bluuebunny/crossref_metadata_2025 huggingface dataset:
Estimation of solute transport and storage parameters in a stream with anthropogenically produced unsteady flow and industrial bromide input
Authors: Ryan Robert J. , Packman Aaron I. , Welty Claire
Published: January 2004

We used an anthropogenic tracer signal to evaluate downstream solute transport and storage in Valley Creek, a 60 km2 watershed stream near Philadelphia, Pennsylvania, that is subject to urbanization and anthropogenically induced unsteady flow. Bromide‐bearing groundwater from an abandoned mineral processing plant enters the upstream reach of Valley Creek through a series of well‐defined seeps and springs, producing a steady and significant concentration of bromide in Valley Creek. In addition, at the time of the study, a quarry located near the center of the watershed discharged accumulated groundwater seepage to the stream on a cyclical basis. The quarry discharge reduced the bromide concentration in the main stream and produced a periodicity in the in‐stream bromide concentration downstream of the quarry. We used these variations in the ambient bromide concentration to assess solute mixing and transport in Valley Creek. We applied the USGS code OTIS to analyze solute advection, dispersion, transient storage, and groundwater inflow over a 7.5 km stream reach. To apply OTIS for unsteady flow conditions, we independently modeled the variation of stream conditions during a flow cycle and then simulated solute transport using temporal and reach average values of the longitudinal dispersion coefficient (D), transient storage exchange rate (α), and transient storage area (As/A). Calibrated values of D ranged from 0.5 m2 s−1 to 1.4 m2 s−1, As/A ranged from 0.03 to 1.3, and α ranged from 8.7 × 10−6 s−1 to 1.0 × 10−2 s−1. Observed temporal variability in the in‐stream bromide concentration was simulated well. The observed transient storage parameters reflect differences in the structure of the three test reaches. Solute storage in the middle reach was dominated by in‐stream storage produced by relict hydraulic structures, while solute transport in the other two reaches reflected differences in bed sediment characteristics between upstream and downstream reaches. However, overparameterization of the model and high sensitivity to the dispersion coefficient made it difficult to assess the magnitude of transient storage or hyporheic exchange.
Spatial and temporal stream temperature prediction: Modeling nonstationary temporal covariance structures
Authors: Gardner Beth , Sullivan Patrick J.
Published: January 2004

Stream temperature is a measure of water quality and directly influences both the biotic and abiotic dynamics within the aquatic system. Because of its importance, there is a need to find better methods of monitoring and modeling stream temperatures. Gardner et al. [2003] derived a networked geostatistical model to explain the spatial patterns of stream temperatures based on 72 temperature loggers recording stream temperatures hourly throughout the Beaverkill watershed network, New York, for a single time period. Because temperatures and temperature relationships between stream sections change over the season, the covariance structure of the system is likely also to change with time. The covariance structure of stream temperature data collected throughout the month of July was examined and found to be nonstationary temporally. However, the observed changes in the covariance structure were found to be highly dependent on main stem stream temperatures over time. Five nested correlation models were created and compared using Mallow's Cp. One model representing large‐scale variation in the sill as a linear function of the main stem temperature for each day was selected as the most parsimonious model.
Activation of group III metabotropic glutamate receptors in selected regions of the basal ganglia alleviates akinesia in the reserpine‐treated rat
Authors: MacInnes Nicholas , Messenger Marcus J , Duty Susan
Published: January 2004

This study examined whether group III metabotropic glutamate (mGlu) receptor agonists injected into the globus pallidus (GP), substantia nigra pars reticulata (SNr) or intracerebroventricularly (i.c.v.) could reverse reserpine‐induced akinesia in the rat.

Male Sprague–Dawley rats, cannulated above the GP, SNr or third ventricle, were rendered akinetic with reserpine (5 mg kg−1 s.c.). 18 h later, behavioural effects of the group III mGlu receptor agonists L‐serine‐O‐phosphate (L‐SOP) or L‐(+)‐2‐amino‐4‐phosphonobutyric acid (L‐AP4) were examined.

In reserpine‐treated rats, unilateral injection of L‐SOP (2000 and 2500 nmol in 2.5 μl) into the GP produced a significant increase in net contraversive rotations compared to vehicle, reaching a maximum of 83±21 rotations 120 min−1 (n=8). Pretreatment with the group III mGlu receptor antagonist methyl‐serine‐O‐phosphate (M‐SOP; 250 nmol in 2.5 μl) inhibited the response to L‐SOP (2000 nmol) by 77%.

Unilateral injection of L‐SOP (250‐1000 nmol in 2.5 μl) into the SNr of reserpine‐treated rats produced a dose‐dependent increase in net contraversive rotations, reaching a maximum of 47±6 rotations 30 min−1 (n=6). M‐SOP (50 nmol in 2.5 μl) inhibited the response to L‐SOP (500 nmol) by 78%.

Following i.c.v. injection, L‐SOP (2000–2500 nmol in 2.5 μl) or L‐AP4 (0.5–100 nmol in 2 μl) produced a dose‐dependent reversal of akinesia, attaining a maximum of 45±17 (n=8) and 72±3 (n=9) arbitrary locomotor units 30 min−1, respectively.

These studies indicate that injection of group III mGlu receptor agonists into the GP, SNr or cerebral ventricles reverses reserpine‐induced akinesia, the mechanism for which remains to be established.

British Journal of Pharmacology (2004) 141, 15–22. doi:10.1038/sj.bjp.0705566
Pharmacological characterisation of a rat model of incisional pain
Authors: Whiteside Garth T , Harrison James , Boulet Jamie , Mark Lilly , Pearson Michelle , Gottshall Susan , Walker Katharine
Published: January 2004

Both clinical and preclinical models of postsurgical pain are being used more frequently in the early evaluation of new chemical entities. In order to assess the validity and reliability of a rat model of postincisional pain, the effects of different classes of clinically effective analgesic drugs were evaluated against multiple behavioural end points.

