bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of cyclic AMP systems in morphine physical dependence in mice: prevention of development of morphine dependence by rolipram, a phosphodiesterase 4 inhibitor
Authors: Mamiya Takayoshi , Noda Yukihiro , Ren Xiuhai , Hamdy Moustafa , Furukawa Shoei , Kameyama Tsutomu , Yamada Kiyofumi , Nabeshima Toshitaka
Published: January 2005

In this study, we examined whether morphine dependence was inhibited by rolipram, a cyclic AMP selective phosphodiesterase inhibitor in mice, since a role for the cyclic AMP systems in the development of morphine dependence has been reported.

Mice, which received morphine (10 mg kg−1 s.c.) twice a day for 5 days showed withdrawal syndromes such as jumping, rearing and forepaw tremor following naloxone challenge (5 mg kg−1 i.p.) on the 6th day.

Such mice exhibited a significant elevation of cyclic AMP levels in the thalamus compared to control mice. However, co‐administration of rolipram (1 mg kg−1 i.p.) with morphine for 5 days significantly attenuated the severity of the withdrawal syndrome and the increase in the cyclic AMP levels after the administration of naloxone.

In naïve mice, acute morphine treatment (10 mg kg−1 s.c.) decreased cyclic AMP levels in the thalamus and cerebral cortex 10 min later. The decrease of cyclic AMP levels induced by acute morphine treatment was blocked by co‐administration of rolipram (1 mg kg−1 i.p.). However, acute rolipram did not affect the naloxone‐precipitated morphine withdrawal syndrome.

These results suggest that the elevation of the cyclic AMP levels is involved in the development of morphine withdrawal syndrome and that blockade of the morphine‐induced reduction of cyclic AMP levels by chronic rolipram inhibits the development of dependence and the behavioural and biochemical changes induced by naloxone. Furthermore, rolipram may be a useful drug for attenuating the development of morphine dependence.

British Journal of Pharmacology (2001) 132, 1111–1117; doi:10.1038/sj.bjp.0703912
The transport activity of the human cationic amino acid transporter hCAT‐1 is downregulated by activation of protein kinase C
Authors: Gräf Petra , Förstermann Ulrich , Closs Ellen I
Published: January 2005

The human cationic amino acid transporter hCAT‐1 contains several consensus sequences for phosphorylation by protein kinase C (PKC). This study investigates the effect of PKC activation on hCAT‐1‐mediated transport.

When expressed in Xenopus laevis oocytes, hCAT‐1‐mediated L‐arginine transport was reduced to 44±3% after a 30 min treatment of the oocytes with 100 nM phorbol‐12‐myristate‐13‐acetate (PMA). 4α‐phorbol‐12,13‐didecanoate (4α‐PDD, 100 nM) had no effect.

In EA.hy926 endothelial cells, maximal inhibition of hCAT‐1‐mediated L‐arginine transport (to 3 – 11% of control) was observed after treatment of the cells with 100 nM PMA for 4 h. A 20 – 30 h exposure of the cells to 100 nM PMA led to the recovery of the L‐arginine uptake rate that was now resistant to a second application of PMA. Phorbol‐12,13‐dibutyrate had similar effects as PMA, whereas 4α‐PDD had no effect. One μM bisindolylmaleimide I reduced the PMA effect significantly.

Interestingly, a 4 h treatment with 100 nM PMA increased the expression of hCAT‐1 mRNA 3 – 5 fold. hCAT‐1 protein levels were unchanged for up to 4 h after PMA treatment and then increased slightly between 8 – 28 h.

It is concluded that PMA downregulates the intrinsic activity of hCAT‐1 by a pathway involving protein kinase C.

British Journal of Pharmacology (2001) 132, 1193–1200; doi:10.1038/sj.bjp.0703921
Pharmacological analysis of cannabinoid receptor activity in the rat vas deferens
Authors: Christopoulos Arthur , Coles Peter , Lay Lucy , Lew Michael J , Angus James A
Published: January 2005

The interaction between the cannabinoid agonists, WIN 55,212‐2 or CP 55,940 with the CB1 receptor‐selective antagonists, SR141716A or LY320135 was investigated using the rat electrically‐stimulated vas deferens bioassay.

Tissues were stimulated by single‐field pulses (150 V, 0.5 ms) delivered every 30 mins. In the presence of nifedipine (3 μM), agonists elicited a concentration‐dependent inhibition of the contractile response, with pEC50 values of 7.93 and 6.84 for WIN 55,212‐2 and CP 55,940, respectively.

SR141716A and LY320135 caused parallel dextral displacements of the agonist concentration‐response curves. However, the shift of the agonist curves by either antagonist was accompanied by a concentration‐dependent enhancement of basal (agonist‐independent) tissue contraction.

Addition of the amidase inhibitor, phenylmethylsulphonylfluoride (200 μM), resulted in a significant reduction of the basal twitch response, an effect consistent with the presence of tonic receptor activation mediated by the endogenous cannabinoid, anandamide.

In light of these findings, we propose a theoretical model of competitive agonist‐antagonist interaction in the presence of endogenous agonist tone that was used to derive an optimized analytical approach for the determination of antagonist potency estimates under conditions of tonic receptor activation.

This approach yielded pKB estimates for SR141716A and LY320135 that were in good agreement with their activity at cannabinoid CB1 receptors.

It is concluded that the rat vas deferens contains prejunctional cannabinoid CB1 receptors that are under tonic activation from endogenous substances; under these conditions our analytical approach is preferable to the standard methods for the determination of antagonist potency.

British Journal of Pharmacology (2001) 132, 1281–1291; doi:10.1038/sj.bjp.0703930
Dual effects of muscarinic M2 acetylcholine receptors on the synthesis of cyclic AMP in CHO cells: dependence on time, receptor density and receptor agonists
Authors: Michal Pavel , Lysíková Michaela , Tuček Stanislav
Published: January 2005

Muscarinic M2 receptors normally inhibit the production of cyclic AMP via Gi proteins, but a stimulatory component occurs in their effect at high agonist concentrations, believed to be based on the activation of Gs proteins. We investigated the conditions which determine the occurrence and extent of the stimulatory component in CHO cells stably expressing muscarinic M2 receptors.

