bluuebunny/crossref_metadata_2025 huggingface dataset:
Histamine H2 receptors mediate the inhibitory effect of histamine on human eosinophil degranulation
Authors: Ezeamuzie Charles I , Philips Elizabeth
Published: January 2005

The effect of histamine on human eosinophil degranulation and the receptor mediating such effect were studied in vitro using the complement C5a‐mediated eosinophil peroxidase (EPO) release model.

Following pre‐treatment with 5 μg ml−1 cytochalasin B(CB), C5a induced a concentration‐dependent release of EPO from eosinophils isolated from healthy donors.

Histamine (0.1–50 μM), but not L‐histidine, inhibited concentration‐dependently C5a‐induced EPO release with IC50 (95% CI) of 0.6 μM (0.3–1.2 μM) and maximal inhibition of ∼60%.

A similar effect was seen with the selective H2 agonists dimaprit (IC50 (95% CI)=6.9 μM (3.2–10.6 μM)) and amthamine (IC50 (95% CI)=0.4 μM (0.2–0.7 μM)). Neither the selective H1 agonist 6‐(2‐(4‐imidazolyl)ethylamino)‐N‐(4‐trifluoromethylphenyl) heptanecarboxamide(HTMT), nor the selective H3 agonists imetit (up to 100 μM) had any significant effect.

The inhibition by histamine was reversed by cimetidine (0.1–30 μM) and other H2 antagonists, but not the H1 antagonist mepyramine (1.0–100 μM), nor the H3 antagonist thioperamide (1.0–100 μM). Cimetidine (1–30 μM) shifted to the right the dimaprit log dose‐response curve, producing a pA2 value of 5.9 and Schild's plot slope of 0.98, thus confirming simple competitive antagonism.

Histamine (10–100 μM) increased intracellular level of adenosine 3′,5′‐cyclic monophosphate, which was completely abolished by cimetidine (30 μM), but not mepyramine or thioperamide. The cyclic AMP analogue – dibutyryl cyclic AMP – also inhibited degranulation (IC50 ∼300 μM). The cyclic AMP phosphodiesterase(PDE) IV inhibitor rolipram (10 μM) synergistically enhanced the inhibition of EPO release by histamine.

These results suggest that histamine, via stimulation of H2 receptors and a consequent elevation of intracellular levels of cyclic AMP, inhibits human eosinophil degranulation.

British Journal of Pharmacology (2000) 131, 482–488; doi:10.1038/sj.bjp.0703556
Induction of COX‐2 and PGE2 biosynthesis by IL‐1β is mediated by PKC and mitogen‐activated protein kinases in murine astrocytes
Authors: Molina‐Holgado Eduardo , Ortiz Sergio , Molina‐Holgado Francisco , Guaza Carmen
Published: January 2005

Interleukin‐1 (IL‐1) is an important mediator of immunoinflammatory responses in the brain. In the present study, we examined whether prostaglandin E2 (PGE2) production after IL‐1β stimulation is dependent upon activation of protein kinases in astroglial cells.

Astrocyte cultures stimulated with IL‐1β or the phorbol ester, PMA significantly increased PGE2 secretion. The stimulatory action of IL‐1β on PGE2 production was totally abolished by NS‐398, a specific inhibitor of cyclo‐oxygenase‐2 activity, as well as by the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone. Furthermore, IL‐1β induced the expression of COX‐2 mRNA. This occurred early at 2 h, with a maximum at 4 h and declined at 12 h. IL‐1 β treatment also induced the expression of COX‐2 protein as determined by immunoblot analysis. In that case the expression of the protein remained high at least up to 12 h.

Treatment of cells with protein kinase C inhibitors (H‐7, bisindolylmaleimide and calphostin C) inhibited IL‐1β stimulation of PGE2. In addition, PKC‐depleted astrocyte cultures by overnight treatment with PMA no longer responded to PMA or IL‐1. The ablation of the effects of PMA and IL‐1β on PGE2 production, likely results from down‐regulation of phorbol ester sensitive‐PKC isoenzymes. Immunoblot analysis demonstrated the translocation of the conventional isoform cPKC‐α from cytosol to membrane following treatment with IL‐1β.

In addition, IL‐1β treatment led to activation of extracellular signal‐regulated kinase (ERK1/2) and p38 subgroups of MAP kinases in astroglial cells. Interestingly, the inhibition of ERK kinase with PD 98059, as well as the inhibition of p38 MAPK with SB 203580, prevented IL‐1β‐induced PGE2 release.

ERK1/2 activation by IL‐1β was sensitive to inhibition by the PKC inhibitor bisindolylmaleimide suggesting that ERK phosphorylation is a downstream signal of PKC activation.

These results suggest key roles for PKC as well as for ERK1/2 and p38 MAP kinase cascades in the biosynthesis of PGE2, likely by regulating the induction of cyclo‐oxygenase‐2, in IL‐1β‐stimulated astroglial cells.

British Journal of Pharmacology (2000) 131, 152–159; doi:10.1038/sj.bjp.0703557
Effect of saikosaponin, a triterpene saponin, on apoptosis in lymphocytes: association with c‐myc, p53, and bcl‐2 mRNA
Authors: Hsu Ming‐Jen , Cheng Jur‐Shan , Huang Huei‐Chen
Published: January 2005

The mechanisms involved in the apoptotic effect of saikosaponin‐d, a triterpene saponin from Bupleurum falcatum L., were studied in human CEM lymphocytes and compared with those of dexamethasone (3×10−7 M).

Saikosaponin‐d (10−8 to 10−5 M) inhibited the serum‐stimulated [3H]‐thymidine incorporation in a concentration‐dependent manner. Dexamethasone also inhibited serum‐stimulated [3H]‐thymidine incorporation.

Cell viability was unaffected by saikosaponin‐d until 10−5–10−4 M. Dexamethasone significantly reduced the number of viable cells.

Following saikosaponin‐d (10−5–10−4 M) treatment, flow cytometry analysis of propidium iodide‐stained cells showed a significant increase in the percentage of cells in the apoptotic region. Dexamethasone also significantly increased the percentage of apoptotic cells. The supravital exposure to propidium iodide and annexin V labelling demonstrated that saikosaponin‐d (10−5–10−4 M) induced apoptosis as well as necrosis.

