bluuebunny/crossref_metadata_2025 huggingface dataset:
Cigarette smoke‐inhibition of neurogenic bronchoconstriction in guinea‐pigs in vivo: involvement of exogenous and endogenous nitric oxide
Authors: Emms Joanne C. , Rogers Duncan F.
Published: January 2005

We investigated the effect of acute inhalation of cigarette smoke on subsequent non‐adrenergic, non‐cholinergic (NANC) neural bronchoconstriction in anaesthetized guinea‐pigs in vivo by use of pulmonary insufflation pressure (PIP) as an index of airway tone. The contribution of endogenous nitric oxide (NO) was investigated with the NO synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME). The contribution of plasma exudation to the response was investigated with Evans blue dye as a plasma marker.

Inhalation of 50 tidal volumes of cigarette smoke or air had no significant effect on baseline PIP. In the presence of propranolol and atropine (1 mg kg−1 each), electrical stimulation of the vagus nerves in animals given air 30 min previously induced a frequency‐dependent increase in PIP above sham stimulated controls (16 fold increase at 2.5 Hz, 24 fold increase at 10 Hz). In contrast, in smoke‐exposed animals, the increase in subsequent vagally‐induced PIP was markedly less than in the air controls (90% less at 2.5 Hz, 76% less at 10 Hz).

L‐NAME (10 mg kg−1), given 10 min before air or smoke, potentiated subsequent vagally‐induced (2.5 Hz) NANC bronchoconstriction by 338% in smoke‐exposed animals, but had no significant effect in air‐exposed animals. The inactive enantiomer D‐NAME (10 mg kg−1) had no effect, and the potentiation by L‐NAME was partially reversed by the NO‐precursor L‐arginine (100 mg kg−1). Vagal stimulation did not affect the magnitude of vagally‐induced bronchoconstriction 30 min later.

Cigarette smoke exposure reduced the magnitude of subsequent bronchoconstriction induced by neurokinin A (NKA) by 37% compared with the effect of NKA in air‐exposed animals. L‐NAME had no significant effect on the smoke‐induced inhibition of NKA‐induced bronchoconstriction.

Vagally‐induced plasma exudation in the main bronchi was greater in smoke‐exposed animals compared with air‐exposed animals (120% greater at 2.5 Hz, 82% greater at 10 Hz).

We conclude that cigarette smoke‐induced inhibition of subsequent NANC neurogenic bronchoconstriction is not associated with inhibition of airway plasma exudation and is mediated in part via exogenous smoke‐derived NO, or another bronchoprotective molecule, and by endogenous NO.

British Journal of Pharmacology (1997) 122, 779–785; doi:10.1038/sj.bjp.0701440
Aminoguanidine and the prevention of leukocyte dysfunction in diabetes mellitus: a direct vital microscopic study
Authors: Sannomiya Paulina , Oliveira Maria A. , Fortes Zuleica B.
Published: January 2005

Defective leukocyte‐endothelial interactions are observed in experimental diabetes mellitus. The present study investigated the effect of aminoguanidine, an inhibitor of advanced glycation end products formation, on leukocyte‐endothelial interactions in alloxan‐induced diabetic rats.

In rats anaesthetized with sodium pentobarbitone, the internal spermatic fascia was exteriorized and the microcirculation was observed by a closed‐circuit TV coupled to a microscope. The number of leukocytes rolling along the venular endothelium and sticking to the vascular wall was determined after topical application of zymosan‐activated plasma (1 mg ml−1), as well as the number of adherent and migrated cells after an irritative stimulus (carrageenan 100 μg).

The diabetic state decreased the number of rolling, sticking and migrated leukocytes. Pretreatment of diabetic animals with aminoguanidine (250 mg kg−1 day−1, for 18 days) normalized these values. To be effective, aminoguanidine had to be administered chronically, starting treatment before induction of the diabetic state.

The preventive effect was unrelated to the number of circulating leukocytes, or to the hyperglycaemia or to the hyperosmolality secondary to hyperglycaemia.

A non‐dialyzed (>12,000‐Mr) material in plasma from diabetic, but not normal animals, decreased the number of rolling, sticking and migrated leukocytes in recipient rats. This effect was completely abolished by chronic treatment of diabetic plasma donors with aminoguanidine.

The results suggest that a protein modified by glycosylation (>12 kDa) is associated with leukocyte dysfunction in diabetes mellitus and that the ability of aminoguanidine to prevent such dysfunction is related to an inhibitory effect on advanced glycation end products formation.

British Journal of Pharmacology (1997) 122, 894–898; doi:10.1038/sj.bjp.0701448
Inhibition of trigeminal neurones after intravenous administration of naratriptan through an action at 5‐hydroxy‐tryptamine (5‐HT1B/1D) receptors
Authors: Goadsby Peter J. , Knight Yolande
Published: January 2005

The observation that 5‐hydroxytryptamine (5‐HT) is effective in treating acute attacks of migraine when administered intravenously resulted in a research effort that led to the discovery of the 5‐HT1B/1D receptor agonist sumatriptan.

Clinical experience has shown sumatriptan to be an effective treatment with some limitations, such as relatively poor bioavailability, which naratriptan was developed to address. Increasing bioavailability has been achieved with greater lipophilicity and thus the potential for greater activity in the central nervous system.

In this study the increased access to central sites has been exploited in an attempt to characterize the pharmacology of those central receptors with the newer tools available. Trigeminovascular activation was examined in the model of superior sagittal sinus stimulation.

Cats were anaesthetized with α‐chloralose (60 mg kg−1, intraperitoneal), paralyzed (gallamine 6 mg kg−1, intravenously) and ventilated. The superior sagittal sinus was accessed and isolated for electrical stimulation (250 μs pulses, 0.3 Hz, 100 V) by a mid‐line circular craniotomy. The region of the dorsal surface of C2 spinal cord was exposed by a laminectomy and an electrode placed for recording evoked activity from sinus stimulation.