Biphasic concentration‐response curves (decline followed by return towards control values) were obtained after 10 min incubation with carbachol, oxotremorine‐M, acetylcholine, arecoline and arecaidine propargyl ester, but the upward phase was missing with oxotremorine, methylfurmethide, furmethide and pentylthio‐TZTP. Shortening the incubation favoured the occurrence of the stimulatory component. Carbachol (1 mM) and oxotremorine‐M (1 mM) brought about net stimulation (above 100% of control) of cyclic AMP synthesis during 2 min incubations. The stimulatory components disappeared after the density of receptors had been lowered with oxyphenonium mustard.

All agonists stimulated the synthesis of cyclic AMP in cells pretreated with pertussis toxin.

Most differences between agonists regarding the stimulatory component of their effect on cyclic AMP synthesis could be explained by differences in their efficacy and the induced receptor internalization.

We propose that the Gs‐mediated stimulatory component of the effect of muscarinic M2 receptors on cyclic AMP synthesis only occurs if the density of activated receptors is high enough to saturate the Gi proteins and proportionate to the receptors' low affinity for the Gs proteins. It tends to be abolished by receptor internalization.

British Journal of Pharmacology (2001) 132, 1217–1228; doi:10.1038/sj.bjp.0703931
Tonabersat (SB‐220453) a novel benzopyran with anticonvulsant properties attenuates trigeminal nerve‐induced neurovascular reflexes
Authors: Parsons Andrew A , Bingham Sharon , Raval Pravin , Read Simon , Thompson Mervyn , Upton Neil
Published: January 2005

The effects of tonabersat (SB‐220453) were evaluated on trigeminal nerve ganglion stimulation‐induced sensory‐autonomic neurovascular reflexes in the anaesthetized cat. Comparisons were made to intravenous administration of carabersat (SB‐204269), and to valproate, gabapentin and lamotrigine following intraduodenal administration.

There were no effects on resting blood pressure, heart rate, carotid blood flow or carotid vascular resistance for any compound evaluated.

Trigeminal nerve ganglion stimulation increased carotid blood flow by 65% and reduced vascular resistance by 41% with minimal effect on blood pressure (<10%) and no effect on heart rate. Intravenous infusion of tonabersat or carabersat (both 3.4 μmol h−1) produced time related reductions in stimulation‐induced responses with a maximal inhibition (relative to control) of 30±7% (n=4), at 240 min for tonabersat and 33±4% (n=3) at 180 min for carabersat. Tonabersat (11.5 μmol h−1) produced a similar inhibitory effect (32±9%, n=4) after 120 min of infusion.

Following intraduodenal administration of tonabersat, the maximal inhibition of nerve stimulation‐induced responses was 55±4% at 120 min (n=4) for tonabersat 10 mg kg−1, and 24±2% after 180 min for 1 mg kg−1 (n=4).

Intraduodenal administration of sodium valproate (10 or 100 mg kg−1 n=4/group) had no effect on neurovascular reflexes. Maximal inhibition of nerve ganglion‐stimulated reductions in carotid vascular resistance were observed at 150 min for lamotrigine (50 mg kg−1, 52±12%, n=4) and gabapentin (100 mg kg−1, 17±13%, n=3). Lamotrigine 10 mg kg−1 produced 22±11% (n=3) inhibition after 180 min.

These data demonstrate blockade of trigeminal parasympathetic reflexes with tonabersat, carabersat and other anticonvulsants. These agents may therefore have therapeutic benefit in conditions where this type of reflex is evident.

British Journal of Pharmacology (2001) 132, 1549–1557; doi:10.1038/sj.bjp.0703932
Effects of some sterically hindered phenols on whole‐cell Ca2+current of guinea‐pig gastric fundus smooth muscle cells
Authors: Fusi Fabio , Saponara Simona , Gagov Hristo , Sgaragli Giampietro
Published: January 2005

The aim of the present study was to investigate the effects of extracellular application of some sterically‐hindered phenols, namely 3‐t‐butyl‐4‐hydroxyanisole (BHA), 3,5‐di‐t‐butyl‐4‐hydroxyanisole (DTBHA) and the dimer of BHA, 2,2′‐dihydroxy‐3,3′‐di‐t‐butyl‐5,5′‐dimethoxydiphenyl (DIBHA), on the whole‐cell Ca2+current (ICa) of freshly isolated smooth muscle cells from the guinea‐pig gastric fundus, in the presence of a range of Ca2+concentrations (1 – 5 mM) using the patch‐clamp technique. The influx of Ca2+had characteristics of L‐typeICa(ICa(L)).

BHA as well as DTBHA inhibitedICa(L)in a concentration‐dependent manner, during depolarization to 10 mV from a holding potential of −50 mV. Bath application of BHA (50 μM) and DTBHA (30 μM) decreasedICa(L)by 48.9% and 45.2%, respectively. This inhibition was only partially reversible. In contrast, DIBHA (up to 50 μM) was devoided of effects onICa(L).

BHA inhibition ofICa(L)was voltage‐dependent and inversely related to the external concentration of Ca2+. On the other hand, DTBHA inhibition was only voltage‐dependent.

BHA and DTBHA shifted the voltage range of the steady‐state inactivation curve to more negative potentials by 8 mV at the mid‐potential of the curve, without affecting the activation curve. Furthermore, BHA and DTBHA did not modify the time‐course of the current decay.

We conclude that the inhibition ofICa(L)by BHA and DTBHA is qualitatively similar to that of a Ca2+channel blocker and is characterized by the stabilizing effect of the inactivated state of the channel.

British Journal of Pharmacology(2001)132, 1326–1332; doi:10.1038/sj.bjp.0703935
Inhibition of the cyclin D1/E2F pathway by PCA‐4230, a potent repressor of cellular proliferation
Authors: Goukassian David , Sanz‐González Silvia M , Pérez‐Roger Ignacio , Font de Mora Jaime , Ureña Jesús , Andrés Vicente
Published: January 2005

Tight control of cellular growth is essential to ensure normal tissue patterning and prevent pathological responses. Excessive vascular smooth muscle cell (VSMC) proliferation is associated with the pathophysiology of atherosclerosis and restenosis post‐angioplasty. Thus, drug targeting of pathological VSMC growth may be a suitable therapeutic intervention in vascular proliferative diseases.

