bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of recombinant human orexin receptor pharmacology in a Chinese hamster ovary cell‐line using FLIPR
Authors: Smart D , Jerman J C , Brough S J , Rushton S L , Murdock P R , Jewitt F , Elshourbagy N A , Ellis C E , Middlemiss D N , Brown F
Published: January 2005

The cellular mechanisms underlying the physiological effects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca2+]i) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin‐1 (OX1) or orexin‐2 (OX2) receptors using Fluo‐3AM. Orexin‐A and orexin‐B increased [Ca2+]i in a concentration dependent manner in CHO‐OX1 (pEC50=8.03±0.08 and 7.30±0.08 respectively, n=5) and CHO‐OX2 (pEC50=8.18±0.10 and 8.43±0.09 respectively, n=5) cells. This response was typified as a rapid peak in [Ca2+]i (maximal at 6–8 s), followed by a gradually declining secondary phase. Thapsigargin (3 μM) or U73122 (3 μM) abolished the response. In calcium‐free conditions the peak response was unaffected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C‐mediated release of calcium from intracellular stores, with subsequent calcium influx.

British Journal of Pharmacology (1999) 128, 1–3; doi:10.1038/sj.bjp.0702780
Iodinated radiographic contrast media inhibit shear stress‐ and agonist‐evoked release of NO by the endothelium
Authors: Hutcheson I R , Griffith T M , Pitman M R , Towart R , Gregersen M , Refsum Helge , Karlsson J O G
Published: January 2005

We have used isolated arterial preparations from the rabbit and dog to investigate whether non‐ionic iodinated radiographic contrast media (IRCM) modulate nitric oxide (NO) release. The tri‐iodinated monomers iopromide and iohexol were compared with the hexa‐iodinated dimer iodixanol.

The vasodilator effects of iohexol (300 mg ml−1) and iodixanol (320 mg ml−1) were assessed in cascade bioassay. Increasing concentrations of iohexol or iodixanol caused concentration‐dependent relaxations of the detector tissue which were insensitive to 100 μM NG‐nitro L‐arginine methyl ester (L‐NAME) and 10 μM indomethacin, whereas viscosity‐associated relaxations induced by the ‘inert’ agent dextran (MW 80,000; 1–4%) were attenuated by inhibition of NO synthesis.

Relaxations of endothelium‐intact rings to acetylcholine (ACh) were attenuated by preincubation with iohexol or iodixanol, whereas relaxations to sodium nitroprusside (SNP) in endothelium‐denuded rings were unaffected. Inhibitory activity did not correlate with either molarity or iodine concentration. Mannitol caused inhibition of both ACh‐ and SNP‐induced responses.

In isolated perfused arteries the depressor responses to iodixanol (320 mg ml−1) and iopromide (300 mg ml−1) administered as close arterial bolus attained a plateau with maximal dilatations of ∼25% and ∼60%, respectively. Addition of 100 μM NG‐nitro L‐arginine (L‐NOARG) and/or 10 μM indomethacin to the perfusate had no effect on the responses to either agent.

We conclude that IRCM exert direct effects on the endothelium that inhibit NO production rather than its action on vascular smooth muscle. Shear stress‐induced stimulation of NO production by IRCM is unlikely to contribute to their vasodilator activity in vivo when administered during angiography despite high intrinsic viscosity.

British Journal of Pharmacology (1999) 128, 451–457; doi:10.1038/sj.bjp.0702781
Cyclosporin A and tacrolimus (FK506) suppress expression of inducible nitric oxide synthase in vitro by different mechanisms
Authors: Dusting Gregory J , Akita Kazuhiro , Hickey Haruyo , Smith Melanie , Gurevich Vladimir
Published: January 2005

The effects of the immunosuppressant drugs cyclosporin A and tacrolimus (FK506) on nitric oxide synthesis were examined in a murine macrophage cell line (J774) and rat vascular smooth muscle cells (VSMC) in culture for 24 and 48 h, respectively.

