐acetate) showed a dose‐dependent inhibition (0.5, 5 and 50 μg per sponge per day) of sponge‐induced angiogenesis. Tetrahydro‐S was also effective at 5 μg doses, but medroxyprogesterone failed to affect the angiogenic response. None of these steroids caused atrophies of the spleen and thymus.

. Daily local injection of dexamethasone (0.5 μg per sponge) inhibited the basal sponge‐induced angiogenesis almost completely. Although higher doses of dexamethasone (5 and 50 μg per sponge) did not produce further inhibition of angiogenesis, they caused severe spleen and thymus weight losses, indicative of immunosuppression.

. At the daily dose of 5 μg per sponge, dexamethasone inhibited angiogenesis and produced a marked reduction in the levels of TNF‐α and IL‐6 at day 14. In contrast, hydrocortisone, U‐24067 and tetrahydro‐S did not influence the levels of TNF‐α and IL‐6.

. We concluded that the anti‐angiogenic activity of angiostatic steroids and anti‐inflammatory steroids in the rat sponge model is independent of their ability to reduce the production of TNF‐α and IL‐6. The differential effects of angiostatic and anti‐inflammatory steroids suggest that U‐24067 and its derivatives may have therapeutic potential in the management of angiogenic diseases such as rheumatoid arthritis.
Effects of theophylline and rolipram on leukotriene C4 (LTC4) synthesis and chemotaxis of human eosinophils from normal and atopic subjects
Authors: Tenor Hermann , Hatzelmann Armin , Church Martin K. , Schudt Christian , Shute Janis K.
Published: July 2012

. The effects of the non‐selective phosphodiesterase (PDE) inhibitor theophylline and the selective PDE4 inhibitor rolipram on leukotriene C4 (LTC4) synthesis and chemotaxis of complement 5a (C5a)‐and platelet‐activating factor (PAF)‐stimulated human eosinophils obtained from normal and atopic donors were investigated.

. Eosinophils were purified from peripheral venous blood of normal and atopic subjects by an immunomagnetic procedure to a purity > 99%. Eosinophils were stimulated with PAF (0.1 μm) or C5a (0.1 μm) for 15 min and LTC4 was measured by radioimmunoassay (RIA). Eosinophil chemotaxis in response to PAF and C5a was assessed with 48‐well microchambers (Boyden).

. Under these conditions substantial amounts of LTC4 (about 300–1000 pg per 106 cells) were only detectable in the presence of indomethacin (0.1–10 μm). To explain this finding it was hypothesized that indomethacin reversed the inhibition of LTC4 synthesis by endogenously synthesized prostaglandins, in particular prostaglandin E2 (PGE2). In fact, eosinophils release 23 pg PGE2 per 106 cells following PAF stimulation; this PGE2 synthesis was completely inhibited by indomethacin and readdition of PGE2 inhibited eosinophil LTC4 synthesis (IC50 = 3 nM). The following experiments were performed in the presence of 10 μm indomethacin.

. Theophylline (IC50∼50 μm) and rolipram (IC50∼ 0.03‐0.2 μm) suppressed PAF‐ and C5a‐stimulated LTC4 synthesis. This PDE inhibitor‐induced suppression of LTC4 generation is mediated by activation of protein kinase A, since it was reversed by the protein kinase A inhibitor Rp‐8‐Br‐cyclic AMPS. In addition, exogenous arachidonic acid concentration‐dependently (0.3 μm‐3 μm) reversed the inhibition of LTC4 synthesis by the PDE inhibitors, indicating that theophylline and rolipram suppress the mobilization of arachidonic acid. The β‐adrenoceptor agonist salbutamol inhibited eosinophil LTC4 synthesis (IC50 = 0.08 μm). The combination of salbutamol with theophylline (10 μm) or rolipram (3 nM) appeared to be additive.

. Theophylline (IC50∼40 μm), rolipram (IC50∼0.02 μm [C5a], ∼0.6 μm [PAF]) and PGE2 (IC50∼3 nM) inhibited C5a‐ and PAF‐stimulated eosinophil chemotaxis. The combination of PGE2 with theophylline resulted in an additive effect.