In the present study, we investigated the mechanisms underlying VSMC growth arrest induced by the pharmacological agent PCA‐4230. Addition of PCA‐4230 to cultured VSMCs blocked the induction of cyclin D1 and cyclin A expression normally seen in serum‐restimulated cells. Moreover, PCA‐4230 inhibited cyclin‐dependent kinase 2 (CDK2) activity and abrogated hyperphosphorylation of the retinoblastoma (Rb) gene product. Similarly, PCA‐4230‐dependent growth arrest of transformed cell lines correlated with reduced level of cyclin D1 protein and inhibition of CDK2 activity. Consistent with these findings, PCA‐4230 repressed serum‐inducible cyclin A promoter activity, and overexpression of either cyclin D1 or E2F1 efficiently circumvented this inhibitory effect. Importantly, adenovirus‐mediated overexpression of E2F1 restored S‐phase entry in PCA‐4230‐treated VSMCs, demonstrating that PCA‐4230 represses cyclin A gene expression and VSMC growth via inhibition of the cyclin D1/E2F pathway.

Because of its ability to inhibit the growth of human VSMCs and transformed cell lines, future studies are warranted to assess whether PCA‐4230 may be a suitable therapeutic intervention for the treatment of hyperproliferative disorders, including cardiovascular disease and cancer.

British Journal of Pharmacology (2001) 132, 1597–1605; doi:10.1038/sj.bjp.0703945
Cyclo‐oxygenase and lipoxygenase pathways in mast cell dependent‐neurogenic inflammation induced by electrical stimulation of the rat saphenous nerve
Authors: Le Filliatre G , Sayah S , Latournerie V , Renaud J F , Finet M , Hanf R
Published: January 2005

We investigated the role of arachidonic acid metabolism and assessed the participation of mast cells and leukocytes in neurogenic inflammation in rat paw skin. We compared the effect of lipoxygenase (LOX) and cyclo‐oxygenase (COX) inhibitors on oedema induced by saphenous nerve stimulation, substance P (SP), and compound 48/80.

Intravenous (i.v.) pre‐treatment with a dual COX/LOX inhibitor (RWJ 63556), a dual LOX inhibitor/cysteinyl‐leukotriene (CysLt) receptor antagonist (Rev 5901), a LOX inhibitor (AA 861), a five‐lipoxygenase activating factor (FLAP) inhibitor (MK 886), or a glutathione S‐transferase inhibitor (ethacrynic acid) significantly inhibited (40 to 60%) the development of neurogenic oedema, but did not affect cutaneous blood flow. Intradermal (i.d.) injection of LOX inhibitors reduced SP‐induced oedema (up to 50% for RWJ 63556 and MK 886), whereas ethacrynic acid had a potentiating effect.

Indomethacin and rofecoxib, a highly selective COX‐2 inhibitor, did not affect neurogenic and SP‐induced oedema. Surprisingly, the structurally related COX‐2 inhibitors, NS 398 and nimesulide, significantly reduced both neurogenic and SP‐induced oedema (70% and 42% for neurogenic oedema, respectively; 49% and 46% for SP‐induced oedema, respectively).

COX‐2 mRNA was undetectable in saphenous nerves and paw skin biopsy samples, before and after saphenous nerve stimulation.

A mast cell stabilizer, cromolyn, and a H1 receptor antagonist, mepyramine, significantly inhibited neurogenic (51% and 43%, respectively) and SP‐induced oedema (67% and 63%, respectively).

The co‐injection of LOX inhibitors and compound 48/80 did not alter the effects of compound 48/80. Conversely, ethacrynic acid had a significant potentiating effect. The pharmacological profile of the effect of COX inhibitors on compound 48/80‐induced oedema was similar to that of neurogenic and SP‐induced oedema.

The polysaccharide, fucoidan (an inhibitor of leukocyte rolling) did not affect neurogenic or SP‐induced oedema.

Thus, (i) SP‐induced leukotriene synthesis is involved in the development of neurogenic oedema in rat paw skin; (ii) this leukotriene‐mediated plasma extravasation might be independent of mast cell activation and/or of the adhesion of leukocytes to the endothelium; (iii) COX did not appear to play a significant role in this process.

British Journal of Pharmacology (2001) 132, 1581–1589; doi:10.1038/sj.bjp.0703950
Role of the epithelium in opposing H2O2‐induced modulation of acetylcholine‐induced contractions in rabbit intrapulmonary bronchiole
Authors: Asano Takayuki , Hattori Tomonori , Tada Toyohiro , Kajikuri Junko , Kamiya Toshio , Saitoh Michihiro , Yamada Yasuo , Itoh Makoto , Itoh Takeo
Published: January 2005

The role played by the epithelium in H2O2‐induced modulation of the mechanical responses induced by acetylcholine (ACh) in rabbit intrapulmonary bronchioles was investigated in epithelium‐intact and ‐denuded strips.

When ACh (3 μM) was applied intermittently, H2O2 (30 μM) enhanced the ACh‐induced contractions in epithelium‐intact strips. In contrast, in epithelium‐denuded strips H2O2 (30 μM) inhibited such contractions. At higher concentrations, H2O2 concentration‐dependently attenuated the ACh‐induced contractions in both epithelium‐intact and ‐denuded strips, its action being more potent in the latter strips than in the former.

Diclofenac (a cyclo‐oxygenase inhibitor; 3 μM) reduced the H2O2‐induced enhancement of ACh‐contractions in epithelium‐intact strips but had no effect on the H2O2‐induced inhibition in epithelium‐denuded strips. NG‐nitro‐L‐arginine did not alter the effect of H2O2 on ACh‐induced contractions in epithelium‐intact strips.

Catalase (500 u ml−1) completely blocked both H2O2‐induced effects on ACh‐contractions (enhancement and inhibition). Neither superoxide dismutase (200 u ml−1) nor deferoxamine (0.5 mM) had any effect on H2O2‐induced inhibition in epithelium‐denuded strips.

Aminotriazole (an inhibitor of catalase; 50 mM) significantly potentiated the H2O2‐induced inhibition of ACh‐contractions in epithelium‐intact strips but not in epithelium‐denuded strips.

The density ratio for catalase (epithelium‐intact over ‐denuded strips) analysed by Western blot was about 2.1, suggesting that epithelium contains more catalase than smooth muscle.