Following surgical incision, under general anaesthesia, of the plantar surface of the rat hind paw, we determined the time course of mechanical hyperalgesia, tactile allodynia and hind limb weight bearing using the Randall–Selitto (paw pressure) assay, electronic von Frey and dual channel weight averager, respectively. Behavioural evaluations began 24 h following surgery, and were continued for 9–14 days.

Mechanical hyperalgesia, tactile allodynia and a decrease in weight bearing were present on the affected limb within 1 day of surgery with maximum sensitivity 1–3 days postsurgery. Accordingly, we examined the effect of nonsteroidal antiinflammatory drugs (NSAIDs), morphine and gabapentin, on established hyperalgesia and allodynia, 1 day following plantar incision.

In accordance with previous reports, both systemic morphine and gabapentin administration reversed mechanical hyperalgesia and tactile allodynia in the incised rat hind paw. Both drugs were more potent against mechanical hyperalgesia than tactile allodynia.

All of the NSAIDs tested, including cyclooxygenase 2 selective inhibitors, reversed mechanical hyperalgesia and tactile allodynia in the incised rat hind paw. The rank order of potency for both hyperalgesia and allodynia was indomethacin > celecoxib > etoricoxib > naproxen.

We have investigated the potency and efficacy of different classes of analgesic drugs in a rat model of postincisional pain. The rank order of potency for these drugs reflects their utility in treating postoperative pain in the clinic. As these compounds showed reliable efficacy across two different behavioural end points, the Randall–Selitto (paw pressure) assay and electronic von Frey, these methods may prove useful in the study of postsurgical pain and the assessment of novel treatments.

British Journal of Pharmacology (2004) 141, 85–91. doi:10.1038/sj.bjp.0705568
Autoradiographic distribution of 5‐HT7 receptors in the human brain using [3H]mesulergine: comparison to other mammalian species
Authors: Martín‐Cora Francisco J , Pazos Angel
Published: January 2004

The main aim of this investigation was to delineate the distribution of the 5‐HT7 receptor in human brain. Autoradiographic studies in guinea‐pig and rat brain were also carried out in order to revisit and compare the anatomical distribution of 5‐HT7 receptors in different mammalian species.

Binding studies were performed in rat frontal cortex membranes using 10 nM [3H]mesulergine in the presence of raclopride (10 μM) and DOI (0.8 μM). Under these conditions, a binding site with pharmacological characteristics consistent with those of the 5‐HT7 receptors was identified (rank order of binding affinity values: 5‐CT>5‐HT>5‐MeOT>mesulergine ∼methiothepin>8‐OH‐DPAT=spiperone ∼(+)‐butaclamol≫imipramine ∼(±)‐pindolol≫ondansetron ∼clonidine ∼prazosin).

The autoradiographic studies revealed that the anatomical distribution of 5‐HT7 receptors throughout the human brain was heterogenous. High densities were found over the caudate and putamen nuclei, the pyramidal layer of the CA2 field of the hippocampus, the centromedial thalamic nucleus, and the dorsal raphe nucleus. The inner layer of the frontal cortex, the dentate gyrus of the hippocampus, the subthalamic nucleus and superior colliculus, among others, presented intermediate concentrations of 5‐HT7 receptors. A similar brain anatomical distribution of 5‐HT7 receptors was observed in all three mammalian species studied.

By using [3H]mesulergine, we have mapped for the first time the anatomical distribution of 5‐HT7 receptors in the human brain, overcoming the limitations previously found in radiometric studies with other radioligands, and also revisiting the distribution in guinea‐pig and rat brain.

British Journal of Pharmacology (2004) 141, 92–104. doi:10.1038/sj.bjp.0705576
The superoxide dismutase mimetic, tempol, blunts right ventricular hypertrophy in chronic hypoxic rats
Authors: Elmedal Britt , De Dam Mette Y , Mulvany Michael John , Simonsen Ulf
Published: January 2004

The purpose of this study was to investigate whether a membrane‐permeable superoxide dismutase mimetic, tempol, added either alone or in combination with the nitric oxide (NO) donor molsidomine, prevents the development of pulmonary hypertension (PH) in chronic hypoxic rats.

Chronic hypobaric hypoxia (10% oxygen) for 2 weeks increased the right ventricular systolic pressure (RVSP), right ventricle and lung wet weight. Relaxations evoked by acetylcholine (ACh) and the molsidomine metabolite SIN‐1 were impaired in isolated proximal, but not distal pulmonary arteries, from chronic hypoxic rats.

Treatment with tempol (86 mg kg−1 day−1 in drinking water) normalized RVSP and reduced right ventricular hypertrophy, while systemic blood pressure, lung and liver weights, and blunted ACh relaxation of pulmonary arteries were unchanged.

Treatment with molsidomine (15 mg kg−1 day−1 in drinking water) had the same effects as tempol, except that liver weight was reduced, and potassium and U46619‐evoked vasoconstrictions in pulmonary arteries were increased. Combining tempol and molsidomine did not have additional effects compared to tempol alone. ACh relaxation in pulmonary arteries was not normalized by these treatments.

The media to lumen diameter ratio of the pulmonary arteries was greater for the hypoxic rats compared to the normoxic rats, and was not reversed by treatment with tempol, molsidomine, or the combination of tempol and molsidomine.

We conclude that tempol, like molsidomine, is able to correct RVSP and reduce right ventricular weight in the rat hypoxic model. Functional and structural properties of pulmonary small arteries were little affected. The results support the possibility that superoxide dismutase mimetics may be a useful means for the treatment of PH.

British Journal of Pharmacology (2004) 141, 105–113. doi:10.1038/sj.bjp.0705580
Haemodynamic effects of the selective phosphodiesterase 5 inhibitor, UK‐357,903, in conscious SHR
Authors: Gardiner Sheila M , March Julie E , Kemp Philip A , Ballard Stephen A , Hawkeswood Ed , Hughes Bernadette , Bennett Terence
Published: January 2004

Regional haemodynamic responses to a continuous, 4‐day infusion of the selective phosphodiesterase type 5 inhibitor, UK‐357,903 (0.133 or 1.33 mg kg−1 h−1) were measured in conscious spontaneously hypertensive rats, and compared with those of enalapril (1 mg kg−1 h−1).