. Both C5a‐ and PAF‐stimulated eosinophil chemotaxis and LTC4 generation were significantly elevated in eosinophils from atopic individuals compared to normal subjects. However, eosinophils from normal and atopic individuals were not different with respect to their total cyclic AMP‐PDE and PDE4 isoenzyme activities as well as the potencies of theophylline and rolipram to suppress LTC4 generation and chemotaxis.
Effects of protein tyrosine kinase inhibitors on cytokine‐induced adhesion molecule expression by human umbilical vein endothelial cells
Authors: May Michael J. , Wheeler‐Jones Caroline P.D. , Pearson Jeremy D.
Published: July 2012

. Endothelial cells can be stimulated by the pro‐inflammatory cytokines interleukin (IL)‐1α and tumour necrosis factor (TNF)α to express the leukocyte adhesion molecules E‐selectin, vascular cell adhesion molecule (VCAM)‐1 and intercellular adhesion molecule (ICAM)‐1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine‐activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate.

. Maximal E‐selectin expression induced by incubation of HUVEC for 4 h with IL‐1α (100 u ml−1) and TNFα (100 u ml−1) was dose‐dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12‐myristate, 13‐acetate (PMA)‐induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31‐7549 or Ro31‐8220 did not affect IL‐1α‐ or TNFα‐induced E‐selectin expression at concentrations which maximally inhibited PMA‐induced expression.

. Genistein inhibited VCAM‐1 expression induced by incubation of HUVEC for 24 h with TNFα or IL‐1α whereas it did not affect ICAM‐1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM‐1 and ICAM‐1 expression induced by TNFα.

. Basal expression of E‐selectin, VCAM‐1 and ICAM‐1 was dose‐dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNFα‐induced expression of these molecules with maximal E‐selectin and ICAM‐1 expression being slightly enhanced and VCAM‐1 expression dose‐dependently reduced.

. We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre‐myeloid cell line U937 to TNFα‐stimulated HUVEC. Adhesion of U937 cells to HUVEC pretreated for 4 or 24 h with TNFα was dose‐dependently inhibited by genistein and herbimycin A but unaffected by daidzein. Adhesion of U937 cells after 4 h was partially inhibited by blocking antibodies against both E‐selectin and VCAM‐1 but after 24 h was only inhibited by anti‐VCAM‐1.

. Sodium orthovanadate had no effect on TNFα‐induced U937 adhesion but dose‐dependently enhanced adhesion to unstimulated HUVEC. Vanadate‐induced adhesion was inhibited by an antibody against VCAM‐1.

. These results demonstrate that PTK‐mediated phosphorylation events are important for the regulation of adhesion molecule expression by human endothelial cells, and additionally show that PTK inhibitors differentially affect upregulation of different adhesion molecules, implicating divergent regulatory pathways for cytokine‐induced adhesion molecule expression.
Cardiac and gastric effects of histamine H2 receptor antagonists: no evidence for a correlation between lipophilicity and receptor affinity
Authors: Coruzzi G. , Adami M. , Pozzoli C. , Giorgi F. , Bertaccini G.
Published: July 2012

. A series of histamine H2 receptor antagonists with different lipophilicity were tested in cardiac and gastric assays in order to reveal possible differences in receptor affinity. Lipophilicity of the compounds was expressed as CLOG P (theoretically‐determined logarithm of octanol:water partition coefficient) and log k' (logarithm of capacity factor, experimentally‐determined by reverse‐phase high performance liquid chromatography).

. Aminopotentidine (APT) and iodoaminopotentidine (I‐APT), which are both lipophilic compounds, behaved as insurmountable antagonists of histamine responses in rat isolated gastric fundus (pKB = 6.20 ± 0.16 and 6.89 ± 0.19, respectively) and guinea‐pig isolated papillary muscle (pKB = 6.34 ± 0.37 and 6.81 ± 0.26, respectively). They were approximately as effective as ranitidine (RAN) in reducing histamine‐induced acid secretion in the anaesthetized rat, ID50 values being 0.018 ± 0.02, 0.020 ± 0.03 and 0.036 ± 0.01 μmol kg−1 i.v. for APT, I‐APT and RAN, respectively. Both APT and I‐APT had a significantly longer duration of action than RAN.