It is concluded that in rabbit intrapulmonary bronchioles, H2O2 has dual actions on ACh‐contractions. It is suggested that the epithelium may act as a powerful biochemical barrier via both the action of catalase (scavenging H2O2) and the release of bronchoconstrictor prostaglandins, thus attenuating the H2O2‐induced modulation of ACh‐contractions.

British Journal of Pharmacology (2001) 132, 1271–1280; doi:10.1038/sj.bjp.0703956
Agonist trafficking of Gi/o‐mediated α2A‐adrenoceptor responses in HEL 92.1.7 cells
Authors: Kukkonen Jyrki P , Jansson Christian C , Åkerman Karl E O
Published: January 2005

The ability of 19 agonists to elevate Ca2+ and inhibit forskolin‐induced cyclic AMP elevation through α2A‐adrenoceptors in HEL 92.1.7 cells was investigated. Ligands of catecholamine‐like‐ (five), imidazoline‐ (nine) and non‐catecholamine‐non‐imidazoline‐type (five) were included.

The relative maximum responses were similar in both assays. Five ligands were full or nearly full agonists, six produced 20 – 70% of the response to a full agonist and the remaining eight gave lower responses (<20%) so that their potencies were difficult to evaluate.

Marked differences in the potencies of the agonists with respect to the two measured responses were seen. The catecholamines were several times less potent in decreasing cyclic AMP than in increasing Ca2+, whereas the other, both imidazoline and ox‐/thiazoloazepine ligands, were several times more potent with respect to the former than the latter response. For instance, UK14,304 was more potent than adrenaline with respect to the cyclic AMP response but less potent than adrenaline with respect to the Ca2+ response.

All the responses were sensitive to pertussis toxin‐pretreatment. Also the possible role of PLA2, β‐adrenoceptors or ligand transport or metabolism as a source of error could be excluded. The results suggest that the active receptor states produced by catecholamines and the other agonists are markedly different and therefore have different abilities to activate different signalling pathways.

British Journal of Pharmacology (2001) 132, 1477–1484; doi:10.1038/sj.bjp.0703964
Activity of mu‐ and delta‐opioid agonists in vas deferens from mice deficient in MOR gene
Authors: Maldonado Rafael , Severini Cintia , Matthes Hans W D , Kieffer Brigitte L , Melchiorri Pietro , Negri Lucia
Published: January 2005

Mice lacking the mu‐opioid receptor have been recently generated. Centrally mediated responses of mu‐opioid agonists are suppressed whereas some of the delta‐opioid responses are preserved in these mutant mice.

The vas deferens bioassay has been used in this study to investigate the functional activity at a peripheral level of mu‐ and delta‐opioid agonists in mice lacking mu‐opioid receptors.

The different mu‐opioid agonists evaluated, morphine, DAMGO, dermorphin and [Lys7]‐dermorphin produced an inhibitory response in vas deferens from wild‐type mice but had no relevant activity on vas deferens from mutant mice.

The selective delta‐opioid agonists DPDPE, BUBU, deltorphin I, deltorphin II and [D‐Met2]‐deltorphin induced inhibitory effects in vas deferens from both wild‐type and mutant mice. However, the biological activities of these ligands were slightly reduced in preparations from mutant mice. The inhibitory responses of all these delta‐opioid agonists were prevented by the administration of the selective delta‐opioid antagonist naltrindole.

These data indicate that delta‐opioid agonists, but not mu‐opioid agonists, are biologically active in vas deferens from mice lacking mu‐opioid receptors. The decreased response of delta‐agonists in mutant mice suggests that some cooperativity may exist between mu‐ and delta‐opioid receptors in these vas deferens preparations.

British Journal of Pharmacology (2001) 132, 1485–1492; doi:10.1038/sj.bjp.0703966
Direct inhibitory effect of digitalis on progesterone release from rat granulosa cells
Authors: Chen Jiann‐Jong , Wang Paulus S , Chien Eileen‐Jea , Wang Shyi‐Wu
Published: January 2005

Digoxin (10−7 – 10−5 M) or digitoxin (10−7 – 10−5 M) decreased the basal and human chorionic gonadotropin (hCG)‐stimulated release of progesterone from rat granulosa cells.

Digoxin (10−5 M) or digitoxin (10−5 M) attenuated the stimulatory effects of forskolin and 8‐bromo‐cyclic 3′ : 5′‐adenosine monophosphate (8‐Br‐cAMP) on progesterone release from rat granulosa cells.

Digoxin (10−5 M) or digitoxin (10−5 M) inhibited cytochrome P450 side chain cleavage enzyme (cytochrome P450scc) activity (conversion of 25‐hydroxyl cholesterol to pregnenolone) in rat granulosa cells but did not influence the activity of 3β‐hydroxysteroid dehydrogenase (3β‐HSD).

Neither progesterone production nor P450scc activity in rat granulosa cells was altered by the administration of ouabain.

Digoxin (10−5 M) or digitoxin (10−5 M), but not ouabain, decreased the expression of P450scc and steroidogenic acute regulatory (StAR) protein in rat granulosa cells.

The present results suggest that digoxin and digitoxin decrease the progesterone release by granulosa cells via a Na+,K+‐ATPase‐independent mechanism involving the inhibition of post‐cyclic AMP pathway, cytochrome P450scc and StAR protein functions.

British Journal of Pharmacology (2001) 132, 1761–1768; doi:10.1038/sj.bjp.0703974
Mitogenic effect of oxidized low‐density lipoprotein on vascular smooth muscle cells mediated by activation of Ras/Raf/MEK/MAPK pathway
Authors: Yang Chuen‐Mao , Chien Chin‐Sung , Hsiao Li‐Der , Pan Shiow‐Lin , Wang Chuan‐Chawn , Chiu Chi‐Tso , Lin Chih‐Chung
Published: January 2005

It has been demonstrated that oxidized low‐density lipoprotein (OX‐LDL) is a risk factor in atherosclerosis by stimulating vascular smooth muscle cell (VSMC) proliferation. However, the mechanisms of OX‐LDL‐induced cell proliferation are not completely understood. Therefore, we investigated the effect of OX‐LDL on cell proliferation associated with mitogen‐activated protein kinase (MAPK) activation in rat cultured VSMCs.

