bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of adenosine receptors evoking excitation of mesenteric afferents in the rat
Authors: Kirkup A J , Eastwood C , Grundy D , Chessell I P , Humphrey P P A
Published: January 2005

We examined the effects of adenosine receptor agonists and antagonists on the discharge of mesenteric afferent nerves supplying the jejunum in pentobarbitone sodium‐anaesthetized rats.

Adenosine (0.03–10 mg kg−1, i.v.), NECA (0.3–300 μg kg−1, i.v.) and the A1 receptor agonist, GR79236 (0.3–1000 μg kg−1, i.v.), each induced dose‐dependent increases in afferent nerve activity and intrajejunal pressure, hypotension and bradycardia. The A1 receptor antagonist, DPCPX (3 mg kg−1, i.v.), antagonized all the effects of GR79236 but only the haemodynamic effects of adenosine and NECA. The A2A receptor antagonist, ZM241385 (3 mg kg−1, i.v.), antagonized the hypotensive effect of NECA but none of the effects of GR79236.

The A2A receptor agonist, CGS21680 (0.3–300 μg kg−1, i.v.), and the A3 receptor agonist, IB‐MECA (0.3–300 μg kg−1, i.v.), each induced only a dose‐dependent hypotension. Subsequent administration of adenosine (3 mg kg−1, i.v.) induced increases in afferent nerve activity and intrajejunal pressure and bradycardia. ZM241385 (3 mg kg−1, i.v.) antagonized the hypotensive effect of CGS21680 but not the effects of adenosine.

Bethanechol (300 μg kg−1, i.v.) evoked increases in afferent nerve activity and intrajejunal pressure, hypotension and bradycardia. However, adenosine (3 mg kg−1, i.v.) evoked greater increases in afferent nerve activity than bethanechol despite inducing smaller increases in intrajejunal pressure.

In summary, A1 and A2B and/or A2B‐like receptors evoke adenosine‐induced increases in mesenteric afferent nerve activity and intrajejunal pressure in the anaesthetized rat. Furthermore, elevations in intrajejunal pressure do not wholly account for adenosine‐evoked excitation of mesenteric afferent nerves.

British Journal of Pharmacology (1998) 125, 1352–1360; doi:10.1038/sj.bjp.0702202
Inhibition by inorganic ions of a sustained calcium signal evoked by activation of mGlu5 receptors in rat cortical neurons and glia
Authors: Prothero Larissa S , Richards Christopher D , Mathie Alistair
Published: January 2005

The effect of mGlu receptor agonists on intracellular calcium (Ca2+) in rat cortical neurons and glial cells was studied. The responses evoked consisted of two phases; an initial transient response followed by a sustained plateau. In both cell types the order of potency of group I mGlu receptor agonists was DHPG>1S,3R ACPD>3‐HPG.

The selective mGlu5 agonist CHPG elicited responses in both cell types as did S4C3‐HPG which is thought to be an mGlu5 agonist at high concentrations. S4‐CPG had no effect on intracellular Ca2+ levels nor did it inhibit the action of 1S,3R ACPD. These results suggest that the responses in both cell types are mediated by mGlu5 receptors.

In the absence of extracellular Ca2+ ions, 1S,3R ACPD (100 μm) induced only a transient Ca2+ response which decayed to baseline with a time constant of approximately 20 s in both cell types. Subsequent readdition of Ca2+ (2 mm) to the external solution in the continued presence of 1S,3R ACPD induced a sustained Ca2+ plateau.

The sustained Ca2+ plateau could be blocked by a number of inorganic cations, with an order of potency of Zn2+La3+>Cd2+Co2+>Ni2+>Mg2+. Similar concentrations of Zn2+ had little effect on Ca2+‐influx evoked by 25 mm K+.

It is concluded that the Ca2+‐entry pathway activated by mGlu5 receptors resembles store‐operated Ca2+‐entry pathways that have been described in other cell types.

British Journal of Pharmacology (1998) 125, 1551–1561; doi:10.1038/sj.bjp.0702203
Comparison of cannabinoid binding sites in guinea‐pig forebrain and small intestine
Authors: Ross Ruth A , Brockie Heather C , Fernando Susanthi R , Saha Bijali , Razdan Raj K , Pertwee Roger G
Published: January 2005

We have investigated the nature of cannabinoid receptors in guinea‐pig small intestine by establishing whether this tissue contains cannabinoid receptors with similar binding properties to those of brain CB1 receptors. The cannabinoids used were the CB1‐selective antagonist SR141716A, the CB2‐selective antagonist SR144528, the novel cannabinoid receptor ligand, 6′‐azidohex‐2′‐yne‐Δ8‐tetrahydrocannabinol (O‐1184), and the agonists CP55940, which binds equally well to CB1 and CB2 receptors, and WIN55212‐2, which shows marginal CB2 selectivity.

[3H]‐CP55940 (1 nm) underwent extensive specific binding both to forebrain membranes (76.3%) and to membranes obtained by sucrose density gradient fractionation of homogenates of myenteric plexus‐longitudinal muscle of guinea‐pig small intestine (65.2%).

Its binding capacity (Bmax) was higher in forebrain (4281 fmol mg−1) than in intestinal membranes (2092 fmol mg−1). However, the corresponding KD values were not significantly different from each other (2.29 and 1.75 nm respectively). Nor did the Ki values for its displacement by CP55940, WIN55212‐2, O‐1184, SR141716A and SR144528 from forebrain membranes (0.87, 4.15, 2.85, 5.32 and 371.9 respectively) differ significantly from the corresponding Ki values determined in experiments with intestinal membranes (0.99, 5.03, 3.16, 4.95 and 361.5 nm respectively).

The Bmax values of [3H]‐CP55940 and [3H]‐SR141716A in forebrain membranes did not differ significantly from each other (4281 and 5658 fmol mg−1) but were both greater than the Bmax of [3H]‐WIN55212‐2 (2032 fmol mg−1).