Both doses of UK‐357,903 caused modest reductions in mean blood pressure that were not dose‐dependent and only significantly different from the vehicle effects on Day 1 of the study (mean −11.8 and −15.3 mmHg for low and high doses, respectively). UK‐357,903 had mesenteric and hindquarters vasodilator effects, which were, again, similar for both dose levels and only significantly different from vehicle on Day 1. Neither dose of UK‐357,903 affected renal vascular conductance or heart rate.

Although the haemodynamic effects of UK‐357,903 were not clearly dose‐related and some appeared to wane with time, geometric mean plasma levels of UK‐357,903 increased in proportion to dose, and were sustained throughout the infusion period. Furthermore, plasma cyclic guanosine monophosphate, a biomarker of phosphodiesterase 5 inhibition, was persistently elevated, and increased with increasing dose.

Enalapril caused a fall in mean blood pressure on day 1 (−14.1 mmHg) that was associated with dilatation in renal, mesenteric and hindquarters vascular beds. The haemodynamic effects of enalapril were sustained or increased over the 4‐day infusion, although plasma free drug levels were stable.

In conclusion, we have shown regional and temporal changes in the haemodynamic effects of UK‐357,903, which may be due to activation of compensatory mechanisms, but there were no signs of functional compensation to the cardiovascular effects of enalapril.

British Journal of Pharmacology (2004) 141, 114–122. doi:10.1038/sj.bjp.0705581
Heterogeneity of neuronal and smooth muscle receptors involved in the VIP‐ and PACAP‐induced relaxations of the pig intravesical ureter
Authors: Hernández Medardo , Barahona María Victoria , Recio Paz , Rivera Luis , Benedito Sara , Martínez Ana Cristina , García‐Sacristán Albino , Orensanz Luis M , Prieto Dolores
Published: January 2004

The mechanisms and receptors involved in the vasoactive intestinal peptide (VIP)‐ and pituitary adenylate cyclase‐activating polypeptide (PACAP)‐induced relaxations of the pig intravesical ureter were investigated.

VIP, PACAP 38 and PACAP 27 concentration‐dependently relaxed U46619‐contracted ureteral strips with a similar potency. [Ala11,22,28]‐VIP, a VPAC1 agonist, showed inconsistent relaxations.

The neuronal voltage‐gated Ca2+ channel inhibitor, ω‐conotoxin GVIA (ω‐CgTX, 1 μM), reduced the VIP relaxations. Urothelium removal or blockade of capsaicin‐sensitive primary afferents, nitric oxide (NO) synthase and guanylate cyclase with capsaicin (10 μM), NG‐nitro‐L‐arginine (L‐NOARG, 100 μM) and 1H‐[1,2,4]‐oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 5 μM), respectively, did not change the VIP relaxations. However, the PACAP 38 relaxations were reduced by ω‐CgTX, capsaicin, L‐NOARG and ODQ.

The VIP and VIP/PACAP receptor antagonists, [Lys1, Pro2,5, Arg3,4, Tyr6]‐VIP (1 μM) and PACAP (6–38) (0.4 μM), inhibited VIP and VIP and PACAP 38, respectively, relaxations.

The nonselective and large‐conductance Ca2‐activated K+ channel blockers, tetraethylammonium (3 mM) and charybdotoxin (0.1 μM), respectively, and neuropeptide Y (0.1 μM) did not modify the VIP relaxations. The small‐conductance Ca2‐activated K+ channel blocker apamin (1 μM) did not change the PACAP 27 relaxations.

The cAMP‐dependent protein kinase A (PKA) blocker, 8‐(4‐chlorophenylthio)adenosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐CPT‐cAMPS, 100 μM), reduced VIP relaxations. The phosphodiesterase 4 inhibitor rolipram and the adenylate cyclase activator forskolin relaxed ureteral preparations. The rolipram relaxations were reduced by Rp‐8‐CPT‐cAMPS. Forskolin (30 nM) evoked a potentiation of VIP relaxations.

These results suggest that VIP and PACAP relax the pig ureter through smooth muscle receptors, probably of the VPAC2 subtype, linked to a cAMP‐PKA pathway. Neuronal VPAC receptors localized at motor nerves and PAC1 receptors placed at sensory nerves and coupled to NO release, seem also to be involved in the VIP and PACAP 38 relaxations.

British Journal of Pharmacology (2004) 141, 123–131. doi:10.1038/sj.bjp.0705582
Evidence in locomotion test for the functional heterogeneity of ORL‐1 receptors
Authors: Kuzmin Alexander , Sandin Johan , Terenius Lars , Ögren Sven Ove
Published: January 2004

The ORL1 agonists nociceptin and Ro 64‐6198 were compared in their ability to modify spontaneous locomotor activity in male NMRI mice not habituated to the test environment.

Higher doses of nociceptin (>5 nmol i.c.v.) reduced whereas lower doses (<1 nmol i.c.v.) stimulated locomotor activity. Both effects were blocked by the putative ORL1 antagonists [NPhe1]nociceptin(1–13)NH2 (10 nmol i.c.v.) and UFP101 (10 nmol, i.c.v.). The effects were also blocked by naloxone benzoylhydrazone (1 mg kg−1 s.c.), but not by the nonselective opioid antagonist naloxone (1 mg kg−1 s.c.).

In contrast to nociceptin, the synthetic ORL1 agonist Ro 64‐6198 (0.01–1.0 mg kg−1 i.p.) produced monophasic inhibition of locomotor activity, which was insensitive to the treatment with [NPhe1]nociceptin(1–13)NH2 or naloxone benzoylhydrazone. Treatment with UFP101 abolished the locomotor inhibition induced by Ro 64‐6198 (1.0 mg kg−1), whereas naloxone (1.0 mg kg−1, s.c.) further increased the locomotor‐inhibitory effects.

Naloxone benzoylhydrazone (0.3; 1.0 and 3.0 mg kg−1 s.c.) increased locomotor activity, although the effect was statistically significant only with the highest dose used.