Both native‐LDL (N‐LDL) and OX‐LDL induced a time‐ and concentration‐dependent incorporation of [3H]‐thymidine in VSMCs.

OX‐LDL induced time‐ and concentration‐dependent phosphorylation of p42/p44 MAPK. Pretreatment of these cells with pertussis toxin or U73122 attenuated the OX‐LDL‐induced responses.

Pretreatment with PMA for 24 h, preincubation with a PKC inhibitor staurosporine or the tyrosine kinase inhibitors, genistein and herbimycin A for 1 h, substantially reduced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation induced by OX‐LDL.

Removal of Ca2+ by BAPTA/AM or depletion of the internal Ca2+ pool by thapsigargin significantly inhibited OX‐LDL‐induced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation.

OX‐LDL‐induced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation was inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK) in a concentration‐dependent manner.

Overexpression of dominant negative mutants of Ras (H‐Ras‐15A) and Raf (Raf‐N4) significantly suppressed MEK1/2 and p42/p44 MAPK activation induced by OX‐LDL and PDGF‐BB, indicating that Ras and Raf may be required for activation of these kinases.

These results suggest that the mitogenic effect of OX‐LDL is mediated through a PTX‐sensitive G protein‐coupled receptor that involves the activation of the Ras/Raf/MEK/MAPK pathway similar to that of PDGF‐BB in rat cultured VSMCs.

British Journal of Pharmacology (2001) 132, 1531–1541; doi:10.1038/sj.bjp.0703976
The effect of SB‐269970, a 5‐HT7 receptor antagonist, on 5‐HT release from serotonergic terminals and cell bodies
Authors: Roberts Claire , Allen Lucy , Langmead Christopher J , Hagan Jim J , Middlemiss Derek N , Price Gary W
Published: January 2005

The presence of 5‐HT7 receptor mRNA and protein in 5‐HT neurons suggests that this receptor may act as a 5‐HT autoreceptor. In this study, the effect of the 5‐HT7 receptor antagonist, SB‐269970 ((R)‐1‐[3‐hydroxy phenyl)sulfonyl]‐2‐[2‐(4‐methyl‐1‐piperidinyl)ethyl]pyrrolidine), was investigated on 5‐HT release in the guinea‐pig and rat cortex and the rat dorsal raphe nucleus (DRN), using the techniques of in vitro [3H]‐5‐HT release or fast cyclic voltammetry, respectively.

Cortical slices were loaded with [3H]‐5‐HT and release was evoked by electrical stimulation. 5‐CT inhibited the evoked release of [3H]‐5‐HT in a concentration‐dependent manner. SB‐269970 had no significant effect on [3H]‐5‐HT release while the 5‐HT1B receptor antagonist, SB‐224289 significantly potentiated [3H]‐5‐HT release. In addition, SB‐269970 was unable to attenuate the 5‐CT‐induced inhibition of release while SB‐224289 produced a rightward shift of the 5‐CT response, generating estimated pKB values of 7.8 and 7.6 at the guinea‐pig and rat terminal 5‐HT autoreceptors respectively.

Rat DRN slices were electrically stimulated and the evoked 5‐HT efflux detected by voltammetric analysis. 8‐OH‐DPAT inhibited evoked 5‐HT efflux and was fully reversed by WAY 100635. SB‐269970 had no effect on either 5‐HT efflux per se or 8‐OH‐DPAT‐induced inhibition of 5‐HT efflux. In addition, 5‐CT inhibited 5‐HT efflux in a concentration‐dependent manner. SB‐269970 was unable to attenuate the 5‐CT‐induced inhibition of 5‐HT efflux.

In conclusion, we were unable to provide evidence to suggest a 5‐HT autoreceptor role for 5‐HT7 receptors. However, investigations with more selective 5‐HT7 receptor agonists are needed to confirm the data reported here.

British Journal of Pharmacology (2001) 132, 1574–1580; doi:10.1038/sj.bjp.0703979
Phenolic antioxidants trolox and caffeic acid modulate the oxidized LDL‐induced EGF‐receptor activation
Authors: Vacaresse Nathalie , Vieira Otília , Robbesyn Fanny , Jürgens Günther , Salvayre Robert , Negre‐Salvayre Anne
Published: January 2005

Oxidized low density lipoproteins (oxLDL) are thought to play a major role in atherosclerosis. OxLDL act in part through alteration of intracellular signalling pathways in cells of the vascular wall. We recently reported that the EGF receptor (EGFR) signalling pathway is activated by lipid peroxidation products (among them 4‐hydroxynonenal, 4‐HNE) contained in oxLDL.

The use of phenolic antioxidants, such as trolox, alpha‐tocopherol, caffeic acid and tyrphostins A‐25, A‐46 or A‐1478, showed that the oxLDL‐induced EGFR activation is constituted by two separate components, the first (early) one being antioxidant‐insensitive, the second (late) being antioxidant‐sensitive.

4‐HNE derivatization of EGFR and EGFR activation induced by exogenous 4‐HNE, suggest that the early (0.5 – 3 h) component of oxLDL‐induced EGFR activation is mediated (at least in part) by 4‐HNE (and possibly by other oxidized lipids). This early component is antioxidant‐insensitive.

The second component (4 – 5 h) of the oxLDL‐induced EGFR activation is antioxidant‐sensitive, since it is blocked by antioxidants such as trolox, caffeic acid or PDTC, which act by blocking the cellular oxidative stress (H2O2 generation) evoked by oxLDL. Conversely, exogenous H2O2 induced EGFR autophosphorylation (thus mimicking the second component) and was also inhibited by antioxidants. This effect is mediated in part through inhibition by oxidative stress of protein tyrosine phosphatases involved in EGFR dephosphorylation.

British Journal of Pharmacology (2001) 132, 1777–1788; doi:10.1038/sj.bjp.0703981
Long‐lasting antinociceptive effects of a novel dynorphin analogue, Tyr‐D‐Ala‐Phe‐Leu‐Arg ψ (CH2NH) Arg‐NH2, in mice
Authors: Hiramatsu Masayuki , Inoue Kaori , Ambo Akihiro , Sasaki Yusuke , Kameyama Tsutomu
Published: January 2005

Tyr‐D‐Ala‐Phe‐Leu‐Arg ψ (CH2NH) Arg‐NH2 (SK‐9709) is a dynorphin derivative in which the peptide bond was replaced with a ψ (CH2NH) bond. In the present study, the antinociceptive effects of SK‐9709 were determined in an acetic acid‐induced writhing test and a hot‐plate test.