The apoptotic effect of saikosaponin‐d (3×10−6–10−4 M) was also demonstrated by TUNEL analysis and DNA laddering. The percentage of apoptotic cells induced by saikosaponin‐d (3×10−6–10−5 M) was unaffected by the presence of Z‐VAD‐FMK, indicating that saikosaponin‐d‐induced apoptosis may not be mediated by caspase activity. However, the percentage of apoptotic cells induced by dexamethasone was significantly reduced by the presence of Z‐VAD‐FMK.

Levels of c‐myc, p53, and bcl‐2 mRNA were analysed by the reverse transcription‐polymerase chain reaction. Levels of c‐myc and p53 mRNA were significantly increased, while the level of bcl‐2 mRNA was decreased, by saikosaponin‐d (10−5 M) treatment. Dexamethasone did not significantly change the expression of these genes.

It is suggested that the apoptotic effect of saikosaponin‐d may be partly mediated by increases in c‐myc and p53 mRNA levels accompanied by a decrease in bcl‐2 mRNA level.

British Journal of Pharmacology (2000) 131, 1285–1293; doi:10.1038/sj.bjp.0703559
Anticonvulsant and proconvulsant effects of tramadol, its enantiomers and its M1 metabolite in the rat kindling model of epilepsy
Authors: Potschka Heidrun , Friderichs Elmar , Löscher Wolfgang
Published: January 2005

The centrally acting analgesic tramadol has recently been reported to cause seizures at re‐commended dosages in patients, whereas animal experiments had indicated that seizures only occur in high, toxic doses. Tramadol has a dual mechanism of action that includes weak agonistic effects at the mu‐opioid receptor as well as inhibition of monoamine (serotonin, norepinephrine) re‐uptake. Its major (M1) metabolite mono‐O‐desmethyltramadol, which is rapidly formed in vivo, has a markedly higher affinity for mu receptors and may thus contribute to the effects of the parent compound. Furthermore, the pharmacological effects of tramadol appear to be related to the different, but complementary and interactive pharmacologies of its enantiomers. In the present study, we evaluated (±)‐tramadol, its enantiomers, and its M1 metabolite ((+)‐enantiomer) in the amygdala kindling model of epilepsy in rats. Adverse effects determined in kindled rats were compared to those in nonkindled rats.

At doses within the analgesic range, (±)‐tramadol and its enantiomers induced anticonvulsant effects in kindled rats. However, at only slightly higher doses seizures occurred. With (±)‐tramadol, generalized seizures were observed at 30 mg kg−1 in most kindled but not in nonkindled rats. The (−)‐enantiomer induced myoclonic seizures at 30 mg kg−1 in most kindled but not in nonkindled rats, although myoclonic seizure activity was observed in some nonkindled rats at 10 or 20 mg kg−1. Seizures were also observed after the (+)‐enantiomer and the (+)‐enantiomer of the M1 metabolite, but experiments with higher doses of these compounds were limited by marked respiratory depression.

The data demonstrate that kindling enhances the susceptibility of rats to convulsant adverse effects of tramadol and its enantiomers, indicating that a preexisting lowered seizure threshold increases the risk of tramadol‐induced seizures.

British Journal of Pharmacology (2000) 131, 203–212; doi:10.1038/sj.bjp.0703562
Extracellular ATP‐dependent activation of plasma membrane Ca2+ pump in HEK‐293 cells
Authors: Qi Z , Murase K , Obata S , Sokabe M
Published: January 2005

It is well known that extracellular ATP (ATPo) elevates the intracellular Ca2+ concentration ([Ca2+]i) by inducing Ca2+ influx or mobilizing Ca2+ from internal stores via activation of purinoceptors in the plasma membrane. This study shows that ATPo also activates the plasma membrane Ca2+ pumps (PMCPs) to bring the elevated [Ca2+]i back to the resting level in human embryonic kidney‐293 (HEK‐293) cells.

The duration of ATPo‐induced intracellular Ca2+ transients was significantly increased by PMCP blockers, La3+ or orthovanadate. In contrast, replacement of extracellular Na+ with NMDG+, a membrane‐impermeable cation, had no significant effect on duration, thus suggesting that Na+/Ca2+ exchangers do not participate in the ATPo‐induced Ca2+ transient.

A rapid and significant decrease in [Ca2+]i, which was not dependent on extracellular Na+, was induced by ATPo in cells pretreated with thapsigargin (TG). This decrease was blocked by orthovanadate, indicating that it was caused by PMCPs rather than sarco/endoplasmic reticulum Ca2+ pumps (SERCPs).

UTP and ATPγS also caused a decrease in [Ca2+]i in cells pretreated with TG, although they were less effective than ATP. The effect of UTP implies the involvement of both P2Y1 and P2Y2 receptors, while the effect of ATPγS implies no significant role of ectophosphorylation and agonist hydrolysis in the agonist‐induced [Ca2+]i decreases.

These results point to a role of PMCPs in shaping the Ca2+ signal and in restoring the resting [Ca2+]i level to maintain intracellular Ca2+ homeostasis after agonist stimulation.

British Journal of Pharmacology (2000) 131, 370–374; doi:10.1038/sj.bjp.0703563
Effects of halothane on the transient outward K+ current in rat ventricular myocytes
Authors: Davies Lucinda A , Hopkins Philip M , Boyett Mark R , Harrison Simon M
Published: January 2005

Halothane has been shown to affect several membrane currents in cardiac tissue including the L‐type calcium current (ICa), sodium current and a variety of potassium currents. However, little is known about the effects of halothane on the transient outward K+ current (Ito).

Single ventricular myocytes from rat hearts were voltage clamped using the whole cell patch configuration and an EGTA‐containing pipette solution to record the Ca2+‐independent, 4‐aminopyridine sensitive component of Ito. 300 μM Cd2+ or 10 μM nifedipine was used to block ICa.

At +80 mV, Ito (peak current minus current at the end of the pulse) was 1.8±0.2 nA under control conditions which was reduced to 1.3±0.2 nA by 1 mM halothane (P<0.001, mean±s.e.mean, n=9). The inhibition of Ito by halothane was concentration‐dependent (K0.5, 1.1±0.2 mM). One mM halothane led to a 16 mV shift in the steady‐state inactivation curve towards negative membrane potentials (P=0.005, n=8) but had no significant effect on the activation‐voltage relationship (P=0.724).