Stimulation of the superior sagittal sinus resulted in activation of cells in the dorsal horn of C2. Cells fired with a probability of 0.69±0.1 at a latency of 9.2±0.2 ms. Intravenous (i.v.) administration of naratriptan at clinically relevant doses (30 and 100 μg kg−1), inhibited neuronal activity in trigeminal neurones of the C2 dorsal horn, reducing probability of firing without affecting latency.

The effect of naratriptan could be reversed by administration of the selective 5‐HT1B/1D receptor antagonist GR127935 (100 μg kg−1, i.v.).

These data establish that naratriptan acts on central trigeminal neurones since sagittal sinus stimulation activates axons within the tentorial nerve and there are no inhibitory effects mediated within the trigeminal ganglion. Furthermore, given that this inhibition could be reversed by the relatively selective 5‐HT1B/1D receptor antagonist GR127935, it is highly likely that the anti‐migraine effects of drugs of this class with central nervous system access are mediated, at least in part, by 5‐HT1B/1D receptors within the trigeminal nucleus.

British Journal of Pharmacology (1997) 122, 918–922; doi:10.1038/sj.bjp.0701456
The role of cyclic AMP production, calcium channel activation and enzyme activities in the inhibition of testosterone secretion by amphetamine
Authors: Tsai Shiow‐Chwen , Chen Jiann‐Jong , Chiao Yu‐Chung , Lu Chien‐Chen , Lin Ho , Yeh Jiun‐Yih , Lo Ming‐Jae , Kau Mei‐Mei , Wang Shyi‐Wu , Wang Paulus S.
Published: January 2005

The aim of this study was to investigate the mechanism by which amphetamine exerts its inhibitory effect on testicular interstitial cells of male rats.

Administration of amphetamine (10−12–10−6 M) in vitro resulted in a dose‐dependent inhibition of both basal and human chorionic gonadotropin (hCG, 0.05 iu ml−1)‐stimulated release of testosterone.

Amphetamine (10−9 M) enhanced the basal and hCG‐increased levels of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) accumulation in vitro (P<0.05) in rat testicular interstitial cells.

Administration of SQ22536, an adenylyl cyclase inhibitor, decreased the basal release (P<0.05) of testosterone in vitro and abolished the inhibitory effect of amphetamine.

Nifedipine (10−6 M) alone decreased the secretion of testosterone (P<0.01) but it failed to modify the inhibitory action of amphetamine (10−10–10−6 M).

Amphetamine (10−10–10−6 M) significantly (P<0.05 or P<0.01) decreased the activities of 3β‐hydroxysteroid dehydrogenase (3β‐HSD), P450c17, and 17‐ketosteroid reductase (17‐KSR) as indicated by thin‐layer chromatography (t.l.c.).

These results suggest that increased cyclic AMP production, decreased Ca2+ channel activity and decreased activities of 3β‐HSD, P450c17, and 17‐KSR are involved in the inhibition of testosterone production induced by the administration of amphetamine.

British Journal of Pharmacology (1997) 122, 949–955; doi:10.1038/sj.bjp.0701463
Effect of descarboethoxyloratadine, the major metabolite of loratadine, on the human cardiac potassium channel Kv1.5
Authors: Caballero Ricardo , Delpón Eva , Valenzuela Carmen , Longobardo Mónica , Franqueza Laura , Tamargo Juan
Published: January 2005

The effects of descarboethoxyloratadine (DCL), the major metabolite of loratadine, were studied on a human cardiac K+ channel (hKv1.5) cloned from human ventricle and stably expressed in a mouse cell line by means of the patch‐clamp technique. DCL (1–100 μM) inhibited hKv1.5 current in a concentration‐dependent manner with an apparent affinity constant of 12.5±1.2 μM. The blockade increased steeply over the voltage range of channel opening, which indicated that DCL binds preferentially to the open state of the channel. At more depolarized potentials a weaker voltage‐dependence was observed consistent with a binding reaction sensing ∼amp;20% of the transmembrane electrical field. DCL, 20 μM, increased the time constant of deactivation of tail currents, thus inducing a ‘crossover’ phenomenon. The present results demonstrated that DCL blocked hKv1.5 channels in a concentration‐, voltage‐, and time‐dependent manner.

British Journal of Pharmacology (1997) 122, 796–798; doi:10.1038/sj.bjp.0701468
Reversal of tolerance to the antitransit effects of morphine during acute intestinal inflammation in mice
Authors: Pol Olga , Puig Margarita M.
Published: January 2005

The aim of investigation was to establish and compare the reversibility of tolerance to the antitransit effects of morphine by three different procedures: (a) acute inflammation of the gut, (b) lorglumide a cholecystokininA (CCKA) receptor antagonist, or (c) MK‐801, an N‐methyl‐D‐aspartate (NMDA) receptor ion channel blocker. The type of interaction between morphine and lorglumide or MK‐801 on the inhibition of gastrointestinal transit (GIT) in naive animals was also evaluated.

Male Swiss CD‐1 mice were implanted with 75 mg of morphine base or placebo pellets. Gastrointestinal transit was assessed with a charcoal meal and results expressed as % inhibition of GIT. Inflammation was induced by the intragastric (p.o.) administration of croton oil (CO), while controls received castor oil (CA) or saline (SS). Morphine was administered by subcutaneous (s.c.) or intracerebroventricular (i.c.v.) injection, to naive and tolerant animals treated with CO, CA or SS. Dose‐response curves for s.c. morphine were also perforrmed in naive and tolerant mice receiving 5.2 or 7.4 nmol (s.c.) lorglumide or MK‐801, respectively.