Cyclosporin A (0.01–10 μM) inhibited by up to 90% accumulation of nitrite induced by lipopolysaccharide (LPS) in both cell lines, but FK506 (0.01–10 μM) had a weaker effect on nitrite accumulation in these cells. Cyclosporin A and FK506 (at 1 μM) also significantly inhibited nitrite production induced by recombinant murine interferon‐γ (rIFNγ) and recombinant murine interleukin‐1β (rIL‐1β) in J774 and VSMC, respectively.

In J774 cells, cyclosporin A (but not FK506) at 1 μM was inhibitory when co‐incubated with the inducing agents but not when the cells were treated with the immunosuppressant before or after the inducer. In VSMC, nitrite production was inhibited by co‐incubation of cyclosporin A or FK506 with the inducer, or when the immunosuppressants were pre‐incubated with cells. In contrast, N‐monomethyl L‐arginine (NMMA) abolished nitrite production when incubated with either cell type during or after addition of inducing agent, but not if cells were preincubated with NMMA.

RNA extracted from treated J774 and VSMC was subjected to reverse transcription–polymerase chain reaction (RT–PCR). Cyclosporin A, but not FK506, suppressed expression of mRNA for NOS2 in a concentration‐dependent manner when co‐incubated with LPS.

The fact that the potency difference between cyclosporin A and FK506 for NO suppression is the opposite to that for inhibition of interleukin‐2 generation suggests that the immunosuppressants act in J774 macrophages and VSMC through intracellular mechanisms that differ from those elucidated in T‐cells. Cyclosporin A suppresses NOS2 gene transcription, but FK506 acts post‐transcriptionally to suppress NO generation in VSMC.

Taken together the present data suggest that therapeutic concentrations of cyclosporin A, but not FK506, might well suppress NO production, but FK506 would not have this effect. Suppression of NO might contribute to the side effects of hypertension and nephrotoxicity associated with long‐term use of cyclosporin A to prevent transplant rejection.

British Journal of Pharmacology (1999) 128, 337–344; doi:10.1038/sj.bjp.0702782
Stimulation of bradykinin B2‐receptors on endothelial cells induces relaxation and contraction in porcine basilar artery in vitro
Authors: Miyamoto Atsushi , Ishiguro Shigeru , Nishio Akira
Published: January 2005

The aim of the present study was to characterize the subtypes of bradykinin (BK) receptors that evoke the relaxation and contraction induced by BK and to identify the main contracting and relaxing factors in isolated porcine basilar artery by measuring changes in isometric tension and a thromboxane (TX) metabolite.

Endothelial denudation completely abolished both responses. [Thi5,8, D‐Phe7]‐BK (a B2‐receptor antagonist) inhibited the BK‐induced relaxation and contraction, whereas des‐Arg9, [Leu8]‐BK (a B1‐receptor antagonist) had no effect.

L‐nitro‐arginine (L‐NA, a nitric oxide synthase inhibitor) completely inhibited BK‐induced relaxation. Indomethacin (a cyclo‐oxygenase inhibitor) completely and ONO‐3708 (a TXA2/prostaglandin H2 receptor antagonist) partially inhibited BK‐induced contraction, whereas OKY‐046 (a TXA2 synthase inhibitor) and nordihydroguaiaretic acid (a lipoxygenase inhibitor) did not.

In the presence of L‐NA, the contractile response to BK was inhibited by indomethacin or ONO‐3708 and was competitively antagonized by [Thi5,8, D‐Phe7]‐BK (pA2=7.50). In the presence of indomethacin, the relaxant response to BK was inhibited by L‐NA and was competitively antagonized by [Thi5,8, D‐Phe7]‐BK (pA2=7.59).

TXA2 release was not induced by BK‐stimulation.

These results suggest that the endothelium‐dependent relaxation and contraction to BK in the porcine basilar artery is mediated via activation of endothelial B2‐receptors. The main relaxing factor may be NO and the main contracting factor may be prostaglandin H2.

British Journal of Pharmacology (1999) 128, 241–247; doi:10.1038/sj.bjp.0702783
Characterization of non‐adrenergic, non‐cholinergic inhibitory responses of the isolated guinea‐pig trachea: differences between pre‐ and post‐ganglionic nerve stimulation
Authors: Moffatt James D , Dumsday Bryan , McLean John R
Published: January 2005

Differences in the mechanism of non‐adrenergic, non‐cholinergic (NANC) inhibitory responses to preganglionic‐ and post‐ganglionic nerve stimulation were investigated in the guinea‐pig isolated trachea.