Pretreatment with the tyrosine hydroxylase inhibitor H44‐68 totally eliminated the motor‐stimulatory effects of low doses of nociceptin, probably via dopamine depletion.

The results suggest that nociceptin stimulates locomotor activity at low doses if dopamine activity is intact. High doses of nociceptin and all the tested doses of Ro 64‐6198 seem to interact with a functionally different subset of ORL1 receptors. In addition, the effects of Ro 64‐6198 are modulated by tonic opioid receptor activity.

British Journal of Pharmacology (2004) 141, 132–140. doi:10.1038/sj.bjp.0705583
Phospholipase C, but not InsP3 or DAG, ‐dependent activation of the muscarinic receptor‐operated cation current in guinea‐pig ileal smooth muscle cells
Authors: Zholos Alexander V , Tsytsyura Yaroslav D , Gordienko Dmitri V , Tsvilovskyy Vladimir V , Bolton Tom B
Published: January 2004

In visceral smooth muscles, both M2 and M3 muscarinic receptor subtypes are found, and produce two major metabolic effects: adenylyl cyclase inhibition and PLCβ activation. Thus, we studied their relevance for muscarinic cationic current (mICAT) generation, which underlies cholinergic excitation. Experiments were performed on single guinea‐pig ileal cells using patch‐clamp recording techniques under conditions of weakly buffered [Ca2+]i (either using 50 μM EGTA or 50–100 μM fluo‐3 for confocal fluorescence imaging) or with [Ca2+]i ‘clamped’ at 100 nM using 10 mM BAPTA/CaCl2 mixture.

Using a cAMP‐elevating agent (1 μM isoproterenol) or a membrane‐permeable cAMP analog (10 μM 8‐Br‐cAMP), we found no evidence for mICAT modulation through a cAMP/PKA pathway.

With low [Ca2+]i buffering, the PLC blocker U‐73122 at 2.5 μM almost abolished mICAT, in some cases without any significant effect on [Ca2+]i. When [Ca2+]i was buffered at 100 nM, U‐73122 reduced both carbachol‐ and GTPγS‐induced mICAT maximal conductances (IC50=0.5–0.6 μM) and shifted their activation curves positively.

U‐73343, a weak PLC blocker, had no effect on GTPγS‐induced mICAT, but weakly inhibited carbachol‐induced current, possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to 1 mM. Aristolochic acid and D‐609, which inhibit PLA2 and phosphatidylcholine‐specific PLC, respectively, had no or very small effects on mICAT, suggesting that these enzymes were not involved.

InsP3 (1 μM) in the pipette or OAG (20 μM) applied externally had no effect on mICAT or its inhibition by U‐73122. Ca2+ store depletion (evoked by InsP3, or by combined cyclopiazonic acid, ryanodine and caffeine treatment) did not induce any significant current, and had no effect on mICAT in response to carbachol when [Ca2+]i was strongly buffered to 100 nM.

It is concluded that phosphatidylinositol‐specific PLC modulates mICAT via Ca2+ release, but also does so independently of InsP3, DAG, Ca2+ store depletion or a rise of [Ca2+]i. Our present results explain the previously established ‘permissive’ role of the M3 receptor subtype in mICAT generation, and provide a new insight into the molecular mechanisms underlying the shifts of the cationic conductance activation curve.

British Journal of Pharmacology (2004) 141, 23–36. doi:10.1038/sj.bjp.0705584
Peroxisome proliferator activated‐receptor agonism and left ventricular remodeling in mice with chronic myocardial infarction
Authors: Frantz Stefan , Hu Kai , Widder Julian , Bayer Barbara , Witzel Catharina Clara , Schmidt Isabel , Galuppo Paolo , Strotmann Jörg , Ertl Georg , Bauersachs Johann
Published: January 2004

Peroxisome proliferator activated receptor γ (PPARγ) has been implicated in several cellular pathways assumed to beneficially affect heart failure progression. In contrast, population‐based studies demonstrate an increased incidence of heart failure in patients treated with PPARγ agonists. Therefore, we examined the effect of pioglitazone, a PPARγ agonist, on chronic left ventricular remodeling after experimental myocardial infarction (MI) in mice.

Mice were treated with placebo or pioglitazone (20 mg kg−1 by gavage) from week 1 to week 6 after ligation of the left anterior descending artery. Serial transthoracic echocardiography was performed at weeks 1, 3, and 6.

Over 6 weeks, there was no difference in mortality (placebo 12%, pioglitazone 10%). Echocardiography showed significant left ventricular dilatation in animals with MI (week 6, end‐systolic area, placebo sham 9.6±1.3 vs placebo MI 14.4±2.5 mm2). However, there was no difference between the placebo and pioglitazone groups (week 6, end‐systolic area, pioglitazone MI 14.8±2.9 mm2, P=NS vs placebo).

Moreover, there were no changes in metabolic parameters, inflammation, and collagen deposition. Endothelial function in the aorta was not changed by PPARγ activation.

In conclusion, PPARγ activation did not adversely affect left ventricular remodeling and survival in mice with chronic MI. However, we were also not able to identify a protective effect of pioglitazone.

British Journal of Pharmacology (2004) 141, 9–14. doi:10.1038/sj.bjp.0705585
Ligands of the peroxisome proliferator‐activated receptor‐γ and heart failure
Authors: Thiemermann C
Published: January 2004

Peroxisome proliferator‐activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand‐activated transcription factors that are related to retinoid, steroid and thyroid hormone receptors. The PPAR subfamily comprises of three members, PPAR‐α, PPAR‐β and PPAR‐γ. There is good evidence that ligands of PPAR‐γ, including certain thiazolinediones, reduce myocardial tissue injury and infarct size. The use of PPAR‐γ agonists in the treatment of heart failure is, however, controversial.