. The hydrophilic compound, SK&F 92857, was inactive up to 10 μm in modifying histamine‐induced acid secretion in the isolated rat stomach. In the papillary muscle, low concentrations (0.1‐1 μm) of this compound produced a competitive antagonism of the histamine responses (pA2 value = 7.38 ± 0.11), while a higher concentration (10 μm) significantly reduced the maximal response to histamine.

. RAN competitively antagonized histamine effects with a comparable affinity in cardiac and gastric preparations (pA2 values were 6.42 ± 0.09 and 6.78 ± 0.38 in heart and stomach, respectively).

. Results obtained in this study clearly showed that the discrepancies between gastric and cardiac effects observed for some H2 antagonists are not explained solely by differences in lipophilicity of compounds. Moreover, the significant correlation found between CLOG P and log k' parameter, which takes into account, besides their lipophilicity, the ionization of the molecules, suggests that ionization has a similar influence for all the molecules on the partition between the lipophilic and aqueous phase.
Influence of aminoguanidine and the endothelin antagonist, SB 209670, on the regional haemodynamic effects of endotoxaemia in conscious rats
Authors: Gardiner S.M. , Kemp P.A. , March J.E. , Bennett T.
Published: July 2012

. We compared the regional haemodynamic responses to lipopolysaccharide (LPS; 150 μg kg−1 h−1, i.v.) in the presence of saline, aminoguanidine (AG; 45 mg kg−1 bolus, 45 mg kg−1 h−1 infusion), or AG and the non‐selective endothelin receptor antagonist, SB 209670 (600 μg kg−1 h−1), in conscious, chronically instrumented, Long Evans rats (350–450 g; n = 8 in all groups). We used AG because there is evidence that it is a selective inhibitor of inducible nitric oxide synthase (iNOS), although recently it has been claimed AG also inhibits constitutive NOS.

. Infusion of LPS in the presence of saline caused an early, transient hypotension (1–2 h) and a renal vasodilatation, with a secondary, delayed fall in mean arterial blood pressure (MAP), progressive tachycardia, and renal and hindquarters vasodilatation.

. AG alone caused a rapid (within 30 s) transient rise in MAP (Δ 27 ± 3 mmHg), accompanied by tachycardia and regional vasoconstrictions, but no reduction in regional flows, indicating the pressor effect of AG was, probably, largely due to an increase in cardiac output. These effects are not consistent with AG inhibiting constitutive NOS. In the presence of AG, LPS still caused an early, transient fall in MAP accompanied by a renal vasodilatation, but thereafter there was a significant rise in MAP (17 ± 3 mmHg, 3 h after onset of LPS infusion) accompanied by bradycardia and marked mesenteric and hindquarters vasoconstrictions. However, 23 h after the onset of co‐infusion of AG and LPS all variables were not different from baseline, except heart rate and renal vascular conductance, which were increased.

. In the presence of AG and SB 209670, LPS caused progressive hypotension and increases in renal, mesenteric and hindquarters vascular conductances. Hence, SB 209670 prevented the rise in MAP and the regional vasoconstrictions seen with AG and LPS, indicating an involvement of endothelin in these events.

. In the presence of AG and SB 209670, 23 h after the onset of LPS infusion, the AT1‐receptor antagonist, losartan (10 mg kg−1), and the V1‐receptor antagonist, d(CH2)5‐O‐Me‐Tyr‐AVP (10 μg kg−1, 10 μg kg−1 h−1) caused additional incremental falls in MAP and increases in renal, mesenteric and hindquarters vascular conductances. Under these circumstances, MAP was lower and regional vascular conductances higher than in the other experiments following administration of losartan and d(CH2)5‐O‐Me‐Tyr‐AVP. Thus, although the findings are consistent with AG inhibiting iNOS, thereby revealing the pressor and vasoconstrictor actions of endothelin released by LPS, it is clear that LPS activates a very powerful hypotensive/vasodilator mechanism(s) which is resistant to AG, and whose full influence is only unmasked when the actions of endothelin, angiotensin II and vasopressin are inhibited.
Regional differences in the arterial response to vasopressin: role of endothelial nitric oxide
Authors: García‐Villalón Angel Luis , García José Luis , Fernández Nuria , Monge Luis , Gómez Bernardino , Diéguez Godofredo
Published: July 2012

. The isometric response to arginine‐vasopressin (10−10‐10−7 m) was studied in 2 mm long rabbit arterial segments isolated from several vascular beds (cutaneous, pial, renal, coronary, muscular, mesenteric and pulmonary).