O‐1184 (10 or 100 nm) produced parallel dextral shifts in the log concentration‐response curves of WIN55212‐2 and CP55940 for inhibition of electrically‐evoked contractions of the myenteric plexus‐longitudinal muscle preparation, its KD values being 0.20 nm (against WIN55212‐2) and 0.89 nm (against CP55940).

We conclude that cannabinoid binding sites in guinea‐pig small intestine closely resemble CB1 binding sites of guinea‐pig brain and that O‐1184 behaves as a cannabinoid receptor antagonist in the guinea‐pig myenteric plexus‐longitudinal muscle preparation.

British Journal of Pharmacology (1998) 125, 1345–1351; doi:10.1038/sj.bjp.0702204
Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO‐K1 cells
Authors: Michel A D , Chessell I P , Hibell A D , Simon J , Humphrey P P A
Published: January 2005

CHO‐K1 cells were examined for their cellular responses to the P2 receptor agonist, 2′‐ and 3′‐O‐(4‐benzoylbenzoyl)‐ATP (DbATP), and for the presence of mRNA for P2X receptors.

Reverse transcriptase‐polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1‐P2X6 subunits.

DbATP (EC50∼100 μm) evoked non‐desensitizing inward currents which reversed at ∼0mV, suggesting activation of a non‐selective cation channel. ATP also evoked inward currents but was less potent than DbATP.

DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO‐PRO‐1, in CHO‐K1 cells. Both responses were inhibited by NaCl and MgCl2. In 280 mm sucrose buffer, 45Ca2+ accumulation was measurable within 10–20 s of agonist addition, whereas YO‐PRO‐1 accumulation was only detectable after 8 min. ATP and ATPγS were also agonists but were less potent than DbATP, while UTP, 2‐methylthio ATP, ADP and αβmethylene ATP were inactive at concentrations up to 100 μm.

DbATP increased lactate dehydrogenase release from CHO‐K1 cells, suggesting cell lysis, although this effect was only pronounced after 60–90 min.

These data suggest that CHO‐K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO‐PRO‐1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.

British Journal of Pharmacology (1998) 125, 1194–1201; doi:10.1038/sj.bjp.0702205
Effect of nimesulide and indomethacin on contractility and the Ca2+ channel current in myometrial smooth muscle from pregnant women
Authors: Sawdy Robert , Knock Gregory A , Bennett Philip R , Poston Lucilla , Aaronson Philip I
Published: January 2005

The non‐steroidal anti‐inflammatory drug (NSAID) indomethacin inhibits both constitutive and inducible forms of cyclo‐oxygenase (COX‐1 and COX‐2, respectively), while nimesulide is a selective COX‐2 inhibitor. Uterine COX‐2 is upregulated before and during term and pre‐term labour, and prostaglandins play a crucial role in parturition. We therefore evaluated the effects of these drugs on myometrial contractility and the voltage‐gated Ca2+ channel current in tissue strips and isolated human myometrial smooth muscle cells (HMSMC) from myometrial biopsies taken with informed consent from women undergoing caesarean section at term (not in labour).

Nimesulide and indomethacin caused almost complete inhibition of spontaneous myometrial contractions at concentrations of 100 and 300 μm, respectively. The Ca2+ channel current was inhibited in a concentration‐dependent manner by both drugs, with a 40% reduction of the current at 100 μm nimesulide and 300 μm indomethacin. Nimesulide also accelerated the decay of the Ca2+ channel current.

The inhibition of the Ca2+ channel current by 100 μm nimesulide and 300 μm indomethacin was unaffected by the presence of either PGF2α or PGE2 (30 μm), and was of similar magnitude whether 10 mm Ba2+ or 1.5 mm Ca2+ was used as the charge carrier.

The concentrations of indomethacin and nimesulide required to suppress spontaneous contractility in human pregnant myometrium were much higher than those necessary to inhibit prostaglandin production. The results suggest that both nimesulide and indomethacin inhibit myometrial contractility via mechanisms independent of cyclo‐oxygenase inhibition. Blockade of the Ca2+ current may contribute to this effect.

British Journal of Pharmacology (1998) 125, 1212–1217; doi:10.1038/sj.bjp.0702211
Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase‐1
Authors: Sammut Ivan A. , Foresti Roberta , Clark James E. , Exon David J. , Vesely Martin J. J. , Sarathchandra Padmini , Green Colin J. , Motterlini Roberto
Published: January 2005

The contribution of haeme oxygenase‐derived carbon monoxide (CO) to the regulation of vascular tone in thoracic aorta was investigated following induction of the inducible isoform of haeme oxygenase (HO‐1).

Isometric smooth muscle contractions were recorded in isolated rat aortic ring preparations. Rings were incubated in the presence of the nitric oxide (NO) donor S‐nitroso‐N‐acetyl penicillamine (SNAP, 500 μm) for 1 h, then repetitively washed and maintained for a further 4 h prior to producing a concentration‐response curve to phenylephrine (PE, 1–3000 nm).

Treatment with SNAP resulted in increased mRNA and protein expression of aortic HO‐1 and was associated with a significant suppression of the contractile response to PE (P<0.05 vs control). Immunohistochemical staining procedures revealed marked HO‐1 expression in the endothelial layer and, to a lesser extent, in smooth muscle cells.

Induction of HO‐1 in SNAP‐treated rings was associated with a higher 14CO release compared to control, as measured by scintillation counting after incubation of aortas with [2‐14C]‐L‐glycine, the precursor of haeme. Guanosine 3′,5′‐monophosphate (cyclic GMP) content was also greatly enhanced in aortas expressing high levels of HO‐1.

Incubation of aortic rings with the NO synthase inhibitor, NG‐monomethyl‐L‐arginine (100 μm), significantly (P<0.05) increased the contractile response to PE in controls but failed to restore PE‐mediated contractility in SNAP‐treated rings. In contrast, the selective inhibitor of haeme oxygenase, tin protoporphyrin IX (SnPP‐IX, 10 μm), restored the pressor response to PE in SNAP‐treated rings whilst markedly reducing CO and cyclic GMP production.