Stimulation of the vagus nerve at frequencies above 4 Hz elicited NANC relaxation of the trachealis muscle. Responses to low frequencies of stimulation (4–8 Hz) were abolished by the nitric oxide (NO) synthase inhibitor L‐NOARG (10 μM), while a L‐NOARG resistant component was observed at higher stimulus frequencies. The L‐NOARG‐resistant component of NANC inhibitory responses to higher frequencies of vagus nerve stimulation were significantly attenuated by the proteinase α‐chymotrypsin (2 U/ml), suggesting that a neuropeptide such as VIP may contribute to NANC responses.

When postganglionic nerves were stimulated by electrical field stimulation (EFS), responses were readily elicited at frequencies below 4 Hz. Like responses to vagus nerve stimulation, responses to low frequency (<4 Hz) EFS were abolished by L‐NOARG while a L‐NOARG‐resistant component was apparent at higher stimulus frequencies.

The L‐NOARG‐resistant component of NANC inhibitory responses to EFS was sensitive to α‐chymotrypsin only if stimuli were delivered in either long trains at a low frequency (4 Hz for 10–30 s) or short trains of high frequency (16 Hz for 2.5–7.5 s).

Responses to preganglionic nerve stimulation were approximately 35% of the amplitude of responses to EFS in the same preparations.

In conclusion, responses to preganglionic and postganglionic NANC inhibitory nerve stimulation in the guinea‐pig trachea differ in maximum amplitude, frequency‐response characteristics and the contributions of cotransmitters. We suggest that these differences may be explained by filtering of preganglionic input to postganglionic NANC neurons. These results have implications in all studies where EFS is considered to be representative of physiological stimulation of post‐ganglionic nerve stimulation.

British Journal of Pharmacology (1999) 128, 458–464; doi:10.1038/sj.bjp.0702786
Rat chromaffin cells lack P2X receptors while those of the guinea‐pig express a P2X receptor with novel pharmacology
Authors: Liu M , Dunn P M , King B F , Burnstock G
Published: January 2005

Whole‐cell patch‐clamp recording was used to determine the functional expression and pharmacological properties of P2X receptors in chromaffin cells dissociated from adrenal medullae of rats and guinea‐pigs.

In rat chromaffin cells maintained in culture for 1–7 days, ATP and UTP failed to evoke any detectable response.

Guinea‐pig chromaffin cells responded to ATP (100 μM) with a rapidly activating inward current. The amplitude of the response to ATP increased over the period cells were maintained in culture and so did the number of cells giving a detectable response, with 69% of cells responding after 4 days of culture.

The response to ATP desensitized slowly, and had a reversal potential of 2.5 mV. The EC50 for ATP was 43 μM. The potency order for ATP analogues was 2‐MeSATP>ATP>ADP. Adenosine, UTP and α,β‐meATP were inactive.

Suramin (100 μM) and Cibacron blue (50 μM) inhibited the ATP (100 μM)‐activated current by 51 and 47%, respectively. PPADS antagonized the response to ATP (100 μM) with an IC50 of 3.2 μM.

The ATP concentration‐response curve shifted to the left at pH 6.8 (EC50, 19 μM) and right at pH 8.0 (EC50, 96 μM), without changing the maximal response. Zn2+ inhibited the response to ATP (100 μM) with an IC50 of 48 μM.

This study indicates that expression of ATP‐gated cation channels in chromaffin cells is species dependent. The P2X receptors in guinea‐pig chromaffin cells show many characteristics of the P2X2 receptor subtype.

British Journal of Pharmacology (1999) 128, 61–68; doi:10.1038/sj.bjp.0702790
Multiple calcium channels regulate neurotransmitter release from vagus nerve terminals in the cat bronchiole
Authors: Fujisawa Kanichiro , Onoue Hitoshi , Abe Kihachiro , Ito Yushi
Published: January 2005

Twitch‐like contractions and non‐adrenergic non‐cholinergic (NANC) relaxations evoked by electrical field stimulation (EFS) of the cat bronchiole were used to examine the voltage‐activated calcium channels involved in excitatory and inhibitory neurotransmission in the cat bronchiole.