One mM halothane also increased the rate of inactivation of Ito; the dominant time constant of inactivation was reduced from 14±1 to 9±1 ms (P=0.017, mean±s.e.mean, n=6).

These data show that halothane reduced Ito; 0.3 mM, close to the MAC50 value for halothane, inhibited the current by 15% and as such, the inhibition of Ito will be relevant to the clinical situation. Halothane induced a shift in the steady‐state inactivation curve and accelerated the inactivation process of Ito which could be responsible for its inhibitory effect.

Due to the differential transmural expression of Ito in ventricular tissue, inhibition of Ito would reduce the transmural dispersion of refractoriness which could contribute to the arrhythmogenic properties of halothane.

British Journal of Pharmacology (2000) 131, 223–230; doi:10.1038/sj.bjp.0703565
Effect of MEN 10755, a new disaccharide analogue of doxorubicin, on sarcoplasmic reticulum Ca2+ handling and contractile function in rat heart
Authors: Zucchi Riccardo , Yu Gongyuan , Ghelardoni Sandra , Ronca Francesca , Ronca‐Testoni Simonetta
Published: January 2005

The use of anthraquinone antineoplastic agents is limited by their cardiac toxicity, which is largely due to activation of the sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor). MEN 10755 is a new disaccharide analogue of doxorubicin. We have evaluated its effects on SR function and its toxicity in isolated working rat hearts.

In rat SR vesicles, doxorubicin stimulated [3H]‐ryanodine binding by increasing its Ca2+‐sensitivity. At 1 μM Ca2+, ryanodine binding increased by 15.3±2.5 fold, with EC50=20.6 μM. Epirubicin produced a similar effect, i.e. 9.7±0.6 fold stimulation with EC50=11.1 μM. MEN 10755 increased ryanodine binding by 1.9±0.3 fold (P<0.01 vs doxorubicin and epirubicin), with EC50=38.9 μM.

Ca2+‐induced Ca2+ release experiments were performed by quick filtration technique, after SR loading with 45Ca2+. At 2 μM Ca2+, doxorubicin (50 μM) increased the rate constant of Ca2+ release to 82±5 s−1 vs a control value of 22±2 s−1 (P<0.01), whereas 50 μM MEN 10755 did not produce any significant effect (24±3 s−1).

Ca2+‐ATPase activity and 45Ca2+‐uptake were not significantly affected by doxorubicin, its 13‐dihydro‐derivative, epirubicin, MEN 10755 and the 13‐dihydro‐derivative of MEN 10755, at concentrations 100 μM.

In isolated heart experiments, administration of 30 μM doxorubicin or epirubicin caused serious contractile impairment, whereas 30 μM MEN 10755 produced only minor effects.

In conclusion, in acute experiments MEN 10755 was much less cardiotoxic than equimolar doxorubicin or epirubicin. This result might be accounted for by reduced activation of SR Ca2+ release.

British Journal of Pharmacology (2000) 131, 342–348; doi:10.1038/sj.bjp.0703575
Amphotericin B severely affects expression and activity of the endothelial constitutive nitric oxide synthase involving altered mRNA stability
Authors: Suschek Christoph Viktor , Bonmann Eckhard , Kleinert Hartmut , Wenzel Michael , Mahotka Csaba , Kolb Hubert , Förstermann Ulrich , Gerharz Claus‐Dieter , Kolb‐Bachofen Victoria
Published: January 2005

The therapeutic use of the antifungal drug amphotericin B (AmB) is limited due to severe side effects like glomerular vasoconstriction and risk of renal failure during AmB administration. As nitric oxide (NO) has substantial functions in renal autoregulation, we have determined the effects of AmB on endothelial constitutive NO synthase (ecNOS) expression and activity in human and rat endothelial cell cultures.

AmB used at concentrations of 0.6 to 1.25 μg ml−1 led to increases in ecNOS mRNA and protein expression as well as NO production. This was the result of an increased ecNOS mRNA half‐life. In contrast, incubation of cells with higher albeit subtoxic concentrations of AmB (2.5–5.0 μg ml−1) resulted in a decrease or respectively in completely abolished ecNOS mRNA and protein expression with a strongly reduced or inhibited ecNOS activity, due to a decrease of ecNOS mRNA half‐life. None of the AmB concentrations affected promoter activity as found with a reporter gene construct stably transfected into ECV304 cells.

Thus, our experiments show a concentration‐dependent biphasic effect of AmB on expression and activity of ecNOS, an effect best explained by AmB influencing ecNOS mRNA stability. In view of the known renal accumulation of this drug the results reported here could help to elucidate its renal toxicity.

British Journal of Pharmacology (2000) 131, 473–481; doi:10.1038/sj.bjp.0703576
Curcumin inhibition of bleomycin‐induced pulmonary fibrosis in rats
Authors: Punithavathi Durairaj , Venkatesan Narayanan , Babu Mary
Published: January 2005

Curcumin, an anti‐inflammatory, antioxidant, was evaluated for its ability to suppress bleomycin (BLM)‐induced pulmonary fibrosis in rats. A single intratracheal instillation of BLM (0.75 U 100−1 g, sacrificed 3, 5, 7, 14 and 28 days post‐BLM) resulted in significant increases in total cell numbers, total protein, and angiotensin‐converting enzyme (ACE), and alkaline phosphatase (AKP) activities in bronchoalveolar lavage fluid. Animals with fibrosis had a significant increase in lung hydroxyproline content. Alveolar macrophages from BLM‐administered rats elaborated significant increases in tumour necrosis factor (TNF)‐α release, and superoxide and nitric oxide production in culture medium. Interestingly, oral administration of curcumin (300 mg kg−1 10 days before and daily thereafter throughout the experimental time period) inhibited BLM‐induced increases in total cell counts and biomarkers of inflammatory responses in BALF. In addition, curcumin significantly reduced the total lung hydroxyproline in BLM rats. Furthermore, curcumin remarkably suppressed the BLM‐induced alveolar macrophage production of TNF‐α, superoxide and nitric oxide. These findings suggest curcumin as a potent anti‐inflammatory and anti‐fibrotic agent against BLM‐induced pulmonary fibrosis in rats.