We conclude that up‐regulation of the HO‐1/CO pathway significantly contributes to the suppression of aortic contractility to PE. This effect appears to be mediated by the elevation of cyclic GMP levels and can be reversed by inhibition of the haeme oxygenase pathway.

British Journal of Pharmacology (1998) 125, 1437–1444; doi:10.1038/sj.bjp.0702212
Evidence for two different types of P2 receptors stimulating insulin secretion from pancreatic B cell
Authors: Petit P , Hillaire‐Buys D , Manteghetti M , Debrus S , Chapal J , Loubatières‐Mariani M M
Published: January 2005

Adenine nucleotides have been shown to stimulate insulin secretion by acting on P2 receptors of the P2Y type. Since there have been some discrepancies in the insulin response of different analogues of ATP and ADP, we investigated whether two different types of P2 receptors exist on pancreatic B cells. The effects of α,β‐methylene ATP, which is more specific for the P2X subtype, were studied in vitro in pancreatic islets and isolated perfused pancreas from rats, in comparison with the potent P2Y receptor agonist ADPβS.

In isolated islets, incubated with a slightly stimulating glucose concentration (8.3 mm), α,β‐me ATP (200 μm) and ADPβS (50 μm) similarly stimulated insulin secretion; by contrast, under a non stimulating glucose concentration (3 mm), α,β‐me ATP was still effective whereas ADPβS was not. In the same way, in islets perifused with 3 mm glucose, α,β‐me ATP but not ADPβS induced a partial but significant reduction in the peak 86Rb efflux induced by the ATP‐dependent potassium channel opener diazoxide.

In the isolated pancreas, perfused with a non stimulating glucose concentration (4.2 mm), ADPβS and α,β‐me ATP (5–50 μm), administered for 10 min, induced an immediate, transient and concentration‐dependent increase in the insulin secretion; their relative potency was not significantly different. In contrast, with a slightly stimulating glucose concentration (8.3 mm), ADPβS was previously shown to be 100 fold more potent than α,β‐me ATP. Furthermore, at 4.2 mm glucose a second administration of α,β‐me ATP was ineffective. In the same way, ADPβS was also able to desensitize its own insulin response. At 3 mm glucose, α,β‐me ATP as well as ADPβS (50 μm) induced a transient stimulation of insulin secretion and down regulated the action of each other.

These results give evidence that pancreatic B cells, in addition to P2Y receptors, which potentiate glucose‐induced insulin secretion, are provided with P2X receptors, which transiently stimulate insulin release at low non‐stimulating glucose concentration and slightly affect the potassium conductance of the membrane.

British Journal of Pharmacology (1998) 125, 1368–1374; doi:10.1038/sj.bjp.0702214
Effect of lercanidipine and its (R)‐enantiomer on atherosclerotic lesions induced in hypercholesterolemic rabbits
Authors: Soma Maurizio R. , Natali Malvina , Donetti Elena , Baetta Roberta , Farina Pierluigi , Leonardi Amedeo , Comparato Carmen , Barberi Laura , Catapano Alberico L.
Published: January 2005

The in vivo antiatherogenic activity of the calcium antagonist lercanidipine and its (R)‐enantiomer was investigated in two different types of atherosclerotic lesions (hyperplastic and fatty‐streak lesions) in rabbits.

Lercanidipine (0.3, 1, and 3 mg kg−1 week−1) as well as its (R)‐enantiomer at 3 mg kg−1 week−1 were given by subcutaneous injection for 10 weeks to White New Zealand rabbits, with cholesterol feeding beginning at week 2. The hyperplastic lesion was obtained by positioning a hollow silastic collar around one carotid artery, while aortic fatty streak lesions were induced by cholesterol feeding. In untreated animals (n=5), 14 days after collar positioning an intimal hyperplasia was clearly detectable: the arteries without collar showed a intima/media (I/M) ratio of 0.03±0.02, whereas in carotids with a collar the ratio was 2±0.42. In lercanidipine‐treated animals a significant and dose‐dependent effect on intimal hyperplasia was observed. I/M ratios were 0.73±0.4, 0.42±0.1, 0.32±0.1 for 0.3, 1, and 3 mg kg−1 week−1, respectively (P<0.05). The lercanidipine enantiomer (3 mg kg−1 week−1) was as effective as the racemate (0.41±0.11). Proliferation of smooth muscle cells, assessed by incorporation of BrdU into DNA, was reduced by about 50%, 70%, 85%, and 80% by lercanidipine (0.3, 1, and 3 mg kg−1 week−1) and its (R)‐enantiomer, respectively.

The area of fatty‐streaks in the aorta (n=11–15) was significantly reduced by lercanidipine (3 mg kg−1 week−1, 16% vs 27%, P<0.05), a trend was observed also with lower doses. When different segments of the aorta were considered (arch, thoracic, abdominal) a significant and dose‐dependent effect in the thoracic and abdominal aorta was observed also at lower doses. The (R)‐enantiomer was as effective as lercanidipine.

These results suggest a direct antiatherosclerotic effect of lercanidipine, independent of modulation of risk factors such as hypercholesterolemia and/or hypertension as demonstrated by the absence of stereoselectivity.

British Journal of Pharmacology (1998) 125, 1471–1476; doi:10.1038/sj.bjp.0702221
Insensitivity of volume‐sensitive chloride currents to chromones in human airway epithelial cells
Authors: Zegarra‐Moran Olga , Lantero Sabina , Sacco Oliviero , Rossi Giovanni A , Galietta Luis J V
Published: January 2005

Chromones (sodium cromoglycate and sodium nedocromil) block cell swelling‐activated Cl− channels in NIH‐3T3 fibroblasts and endothelial cells. This has led to hypothesize that cell volume regulation might be involved in asthma pathogenesis.

