bluuebunny/crossref_metadata_2025 huggingface dataset:
KATP‐channel activation: effects on myocardial recovery from ischaemia and role in the cardioprotective response to adenosine A1‐receptor stimulation
Authors: Ford William R , Lopaschuk Gary D , Schulz Richard , Clanachan Alexander S
Published: January 2005

Optimization of myocardial energy substrate metabolism improves the recovery of mechanical function of the post‐ischaemic heart. This study investigated the role of KATP‐channels in the regulation of the metabolic and mechanical function of the aerobic and post‐ischaemic heart by measuring the effects of the selective KATP‐channel activator, cromakalim, and the effects of the KATP‐channel antagonist, glibenclamide, in rat fatty acid perfused, working hearts in vitro. The role of KATP channels in the cardioprotective actions of the adenosine A1‐receptor agonist, N6‐cyclohexyladenosine (CHA) was also investigated.

Myocardial glucose metabolism, mechanical function and efficiency were measured simultaneously in hearts perfused with modified Krebs‐Henseleit solution containing 2.5 mM Ca2+, 11 mM glucose, 1.2 mM palmitate and 100 mu l−1 insulin, and paced at 300 beats min−1. Rates of glycolysis and glucose oxidation were measured from the quantitative production of 3H2O and 14CO2, respectively, from [5‐3H/U‐14C]‐glucose.

In hearts perfused under aerobic conditions, cromakalim (10 μM), CHA (0.5 μM) or glibenclamide (30 μM) had no effect on mechanical function. Cromakalim did not affect glycolysis or glucose oxidation, whereas glibenclamide significantly increased rates of glycolysis and proton production. CHA significantly reduced rates of glycolysis and proton production but had no effect on glucose oxidation. Glibenclamide did not alter CHA‐induced inhibition of glycolysis and proton production.

In hearts reperfused for 30 min following 30 min of ischaemia, left ventricular minute work (LV work) recovered to 24% of aerobic baseline values. Cromakalim (10 μM), administered 5 min before ischaemia, had no significant effect on mechanical recovery or glucose metabolism. CHA (0.5 μM) significantly increased the recovery of LV work to 67% of aerobic baseline values and also significantly inhibited rates of glycolysis and proton production. Glibenclamide (30 μM) significantly depressed the recovery of mechanical function to <1% of aerobic baseline values and stimulated glycolysis and proton production.

Despite the deleterious actions of glibenclamide per se in post‐ischaemic hearts, the beneficial effects of CHA (0.5 μM) on the recovery of mechanical function and proton production were not affected by glibenclamide.

The data indicate that the cardioprotective mechanism of adenosine A1‐receptor stimulation does not involve the activation of KATP‐channels. Furthermore, in rat fatty acid perfused, working hearts, stimulation of KATP‐channels is not cardioprotective and has no significant effects on myocardial glucose metabolism.
Curcumin attenuation of acute adriamycin myocardial toxicity in rats
Authors: Venkatesan Narayanan
Published: January 2005

The protective effect of curcumin on acute adriamycin (ADR) myocardial toxicity was analysed in rats. ADR toxicity, induced by a single intraperitoneal injection (30 mg kg−1), was revealed by elevated serum creatine kinase (CK) and lactate dehydrogenase (LDH). The level of the lipid peroxidation products, conjugated dienes and malondialdehyde, was markedly elevated by ADR. ADR caused a decrease in myocardial glutathione content and glutathione peroxidase activity. In contrast, cardiac catalase activity was increased in ADR rats. Curcumin treatment (200 mg kg−1, seven days before and two days following ADR) significantly ameliorated the early manifestation of cardiotoxicity (ST segment elevation and an increase in heart rate) and prevented the rise in serum CK and LDH exerted by ADR. ADR rats that received curcumin displayed a significant inhibition of lipid peroxidation and augmentation of endogenous antioxidants. These results suggest that curcumin inhibits ADR cardiotoxicity and might serve as novel combination chemotherapeutic agent with ADR to limit free radical‐mediated organ injury.
Bronchodilatationin vivoby carbon monoxide, a cyclic GMP related messenger
Authors: Cardell Lars‐Olaf , Ueki Iris F. , Stjärne Pär , Agusti Carlos , Takeyama Kiyoshi , Lindén Anders , Nadel Jay A.
Published: January 2005

Recent studies suggest that gaseous carbon monoxide (CO) is involved in neurotransmission and that this molecule also is an important vasodilatorin vivo. In the present study we evaluated the effect of inhaled CO on guinea‐pig airway smooth muscle tone. The mechanisms involved were characterized by use of a cyclic GMP antagonist, Rp‐8Br‐cyclic GMPS, and a nitric oxide synthase inhibitor,l‐NAME.

Anaesthetized, ventilated guinea‐pigs were given a bolus injection of histamine (0.12 mg kg−1, i.v.), followed by a continuous infusion of histamine (0.30 μg kg−1min−1) to increase total pulmonary resistance (RL). Subsequent exposure to 7, 15 or 30 breaths of CO (100%), resulted in a dose‐dependent inhibition of the bronchoconstriction. In the highest dose tested (30 breaths), CO inhibited 80% of the histamine‐induced increase inRL.

In separate experiments, animals receiving histamine infusions followed by 30 breaths of CO, were pretreated with Rp‐8Br‐cyclic GMPS (0.05 mg kg−1). This pretreatment abolished >60% of the CO‐induced reduction inRL, but it had no effect on the bronchodilator response induced by salbutamol. In another set of experiments animals were pretreated withl‐NAME (1.60 mg kg−1). In contrast to the Rp‐8Br‐cyclic GMPS pretreatment, the pretreatment withl‐NAME did not affect the CO‐induced reduction inRL.

The present findings indicate that CO causes bronchodilatationin vivovia cyclic GMP.

