bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages
Authors: Lin Wan‐W , Chen Bin C
Published: January 2005

The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin‐induced arachidonic acid (AA) release and prostagladin E1 (PGE1)‐stimulated cyclic AMP production, respectively.

PMA at 1 μm caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%.

Treatment of cells with staurosporine and Ro 31‐8220 inhibited the PMA‐induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCβ) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA.

Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%.

Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production.

The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane.

Western blot analysis revealed the presence of eight PKC isoforms (α, βI, βII, δ, ε, μ λ and ξ) in RAW 264.7 cells and PMA was shown to induce the translocation of the α, βI, βII, δ, ε and μ isoforms from the cytosol to the cell membrane within 2 min.

Pretreatment of cells with PMA for 2–24 h resulted in a time‐dependent down‐regulation of PKCα, βI, βII, and δ expression, while the levels of the other four PKC isozymes were unchanged after PMA treatment for 24 h. A decrease in the potentiation of AA release by PMA was observed, concomitant with the time‐dependent down‐regulation of PKC.

These results indicate that PKCβ has a crucial role in the mediation of cPLA2 activation by the phorbol ester PMA, whereas PMA utilizes PKC ε and/or μ to up‐regulate AC activity.

British Journal of Pharmacology (1998) 125, 1601–1609; doi:10.1038/sj.bjp.0702219
Pharmacological characterization of the human vasopressin receptor subtypes stably expressed in Chinese hamster ovary cells
Authors: Tahara Atsuo , Saito Masayuki , Sugimoto Toru , Tomura Yuichi , Wada Koh‐ichi , Kusayama Toshiyuki , Tsukada Junko , Ishii Noe , Yatsu Takeyuki , Uchida Wataru , Tanaka Akihiro
Published: January 2005

Three subtypes of human (h) arginine vasopressin (AVP) receptors, hV1A, hV1B and hV2, were stably expressed in Chinese hamster ovary (CHO) cells and characterized by [3H]‐AVP binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated.

Scatchard analysis of saturation isotherms for the specific binding of [3H]‐AVP to membranes, prepared from CHO cells transfected with hV1A, hV1B and hV2 receptors, yielded an apparent equilibrium dissociation constant (Kd) of 0.39, 0.25 and 1.21 nm and a maximum receptor density (Bmax) of 1580 fmol mg−1 protein, 5230 fmol mg−1 protein and 7020 fmol mg−1 protein, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogenous, non‐interacting receptor populations.

Pharmacological characterization of the transfected human AVP receptors was undertaken by measuring the relative ability of nonpeptide AVP receptor antagonists, YM087, OPC‐21268, OPC‐31260, SR 49059 and SR 121463A, to inhibit binding of [3H]‐AVP. At hV1A receptors, the relative order of potency was SR49059>YM087>OPC‐31260>SR 121463A>>OPC‐21268 and at hV2 receptors, YM087=SR 121463A>OPC‐31260>SR 49059>>OPC‐21268. In contrast, the relative order of potency, at hV1B receptors, was SR 49059>>SR 121463A=YM087=OPC‐31260=OPC‐21268.

In CHO cells expressing either hV1A or hV1B receptors, AVP caused a concentration‐dependent increase in intracellular Ca2+ concentration ([Ca2+]i) with an EC50 value of 1.13 nm and 0.90 nm, respectively. In contrast, stimulation of CHO cells expressing hV2 receptors resulted in an accumulation of cyclic AMP with an EC50 value of 2.22 nm. The potency order of antagonists in inhibiting AVP‐induced [Ca2+]i or cyclic AMP response was similar to that observed in radioligand binding assays.

In conclusion, we have characterized the pharmacology of human cloned V1A, V1B and V2 receptors and used these to determine the affinity, selectivity and potency of nonpeptide AVP receptor antagonists. Thus they may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of AVP.

British Journal of Pharmacology (1998) 125, 1463–1470; doi:10.1038/sj.bjp.0702220
Desensitization of the histamine H1‐receptor and transcriptional down‐regulation of histamine H1‐receptor gene expression in bovine tracheal smooth muscle
Authors: Pype J. L. , Mak J. C. W. , Dupont L. J. , Verleden G. M. , Barnes P. J.
Published: January 2005

We have investigated the role of protein kinase C (PKC) in the desensitization of histamine H1‐receptors and in the expression of the histamine H1‐receptor gene in airway smooth muscle.

Prolonged 4β‐phorbol 12,13 dibutyrate (PDBu) pretreatment (4 h, 100 nm–1 μm) of bovine trachealis caused a concentration‐dependent loss of contraction in response to histamine H1‐receptor stimulation, which was associated with a concentration‐dependent decrease in histamine‐induced total [3H]‐inositol phosphates accumulation. In contrast, the responses to sodium fluoride, a direct G‐protein activator, were unalterd by PDBu (100–300 nm) pre‐incubation and only slightly reduced following incubation with 1 μm PDBu.

A selective PKC inhibitor, GF 109203X, partially blocked the PDBu (1 μm)‐induced desensitization and completely blocked the effect of 100 nm PDBu, confirming the involvement of PKC.

Binding experiments using [3H]‐pyrilamine revealed a class of high‐affinity binding sites within the range for the histamine H1 receptor in airway smooth muscle. PDBu (1 μm) pretreatment for 4 h did not change the number of histamine H1 receptors.

PDBu (1 μm) exposure caused a time‐dependent reduction in the steady‐state levels of histamine H1‐receptor mRNA, which was inhibited by pre‐incubation with GF 109203X and by cycloheximide, a protein synthesis inhibitor.

Nuclear run‐on assays revealed a 50% reduction in the rate of histamine H1‐receptor gene transcription after 17 h PDBu pretreatment, whereas mRNA stability was not affected by PDBu pretreatment (17 h).