British Journal of Pharmacology (2000) 131, 169–172; doi:10.1038/sj.bjp.0703578
Advantages of heterologous expression of human D2long dopamine receptors in human neuroblastoma SH‐SY5Y over human embryonic kidney 293 cells
Authors: Alberts Glen L , Pregenzer Jeffrey F , Im Wha Bin
Published: January 2005

The human D2long dopamine receptor when expressed heterologously in a human neuronal cell line, SH‐SY5Y, produced more robust functional signals than when expressed in a human embryonic kidney cell line, HEK293. Quinpirole (agonist)‐induced GTPγ35S binding and high affinity sites were 3–4 fold greater in SH‐SY5Y than in HEK293 cells.

N‐type Ca2+ channel currents present in SH‐SY5Y cells, but not HEK293 cells, were inhibited potently by quinpirole with a half‐maximal inhibitory concentration of 0.15±0.03 nM. Inhibition of adenylyl cyclases by agonists, on the other hand, was of similar potency and efficacy in the two cell lines.

GTPγ35S‐Bound Gα subunits from quinpirole‐activated and solubilized membranes were monitored upon immobilization with various Gα‐specific antibodies. Gαi and Gαo subunits were highly labelled with GTPγ35S in SH‐SY5Y cells, but only Gαi subunits were labelled in HEK293 cells. The additional Go coupling in SH‐SY5Y cells could arise, at least in part, from the presence of Go coupled‐effectors, such as the N‐type Ca2+ channel, and may contribute to robust agonist‐induced GTPγ35S binding, which is a reliable means for measuring ligand intrinsic efficacy.

It appears that expression of neuronal G protein‐coupled receptors in neuronal environments could reveal additional functional characteristics that are absent in non‐neuronal cell lines. This appears to be due to, at least in part, to the presence of neuron‐specific effectors. These findings underscore the importance of the cellular environment in which drug actions are examined, particularly in the face of intensive efforts to develop drugs for G protein‐coupled receptors of various origins.

British Journal of Pharmacology (2000) 131, 514–520; doi:10.1038/sj.bjp.0703580
Differential regulation of P2Y11 receptor‐mediated signalling to phospholipase C and adenylyl cyclase by protein kinase C in HL‐60 promyelocytes
Authors: Suh Byung‐Chang , Kim Tae‐Don , Lee Ihn‐Soon , Kim Kyong‐Tai
Published: January 2005

The regulatory mode of the P2Y11 purinoceptor‐mediated signalling cascades towards phospholipase C and adenylyl cyclase was studied in HL‐60 promyelocytes.

Treatment with the potent P2Y11 receptor activator dATP evoked an elevated intracellular Ca2+ concentration ([Ca2+]i) and inositol 1,4,5‐trisphosphate (IP3) production that was sustained for longer than 30 min. However, the dATP‐induced responses were significantly inhibited by the activation of protein kinase C after a short exposure to phorbol 12‐myristate 13‐acetate (PMA).

dATP also potently stimulated cyclic AMP production with half maximum effect seen at 23±7 μM dATP. In addition, a 5‐min pretreatment with PMA enhanced the dATP‐stimulated cyclic AMP accumulation.

PMA potentiated the cyclic AMP production when adenylyl cyclase was activated directly by forskolin or indirectly by G protein activation after cholera toxin treatment. dATP also enhanced the forskolin‐mediated cyclic AMP generation.

Treatment of the cells with 10 μM U‐73122, which almost completely blocked the dATP‐stimulated IP3 production and [Ca2+]i rise, had no effect on cyclic AMP accumulation, while 10 μM 9‐(tetrahydro‐2‐furyl)adenine (SQ 22536), which inhibited the adenylyl cyclase activation, did not effect the dATP‐stimulated phosphoinositide turnover.

Taken together, the results indicate that P2Y11 receptor‐mediated activation of phospholipase C and adenylyl cyclase occurs through independent pathways and is differentially regulated by protein kinase C in HL‐60 cells.

British Journal of Pharmacology (2000) 131, 489–497; doi:10.1038/sj.bjp.0703581
Characterization of the ORL1 receptor on adrenergic nerves in the rat anococcygeus muscle
Authors: Ho M , Corbett A D , McKnight A T
Published: January 2005

Nociceptin, the endogenous ORL1 receptor agonist inhibited the motor response to electrical‐field stimulation in the rat anococcygeus muscle. This effect was characterized using the peptide ligands acetyl‐Arg‐Tyr‐Tyr‐Arg‐Trp‐Lys‐NH2 (Ac‐RYYRWK‐NH2), acetyl‐Arg‐Tyr‐Tyr‐Arg‐Ile‐Lys‐NH2 (Ac‐RYYRIK‐NH2) and [Phe1ψ(CH2‐NH)Gly2]nociceptin(1‐13)NH2 ([F/G]NC(1‐13)NH2), and the non‐selective opioid antagonist naloxone benzoylhydrazone (NalBzOH).

Nociceptin produced a concentration‐dependent inhibition of the adrenergic motor response to electrical‐field stimulation (EC50 19 nM, pEC50 7.7±0.1, n=8), but the response to exogenous noradrenaline (0.2–1 μM) was unaffected. The inhibitory nerve response was not affected by up to 1 μM nociceptin.

After inhibition of nitric oxide synthase (Nω‐nitro‐L‐arginine 100 μM), and in the presence of peptidase inhibitors, nociceptin produced full inhibition of the pure adrenergic motor response (EC50 4 nM; pEC50 8.4±0.1, Emax 98.3±1.2%, n=12). Ac‐RYYRWK‐NH2 was a potent partial‐agonist (pEC50 9.0±0.1, Emax 66.4±5.2; n=11) but the efficacy of Ac‐RYYRIK‐NH2 (pEC50 8.0±0.2, Emax 36.7±3.5; n=12) was lower and the peptide could be tested as an antagonist (pA2 9.01).