British Journal of Pharmacology(1998)124, 1065–1068; doi:10.1038/sj.bjp.0701878
Molecular cloning and characterization of rat P2Y4 nucleotide receptor
Authors: Bogdanov Y D , Wildman S S , Clements M P , King B F , Burnstock G
Published: January 2005

An intronless open reading frame encoding a protein (361aa in length) was isolated from a rat genomic library probed with a DNA fragment from rat heart. This protein showed 83% sequence identity with the human P2Y4 (hP2Y4) receptor and represents a homologue of the human pyrimidinoceptor. However, the rP2Y4 receptor is not selective for uridine nucleotides and, instead, shows an agonist potency order of ITP=ATP=ADP(pure)=UTP=ATPγS=2‐MeSATP=Ap4A>UDP(pure). ADP, ATPγS, 2‐MeSATP and UDP are partial agonists. Thus, in terms of agonist profile, rP2Y4 is more like the P2U receptor subtype. The rP2Y4 receptor was reversibly antagonized by Reactive blue 2 but not by suramin which, otherwise, inhibits the hP2Y2 receptor (a known P2U receptor). Thus, rP2Y4 and the P2Y2 subtype appear to be structurally distinct forms of the P2U receptor (where ATP and UTP are equi‐active) but can be distinguished as suramin‐insensitive and suramin‐sensitive P2U receptors, respectively.
Neurogenic contraction and relaxation of human penile deep dorsal vein
Authors: Segarra Gloria , Medina Pascual , Domenech Cristina , Martínez León Juan B , Vila José M , Aldasoro Martin , Lluch Salvador
Published: January 2005

The aim of the present study was to characterize neurogenic and pharmacological responses of human penile deep dorsal vein and to determine whether the responses are mediated by nitric oxide from neural or endothelial origin.

Ring segments of human penile deep dorsal vein were obtained from 22 multiorgan donors during procurement of organs for transplantation. The rings were suspended in organ bath chambers for isometric recording of tension. We then studied the contractile and relaxant responses to electrical field stimulation and to vasoactive agents.

Electrical field stimulation (0.5–2 Hz) and noradrenaline (3×10−10–3×10−5 M) caused frequency‐ and concentration‐dependent contractions that were of greater magnitude in veins denuded of endothelium. The inhibitor of nitric oxide synthesis NG‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME, 10−4 M) increased the adrenergic responses only in rings with endothelium.

In preparations contracted with noradrenaline in the presence of guanethidine (10−6 M) and atropine (10−6 M), electrical stimulation induced frequency‐dependent relaxations. This neurogenic relaxation was prevented by L‐NAME, methylene blue (3×10−5 M) and tetrodotoxin (10−6 M), but was unaffected by removal of endothelium.

Acetylcholine (10−8–3×10−5 M) and substance P (3×10−11–3×10−7 M) induced endothelium‐dependent relaxations. In contrast, sodium nitroprusside (10−9–3×10−5 M) and papaverine (10−8–3×10−5 M) caused endothelium‐independent relaxations.

The results provide functional evidence that the human penile deep dorsal vein is an active component of the penile vascular resistance through the release of nitric oxide from both neural and endothelial origin. Dysfunction in any of these sources of nitric oxide should be considered in some forms of impotence.
The effect of the nonpeptide neurotrophic compound SR 57746A on the progression of the disease state of the pmn mouse
Authors: Duong F , Fournier J , Keane P E , Guénet J L , Soubrié P , Warter J M , Borg J , Poindron P
Published: January 2005

The progressive motor neuronopathy (pmn) mouse is an autosomal recessive mutant, in which the homozygotes suffer caudio‐cranial degeneration of motor axons and die several weeks after birth. This strain provides the opportunity of testing potential therapeutic strategies for the treatment of motor neurone diseases such as amyotrophic lateral sclerosis. We have performed a study of the effects on the pmn mouse of SR 57746A, an orally‐active, non‐peptide compound which has been found to exhibit neurotrophic effects in vitro and in vivo. In order to treat the affected mice from birth, the mothers were administered 2.5 mg kg−1, p.o., SR 57746A every two days until the weaning of the offspring (at day 20); then the offspring were given every two days a dose of 30 μg kg−1, p.o., until their death.

Affected mice treated with SR 57746A had a lifespan 50% longer than that of the vehicle‐treated mice (P=0.01). Compared to vehicle‐treated pmn mice, SR 57746A improved the performance of the pmn mice in three different behavioural tasks. SR 57746A also maintained the amplitude of the motor evoked response of the gastrocnemius muscle, reduced the distal motor latency, and delayed the occurrence of the spontaneous denervation activity in this muscle. Histological studies indicated that at 20 days of age the mean surface areas of the fibres of the sciatic nerve were higher in SR 57746A‐treated than in vehicle‐treated mice.

At present, SR 57746A is the only orally active, nonpeptide compound known to be capable of delaying the progression of the motor neurone degeneration in pmn mice.
Nucleotide‐evoked calcium signals and anion secretion in equine cultured epithelia that express apical P2Y2 receptors and pyrimidine nucleotide receptors
Authors: Wilson S M , Law V W Y , Pediani J D , Allen E A , Wilson G , Khan Z E , Ko W H
Published: January 2005

Experiments with a spontaneously transformed equine epithelial cell line showed that certain nucleotides increased intracellular free calcium ([Ca2+]i) in cells plated on glass coverslips. The rank order of potency was ATP=UTP>5‐Br‐UTP, whilst UDP and ADP were ineffective. The response thus appears to be mediated by P2Y2 receptors.

Nucleotides also increased short circuit current (ISC) in cells grown into epithelial monolayers and the rank order of potency was UDP>UTP>5‐Br‐UTP>ATP>ADP. The increase in [Ca2+]i and the rise in ISC thus have different pharmacological properties. Cross‐desensitization experiments indicated that, as well as P2Y2 receptors, the monolayer cultures express at least one additional receptor population that allowed nucleotides to increase ISC.

