bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancement of NMDA responses by group I metabotropic glutamate receptor activation in striatal neurones
Authors: Pisani Antonio , Calabresi Paolo , Centonze Diego , Bernardi Giorgio
Published: August 2006

The interactions between N‐methyl‐d‐aspartate (NMDA) and metabotropic glutamate receptors (mGluRs) were investigated in striatal slices, by utilizing intracellular recordings, both in current‐ and voltage‐clamp mode.

Bath‐application (50 μm) or focal application of NMDA induced a transient membrane depolarization, while in the voltage‐clamp mode, NMDA (50 μm) caused a transient inward current. Following bath‐application of the non‐selective mGluR agonist 1S,3R‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD, 10 μm), NMDA responses were reversibly potentiated both in current (197±15% of control) and voltage‐clamp experiments (200±18% of control).

Bath‐application of the group I mGluR agonist (RS)‐3,5‐dihydroxyphenylglycine (3,5‐DHPG, 10–300 μm) resulted in a dose‐dependent potentiation of NMDA‐induced membrane depolarization (up to 400±33% of control). This potentiation was either prevented by preincubation with (RS)‐α‐methyl‐4‐carboxyphenylglycine (RS‐α‐MCPG, 300 μm), or blocked when applied immediately after 3,5‐DHPG wash‐out.

Neither (2S,1′S,2′S)2‐(2′‐carboxycyclopropyl)glycine (L‐CCG I, up to 100 μm) nor (2S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycyclopropyl)‐glycine (DCG‐IV, 1 μm), agonists for group II mGluRs caused any change in NMDA responses. Likewise, l‐serine‐O‐phosphate (l‐SOP, 30 μm), agonist for group III mGluRs, did not affect the NMDA‐induced depolarization.

The enhancement of the NMDA responses was mimicked by phorbol‐12,13‐diacetate (PDAc, 1 μm) which activates protein kinase C (PKC). The 3,5‐DHPG‐mediated potentiation of the NMDA‐induced depolarization was prevented by preincubation with staurosporine (100 nm) or calphostin C (1 μm), antagonists of PKC.

Electrophysiological responses to α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor activation were not affected by agonists for the three‐classes of mGluRs.

The present data suggest that group I mGluRs exert a positive modulatory action on NMDA responses, probably through activation of PKC. This functional interaction in the striatum appears of crucial importance in the understanding of physiological and pathological events, such as synaptic plasticity and neuronal death, respectively.

British Journal of Pharmacology (1997) 120, 1007–1014; doi:10.1038/sj.bjp.0700999
Role of eicosanoids in alteration of membrane electrical properties in isolated mesenteric arteries of salt‐loaded, Dahl salt‐sensitive rats
Authors: Fujii Koji , Onaka Uran , Ohya Yusuke , Ohmori Susumu , Tominaga Mitsuhiro , Abe Isao , Takata Yutaka , Fujishima Masatoshi
Published: August 2006

The role of eicosanoids in altered membrane electrical properties of Dahl salt‐sensitive (DS) rats was investigated, by use of conventional microelectrodes technique, in isolated superior mesenteric arteries of DS rats and Dahl salt‐resistant (DR) rats fed either a high or low salt diet.

The membrane was significantly depolarized in salt‐loaded DS rats compared with the other three groups. In addition, the arteries of salt‐loaded DS rats exhibited spontaneous electrical activity.

Spontaneous electrical activity in salt‐loaded DS rats was inhibited by the following: indomethacin, a cyclo‐oxygenase inhibitor; ONO‐3708, a prostaglandin H2/thromboxane A2 receptor antagonist; OKY‐046, a thromboxane A2 synthase inhibitor; nicardipine, a Ca2+‐channel antagonist and by Ca2+‐free solution. In addition, spontaneous electrical activity was enhanced by a thromboxane A2 analogue and by prostaglandin H2. Spontaneous electrical activity was unaffected by phentolamine, atropine and tetrodotoxin.

Membrane potential in arteries of salt‐loaded DS rats was not affected by either indomethacin or ONO‐3708.

Spontaneous contraction, sensitive to indomethacin, was present, and contractile sensitivity to high potassium solution was enhanced in arteries of salt‐loaded DS rats.

These findings suggest that eicosanoid action, together with membrane depolarization, may lead to the activation of voltage‐dependent Ca2+‐channels, thereby causing spontaneous electrical activity in mesenteric arteries of salt‐loaded DS rats. In addition, tension data suggest that these changes in membrane properties are related to enhanced contractile activities in salt‐loaded DS rats. Mechanisms of depolarization remain to be determined.

British Journal of Pharmacology (1997) 120, 1207–1214; doi:10.1038/sj.bjp.0701023
SCA 40: studies of the relaxant effects on cryopreserved human airway and vascular smooth muscle
Authors: Müller‐Schweinitzer E. , Fozard J. R.
Published: August 2006

6‐Bromo‐8‐methylaminoimidazol[1,2‐a]pyrazine‐2carbonitrile (SCA 40) has been claimed to induce relaxation in guinea‐pig trachea by opening high conductance, calcium‐activated potassium (BKCa) channels. The mechanism of action of SCA 40 has now been further investigated in ring preparations from cryopreserved human airway and vascular smooth muscle preparations in vitro.

Human bronchi with spontaneous tone relaxed in response to SCA 40 in a biphasic way. A high affinity component (pD2 8.61±0.21; mean±s.e.mean) accounted for 30% of the response and a low affinity component (pD2 6.53±0.14) for the remaining 70%. In contrast, in bronchi contracted with carbachol, 1 μm, the concentration‐response curve to SCA 40 was monophasic and yielded a pD2 of 6.31±0.29.

SCA 40 relaxed pulmonary and mesenteric arteries and peripheral veins which had been precontracted by 10 nm U46619 nearly completely and in a monophasic way; the pD2 values were 6.37±0.08, 6.17±0.15 and 5.45±0.25, respectively.