In conclusion, we have shown a PKC‐mediated desensitization of the histamine H1‐receptor in BTSM and a transcriptional down‐regulation of the histamine H1‐receptor gene expression, which requires new protein synthesis.

British Journal of Pharmacology (1998) 125, 1477–1484; doi:10.1038/sj.bjp.0702222
Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo
Authors: He Shaoheng , Walls Andrew F.
Published: January 2005

The roles of chymase in acute allergic responses are not clear, despite the relative abundance of this serine proteinase in the secretory granules of human mast cells. We have isolated chymase to high purity from human skin tissue by heparin‐agarose affinity chromatography and Sephacryl S‐200 gel filtration procedures, and have investigated the ability of human mast cell chymase to stimulate cell accumulation following injection into laboratory animals.

Injection of chymase provoked marked neutrophilia and eosinophilia in the skin of Dunkin Hartley guinea‐pigs. Compared with saline injected control animals, there were some 60 fold more neutrophils and 12 fold more eosinophils present at the injection site.

Following injection of chymase into the peritoneum of BALB/c mice, there were up to 700 fold more neutrophils, 21 fold more eosinophils, 19 fold more lymphocytes and 7 fold more macrophages recovered than from saline injected controls at 16 h. Doses of chymase as low as 5 ng (1.7×10−13 mole) stimulated an inflammatory infiltrate, and significant neutrophilia was elicited within 3 h.

The chymase induced cell accumulation in both the guinea‐pig and mouse models was dependent on an intact catalytic site, being reduced by co‐injection of proteinase inhibitors or heat inactivation of the enzyme.

Co‐injection of histamine or heparin significantly reduced the chymase induced neutrophil accumulation, whereas neither histamine nor heparin by themselves had any effect on the accumulation of nucleated cells. No synergistic or antagonist interactions between chymase and tryptase were observed when these two major mast cell proteinases were co‐injected into the mouse peritoneum.

Our findings suggest that chymase may provide an potent stimulus for inflammatory cell recruitment following mast cell activation.

British Journal of Pharmacology (1998) 125, 1491–1500; doi:10.1038/sj.bjp.0702223
Inhibitory effect of digoxin on testosterone secretion through mechanisms involving decreases of cyclic AMP production and cytochrome P450scc activity in rat testicular interstitial cells
Authors: Lin Ho , Wang Shyi‐Wu , Tsai Shiow‐Chwen , Chen Jiann‐Jong , Chiao Yu‐Chung , Lu Chien‐Chen , Ji‐Sien Huang William , Wang Guei‐Jane , Chen Chieh‐Fu , Wang Paulus S.
Published: January 2005

In vivo and in vitro experiments were performed to examine inhibitory effects of digoxin on testosterone secretion and to determine possible underlying mechanisms.

A single intravenous injection of digoxin (1 μg kg−1) decreased the basal and human chorionic gonadotropin (hCG)‐stimulated plasma testosterone concentrations in adult male rats.

Digoxin (10−7–10−4 M) decreased the basal and hCG‐stimulated release of testosterone from rat testicular interstitial cells in vitro.

Digoxin (10−7–10−4 M) also diminished the basal and hCG‐stimulated production of cyclic 3′ : 5′‐adenosine monophosphate (AMP) and attenuated the stimulatory effects of forskolin and 8‐Br‐cyclic AMP on testosterone production by rat testicular interstitial cells.

Digoxin (10−4 M) inhibited cytochrome P450 side chain cleavage enzyme (cytochrome P450scc) activity (conversion of 25‐hydroxy cholesterol to pregnenolone) in the testicular interstitial cells but did not influence the activity of other steroidogenic enzymes.

These results suggest that digoxin inhibits the production of testosterone in rat testicular interstitial cells, at least in part, via attenuation of the activities of adenylyl cyclase and cytochrome P450scc.
Involvement of 5‐HT6 receptors in nigro‐striatal function in rodents
Authors: Bourson Anne , Boess Frank G , Bös Michael , Sleight Andrew J
Published: January 2005

4‐Amino‐N‐(2,4 bis‐methylamino‐pyrimidin‐4‐yl) benzene sulphonamide (Ro 04‐6790) is a potent, selective and competitive antagonist for the 5‐HT6 receptor which can be detected in the cerebro‐spinal fluid (CSF) of rats following intraperitoneal administration. Since 5‐HT6 receptor mRNA and 5‐HT6 receptor‐like immunoreactivity have been shown to be present in the striatum, the purpose of the present study was to evaluate the effect of 5‐HT6 receptor antagonism on haloperidol‐ and SCH 23390‐induced catalepsy in mice and on the turning behaviour of rats with unilateral 6‐hydroxydopamine (6‐OHDA) lesions of the medial forebrain bundle.

Ro 04‐6790 (3, 10 and 30 mg kg−1 i.p.) did not induce catalepsy and had no effect on catalepsy induced by either haloperidol or SCH 23390.

Ro 04‐6790 (3, 10 and 30 mg kg−1 i.p.) did not itself induce rotational behaviour in rats with unilateral 6‐hydroxydopamine (6‐OHDA) lesions of the medial forebrain bundle nor did it affect the rotational behaviour induced by either L‐Dopa or amphetamine.

5‐HT6 receptor antagonism inhibited the rotational behaviour of 6‐OHDA lesioned rats induced by treatment with the muscarinic antagonists scopolamine and atropine.

The data support earlier conclusions from experiments with antisense oligonucleotides that the 5‐HT6 receptor is involved in the control of acetylcholine neurotransmission in the rat brain.

British Journal of Pharmacology (1998) 125, 1562–1566; doi:10.1038/sj.bjp.0702230
Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide
Authors: Weisbrod Robert M. , Griswold Mark C. , Yaghoubi Mohammad , Komalavilas Padmini , Lincoln Thomas M. , Cohen Richard A.
Published: January 2005

The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca2+]i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10−7 M), NO (10−10–10−6 M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca2+]i and cation influx as indicated by fura‐2 fluorimetry.