British Journal of Pharmacology (2004) 141, 1–3. doi:10.1038/sj.bjp.0705586
Repeated treatment with the synthetic cannabinoid WIN 55,212‐2 reduces both hyperalgesia and production of pronociceptive mediators in a rat model of neuropathic pain
Authors: Costa B , Colleoni M , Conti S , Trovato A E , Bianchi M , Sotgiu M L , Giagnoni G
Published: January 2004

The antinociceptive properties of cannabinoids in persistent pain are not fully elucidated. We investigated the effect of repeated treatment with the synthetic cannabinoid receptor agonist WIN 55,212‐2 on the neuropathic pain induced in rats by chronic constriction of the sciatic nerve. WIN 55,212‐2 administered daily throughout the development of neuropathy reversed the hyperalgesia, at a dose (0.1 mg kg−1, s.c.) that had no effect on the nociceptive responses of either paw contralateral to the sciatic ligation or of animals subjected to sham surgery. At 14 days after injury, the levels of mediators known to be involved in neuropathic pain, such as prostaglandin E2, NO and the neuronal NOS, were increased. Repeated treatment with WIN 55,212‐2 abolished these increases. In the light of the current clinical need for neuropathic pain treatments, these findings indicate that cannabinoid agonists, at doses devoid of psychoactive effects, could constitute important compounds for the development of new analgesics.

British Journal of Pharmacology (2004) 141, 4–8. doi:10.1038/sj.bjp.0705587
Adenine nucleotides inhibit recombinant N‐type calcium channels via G protein‐coupled mechanisms in HEK 293 cells; involvement of the P2Y13 receptor‐type
Authors: Wirkner Kerstin , Schweigel Joana , Gerevich Zoltan , Franke Heike , Allgaier Clemens , Barsoumian Edward Leon , Draheim Henning , Illes Peter
Published: January 2004

N‐type Ca2+ channel modulation by an endogenous P2Y receptor was investigated by the whole‐cell patch‐clamp method in HEK 293 cells transfected with the functional rabbit N‐type calcium channel.

The current responses (ICa(N)) to depolarizing voltage steps were depressed by ATP in a concentration‐dependent manner. Inclusion of either guanosine 5′‐O‐(3‐thiodiphosphate) or pertussis toxin into the pipette solution as well as a strongly depolarizing prepulse abolished the inhibitory action of ATP.

In order to identify the P2Y receptor subtype responsible for this effect, several preferential agonists and antagonists were studied. Whereas the concentration–response curves of ADP and adenosine 5′‐O‐(2‐thiodiphosphate) indicated a higher potency of these agonists than that of ATP, α,β‐methylene ATP, UTP and UDP were considerably less active. The effect of ATP was abolished by the P2Y receptor antagonists suramin and N6‐(2‐methylthioethyl)‐2‐(3,3,3‐trifluoropropylthio)‐β,γ‐dichloromethylene‐ATP, but not by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid, 2′deoxy‐N6‐methyladenosine‐3′,5′‐diphosphate or 2‐methylthio AMP.

Using reverse transcription and polymerase chain reaction, mRNA for the P2Y1, P2Y4, P2Y6, P2Y11 and P2Y13 receptor subtypes, but not the P2Y2, and P2Y12 subtypes, was detected in HEK 293 cells.

Immunocytochemistry confirmed the presence of P2Y1, and to a minor extent that of P2Y4, but not of P2Y2 receptors.

Hence, it is tempting to speculate that P2Y13 receptors may inhibit N‐type Ca2+ channels via the βγ subunits of the activated Gi protein.

British Journal of Pharmacology (2004) 141, 141–151. doi:10.1038/sj.bjp.0705588
A truncated form of CKβ8‐1 is a potent agonist for human formyl peptide‐receptor‐like 1 receptor
Authors: Elagoz Aram , Henderson Duncan , Babu Poda Suresh , Salter Sylvia , Grahames Caroline , Bowers Lorna , Roy Marie‐Odile , Laplante Patricia , Grazzini Eric , Ahmad Sultan , Lembo Paola M C
Published: January 2004

Human formyl peptide‐receptor‐like‐1 (FPRL‐1) is a promiscuous G protein‐coupled receptor (GPCR), and belongs to a chemoattractant receptor family protein. This receptor has been reported to interact with various host‐derived peptides and lipids involved in inflammatory responses. We described here, a novel role for FPRL‐1 as a high‐affinity β‐chemokine receptor for an N‐terminally truncated form of the CKβ8 (CCL23/MPIF‐1) splice variant CKβ8‐1 (22–137 aa).

RT‐PCR analysis of mRNA derived from human tissues and cells revealed a predominant expression of FPRL‐1 in inflammatory cells, particularly in neutrophils.

Intracellular calcium mobilisation assay, used as screening tool, in recombinant Chinese hamster ovary (CHO‐K1) and human embryonic kidney (HEK293s) cells coexpressing FPRL‐1 and Gα16, demonstrated FPRL‐1 is a functional high‐affinity receptor for CKβ8‐1 (46–137 aa, sCKβ8‐1), with pEC50 values of 9.13 and 8.85, respectively.

The FPRL‐1 activation in CHO‐K1 cells is mediated by Gαi/Gαo proteins, as assessed by pertussis toxin sensitivity and inhibition of forskolin‐induced cyclic AMP accumulation.

Binding experiments were performed with a radio‐iodinated synthetic peptide, [125‐I]‐WKYMVm, a known potent FPRL‐1 agonist. CHO‐K1 cell membranes expressing FPRL‐1 bound [125‐I]‐WKYMVm with a Kd value of 9.34. Many known FPRL‐1 agonists were tested and sCKβ8‐1 was the most effective nonsynthetic ligand in displacing the radiolabelled agonist, with a pIC50 of 7.97.

The functional significance of sCKβ8‐1 interaction with FPRL‐1 was further demonstrated by the activation of polymorphonuclear leukocytes (PMNs) calcium mobilisation and chemotaxis. These interactions were shown to be via FPRL‐1 by specific blockade of PMNs activation in the presence of an FPRL‐1 antibody.