Using whole‐cell patch‐clamp experiments, we studied the effect of chromones on volume‐sensitive Cl− currents in transformed human tracheal epithelial cells (9HTEo‐) and in primary cultures of human bronchial epithelial cells (BE).

Cl− currents activated by hypotonic shock were poorly blocked by extracellular nedocromil or cromoglycate. The block was voltage‐dependent since it was observed only at positive membrane potentials. At the concentration of 5 mm, the current inhibition by both chromones at +80 mV was about 40% for 9HTEo‐ and only 20% for BE.

Intracellular application of chromones elicited a voltage‐independent inhibition in 9HTEo‐ cells. Under this condition, volume‐sensitive Cl− currents were reduced at all membrane potentials (60 and 45% inhibition by 2 mm nedocromil and cromoglycate respectively). In contrast intracellular chromones were ineffective in BE cells.

The relative refractoriness to chromones, in contrast with the high sensitivity shown by other Cl− channels, suggests that the epithelial volume‐sensitive Cl− channel is not involved in asthma.

British Journal of Pharmacology (1998) 125, 1382–1386; doi:10.1038/sj.bjp.0702225
Expression of cyclic GMP‐inhibited phosphodiesterases 3A and 3B (PDE3A and PDE3B) in rat tissues: Differential subcellular localization and regulated expression by cyclic AMP
Authors: Liu H , Maurice D H
Published: January 2005

A combination of pharmacological, molecular biological and biochemical approaches were used to investigate the differential expression of two cyclic GMP‐inhibited cyclic nucleotide phosphodiesterase genes (PDE3A and PDE3B) in the rat.

RT–PCR using PDE3A‐ or PDE3B‐specific oligonucleotide primers allowed amplification of products encoding PDE3A (508 bp) or PDE3B (499 bp) sequences from several rat tissues (heart, aorta, liver, kidney and epididymal fat), from primary cultures of aortic vascular smooth muscle cells (VSMC) as well as from an SV40 large T‐antigen immortalized aortic VSMC line.

Immunoblotting experiments with PDE3‐selective antisera allowed the detection of both PDE3A and PDE3B immunoreactive proteins in several rat tissues, including tissues of the cardiovascular system, in primary cultures of aortic VSMC and in an SV40 large T‐antigen immortalized aortic VSMC line. In all cases, PDE3A was expressed as a 120 kDa protein which was only detected in the cytosolic fraction. PDE3B was expressed as a 135 kDa protein and its expression was limited to the particulate fraction of all tissues and cells studied.

Prolonged incubation of cultured aortic VSMC with agents that increase VSMC cyclic AMP (forskolin or 8‐bromo‐cyclic AMP) produced marked time‐dependent increases in PDE3 activity which correlated with increases in PDE3A and PDE3B RT–PCR signals and a marked increase in particulate PDE3 activity and PDE3B protein.

The physiological, pharmacological and biochemical implications of these findings are discussed based on previous reports of the effects of PDE3 inhibitors in the cardiovascular system and the relevance of our findings are presented in the context of the development of PDE3A and/or PDE3B‐selective pharmacological agents.

British Journal of Pharmacology (1998) 125, 1501–1510; doi:10.1038/sj.bjp.0702227
Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS‐activated J774 cells
Authors: Baydoun Anwar R , Morgan David M L
Published: January 2005

We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by α‐difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages.

DFMO potentiated LPS‐stimulated nitrite production in both a concentration‐ and time‐dependent manner, increasing nitrite levels by 48±5% at 10 mm. This effect was observed in cells pre‐treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS‐induced nitrite production.

Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS‐induced nitrite production or in the ability of DFMO (10 mm) to potentiate LPS‐induced NO synthesis.

Metabolism of L‐[3H]arginine to L‐[3H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1–10 mm) or by putrescine (0.001–1 mm), spermidine (0.001–1 mm) or spermine (0.001–1 mm). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70±0.07%) by L‐NMMA (100 μm).

Pre‐incubation of cells with DFMO (10 mm; 24 h) prior to activation with LPS resulted in enhanced (∼2 fold) iNOS protein expression.

These results show that DFMO potentiates LPS‐induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.

British Journal of Pharmacology (1998) 125, 1511–1516; doi:10.1038/sj.bjp.0702231
Potassium channel activation and relaxation by nicorandil in rat small mesenteric arteries
Authors: Davie C. S. , Kubo M , Standen N. B.
Published: January 2005

We used whole‐cell patch clamp to investigate the currents activated by nicorandil in smooth muscle cells isolated from rat small mesenteric arteries, and studied the relaxant effect of nicorandil using myography.

Nicorandil (300 μM) activated currents with near‐linear current‐voltage relationships and reversal potentials near to the equilibrium potential for K+.

The nicorandil‐activated current was blocked by glibenclamide (10 μM), but unaffected by iberiotoxin (100 nM) and the guanylyl cyclase inhibitor LY 83583 (1 μM). During current activation by nicorandil, openings of channels with a unitary conductance of 31 pS were detected.

One hundred μM nicorandil had no effect on currents through Ca2+ channels recorded in response to depolarizing voltage steps using 10 mM Ba2+ as a charge carrier. A small reduction in current amplitude was seen in 300 μM nicorandil, though this was not statistically significant.

In arterial rings contracted with 20 mM K+ Krebs solution containing 200 nM BAYK 8644, nicorandil produced a concentration‐dependent relaxation with mean pD2=4.77±0.06. Glibenclamide (10 μM) shifted the curve to the right (pD2=4.32±0.05), as did 60 mM K+. LY 83583 caused a dose‐dependent inhibition of the relaxant effect of nicorandil, while LY 83583 and glibenclamide together produced greater inhibition than either alone.

Metabolic inhibition with carbonyl cyanide m‐chlorophenyl hydrazone (30 nM), or by reduction of extracellular glucose to 0.5 mM, increased the potency of nicorandil.