Zaprinast (10−4 M), increased NO‐stimulated levels of cyclic GMP by 3–20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca2+]i reached after administration of NO, the initial decreases in [Ca2+]i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast.

The guanylyl cyclase inhibitor, H‐(1,2,4) oxadiazolo(4,3‐a) quinoxallin‐1‐one (ODQ, 10−5 M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp‐8‐Br‐cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10−10–10−8 M, but the decrease in [Ca2+]i or cation influx caused by higher concentrations of NO (10−7–10−6 M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10−7–10−5 M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp‐8‐Br‐cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO.

Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca2+]i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP‐independent mechanisms also contribute significantly to the decrease in [Ca2+]i and smooth muscle relaxation to NO.
Modelling of the pharmacodynamic interaction between phenytoin and sodium valproate
Authors: Paschoa O E Della , Voskuyl R A , Danhof M
Published: January 2005

Treatment of epilepsy with a combination of antiepileptic drugs remains the therapeutic choice when monotherapy fails. In this study, we apply pharmacokinetic‐pharmacodynamic modelling to characterize the interaction between phenytoin (PHT) and sodium valproate (VPA).

Male Wistar rats received a 40 mg kg−1 intravenous dose of PHT over 5 min either alone or in combination with an infusion of VPA resulting in a steady‐state concentration of 115.5±4.9 μg ml−1. A control group received only the infusion of VPA. The increase in the threshold for generalized seizure activity (ΔTGS) was used as measure of the anticonvulsant effect.

PHT pharmacokinetics was described by a pharmacokinetic model with Michaelis‐Menten elimination. The concentration‐time course and plasma protein binding of PHT were not altered by VPA. The pharmacokinetic parameters Vmax and Km were, respectively, 294±63 μg min−1 and 7.8±2.4 μg ml−1 in the absence of VPA and 562±40 μg min−1 and 15.6±0.9 μg ml−1 upon administration in combination with VPA.

A delay of the onset of the effect relative to plasma concentrations of PHT was observed. The assessment of PHT concentrations at the effect site was based on the effect‐compartment model, yielding mean ke0 values of 0.128 and 0.107 min−1 in the presence and absence of VPA, respectively.

A nonlinear relationship between effect‐site concentration and the increase in the TGS was observed. The concentration that causes an increase of 50% in the baseline TGS (EC50%TGS) was used to compare drug potency. A shift of EC50%TGS from 13.27±3.55 to 4.32±0.52 μg ml−1 was observed upon combination with VPA (P<0.01).

It is concluded that there is a synergistic pharmacodynamic interaction between PHT and VPA in vivo.

British Journal of Pharmacology (1998) 125, 1610–1616; doi:10.1038/sj.bjp.0702235
Studies of the antagonist actions of (RS)‐2‐amino‐3‐[5‐tert‐butyl‐3‐(phosphonomethoxy)‐4‐isoxazolyl]propionic acid (ATPO) on non‐NMDA receptors in cultured rat neurones
Authors: Dai W‐M , Ebert B , Madsen U , Lambert J D C
Published: January 2005

Whole‐cell patch‐clamp recordings from single cultured cortical neurones have been used to study the action of (RS)‐2‐amino‐3‐[5‐tert‐butyl‐3‐(phosphonomethoxy)‐4‐isoxazolyl]propionic acid (ATPO), which has previously been proposed to be a potent selective antagonist of 2‐amino‐3‐(3‐hydroxy‐5‐methyl‐4‐isoxazolyl)propionic acid (AMPA) receptors.

ATPO competitively reduced peak responses evoked by semi‐rapid applications of AMPA (Ki=16 μm) but had variable effects on plateau responses, which were on average unchanged. Following blockade of AMPA receptor desensitization by cyclothiazide (CTZ, 100 μm), the plateau responses were reduced by ATPO to a similar extent as the peak responses, indicating that ATPO reduces desensitization of AMPA receptors.

Semi‐rapid application of kainic acid (KA) and the KA receptor‐selective agonist, (2S,4R)‐4‐methylglutamic acid (MeGlu) evoked non‐desensitizing responses which were competitively antagonized by ATPO (Ki values: 27 and 23 μm, respectively).

Responses to MeGlu were unaffected by CTZ (100 μm), but potentiated 3 fold following blockade of KA receptor desensitization by concanavalin A (Con A, 300 μg ml−1). Responses of spinal cord neurones to MeGlu were blocked by ATPO to a similar extent before and after blockade of KA receptor desensitization by Con A.

Although selectively potentiated by Con A, plateau responses to MeGlu were reduced by 69.6% by the AMPA selective antagonist, GYKI 53655 (10 μm). The remaining component was further reduced by ATPO with a Ki of 36 μm, which was not significantly different from that in the absence of GYKI 53655, but was greater than that on responses to AMPA.

It is concluded that ATPO is a moderate‐potency competitive inhibitor of naturally expressed non‐NMDA receptors.

British Journal of Pharmacology (1998) 125, 1517–1528; doi:10.1038/sj.bjp.0702236
Facilitation and inhibition of male rat ejaculatory behaviour by the respective 5‐HT1Aand 5‐HT1Breceptor agonists 8‐OH‐DPAT and anpirtoline, as evidenced by use of the corresponding new and selective receptor antagonists NAD‐299 and NAS‐181
Authors: Hillegaart Viveka , Ahlenius Sven
Published: January 2005

Ejaculatory problems and anorgasmia are well‐known side‐effects of the SSRI antidepressants, and a pharmacologically induced increase in serotonergic neurotransmission inhibits ejaculatory behaviour in the rat. In the present study the role of 5‐HT1Aand 5‐HT1Breceptors in the mediation of male rat ejaculatory behaviour was examined by use of selective agonists and antagonists acting at these 5‐HT receptor subtypes.