British Journal of Pharmacology (2004) 141, 37–46. doi:10.1038/sj.bjp.0705592
Changes in rectal temperature and ECoG spectral power of sensorimotor cortex elicited in conscious rabbits by i.c.v. injection of GABA, GABAA and GABAB agonists and antagonists
Authors: Frosini Maria , Valoti Massimo , Sgaragli Giampietro
Published: January 2004

In order to ascertain whether both GABAA and GABAB, or only GABAB receptors, directly modulate thermoregulation in conscious rabbits, GABAA/GABAB agonist and antagonist agents were injected intracerebroventricularly in conscious rabbits while monitoring changes in rectal temperature (RT), gross motor behaviour (GMB) and electrocorticogram (ECoG) power spectra (ps) from sensorimotor cortices.

GABA (48 μmol), nipecotic acid (50 nmol), THIP (60 nmol), muscimol (18 nmol) and baclofen (8 nmol) induced hypothermia (−ΔRTmax values of 1.70±0.1, 1.4±0.2, 1.0±0.4, 1.1±0.2 and 1.6±0.3°C, respectively), accompanied by inhibition of GMB and ECoG synchronization. THIP increased ps at δ frequency band (1.1−3.3 Hz), while GABA, nipecotic acid, muscimol and baclofen did the same at both δ and θ (4.6−6.5 Hz) frequency bands. ECoG ps changes were concomitant or even preceded hypothermia.

Bicuculline (1.8 nmol) induced hyperthermia (ΔRTmax 1.2±0.5°C) and slight excitation of GMB, while CGP35348 (1.2 μmol) did not affect RT nor GMB. Both compounds did not affect ECoG ps.

Bicuculline potentiated muscimol‐induced hypothermia, inhibition of GMB and synchronization of ECoG, while CGP35348 fully antagonized these effects.

In conclusion, the present results, while confirming the prevailing role of GABAB, also outline a direct involvement of GABAA receptors in the central mechanisms of thermoregulation. Ascending inhibition towards discrete cortical areas controlling muscular activity and thermogenesis may result from GABA receptor activation in neurones proximal to the ventricles, thus contributing to hypothermia, although hypothermia‐induced reduction of neuronal activity of these cortical areas cannot be ruled out.

British Journal of Pharmacology (2004) 141, 152–162. doi:10.1038/sj.bjp.0705593
An improved model for the binding of lidocaine and structurally related local anaesthetics to fast‐inactivated voltage‐operated sodium channels, showing evidence of cooperativity
Authors: Leuwer Martin , Haeseler Gertrud , Hecker Hartmut , Bufler Johannes , Dengler Reinhard , Aronson Jeffrey K
Published: January 2004

The interaction of lidocaine‐like local anaesthetics with voltage‐operated sodium channels is traditionally assumed to be characterized by tighter binding of the drugs to depolarized channels. As inactivated and drug‐bound channels are both unavailable on depolarization, an indirect approach is required to yield estimates for the dissociation constants from channels in inactivated states. The established model, originally described by Bean et al., describes the difference in affinity between resting and inactivated states in terms of the concentration dependence of the voltage shift in the availability curve. We have tested the hypothesis that this model, which assumes a simple Langmuir relationship, could be improved by introducing a Hill‐type exponent, which would take into account potential sources of cooperativity.

Steady‐state block by lidocaine was studied in heterologously (HEK 293) expressed human skeletal muscle sodium channels and compared with experimental data previously obtained for 2,6‐dimethylphenol, 3,5‐dimethyl‐4‐chlorophenol, and 4‐chlorophenol. Cells were clamped to membrane potentials from −150 to −5 mV, and a subsequent test pulse was used to assess the number of channels available to open.

All compounds shifted the voltage dependence of channel availability in the direction of negative prepulse potentials. Prediction of the concentration dependence of the voltage shift in the availability curve was improved by the modified model, as shown by a marked reduction in the residual sum of squares.

For all compounds, the Hill‐type exponent was significantly greater than one. These results could be interpreted in the light of the contemporary hypothesis that lidocaine functions as an allosteric gating effector to enhance sodium channel inactivation by strengthening the latch mechanism of inactivation, which is considered to be a particle‐binding process allosterically coupled to activation. Alternatively, they could be interpreted by postulating additional binding sites for lidocaine on fast‐inactivated sodium channels.

British Journal of Pharmacology (2004) 141, 47–54. doi:10.1038/sj.bjp.0705594
G‐protein receptor kinase 3 (GRK3) influences opioid analgesic tolerance but not opioid withdrawal
Authors: Terman Gregory W , Jin Wenzhen , Cheong Young‐Pyo , Lowe Janet , Caron Marc G , Lefkowitz Robert J , Chavkin Charles
Published: January 2004

Tolerance to opioids frequently follows repeated drug administration and affects the clinical utility of these analgesics. Studies in simple cellular systems have demonstrated that prolonged activation of opioid receptors produces homologous receptor desensitization by G‐protein receptor kinase mediated receptor phosphorylation and subsequent β‐arrestin binding. To define the role of this regulatory mechanism in the control of the electrophysiological and behavioral responses to opioids, we used mice having a targeted disruption of the G‐protein receptor kinase 3 (GRK3) gene.

Mice lacking GRK3 did not differ from wild‐type littermates neither in their response latencies to noxious stimuli on the hot‐plate test nor in their acute antinociceptive responses to fentanyl or morphine.

Tolerance to the electrophysiological response to the opioid fentanyl, measured in vitro in the hippocampus, was blocked by GRK3 deletion. In addition, tolerance to the antinociceptive effects of fentanyl was significantly reduced in GRK3 knockouts compared to wild‐type littermate controls.

Tolerance to the antinociceptive effects of morphine was not affected by GRK3 deletion although morphine tolerance in hippocampal slices from GRK3 knockout mice was significantly inhibited. Tolerance developed more slowly in vitro to morphine than fentanyl supporting previous work in in vitro systems showing a correlation between agonist efficacy and GRK3‐mediated desensitization.

The results of these studies suggest that GRK3‐mediated mechanisms are important components of both electrophysiologic and behavioral opioid tolerance. Fentanyl, a high efficacy opioid, more effectively produced GRK3‐dependent effects than morphine, a low efficacy agonist.