In the acetic acid‐induced writhing test, significant antinociceptive effects were observed after subcutaneous (s.c.), intracerebroventricular (i.c.v.) and intrathecal (i.t.) injection of SK‐9709, with maximal effects at 120, 30 and 15 min, respectively. The antinociceptive effects were dose‐dependent and ED50 values (range of 95% confidence limits) after s.c., i.c.v. and i.t. injection were 1.36 (0.61 – 3.02) μmol kg−1, 2.11 (1.18 – 3.79) and 0.79 (0.61 – 1.03) nmol per mouse, respectively.

The effects of SK‐9709 (s.c., i.c.v. and i.t.) were reversed by the opioid receptor antagonist naloxone (1.36 μmol kg−1, s.c.). The effects of SK‐9709 (s.c.) were also reversed by the selective μ‐opioid receptor antagonist β‐funaltrexamine (4.7 nmol per mouse, i.c.v.), and κ‐opioid receptor antagonist nor‐binaltorphimine (4.9 nmol per mouse, i.t.).

In the hot‐plate test, the antinociceptive effect of SK‐9709 (s.c., i.c.v. and i.t.) was also dose‐dependent with the maximal peak effect at 120, 15 and 15 min similarly to the acetic acid‐induced writhing test. The antinociceptive effects were dose‐dependent and ED50 values (range of 95% confidence limits) after s.c., i.c.v. and i.t. injection were 39.1 (5.4 – 283.0) μmol kg−1, 6.5 (4.0 – 10.7) and 7.4 (5.0 – 11.0) nmol per mouse, respectively.

These findings indicated that systemically administered SK‐9709 produced long‐lasting antinociceptive effects and these effects were mediated by both supra‐spinal μ‐ and spinal κ‐opioid receptors.

British Journal of Pharmacology (2001) 132, 1948–1956; doi:10.1038/sj.bjp.0703982
Pharmacological characterization of serotonin receptor subtypes modulating primary afferent input to deep dorsal horn neurons in the neonatal rat
Authors: Garraway Sandra M , Hochman Shawn
Published: January 2005

Spinal cord slices and whole‐cell patch clamp recordings were used to investigate the effects of serotonergic receptor ligands on dorsal root‐evoked synaptic responses in deep dorsal horn (DDH) neurons of the neonatal rat at postnatal days (P) 3 – 6 and P10 – 14.

Bath applied 5‐hydroxytryptamine (5‐HT) potently depressed synaptic responses in most neurons. Similarly, the 5‐HT1/7 receptor agonist, 5‐carboxamidotryptamine (5‐CT) depressed synaptic responses. This action was probably mediated by 5‐HT1A receptor activation, since it occurred in the presence of the 5‐HT7 receptor antagonist clozapine and was not observed in the presence of NAN‐190, a 5‐HT1A receptor antagonist. In the absence of any agonist, 5‐HT1A receptor antagonists often facilitated synaptic responses, suggesting that there is sufficient endogenous 5‐HT to tonically activate 5‐HT1A receptors.

8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT), the 5‐HT1A/7 receptor agonist, facilitated synaptic responses, an action probably mediated by 5‐HT7 receptors, since the facilitation could be reversed by subsequent application of the 5‐HT7 receptor antagonist clozapine.

Agonists for the 5‐HT1B, 5‐HT2 and 5‐HT3 receptors exerted only modest modulatory actions.

A pharmacological analysis of the depression evoked by 5‐HT suggested an action partly mediated by 5‐HT1A receptor activation, since antagonism of the 5‐HT1A receptor with NAN‐190 or WAY‐100635 partly reversed 5‐HT‐evoked depression. In comparison, 5‐HT7 receptor activation could account for much of the 5‐HT‐evoked facilitation.

We conclude that 5‐HT is capable of modulating sensory input onto DDH neurons via several receptor subtypes, producing both facilitatory and depressant actions. Also, the actions of most receptor ligands on the evoked responses were similar within the first 2 postnatal weeks.

British Journal of Pharmacology (2001) 132, 1789–1798; doi:10.1038/sj.bjp.0703983
An endogenous 5‐HT7 receptor mediates pigment granule dispersion in Xenopus laevis melanophores
Authors: Teh Muy‐Teck , Sugden David
Published: January 2005

Melatonin (5‐methoxy N‐acetyltryptamine) and serotonin (5‐HT) exert rapid, but opposite effects on pigment granule distribution in Xenopus laevis melanophores. Low concentrations of melatonin (10−11 – 10−9 M) cause a dramatic perinuclear aggregation of the melanin‐containing granules, while 5‐HT (10−8 – 10−5 M) disperses pigment granules throughout the cell.

The present study found that pharmacological doses of melatonin (10−6 M) induced a time‐ and concentration‐dependent pigment granule dispersion, which was mediated by an endogenous melanophore 5‐HT receptor.

5‐HT produced a concentration‐dependent elevation of melanophore cyclic AMP, and 5‐HT‐induced dispersion was blocked by H89 (10−4 M), an inhibitor of protein kinase A (PKA), but not by a PKC inhibitor (Ro 31‐8220, 10−5 M), indicating a vital role for cyclic AMP in 5‐HT‐induced dispersion.

5‐HT‐mediated dispersion was not blocked by antagonists selective for Gs‐coupled 5‐HT4 (GR113808) or 5‐HT6 (Ro 04‐6790, Ro 63‐0563, olanzepine) receptors, nor by 5‐HT1 – 3 (pindolol, ketanserine, metoclopramide, MDL72222, tropisetron) receptor antagonists, but was inhibited by a selective 5‐HT7 receptor antagonist, DR4004, and other antagonists with a high affinity for 5‐HT7 receptors. The rank order of antagonist potency was: risperidone (mean pKB 7.82)>methiothepin (7.43)>DR4004 (6.92)>mesulergine (6.83)>methysergide (6.60)>[±]‐sulpiride (5.81)>spiperone (5.52). The agonist potency order [mean pEC50, 5‐CT (8.68)>5‐HT (7.13)>5‐MT (6.94)>8‐OH‐DPAT (4.79)>sumatriptan (<4)] was also consistent with an action on 5‐HT7 receptors.