The 5‐HT1Areceptor agonist 8‐OH‐DPAT (0.25–4.00 μmol kg−1s.c.) produced an expected facilitation of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the new selective 5‐HT1Areceptor antagonist (R)‐3‐N,N‐dicyclobutylamino‐8‐fluoro‐3,4‐dihydro‐2H‐1‐benzopyran‐5‐carboxamide hydrogen (2R,3R) tartrate monohydrate (NAD‐299) (1.0 μmol kg−1s.c.). NAD‐299 by itself (0.75–3.00 μmol kg−1s.c.) did not affect the male rat ejaculatory behaviour.

The 5‐HT1Breceptor agonist anpirtoline (0.25–4.00 μmol kg−1s.c.) produced a dose‐dependent inhibition of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the 5‐HT1Breceptor antagonist isamoltane (16 μmol kg−1s.c.) as well as by the new and selective antagonist (R)‐(+)‐2‐(3‐morpholinomethyl‐2H‐chromene‐8‐yl)oxymethylmorpholino methansulphonate (NAS‐181) (16 μmol kg−1s.c.). Isamoltane (1.0–16.0 μmol kg−1s.c.) and NAD‐181 (1.0–16.0 μmol kg−1s.c.) had no, or weakly facilitatory effects on the male rat ejaculatory behaviour. The non‐selective 5‐HT1receptor antagonist (−)‐pindolol (8 μmol kg−1s.c.), did not antagonize the inhibition produced by anpirtoline.

The present results demonstrate opposite effects, facilitation and inhibition, of male rat ejaculatory behaviour by stimulation of 5‐HT1Aand 5‐HT1Breceptors, respectively, suggesting that the SSRI‐induced inhibition of male ejaculatory dysfunction is due to 5‐HT1Breceptor stimulation.
Electrophysiological studies on oxindole, a neurodepressant tryptophan metabolite
Authors: Mannaioni Guido , Carpenedo Raffaella , Maria Pugliese Anna , Corradetti Renato , Moroni Flavio
Published: January 2005

The aim of the present work was to investigate the electrophysiological effects of oxindole, a tryptophan metabolite present in rat blood and brain, and recently proposed as a contributing factor in the pathogenesis of hepatic encephalopathy.

Using rat hippocampal slices in vitro and extra‐ or intracellular recordings, we evaluated oxindole effects on the neurotransmission of the CA1 region following orthodromic stimulation of the Schaffer collaterals.

Oxindole (0.3–3 mM) decreased the amplitude of population spikes extracellularly recorded at the somatic level and of the fEPSPs recorded at the dendritic level. In intracellular recordings, oxindole (0.1–3 mM) did not affect the resting membrane potential or the neuronal input resistance, but reduced the probability of firing action potentials upon either synaptic or direct activation of the pyramidal cells.

Oxindole (0.3–3 mM) increased the threshold and the latency of firing action potentials elicited by depolarizing steps without changing the duration or the peak amplitude of the spikes. It also significantly increased the spike frequency adaptation induced by long lasting (400 ms) depolarizing stimuli.

In separate experiments, performed by measuring AMPA or NMDA‐induced responses in cortical slices, oxindole (1–3 mM) did not modify glutamate receptor agonist responses.

Our results show that concentrations of oxindole which may be reached in pathological conditions, significantly decrease neuronal excitability by modifying the threshold of action potential generation.
Pharmacological characterization of β2‐adrenoceptor in PGT‐β mouse pineal gland tumour cells
Authors: Suh Byung‐Chang , Chae Hee‐Don , Chung Joo‐Ho , Kim Kyong‐Tai
Published: January 2005

The adrenoceptor in a mouse pineal gland tumour cell line (PGT‐β) was identified and characterized using pharmacological and physiological approaches.

Adrenaline and noradrenaline, adrenoceptor agonists, stimulated cyclic AMP generation in a concentration‐dependent manner, but had no effect on inositol 1,4,5‐trisphosphate production. Adrenaline was a more potent activator of cyclic AMP generation than noradrenaline, with half maximal‐effective concentrations (EC50) seen at 175±22 nM and 18±2 μM for adrenaline and noradrenaline, respectively.

The addition of forskolin synergistically stimulated the adrenaline‐mediated cyclic AMP generation in a concentration‐dependent manner.

The pA2 value for the specific β2‐adrenoceptor antagonist ICI‐118,551 (8.7±0.4) as an antagonist of the adrenaline‐stimulated cyclic AMP generation were ∼3 units higher than the value for the βI‐adrenoceptor antagonist atenolol (5.6±0.3).

Treatment of the cells with adrenaline and forskolin evoked a ∼3 fold increase in the activity of serotonin N‐acetyltransferase with the peak occurring 6 h after stimulation.

These results suggest the presence of β2‐adrenoceptors in mouse pineal cells and a functional relationship between the adenylyl cyclase system and the regulation of N‐acetyltransferase expression.

British Journal of Pharmacology (1999) 126, 399–406; doi:10.1038/sj.bjp.0702248
Relaxation of corpus cavernosum and raised intracavernous pressure by berberine in rabbit
Authors: Chiou Wen‐Fei , Chen James , Chen Chieh‐Fu
Published: January 2005

In the present study, we have investigated the effect of berberine in rabbit isolated corpus cavernosum and measured the intracavernous pressure (ICP) change after intracavernosal injection of berberine in rabbit.

Berberine alone suppressed the basal tone and induced a concentration (0.1–100 μM)‐dependent relaxation in phenylephrine (PE)‐precontracted corpus cavernosum.