British Journal of Pharmacology (2004) 141, 55–64. doi:10.1038/sj.bjp.0705595
A nitric oxide (NO)‐releasing derivative of gabapentin, NCX 8001, alleviates neuropathic pain‐like behavior after spinal cord and peripheral nerve injury
Authors: Wu Wei‐Ping , Hao Jing‐Xia , Ongini Ennio , Impagnatiello Francesco , Presotto Cristina , Wiesenfeld‐Hallin Zsuzsanna , Xu Xiao‐Jun
Published: January 2004

Nitric oxide (NO) participates, at least in part, to the establishment and maintenance of pain after nerve injury. Therefore, drugs that target the NO/cGMP signaling pathway are of interest for the treatment of human neuropathic pain. Various compounds endowed with NO‐releasing properties modulate the expression and function of inducible nitric oxide synthase (iNOS), the key enzyme responsible for sustained NO production under pathological conditions including neuropathic pain.

With this background, we synthesized a new chemical entity, [1‐(aminomethyl)cyclohexane acetic acid 3‐(nitroxymethyl)phenyl ester] NCX8001, which has a NO‐releasing moiety bound to gabapentin, a drug currently used for the clinical management of neuropathic pain. We examined the pharmacological profile of this drug with respect to its NO‐releasing properties in vitro as well as to its efficacy in treating neuropathic pain conditions (allodynia) consequent to experimental sciatic nerve or spinal cord injuries.

NCX8001 (1–30 μM) released physiologically relevant concentrations of NO as it induced a concentration‐dependent activation of soluble guanylyl cyclase (EC50=5.6 μM) and produced consistent vasorelaxant effects in noradrenaline‐precontracted rabbit aortic rings (IC50=1.4 μM).

NCX8001, but not gabapentin, counteracted in a concentration‐dependent fashion lipopolysaccharide‐induced overexpression and function of iNOS in RAW264.7 macrophages cell line. Furthermore, NCX8001 also inhibited the release of tumor necrosis factor alpha (TNFα) from stimulated RAW264.7 cells.

NCX8001 (28–280 μmol kg−1, i.p.) reduced the allodynic responses of spinal cord injured rats in a dose‐dependent fashion while lacking sedative or motor effects. In contrast, gabapentin (170–580 μmol kg−1, i.p.) resulted less effective and elicited marked side effects.

NCX8001 alleviated the allodynia‐like responses of rats to innocuous mechanical or cold stimulation following lesion of the sciatic nerve. This effect was not shared by equimolar doses of gabapentin.

Potentially due to the slow releasing kinetics of NO, NCX8001 alleviated pain‐like behaviors in two rat models of neuropathic pain in a fashion that is superior to its parent counterpart gabapentin. This new gabapentin derivative, whose mechanism deserves to be explored further, offers new hopes to the treatment of human neuropathic pain.

British Journal of Pharmacology (2004) 141, 65–74. doi:10.1038/sj.bjp.0705596
Paradoxical early glucocorticoid induction of stem cell factor (SCF) expression in inflammatory conditions
Authors: Da Silva Carla Alexandra , Kassel Olivier , Lebouquin Renaud , Lacroix Emmanuel Jean , Frossard Nelly
Published: January 2004

Stem cell factor (SCF) is a major growth factor for mast cells, promoting their differentiation and chemotaxis. Its expression is regulated by glucocorticoids in inflammatory conditions, showing an early increased protein expression, before the expected anti‐inflammatory decrease (Da Silva et al., Br. J. Pharmacol. 2002:135,1634).

We here evaluated the early kinetic of SCF expression regulated by interleukin (IL)‐1β, budesonide and the combination of both in human lung fibroblasts in culture.

Budesonide potentiated the IL‐1β‐enhanced expression of SCF mRNA (+103%) and protein (+98%) very shortly after treatment (at 30 min and 1 h, respectively). A gentle downregulation followed. This potentiating effect of budesonide was related to increased SCF mRNA stability and SCF gene transcription.

Deletion of a κB‐like site that we identified in the first intron of the SCF gene, in a luciferase reporter system, abolished the potentiation by budesonide, as well as the effect of IL‐1β alone, as compared to the wild‐type construction activity.

All budesonide‐induced effects were glucocorticoid‐receptor dependent, since they were reproduced by dexamethasone and blocked by RU486.

IL‐1β+budesonide did not affect the relative expression of the soluble and membrane‐bound forms of SCF.

In conclusion, our results clearly show that glucocorticoids act very early to adversely increase the expression of SCF mRNA and protein in the inflammatory conditions created by IL‐1β, and that this effect involves increased mRNA stability and increased gene expression through activation of the NF‐κB‐like responsive element.

British Journal of Pharmacology (2004) 141, 75–84. doi:10.1038/sj.bjp.0705598
Desensitisation of mast cell β2‐adrenoceptor‐mediated responses by salmeterol and formoterol
Authors: Scola Anne‐Marie , Chong Lee K , Suvarna S Kim , Chess‐Williams Russell , Peachell Peter T
Published: January 2004

The long‐acting β2‐adrenoceptor agonist formoterol (10−10–10−6 M) inhibited the IgE‐dependent release of histamine from human lung mast cells in a concentration‐dependent manner. Formoterol was more potent and a full agonist relative to the nonselective β‐adrenoceptor agonist isoprenaline. By contrast, the long‐acting β2‐adrenoceptor agonist salmeterol (10−10–10−6 M) was about two‐thirds less efficacious than either formoterol or isoprenaline as an inhibitor of histamine release.

Isoprenaline, formoterol and salmeterol (all at 10−5 M) increased total cell cAMP levels in mast cells over basal by 361±90 (P<0.05), 321±89 (P<0.05) and 64±24% (P>0.05), respectively.

Long‐term (24 h) incubation of mast cells with formoterol (10−6 M) or salmeterol (10−6 M) essentially abolished the subsequent ability of isoprenaline to inhibit histamine release. Both formoterol and salmeterol were more effective at inducing the functional desensitisation than isoprenaline (10−6 M) or the short‐acting β2‐adrenoceptor agonist salbutamol (10−6 M).