The ED50 values for inhibition of GIT by s.c. morphine were: 45.9±2.7 and 250.1±3.1 nmol in naive and tolerant animals, respectively, demonstrating a five fold decrease in the potency of morphine. In naive animals, inflammation (CO) decreased the ED50 of morphine three times (14.4±2.2 nmol). However, no tolerance to s.c. morphine (ED50 16.4±2.6 nmol) was manifested during intestinal inflammation. After i.c.v. administration, a similar degree of tolerance to morphine was observed (4.8 fold decrease in potency). Intestinal inflammation had no effect on the ED50 values of i.c.v. morphine in naive and tolerant animals, showing that reversal of tolerance is related to local mechanism/s. Mean values for intestinal pH were 6.9±0.04 and 6.2±0.04 in SS and CO treated mice, respectively. In addition, morphine was 74 times more potent by the i.c.v. than by the s.c. route (naive‐SS).

Morphine and lorglumide interacted synergistically in naive animals; in addition, the administration of lorglumide reversed tolerance to s.c. morphine. No interaction (additivity) was observed in naive animals when morphine and MK‐801 were administered in combination. However, the drug completely reversed tolerance to the antitransit effects of morphine.

The present investigation shows that acute inflammation of the gut reverses tolerance to the antitransit effects of s.c. morphine by a peripheral mechanism. Qualitatively similar results were obtained after the administration of lorglumide or MK‐801. Our results suggest that a local decrease in pH could play an important role during inflammation, while antagonism of endogenous compensatory systems would explain the reversal of tolerance induced by lorglumide or MK‐801.

British Journal of Pharmacology (1997) 122, 1216–1222; doi:10.1038/sj.bjp.0701472
The pacemaker current If in single human atrial myocytes and the effect of β‐adrenoceptor and A1‐adenosine receptor stimulation
Authors: Porciatti Francesco , Pelzmann Brigitte , Cerbai Elisabetta , Schaffer Peter , Pino Roberto , Bernhart Eva , Koidl Bernd , Mugelli Alessandro
Published: January 2005

We used single human atrial myocytes to study If occurrence, properties and pharmacological modulation. Cells were obtained by chunk enzymatic digestion from samples of right atrial appendages of patients undergoing corrective cardiac surgery.

Patch‐clamped cells in the whole‐cell configuration were superfused with a modified Tyrode solution to reduce contamination by interfering currents and to amplify If. The average cell membrane capacitance was 85.06±2.41 pF (n=531). Data were consistent with the geometrical dimensions of the cells (length 94.2±1.89 μm, width 17.9±0.42 μm, n=126).

When hyperpolarizing to −120 mV from a holding potential of −40 mV, 252 of 306 tested cells (82%) expressed a hyperpolarization‐activated inward current (If density =3.77±0.25 pA pF−1); the current was considered to be present in a given cell if its density at −120 mV was larger than 0.5 pA pF−1.

Current activation was sigmoidal and fitted a Boltzmann model; the average activation curve (n=25) showed a maximum current amplitude of 205.97±19.94 pA, corresponding to 3.87±0.63 pA pF−1, voltage of half‐maximal activation (V1/2) at −86.68±2.19 mV and a slope of −11.39±0.69 mV. The reversal potential of If measured by tail‐current analysis was −13.07±1.92 mV (n=6). The addition of CsCl (5 mM) fully and reversibly blocked the current.

In the presence of the β‐adrenoceptor agonist isoprenaline (Iso, 1 μM), V1/2 was significantly shifted toward less negative potentials by 6.06±1.96 mV (n=16, P=0.0039). The selective A1‐adenosine receptor agonist cyclopentyladenosine (CPA, 1 μM) caused a statistically significant shift of V1/2 toward more negative potentials with respect to the control curve, both in the absence (−7.37±1.83 mV, P=0.0005, n=11) and in the presence of 1 μM Iso (−4.97±1.78, P=0.031, n=6).

These results demonstrate that a current with the properties of If described in cardiac primary and secondary pacemakers occurs in the majority of human atrial cells. While the pathophysiological relevance of If in human atrial tissue remains to be defined, our data clearly show that it is modulated through stimulation of β‐adrenoceptors and A1‐adenosine receptors.

British Journal of Pharmacology (1997) 122, 963–969; doi:10.1038/sj.bjp.0701473
Fidelity in functional coupling of the rat P2Y1 receptor to phospholipase C
Authors: Schachter Joel B. , Boyer José L. , Li Qing , Nicholas Robert A. , Harden T. Kendall
Published: January 2005

The rat homologue of the P2Y1 receptor has been heterologously expressed in 1321N1 human astrocytoma cells and in C6 rat glioma cells.

As has been shown previously for the turkey and human P2Y1 receptors, the rat P2Y1 receptor expressed in either cell type responded to 2MeSATP with increases in inositol phosphate accumulation that were competitively blocked by the antagonist PPADS. Neither of the wild type cell lines exhibited inositol phosphate responses to P2Y1 receptor agonists.

Expression of the rat P2Y1 receptor did not confer a capacity of 2MeSATP to inhibit adenylyl cyclase activity in 1321N1 cells. Moreover, the inhibition of adenylyl cyclase mediated by an endogenous P2Y receptor of C6 glioma cells was not enhanced by expression of the rat P2Y1 receptor. The P2Y receptor‐mediated inhibition of adenylyl cyclase in C6 glioma cells expressing both the endogenous P2Y receptor and the rat P2Y1 receptor remained unaffected by PPADS.

Since the P2Y receptor responsible for inhibition of adenylyl cyclase in C6 glioma cells does not share the pharmacological or functional properties of the P2Y1 receptor, even when both receptors originate from the same species and are simultaneously expressed in the same cell line, it is concluded that the P2Y1 receptor is distinct from an endogenous P2Y receptor in C6 cells that couples to inhibition of adenylyl cyclase.

British Journal of Pharmacology (1997) 122, 1021–1024; doi:10.1038/sj.bjp.0701479
Activation of the cAMP transduction cascade contributes to the mechanical hyperalgesia and allodynia induced by intradermal injection of capsaicin
Authors: Sluka Kathleen A.
Published: January 2005

The spinal role of the cAMP transduction cascade in nociceptive processing was investigated in awake behaving rats (male, Sprague‐Dawley) by activating or inhibiting this pathway spinally. Microdialysis fibres were implanted into the dorsal horn to infuse drugs directly to the spinal cord.