Tetrodotoxin (0.1 and 1 μM) treatment had no significant effect on the berberine‐induced relaxation. Phentolamine (1 and 10 μM), propranolol (1 and 3 μM) and atropine (1 and 3 μM) were also without effect. These results suggest that berberine might cause relaxation of the cavernosal strip by direct action on the corpus cavernosum, not by a neuronal effect. Furthermore, muscarinic‐ and β‐adrenoceptors were not involved.

Berberine‐induced relaxations were significantly reduced by endothelium removal and by exposure to L‐NG‐nitro arginine methyl ester (0.1 and 0.3 mM), but not indomethacin (30 μM).

In endothelium‐deprived corpus cavernosal tissues, berberine‐induced relaxations were significantly reduced in high K+ medium (KCl=60 mM), by charybdotoxin (ChTX) and 4‐aminopyridine (4‐AP) but not by glibenclamide and apamin.

After intracavernous injection of berberine (1, 2, 3 and 5 mg kg−1), the ICP rose from 12.7 ± 3.6 to 13.2 ± 5.4, 25.3 ± 6.1, 46.5 ± 8.2, and 63.4 ± 10.2 mmHg, respectively. The duration of tumescence ranged from 11.5–43.7 min.

The results show that berberine possesses a relaxant effect on rabbit corpus cavernosal tissues which is attributable to both endothelium‐dependent and‐independent properties. While the former component is apparently due to the release of NO from sinusoidal endothelium, the endothelium‐independent mechanism involved in berberine relaxation is probably linked to ChTX‐ and 4‐AP‐sensitive K+ channel activation in the cavernosal vasculature.
Brain‐to‐blood partition and in vivo inhibition of 5‐hydroxytryptamine reuptake and quipazine‐mediated behaviour of nefazodone and its main active metabolites in rodents
Authors: Nacca Angelo , Guiso Giovanna , Fracasso Claudia , Cervo Luigi , Caccia Silvio
Published: January 2005

The brain/plasma partition of nefazodone, hydroxynefazodone (OHNFZ) and m‐chlorophenyl‐piperazine (mCPP) and their antagonism of p‐chloroamphetamine (PCA)‐induced 5‐hydroxytryptamine (5‐HT) depletion and quipazine‐induced head twitches were compared in rodents.

Nefazodone (30 mg kg−1, i.p.) rapidly entered the brain but concentrations were exceeded by mCPP, the metabolic ratio being 47 and 10 in the mouse and rat respectively. OHNFZ was detectable in plasma but never in brain.

Brain concentrations of OHNFZ in the mouse (30 mg kg−1, i.p.) were less than 10% of those in plasma, confirming a poor blood‐brain barrier penetration. Concentrations of its metabolite mCPP were similar to those after 5 mg kg−1(i.p.) mCPP.

In the mouse, nefazodone (30 mg kg−1) antagonized the 5‐HT depleting effect of PCA 2 h after dosing, when it had disappeared from brain but when mCPP concentrations were similar to those after 5 mg kg−1 (i.p.) mCPP. However, mCPP antagonized PCA less than nefazodone.

In the rat, nefazodone pretreatment (30 mg kg−1, 15 min) prevented (97% of inhibition) quipazine‐induced head twitches. The effect was weaker (65% of inhibition) but significant when only mCPP was detected in brain. Analysis of brain concentrations of the two compounds after their ED50 against quipazine indicated that both contributed to the effect, although nefazodone was more active than mCPP in terms of concentrations required to obtain a comparable reduction of twitches.

These findings show that mCPP concentrates in the brain following injection of nefazodone and may play a role in preventing quipazine‐induced behaviour and PCA‐induced 5‐HT depletion. In contrast OHNFZ poorly enters the brain and its in vivo activity is mostly due to its biotransformation to mCPP.

British Journal of Pharmacology (1998) 125, 1617–1623; doi:10.1038/sj.bjp.0702251
Mechanisms involved in the metabotropic glutamate receptor‐enhancement of NMDA‐mediated motoneurone responses in frog spinal cord
Authors: Holohean Alice M , Hackman John C , Davidoff Robert A
Published: January 2005

The metabotropic glutamate receptor (mGluR) agonist trans‐(±)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid (trans‐ACPD) (10–100 μM) depolarized isolated frog spinal cord motoneurones, a process sensitive to kynurenate (1.0 mM) and tetrodotoxin (TTX) (0.783 μM).

In the presence of NMDA open channel blockers [Mg2+; (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate (MK801); 3,5‐dimethyl‐1‐adamantanamine hydrochloride (memantine)] and TTX, trans‐ACPD significantly potentiated NMDA‐induced motoneurone depolarizations, but not α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐proprionate (AMPA)‐ or kainate‐induced depolarizations.

NMDA potentiation was blocked by (RS)‐α‐methyl‐4‐carboxyphenylglycine (MCPG) (240 μM), but not by α‐methyl‐(2S,3S,4S)‐α‐(carboxycyclopropyl)‐glycine (MCCG) (290 μM) or by α‐methyl‐(S)‐2‐amino‐4‐phosphonobutyrate (L‐MAP4) (250 μM), and was mimicked by 3,5‐dihydroxyphenylglycine (DHPG) (30 μM), but not by L(+)‐2‐amino‐4‐phosphonobutyrate (L‐AP4) (100 μM). Therefore, trans‐ACPD's facilitatory effects appear to involve group I mGluRs.

Potentiation was prevented by the G‐protein decoupling agent pertussis toxin (3–6 ng ml−1, 36 h preincubation). The protein kinase C inhibitors staurosporine (2.0 μM) and N‐(2‐aminoethyl)‐5‐isoquinolinesulphonamide HCl (H9) (77 μM) did not significantly reduce enhanced NMDA responses. Protein kinase C activation with phorbol‐12‐myristate 13‐acetate (5.0 μM) had no effect.