. Vasopressin induced contraction in central ear (cutaneous), basilar (pial), renal, coronary and saphenous (muscular) arteries, but had no effect in mesenteric and pulmonary arteries; the order of potency for the contraction was: ear > basilar > renal > coronary > saphenous arteries.

. Treatment with the blocker of nitric oxide synthesis NG‐nitro‐L‐arginine methyl ester (L‐NAME; 10−6‐10−4 m) increased significantly (P < 0.05) the contraction to vasopressin in ear (148% of control), basilar (150% of control), renal (304% of control), coronary (437% of control) and saphenous (235% of control) arteries. Removal of the endothelium increased significantly (P < 0.05) the contraction to vasopressin in basilar (138% of control), renal (253% of control), coronary (637% of control) and saphenous (662% of control) arteries, but not in ear artery. Mesenteric and pulmonary arteries in the presence of L‐NAME or after endothelium removal did not respond to vasopressin, as occurred in control conditions.

. The specific antagonist for V1 vasopressin receptors d(CH2)5Tyr(Me)AVP (3 × 10−9‐10−7 m) was more potent (pA2 = 9.3–10.1) than the antagonist for both V1 and V2 vasopressin receptors desGly‐d(CH2)5‐D‐Tyr(Et)ValAVP (10−7‐10−6 m) (pA2 = 7.4–8.4) to block the contraction to vasopressin of ear, basilar, renal and coronary arteries.

. The specific V2 vasopressin agonist [deamino‐Cys1, D‐Arg8]‐ vasopressin (desmopressin) (10−10‐10−7 m) did not produce any effect in any of the arteries studied, with or without endothelium.

. In arteries precontracted with endothelin‐1, vasopressin or desmopressin did not produce relaxation.

. These results suggest: (a) most arterial beds studied (5 of 7) exhibit contraction to vasopressin with different intensity; (b) the vasoconstriction to this peptide is mediated mainly by stimulation of V1 vasopressin receptors, and (c) endothelial nitric oxide may inhibit the vasoconstriction to this peptide, especially in coronary and renal vasculatures.
EndothelinB (ETB) receptor‐activated potentiation of cholinergic nerve‐mediated contraction in human bronchus
Authors: Fernandes Lynette B. , Henry Peter J. , Rigby Paul J. , Goldie Roy G.
Published: July 2012

In human isolated bronchial preparations, the endothelinB (ETB) receptor‐selective agonist, sarafotoxin S6c (Stx6c; 1 nM) increased nerve‐mediated contraction in response to electrical field stimulation (EFS) at 0.5‐1 Hz from 19±4% to 42±7% (n = 9). This effect was blocked in the presence of the ETB receptor‐selective antagonist, BQ‐788 (10 μm). These data are consistent with findings in some animal species that ET‐1 and related peptides have marked neuromodulatory influences on the cholinergic system. Furthermore, they provide additional support for the concept that ET‐1 may have a mediator role in bronchial obstruction in asthma.
Modulation by atrial natriuretic factor of receptor‐mediated cyclic AMP‐dependent responses in canine pulmonary artery during heart failure
Authors: Mathew Rajamma , Omar Hatim A. , Fayngersh Raisa , Shen Weiqun , Wang Jie , Gewitz Michael H. , Hintze Thomas H. , Wolin Michael S.
Published: July 2012

Pacing‐induced congestive heart failure (CHF) in dogs is associated with increased plasma levels of atrial natriuretic factor (ANF) and inhibiton of receptor‐mediated cyclic AMP‐dependent relaxation in isolated pulmonary arteries (PA). Since ANF is known to be negatively coupled to adenylate cyclase, we studied cyclic AMP‐mediated relaxation to isoprenaline (Iso) and arachidonic acid (AA) in PA from control dogs (C), dogs with pacing‐induced CHF (CHF) and dogs with bilateral atrial appendectomy and CHF (ATR APP + CHF).