RT – PCR confirmed that melanophores express 5‐HT7 receptor mRNA. The pigment dispersing effect of high melatonin concentrations in melanophores is most likely mediated by activation of 5‐HT7 receptors. Conceivably some of the effects attributed to pharmacological doses of melatonin in mammals may be mediated by activation of 5‐HT7 receptors.

British Journal of Pharmacology (2001) 132, 1799–1808; doi:10.1038/sj.bjp.0703988
Reduction of opioid dependence by the CB1 antagonist SR141716A in mice: evaluation of the interest in pharmacotherapy of opioid addiction
Authors: Mas‐Nieto Magdalena , Pommier Blandine , Tzavara Eleni T , Caneparo Anne , Nascimento Sophie Da , Fur Gérard Le , Roques Bernard P , Noble Florence
Published: January 2005

Several compounds, mainly opioid agonists such as methadone, are currently used for long term medication of heroin addicts. Nevertheless, these maintenance treatments have the disadvantage to induce a dependence to another opiate. As interactions between opioid and cannabinoid systems have been demonstrated, the ability of the CB1 antagonist, SR141716A to reduce morphine‐induced addiction was investigated.

The effects of SR141716A on the rewarding responses of morphine were evaluated in the place conditioning paradigm. No significant conditioned preference or aversion were observed after repeated treatment with the CB1 antagonist alone. However, SR141716A was able to antagonize the acquisition of morphine‐induced conditioned place preference.

SR141716A was co‐administered with morphine for 5 days, and the withdrawal syndrome was precipitated by naloxone administration. A reduction in the incidence of two main signs of abstinence: wet dog shakes and jumping was observed while the other were not significantly modified. In contrast, an acute injection of the CB1 antagonist just before naloxone administration was unable to modify the incidence of the behavioural manifestations of the withdrawal, suggesting that only chronic blockade of CB1 receptors is able to reduce morphine‐induced physical dependence.

Several biochemical mechanisms could explain the reduction of opioid dependence by CB1 antagonists. Whatever the hypotheses, this study supports the reported interaction between the endogenous cannabinoid and opioid systems, and suggests that SR 141716A warrants further investigations for a possible use in opioid addiction.

British Journal of Pharmacology (2001) 132, 1809–1816; doi:10.1038/sj.bjp.0703990
Role of the haeme oxygenase/carbon monoxide pathway in mechanical nociceptor hypersensitivity
Authors: Steiner A A , Branco L G S , Cunha F Q , Ferreira S H
Published: January 2005

The cleavage of haeme by haeme oxygenase (HO) yields carbon monoxide (CO), a biologically active molecule which exerts most of its effects via activation of soluble guanylate cyclase (sGC). In the present study, we tested the hypothesis that endogenous CO could modulate inflammatory hyperalgesia. The intensity of hyperalgesia was investigated in a model of mechanical nociceptor hypersensitivity in rats.

The intra‐plantar (i.pl.) administration of the HO inhibitor, ZnDPBG (Zinc deuteroporphyrin 2,4‐bis glycol), potentiated in a dose‐dependent manner the mechanical nociceptor hypersensitivity evoked by i.pl. administration of carrageenan.

The mechanical hypersensitivity evoked by i.pl. injection of interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α), but not interleukin‐8 (IL‐8), prostaglandin E2 (PGE2) or dopamine, was also enhanced by ZnDPBG.

Moreover, the haeme (HO substrate) injection in the paws reduced the hypersensitivity evoked by IL‐1β, but not PGE2. Furthermore, i.pl. administration of the gas CO reduced the hypersensitivity elicited by PGE2.

The inhibitory effect of haeme and CO upon mechanical nociceptor hypersensitivity were counteracted by a soluble guanylate cyclase (sGC) inhibitor, ODQ (1H‐[1,2,4]‐oxadiazolo[4,3‐a]quinoxalin‐1‐one), suggesting that this effect of CO is mediated via cyclic GMP.

Finally, the inhibitory effect of CO upon mechanical nociceptor hypersensitivity was prevented by the NO synthase blocker, L‐NMMA (NG‐monomethyl L‐arginine), suggesting that the impairment of mechanical hypersensitivity elicited by CO depends on the integrity of the NO pathway.

In conclusion, the results presented in this paper imply that endogenously CO produced by HO plays an anti‐hyperalgesic role in inflamed paws, probably by increasing the intracellular levels of cyclic GMP in the primary afferent neurone.

British Journal of Pharmacology (2001) 132, 1673–1682; doi:10.1038/sj.bjp.0704014
Characterization of the effects of isoprostanes on platelet aggregation in human whole blood
Authors: Cranshaw Julius H , Evans Timothy W , Mitchell Jane A
Published: January 2005

We tested the effects of 11 commercially‐available isoprostanes on platelet aggregation directly or when triggered by the thromboxane receptor agonist U46619 or collagen in healthy human citrated blood using a whole blood aggregometer.

None of the isoprostanes tested triggered aggregation alone, nor facilitated aggregation by a sub‐threshold dose of U46619 or collagen. Five isoprostanes inhibited aggregation (rank order of potency 8‐iso PGE1>8‐iso PGE2>8‐iso PGF2α>8‐iso PGF3α>8‐iso‐13,14‐dihydro‐15‐keto PGF2α).

Blood incubated with LPS to induce a gross inflammatory response exhibited a time dependent (2 – 12 h) reduction in aggregation to U46619 but maintained a consistent response to collagen. Under these conditions, as in control blood, none of the isoprostanes tested induced aggregation. In fact, the inhibitory actions of isoprostanes on U46619‐induced aggregation were enhanced in blood treated with LPS.

L‐NAME inhibited aggregation induced by U46619 in fresh blood and in blood treated with LPS. In the presence of L‐NAME, (with or without LPS) none of the isoprostanes tested induced aggregation but retained their inhibitory action.

Thus, in human whole blood the action of 8‐iso PGE1, 8‐iso PGE2, 8‐iso PGF2α, 8‐iso PGF3α, and 8‐iso‐13,14‐dihydro‐15‐keto PGF2α is antiaggregatory. Moreover, this inhibitory capacity is still apparent and may be enhanced in blood subjected to inflammatory stimulation.