Nifedipine (50 μM), the L‐type calcium channel antagonist, did not affect the twitch‐like contraction and NANC relaxations. However, low concentrations of the N‐type calcium channel blocker ω‐conotoxin GVIA (ω‐CgTX GVIA) (0.1 μM) irreversibly abolished twitch‐like contractions evoked by trains of EFS 10 stimuli at 20 Hz.

After the prolonged treatment with 0.1 μM ω‐CgTX GVIA, EFS evoked initial fast and later slow NANC relaxations in the presence of 5‐HT (10 μM), atropine and guanethidine (1 μM each). However increased concentration of ω‐CgTX GVIA (1 μM) completely suppressed the slow NANC relaxation without affecting the initial fast component.

ω‐agatoxin IVA (100 nM), the P‐ and Q‐type calcium channel inhibitor, and nimodipine (10 μM), the L‐ and T‐type calcium channel blocker, did not affect the amplitude of the initial fast NANC relaxation in the absence or presence of ω‐CgTX GVIA (1 μM).

The contraction or relaxation induced by exogenous acetylcholine (ACh) (0.5 μM) or the nitric oxide donor, s‐nitroso‐N‐acetyl penicillamine (SNAP) (1 μM) were not affected by ω‐CgTX GVIA (1 μM).

Taken together, these results suggest that generation of twitch‐like contraction and later slow NANC relaxation are regulated by N‐type calcium channels, whereas generation of the initial fast NANC relaxation possibly involves R‐type calcium channel.

British Journal of Pharmacology (1999) 128, 262–268; doi:10.1038/sj.bjp.0702791
Modulatory activity of extracellular H+ and Zn2+ on ATP‐responses at rP2X1 and rP2X3 receptors
Authors: Wildman S S , King B F , Burnstock G
Published: January 2005

The modulatory activity of extracellular H+ and Zn2+ was examined on ATP‐responses at rat P2X1 (rP2X1) and rat P2X3 (rP2X3) receptors expressed in Xenopus oocytes and studied under voltage‐clamp conditions.

Superfused ATP (0.03–30 μM, at pH 7.5) evoked inward currents at rP2X1 receptors (EC50 value, 300±7 nM). ATP potency was reduced 2 fold at pH 6.5, and 6 fold at pH 5.5, without altering the maximum ATP effect. Alkaline conditions (pH 8.0) did not alter ATP activity.

Superfused ATP (0.01–300 μM, at pH 7.5) evoked inward currents at rP2X3 receptors (EC50 value, 1.8±0.3 μM). ATP activity was affected only at pH 5.5, reducing agonist potency 15 fold without altering the maximum ATP effect.

Extracellular Zn2+ inhibited ATP‐responses at rP2X1 receptors in a time‐dependent manner, a 20 min pre‐incubation being optimal (IC50 value, 1.0±0.2 μM). However, the Zn2+ effect was pH‐independent, suggesting Zn2+‐ and H+‐inhibition of ATP‐responses occur through independent processes.

Extracellular Zn2+ weakly potentiated ATP‐responses at rP2X3 receptors (EC50 value, 11±1 μM). The Zn2+ effect was dependent on pre‐incubation time and, with 20 min pre‐incubation periods, Zn2+ potentiated then inhibited ATP‐responses in a concentration‐dependent, but pH‐independent, manner.

In summary, ATP activity at rP2X1 receptors was decreased by both extracellular H+ and Zn2+ and their effects were additive. ATP activity at rP2X3 receptors was less sensitive to H+‐inhibition and, in contrast, was potentiated by Zn2+ in a pH‐independent manner. These differential effects may help distinguish P2X1 and P2X3 receptors in whole tissues.