The UDP‐evoked increase in ISC was essentially abolished in BAPTA‐loaded epithelia suggesting that this response is dependent upon increased [Ca2+]i. Moreover, experiments in which ISC and [Ca2+]i were measured simultaneously showed that the UDP‐ and ADP‐evoked increases in ISC were accompanied by increases in [Ca2+]i.

When grown under conditions which favour the development of a polarized phenotype, these epithelial cells thus appear to express [Ca2+]i‐mobilizing receptors sensitive to UDP and ADP that are not present in non‐polarized cells on coverslips.
Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP
Authors: Harper Simon , Webb Tania E , Charlton Steven J , Ng Leong L , Boarder Michael R
Published: January 2005

Previous studies have shown that ATP and UTP are able to stimulate phospholipase C (PLC) and proliferation in cultured aortic smooth muscle cells. Here we set out to characterize the receptor responsible, and investigate a possible role for p42 and p44 mitogen activated protein kinase (MAPK) in the proliferative response.

The phospholipase C response of spontaneously hypertensive rat (SHR) derived aortic smooth muscle cells in culture showed that the response to ATP was partial compared to the response to UTP.

Further studies characterized the responses of the SHR derived cells. UTP was the only full agonist with the SHR cells; UDP gave a partial response while ADP, 2‐methythio‐ATP and α,β‐methylene ATP were essentially ineffective. The response to UDP was almost lost in the presence of hexokinase, consistent with this being due to extracellular conversion to UTP. These observations are inconsistent with the response being mediated by either P2Y1 or P2Y6 receptors.

When increasing concentrations of ATP were present with a maximally effective concentration of UTP, the size of the response diminished, consistent with UTP and ATP acting at a single population of receptors for which ATP was a partial agonist. This is inconsistent with a response mainly at P2Y2 receptors.

1321N1 cells transfected with human P2Y4 receptors gave a similar agonist response profile, with ATP being partial compared to UTP, loss of response to UDP with hexokinase treatment, and with the response to UTP diminishing in the presence of increasing concentrations of ATP.

Use of the reverse transcriptase‐polymerase chain reaction confirmed the presence of mRNA encoding P2Y4 receptors in SHR derived vascular smooth muscle cells. Transcripts for P2Y2, P2Y4 and P2Y6 receptors, but not P2Y1 receptors, were detected.

Stimulation of SHR derived cells with UTP enhanced the tyrosine phosphorylation of both p42 and p44 MAPK, and the incorporation of [3H]‐thymidine into DNA. Both these responses were diminished in the presence of an inhibitor of activation of MAPK.

These results lead to the conclusion that in SHR derived cultured aortic smooth muscle cells, PLC responses to extracellular UTP and ATP are predominantly at P2Y4 receptors, and suggest that these receptors are coupled to mitogenesis via p42/p44 MAPK.
Pharmacological characterization of thromboxane and prostanoid receptors in human isolated urinary bladder
Authors: Palea S , Toson G , Pietra C , Trist D G. , Artibani W , Romano O , Corsi M
Published: January 2005

Cumulative concentration‐response curves (CRC) to prostaglandin E1 (PGE1), PGE2, PGD2 and PGF2α (0.01–30 μM) and to the thromboxane A2 (TXA2) receptor agonist U‐46619 (0.01–30 μM) were constructed in human isolated detrusor muscle strips both in basal conditions and during electrical field stimulation.

All the agonists tested contracted the detrusor muscle. The rank order of agonist potency was: PGF2α>U‐46619>PGE2 whereas weak contractile responses were obtained with PGD2 and PGE1. Any of the agonists tested was able to induce a clear plateau of response even at 30 μM.

The selective TXA2 antagonist, GR 32191B (vapiprost), antagonized U‐46619‐induced contractions with an apparent pKB value of 8.27±0.12 (n=4 for each antagonist concentration). GR 32191B (0.3 μM) did not antagonize the contractile responses to PGF2α and it was a non‐surmountable antagonist of PGE2 (apparent pKB of 7.09±0.04; n=5). The EP receptor antagonist AH 6809 at 10 μM shifted to the right the CRC to U‐46619 (apparent pKB value of 5.88±0.04; n=4).

Electrical field stimulation (20 Hz, 70 V, pulse width 0.1 ms, trains of 5 s every 60 s) elicited contractions fully sensitive to TTX (0.3 μM) and atropine (1 μM). U‐46619 (0.01–3 μM) potentiated the twitch contraction in a dose‐dependent manner and this effect was competitively antagonized by GR 32191B with an estimated pKB of 8.54±0.14 (n=4 for each antagonist concentration). PGF2α in the range 0.01–10 μM (n=7), but not PGE2 and PGE1 (n=3 for each), also potentiated the twitch contraction of detrusor muscle strips (23.5±0.3% of KCl 100 mM‐induced contraction) but this potentiation was unaffected by 0.3 μM GR 32191B (n=5).

Cumulative additions of U‐46619 (0.01–30 μM) were without effect on contractions induced by direct smooth muscle excitation (20 Hz, 40 V, 6 ms pulse width, trains of 2 s every 60 s, in the presence of TTX 1 μM; n=3). Moreover, pretreatment of the tissue with 0.3 μM U‐46619 did not potentiate the smooth muscle response to 7 μM bethanecol (n=2).

We concluded that TXA2 can induce direct contraction of human isolated urinary bladder through the classical TXA2 receptor. Prostanoid receptors, fully activated by PGE2 and PGF2α are also present. All these receptors are probably located post‐junctionally. The rank order of agonist potency and the fact that GR32191B, but not AH6809, antagonized responses to PGE2 seem to indicate the presence of a new EP receptor subtype. Moreover, we suggest the presence of prejuctional TXA2 and FP receptors, potentiating acetylcholine release from cholinergic nerve terminals.
Characterization and localization of thromboxane A2 receptor in human and guinea‐pig nasal mucosa using radiolabelled (+)‐S‐145
Authors: Arimura A , Miwa M , Hasegawa H , Kishino J , Notoya M , Yasui K , Komori M , Iwata S
Published: January 2005

TxA2 receptor (TP‐receptor) antagonists such as S‐1452 and Bay u 3405 have been shown to be effective in alleviating nasal blockage in patients with allergic rhinitis as well as guinea‐pig allergic rhinitis models. The present study was conducted to examine the existence and localization of the TP‐receptor in human and guinea‐pig nasal mucosa by in vitro receptor binding autoradiography using radiolabelled (+)‐S‐145, which is a potent and specific TP‐receptor antagonist with an extremely slow dissociation rate.