[F/G]NC(1‐13)NH2 and NalBzOH had little or no efficacy and were competitive antagonists with pKB values of 7.4 (95% c.l. 7.1, 7.7) and 6.9 (95% c.l. 6.7, 7.1) respectively. Both increased the response to field stimulation at high concentrations, suggesting the release of an endogenous agonist for the ORL1 receptor during stimulation.

Rat nocistatin did not affect the response to electrical‐field stimulation, nor did it modify the inhibitory action of nociceptin.

Our findings suggest there is a significant endowment of ORL1 receptors on sympathetic terminals of the rat anococcygeus, where nociceptin mediates a powerful inhibitory effect on adrenergic neuromuscular transmission.

British Journal of Pharmacology (2000) 131, 349–355; doi:10.1038/sj.bjp.0703583
Inverse agonism and neutral antagonism at a constitutively active alpha‐1a adrenoceptor
Authors: Zhu Jun , Taniguchi Takanobu , Takauji Rumiko , Suzuki Fumiko , Tanaka Takashi , Muramatsu Ikunobu
Published: January 2005

We have studied the antagonist action of prazosin and KMD‐3213 in a constitutively active mutant of the human alpha‐1a adrenoceptor in which Ala271 was substituted to Thr and was expressed in CHO cells. Inverse agonism was characterized by up‐regulation of receptor density, a decrease in basal GTPγS binding, and a reduction in basal inositol‐1,4,5‐trisphosphate (IP3) level.

According to the above criteria, prazosin acted as an inverse agonist, whilst KMD‐3213 behaved as a neutral antagonist.

Compared with the wild‐type receptor, mutant receptor exhibited single affinity sites for [3H]‐prazosin, [3H]‐KMD and the non‐radioactive ligands tested, and displayed significantly higher affinities for several agonists but not for the two antagonists.

Administration of KMD‐3213 to prazosin‐treated CHO cells expressing the mutant receptor reversed the inverse agonism of prazosin resulting in rapid increases in cellular IP3, in intracellular [Ca2+] and in the rate of extracellular acidification.

These results indicated that a neutral antagonist can reverse the action of an inverse agonist at the receptor site. The distinct properties of inverse agonist and neutral antagonist in affecting receptor function may be important for the clinical use of such antagonists.

British Journal of Pharmacology (2000) 131, 546–552; doi:10.1038/sj.bjp.0703584
RSD1019 suppresses ischaemia‐induced monophasic action potential shortening and arrhythmias in anaesthetized rabbits
Authors: Barrett Terrance D , MacLeod Bernard A , Walker Michael J A
Published: January 2005

The electrophysiological actions of lidocaine, tedisamil and RSD1019 were assessed on normal and ischaemic cardiac tissue using monophasic action potentials (MAPs) recorded from the epicardium of anaesthetized rabbits. Drug effects on ischaemia‐induced arrhythmias were assessed simultaneously in the same rabbits.

Lidocaine, infused at 2.5, 5 and 10 μmol kg−1 min−1 i.v., accelerated and worsened the electrophysiological derangement caused by ischaemia, had profibrillatory actions and reduced the time to the occurrence of ventricular fibrillation (VF) relative to controls.

Tedisamil, infused at 0.063, 0.125 and 0.25 μmol kg−1 min−1 i.v., prolonged MAP duration at 90% repolarization (MAPD90%) before induction of ischaemia in a dose‐related manner; however, this effect was not maintained 5 min after induction of ischaemia. Tedisamil had no significant antiarrhythmic actions over the dose‐range tested.

RSD1019, infused at 2, 4 and 8 μmol kg−1 min−1 i.v., produced a small increase in MAPD90% before induction of ischaemia and only at the highest dose tested. In contrast to tedisamil, RSD1019 suppressed ischaemia‐induced MAP shortening assessed 5 min after induction of ischaemia. This effect was dose‐related. RSD1019 completely prevented ischaemia‐induced tachyarrhythmias at the mid and highest infusion levels tested.

The results of this study illustrate a pathologically targeted approach for preventing ischaemia‐induced arrhythmias. Suppression of ischaemia‐induced MAP shortening, demonstrated herein for RSD1019, represents a novel antifibrillatory approach.

British Journal of Pharmacology (2000) 131, 405–414; doi:10.1038/sj.bjp.0703592
Effects of (+)‐pentazocine and 1,3‐di‐o‐tolylguanidine (DTG), sigma (σ) ligands, on micturition in anaesthetized rats
Authors: Shimizu Isao , Kawashima Katsuyoshi , Ishii Daisuke , Oka Makoto
Published: January 2005

The effects of two sigma (σ) binding site ligands, (+)‐pentazocine and 1,3‐di‐o‐tolylguanidine (DTG), on bladder functions were examined in rats.

Cystometry using urethane‐anaesthetized rats showed that (+)‐pentazocine (1–5 mg kg−1, i.v.) and DTG (1–5 mg kg−1, i.v.) prolonged micturition intervals, indicating increased bladder capacity and raised the threshold pressure.

The effects of (+)‐pentazocine (2 mg kg−1, i.v.) on micturition were not influenced by naloxone (0.5 mg kg−1, i.v.), which antagonized similar effects of morphine (2 mg kg−1, i.v.).

When administered intracerebroventricularly (i.c.v.), DTG (1 μg) and (+)‐pentazocine (30 μg) prolonged micturition intervals with increased threshold pressure on the cystometrogram.

In isolated bladder detrusor strips of rats, (+)‐pentazocine (3 μM) and DTG (1 μM) did not affect contractile responses to electrical field stimulation. A higher concentration of DTG (3 μM) slightly suppressed the response induced by 30 Hz stimulation.

The effects of (+)‐pentazocine and DTG on micturition were abolished by pre‐treatment with pertussis toxin (PTX, 1 μg, i.c.v.).

These results indicate that typical σ ligands, such as (+)‐pentazocine and DTG, increase bladder capacity in anaesthetized rats. Moreover, the mechanism by which σ ligands change the urinary storage properties in rats may involve pathways in which the function of Gi/o proteins is necessary.