Lemakalim, an opener of ATP‐dependent potassium (KATP) channels, also relaxed human bronchi under spontaneous tone and the vascular tissues. NS 1619, a recognised opener of BKca channels, was inactive up to 10 μm on bronchial and vascular tissues.

The SCA 40‐induced relaxation of human bronchi was reduced concentration‐dependently in the presence of high potassium chloride (20 and 80 mm). However, in the presence of 80 mm KCl and nifedipine, 30 nm, SCA 40 fully relaxed the remaining contractile response with pD2 values of 8.08±0.13 and 5.27±0.13 for the high and low affinity component, respectively.

Relaxation responses to SCA 40 in human bronchi were resistant to blockade by glibenclamide at concentrations up to 10 μm (which blocked the relaxant response to lemakalim), quinine (30 μm), apamin (100 nm), tetraethylammonium (0.11 mm) and charybdotoxin (10100 nm), thus excluding the involvement of a variety of K+ channels including KATP and KCa channels.

In bronchi contracted with carbachol, 1 μm, the nature of the interaction between SCA 40 and the β2‐adrenoceptor agonist, salbutamol, was synergistic.

These experiments establish that SCA 40 is a potent relaxant of human bronchial smooth muscle manifesting spontaneous tone. A low affinity relaxant component has its counterpart in the relaxation seen in both human arterial and venous smooth muscle. The consensus of the evidence suggests that K+ channel opening is not the basis of the relaxant response to SCA 40. Furthermore, BKCa channels appear to be of minor importance in the regulation of human airway smooth muscle tone. Our data suggest that inhibition of an adenosine 3′ : 5′‐cyclic monophosphate phosphodiesterase may contribute, at least to the low affinity relaxant component of SCA 40. However, the exact mechanism mediating the SCA 40‐induced relaxation of human airways remains to be defined.

British Journal of Pharmacology (1997) 120, 1241–1248; doi:10.1038/sj.bjp.0701037
Analysis of two pharmacologically predicted endothelin B receptor subtypes by using the endothelin B receptor gene knockout mouse
Authors: Mizuguchi Tadahiko , Nishiyama Mariko , Moroi Kayoko , Tanaka Hirokazu , Saito Toshihiro , Masuda Yoshiaki , Masaki Tomoh , De Wit Damiane , Yanagisawa Masashi , Kimura Sadao
Published: August 2006

This study was performed to clarify whether the endothelin (ET) receptor subtypes mediating two pharmacologically heterogeneous response to ETB receptor agonists in normal mice are the product(s) of a single ETB receptor gene.

Vasodilator responses to sarafotoxin S6c (S6c) in the thoracic aorta and contractile responses to ET‐1 and IRL1620 in the stomach were examined in tissues from normal and ETB receptor gene knockout mice, in the absence and presence of an ETA receptor antagonist, BQ‐123, or an ETA/ETB receptor antagonist, PD142893.

In the normal mouse aorta precontracted with phenylephrine, S6c (0.1100 nm) caused concentration‐dependent relaxations (pD2=8.4). BQ‐123 had no effect on these responses. However, PD142893 almost abolished the relaxations induced by 0.1300 nm S6c.

In aortae taken from ETB receptor gene knockout mice, S6c up to 1 μm failed to cause relaxations, confirming that ETB receptors are involved in mediating this response.

In normal mouse gastric fundus, 0.1 nm1 μm ET‐1, S6c or IRL1620 caused dose‐dependent, BQ‐123‐insensitive contractions, which were much more resistant to PD142893 than S6c‐induced relaxations of the aorta. The pD2 values for S6c in the absence and presence of PD142893 (10 μm) were 8.12±0.11 and 7.70±0.11, respectively.

In the gastric fundus of the ETB receptor gene knockout mouse, S6c and IRL1620 caused no contractions. ET‐1 (0.1 nm1 μm) caused contractions sensitive to both BQ‐123 and PD142893, indicating that only ETA receptors mediate ET‐1‐induced contractions of the knockout mouse gastric fundus.

Since both the PD142893‐sensitive vasodilator response of the aorta and the PD142893‐resistant contractile response of the gastric fundus to S6c were completely absent in the ETB receptor gene knockout mouse, we conclude that the two pharmacologically heterogeneous responses to S6c are mediated by receptors derived from the same ETB receptor gene.

British Journal of Pharmacology (1997) 120, 1427–1430; doi:10.1038/sj.bjp.0701054
Role of intracellular calcium in fast and slow desensitization of P2‐receptors in PC12 cells
Authors: Khiroug L. , Giniatullin R. , Talantova M. , Nistri A.
Published: August 2006

Combined whole‐cell patch clamp recording and confocal laser scanning microscopy of [Ca2+]i transients were performed on single PC12 cells to study any correlation between membrane currents induced by ATP and elevation in [Ca2+]i. ATP was applied by pressure from micropipettes near the recorded PC12 cells continuously superfused at a fast rate.

Brief (20 ms) pulses of ATP elicited monophasic inward currents and [Ca2+]i increases. Long applications (2 s) of ATP (5 mm) evoked peak currents which rapidly faded during the pulse and were followed by a large rebound current, interpreted as due to rapid desensitization and recovery of P2‐ receptors. The associated [Ca2+]i increase grew monotonically to a peak reached only after the occurrence of the current rebound, indicating that it is unlikely this cation has a role in fast desensitization.

Both membrane currents and [Ca2+]i transients were linearly dependent on holding membrane potential, suggesting that Ca2+ influx is the predominant cause of [Ca2+]i elevation. This view was supported by experiments carried out in Ca2+‐free solution.

Brief pulses of ATP applied after a desensitizing pulse (2 s) of the same elicited smaller inward currents and [Ca2+]i rises indicating a role for [Ca2+]i in controlling slow desensitization of P2‐receptors.

This notion was confirmed in experiments with various [Ca2+]i chelators which differentially affected slow desensitization in relation to their buffering capacity, while sparing fast receptor desensitization.