British Journal of Pharmacology (2001) 132, 1699–1706; doi:10.1038/sj.bjp.0704019
Role of p38 MAP kinase in LPS‐induced airway inflammation in the rat
Authors: Haddad El‐Bdaoui , Birrell Mark , McCluskie Kerryn , Ling Andrea , Webber Stephen E , Foster Martyn L , Belvisi Maria G
Published: January 2005

We investigated the effect of the p38 kinase inhibitor SB 203580 on airway inflammation induced by aerosolized lipopolysaccharide (LPS) in male Wistar rats. SB 203580 significantly inhibited (ED50=15.8 mg kg−1) plasma levels of TNF‐α in rats challenged with LPS (1.5 mg kg−1, i.p.).

Aerosolized LPS induced a peak in TNF‐α levels and the initiation of a neutrophilic response in bronchoalveolar lavage (BAL) fluid at the 2 h time point. Furthermore, the 4 h time point was associated with the peak in IL‐1β levels and the initial plateau of neutrophilia observed in the BAL fluid.

SB 203580 (100 mg kg−1), had no effect on peak TNF‐α levels or the associated neutrophilia in the BAL. Interestingly, the PDE 4 inhibitor RP 73401 (100 mg kg−1) significantly reduced both TNF‐α levels and neutrophilic inflammation. However, the BAL fluid from rats pre‐treated with either compound significantly inhibited TNF‐α release from cultured human monocytes 18 h after LPS treatment (83.6 and 44.5% inhibition, respectively).

Alternatively, SB 203580 (100 mg kg−1) produced dose‐related inhibition of BAL IL‐1β levels (67.5% inhibition, P<0.01) and BAL neutrophilia (45.9% inhibition, P<0.01) 4 h after LPS challenge.

P38 protein was present in lung tissue and the level of expression was not affected by LPS treatment.

P38 kinase appears to be involved in the release of IL‐1β and the sustained neutrophilic response in the BAL fluid. This data may suggest a role for p38 inhibitors in the treatment of airway inflammatory diseases in which neutrophilia is a feature of the lung pathology.

British Journal of Pharmacology (2001) 132, 1715–1724; doi:10.1038/sj.bjp.0704022
Effects of authentic and VLDL hydrolysis‐derived fatty acids on vascular smooth muscle cell growth
Authors: Gouni‐Berthold Ioanna , Berthold Heiner K , Seul Claudia , Ko Yon , Vetter Hans , Sachinidis Agapios
Published: January 2005

There are contradictory findings regarding the effects of free fatty acids on vascular smooth muscle cell (VSMC) growth. In the present study we investigated the effects of fatty acids released from hydrolysis of human VLDL triglycerides by lipoprotein lipase and of the fatty acids most abundant in the hydrolysed VLDL, namely oleic, linoleic, palmitic and myristic acid, all non albumin‐bound, on VSMC growth.

The effect of fatty acids on VSMC growth was assessed by [3H]‐thymidine incorporation, colourimetrically, by cell counting, by determination of the cytoplasmic histone‐associated DNA fragments and the caspase 3 activity. The fatty acid concentrations were determined by gas chromatography‐mass spectrometry. Stimulation of ERK1/2 and p38 was determined by the chemiluminescence Western blotting method.

Incubation of VSMC with purified VLDL (100 μg ml−1) and lipoprotein lipase (35 u ml−1) led to almost complete cell death although the ERK1/2 and the p38 MAP kinases were stimulated. The EC50 of oleic, linoleic, myristic and palmitic acid were 4.6±1.3, 2.4±0.2, 116±10 and 287±30 μM, respectively. The estimated EC50 of myristic and palmitic acid when derived from hydrolysed VLDL were 10 and 8 times, respectively, lower than when used alone. Apoptosis was not involved in the fatty acid‐induced VSMC growth suppression/death.

We conclude that (a) non albumin‐bound fatty acids cause VSMC necrosis in a dose‐dependent manner with a parallel ERK1/2 and p38 stimulation, (b) unsaturated fatty acids are more toxic to VSMC than saturated, and (c) saturated fatty acids are more toxic to VSMC in the hydrolysed VLDL than when used individually.

British Journal of Pharmacology (2001) 132, 1725–1734; doi:10.1038/sj.bjp.0704026
Regulation of prostacyclin and prostaglandin E2 receptor mediated responses in adult rat dorsal root ganglion cells, in vitro
Authors: Rowlands Dewi K , Kao Chung‐lei , Wise Helen
Published: January 2005

Primary cultures of adult rat dorsal root ganglia (DRG) were prepared to examine the properties of prostacyclin (IP) receptors and prostaglandin E2 (EP) receptors in sensory neurones.

IP receptor agonists, cicaprost and iloprost, stimulated adenylyl cyclase activity with EC50 values of 22 and 28 nM, respectively. Prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) were 7 fold less potent than cicaprost and iloprost, with PGE2 displaying a lower maximal response.

Adenylyl cyclase activation by iloprost, PGE1 and PGE2, but not by forskolin, was highly dependent on DRG cell density. Although the potency of iloprost and PGE2 for stimulating adenylyl cyclase was unchanged, their maximal responses were significantly increased at low cell density.

Both IP and EP2/4 receptors could be down‐regulated by agonist pretreatment, however the presence of cyclo‐oxygenase (COX) inhibitors did not prevent this apparent down‐regulation of IP and EP2/4 receptors at high DRG cell densities.

Stimulation of adenylyl cyclase by the neuropeptide calcitonin gene‐related peptide was also decreased at high DRG cell density, whereas the responses to β‐adrenoceptor agonists were increased at high DRG cell density.

Addition of nerve growth factor (NGF), or the addition of anti‐neurotrophin antibodies during the 5‐day culture of DRG cells, had no effect on IP receptor‐mediated responses.

These results indicate that Gs‐coupled receptors involved in nociception are regulated in a variable manner in adult rat sensory neurones, and that this cell density‐dependent regulation may be agonist‐independent for IP and EP2/4 receptors.

British Journal of Pharmacology (2001) 133, 13–22; doi:10.1038/sj.bjp.0704028