We ascertained the binding specificity of [3H]‐(+)‐S‐145 in human and guinea‐pig platelet membranes by comparing the ability of four TP‐receptor ligands of U‐46619, (+)‐S‐145, I‐(+)‐S‐145 and Bay u 3405 to displace the specific binding of [3H]‐(+)‐S‐145 and [3H]‐U‐46619. The rank order of potency (Ki) for the displacement was correlated highly with that determined from [3H]‐U‐46619 binding to the same preparations.

Quantitative autoradiography using a radioluminographic imaging plate system, in which the radioactivity of [3H]‐(+)‐S‐145 is expressed as photostimulated luminescence (PSL) per area (mm2), revealed that specific binding of [3H]‐(+)‐S‐145 to human and guinea‐pig nasal mucosa was saturable. Scatchard analysis showed about three fold higher affinity and two fold greater maximal binding to the nasal mucosa of humans than that of guinea‐pigs: the KD and Bmax values in human mucosa were 2.82±0.35 nM and 6.47±0.33 PSL mm−2 and those in guinea‐pig mucosa were 8.23±1.93 nM and 3.37±0.66 PSL mm−2, respectively.

Specific [3H]‐(+)‐S‐145 binding to cryostat sections of human and guinea‐pig nasal mucosa was displaced by another TP‐receptor antagonist, Bay u 3405, and a TP‐receptor agonist, U‐46619. The order of potency (Ki value: nM) was (+)‐S‐145 (2.5) > Bay u 3405 (15.4) >> U‐46619 (359.6) in human nasal mucosa and (+)‐S‐145 (22.8) > U‐46619 (49.8) & Bay u 3405 (62.1) in guinea‐pig nasal mucosa. These rank orders showed rather good correlation with those obtained for the respective platelet membranes.

Autoradiographs of human nasal mucosa demonstrated that specific [125I]‐(+)‐S‐145 binding sites mainly exist on the smooth muscle layers of venous sinusoids and arterioles in the lamina propria, with few or no binding sites in the epithelium and nasal gland.

We concluded that radiolabelled (+)‐S‐145 can be used as a TP‐receptor ligand for autoradiographic study, and that the TP‐receptor is exclusively located on smooth muscle layers of venous sinusoids and arterioles in the nasal mucosa. The potent vasoconstrictive activity of TxA2 may cause reduction of local blood flow followed by mucosal oedema probably through mechanisms of vascular injury such as ischaemia‐reperfusion.
AT2 antagonist‐sensitive potentiation of angiotensin II‐induced constriction by NO blockade and its dependence on endothelium and P450 eicosanoids in rat renal vasculature
Authors: Muller Catherine , Endlich Karlhans , Helwig Jean‐Jacques
Published: January 2005

We showed earlier that NO inhibition caused a left‐shift and augmented Emax of the concentration‐response curve of AT1‐mediated (angiotensin II)‐induced vasoconstrictions (AII‐VC) in the rat kidney. The 0.01–0.1 nM AII‐VC unmasked by the potentiating effect of NO inhibition, were sensitive not only to AT1 (L158809), but also to AT2 receptor (PD123319) antagonists. We now demonstrate the role of endothelium and eicosanoids in the NO‐masked AT1/AT2‐mediated component of the AII‐VC in isolated indomethacin‐perfused kidneys of the rat.

L‐NAME increased 0.1 nM AII‐VC 7.2 fold. Pretreatment of the kidneys with factor VIII antibody/complement or with the detergent CHAPS to damage endothelium, decreased carbachol‐induced vasodilatation and blunted by 60 and 30% respectively, the enhancement of AII‐VC during NO inhibition.

L‐NAME also increased 3 μM noradrenaline (NA)‐induced vasoconstriction (NA‐VC) 8.1 fold. In contrast to AII‐VC, endothelium damage was without effect on the enhancement of NA‐VC by L‐NAME, suggesting a dominant role of endothelium‐derived NO in the enhancement of NA‐VC.

During NO inhibition, ETYA (2 μM; an inhibitor of all arachidonic acid derived pathways) and α‐naphtoflavone (10 μM; an inhibitor of the cytochrome P450 isozymes), decreased by 85% the 0.1 nM AII‐VC.

In conclusion, during NO inhibition, the AT1‐mediated constriction to low concentrations of AII, which is sensitive to AT2 antagonists, depends on intact endothelium, and can be blocked by inhibition of eicosanoid synthesis. The results suggest that the AII‐mediated vasoconstriction through AT1 receptors is potentiated in the absence of NO, by the release of eicosanoids from the endothelium through AT2 receptors.
Epigallocatechin suppression of proliferation of vascular smooth muscle cells: correlation with c‐jun and JNK
Authors: Lu Liang‐Huei , Lee Shoei‐Sheng , Huang Huei‐Chen
Published: January 2005

The mechanisms of the antiproliferative effect of epigallocatechin, one of the catechin derivatives found in green tea, in vascular smooth muscle cells were studied. The proliferative response was determined from the uptake of tritiated thymidine.

In the concentration range of 10−6 to 10−4 m, catechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin, epigallocatechin gallate, concentration‐dependently inhibited the proliferative response stimulated by serum in rabbit cultured vascular smooth muscle cells. Catechin and epicatechin were less effective in inhibiting the serum‐stimulated smooth muscle cell proliferation, indicating that the galloyl group may be important for full inhibitory activity.