In CHF, plasma ANF levels increased from a baseline of 80±8 pg ml−1 to 283±64 pg ml−1 (P<0.05), but the ATR APP + CHF group failed to show this increase (67±7 pg ml−1 vs 94±15 pg ml−1, P = NS). Plasma ANF levels, however, did not influence myocardial dysfunction in CHF.

The relaxation of 49±5% to 1 μm Iso in C was reduced to 23±4% in CHF (P<0.05), but relaxation of 49±12% was observed in the ATR APP+CHF group (P = NS vs C). Relaxation responses to 10 μm AA were as follows: 77±5% (C, n = 8), 27±8% (CHF, n = 10, P<0.05 vs C), and 93±5% (ATR APP + CHF, n = 5). The presence of CHF, or the plasma ANF levels, did not affect responses to cyclic GMP‐mediated relaxing agents in PA.

These data indicate that the myocardial performance in CHF is not influenced by plasma ANF levels. However, altered cyclic AMP‐mediated relaxation in PA during CHF is, in part, modulated by circulating ANF levels.
Effect of Hoe 694, a novel Na+‐H+ exchange inhibitor, on intracellular pH regulation in the guinea‐pig ventricular myocyte
Authors: Loh Shih‐Hurng , Sun Bing , Vaughan‐Jones Richard D.
Published: July 2012

Hoe 694 (3‐methylsulphonyl‐4‐piperidinobenzoyl, guanidine hydrochloride) is a Na+/H+ exchange (NHE) inhibitor exhibiting cardioprotective properties during ischaemia and reperfusion in animal hearts. We have (i) tested the selectivity of Hoe 694 for NHE over other pHi‐regulating mechanisms in the myocardium, and (ii) tested if the functionally important NHE isoform contributing to intracellular pH regulation in heart is NHE‐1, as suggested from molecular biology studies of this protein.

pHi was recorded by fluorescence microscopy with carboxy SNARF‐1, AM‐loaded into single ventricular myocytes of guinea‐pig.

In nominally HCO3−‐free media, recovery of pHi from an intracellular acid load is mediated by NHE, and was inhibited by Hoe 694, amiloride (an NHE inhibitor) or dimethyl amiloride (DMA, a high affinity NHE inhibitor) with potency values of 2.05, 87.3 and 1.96 μm respectively, giving the potency series: Hoe 694 ⋍ DMA > > amiloride. This potency series, and the potency values (corrected for drug competition with extracellular Na+) match those determined previously for cloned NHE‐1 expressed in mutant fibroblasts. In the absence of extracellular Na+ (to inhibit NHE), Hoe 694 had no effect on pHi.

In 5% CO2/HCO3−‐buffered solution containing DMA, pHi recovery from acidosis is mediated by Na+‐HCO3− symport and was unaffected by Hoe 694. The drug also had no effect on pHi recovery from an alkali‐load, a process largely mediated by Cl−‐HCO3− exchange. Finally, the fall of pHi upon adding extracellular Na‐lactate is assisted by H+‐lactate symport, and this too was unaffected by Hoe 694.

We conclude (i) Hoe 694 has no detectable inhibitory potency for pH‐regulating carriers in heart other than NHE. (ii) native NHE functioning during pHi‐regulation in the cardiomyocyte is the NHE‐1 isoform. These data strengthen the case for NHE‐1 being the receptor for mediating the cardioprotective effects of Hoe 694.
Intranasal administration of neuropeptide Y in man: systemic absorption and functional effects
Authors: Lacroix J.S. , Ricchetti A.P. , Morel D. , Mossimann B. , Waeber B. , Grouzmann E.
Published: July 2012

Exogenous neuropeptide Y (NPY, 10 nmol, 50 nmol and 100 nmol) and its vehicle (NaCl 0.9%) were administered in a double blind, randomized and controlled manner by intranasal spray in 7 healthy volunteers. Variations of plasma NPY concentration over time were measured during 120 min. Forty min after the administration of 50 nmol and 100 nmol of exogenous NPY, plasma NPY increased from 5.5 ± 1.1 pM to 9.8 ± 2.3 pM (P < 0.05) and from 9.06 ± 5.1 pM to 20.8 ± 6.16 pM (P < 0.001), respectively. There was no significant modification of the mean arterial blood pressure and no subjective discomfort was reported.