We conclude that nicorandil activates KATP channels in these vessels and also acts through guanylyl cyclase to cause vasorelaxation, and that the potency of nicorandil is increased during metabolic inhibition.
The effect of inhibitors of receptor internalization on the desensitization and resensitization of three Gs‐coupled receptor responses
Authors: Mundell Stuart J , Kelly Eamonn
Published: January 2005

Many G protein‐coupled receptors (GPCRs) are known to internalize following agonist exposure, however the relative importance of this mechanism for the desensitization and resensitization of different GPCRs is unclear. In the present study, we have pretreated NG108‐15 cells with hypertonic sucrose or concanavalin A (con A), to investigate the effects of these inhibitors of internalization on the agonist‐induced desensitization and subsequent resensitization of three Gs‐coupled receptor responses.

Incubation of cells with sucrose or con A did not affect subsequent acute agonist stimulation of the A2A adenosine receptor or the agonist‐induced desensitization of this receptor response. However, the resensitization of the A2A adenosine receptor response following agonist removal was abolished in the presence of sucrose or con A.

Sucrose or con A treatment affected neither the desensitization nor resensitization of IP‐prostanoid receptor responsiveness. On the other hand con A but not sucrose reduced the agonist‐induced desensitization of secretin receptor responsiveness. However, secretin receptor responsiveness did not resensitize within the time period studied whether or not inhibitors of internalization were present.

These results indicate that receptor internalization appears to subserve different functions for different GPCRs.

British Journal of Pharmacology (1998) 125, 1594–1600; doi:10.1038/sj.bjp.0702234
Investigation of the interaction between cholinergic and nitrergic neurotransmission in the pig gastric fundus
Authors: Leclere P. G. , Lefebvre R. A.
Published: January 2005

The interaction between the cholinergic and nitrergic innervation was investigated in circular muscle strips of the pig gastric fundus.

In physiological salt solution containing 4×10−6 M guanethidine, electrical field stimulation (EFS; 40 V, 0.5 ms, 0.5–32 Hz, 10 s at 4 min intervals) induced small transient relaxations at 0.5–4 Hz, and large frequency‐dependent contractions, sometimes followed by off‐relaxations, at 8–32 Hz.

In the presence of L‐NG‐nitroarginine methyl ester (L‐NAME; 3×10−4 M) or physostigmine (10−6 M), relaxations were reversed into contractions and contractions were enhanced. Physostigmine added to L‐NAME further enhanced contractions, while addition of L‐NAME to physostigmine had no additional effect. Off‐relaxations were enhanced in the presence of L‐NAME and physostigmine. L‐NAME and physostigmine consistently increased basal tone.

Tissues contracted by 5‐hydroxytryptamine or by acetylcholine responded to EFS in a similar way as in basal conditions and L‐NAME reversed the relaxations at the lower stimulation frequencies into contractions and enhanced the contractions at the higher stimulation frequencies.

Off‐relaxations in the presence of L‐NAME were partially reduced by α‐chymotrypsin (10 U ml−1).

In the absence of physostigmine, the concentration‐response curve to exogenous acetylcholine was not influenced by L‐NAME.

Contractions of the same amplitude induced by EFS at 4 Hz and by exogenous acetylcholine were either decreased or enhanced to the same extent by sodium nitroprusside (SNP; 10−5 M), depending upon the degree of relaxation by SNP.

These experiments suggest that endogenous nitric oxide interferes with cholinergic neurotransmission in the pig gastric fundus by functional antagonism at the postjunctional level. The interaction is independent of the degree of contraction.
Improved metabolic status and insulin sensitivity in obese fatty (fa/fa) Zucker rats and Zucker Diabetic Fatty (ZDF) rats treated with the thiazolidinedione, MCC‐555
Authors: Upton R , Widdowson P. S. , Ishii S , Tanaka H , Williams G
Published: January 2005

We examined the effect of chronic (21 days) oral treatment with the thiazolidinedione, MCC‐555 ((±)‐5‐[{6‐(2‐fluorbenzyl)‐oxy‐2‐naphy}methyl]‐2,4‐thiazolidinedione) on metabolic status and insulin sensitivity in obese (fa/fa) Zucker rats and Zucker Diabetic Fatty (ZDF) rats which display an impaired glucose tolerance (IGT) or overt diabetic symptoms, respectively.

MCC‐555 treatment to obese Zucker rats (10 and 30 mg kg−1) and diabetic ZDF rats (10 mg kg−1) reduced non‐esterified fatty acid concentrations in both rat strains and reduced plasma glucose and triglyceride concentrations in the obese Zucker rats. Liver glycogen concentrations were significantly increased by chronic MCC‐555 treatment in both obese Zucker rats (30 mg kg−1 day−1) and diabetic ZDF rats (10 mg kg−1 day−1), as compared with vehicle‐treated lean and obese rats and there was a significant increase in hepatic glycogen synthase activity in MCC‐555‐treated diabetic ZDF rats as compared to vehicle‐treated controls.

During a euglycaemic hyperinsulinaemic clamp, MCC‐555‐treated obese Zucker rats and diabetic ZDF rats required significantly higher glucose infusion rates to maintain stable glucose concentrations (2.01 ± 0.19 mg min−1 and 6.42 ± 1.03 mg min−1, respectively) than vehicle‐treated obese controls (0.71 ± 0.17 mg min−1 and 2.09 ± 0.71 mg min−1; P<0.05), demonstrating improved insulin sensitivity in both Zucker and ZDF rats. MCC‐555 treatment also enhanced insulin‐induced suppression of hepatic glucose production in ZDF rats as measured using infusions of [6‐3H]‐glucose under clamp conditions.

In conclusion, we have demonstrated that MCC‐555 improves metabolic status and insulin sensitivity in obese Zucker and diabetic ZDF rats. MCC‐555 may prove a useful compound for alleviating the metabolic disturbances and IGT associated with insulin resistance in man.
In vivo demonstration of H3‐histaminergic inhibition of cardiac sympathetic stimulation by R‐α‐methyl‐histamine and its prodrug BP 2.94 in the dog
Authors: Mazenot Catherine , Ribuot Christophe , Durand Andrée , Joulin Yves , Demenge Pierre , Godin‐Ribuot Diane
Published: January 2005

The aim of this study was to investigate whether histamine H3‐receptor agonists could inhibit the effects of cardiac sympathetic nerve stimulation in the dog.