Epigallocatechin (EGC) inhibited the proliferative responses in different cells including rat aortic smooth muscle cells (A7r5 cells), rabbit cultured aortic smooth muscle cells, human coronary artery smooth muscle cells, and human CEM lymphocytes in a concentration‐dependent manner. The possible mechanisms of the antiproliferative effect of EGC were further studied in A7r5 cells.

The membranous protein tyrosine kinase activity stimulated by serum in A7r5 cells was significantly reduced by 10−5 m EGC. In contrast, the cytosolic protein kinase C activity stimulated by phorbol ester was unaffected by directly incubating with EGC (10−6−10−4 m).

We also performed Western blot analysis using the anti‐phosphotyrosine monoclonal antibody PY‐20. EGC (10−5 m) reduced the levels of tyrosine phosphorylated proteins with different molecular weights, indicating that EGC may inhibit the protein tyrosine kinase activity or stimulate the protein phosphatase activity.

Reverse transcription‐polymerase chain reaction analysis of c‐fos, c‐jun and c‐myc mRNA levels demonstrated that c‐jun mRNA level after serum‐stimulation was significantly reduced by 10−5 m EGC. However, the reduction of c‐fos and c‐myc mRNA levels by 10−5 m EGC did not achieve significance.

Western blot analysis using the antibody against JNK (c‐jun N‐terminal kinase) and ERK (extracellular signal‐regulated kinase) demonstrated that the level of phosphorylated JNK1, but not phosphorylated ERK1 and ERK2, was reduced by 10−5 m EGC. Direct measurement of kinase activity by immune complex kinase assay confirmed that JNK1 activity was inhibited by EGC treatment. These results demonstrate that EGC preferentially reduced the activation of JNK/SAPK (stress‐activated protein kinase) signal transduction pathway.

It is suggested that the antiproliferative effect of epigallocatechin on vascular smooth muscle cells may partly be mediated through inhibition of protein tyrosine kinase activity, reducing c‐jun mRNA expression and inhibiting JNK1 activation. Tea catechins may be useful as a template for the development of drugs to prevent the pathological changes of atherosclerosis and post‐angioplasty restenosis.

British Journal of Pharmacology (1998) 124, 1227–1237; doi:10.1038/sj.bjp.0701912
Mutational analysis of the potential phosphorylation sites for protein kinase C on the CCKA receptor
Authors: Smeets R L L. , Fouraux M A. , Pouwels W , van Emst‐de Vries S E. , Ronken E , De Pont J J H H M. , Willems P H G M.
Published: January 2005

Many G protein‐coupled receptors contain potential phosphorylation sites for protein kinase C (PKC), the exact role of which is poorly understood. In the present study, a mutant cholecystokininA (CCKA) receptor was generated in which the four consensus sites for PKC action were changed in an alanine. Both the wild‐type (CCKAWT) and mutant (CCKAMT) receptor were stably expressed in Chinese hamster ovary (CHO) cells.

Binding of [3H]‐cholecystokinin‐(26‐33)‐peptide amide (CCK‐8) to membranes prepared from CHO‐CCKAWT cells and CHO‐CCKAMT cells revealed no difference in binding affinity (Kd values of 0.72 nM and 0.86 nM CCK‐8, respectively).

The dose‐response curves for CCK‐8‐induced cyclic AMP accumulation and inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) formation were shifted to the left in CHO‐CCKAMT cells. This leftward shift was mimicked by the potent inhibitor of protein kinase activity, staurosporine. However, the effect of staurosporine was restricted to CHO‐CCKAWT cells. This demonstrates that attenuation of CCK‐8‐induced activation of adenylyl cyclase and phospholipase C‐β involves a staurosporine‐sensitive kinase, which acts directly at the potential sites of PKC action on the CCKA receptor in CCK‐8‐stimulated CHO‐CCKAWT cells.

The potent PKC activator, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), evoked a rightward shift of the dose‐response curve for CCK‐8‐induced cyclic AMP accumulation in CHO‐CCKAWT cells but not CHO‐CCKAMT cells. This is in agreement with the idea that PKC acts directly at the CCKA receptor to attenuate adenylyl cyclase activation.

In contrast, TPA evoked a rightward shift of the dose‐response curve for CCK‐8‐induced Ins(1,4,5)P3 formation in both cell lines. This demonstrates that high‐level PKC activation inhibits CCK‐8‐induced Ins(1,4,5)P3 formation also at a post‐receptor site.

TPA inhibition of agonist‐induced Ca2+ mobilization was only partly reversed in CHO‐CCKAMT cells. TPA also inhibited Ca2+ mobilization in response to the G protein activator, Mas‐7. These findings are in agreement with the idea that partial reversal of agonist‐induced Ca2+ mobilization is due to the presence of an additional site of PKC inhibition downstream of the receptor and that the mutant receptor itself is not inhibited by the action of PKC.

The data presented demonstrate that the predicted sites for PKC action on the CCKA receptor are the only sites involved in TPA‐induced uncoupling of the receptor from its G proteins. In addition, the present study unveils a post‐receptor site of PKC action, the physiological relevance of which may be that it provides a means for the cell to inhibit phospholipase C‐β activation by receptors that are not phosphorylated by PKC.
Evidence for inverse agonism of SR141716A at human recombinant cannabinoid CB1 and CB2 receptors
Authors: MacLennan S J , Reynen P H , Kwan J , Bonhaus D W
Published: January 2005

The cannabinoid receptor antagonist SR141716A has been suggested to be an inverse agonist at CB1 receptors in some isolated intact tissues. We found that the basal incorporation of [35S]‐GTPγS in Chinese hamster ovary cells expressing human recombinant CB1 and CB2 receptors was inhibited by SR141716A (mean pEC50s 8.26 and 6.00, respectively), whereas cannabinol (10 μM) had no significant effect at hCB1 receptors but inhibited the binding at hCB2 receptors. As cannabinol had no effect on basal [35S]‐GTPγS binding at hCB1 at a concentration 100 fold higher than its binding affinity (Ki=0.1 μM), we conclude that endogenous cannabinoid receptor agonists are not a confounding factor and suggest the actions of SR141716A at the hCB1 receptor, and the actions of SR141716A and cannabinol at the hCB2 receptor, are due to inverse agonism.
Role of adventitial nitric oxide in vascular hyporeactivity induced by lipopolysaccharide in rat aorta
Authors: Kleschyov Andrei L , Muller Bernard , Schott Christa , Stoclet Jean‐Claude
Published: January 2005