The desensitisation induced by long‐term treatments with salmeterol and formoterol was specific for β2‐adrenoceptor‐mediated inhibition of histamine release as the inhibitory effects of alternative cAMP‐elevating compounds, prostaglandin E2, a receptor‐mediated activator of adenylate cyclase, and forskolin, a direct activator of adenylate cyclase, were unaffected by desensitising treatments.

Radioligand binding studies were performed to determine β2‐adrenoceptor density in cell membranes after pretreatment (24 h) of cells with agonists. Isoprenaline, formoterol and salmeterol (all at 10−6 M) reduced β2‐adrenoceptor density by 13±5 (P>0.05), 49±13 (P<0.05) and 35±17% (P>0.05), respectively.

These data indicate that long‐term exposure of mast cells to both salmeterol and formoterol can cause substantial levels of desensitisation to β2‐adrenoceptor‐mediated responses in mast cells.

British Journal of Pharmacology (2004) 141, 163–171. doi:10.1038/sj.bjp.0705599
Reactive nitrogen species scavenging, rather than nitric oxide inhibition, protects from articular cartilage damage in rat zymosan‐induced arthritis
Authors: Bezerra Mirna Marques , Brain Susan D , Greenacre Stan , Jerônimo Selma Maria Bezerra , De Melo Liana Batista , Keeble Julie , Da Rocha Francisco Airton Castro
Published: January 2004

The contribution of nitric oxide (NO) and peroxynitrite (PN) to inflammation in a zymosan‐induced (1 mg, intra‐articular, i.art.) rat model of arthritis was assessed by histopathology and by measuring the glycosaminoglycan (GAG) content of the articular cartilage.

Progression of the chronic synovitis in zymosan‐induced arthritis (ZYA) was associated with increased nitrite and nitrotyrosine (3‐NT) levels in the joint exudates that paralleled a progressive loss of the GAG content. An increase in 3‐NT was also observed after i.art. PN.

The nonselective nitric oxide synthase (NOS) inhibitor L‐NG‐nitroarginine methyl ester (25–75 mg kg−1day−1) or the selective inducible NOS inhibitor aminoguanidine (50–100 mg kg−1day−1) given 1 h before (prophylactic) or 3 days after (therapeutic) injection of the zymosan ameliorated the synovitis, but worsened the GAG loss, as measured at the end of the experiment (day 7).

The PN scavenger uric acid (100–250 mg kg−1 i.p. four times daily) given prophylactically until the end of the experiment (day 14), in a dose compatible with its PN scavenging activity, significantly decreased both the synovitis and the GAG loss.

In conclusion, PN formation is associated with cartilage damage in addition to proinflammatory activity in ZYA. NOS inhibitors and a PN scavenger were able to reduce the cellular infiltration, while displaying opposite effects on cartilage homeostasis either by enhancing or ameliorating the damage, respectively.

British Journal of Pharmacology (2004) 141, 172–182. doi:10.1038/sj.bjp.0705600
Correlation between spontaneous electrical, calcium and mechanical activity in detrusor smooth muscle of the guinea‐pig bladder
Authors: Hashitani Hikaru , Brading Alison F , Suzuki Hikaru
Published: January 2004

To investigate the cellular mechanisms underlying spontaneous excitation of smooth muscle of the guinea‐pig urinary bladder, isometric tension was measured in muscle bundles while recording the membrane potential from a cell in the bundle with a microeletrode. Changes in the intracellular calcium concentration ([Ca2+]i; calcium transients) were recorded in strips loaded with the fluorescent dye, fura‐PE3.

In 40% of preparations, individual action potentials and contractions, which were abolished by nifedipine (1 μM), were generated. In the remaining preparations, bursting action potentials and contractions were generated. Contractions were again abolished by nifedipine (1 μM), while higher concentrations of nifedipine (10–30 μM) were required to prevent the electrical activity.

Carbachol (0.1 μM) increased the frequency of action potentials and corresponding contractions. Apamin (0.1 μM) potentiated bursting activity and enhanced phasic contraction. Charybdotoxin (CTX, 50 nM) induced prolonged action potentials that generated enlarged contractions. In contrast, levcromakalim (0.1 μM) reduced the frequency of action potentials, action potential bursts and the size of the contractions.

Forskolin (0.1 μM), 8‐bromoguanosin 3′, 5′ cyclic monophosphate (8Br‐cGMP, 0.1 mM) and Y‐26763 (10 μM) suppressed contractions without reducing the amplitude of either action potentials or Ca transients.

This paper confirms that action potentials and associated calcium transients are fundamental mechanisms in generating spontaneous contractions in smooth muscles of the guinea‐pig bladder. However, in parallel with the excitation–contraction coupling, the sensitivity of the contractile proteins for Ca2+ may play an important role in regulating spontaneous excitation and can be modulated by cyclic nucleotides and Rho kinase.

British Journal of Pharmacology (2004) 141, 183–193. doi:10.1038/sj.bjp.0705602
Protein kinase‐independent activation of CFTR by phosphatidylinositol phosphates
Authors: Himmel Bettina , Nagel Georg
Published: January 2004

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed in many epithelia and in the heart. Phosphorylation of CFTR by protein kinases is thought to be an absolute prerequisite for the opening of CFTR channels. In addition, nucleoside triphosphates were shown to regulate the opening of phosphorylated CFTR. Here, we report that phosphatidylinositol 4,5‐bisphosphate (PIP2) activates human CFTR, resulting in ATP responsiveness of PIP2‐treated CFTR. PIP2 alone is not sufficient to open CFTR, but ATP opens nonphosphorylated CFTR after application of PIP2. The effect of PIP2 is independent of protein kinases, as PIP2 activates CFTR in the complete absence of Mg. Phosphatidylinositol and phosphatidylinositol monophosphate activate CFTR less efficiently than PIP2. PIP2 application to phosphorylated CFTR may inhibit the CFTR chloride current. We suggest that regulation of CFTR by PIP2 is a previously unrecognized, alternative mechanism to control chloride conductance.