British Journal of Pharmacology (2000) 131, 610–616; doi:10.1038/sj.bjp.0703593
The effect of nucleotides and adenosine on stimulus‐evoked glutamate release from rat brain cortical slices
Authors: Bennett Gillian C , Boarder Michael R
Published: January 2005

Evidence has previously been presented that P1 receptors for adenosine, and P2 receptors for nucleotides such as ATP, regulate stimulus‐evoked release of biogenic amines from nerve terminals in the brain. Here we investigated whether adenosine and nucleotides exert presynaptic control over depolarisation‐elicited glutamate release.

Slices of rat brain cortex were perfused and stimulated with pulses of 46 mM K+ in the presence of the glutamate uptake inhibitor L‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (0.2 mM). High K+ substantially increased efflux of glutamate from the slices.

Basal glutamate release was unchanged by the presence of nucleotides or adenosine at concentrations of 300 μM.

Adenosine, ATP, ADP and adenosine 5′‐O‐(3‐thiotriphoshate) at 300 μM attenuated depolarisation‐evoked release of glutamate. However UTP, 2‐methylthio ATP, 2‐methylthio ADP, and α,β‐methylene ATP at 300 μM had no effect on stimulated glutamate efflux.

Adenosine deaminase blocked the effect of adenosine, but left the response to ATP unchanged.

The A1 antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine antagonised the inhibitory effect of both adenosine and ATP.

Cibacron blue 3GA inhibited stimulus‐evoked glutamate release when applied alone. When cibacron blue 3GA was present with ATP, stimulus‐evoked glutamate release was almost eliminated. However, this P2 antagonist had no effect on the inhibition by adenosine.

These results show that the release of glutamate from depolarised nerve terminals of the rat cerebral cortex is inhibited by adenosine and ATP. ATP appears to act directly and not through conversion to adenosine.

British Journal of Pharmacology (2000) 131, 617–623; doi:10.1038/sj.bjp.0703598
Suppression of the development of hypertension by the inhibitor of inducible nitric oxide synthase
Authors: Hong Hong‐Jye , Loh Shih‐Hurng , Yen Mao‐Hsiung
Published: January 2005

Our previous study demonstrated that the aortic inducible nitric oxide synthase (iNOS) expression and the plasma nitrite level in spontaneously hypertensive rats (SHR) were greater than that in age‐matched Wistar‐Kyoto rats (WKY). We subsequently hypothesized that the over‐expression of iNOS might play an important role in the pathogenesis of hypertension in SHR.

In the present study, pyrrolidinedithiocarbamate (PDTC, 10 mg kg−1 day−1, p.o., antioxidant and nuclear factor‐κ B inhibitor) and aminoguanidine (15 mg kg−1 day−1, p.o., selective inhibitor of iNOS) was used to treat SHR and WKY from age of 5 weeks through 16 weeks.

We found that PDTC and aminoguanidine significantly suppressed the development of hypertension and improved the diminished vascular responses to acetylcholine in SHR but not in WKY. Likewise, the increase of iNOS expression, nitrotyrosine immunostaining, nitric oxide production and superoxide anion formation in adult SHR were also significantly suppressed by chronic treatment with PDTC and aminoguanidine.

In conclusion, this study demonstrated that both PDTC and aminoguanidine significantly attenuated the development of hypertension in SHR. The results suggest that PDTC suppresses iNOS expression due to its anti‐oxidant and/or nuclear factor‐κ B inhibitory properties. However, the effect of aminoguanidine was predominantly mediated by inhibition of iNOS activity, thereby reducing peroxynitrite formation. We propose that the development of a more specific and potent inhibitor of iNOS might be beneficial in preventing pathological conditions such as the essential hypertension.

British Journal of Pharmacology (2000) 131, 631–637; doi:10.1038/sj.bjp.0703603
Effect of the p38 kinase inhibitor, SB 203580, on allergic airway inflammation in the rat
Authors: Escott K J , Belvisi M G , Birrell M A , Webber S E , Foster M L , Sargent C A
Published: January 2005

Tumour necrosis factor‐α (TNF‐α) and interleukin 1β (IL‐1β) have been implicated in the pathogenesis of asthma. The p38 kinase inhibitor, SB 203580 inhibits TNF‐α and IL‐1β production in vitro and in vivo. In this study the effect of SB 203580 on allergen‐induced airway TNF‐α production and inflammatory cell recruitment was investigated in sensitized Brown Norway rats. The allergen‐induced increase in bronchoalveolar lavage (BAL) TNF‐α was inhibited by SB 203580 at every dose tested (10–100 mg kg−1, p.o.). In contrast, neither ovalbumin‐induced eosinophilia or neutrophilia were inhibited by SB 203580 (10–100 mg kg−1, p.o.). In conclusion, SB 203580 inhibits BAL TNF‐α production by 95% without inhibiting either antigen‐induced airway eosinophilia or neutrophilia. This data suggests that either the residual TNF‐α is sufficent to drive allergen‐induced inflammatory cell recruitment into the lung or that TNF‐α is not involved in allergen‐induced inflammatory cell recruitment.

British Journal of Pharmacology (2000) 131, 173–176; doi:10.1038/sj.bjp.0703605
Inhibition of acetylcholine muscarinic M1 receptor function by the M1‐selective ligand muscarinic toxin 7 (MT‐7)
Authors: Olianas Maria C , Maullu Carlo , Adem Abdu , Mulugeta Ezra , Karlsson Evert , Onali Pierluigi
Published: January 2005

MT‐7 (1–30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M1 receptor, inhibited the acetylcholine (ACh)‐stimulated [35S]‐guanosine‐5′‐O‐(3‐thio)triphosphate ([35S]‐GTPγS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M1 receptor subtype.

MT‐7 failed to affect the ACh‐stimulated [35S]‐GTPγS binding in membranes of CHO cells expressing either the M2, M3 or M4 receptor subtype.

In N1E‐115 neuroblastoma cells endogenously expressing the M1 and M4 receptor subtypes, MT‐7 (0.3–3.0 nM) inhibited the carbachol (CCh)‐stimulated inositol phosphates accumulation, but failed to affect the CCh‐induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38‐stimulated cyclic AMP accumulation.

In both CHO/M1 and N1E‐115 cells the MT‐7 inhibition consisted in a decrease of the maximal agonist effect with minimal changes in the agonist EC50 value.