Intracellular Ca2+ depletion with thapsigargin (0.1 μM) (which inhibits Ca2+/ATPase), 1,2‐bis(O‐aminophenoxy)ethane‐N,N,N′,N′‐tetracetic acid acetyl methyl ester (BAPTA‐AM) (50 μM) (which buffers elevations of [Ca2+]i), and bathing spinal cords in nominally Ca2+‐free medium all reduced trans‐ACPD's effects.

The calmodulin antagonists N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide (W7) (100 μM) and chlorpromazine (100 μM) diminished the potentiation.

In summary, group I mGluRs selectively facilitate NMDA‐depolarization of frog motoneurones via a G‐protein, a rise in [Ca2+]i from the presumed generation of phosphoinositides, binding of Ca2+ to calmodulin, and lessening of the Mg2+‐produced channel block of the NMDA receptor.

British Journal of Pharmacology (1999) 126, 333–341; doi:10.1038/sj.bjp.0702263
Dual actions of the metabolic inhibitor, sodium azide on KATP channel currents in the rat CRI‐G1 insulinoma cell line
Authors: Harvey J , Hardy S C , Ashford M L J
Published: January 2005

The effects of various inhibitors of the mitochondrial electron transport chain on the activity of ATP‐sensitive K+ channels were examined in the Cambridge rat insulinoma G1 (CRI‐G1) cell line using a combination of whole cell and single channel recording techniques.

Whole cell current clamp recordings, with 5 mM ATP in the pipette, demonstrate that the mitochondrial uncoupler sodium azide (3 mM) rapidly hyperpolarizes CRI‐G1 cells with a concomitant increase in K+ conductance. This is due to activation of KATP channels as the sulphonylurea tolbutamide (100 μM) completely reversed the actions of azide. Other inhibitors of the mitochondrial electron transport chain, rotenone (10 μM) or oligomycin (2 μM) did not hyperpolarize CRI‐G1 cells or increase K+ conductance.

In cell‐attached recordings, bath application of 3 mM sodium azide (in the absence of glucose) resulted in a rapid increase in KATP channel activity, an action readily reversible by tolbutamide (100 μM). Application of sodium azide (3 mM), in the presence of Mg‐ATP, to the intracellular surface of excised inside‐out patches also increased KATP channel activity, in a reversible manner.

In contrast, rotenone (10 μM) or oligomycin (2 μM) did not increase KATP channel activity in either cell‐attached, in the absence of glucose, or inside‐out membrane patch recordings.

Addition of sodium azide (3 mM) to the intracellular surface of inside‐out membrane patches in the presence of Mg‐free ATP or the non‐hydrolysable analogue 5′‐adenylylimidodiphosphate (AMP‐PNP) inhibited, rather than increased, KATP channel activity.

In conclusion, sodium azide, but not rotenone or oligomycin, directly activates KATP channels in CRI‐G1 insulin secreting cells. This action of azide is similar to that reported previously for diazoxide.

British Journal of Pharmacology (1999) 126, 51–60; doi:10.1038/sj.bjp.0702267
Homologous regulation of the α2C‐adrenoceptor subtype in human hepatocarcinoma, HepG2
Authors: Cayla Cécile , Schaak Stéphane , Roquelaine Cyril , Gales Céline , Quinchon Françoise , Paris Hervé
Published: January 2005

Previous studies of the regulation of the α2C‐adrenoceptor in OK and in transfected cells have led to discrepant conclusions. In the present work, we examined the homologous regulation of the human α2C‐adrenoceptor in the hepatocarcinoma cell‐line, HepG2; a model which expresses this subtype spontaneously.

Short‐period treatment of the cells with UK14304 provoked neither a diminution of the potency of the α2‐agonist to inhibit forskolin‐induced cyclic AMP‐accumulation nor a change in the degree of receptor coupling to G‐proteins.

Long‐period exposure to UK14304 resulted in a large reduction of [3H]MK912 binding sites (55% decrease). The action of UK14304 was dose‐dependent (EC50=190±45 nM), rapid (t1/2 =4.2 h) and reversible. Receptor down‐regulation was also observed with clonidine or (−)adrenaline (38 and 36% decrease, respectively) and was blocked by the addition of α2‐antagonists.

Conversely to that observed with α2‐agonists, treatment of the cells with RX821002 or yohimbine alone, but not with phentolamine, promoted a significant increase of the receptor expression.

The observed alterations of receptor density are not the reflection of changes at the α2C4 mRNA level. Estimation of the receptor protein turnover and measurement of its half‐life demonstrated that down‐regulation by α2‐agonists and up‐regulation by α2‐antagonists, with inverse‐agonist efficacy, are respectively the consequence of increased and decreased rate of receptor degradation.

In conclusion, our data show that α2C‐adrenoceptor does not undergo desensitization but is down‐regulated in HepG2. The lack of desensitization agrees with previous results obtained in cells transfected with the α2C4 gene, but not with observations made in OK cells. Inversely, down‐regulation fits with results obtained in OK but not in transfected cells. The reasons for these discrepancies are discussed. Our results also demonstrated that certain α2‐antagonists behave as inverse agonist on the HepG2 model and thus provide for the first time evidence of inverse efficacy of antagonists on a cellular model expressing physiological level of a wild‐type α2‐adrenoceptor.

British Journal of Pharmacology (1999) 126, 69–78; doi:10.1038/sj.bjp.0702269
Modulation of chloride, potassium and bicarbonate transport by muscarinic receptors in a human adenocarcinoma cell line
Authors: Holliday Nicholas D , Cox Helen M
Published: January 2005

Short‐circuit current (ISC) responses to carbachol (CCh) were investigated in Colony 1 epithelia, a subpopulation of the HCA‐7 adenocarcinoma cell line. In Krebs‐Henseleit (KH) buffer, CCh responses consisted of three ISC components: an unusual rapid decrease (the 10 s spike) followed by an upward spike at 30 s and a slower transient increase (the 2 min peak). This response was not potentiated by forskolin; rather, CCh inhibited cyclic AMP‐stimulated ISC.