Nasal airway resistance (NAR) was measured by anterior rhinomanometry and was reduced by 25 ± 3% and 32 ± 5% after the spray of 50 nmol and 100 nmol, respectively, for about 90 min.

Double‐blind, randomized, placebo‐controlled and 3‐way crossover design experiments were performed in 8 healthy volunteers to evaluate the influence of intranasal pretreatment with NPY (20 nmol) and the mixed α1/α2‐adrenoceptor agonist oxymetazoline (20 nmol) on the functional effects of subsequent local irritation evoked by capsaicin (3.3 × 10−4 mol). Subjective evaluation of NAR and local intensity of discomfort were evaluated by means of a visual analogue scale. Nasal secretions were collected and objective NAR was recorded by rhinomanometry.

Subjective NAR, nasal secretions and rhinomanometry recordings were not modified by intranasal application of saline, NPY or oxymetazoline. Subjective nasal obstruction, local discomfort, nasal secretions and NAR increase evoked by capsaicin were markedly reduced by NPY pretreatment (P < 0.05) when compared to saline or oxymetazoline.

It is concluded that intranasal application of exogenous NPY has very low systemic absorption but induced long lasting nasal vasoconstriction without cardiovascular effects. Pretreatment of the nasal mucosa with exogenous NPY reduces both secretagogue and vasodilator responses to subsequent application of capsaicin.
Characterization of [125I]‐endothelin‐1 and [125I]‐BQ3020 binding to rat cerebellar endothelin receptors
Authors: Widdowson P.S. , Kirk C.N.P.
Published: July 2012

We performed radioligand binding experiments on rat cerebellar homogenates using [125I]‐endothelin‐1 ([125I]‐ET‐1) and [125I]‐BQ3020 to examine the pharmacology of endothelin receptors in rat brain. Saturation experiments demonstrated a single population of binding sites with high affinity for both radioligands ([125I]‐ET‐1, pKd = 8.94 ± 0.17; [125I]‐BQ3020, pKd = 9.18 ± 0.14 nM; mean ± s.e.mean). However, [125I]‐BQ3020 only recognised approximately one third the number of endothelin receptors measured with [125I]‐ET‐1.

Saturation binding experiments with [125I]‐PD151242 revealed high affinity binding to a single population of ETA receptors in the cerebellar homogenates (pKd = 9.95 ± 0.14; Bmax = 30 ± 15 fmol mg−1 protein).

Competition experiments were performed with ligands that are either non‐selective or selective for endothelin receptor subtypes. The rat cerebellar endothelin receptor displayed a high affinity for endothelin‐1 (ET‐1), endothelin‐3 (ET‐3) and sarafotoxin‐S6c (STX‐6c) although the affinity for ET‐3 was slightly higher than the affinity for ET‐1 using both radioligands. The selective ETA antagonists, BQ123, BMS‐182,874 and JKC‐301 all displayed low affinities at the endothelin receptors. In contrast the selective ETB agonists, IRL1620 and [Ala1,3,11,15]ET‐1 and the selective ETB antagonist, BQ‐788 had moderate affinities at the endothelin receptor, in the low nanomolar range. The ETB agonist, BQ3020, had approximately 10 fold higher affinity than IRL1620 and [Ala1,3,11,15]ET‐1 at the rat cerebellar endothelin receptors. The non‐selective antagonists, Ro‐46,2005, Ro‐47,0203 and PD‐142,893 displayed moderate affinities at the cerebellar receptor.

Since [125I]‐BQ3020 recognises only a fraction of the [125I]‐ET‐1 binding sites, the majority of the endothelin receptors in the cerebellum cannot be classed as ETB. Although [125I]‐PD151242 was able to detect ETA receptors in the rat cerebellar homogenates, the small population of ETA receptors (2% of the total endothelin population as measured with [125I]‐ET‐1) could not account for the non‐ETB receptor population. We conclude that the rat brain cerebellar receptor has a profile similar to the ETB1 receptor as it has a high affinity for ET‐1, ET‐3, STX‐6c and was moderately sensitive to PD‐142,893. However, as the ETB ligands BQ‐788, IRL1620 and [Ala1,3,11,15]ET‐1 have only a moderate affinity for the rat cerebellar endothelin receptor and since ET‐3 has a higher affinity as compared to ET‐1, our findings suggest that the rat cerebellum contains predominately ETC receptors.
Rapid nitric oxide‐ and prostaglandin‐dependent release of calcitonin gene‐related peptide (CGRP) triggered by endotoxin in rat mesenteric arterial bed
Authors: Wang Xian , Wu Zhongxin , Tang Yueming , Fiscus Ronald R. , Han Chide
Published: July 2012