This study was designed to elucidate the role of the adventitia in NO‐mediated vascular effects of lipopolysaccharide (LPS). After incubation of rat aorta with LPS, the adventitia generated 3.5 times more nitrite plus nitrate than a corresponding segment of media. Control media covered by adventitia from LPS‐treated aortic rings exhibited a 4 fold elevated level of cyclic GMP. Medial layers from LPS‐treated aortic rings (like LPS‐treated adventitia‐intact rings) exhibited a decrease in sensitivity to noradrenaline (NA) that was reversed by 1H‐(1,2,4)oxadiazolo(4,3‐a)quinoxalin‐1‐one (1 μM) or Nω‐nitro‐L‐arginine methylester (0.3 mM). However, in contrast to LPS‐treated adventitia‐intact rings, medial layers showed no reduction in maximal contraction to NA and virtually no relaxation to L‐arginine. These data indicate that in blood vessels exposed to LPS, the adventitia is a more powerful source of NO than the media. The adventitia‐derived NO can reach soluble guanylyl cyclase in the medial layer and contribute greatly to vascular hyporeactivity and L‐arginine‐induced relaxation observed in blood vessels exposed to LPS.
Modulation of GABAA receptors and inhibitory synaptic currents by the endogenous CNS sleep regulator cis‐9,10‐octadecenoamide (cOA)
Authors: Lees George , Edwards Michelle D. , Hassoni Abdul A. , Robin Ganellin C , Galanakis Dimitrious
Published: January 2005

Cis‐9,10‐octadecenoamide (cOA) accumulates in the CSF of sleep‐deprived cats and may represent a novel signalling molecule. Synthetic cOA has been shown to induce physiological sleep when injected into laboratory rats. Here we assess the cellular mode of action of cOA in vitro.

In all rat cultured cortical neurones (pyramidal cells) examined, the synthetic brain lipid (3.2–64 μM) enhanced the responses to subsaturating GABA concentrations (up to circa 2×) in a concentration‐dependent manner (EC50, circa 15 μM).

(20 μM) cOA significantly enhanced the affinity of exogenous GABA for its receptor without changing the Hill slope or the maximal response. These effects were not voltage‐dependent or secondary to shifts in ECl.

In the absence of GABA, cOA directly evoked small inhibitory currents in a subpopulation (<7%) of sensitive cells.

20 μM cOA reversibly enhanced the duration of spontaneous inhibitory post synaptic currents (circa 2 fold) without significantly altering their amplitude.

At 32–64 μM, cOA reversibly reduced the incidence and amplitude of both inhibitory post synaptic currents (i.p.s.cs) and excitatory post synaptic currents (e.p.s.cs) in the cultured neuronal circuits in common with other depressant drugs acting at the GABAA receptor.

32 μM Oleic acid did not modulate exogenous GABA currents or synaptic activity suggesting that cOAs actions are mediated through a specific receptor.

A specific, protein‐dependent interaction with GABAA receptors was confirmed in Xenopus oocytes. Recombinant human receptors were modulated by 10 μM cOA (and diazepam) only when a γ2 subunit was co‐expressed with α1β2: the cOA response was not sensitive to the specific benzodiazepine antagonist flumazenil (1 μM).

cOA may represent an endogenous ligand for allosteric modulatory sites on isoforms of GABAA receptors which are crucial for the regulation of arousal and have recently been implicated in the circadian control of physiological sleep.
Tandospirone‐induced K+ current in acutely dissociated rat dorsal raphe neurones
Authors: Jin Young‐Ho , Akaike Norio
Published: January 2005

The effects of tandospirone (TDS) on dissociated rat dorsal raphe neurones were investigated using the patch‐clamp method.

Under current‐clamp conditions, TDS hyperpolarized the cell membrane, resulting in the reduction of firing rates.

Under voltage‐clamp conditions, TDS induced an inward rectifying K+ current in a concentration‐dependent manner.

The TDS‐induced K+ currents (ITDS) were mimicked by 8‐OH‐DPAT, a 5‐HT1A agonist. The ITDS was blocked by spiperone, a 5‐HT1A receptor antagonist, in a concentration‐dependent manner.

N‐Ethylmaleimide, an agent which uncouples between the receptor and the G‐protein, irreversibly blocked the ITDS.

In neurones perfused intracellularly with a pipette‐solution containing GTP using the conventional whole‐cell patch recording, the ITDS showed a gradual rundown. When the neurones were perfused with GTPγS, TDS activated the inwardly rectifying K+ current in an irreversible manner.

In the inside‐out patch recording mode, TDS‐activated single K+ channel currents (iTDS) which also showed an inward rectification. When the GDP in cytosolic side was completely replaced with GTP, the open probability of iTDS significantly increased.

These results indicate that the activation of 5‐HT1A receptors by TDS directly opens the inward rectifying K+ channels via a G‐protein mediated process.
Agonist action at D2(long) dopamine receptors: ligand binding and functional assays
Authors: Gardner B , Strange P G.
Published: January 2005

The activities of a range of agonists at D2(long) dopamine receptors expressed in CHO cells have been determined in ligand binding and in a functional assay, the stimulation of [35S]‐GTPγS binding.

For several agonists (apomorphine, dopamine, pergolide, quinpirole, NPA, ropinirole, talipexole) binding in the absence of added guanine nucleotides was best described in terms of interaction at higher and lower affinity states, whereas for other agonists (bromocriptine, DHEC, lisuride, 3‐PPP) a one binding site model was a good description of the data. In the presence of GTP (100 μM) all agonist binding data were best described by a one site model.