These results suggest a role for [Ca2+]i in slow rather than fast desensitization of P2‐receptors, thus proposing this divalent cation as an intracellular factor able to provide an efficient and reversible control over receptor activity induced by ATP.

British Journal of Pharmacology (1997) 120, 1552–1560; doi:10.1038/sj.bjp.0701060
The effect of ketotifen on nitric oxide synthase activity
Authors: Heyman Samuel N. , Karmeli Fanny , Brezis Mayer , Rachmilewitz Daniel
Published: August 2006

We studied the effect of ketotifen, a second generation H1‐receptor antagonist on nitric oxide synthase (NOS) activity in colonic mucosa and in renal tissues, and on rat renal haemodynamics in vivo.

Ketotifen (100 μg ml−1) increased human colonic NOS activity from 3.7±0.6 to 14.5±1.3 nmol g−1 min−1 (P<0.005, ANOVA). In rat renal cortical and medullary tissues ketotifen increased NOS activity by 55% and 86%, respectively (P<0.001). The stimulation of NOS activity was attenuated by NADPH deletion and by the addition of Nω nitro‐l‐arginine methyl ester (l‐NAME) or aminoguanidine, but not by [Ca2+] deprivation. NOS activity was unaffected by two other H1‐antagonists, diphenhydramine and astemizole, or by the structurally related cyproheptadine. Renal cortical NOS activity was also significantly stimulated 90 min after intravenous administration of ketotifen to anaesthetized rats.

Ketotifen administration to anaesthetized rats induced modest declines in blood pressure and reduced total renal, cortical and outer medullary vascular resistance. This is in contrast to diphenhydramine, which did not induce renal vasodilatation.

We conclude that ketotifen stimulates NOS activity by mechanisms other than H1‐receptor antagonism. The association of this effect with therapeutic characteristics of ketotifen and the clinical implications of these findings are yet to be defined.

British Journal of Pharmacology (1997) 120, 1545–1551; doi:10.1038/sj.bjp.0701063
Absence of prostaglandin E2‐induced hyperalgesia in NMDA receptor ε subunit knockout mice
Authors: Minami Toshiaki , Sugatani Junko , Sakimura Kenji , Abe Manabu , Mishina Masayoshi , Ito Seiji
Published: August 2006

We have previously found that intrathecal administration of prostaglandins E2 (PGE2) and D2 (PGD2) into conscious mice induced hyperalgesia by the hot plate test. The present study investigated the involvement of N‐methyl‐d‐aspartate (NMDA) receptor in the prostaglandin‐induced hyperalgesia by use of mice lacking NMDA receptor ε1, ε4, or ε1/ε4 subunits.

PGE2 induced hyperalgesia over a wide range of doses from 50 pg to 500 ng kg−1 in wild‐type mice. But PGE2 could not induce hyperalgesia in ε1, ε4, or ε1/ε4 subunit knockout mice.

The NMDA receptor antagonist d‐(−)‐2‐amino‐5‐phosphonovaleric acid (d‐AP5), the non‐NMDA receptor antagonist γ‐d‐glutamylaminomethyl sulphonic acid (GAMS), and the nitric oxide synthase inhibitor Nω‐nitro‐l‐arginine methyl ester (l‐NAME) inhibited the PGE2‐induced hyperalgesia in wild‐type mice.

PGD2 induced hyperalgesia at doses of 25 ng to 250 ng kg−1 in both wild‐type and ε1/ε4 subunit knockout mice. The substance P receptor antagonist CP 96,345 blocked the PGD2‐induced hyperalgesia in wild‐type and ε1/ε4 subunit knockout mice.

These results demonstrate that the pathways leading to hyperalgesia are different between PGD2 and PGE2 and that both ε1 and ε4 subunits of the NMDA receptor are involved in the PGE2‐induced hyperalgesia.

British Journal of Pharmacology (1997) 120, 1522–1526; doi:10.1038/sj.bjp.0701067
Species‐dependent differences in inotropic effects and phosphoinositide hydrolysis induced by endothelin‐3 in mammalian ventricular myocardium
Authors: Yang Huang‐Tian , Zhu Yi , Endoh Masao
Published: August 2006

Species‐dependent variations in the positive inotropic effect (PIE) of endothelin‐3 (ET‐3), and the relationships between the PIE and specific binding sites for [125I]‐ET‐3 and the PIE and the acceleration of phosphoinositide hydrolysis by ET‐3, were studied in ventricular muscles from the rat, guinea‐pig, rabbit, ferret and dog.

ET‐3 in the presence of (±)‐bupranolol (0.3 μm) and prazosin (0.3 μm) elicited a concentration‐dependent PIE in the ventricular muscle from the rat, guinea‐pig, rabbit and ferret. The potency of ET‐3 and its efficacy in inducing a PIE were highest in the rabbit, intermediate in the rat and guinea‐pig and lowest in the ferret. ET‐3 did not have any inotropic effect on ventricular muscle from the dog.

Specific high‐affinity binding of [125I]‐ET‐3 was observed with membrane fractions derived from the ventricular muscle of the five species. The maximal specific binding (Bmax) of ET‐3 was highest in the rat and guinea‐pig, intermediate in the rabbit and ferret and lowest in the dog. The values of KD in the rabbit and dog (33 and 52 pm) were lower than those in the rat, guinea‐pig and ferret (141221 pm).

In slices of ventricular muscle from all five species, ET‐3 increased the accumulation of [3H]‐inositol monophosphate (IP1) in a concentration‐dependent manner. The extent of accumulation of IP1 was highest in the rat, intermediate in the guinea‐pig and rabbit and lowest in the ferret and dog.

The results demonstrate the wide range of variations in the PIE of ET‐3 on mammalian ventricular muscles. The variations in the coupling processes subsequent to the acceleration of the hydrolysis of PI, triggered by the binding of ET‐3 to its receptor, might be important in these species‐dependent differences in the PIE of ET‐3.