Animals, without peripheral tissue injury, were tested for responses to repeated applications (10 trials) of von Frey filaments and threshold to mechanical stimulation before and after infusion of 8‐bromo‐cAMP. In this group of animals treated spinally with 8‐br‐cAMP (1–10 mM) a dose‐dependent hyperalgesia and allodynia were produced. This was manifested as an increased number of responses to 10 trials of von Frey filaments (10, 50, 150, 250 mN) and a decrease in mechanical threshold.

A second series of experiments studied the manipulation of the cAMP pathway spinally in a model of tissue injury induced by intradermal injection of capsaicin. Animals were either pre‐ or post‐treated spinally with the adenylate cyclase inhibitor, tetrahydrofuryl adenine (THFA) or the protein kinase A inhibitor, myrosilated protein kinase (14–22) amide (PKI). Injection of capsaicin resulted in an increased number of responses to repeated applications of von Frey filaments and a decrease in threshold to mechanical stimuli outside the site of injection, secondary mechanical hyperalgesia and allodynia.

Pre‐treatment with either THFA (1 mM) or PKI (5 mM) had no effect on the capsaicin‐evoked secondary hyperalgesia and allodynia.

In contrast, post‐treatment spinally with THFA (0.01–1 mM) or PKI (0.05–50 mM) dose‐dependently reduced the mechanical hyperalgesia and allodynia produced by capsaicin injection. Furthermore, the mechanical hyperalgesia and allodynia blocked by the adenylate cyclase inhibitor, THFA (1 mM), was reversed by infusion of 8‐bromo‐cAMP (0.01–10 mM) in a dose‐dependent manner.

Thus, this study demonstrates that activation of the cAMP transduction cascade at the spinal cord level results in mechanical hyperalgesia and allodynia and that the secondary mechanical hyperalgesia and allodynia following intradermal injection of capsaicin is mediated by this same transduction cascade.

British Journal of Pharmacology (1997) 122, 1165–1173; doi:10.1038/sj.bjp.0701486
Effects of a novel guanylate cyclase inhibitor on nitric oxide‐dependent inhibitory neurotransmission in canine proximal colon
Authors: Franck H. , Sweeney K. M. , Sanders K. M. , Shuttleworth C. W. R.
Published: January 2005

Previous studies suggested that nitric oxide (NO) may cause hyperpolarization and relaxation of canine colonic smooth muscle by both cGMP‐dependent and cGMP‐independent mechanisms. This hypothesis was tested using 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (ODQ), a novel inhibitor of NO‐stimulated guanylate cyclase.

In the presence of histamine (30 μM), atropine and indomethacin (both at 1 μM), electrical field stimulation of intrinsic neurons (EFS; 5 Hz) produced inhibition of phasic contractile activity that is due to NO synthesis. ODQ caused a concentration‐dependent block of this response (10 nM to 10 μM).

Inhibitory junction potentials (IJPs) due to NO synthesis were recorded from muscle cells located near the myenteric border of the circular muscle layer, using intracellular microelectrodes. IJPs were abolished by ODQ (1–10 μM).

EFS (10–20 Hz) produced frequency‐dependent inhibition of electrical slow waves recorded from cells located near the submucosal surface of the circular muscle layer. This inhibition is due to NO synthesis, and it was abolished by ODQ (1–10 μM).

Hyperpolarization and relaxation produced by an NO donor, sodium nitroprusside, were abolished by ODQ pretreatment (1–10 μM). In contrast, inhibitory responses to 8‐Br‐cGMP (1 mM) were unaffected by ODQ.

ODQ alone (1–10 μM) had no significant effect on spontaneous electrical or phasic contractile activity. In tissues pre‐treated with L‐NAME (300 μM), ODQ decreased the amplitude of spontaneous or histamine‐stimulated phasic contractile activity.

These results suggest that electrical and mechanical effects of endogenously released and exogenously applied NO in canine colon are largely due to cGMP synthesis by ODQ‐sensitive soluble guanylate cyclase. No evidence to support a direct (cGMP‐independent) mechanism of NO action was found. ODQ also appears to cause a non‐specific inhibition of muscle contractile activity; however, this effect does not contribute to block of NO‐dependent effects.

British Journal of Pharmacology (1997) 122, 1223–1229; doi:10.1038/sj.bjp.0701487
Structural requirements for roxatidine in the stimulant effect of rat gastric mucin synthesis and the participation of nitric oxide in this mechanism
Authors: Ichikawa Takafumi , Ishihara Kazuhiko , Saigenji Katsunori , Hotta Kyoko
Published: January 2005

The structural requirements of the histamine H2‐receptor antagonist, roxatidine (2‐acetoxy‐N‐(3‐[m‐(1‐piperidinylmethyl)phenoxy]‐propyl)acetamide hydrochloride), for the stimulant effect on mucin biosynthesis and their relation to histamine H2‐receptor antagonism were identified by considering the structural analogues of this drug using an organ culture system of the rat stomach and competition studies with [125I]iodoaminopotentidine ([125I]‐APT) binding to membranes of the guinea pig striatum.

[3H]Glucosamine incorporation into mucin during 5 h incubation period was stimulated by roxatidine and its structural analogues A (2‐hydroxy‐N‐(3‐[m‐(1‐piperidinylmethyl)phenoxy]‐propyl)acetamide) and B (N‐(3‐[m‐(1‐piperidinylmethyl)phenoxy]‐propyl)acetamide). This effect was seen in mucosal cultures of the corpus, but not antrum, region.

Structural analogues, in which the length of the flexible chain between the benzene ring and the amide structure differs from that of roxatidine, failed to activate mucin synthesis. No significant change in mucus synthesis occurred with the addition of analogues in which the piperidine ring attached to the benzene ring via a methylene bridge was changed.