Catecholamine release by the heart and the associated variation of haemodynamic parameters were measured after electrical stimulation of the right cardiac sympathetic nerves (1–4 Hz, 10 V, 10 ms) in the anaesthetized dog treated with R‐α‐methyl‐histamine (R‐HA) and its prodrug BP 2.94 (BP).

Cardiac sympathetic stimulation induced a noradrenaline release into the coronary sinus along with a tachycardia and an increase in left ventricular pressure and contractility without changes in mean arterial pressure. Intravenous administration of H3‐receptor agonists significantly decreased noradrenaline release by the heart (R‐HA at 2 μmol kg−1 h−1: +77±25 vs +405±82; BP 2.94 at 1 mg kg−1: +12±11 vs +330±100 pg ml−1 in control conditions, P0.05), and increases in heart rate (R‐HA at 2 μmol kg−1 h−1: +26±8 vs +65±10 and BP 2.94 at 1 mg kg−1: +30±8 vs 75±6 beats min−1, in control conditions P0.05), left ventricular pressure, and contractility. Treatment with SC 359 (1 mg kg−1) a selective H3‐antagonist, reversed the effects of H3‐receptor agonists. Treatment with R‐HA at 2 μmol kg−1 h−1 and BP 2.94 at 1 mg kg−1 tended to decrease, while that with SC 359 significantly increased basal heart rate (from 111±3 to 130±5 beats min−1, P0.001).

Functional H3‐receptors are present on sympathetic nerve endings in the dog heart. Their stimulation by R‐α‐methyl‐histamine or BP 2.94 can inhibit noradrenaline release by the heart and its associated haemodynamic effects.

British Journal of Pharmacology (1999) 126, 264–268; doi:10.1038/sj.bjp.0702257
The effects of inflammation and inflammatory mediators on nociceptive behaviour induced by ATP analogues in the rat
Authors: Hamilton Sara G , Wade Alex , McMahon Stephen B
Published: January 2005

We have studied the behavioural effects of intraplantar injections of adenosine 5′‐triphosphate (ATP) and related compounds in freely moving rats and investigated whether these nociceptive effects are augmented in the presence of inflammatory mediators.

We find that in normal animals ATP and analogues produce dose‐dependent nocifensive behaviour (seen as bursts of elevation of the treated hindpaw), and localized thermal hyperalgesia. The rank order of potency was: α,β‐methyleneadenosine 5′‐triphosphate (α,β‐methylene ATP) >2‐methylthioadenosine triphosphate (2‐methylthio ATP)>ATP. After neonatal treatment with capsaicin, to destroy small calibre primary sensory neurones, nocifensive behaviour was largely absent.

The effects of ATP analogues were assessed in three models of peripheral sensitization: 2 h after dilute intraplantar carrageenan (0.25% w v−1); 24 h after irradiation of the hindpaw with ultraviolet (U.V.) B; immediately following prostaglandin E2 (PGE2) treatment. In all models the effect of α,β‐methylene ATP was greatly augmented. After carrageenan, significant hindpaw‐lifting behaviour activity was induced by injection of only 0.05 nmol of α,β‐methylene ATP, some 100 times less than necessary in normal skin.

Our data suggest that it is much more likely that endogenous levels of ATP will reach levels capable of exciting nociceptors in inflamed versus normal skin. Our data also suggest the involvement of P2X3 receptor subunits in ATP‐induced nociception.

British Journal of Pharmacology (1999) 126, 326–332; doi:10.1038/sj.bjp.0702258
Endogenous angiotensin II and the reflex response to stimulation of cardiopulmonary serotonin 5HT3 receptors
Authors: Veelken R , Hilgers K. F. , Scrogin K. E. , Mann J. F. E. , Schmieder R. E.
Published: January 2005

Angiotensin (Ang) II modulates cardiovascular baroreflexes; whether or not the peptide influences chemosensitive cardiovascular reflexes is not known. We tested the hypothesis that Ang II modulates the reflex control of sympathetic nerve activity exerted by 5‐hydroxytryptamine 3 (5HT3) cardiopulmonary receptors.

The 5HT3 receptor agonist phenylbiguanide (PBG), infused intravenously for 15 min, elicited a sustained reflex decrease of renal sympathetic nerve activity (RSNA) but only transient (<3 min) changes of arterial blood pressure (BP) and heart rate (HR) in methohexital‐anaesthesized rats.

Infusion of Ang II at a dose that did not affect baseline BP, HR and RSNA enhanced the PBG‐evoked reflex decrease of RSNA (−54±5% in Ang II treated versus −33±6% in control rats after 15 min PBG, P<0.05, n = 6 each) in methohexital‐anaesthetized rats.

The angiotensin converting enzyme (ACE) inhibitor lisinopril blunted the reflex responses to PBG in anaesthetized as well as conscious animals. The effect of the ACE inhibitor was abolished by concomitant infusion of Ang II.

The reflex response to stimulation of cardiopulmonary 5HT3 afferents was also impaired by the Ang II type 1 receptor (AT1) blocker ZD7155 but not by the type 2 (AT2) blocker PD 123319.

Infusion of a volume load to stimulate cardiopulmonary baroreceptors induced a gradual decrease of RSNA which was impaired by exogenous Ang II (RSNA −26±6% in Ang II treated versus −47±6% in control rats after volume load, P<0.05, n = 6 each) but unaffected by ACE inhibition.