British Journal of Pharmacology (1997) 120, 1497–1504; doi:10.1038/sj.bjp.0701085
Suppressive effect of melatonin administration on ethanol‐induced gastroduodenal injury in rats in vivo
Authors: Melchiorri Daniela , Sewerynek Ewa , Reiter Russel J , Ortiz Genaro G , Poeggeler Burkhard , Nisticò Giuseppe
Published: August 2006

Melatonin protection against ethanol‐induced gastroduodenal injury was investigated in duodenum‐ligated rats.

Melatonin, injected i.p. 30 min before administration of 1 ml of absolute ethanol, given by gavage, significantly decreased ethanol‐induced macroscopic, histological and biochemical changes in the gastroduodenal mucosa.

Ethanol‐induced lesions were detectable as haemorrhagic streaks. Ethanol administration damaged 36% and 25% of the total gastric and duodenal surface, respectively. Melatonin treatment reduced ethanol‐induced gastric and duodenal damage to 14% and 8%, respectively. When indomethacin was given together with ethanol, the gastric damaged area was 44% of the total surface, while the duodenal damaged area was 35%; melatonin administration reduced the damage to only 13% of the total gastric surface and to 12% of total duodenal surface.

Both stomach and duodenum of ethanol‐treated animals showed polymorphonuclear leukocyte (PMN) infiltration. The number of PMN increased more than 600 and 200 times in stomach and duodenum, respectively, following ethanol administration. Melatonin treatment reduced ethanol‐induced PMN infiltration by 38% in the stomach and 20% in the duodenum. In indomethacin‐ethanol‐treated rats, the number of PMN increased by 875% compared to control group in the stomach and by 264% in duodenum. Melatonin administration reduced the indomethacin‐ethanol‐induced PMN rise by 57% in the stomach and 40% in the duodenum.

Gastroduodenal total glutathione (tGSH) concentration and glutathione reductase (GSSG‐Rd) activity were significantly reduced following ethanol and indomethacin‐ethanol administration. Melatonin ameliorated both the decrease in tGSH concentration as well as the reduction of GSSG‐Rd activity elicited by ethanol both in the stomach and duodenum; melatonin was effective against indomethacin‐ethanol‐induced damage only in the stomach.

Ethanol‐induced gastroduodenal damage is believed to be mediated by the generation of free radicals. Recently, a number of in vivo and in vitro experiments have shown melatonin to be an effective antioxidant and free radical scavenger; thus, we conclude that the protection by melatonin against ethanol‐induced gastroduodenal injury is due, at least in part, to its radical scavenging activity.

British Journal of Pharmacology (1997) 121, 264–270; doi:10.1038/sj.bjp.0701104
Central antitussive activity of the NK1 and NK2 tachykinin receptor antagonists, CP‐99,994 and SR 48968, in the guinea‐pig and cat
Authors: Bolser Donald C , DeGennaro Frances C , O'Reilly Sandra , McLeod Robbie L , Hey John A
Published: August 2006

The purpose of this study was to investigate the antitussive activity and sites of action of the NK1 and NK2 tachykinin receptor antagonists, CP‐99,994, SR 48968, and the racemate of SR 48968, SR 48212A in the cat and guinea‐pig.

Guinea‐pigs were dosed subcutaneously (s.c.) with CP‐99,994, SR 48212A or SR 48968 one hour before exposure to aerosols of capsaicin (0.3 mM) to elicit coughing. Coughs were detected with a microphone and counted.

Intracerebroventricular (i.c.v.) cannulae were placed in the lateral cerebral ventricles of anaesthetized guinea‐pigs. Approximately one week later, the animals were dosed with CP‐99,994 or SR 48212A (i.c.v.) and exposed to aerosols of capsaicin (0.3 mM) to elicit coughing.

Cough was produced in anaesthetized cats by mechanical stimulation of the intrathoracic trachea and was monitored from electromyograms of respiratory muscle activity. Cannulae were placed for intravenous (i.v.) or, in separate groups of animals, intravertebral arterial (i.a.) administration of CP‐99,994, SR 48212A or SR 48968. Dose‐response relationships for i.v. and i.a. administration of each drug were generated to determine a ratio of i.v. ED50 to i.a. ED50, known as the effective dose ratio (EDR). The EDR will be 20 or greater for a centrally active drug and less than 20 for a peripherally active drug.

In the guinea‐pig, CP‐99,994 (0.1–30 mg kg−1, s.c.), SR 48212A (1.0–30 mg kg−1, s.c.), and SR 48968 (0.3–3.0 mg kg−1, s.c.) inhibited capsaicin‐induced cough in a dose‐dependent manner. Capsaicin‐induced cough was also inhibited by i.c.v. administration of CP‐99,994 (10 and 100 μg) or SR 48212A (100 μg).

In the cat, both CP‐99,994 (0.0001–0.3 mg kg−1, i.a., n=5; 0.003–3.0 mg kg−1, i.v., n=5) and SR 48212A (0.003–1.0 mg kg−1, i.a., n=5; 0.01–3.0 mg kg−1, i.v., n=5) inhibited mechanically induced cough by either the i.v. or i.a. routes in a dose‐dependent manner. SR 48968 (0.001–0.3 mg kg−1, i.a., n=5; 0.03–1.0 mg kg−1, i.v., n=5) inhibited cough when administered by the i.a. route in a dose‐dependent manner, but had no effect by the i.v. route up to a dose of 1.0 mg kg−1. Intravenous antitussive potencies (ED50, 95% confidence interval (CI)) of these compounds were: CP‐99,994 (0.082 mg kg−1, 95% CI 0.047–0.126), SR 48212A (2.3 mg kg−1, 95% CI 0.5–20), and SR 48968 (>1.0 mg kg−1, 95% CI not determined). The intra‐arterial potencies of these compounds were: CP‐99,994 (1.0 μg kg−1, 95% CI 0.4–1.8), SR 48212A (25 μg kg−1, 95% CI 13–52), and SR 48968 (8.0 μg kg−1, 95% CI 1–32). The derived EDRs for each compound were: CP‐99,994, 82; SR 48212A, 92; and SR 48968, >125.