Our objective was to determine whether endotoxin (ETX) could directly trigger the release of calcitonin gene‐related peptide (CGRP) from perivascular sensory nerves in the isolated mesenteric arterial bed (MAB) of the rat and to determine whether nitric oxide (NO) and prostaglandins (PGs) are involved.

ETX caused time‐ and concentration‐dependent release of CGRP, and as much as a 17 fold increase in CGRP levels in the perfusate at 10–15 min after the administration of ETX (50 μg ml−1).

CGRP‐like immunoreactivity in the perfusate was shown to co‐elute with synthetic rat CGRP by reverse‐phase h.p.l.c.

Pretreatment of MAB with capsaicin or ruthenium red inhibited ETX‐induced CGRP release by 90% and 71%, respectively. ETX‐evoked CGRP release was decreased by 84% during Ca2+‐free perfusion.

The release of CGRP evoked by ETX was enhanced by L‐arginine by 43% and inhibited by Nω‐nitro‐L‐arginine (L‐NOARG) and methylene blue by 37% and 38%, respectively. L‐Arginine reversed the effect of L‐NOARG.

Indomethacin and ibuprofen also inhibited the ETX‐induced CGRP release by 34% and 44%, respectively. No additive inhibition could be found when L‐NOARG and indomethacin were concomitantly incubated.

The data suggest that ETX triggers the release of CGRP from capsaicin‐sensitive sensory nerves innervating blood vessels. The ETX‐induced CGRP release is dependent on extracellular Ca2+ influx and involves a ruthenium red‐sensitive mechanism. Both NO and PGs appear to be involved in the ETX‐induced release of CGRP in the rat mesenteric arterial bed.
Influence of superoxide dismutase inhibition on the discrimination between NO and the nitrergic neurotransmitter in the rat gastric fundus
Authors: Lefebvre R.A.
Published: July 2012

The influence of diethyldithiocarbamate (DETCA), that irreversibly inhibits Cu/Zn‐containing superoxide dismutase, on the inability of 6‐anilino‐5,8‐quinolinedione (LY83583), hypoxanthine/xanthine oxidase, hydroquinone and hydroxocobalamin to reduce electrically‐induced NANC relaxations in the rat gastric fundus was investigated.

Longitudinal muscle strips of the rat gastric fundus were mounted for auxotonic recording in the presence of atropine and guanethidine and tone was raised by administration of prostaglandin F2α. DETCA (3 × 10−3 m) slightly reduced the short‐lasting relaxations induced by 10−5 m exogenous nitric oxide (NO) and transmural electrical stimulation for 10 s at 4 Hz but this effect was not influenced by 1000 u ml−1 superoxide dismutase (SOD).

DETCA (3 × 10−5‐3 × 10−3 m) concentration‐dependently potentiated the inhibitory effect of LY83583 upon the electrically‐induced relaxations, although this was less pronounced than the inhibition of the NO‐induced relaxations. The inhibition of the electrically‐induced non‐adrenergic non‐cholinergic (NANC) relaxations was not reversed by SOD while that of the NO‐induced relaxations was partially reversed.

The inhibitory effect of hypoxanthine/xanthine oxidase, hydroquinone and hydroxocobalamin on the electrically‐induced NANC relaxations in the presence of DETCA (3 × 10−3 m) was not different from the inhibitory effect of DETCA alone.

It was concluded that the differentiating effect of LY83583 between exogenous NO and the endogenous nitrergic neurotransmitter is partially related to protection of the endogenous nitrergic neurotransmitter by high levels of intracellular superoxide dismutase. This mechanism does not hold for hydroquinone and hydroxocobalamin, as they still discriminate between exogenous NO and the endogenous nitrergic neurotransmitter in the presence of DETCA. The possibility that the endogenous nitrergic neurotransmitter is not free NO in the rat gastric fundus therefore remains open.