Specific [125I]‐APT binding to the histamine H2 receptor of guinea pig brain membranes was inhibited by roxatidine and all structural analogues used in this study, except F (N‐(3‐[m‐(N, N‐dimethyl‐aminomethyl)phenoxy]‐propyl)acetamide).

Ranitidine at 10−4 M did not suppress the roxatidine‐induced increase in [3H]glucosamine incorporation into mucin.

Roxatidine‐induced stimulation of [3H]glucosamine incorporation into mucin was completely blocked by the addition of either NG‐nitro‐L‐arginine (10−5 M) or 2‐(4‐carboxyphenyl)‐4,4,5,5,‐tetramethylimidazoline‐1‐oxyl‐3‐oxide sodium salt (10−5 M). The inhibitory action of NG‐nitro‐L‐arginine was totally reversed by L‐arginine (5×10−3 M).

These results suggest that the cardinal chemical features of roxatidine for the activation of mucin biosynthesis in the corpus region of the rat stomach are the appropriate length of the flexible chain between the amide structure and the aromatic ring system bearing the methylpiperidinyl group at the meta position. The activity of roxatidine and its analogues to stimulate mucin synthesis is not related to their histamine H2 receptor antagonistic activity. Roxatidine‐induced activation of mucin biosynthesis in the corpus tissue is mediated by nitric oxide.

British Journal of Pharmacology (1997) 122, 1230–1236; doi:10.1038/sj.bjp.0701488
Effects of dexamethasone and phorbol ester on P2 receptor‐coupled Ca2+ signalling and lipocortin 1 presentation in U937 cells
Authors: Willmott Nicholas J. , Choudhury Qam , Flowers Roderick J.
Published: January 2005

Cell surface bound lipocortin 1 (LC1) is a putative mediator of the antiproliferative and anti‐inflammatory effects of glucocorticoids. This study assessed the hypothesis that the glucocorticoid, dexamethasone‐phosphate (dex‐p), might exert the above effects via an LC1‐mediated downregulation of receptor‐coupled Ca2+ signalling, using P2‐receptor mediated intracellular Ca2+ accumulation in U937 cells as an appropriate model.

Addition of ATP (1–100 μM) to cells resulted in a transient increase in cytosolic Ca2+ ([Ca2+]i). Prior treatment of cells with dex‐p (3–24 h) increased the magnitude of this Ca2+ transient at high, but not low concentrations of ATP, and increased thapsigargin (Tg)‐induced Ca2+ influx, indicating that store‐operated Ca2+ influx was potentiated in these cells. For cells treated with dex‐p for 24 h, cell surface levels of LC1 were significantly reduced by 63%.

Differentiation of cells with 1 nM phorbol ester (PMA) for 24 h resulted in a 2.4 fold increase in the cell surface level of LC1 and inhibition of the ATP‐induced Ca2+ response. However, the Tg‐induced Ca2+ response was unaffected by long‐term PMA treatment, and incubating cells with LC1 did not alter Tg‐induced Ca2+ mobilization and influx, or the ATP‐mediated Ca2+ response.

Data from this study suggest that: (1) dex‐p does not inhibit P2‐receptor‐coupled Ca2+ signalling in this cell line, (2) the observed modulation of the ATP‐induced increase in [Ca2+]i by dex‐p and PMA, and store‐operated Ca2+ influx by dex‐p, is not linked to an increase in the cell surface level of LC1, and (3) differentiation of U937 cells with PMA downregulates the ATP‐induced Ca2+ response, but does not affect the thapsigargin‐sensitive Ca2+ pool or store‐operated Ca2+ influx of these cells.

British Journal of Pharmacology (1997) 122, 1055–1060; doi:10.1038/sj.bjp.0701490
Nitrergic modulation of cholinergic responses in the opossum lower oesophageal sphincter
Authors: Cellek Selim , Moncada Salvador
Published: January 2005

Electrical field stimulation (EFS) of the superfused lower oesophageal sphincter from opossum (Monodelphis domestica) elicited biphasic responses. The first phase (relaxation) was strictly dependent on the duration of the EFS. The second phase (contraction) started following termination of the EFS (15 Hz). EFS at frequencies above 15 Hz led only to contraction, which started immediately upon initiation of the stimulation.

In the presence of NG‐nitro‐L‐arginine (L‐NOARG; 0.1–300 μM), the relaxation phase was abolished and the contractile response started with the initiation of EFS (at all frequencies) and was greater in magnitude. The contractile response to EFS was completely blocked with scopolamine (10 μM).

Exogenous acetylcholine (1–100 μM) elicited concentration‐dependent contractions of the sphincter in the presence of botulinum toxin. These contractions were abolished when EFS was applied during administration of acetylcholine. This inhibitory effect of EFS was completely reversed when the tissue was treated with L‐NOARG (100 μM).

These results suggest that the cholinergic response in the opossum lower oesophageal sphincter is under nitrergic control.

British Journal of Pharmacology (1997) 122, 1043–1046; doi:10.1038/sj.bjp.0701497
Intravascular and interstitial degradation of bradykinin in isolated perfused rat heart
Authors: Dendorfer Andreas , Wolfrum Sebastian , Wellhöner Peter , Korsman Katja , Dominiak Peter
Published: January 2005

Bradykinin (BK) has been shown to exert cardioprotective effects which are potentiated by inhibitors of angiotensin I‐converting enzyme (ACE). In order to clarify the significance of ACE within the whole spectrum of myocardial kininases we investigated BK degradation in the isolated rat heart.

Tritiated BK (3H‐BK) or unlabelled BK was either repeatedly perfused through the heart, or applied as an intracoronary bolus allowing determination of its elution kinetics. BK metabolites were analysed by HPLC. Kininases were identified by ramiprilat, phosphoramidon, diprotin A and 2‐mercaptoethanol or apstatin as specific inhibitors of ACE, neutral endopeptidase 24.11 (NEP), dipeptidylaminopeptidase IV and aminopeptidase P (APP), respectively.