We concluded that CP‐99,994 and SR 48968 inhibit cough in the guinea‐pig and cat by a central site of action. In the cat, the antitussive action of these compounds appears to be solely by a central site.

British Journal of Pharmacology (1997) 121, 165–170; doi:10.1038/sj.bjp.0701111
The stimulation of human neutrophil migration by angiotensin II: its dependence on Ca2+and the involvement of cyclic GMP
Authors: Elferink Jan G. R. , de Koster Ben M.
Published: August 2006

Angiotensin II had a bimodal effect on human neutrophil migration. Low concentrations of angiotensin II stimulated random migration. At a concentration of 10−10Mit caused a maximal increase of migration; migration increased from 47.2±2.1 μm in the absence of angiotensin II, to 73.1±2.2 μm with 10−10Mangiotensin II present in the lower compartment of the Boyden chamber (n=5,P<0.001). Stimulation of migration by angiotensin II was partly chemotactic and partly chemokinetic. Angiotensin II concentrations of 10−8Mand higher inhibited chemotactic peptide‐stimulated chemotaxis.

The stimulant effect of angiotensin II on migration was completely dependent on extracellular Ca2+. In the presence of 1 mMCa2+, angiotensin II stimulated migration to 76.1±1.7 μm, while migration in the absence of Ca2+was 42.2±1.9 μm (n=4,P<0.001). Different types of calcium channel blockers either moderately or strongly inhibited angiotensin II‐activated migration. Stimulation of migration by angiotensin II in intact cells required higher concentrations of Ca2+than in electroporated cells. This supports the view that there is an influx of Ca2+through the plasma membrane, and a requirement of calcium for an intracellular target.

Angiotensin II‐stimulated migration was inhibited by pertussis toxin; from 71.6±2.0 μm in the absence, to 43.6±1.5 μm in the presence of pertussis toxin (n=4,P<0.001). Migration of electroporated neutrophils stimulated by angiotensin II was synergistically enhanced by GTPγS. This suggests that one or more G‐proteins are involved in the activating effect of angiotensin II.

Inhibitors of soluble guanylate cyclase and antagonists of cyclic GMP‐dependent kinase strongly inhibited the activating effect of angiotensin II. The results suggest that the activating effect of angiotensin II is mediated by cyclic GMP and by cyclic GMP‐dependent kinase.

British Journal of Pharmacology(1997)121, 643–648; doi:10.1038/sj.bjp.0701167
Suppression by cyclosporin A of interleukin 1β‐induced expression of group II phospholipase A2 in rat renal mesangial cells
Authors: Walker Gaby , Kunz Dieter , Pignat Werner , van den Bosch Henk , Pfeilschifter Josef
Published: August 2006

We investigated whether cyclosporin A, a potent immunosuppressive drug, affects group II phospholipase A2 (PLA2; EC 3.1.1.4) induction in rat renal mesangial cells.

Previously we showed that the expression of group II PLA2 in rat renal mesangial cells is triggered by exposure of the cells to inflammatory cytokines such as interleukin 1β (IL‐1β) or tumour necrosis factor α and agents that elevate cellular levels of cyclic AMP. Treatment of mesangial cells with IL‐1β for 24 h induced PLA2 activity secreted into cell culture supernatants by about 16 fold. Incubation of mesangial cells with cyclosporin A inhibited IL‐1β‐induced PLA2 section in a dose‐dependent fashion, with an IC50 value of 4.3 μM. Cyclosporin A did not directly inhibit enzymatic activity of PLA2.

Immunoprecipitation of radioactively labelled PLA2 protein from mesangial cell supernatants revealed that the inhibition of PLA2 activity is due to a suppression of PLA2 protein levels. This effect was preceded by a reduction of PLA2 mRNA steady state levels, as demonstrated by Northern blot analyses of total cellular RNA isolated from stimulated mesangial cells.

In order to evaluate whether cyclosporin A would affect the transcriptional activity of the PLA2 gene, we performed nuclear run on transcription experiments and provided evidence that the transcription rate of the PLA2 gene is reduced by cyclosporin A.

Previously we found that the nuclear transcription factor κB (NFκB) is an essential component of the IL‐1β‐dependent upregulation of PLA2 gene transcription. By electrophoretic mobility shift analysis, we demonstrated that cyclosporin A diminishes the formation of NFκB DNA‐binding complexes, thus suggesting that this transcription factor is a target for cyclosporin A‐mediated repression of PLA2 gene transcription.

The data presented in this study strongly suggest that the cellular mechanism involved in the IL1β ‐ dependent transcriptional upregulation of the PLA2 gene in mesangial cells is a target for the action of cyclosporin A.

British Journal of Pharmacology (1997) 121, 787–793; doi:10.1038/sj.bjp.0701170
The involvement of reactive oxygen species and arachidonic acid in α1‐adrenoceptor‐induced smooth muscle cell proliferation and migration
Authors: Nishio Eisuke , Watanabe Yasuhiro
Published: August 2006

In a previous study, we demonstrated phenylephrine‐stimulated arachidonic acid (AA) release in rabbit cultured aortic smooth muscle cells. Therefore, we have investigated the functional implications of AA which are involved in the cellular response to phenylephrine, particularly proliferation and migration of rabbit cultured aortic smooth muscle cells.

First, to determine whether AA directly modifies proliferation and mobility of vascular smooth muscle cells (VSMCs), we exposed the cells to AA. AA induced proliferation and migration of the cells in a dose‐dependent fashion. Concomitantly added catalase inhibited the proliferation and chemotaxis induced by AA of VSMCs. Conversely, aminotriazole enhanced the proliferation and migration induced by AA.

Secondly, we investigated whether the proliferation and migration of VSMCs by phenylephrine were related to AA and hydrogen peroxide (H2O2). The proliferation and chemotaxis of VSMCs by phenylephrine were inhibited by a phospholipase A2 (PLA2) inhibitor, or catalase.