The reflex control of RSNA by cardiopulmonary 5HT3 receptors is enhanced by Ang II via AT1 receptors. Thus, Ang II facilitates a chemosensitive cardiovascular reflex, in contrast to its inhibitory influences on mechanosensitive reflexes.
Studies of the role of endothelium‐dependent nitric oxide release in the sustained vasodilator effects of corticotrophin releasing factor and sauvagine
Authors: Barker Diana M , Corder Roger
Published: January 2005

The mechanisms of the sustained vasodilator actions of corticotrophin‐releasing factor (CRF) and sauvagine (SVG) were studied using rings of endothelium de‐nuded rat thoracic aorta (RTA) and the isolated perfused rat superior mesenteric arterial vasculature (SMA).

SVG was ∼amp;50 fold more potent than CRF on RTA (EC40: 0.9±0.2 and 44±9 nM respectively, P<0.05), and ∼amp;10 fold more active in the perfused SMA (ED40: 0.05±0.02 and 0.6±0.1 nmol respectively, P<0.05). Single bolus injections of CRF (100 pmol) or SVG (15 pmol) in the perfused SMA caused reductions in perfusion pressure of 23±1 and 24±2% that lasted more than 20 min.

Removal of the endothelium in the perfused SMA with deoxycholic acid attenuated the vasodilatation and revealed two phases to the response; a short lasting direct action, and a sustained phase which was fully inhibited.

Inhibition of nitric oxide synthase with L‐NAME (100 μM) L‐NMMA (100 μM) or 2‐ethyl‐2‐thiopseudourea (ETPU, 100 μM) had similar effects on the vasodilator responses to CRF as removal of the endothelium, suggesting a pivotal role for nitric oxide. However the selective guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 10 μM) did not affect the response to CRF.

High potassium (60 mM) completely inhibited the vasodilator response to CRF in the perfused SMA, indicating a role for K+ channels in this response.

Compared to other vasodilator agents acting via the release of NO, the actions of CRF and SVG are strikingly long‐lasting, suggesting a novel mechanism of prolonged activation of nitric oxide synthase.

British Journal of Pharmacology (1999) 126, 317–325; doi:10.1038/sj.bjp.0702261
The cyclo‐oxygenase‐dependent regulation of rabbit vein contraction: evidence for a prostaglandin E2‐mediated relaxation
Authors: Rouaud Céline , Delaforge Marcel , Anger‐Leroy Marielle , Le Filliatre Gaël , Finet Michel , Hanf Rémy
Published: January 2005

Arachidonic acid (0.01–1μM) induced relaxation of precontracted rings of rabbit saphenous vein, which was counteracted by contraction at concentrations higher than 1μM. Concentrations higher than 1μM were required to induce dose‐dependent contraction of vena cava and thoracic aorta from the same animals.

Pretreatment with a TP receptor antagonist (GR32191B or SQ29548, 3μM) potentiated the relaxant effect in the saphenous vein, revealed a vasorelaxant component in the vena cava response and did not affect the response of the aorta.

Removal of the endothelium from the venous rings, caused a 10 fold rightward shift in the concentration‐relaxation curves to arachidonic acid. Whether or not the endothelium was present, the arachidonic acid‐induced relaxations were prevented by indomethacin (10μM) pretreatment.

In the saphenous vein, PGE2 was respectively a 50 and 100 fold more potent relaxant prostaglandin than PGI2 and PGD2. Pretreatment with the EP4 receptor antagonist, AH23848B, shifted the concentration‐relaxation curves of this tissue to arachidonic acid in a dose‐dependent manner.

In the presence of 1μM arachidonic acid, venous rings produced 8–10 fold more PGE2 than did aorta whereas 6keto‐PGF1α and TXB2 productions remained comparable.

Intact rings of saphenous vein relaxed in response to A23187. Pretreatment with L‐NAME (100μM) or indomethacin (10μM) reduced this response by 50% whereas concomitant pretreatment totally suppressed it. After endothelium removal, the remaining relaxing response to A23187 was prevented by indomethacin but not affected by L‐NAME.

We conclude that stimulation of the cyclo‐oxygenase pathway by arachidonic acid induced endothelium‐dependent, PGE2/EP4 mediated relaxation of the rabbit saphenous vein. This process might participate in the A23187‐induced relaxation of the saphenous vein and account for a relaxing component in the response of the vena cava to arachidonic acid. It was not observed in thoracic aorta because of the lack of a vasodilatory receptor and/or the poorer ability of this tissue than veins to produce PGE2.

British Journal of Pharmacology (1999) 126, 35–44; doi:10.1038/sj.bjp.0702265
Enantioselective inhibition of the biotransformation and pharmacological actions of isoidide dinitrate by diphenyleneiodonium sulphate
Authors: Ratz Jodan D , McGuire John J , Bennett Brian M
Published: January 2005

We have shown previously that the D‐ and L‐ enantiomers of isoidide dinitrate (D‐IIDN and L‐IIDN) exhibit a potency difference for relaxation and cyclic GMP accumulation in isolated rat aorta and that this is related to preferential biotransformation of the more potent enantiomer (D‐IIDN). The objective of the current study was to examine the effect of the flavoprotein inhibitor, diphenyleneiodonium sulphate (DPI), on the enantioselectivity of IIDN action.

In isolated rat aortic strip preparations, exposure to 0.3 μM DPI resulted in a 3.6 fold increase in the EC50 value for D‐IIDN‐induced relaxation, but had no effect on L‐IIDN‐induced relaxation.

Incubation of aortic strips with 2 μM D‐ or L‐IIDN for 5 min resulted in significantly more D‐isoidide mononitrate formed (5.0±1.5 pmol mg  protein−1) than L‐isoidide mononitrate (2.1±0.7 pmol mg protein−1) and this difference was abolished by pretreatment of tissues with 0.3 μM DPI. DPI had no effect on glutathione S‐transferase (GST) activity or GSH‐dependent biotransformation of D‐ or L‐IIDN in the 105,000×g supernatant fraction of rat aorta.

Consistent with both the relaxation and biotransformation data, treatment of tissues with 0.3 μM DPI significantly inhibited D‐IIDN‐induced cyclic GMP accumulation, but had no effect on L‐IIDN‐induced cyclic GMP accumulation.