In sequential perfusion passages, 3H‐BK concentrations in the perfusate decreased by 39% during each passage. Ramiprilat reduced the rate of 3H‐BK breakdown by 54% and nearly abolished [1‐5]‐BK generation. The ramiprilat‐resistant kininase activity was for the most part inhibited by the selective APP inhibitor apstatin (IC50 0.9 μM). BK cleavage by APP yielded the intermediate product [2‐9]‐BK, which was rapidly metabolized to [4‐9]‐BK by dipeptidylaminopeptidase IV.

After bolus injection of 3H‐BK, 10% of the applied radioactivity were protractedly eluted, indicating the distribution of this fraction into the myocardial interstitium. In samples of such interstitial perfusate fractions, 3H‐BK was extensively (by 92%) degraded, essentially by ACE and APP. The ramiprilat‐ and mercaptoethanol‐resistant fraction of interstitial kininase activity amounted to 14%, about half of which could be attributed to NEP. Only the product of NEP, [1–7]‐BK, was continuously generated during the presence of 3H‐BK in the interstitium.

ACE and APP are located at the endothelium and represent the predominant kininases of rat myocardium. Both enzymes form a metabolic barrier for the extravasated fraction of BK. Thus, only interstitial, but not intravascular concentrations of BK are increased by kininase inhibitors to the extent that a significant potentiation of BK effects could be explained. NEP contributes less than 5% to the total kininase activity, but is the only enzyme which is exclusively present in the interstitial space.

British Journal of Pharmacology (1997) 122, 1179–1187; doi:10.1038/sj.bjp.0701501
AT2‐antagonist sensitive potentiation of angiotensin II‐induced vasoconstrictions by blockade of nitric oxide synthesis in rat renal vasculature
Authors: Muller Catherine , Endlich Karlhans , Barthelmebs Mariette , Helwig Jean‐Jacques
Published: January 2005

Although the actions of angiotensin II (Ang II) on renal haemodynamics appear to be mediated by activation of the AT1 receptor subtype, AT2 binding sites have also been evidenced in the adult kidney vasculature. As NO is known to mask part of the renal effects of vasoconstrictor drugs, we queried whether the Ang II‐induced vasoconstrictions could occur via multiple receptor subtypes during inhibition of NO synthesis. We explored the effect of AT1 and AT2 receptor (AT‐R) antagonists on Ang II‐induced pressure increases during NO synthase or soluble guanylyl cyclase inhibition in rat isolated kidneys perfused in the presence of indomethacin at constant flow in a single‐pass circuit.

In the absence of NO blockade, the AT1‐R antagonist L‐158809 (500 nM) antagonized the Ang II‐induced vasoconstrictions, while the AT2‐R antagonist PD‐123319 (500 nM) had no effect.

Perfusing kidneys in the presence of either NO synthase inhibitors, L‐NAME (100 μM) or L‐NOARG (1 mM), or soluble guanylyl cyclase inhibitor, LY‐83583 (10 μM), significantly increased both molar pD2 (from 9.40±0.25 to 10.36±0.11) and Emax values (from 24.9±3.1 to 79.9±4.9 mmHg) of the concentration–response curve for Ang II‐induced vasoconstriction.

In the presence of L‐NAME, 500 nM L158809 abolished the Ang II‐induced vasoconstrictions whatever the concentration tested. On the other hand, 500 nM PD‐123319 reversed the left shift of the concentration–response curve for Ang II (molar pD2 value 9.72±0.13) leaving Emax value unaffected (91.3±7.6 mmHg).

In the presence of L‐NAME, the potentiated vasoconstriction induced by 0.1 nM and the augmented vasoconstriction induced by 10 nM Ang II were fully inhibited in a concentration‐dependent manner by L‐158809 (0.05–500 nM). By contrast, PD‐123319 (0.5–500 nM) did not affect the 10 nM Ang II‐induced vasoconstriction and concentration‐dependently decreased the 0.1 nM Ang II‐induced vasoconstriction plateauing at 65% inhibition above 5 nM antagonist.

Similar to PD‐123319, during NO blockade the AT2‐R antagonist CGP‐42112A at 5 nM decreased by 50% the 0.1 nM Ang II‐induced vasoconstriction and at 500 nM had no effect on 10 nM Ang II‐induced vasoconstriction.

In conclusion, the renal Ang II‐induced vasoconstriction, which is antagonized only by AT1‐R antagonist in the presence of endogenous NO, becomes sensitive to both AT1‐ and AT2‐R antagonists during NO synthesis inhibition. While AT1‐R antagonist inhibited both L‐NAME‐potentiated and‐augmented components of Ang II‐induced vasoconstriction, AT2‐R antagonists inhibited only the L‐NAME‐potentiated component.
Pharmacological evidence for α1D‐adrenoceptors in the rabbit ventricular myocardium: analysis with BMY 7378
Authors: Yang Huang‐Tian , Endoh Masao
Published: January 2005

It was examined by means of BMY 7378, a selective antagonist of α1D‐adrenoceptors, whether α1D‐adrenoceptors contribute to the regulation of myocardial contractility and hydrolysis of phosphoinositide (PI) in rabbit ventricular muscle.

BMY 7378 had a biphasic antagonistic action on the positive inotropic effect (PIE) of phenylephrine depending on the concentration. BMY 7378 at 1–10 nm shifted the concentration–response curve (CRC) for the PIE of phenylephrine to the right and downward and at 100 nm to 1 μm it antagonized the PIE in a competitive manner, the slope of Schild plot being 0.93 and the pA2 being 7.17±0.09.

The inhibitory action of BMY 7378 at 1–10 nm is ascribed to the selective action on α1‐adrenoceptors because the PIE of neither isoprenaline nor endothelin‐3 and angiotensin II was affected by this compound over this concentration range.

In the presence of 100 nm WB 4101, the antagonistic action of BMY 7378 at 1–10 nm remained unchanged but the antagonistic action of BMY 7378 at 100–300 nm disappeared. The antagonistic action of BMY 7378 at 1 nm was unaffected by 100 nm (+)‐niguldipine.