Lastly, we investigated the effects of AA and phenylephrine on the content of H2O2 in VSMCs. AA and phenylephrine treatment led to an increase of H2O2 in a dose‐dependent manner.

These results suggest that the addition of phenylephrine to the cells caused the enhancement of proliferation and migration, probably by mediating AA release and reactive oxygen species (ROS) production.

British Journal of Pharmacology (1997) 121, 665–670; doi:10.1038/sj.bjp.0701171
Inhibition of Ca2+ channel current by μ‐ and κ‐opioid receptors coexpressed in Xenopus oocytes: desensitization dependence on Ca2+ channel α1 subunits
Authors: Kaneko Shuji , Yada Nobumichi , Fukuda Koichiro , Kikuwaka Masanobu , Akaike Akinori , Satoh Masamichi
Published: August 2006

Desensitization of μ‐ and κ‐opioid receptor‐mediated inhibition of voltage‐dependent Ca2+ channels was studied in a Xenopus oocyte translation system.

In the oocytes coexpressing κ‐opioid receptors with N‐ or Q‐type Ca2+ channel α1 and β subunits, the κ‐agonist, U50488H, inhibited both neuronal Ca2+ channel current responses in a pertussis toxin‐sensitive manner and the inhibition was reduced by prolonged agonist exposure.

More than 10 min was required to halve the inhibition of Q‐type channels by the κ‐agonist. However, the half‐life for the inhibition of N‐type channels was only 6±1 min. In addition, in the oocytes coexpressing μ‐opioid receptors with N‐type or Q‐type channels, the uncoupling rate of the μ‐receptor‐mediated inhibition of N‐channels was also faster than that of Q‐type channels.

In the oocytes coexpressing both μ‐ and κ‐receptors with N‐type channels, stimulation of either receptor resulted in a cross‐desensitization of the subsequent response to the other agonist. Treatment of oocytes with either H‐8 (100 μM), staurosporine (400 nM), okadaic acid (200 nM), phorbol myristate acetate (5 nM) or forskolin (50 μM) plus phosphodiesterase inhibitor did not affect either the desensitization or the agonist‐evoked inhibition of Ca2+ channels.

These results suggest that the rate of rapid desensitization is dependent on the α1 subtype of the neuronal Ca2+ channel, and that a common phosphorylation‐independent mechanism underlies the heterologous desensitization between opioid receptor subtypes.

British Journal of Pharmacology (1997) 121, 806–812; doi:10.1038/sj.bjp.0701181
Hydrogen peroxide‐induced impairment of reactivity in rat isolated aorta: potentiation by 3‐amino‐1,2,4‐triazole
Authors: Mian Kousar B. , Martin William
Published: August 2006

In this study the impairment induced by hydrogen peroxide of vascular reactivity and the role of endogenous catalase in protection against this impairment was assessed in isolated rings of rat aorta.

Incubation with hydrogen peroxide at 1 mM, but not at 0.1 mM, for 15, 30 or 60 min followed by washout depressed, in a time‐dependent manner, the subsequent ability of endothelium‐containing and endothelium‐denuded rings to contract to phenylephrine.

Incubation with 3‐amino‐1,2,4‐triazole (50 mM, 90 min, followed by washout) to inhibit endogenous catalase had no effect by itself on subsequent phenylephrine‐induced contraction. However, pretreatment with 3‐amino‐1,2,4‐triazole did lead to a profound enhancement of the ability of hydrogen peroxide (1 mM, present for the final 30 min of the 90 min incubation, followed by washout) to depress phenylephrine‐induced contraction in both endothelium‐containing and endothelium‐denuded rings.

Incubation with hydrogen peroxide at 1 mM, but not at 0.1 mM, for 15, 30 or 60 min followed by washout inhibited, in a time‐dependent manner, the subsequent ability of acetylcholine (10 nM–3 μM) to induce endothelium‐dependent relaxation. Furthermore, incubation with hydrogen peroxide 1 mM (30 min, followed by washout) also inhibited relaxation induced by glyceryl trinitrate (1–100 nM) or isoprenaline (10 nM–3 μM) in endothelium‐denuded rings.

Incubation with 3‐amino‐1,2,4‐triazole (50 mM, 90 min, followed by washout) had no effect by itself on relaxation induced by acetylcholine, glyceryl trinitrate or isoprenaline. In contrast, pretreatment with 3‐amino‐1,2,4‐triazole led to profound enhancement of the ability of hydrogen peroxide (1 mM, present for final 30 min of the 90 min incubation) to block relaxation to acetylcholine, glyceryl trinitrate or isoprenaline.

On the basis of the actions of 3‐amino‐1,2,4‐triazole, it is likely that endogenous catalase plays an important role in the protection of vascular reactivity of rat aorta against oxidant damage by high (1 mM) but not lower (0.1 mM) concentrations of hydrogen peroxide. The data are consistent with the promotion of non‐selective damage to the vascular smooth muscle cells by hydrogen peroxide, but endothelial damage may also be sustained.

British Journal of Pharmacology (1997) 121, 813–819; doi:10.1038/sj.bjp.0701187
The involvement of ATP‐sensitive potassium channels in β2‐adrenoceptor agonist‐induced vasodilatation on rat diaphragmatic microcirculation
Authors: Chang Han‐Yu
Published: August 2006

The effects of glibenclamide (GLB), a blocker of ATP‐sensitive potassium (KATP) channels, on diaphragmatic microcirculation in male Sprague‐Dawley rats were assessed under basal conditions and after β2‐adrenoceptor‐agonist stimulation. In addition, forskolin was used to bypass β‐adrenoceptors and GTP‐binding proteins (G‐protein) to explore the possible mechanism of GLB effects. For comparison, the relationships between KATP channel activity and cyclic GMP‐mediated vasodilator responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were also assessed.

Male Sprague‐Dawley rats were anaesthetized with urethane and mechanically ventilated. The left hemi‐diaphragm of each rat was prepared and microvascular blood flow (QLDF) was recorded with laser‐Doppler flowmetry during continuous superfusion with bicarbonate‐buffered, prewarmed Ringer solution. The drugs were topically applied to the surface of the hemi‐diaphragm.