In the intact animal, 2 mg kg−1 DPI significantly inhibited the pharmacokinetic and haemodynamic properties of D‐IIDN, but had no effect L‐IIDN.

These data suggest that the basis for the potency difference for relaxation by the two enantiomers is preferential biotransformation of D‐IIDN to NO, by an enzyme that is inhibited by DPI. Given that DPI binds to and inhibits NADPH‐cytochrome P450 reductase, the data are consistent with a role for the cytochromes P450‐NADPH‐cytochrome P450 reductase system in this enantioselective biotransformation process.

British Journal of Pharmacology (1999) 126, 61–68; doi:10.1038/sj.bjp.0702268
Proliferative effects of cholecystokinin in GH3 pituitary cells mediated by CCK2 receptors and potentiated by insulin
Authors: Smith Alison J , McKernan Ruth M
Published: January 2005

Proliferative effects of CCK peptides have been examined in rat anterior pituitary GH3 cells, which express CCK2 receptors.

CCK‐8s, gastrin(1–17) and its glycine‐extended precursor G(1–17)‐Gly, previously reported to cause proliferation via putative novel sites on AR4‐2J and Swiss 3T3 cells, elicited significant dose dependent increases of similar magnitude in [3H]thymidine incorporation over 3 days in serum‐free medium of 39±10% (P<0.01, n=20), 37±8% (P<0.01, n=27) and 41±6% (P<0.01, n=36) respectively.

CCK‐8s and gastrin potentially stimulated mitogenesis (EC50 values 0.12 nM and 3.0 nM respectively), whilst G‐Gly displayed similar efficacy but markedly lower potency. L‐365,260 consistently blocked each peptide. The CCK2 receptor affinity of G‐Gly in GH3 cells was 1.09 μM (1.01;1.17, n=6) and 5.53 μM (3.71;5.99, n=4) in guinea‐pig cortex.

1 μM G‐Gly weakly stimulated Ca2+ increase, eliciting a 104±21% increase over basal Ca2+ levels, and was blocked by 1 μM L‐365,260 whilst CCK‐8s (100 nM) produced a much larger Ca2+ response (331±14%).

Insulin dose dependently enhanced proliferative effects of CCK‐8s with a maximal leftwards shift of the CCK‐8s curve at 100 ng ml−1 (17 nM) (EC50 decreased 500 fold, from 0.1 nM to 0.2 pM; P<0.0001). 10 μg ml−1 insulin was supramaximal reducing the EC50 to 5 pM (P=0.027) whilst 1 ng ml−1 insulin was ineffective. Insulin weakly displaced [125I]BHCCK binding to GH3 CCK2 receptors (IC50 3.6 μM).

Results are consistent with mediation of G‐Gly effects via CCK2 receptors in GH3 cells and reinforce the role of CCK2 receptors in control of cell growth. Effects of insulin in enhancing CCK proliferative potency may suggest that CCK2 and insulin receptors converge on common intracellular targets and indicates that mitogenic stimuli are influenced by the combination of extracellular factors present.

British Journal of Pharmacology (1999) 126, 79–86; doi:10.1038/sj.bjp.0702271
Nitric oxide limits the eicosanoid‐dependent bronchoconstriction and hypotension induced by endothelin‐1 in the guinea‐pig
Authors: Lewis Karina , Cadieux Alain , Rae Giles A , Gratton Jean‐Philippe , D'Orléans‐Juste Pedro
Published: January 2005

This study attempts to investigate if endogenous nitric oxide (NO) can modulate the eicosanoid‐releasing properties of intravenously administered endothelin‐1 (ET‐1) in the pulmonary and circulatory systems in the guinea‐pig.

The nitric oxide synthase blocker Nω‐nitro‐L‐arginine methyl ester (L‐NAME; 300 μM; 30 min infusion) potentiated, in an L‐arginine sensitive fashion, the release of thromboxane A2 (TxA2) stimulated by ET‐1, the selective ETB receptor agonist IRL 1620 (Suc‐[Glu9,Ala11,15]‐ET‐1(8–21)) or bradykinin (BK) (5, 50 and 50 nM, respectively, 3 min infusion) in guinea‐pig isolated and perfused lungs.

In anaesthetized and ventilated guinea‐pigs intravenous injection of ET‐1 (0.1–1.0 nmol kg−1), IRL 1620 (0.2–1.6 nmol kg−1), BK (1.0–10.0 nmol kg−1) or U 46619 (0.2–5.7 nmol kg−1) each induced dose‐dependent increases in pulmonary insufflation pressure (PIP). Pretreatment with L‐NAME (5 mg kg−1) did not change basal PIP, but increased, in L‐arginine sensitive manner, the magnitude of the PIP increases (in both amplitude and duration) triggered by each of the peptides (at 0.25, 0.4 and 1.0 nmol kg−1, respectively), without modifying bronchoconstriction caused by U 46619 (0.57 nmol kg−1).

The increases in PIP induced by ET‐1, IRL 1620 (0.25 and 0.4 nmol kg−1, respectively) or U 46619 (0.57 nmol kg−1) were accompanied by rapid and transient increases of mean arterial blood pressure (MAP). Pretreatment with L‐NAME (5 mg kg−1; i.v. raised basal MAP persistently and, under this condition, subsequent administration of ET‐1 or IRL 1620, but not of U‐46619, induced hypotensive responses which were prevented by pretreatment with the cyclo‐oxygenase inhibitor indomethacin.

Thus, endogenous NO appears to modulate ET‐1‐induced bronchoconstriction and pressor effects in the guinea‐pig by limiting the peptide's ability to induce, possibly via ETB receptors, the release of TxA2 in the lungs and of vasodilatory prostanoids in the systemic circulation. Furthermore, it would seem that these eicosanoid‐dependent actions of ET‐1 in the pulmonary system and on systemic arterial resistance in this species are physiologically dissociated.

British Journal of Pharmacology (1999) 126, 93–102; doi:10.1038/sj.bjp.0702274