In HCO3− free buffer, the decrease in forskolin‐elevated ISC after CCh was reduced, although the interactions between CCh and forskolin remained at best additive rather than synergistic. When Cl− anions were replaced by gluconate, both Ca2+‐ and cyclic AMP‐mediated electrogenic responses were significantly inhibited.

Basolateral Ba2+ (1–10 mM) and 293B (10 μM) selectively inhibited forskolin stimulation of ISC, without altering the effects of CCh. Under Ba2+‐ or 293B‐treated conditions, CCh responses were potentiated by pretreatment with forskolin.

Basolateral charybdotoxin (50 nM) significantly increased the size of the 10 s spike of CCh responses in both KH and HCO3− free medium, without affecting the 2 min peak. The enhanced 10 s spike was inhibited by prior addition of 5 mM apical Ba2+. Charybdotoxin did not affect forskolin responses.

In epithelial layers prestimulated with forskolin, the muscarinic antagonists atropine and 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP, both at 100 nM) abolished subsequent 10 μM CCh responses. Following addition of p‐fluoro hexahydro‐sila‐difenidol (pF‐HHSiD, 10 μM) or pirenzepine (1 μM), qualitative changes in the CCh response time‐profile also indicated a rightward shift of the agonist concentration‐response curve; however, 1 μM gallamine had no effect. These results suggest that a single M3‐like receptor subtype mediates the secretory response to CCh.

It is concluded that CCh and forskolin activate discrete populations of basolateral K+ channels gated by either Ca2+ or cyclic AMP, but that the Cl− permeability of the apical membrane may limit their combined effects on electrogenic Cl− secretion. In addition, CCh activates a Ba2+‐sensitive apical K+ conductance leading to electrogenic K+ transport. Both agents may also modulate HCO3− secretion through a mechanism at least partially dependent on carbonic anhydrase.

British Journal of Pharmacology (1999) 126, 269–279; doi:10.1038/sj.bjp.0702270
Rate‐dependent blockade of a potassium current in human atrium by the antihistamine loratadine
Authors: Crumb William J
Published: January 2005

The antihistamine loratadine is widely prescribed for the treatment of symptoms associated with allergies. Although generally believed to be free of adverse cardiac effects, there are a number of recent reports suggesting that loratadine use may be associated with arrhythmias, in particular atrial arrhythmias.

Nothing is known regarding the potassium channel blocking properties of loratadine in human cardiac cells. Using the whole‐cell patch clamp technique, the effects of loratadine on the transient outward K current (Ito), sustained current (Isus), and current measured at −100 mV (IK1 and Ins), the major inward and outward potassium currents present in human atrial myocytes, were examined in order to provide a possible molecular mechanism for the observed atrial arrhythmias reported with loratadine use.

Loratadine rate‐dependently inhibited Ito at therapeutic concentrations with 10 nM loratadine reducing Ito amplitude at a pacing rate of 2 Hz by 34.9±6.0%. In contrast, loratadine had no effect on either Isus or current measured at −100 mV.

These results may provide a possible mechanism for the incidences of supraventricular arrhythmias reported with the use of loratadine.

British Journal of Pharmacology (1999) 126, 575–580; doi:10.1038/sj.bjp.0702273
Beneficial effects of raxofelast (IRFI 016), a new hydrophilic vitamin E‐like antioxidant, in carrageenan‐induced pleurisy
Authors: Cuzzocrea Salvatore , Costantino Giuseppina , Mazzon Emanuela , Caputi Achille P
Published: January 2005

Peroxynitrite is a strong oxidant that results from reaction between NO and superoxide. It has been recently proposed that peroxynitrite plays a pathogenetic role in inflammatory processes. Here we have investigated the therapeutic efficacy of raxofelast, a new hydrophilic vitamin E‐like antioxidant agent, in rats subjected to carrageenan‐induced pleurisy.

In vivo treatment with raxofelast (5, 10, 20 mg kg−1 intraperitoneally 5 min before carrageenan) prevented in a dose dependent manner carrageenan‐induced pleural exudation and polymorphonuclear migration in rats subjected to carrageenan‐induced pleurisy. Lung myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels, as well as histological organ injury were significantly reduced by raxofelast.

Immunohistochemical analysis for nitrotyrosine, a footprint of peroxynitrite, revealed a positive staining in lungs from carrageenan‐treated rats. No positive nitrotyrosine staining was found in the lungs of the carrageenan‐treated rats, which received raxofelast (20 mg kg−1) treatment.

Furthermore, in vivo raxofelast (5, 10, 20 mg kg−1) treatment significantly reduced peroxynitrite formation as measured by the oxidation of the fluorescent dihydrorhodamine 123, prevented the appearance of DNA damage, the decrease in mitochondrial respiration and partially restored the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan‐induced pleurisy.

In conclusion, our study demonstrates that raxofelast, a new hydrophilic vitamin E‐like antioxidant agent, exerts multiple protective effects in carrageenan‐induced acute inflammation.