Salbutamol (0.32–32 μM), terbutaline (0.32 μM–0.32 μM) and forskolin (0.32–10 μM) each elicited a concentration‐dependent increase in QLDF.

Baseline microvascular blood flow was unaffected by a 30 min suffusion of 1 μM GLB (295±51 mV vs 325±62 mV, P=0.738).

The vasodilator response elicited by salbutamol (0.32 μM, 1 μM and 3.2 μM), was significantly attenuated by a 30 min superfusion with 1 μM GLB; this salbutamol‐induced vasodilatation was mediated via an interaction with β‐adrenoceptor receptors, as in other experiments it was greatly inhibited by 30‐min superfusion with propranolol (10 μM).

Similarly, following 30‐min superfusion with GLB (1 μM), the terbutaline (1 μM, 3.2 μM and 10 μM)‐induced vasodilator response was almost abolished and the vasodilator responses induced by incremental concentrations of forskolin (0.32 μM, 1 μM and 3.2 μM) were also significantly attenuated.

Cromakalim (1.5 μM, 3 μM and 3.2 μM) produced an increase of QLDF in a dose‐dependent manner, which was virtually abolished by GLB (1 μM). In contrast, the vasodilator responses induced by acetylcholine (32 μM, 0.1 mM, and 0.32 mM) or sodium nitroprusside (3.2 μM, 10 μM and 20 μM) were independent of GLB (1 μM).

In conclusion, KATP channels may be functional, but tonically inactive in the resting diaphragmatic microcirculation and the vasodilator effect of β2‐adrenoceptor agonists may be partly mediated by KATP channels; the activation of KATP channels may involve the accumulation of cyclic AMP in vascular smooth muscle cells.
Inhibition of 5‐hydroxytryptamine‐induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal smooth muscle cells by phorbol ester
Authors: Yang Chuen‐Mao , Fen Lir‐Wan , Tsao Hui‐Liang , Chiu Chi‐Tso
Published: August 2006

Regulation of the increase in inositol‐1,4,5‐trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by 5‐hydroxytryptamine (5‐HT) caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration‐dependent manner.

Pretreatment of TSMCs with phorbol 12‐myristate 13‐acetate (PMA, 1 μM) for 30 min blocked the 5‐HT‐induced IP3 formation and Ca2+ mobilization. This inhibition was reduced after the cells had been incubated with PMA for 8 h, and within 48 h the 5‐HT‐induced Ca2+ mobilization reached the same extent as control cells.

The concentration of PMA that gave half‐maximal inhibition of 5‐HT‐induced increase in [Ca2+]i was 4 nM. Pretreatment of TSMCs with staurosporine (1 μM) of GF109203X (0.1 μM), PKC inhibitors, inhibited the ability of PMA to attenuate 5‐HT‐induced responses, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC.

In parallel with the effect of PMA on 5‐HT‐induced IP3 formation and Ca2+ mobilization, the translocation and down‐regulation of PKC isozymes were determined by Western blot analysis in TSMCs. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TSMCs expressed PKC‐α, βI, βII, δ, ε, θ and ζ. With PMA treatment of the cells for various times, translocation of PKC‐α, βI, βII, δ, ε and θ from the cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 24 h treatment caused a partial down‐regulation of these PKC isozymes PKC‐ζ was not significantly translocated and down‐regulated at any of the times tested.

In conclusion, these results suggest that activation of PKC may inhibit the receptor‐mediated phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit both responses independently. The translocation of PKC‐α, βI, βII, δ, ε, and θ induced by PMA caused an attenuation of 5‐HT‐stimulated IP3 accumulation and Ca2+ mobilization in TSMCs.
Effects of [3H]‐BIDN, a novel bicyclic dinitrile radioligand for GABA‐gated chloride channels of insects and vertebrates
Authors: Rauh James J , Benner Eric , Schnee Michael E , Cordova Daniel , Holyoke Caleb W , Howard Michael H , Bai Donglin , Buckingham Steven D , Hutton Michael L , Hamon Alain , Roush Richard T , Sattelle David B
Published: August 2006

The radiolabelled bicyclic dinitrile, [3H]‐3,3‐bis‐trifluoromethyl‐bicyclo[2.2.1]heptane‐2,2‐dicarbonitrile ([3H]‐BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of γ‐aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35±3 nM) and dieldrin (IC50, 93±7 nM), displaced [3H]‐BIDN from rootworm membranes. When tested at 100 μM, 1‐(4‐ethynylphenyl)‐4‐n‐propyl‐2,6,7‐trioxabicyclo[2.2.2]octane(EBOB), 4‐t‐butyl‐2,6,7‐trioxa‐1‐phosphabicyclo[2.2.2]octane‐1‐thione (TBPS), 1‐phenyl‐4‐t‐butyl‐2,6,7‐trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [3H]‐BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands.

Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and α and β endosulphan displace bound [3H]‐BIDN from rootworm membranes by a competitive mechanism.

Rat brain membranes were also shown to possess a population of saturable, specific [3H]‐BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [3H]‐BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm.

Functional GABA‐gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABAB receptors were unaffected.

Expression in Xenopus oocytes of either rat brain mRNA, or cDNA‐derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo‐oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA‐gated Cl− channels to which a number of insecticidally‐active molecules bind.
Neocuproine, a selective Cu(I) chelator, and the relaxation of rat vascular smooth muscle by S‐nitrosothiols
Authors: Al‐Sa'doni H H , Megson I L , Bisland S , Butler A R , Flitney F W
Published: August 2006

A study has been made of the effect of neocuproine, a specific Cu(I) chelator, on vasodilator responses of rat isolated perfused tail artery to two nitrosothiols: S‐nitroso‐N‐acetyl‐d,l‐penicillamine (SNAP) and S‐nitroso‐glutathione (GSNO).