Following pretreatment with chloroethylclonidine, BMY 7378 at 1 nm inhibited the maximal response to phenylephrine but the pD2 value for phenylephrine was increased in the presence of BMY 7378. The CRC for phenylephrine was shifted to the left in the presence of 10–100 nm BMY 7378 but it was shifted to the right by BMY 7378 at 300 nm.

Stimulation of PI hydrolysis induced by phenylephrine was not affected by BMY 7378 up to 10 nm but it was reduced significantly by BMY 7378 at higher concentrations (100 nm to 1 μm).

BMY 7378 inhibited the [3H]prazosin specific binding to the rabbit ventricular membrane fraction in a monophasic manner with a pKi value of 7.53±0.09.

The results indicate that in rabbit ventricular muscle, BMY 7378 at 1–10 nm suppressed the maximal response to phenylephrine (probably mediated by α1D‐adrenoceptors) and at 10–100 nm it inhibited the negative inotropic effect of phenylephrine, the mechanisms of which remain to be characterized. At higher concentrations (100 nm to 1 μm) BMY 7378 antagonized the functional and biochemical response via a presumed interaction mainly with the α1B‐adrenoceptor and partially with the α1A‐adrenoceptor.
Dobesilate enhances endothelial nitric oxide synthase‐activity in macro‐ and microvascular endothelial cells
Authors: Suschek Christoph , Kolb Hubert , Kolb‐Bachofen Victoria
Published: January 2005

Dobesilate is used for normalizing vascular dysfunction in a number of diseases. In search for an effect on endothelial NO production, macrovascular endothelial cells from rat aorta, microvascular endothelial cells from rat exocrine pancreatic tissue, and capillary endothelial cells from rat islets, were cultured in the presence or absence of Mg‐Dobesilate. The activity of constitutive nitric oxide synthase (ecNOS) in resident cells as well as of inducible nitric oxide synthase (iNOS) in cytokine‐activated cells was measured indirectly by recording the citrulline concentrations in culture supernatants.

In each of the different endothelial cells Mg‐Dobesilate incubation (0.25–1 mM) for 24 h led to a significant and concentration‐dependent increase in ecNOS‐activities. With cytokine‐activated endothelial cell cultures only moderate effects were seen with little or no concentration‐dependency. Addition of the NOS‐inhibitor NG‐monomethyl‐L‐arginine led to a significant suppression of citrulline formation in all cultures as an evidence for the enzyme specificity of these effects.

iNOS‐ and ecNOS‐specific reverse transcription and semi‐quantitative polymerase chain reaction (RT–PCR) with RNA from resident or cytokine‐activated endothelial cells gave no evidence for an increase in NOS‐specific mRNA after Mg‐Dobesilate‐treatment. Furthermore, Dobesilate‐mediated enhancement of NO synthesis in resting endothelial cells was not due to iNOS induction in these cells, as no iNOS‐specific signal was found by RT–PCR.
The influence of 5‐HT2and 5‐HT4receptor antagonists to modify drug induced disinhibitory effects in the mouse light/dark test
Authors: Costall Brenda , Naylor Robert J.
Published: January 2005

The ability of 5‐HT2and 5‐HT4receptor antagonists to modify the disinhibitory profile of diazepam and other agents was investigated in male BKW mice in the light/dark test box.

The 5‐HT2A/2B/2Creceptor antagonists ritanserin, MDL11939 and RP62203 and also methysergide, which failed to modify mouse behaviour when administered alone, caused dose‐related enhancements (4 to 8 fold) in the potency of diazepam to disinhibit behavioural responding to the aversive situation of the test box.

Ritanserin was shown to enhance the disinhibitory potency of other benzodiazepines, chlordiazepoxide (4 fold), temazepam (10 fold) and lorazepam (10 fold), the 5‐HT1Areceptor ligands, 8‐OH‐DPAT (25 fold), buspirone (100 fold) and lesopitron (500 fold), the 5‐HT3receptor antagonists, ondansetron (100 fold)R(+)‐zacopride (100 fold) andS(−)‐zacopride (greater than a 1000 fold), the substituted benzamides, sulpiride (10 fold) and tiapride (5 to 10 fold) and the cholecystokinin (CCK)Areceptor antagonist, devazepide (100 fold). It also reduced the onset of action of disinhibition following treatment with the 5‐HT synthesis inhibitor parachlorophenylalanine. Ritanserin failed to enhance the disinhibitory effects of the CCKBreceptor antagonist CI‐988, the angiotensin AT1receptor antagonist losarten or the angiotensin converting enzyme inhibitor ceranapril.

The 5‐HT4receptor antagonists SDZ205‐557, GR113808 and SB204070 caused dose‐related reductions in the disinhibitory effect of diazepam, returning values to those shown in vehicle treated controls. The antagonists failed to modify mouse behaviour when administered alone.

GR113808 was also shown to cause a dose‐related antagonism of the disinhibitory effects of chlordiazepoxide, lorazepam, 8‐OH‐DPAT, buspirone, lesopitron, ondansetron,R(+)‐zacopride, sulpiride, tiapride, devazepide, CI‐988, losarten, ceranapril and parachlorophenylalanine.

It was concluded that in BKW mice (a) the failure of 5‐HT2and 5‐HT4receptor antagonists when administered alone to modify behaviour in the light/dark test indicates an absence of an endogenous 5‐HT tone at the 5‐HT2and 5‐HT4receptors and (b) the enhancement by the 5‐HT2receptor antagonists and attenuation by the 5‐HT4receptor antagonists of drug‐induced disinhibition indicates a plurality of 5‐HT receptor involvement in the mediation of drug‐induced disinhibitory profiles in the mouse.

British Journal of Pharmacology(1997)122, 1105–1118; doi:10.1038/sj.bjp.0701513
Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT1A receptor
Authors: Balmforth A. J. , Shepherd F. H. , Warburton P. , Ball S. G.
Published: January 2005

The role of protein kinase C (PKC) in the mechanism underlying rapid agonist‐induced desensitization of angiotensin AT1 receptors remains unresolved. A major problem has been to isolate these receptors in a sufficiently purified form to allow study of their phosphorylation state.