In CHO/M1 cell membranes, MT‐7 (0.05–25 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [3H]‐N‐methylscopolamine ([3H]‐NMS) in a concentration‐dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT‐7 (3 nM) decreased the dissociation rate of [3H]‐NMS by about 5 fold.

CHO/M1 cell membranes preincubated with MT‐7 (10 nM) and washed by centrifugation and resuspension did not recover control [3H]‐NMS binding for at least 8 h at 30°C.

It is concluded that MT‐7 acts as a selective noncompetitive antagonist of the muscarinic M1 receptors by binding stably to an allosteric site.

British Journal of Pharmacology (2000) 131, 447–452; doi:10.1038/sj.bjp.0703606
Antidystonic efficacy of nitric oxide synthase inhibitors in a rodent model of primary paroxysmal dystonia
Authors: Richter Angelika , Löschmann Peter‐A , Löscher Wolfgang
Published: January 2005

In a hamster model (genetic symbol dtsz) of primary paroxysmal non‐kinesiogenic dystonic choreoathetosis, recent studies have shown beneficial effects of glutamate and dopamine receptor antagonists. Nitric oxide (NO), synthesized from L‐arginine by NO synthase in response to glutamate receptor activation, elicits cyclic GMP and modulates glutamate‐mediated processes and striatal dopamine release.

Therefore, the effects of NO synthase inhibitors and of L‐arginine on severity of dystonia were investigated in dtsz hamsters in which dystonic attacks, characterized by twisting movements and postures, can be induced by stress.

The NO synthase inhibitors NG‐nitro‐L‐arginine (L‐NNA), NG‐nitro‐L‐arginine methyl ester (L‐NAME) and 7‐nitroindazole significantly reduced the severity of dystonia. At antidystonic effective doses neither L‐NNA nor L‐NAME caused observable side effects, whereas 7‐nitroindazole exerted moderate reduction of locomotor activity.

The antidystonic effect of L‐NAME was reversed by co‐administration of the NO precursor L‐arginine. However, L‐arginine administered alone did not exert any effect on severity of dystonia.

Cerebellar cyclic GMP levels in brains of mutant hamsters in comparison to non‐dystonic control hamsters did not significantly differ, but the cerebellar cyclic GMP levels tended to be increased in dtsz hamsters during a dystonic attack. L‐NAME significantly decreased the cerebellar cyclic GMP levels in both dtsz and control hamsters.

Although an overproduction of NO is probably not critically involved in the pathogenesis of paroxysmal dystonia, it may contribute to the manifestation of dystonic attacks, as indicated by the antidystonic effects of NO synthase inhibitors.

Peripheral side effects may limit the clinical use of NO synthase inhibitors, but more selective inhibitors of the neuronal NO synthase should be considered as interesting candidates for the treatment of paroxysmal dystonia.

British Journal of Pharmacology (2000) 131, 921–926; doi:10.1038/sj.bjp.0703609
Oncostatin M synergises with house dust mite proteases to induce the production of PGE2 from cultured lung epithelial cells
Authors: Knight Darryl A , Asokananthan Nithiananthan , Watkins D Neil , Misso Neil L A , Thompson Philip J , Stewart Geoffrey A
Published: January 2005

The release of PGE2 and nitric oxide (NO) from the respiratory epithelium may act to dampen inflammation. In other tissues, oncostatin M (OSM), a potent inducer of epithelial antiproteases, has also been shown to interact with IL‐1β to stimulate PGE2 release. However, whether OSM interacts with pro‐inflammatory cytokines and proteases in the production of anti‐inflammatory eicosanoids and NO from airway epithelium is unknown.

The effect of OSM and the related cytokine leukaemia inhibitory factor (LIF) on PGE2 and NO production by the respiratory epithelial cell line, A549 in response to pro‐inflammatory cytokines as well as protease‐rich house dust mite (HDM) fractions and a protease‐deficient rye grass pollen extract was examined by immunohistochemistry, cell culture, ELISA and enzyme‐immunoassay.

Cells treated with a mixture of IL‐1β, IFNγ and LPS for 48 h produced a 9 fold increase in PGE2 and a 3 fold increase in NO levels (both P<0.05). Both OSM and LIF were without effect. However, OSM added together with the cytokine mixture synergistically enhanced PGE2 production (22 fold, P<0.05). OSM also synergistically enhanced PGE2 production in response to a cysteine protease‐enriched, but not serine protease‐enriched HDM fraction (P<0.05). Rye grass extract, neither alone nor in combination with OSM, induced PGE2 or NO production, although it did induce the release of GM‐CSF.

These observations suggest that OSM is an important co‐factor in the release of PGE2 and NO from respiratory epithelial cells and may play a role in defense against exogenous proteases such as those derived from HDM.

British Journal of Pharmacology (2000) 131, 465–472; doi:10.1038/sj.bjp.0703612
Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell lines
Authors: Montaner Beatriz , Navarro Sira , Piqué Maria , Vilaseca Marta , Martinell Marc , Giralt Ernest , Gil Joan , Pérez‐Tomás Ricardo
Published: January 2005

The effects of supernatant from the bacterial strain Serratia marcescens 2170 (CS‐2170) on the viability of different haematopoietic cancer cell lines (Jurkat, NSO, HL‐60 and Ramos) and nonmalignant cells (NIH‐3T3 and MDCK) was studied. We examined whether this cytotoxic effect was due to apoptosis, and we purified the molecule responsible for this effect and determined its chemical structure.

Using an MTT assay we showed a rapid (4 h) decrease in the number of viable cells. This cytotoxic effect was due to apoptosis, according to the fragmentation pattern of DNA, Hoechst 33342 staining and FACS analysis of the phosphatidylserine externalization. This apoptosis was blocked by using the caspase inhibitor Z‐VAD.fmk, indicating the involvement of caspases.

Prodigiosin is a red pigment produced by various bacteria including S. marcescens. Using mutants of S. marcescens (OF, WF and 933) that do not synthesize prodigiosin, we further showed that prodigiosin is involved in this apoptosis. This evidence was corroborated by spectroscopic analysis of prodigiosin isolated from S. marcescens.