All of the agonists tested increased [35S]‐GTPγS binding above the basal level and the maximal effects and potencies of the agonists in this test were different. There was no clear relation betwen the ability of an agonist to stabilize the formation of the ternary complex of agonist/receptor/G‐protein and the maximal activity of the agonist or the amplification factor (ratio of dissociation constant for binding to receptor to EC50 in functional assay).

A comparison was made between the profiles of the D2(short) and D2(long) receptor isoforms in these assays.
Functional coupling of a recombinant Human 5‐HT5A receptor to G‐proteins in HEK‐293 cells
Authors: Hurley P. T. , McMahon R. A. , Fanning P. , O'Boyle K. M. , Rogers M. , Martin F.
Published: January 2005

We have cloned, expressed and pharmacologically characterized the Human 5‐HT5A receptor.

We have shown that ligand activation of the Human 5‐HT5A receptor results in functional coupling to G‐proteins in HEK‐293 cells.

Stimulation of the receptor with 5‐CT (5‐carboxamidotryptamine) resulted in a dose‐dependent increase in the % [35S]‐GTPγS binding over the basal level. This is the first study to describe such G‐protein activation for the Human 5‐HT5A receptor in any cell.

A dose‐dependent inhibition of cyclic AMP accumulation was observed in the recombinant Human 5‐HT5A receptor cell line, suggesting a functional coupling to a Gαi, G‐protein in the HEK‐293 cell line.

A ligand‐stimulated reduction in the detectable level of the catalytic domain of protein kinase A (PKA) in nuclear extracts isolated from Human 5‐HT5A expressing cells was observed. This observation was consistent with the reduction in the level of cyclic AMP accumulation, in response to receptor activation.

British Journal of Pharmacology (1998) 124, 1238–1244; doi:10.1038/sj.bjp.0701928
Activation of a nonspecific cation current in rat cultured retinal pigment epithelial cells: involvement of a Gαi subunit protein and the mitogen‐activated protein kinase signalling pathway
Authors: Ryan Jennifer S. , Kelly Melanie E. M.
Published: January 2005

Whole‐cell patch‐clamp recording techniques were used to investigate the G protein subtype and related signalling molecules involved in activation of a nonspecific cation (NSC) current in rat cultured retinal pigment epithelial (RPE) cells.

Under control conditions, in 130 mm NaCl with K+ aspartate in the pipette, cytosolic dialysis with guanosine‐5′‐O‐(3‐triphosphate) (GTPγS, 0.1 mm) activated a large non‐inactivating NSC current in 80% of the cells recorded from.

Loading RPE cells with antibodies (10 μg‐ml−1) against the α subunit of all PTX‐sensitive G proteins (Gαi/o/t/z) reduced NSC current activation to 11%, while loading RPE cells with antibodies directed specifically against the α subunits of the Gi subclass (Gαi‐3) completely abolished current activation. In RPE cells loaded with anti‐Gαs activation of the NSC current was unaffected.

Investigation of the potential downstream mediators in the Gαi NSC channel pathway revealed that activation of the cation conductance was unaffected by treatment of RPE cells with the selective protein kinase C inhibitor GF 109203X (3 μm) or the selective CaM kinase II inhibitor KN‐93 (50 μm). However, NSC current activation was delayed and the current amplitude reduced in the presence of the nonselective kinase inhibitor H‐7 (100 μm) or the selective inhibitor of MAPKK (MEK) activation, PD 98059 (50 μm).

In the absence of GTPγS, the NSC current was not activated by superfusion of the cells with the cyclic GMP kinase activator dibutyryl‐cyclic GMP or with the adenylate cyclase activator forskolin.

These results support the involvement of a G protein of the Gαi subclass in the activation of a NSC current in rat RPE cells, and suggest a potential modulatory role for MAP kinase‐dependent phosphorylation in current regulation.

British Journal of Pharmacology (1998) 124, 1115–1122; doi:10.1038/sj.bjp.0701936
Effects of U‐50,488H withdrawal on catecholaminergic neurones of the rat ventricle
Authors: Milanés M. V. , Laorden M. L.
Published: January 2005

In the present study the changes in noradrenaline (NA) and dopamine (DA) content and turnover during naloxone‐induced withdrawal were analysed in the right ventricle of rats chronically treated with the κ‐agonist U‐50,488H.

Rats were rendered tolerant by administration of U‐50,488H twice a day for 4 days. On the day of death the animals were injected with saline or naloxone (3 mg kg−1, s.c.) to precipitate a withdrawal syndrome.

After naloxone administration to U‐50,488H‐ treated rats we found neither behaviour signs of physical dependence nor changes in the tissue content of noradrenaline (NA). However, naloxone induced a decrease in both cardiac normetanephrine (NM) levels and NA turnover.

Similarly, naloxone enhanced the dopamine content and decreased the 3,4‐dihydroxyphenylacetic acid (DOPAC) concentration and dopamine turnover.

Importantly and in contrast to μ‐agonists, the present results demonstrate that U‐50,488H withdrawal produced a decrease in the NA and dopamine turnover, without behavioural signs of physical dependence.

British Journal of Pharmacology (1998) 124, 1060–1064; doi:10.1038/sj.bjp.0701939
Spatial heterogeneity in the mechanisms contributing to acetylcholine‐induced dilatation in the rabbit isolated ear
Authors: Berman Rodney S. , Griffith Tudor M.
Published: January 2005

Using an X‐ray microangiographic technique in rabbit isolated perfused ears preconstricted with 5‐HT (300 nm) and histamine (300 nm), we investigated the combined actions of Nω‐nitro‐l‐arginine methyl ester (l‐NAME) and indomethacin on acetylcholine‐induced depressor responses.

Under control conditions, acetylcholine (10 nm–30 μm) induced a concentration‐dependent reversal of the pressor response, reaching a maximum of 66.0±13.6% (n = 6). In the presence of l‐NAME (300 μm) and indomethacin (10 μm), this depressor action was reduced, reaching a maximum of 38.6±5.9% (n = 6).