A cleavable (His)6 affinity tag was introduced into the N‐terminus of the recombinant AT1A receptor and stably expressed in human embryonic kidney cells. This affinity tag allowed rapid isolation, purification and determination of the phosphorylation state of the AT1A receptor. Using these cells, we determined the role of PKC in both agonist‐induced receptor phosphorylation and desensitization under identical conditions.

Agonist‐induced phosphorylation of the AT1A receptor was observed at both low and high concentrations of angiotensin II (AII). Preincubation of cells with Ro‐31‐8220 (a PKC specific inhibitor) revealed that at low concentrations of AII (1 nM), PKC appeared to be the main kinase involved in receptor phosphorylation. In contrast, at high concentrations of AII (100 nM), although PKC‐mediated phosphorylation of the receptor was observed, this was overshadowed by a second kinase.

In preliminary desensitization studies we observed that at a low concentration of AII, preincubation with Ro‐31‐8220 attenuated desensitization, whilst at high concentrations of AII (100 nM) it had little or no effect on the level of desensitization observed.

These data directly demonstrate an association between PKC‐induced receptor phosphorylation and desensitization at low concentrations of AII. Since circulating concentrations of AII are in the picomolar range, we propose that PKC is the physiologically relevant mediator of AT1 receptor desensitization.
Inhibitory effects of genistein on ATP‐sensitive K+ channels in rabbit portal vein smooth muscle
Authors: Ogata Rika , Kitamura Kenji , Ito Yushi , Nakano Hitoo
Published: January 2005

Effects on the pinacidil‐induced outward current of inhibitors of tyrosine kinases and phosphatases were investigated by use of a patch‐clamp method in smooth muscle cells of the rabbit portal vein.

A specific tyrosine kinase inhibitor, genistein, inhibited the pinacidil‐induced current in a concentration‐dependent manner with an IC50 of 5.5 μM. Superfusion of Ca2+‐free solution did not affect this inhibitory effect of genistein. At higher concentrations, genistein inhibited the voltage‐dependent Ba2+ and K+ currents with IC50 values of >100 μM and 75 μM respectively. Tyrphostin B46 (30 μM), a tyrosine kinase inhibitor, also inhibited the pinacidil‐induced current by 70 % of the control.

Sodium orthovanadate (100 μM), an inhibitor of tyrosine phosphatase, slightly but significantly enhanced both the pinacidil‐induced and delayed rectifier K+ currents. Daidzein (100 μM), an inactive analogue of genistein, did not inhibit these currents.

Neither herbimycin A (1 μM), lavendustin A (30 μM), tyrphostin 23 (10 μM), which are also tyrosine kinase inhibitors, nor wortmannin (10 μM), a phosphatidylinositol 3‐kinase inhibitor, had an effect on either the pinacidil‐induced or delayed rectifier K+ currents. Epidermal growth factor (EGF; 1 μg ml−1) did not induce an outward current or enhance the pinacidil‐induced current.

Pinacidil alone, in the cell‐attached configuration, or pinacidil with GDP, in the inside‐out configuration, activated a 42 pS channel in the smooth muscle cells of the rabbit portal vein. Genistein (30 μM) reduced the channel's open probability without inducing a change in unitary conductance at any holding potential (−30 to +20 mV).

In the inside‐out configuration, genistein at 30 μM did not change the mean channel open time, but reduced the burst duration. At 100 μM genistein abolished channel opening. The inhibitory potencies with which 30 and 100 μM genistein acted on the unitary current of the ATP‐sensitive K+ channel were similar to those seen in the whole‐cell voltage‐clamp configuration.

Although direct inhibitory actions of genistein on the ATP‐sensitive K+ channels are not ruled out, our results suggest that a protein tyrosine kinase may play a role in the regulation of ATP‐sensitive K+ channel activity in the rabbit portal vein.
Effects of a nonpeptide bradykinin B2 receptor antagonist, FR167344, on different in vivo animal models of inflammation
Authors: Asano Masayuki , Hatori Chie , Inamura Noriaki , Sawai Hiroe , Hirosumi Jiro , Fujiwara Tatsujiro , Nakahara Kunio
Published: January 2005

The effects of a novel, potent and orally active nonpeptide bradykinin B2 receptor antagonist, FR167344 (N ‐[N ‐[3 ‐[(3 ‐ bromo‐ 2 ‐methylimidazo[1,2‐a]pyridin‐8‐yl)oxymethyl]‐2,4‐dichlorophenyl]‐N‐methylaminocarbonylmethyl]‐4‐(dimethylaminocarbonyl) cinnamylamide hydrochloride) were tested in three different in vivo models of inflammation.

Oral administration of FR167344 inhibited carrageenin‐induced paw oedema in rats (carrageenin: 1%, 0.1 ml per animal, intraplantar), with an ID50 of 2.7 mg kg−1 at 2 h after carrageenin injection (n=10 or 11).

Oral administration of the compound also inhibited kaolin‐induced writhing (kaolin: 250 mg kg−1, i.p.) in mice, with ID50 of 2.8 mg kg−1 in 10 min writhing and 4.2 mg kg−1 in 15 min writhing (n=19 or 20).

Additionally, oral administration of FR167344 inhibited caerulein‐induced pancreatic oedema with an ID50 of 13.8 mg kg−1 as well as increases in amylase and lipase of blood samples with ID50 of 10.3 and 7.4 mg kg−1, respectively, in rats (n=10).

These results show that FR167344 is an orally active, anti‐inflammatory and anti‐nociceptive agent in carrageenin‐induced paw oedema, kaolin‐induced writhing and caerulein‐induced pancreatitis. FR167344 may have therapeutic potential against inflammatory diseases by oral administration and it may be a useful tool for studying the involvement of B2 receptors in various in vivo models of inflammation.