Bolus injections (10μl) of SNAP or GSNO (10−7–10−3m) were delivered into the lumen of perfused vessels pre‐contracted with sufficient phenylephrine (1–7μm) to develop pressures of 100–120mmHg. Two kinds of experiment were made: SNAP and GSNO were either (a) pre‐mixed with neocuproine (10−4m) and then injected into arteries; or (b) vessels were continuously perfused with neocuproine (10−5m) and then injected with either pure SNAP or GSNO.

In each case, neocuproine significantly attenuated vasodilator responses to both nitrosothiols, although the nature of the inhibitory effect differed in the two types of experiment. We conclude that the ability of exogenous nitrosothiols to relax vascular smooth muscle in our ex vivo model is dependent upon a Cu(I) catalyzed process. Evidence is presented which suggests that a similar Cu(I)‐dependent mechanism is responsible for the release of NO from endogenous nitrosothiols and that this process may assist in maintaining vasodilator tone in vivo.
Differential contribution of two serine residues of wild type and constitutively active β2‐adrenoceptors to the interaction with β2‐selective agonists
Authors: Kikkawa Hideo , Kurose Hitoshi , Isogaya Masafumi , Sato Yoji , Nagao Taku
Published: August 2006

We have studied the difference in receptor binding activity between partial and full β2‐adrenoceptor agonists and the abilities of the agonists to interact with Ser204 and Ser207 in the fifth transmembrane region of the β2‐adrenoceptor, amino acid residues that are important for activation of the β2‐adrenoceptor.

In the binding study with [125I]‐iodocyanopindolol, the Ki values of (±)‐salbutamol, (±)‐salmeterol, TA‐2005 and (−)‐isoprenaline for the β2‐adrenoceptor expressed in COS‐7 cell membranes were 3340, 21.0, 12.0 and 904nm, respectively. The β1/β2 selectivity of these agonists was in the order of (±)‐salmeterol (332 fold)>TA‐2005 (52.8)>(±)‐salbutamol (6.8)>(−)‐isoprenaline (1.1), and the β3‐/β2‐adrenoceptor selectivity of these agonists was in the order of TA‐2005 (150 fold)>(±)‐salmeterol (88.6)>(±)‐salbutamol (10.4)>(−)‐isoprenaline (3.2).

The maximal activation of adenylyl cyclase by stimulation of the β1‐, β2‐ and β3‐adrenoceptors by TA‐2005 was 32, 100 and 100% of that by (−)‐isoprenaline, respectively, indicating that TA‐2005 is a full agonist at the β2‐ and β3‐adrenoceptors and a partial agonist at the β1‐adrenoceptor. (±)‐Salbutamol and (±)‐salmeterol were partial agonists at both β1‐ (8% and 9% of (−)‐isoprenaline) and β2‐ (83% and 74% of (−)‐isoprenaline) adrenoceptors.

The affinities of full agonists, TA‐2005 and (−)‐isoprenaline, were markedly decreased by substitution of Ala for Ser204 (S204A) of the β2‐adrenoceptor, whereas this substitution slightly reduced the affinities of partial agonists, (±)‐salbutamol and (±)‐salmeterol. Although the affinities of full agonists for the S207A‐β2‐adrenoceptor were decreased, those of partial agonists for the S207A‐β2‐adrenoceptor were essentially the same as for the wild type receptor.

The constitutively active mutant (L266S, L272A) of the β2‐adrenoceptor had an increased affinity for all four agonists. The affinities of full agonists were decreased by substitution of Ser204 of the constitutively active mutant, whereas the degree of decrease was smaller than that caused by the substitution of the wild type receptor. Although the affinities of (±)‐salbutamol and (±)‐salmeterol for the S207A‐β2‐adrenoceptor were essentially the same as those for the wild type β2‐adrenoceptor, the affinities of (±)‐salbutamol and (±)‐salmeterol for the constitutively active β2‐adrenoceptor were decreased by substitution of Ser207.

These results suggest that Ser204 and Ser207 of the wild type and constitutively active β2‐adrenoceptors differentially interacted with β2‐selective agonists.
Noradrenaline release and the effect of endogenous activation of the phospholipase C/protein kinase C signalling pathway in rat atria
Authors: Majewski H , Kotsonis P , Murphy T V , Barrington M
Published: August 2006

It has been proposed that protein kinase C (PKC) in sympathetic nerves is activated during action‐potential evoked release of noradrenaline and helps maintain transmitter output. We studied this phenomenon further in rat atria radiolabelled with [3H]‐noradrenaline.

Noradrenaline release was elevated by continuous electrical stimulation of the atria for 10min at either 5 or 10Hz. Two inhibitors of PKC, polymyxin B (21μm) and Ro 318220 (3μm), markedly inhibited the release of noradrenaline but only at the higher stimulation frequency.

Further experiments were conducted with 10Hz stimulation but for shorter train durations. In this case polymyxin B inhibited noradrenaline release during a 10 or 15s train of impulses but not during a 5s train. This suggests that PKC effects are induced during the stimulation train by some process.

The diacylglycerol kinase inhibitor R59949 (10μm), which prevents the breakdown of diacylglycerol, enhanced noradrenaline release elicited by stimulation at 10Hz for 10 or 15s. This effect was not seen if polymyxin B was present and suggests that diacylglycerol is the endogenous activator of PKC.

The source of the diacylglycerol may be through phospholipase C pathways, since the phospholipase C inhibitor U73122 (3μm) inhibited noradrenaline release at 10Hz for 10s and the effect was not seen if polymyxin B was also present.

It is unlikely that phospholipase D is the source of diacylglycerol. Although the phospholipase D inhibitor wortmannin (1μm) inhibited noradrenaline release, this effect was still observed in the presence of polymyxin B. Furthermore ethanol, which inhibits diacylglycerol formation by phospholipase D, had no effect on noradrenaline release.

We therefore suggest that during a train of high frequency pulses phospholipase C is activated and this results in the production of diacylglycerol which in turn activates PKC. This enables the neurones to maintain transmitter release at a high level.