These results indicate that prodigiosin, an immunosuppressor, induces apoptosis in haematopoietic cancer cells with no marked toxicity in nonmalignant cells, raising the possibility of its therapeutic use as an antineoplastic drug.

British Journal of Pharmacology (2000) 131, 585–593; doi:10.1038/sj.bjp.0703614
Identification of a novel nicotinic binding site in mouse brain using [125I]‐epibatidine
Authors: Whiteaker Paul , Jimenez Melissa , McIntosh J Michael , Collins Allan C , Marks Michael J
Published: January 2005

[125I]‐Epibatidine binds to multiple nicotinic acetylcholine receptor (nAChR) subtypes with high affinity. In this study, [125I]‐epibatidine was used to label and characterize a novel nAChR subtype found in mouse brain inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates.

Binding of [125I]‐epibatidine was saturable and apparently monophasic in each brain region (KD=71±12 pM mean±s.e.mean across regions) but inhibition of [125I]‐epibatidine binding (200 pM) by A85380, cytisine and (−)‐nicotine was biphasic, indicating the presence of multiple binding sites.

The sites with lower agonist affinity comprised 30.0±2.2, 58.6±0.1 and 48.7±3.3% of specific [125I]‐epibatidine (200 pM) binding in inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates, respectively.

The affinity difference between A85380‐sensitive and ‐resistant binding sites was particularly marked (approximately 1000 fold). Thus A85380 was used to differentiate agonist‐sensitive and ‐resistant sites.

The pharmacological profiles of the A85380‐resistant sites in each region were assessed with inhibition binding experiments, using 14 agonists and five antagonists. The profiles were indistinguishable across regions, implying that A85380‐resistant [125I]‐epibatidine binding sites in inferior colliculus, interpeduncular nucleus, and olfactory bulb represent a single nAChR subtype.

The pharmacological profile of the A85380‐resistant sites is very different from that previously reported for high affinity (−)‐[3H]‐nicotine‐, [125I]‐α‐bungarotoxin‐, or [125I]‐α‐conotoxin MII‐binding sites, suggesting that they represent a novel nAChR population in mouse brain.

British Journal of Pharmacology (2000) 131, 729–739; doi:10.1038/sj.bjp.0703616
Regulation of the human prostanoid TPα and TPβ receptor isoforms mediated through activation of the EP1 and IP receptors
Authors: Walsh Marie‐Therese , Kinsella B Therese
Published: January 2005

The intermolecular cross‐regulation mediated by the prostanoid IP‐receptor (IP)/EP1 receptor (EP1) agonists PGI2 and 17 phenyl trinor PGE2 on TP receptor (TP) signalling within platelets was compared to that which occurs to the individual TPα and TPβ receptors over‐expressed in human embryonic kidney (HEK) 293 cells. Ligand mediated TP receptor activation was monitored by analysing mobilization of intracellular calcium ([Ca2+]i) following stimulation with the selective thromboxane (TX) A2 mimetic U46619.

Consistent with previous studies, in platelets, PGI2 acting through endogenous IP receptors completely inhibited U46619‐mediated TP receptor signalling in a protein kinase (PK) A‐dependent, PKC‐independent manner.

In HEK 293 cells, PGI2, acting through endogenous AH6809 sensitive EP1 rather than IP receptors, and the selective EP1 receptor agonist 17 phenyl trinor PGE2 antagonized U46619‐mediated signalling by both TPα and TPβ receptors in a PKC‐dependent, PKA‐independent manner.

The maximum response induced by either ligand was significantly (P<0.005) greater for the TPα receptor than the TPβ receptor, pointing to possible physiologic differences between the TP isoforms, although the potency of each ligand was similar for both TP receptors.

TPΔ328, a truncated variant of TP receptor lacking the C‐tail sequences unique to TPα or TPβ receptors, was not sensitive to EP1 receptor‐mediated regulation by PGI2 or 17 phenyl trinor PGE2

In conclusion, these data confirm that TPα and TPβ receptors are subject to cross regulation by EP1 receptor signalling in HEK 293 cells mediated by PKC at sites unique to the individual TP receptors and that TPα receptor responses are significantly more reduced by EP1 receptor regulation than those of the TPβ receptor.

British Journal of Pharmacology (2000) 131, 601–609; doi:10.1038/sj.bjp.0703624
Neuroprotective abilities of resveratrol and other red wine constituents against nitric oxide‐related toxicity in cultured hippocampal neurons
Authors: Bastianetto Stéphane , Zheng Wen‐Hua , Quirion Rémi
Published: January 2005

Animal and epidemiological studies suggest that polyphenol constituents of red wine possess antioxidant activities that favour protection against cardiovascular disease – the so‐called. ‘French paradox’ – and possibly, central nervous system disorders such as Alzheimer's disease (AD) and ischaemia.

In the present study, the potential of three major red wine derived‐polyphenols to protect against toxicity induced by the nitric oxide free radical donors sodium nitroprusside (SNP) and 3‐morpholinosydnonimine (SIN‐1) was examined in cultured rat hippocampal cells.

Both co‐ and post‐treatments with either the stilbene resveratrol (5–25 μM) or the flavonoids quercetin (5–25 μM) and (+)‐catechin (1–10 μM) were capable of attenuating hippocampal cell death and intracellular reactive oxygen species accumulation produced by SNP (100 μM and 1 mM, respectively). However, among the phenolic compounds tested, only the flavonoids afforded significant protection against 5 mM SIN‐1‐induced toxicity.

The effects of phenolic constituents were shared by Trolox (100 μM), a vitamin E analogue, but not by selective inhibitors of cyclo‐oxygenases (COX) and lipoxygenases (LOX).

Among the phenolic compounds tested, only quercetin (10 μM) inhibited 100 μM SNP‐stimulated protein kinase C (PKC) activation, whereas none of them were able to attenuate nitrite accumulation caused by SNP (100 μM).

Taken together, these data suggest that the neuroprotective abilities of quercetin, resveratrol, and (+)‐catechin result from their antioxidant properties rather than their purported inhibitory effects on intracellular enzymes such as COX, LOX, or nitric oxide synthase. Quercetin, however, may also act via PKC to produce its protective effects.

British Journal of Pharmacology (2000) 131, 711–720; doi:10.1038/sj.bjp.0703626