The control response was associated with substantial vasodilatation in the central ear artery (G0), a smaller dilatory action on first generation branch arteries (G1) and no effect on second generation branch arteries (G2). In the presence of l‐NAME and indomethacin, vasodilatation occurred in G2 with no effect in G0 or G1.

Two calcium‐activated K+ channels blockers, charybdotoxin (ChTX; 10 nm) and penitrem A (100 nm), further inhibited, but did not abolish, the l‐NAME‐ and indomethacin‐resistant response to acetylcholine (10 nm–300 μm). Both agents abolished the vasodilatory action of acetylcholine in G2.

In conclusion, l‐NAME and indomethacin induced a shift in acetylcholine‐induced vasodilatation from G0 and G1 to G2. This is consistent with the suggestion that nitric oxide dominates in larger vessels whilst other mechanisms dominate in smaller vessels. The l‐NAME‐ and indomethacin‐resistant component was inhibited by ChTX and penitrem A, suggesting it is mediated, at least in part, by activation of KCa channels and could therefore involve a hyperpolarising mediator such as endothelium‐derived hyperpolarising factor.

British Journal of Pharmacology (1998) 124, 1245–1253; doi:10.1038/sj.bjp.0701945
Functional characterisation of the human cloned 5‐HT7 receptor (long form); antagonist profile of SB‐258719
Authors: Thomas David R. , Gittins Susan A. , Collin Lissa L. , Middlemiss Derek N. , Riley Graham , Hagan Jim , Gloger Israel , Ellis Catherine E. , Forbes Ian T. , Brown Anthony M.
Published: January 2005

The functional profile of the long form of the human cloned 5‐HT7 receptor (designated h5‐HT7(a)) was investigated using a number of 5‐HT receptor agonists and antagonists and compared with its binding profile. Receptor function was measured using adenylyl cyclase activity in washed membranes from HEK293 cells stably expressing the recombinant h5‐HT7(a) receptor.

The receptor binding profile, determined by competition with [3H]‐5‐CT, was consistent with that previously reported for the h5‐HT7(a) receptor. The selective 5‐HT7 receptor antagonist SB‐258719 ((R)‐3,N‐Dimethyl‐N‐[1‐methyl‐3‐(4‐methylpiperidin‐1‐yl)propyl]benzene sulfonamide) displayed high affinity (pKi 7.5) for the receptor.

In the adenylyl cyclase functional assay, 5‐CT and 8‐OH‐DPAT were both full agonists compared to 5‐HT and the rank order of potency for agonists (5‐CT>5‐HT>8‐OH‐DPAT) was the same in functional and binding studies.

Risperidone, methiothepin, mesulergine, clozapine, olanzapine, ketanserin and SB‐258719 antagonised surmountably 5‐CT‐stimulated adenylyl cyclase activity. Schild analysis of the antagonism by SB‐258719 gave a pA2 of 7.2±0.2 and slope not significantly different from 1, consistent with competitive antagonism.

The same antagonists also inhibited basal adenylyl cyclase activity with a rank order of potency in agreement with those for antagonist potency and binding affinity. Both SB‐258719 and mesulergine displayed apparent partial inverse agonist profiles compared to the other antagonists tested. These inhibitory effects of antagonists appear to be 5‐HT7 receptor‐mediated and to reflect inverse agonism.

It is concluded that in this expression system, the h5‐HT7(a) receptor shows the expected binding and functional profile and displays constitutive activity, revealing inverse agonist activity for a range of antagonists.

British Journal of Pharmacology (1998) 124, 1300–1306; doi:10.1038/sj.bjp.0701946
Direct activation of endothelial NO pathway by Ba2+ in canine coronary artery
Authors: Yamazaki Jun , Sato Kazuo , Ohara Fumihiro , Nagao Taku
Published: January 2005

We have reported that Ba2+ causes endothelium‐dependent relaxation of canine coronary arteries through NO synthesis in Ca2+‐free and depolarizing solution. To determine the cellular mechanisms by which the endothelium‐dependent relaxation occurs, we used fura‐2 fluorometry (F350 and F390; excitation wavelengths, 350 and 390 nm, respectively) and estimated the intracellular Ba2+ concentration in endothelial and vascular smooth muscle cells.

Ba2+ (10−3 m) increased the fura‐2 ratio (F350/F390) recorded from a combined preparation of smooth muscle and endothelium (0.445±0.073, n = 4) and contracted the arteries in the presence of 80 mm K+ (0.22±0.06 g, n = 4).

Diltiazem (3×10−6 m) blocks Ba2+ entry into vascular smooth muscle cells via l‐type Ca2+ channels. In this condition, Ba2+ increased the fura‐2 ratio in endothelial cells (0.141±0.014, n = 5) and relaxed the underlying smooth muscle (0.08±0.01 g, n = 5) by a mechanism which was sensitive to 10−4 m NG‐methyl‐l‐arginine (l‐NMMA).

Ba2+‐induced relaxation was not attenuated with repeated application and was elicited even after endothelium‐dependent relaxations in response to 10−6 m bradykinin were abolished due to tachyphylaxis. Neither 10−2 m caffeine nor 10−6 m thapsigargin had effect upon Ba2+‐induced relaxation.

To further rule out changes in intracellular Ca2+ as a mechanism of Ba2+‐induced relaxation, fura‐2 fluorescence was measured at the isosbestic wavelengths for Ca2+ (360 nm) and Ba2+ (370 nm) in endothelium‐intact arteries. Ba2+ altered F360, but not F370, suggesting little or no contribution of intracellular Ca2+ to the phenomenon of Ba2+‐induced relaxation.

These results suggest that the Ba2+‐induced relaxation is due to its direct activation of endothelial NO synthesis without mobilization of intracellular Ca2+.

British Journal of Pharmacology (1998) 124, 1149–1158; doi:10.1038/sj.bjp.0701948