British Journal of Pharmacology (1999) 126, 407–414; doi:10.1038/sj.bjp.0702275
Nitrergic neurotransmission mediates the non‐adrenergic non‐cholinergic responses in the clitoral corpus cavernosum of the rabbit
Authors: Cellek Selim , Moncada Salvador
Published: January 2005

The corpus cavernosum is the erectile tissue in the penis and clitoris. Although nitrergic neurotransmission has been characterized in detail in the penile corpus cavernosum, functional studies on the inhibitory non‐adrenergic non‐cholinergic (NANC) transmission in the clitoral corpus cavernosum have been lacking. Here we demonstrate that electrical field stimulation (EFS) induces NANC relaxation responses in the clitoral corpus cavernosum of the rabbit. These responses were inhibited by NG‐nitro‐L‐arginine methylester (L‐NAME), 1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one (ODQ) or tetrodotoxin. The inhibitory effect of L‐NAME was partially reversed by L‐arginine but not by D‐arginine. EFS‐induced relaxations were enhanced by an inhibitor of type V cyclic GMP phosphodiesterase, zaprinast. These results suggest that nitrergic neurotransmission is responsible for the NANC relaxation responses in the clitoral corpus cavernosum of the rabbit.
Role of iNOS in the vasodilator responses induced by L‐arginine in the middle cerebral artery from normotensive and hypertensive rats
Authors: Briones Ana M , Alonso María J , Marín Jesús , Salaices Mercedes
Published: January 2005

The substrate of nitric oxide synthase (NOS), L‐arginine (L‐Arg, 0.01 μM−1 mM), induced endothelium‐independent relaxations in segments of middle cerebral arteries (MCAs) from normotensive Wistar‐Kyoto (WKY) and hypertensive rats (SHR) precontracted with prostaglandin F2α (PGF2α). These relaxations were higher in SHR than WKY arteries.

L‐NG‐nitroarginine methyl ester (L‐NAME) and 2‐amine‐5,6‐dihydro‐6‐methyl‐4H‐1,3‐tiazine (AMT), unspecific and inducible NOS (iNOS) inhibitors, respectively, reduced those relaxations, specially in SHR.

Four‐ and seven‐hours incubation with dexamethasone reduced the relaxations in MCAs from WKY and SHR, respectively.

Polymyxin B and calphostin C, protein kinase C (PKC) inhibitors, reduced the L‐Arg‐induced relaxation.

Lipopolysaccharide (LPS, 7 h incubation) unaltered and inhibited these relaxations in WKY and SHR segments, respectively. LPS antagonized the effect polymyxin B in WKY and potentiated L‐Arg‐induced relaxations in SHR in the presence of polymyxin B.

The contraction induced by PGF2α was greater in SHR than WKY arteries. This contraction was potentiated by dexamethasone and polymyxin B although the effect of polymyxin B was higher in SHR segments. LPS reduced that contraction and antagonized dexamethasone‐ and polymyxin B‐induced potentiation, these effects being greater in arteries from SHR.

These results suggest that in MCAs: (1) the induction of iNOS participates in the L‐Arg relaxation and modulates the contraction to PGF2α; (2) that induction is partially mediated by a PKC‐dependent mechanism; and (3) the involvement of iNOS in such responses is greater in the hypertensive strain.

British Journal of Pharmacology (1999) 126, 111–120; doi:10.1038/sj.bjp.0702281
Two affinities for a single antagonist at the neuronal NK1 tachykinin receptor: evidence from quantitation of receptor endocytosis
Authors: Jenkinson Karl M , Southwell Bridget R , Furness John B
Published: January 2005

In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide‐preferring receptor related to the NK1r.

We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist‐induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea‐pig ileum.

SP and septide gave sigmoid log concentration‐response curves and were equipotent in inducing NK1r endocytosis.

The NK1r antagonists, CP‐99994 (2S,3S)‐3‐(2‐methoxybenzyl)amino‐2‐phenylpiperidine dihydrochloride and MEN‐10581, cyclo(Leuψ[CH2NH]Lys(benzyloxycarbonyl)‐Gln‐Trp‐Phe‐βAla) were both more potent in inhibiting endocytosis (50× and 8× greater respectively) against septide than against SP.

The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide‐preferring tachykinin receptor.

British Journal of Pharmacology (1999) 126, 131–136; doi:10.1038/sj.bjp.0702285
A comparison of an A1 adenosine receptor agonist (CVT‐510) with diltiazem for slowing of AV nodal conduction in guinea‐pig
Authors: Snowdy Stephen , Liang Hui Xiu , Blackburn Brent , Lum Robert , Nelson Marek , Wang Lisa , Pfister Jürg , Sharma Bhavender P , Wolff Andrew , Belardinelli Luiz
Published: January 2005

The purpose of this study was to compare the pharmacological properties (i.e. the AV nodal depressant, vasodilator, and inotropic effects) of two AV nodal blocking agents belonging to different drug classes; a novel A1 adenosine receptor (A1 receptor) agonist, N‐(3(R)‐tetrahydrofuranyl)‐6‐aminopurine riboside (CVT‐510), and the prototypical calcium channel blocker diltiazem.

In the atrial‐paced isolated heart, CVT‐510 was approximately 5 fold more potent to prolong the stimulus‐to‐His bundle (S–H interval), a measure of slowing AV nodal conduction (EC50=41 nM) than to increase coronary conductance (EC50=200 nM). At concentrations of CVT‐510 (40 nM) and diltiazem (1 μM) that caused equal prolongation of S–H interval (∼10 ms), diltiazem, but not CVT‐510, significantly reduced left ventricular developed pressure (LVP) and markedly increased coronary conductance. CVT‐510 shortened atrial (EC50=73 nM) but not the ventricular monophasic action potentials (MAP).

In atrial‐paced anaesthetized guinea‐pigs, intravenous infusions of CVT‐510 and diltiazem caused nearly equal prolongations of P–R interval. However, diltiazem, but not CVT‐510, significantly reduced mean arterial blood pressure.

Both CVT‐510 and diltiazem prolonged S–H interval, i.e., slowed AV nodal conduction. However, the A1 receptor‐selective agonist CVT‐510 did so without causing the negative inotropic, vasodilator, and hypotensive effects associated with diltiazem. Because CVT‐510 did not affect the ventricular action potential, it is unlikely that this agonist will have a proarrythmic action in ventricular myocardium.

British Journal of Pharmacology (1999) 126, 137–146; doi:10.1038/sj.bjp.0702287