British Journal of Pharmacology (1999) 128, 486–492; doi:10.1038/sj.bjp.0702802
Monophosphoryl lipid A provides biphasic cardioprotection against ischaemia‐reperfusion injury in rat hearts
Authors: Yamashita Nobushige , Hoshida Shiro , Otsu Kinya , Taniguchi Naoyuki , Kuzuya Tsunehiko , Hori Masatsugu
Published: January 2005

We utilized a rat model of myocardial infarction to investigate whether cardioprotection by monophosphoryl lipid A (MLA) is provided in the early and late phases, as well as to determine whether this cardioprotection may be related to the activation of manganese superoxide dismutase (Mn‐SOD), an intrinsic radical scavenger.

Pretreatment with MLA (0.5 or 1.0 mg kg−1, i.v.) 24 h prior to 20‐min left coronary artery (LCA) occlusion and 48‐h reperfusion significantly decreased the incidence of ventricular fibrillation (VF) during ischaemia, as well as infarct size. Pretreatment with lower concentrations of MLA, however, was ineffective.

When we examined the time course of MLA (0.5 mg kg−1)‐induced cardioprotection, both infarct size and the incidence of VF were significantly reduced in rats pretreated with MLA 0.5 h and 24 h before occlusion. We observed no differences, however, 2 and 72 h after MLA treatment.

The activity of Mn‐SOD paralleled the cardioprotective effects of MLA. Mn‐SOD activity in the myocardium was significantly enhanced in rats pretreated with MLA (0.5 mg kg−1) 0.5 and 24 h before. Mn‐SOD activity was not altered, however, in rats pretreated 2 or 72 h before. Lower MLA concentrations were not effective even 24 h after the treatment.

We conclude that MLA treatment induced a biphasic pattern of cardioprotection. The pattern of Mn‐SOD activity suggests that this enzyme may play a major role in the acquisition of cardioprotection against ischaemia‐reperfusion injury.

British Journal of Pharmacology (1999) 128, 412–418; doi:10.1038/sj.bjp.0702809
Resiniferatoxin‐type phorboid vanilloids display capsaicin‐like selectivity at native vanilloid receptors on rat DRG neurons and at the cloned vanilloid receptor VR1
Authors: Szallasi A , Szabó T , Bíró T , Modarres S , Blumberg P M , Krause J E , Cortright D N , Appendino G
Published: January 2005

Although the cloned rat vanilloid receptor VR1 appears to account for both receptor binding and calcium uptake, the identification of vanilloids selective for one or the other response is of importance because these ligands may induce distinct patterns of biological activities.

Phorbol 12,13‐didecanoate 20‐homovanillate (PDDHV) evoked 45Ca2+‐uptake by rat dorsal root ganglion neurons (expressing native vanilloid receptors) in culture with an EC50 of 70 nM but inhibited [3H]‐resiniferatoxin (RTX) binding to rat dorsal root ganglion membranes with a much lower potency (Ki>10,000 nM). This difference in potencies represents a more than 100 fold selectivity for capsaicin‐type pharmacology.

45Ca2+ influx by PDDHV was fully inhibited by the competitive vanilloid receptor antagonist capsazepine, consistent with the calcium uptake occurring via vanilloid receptors.

PDDHV induced calcium mobilization in CHO cells transfected with the cloned rat vanilloid receptor VR1 with an EC50 of 125 nM and inhibited [3H]‐RTX binding to these cells with an estimated Ki of 10,000 nM. By contrast, PDDHV failed to evoke a measurable calcium response in non‐transfected CHO cells, confirming its action through VR1.

We conclude that PDDHV is two orders of magnitude more potent for inducing calcium uptake than for inhibiting RTX binding at vanilloid receptors, making this novel vanilloid a ligand selective for capsaicin‐type pharmacology. These results emphasize the importance of monitoring multiple endpoints for evaluation of vanilloid receptor structure‐activity relations. Furthermore, PDDHV now provides a tool to explore the biological correlates of capsaicin‐type vanilloid pharmacology.

British Journal of Pharmacology (1999) 128, 428–434; doi:10.1038/sj.bjp.0702810
Influence of nitrovasodilators and endothelin‐1 on rheology of human blood in vitro
Authors: Walter Roland , Mark Michael , Gaudenz Roman , Harris Llinos G , Reinhart Walter H
Published: January 2005

The shear stress of flowing blood profoundly influences the release of endothelium‐dependent vasodilative and constrictive factors. Conversely, the influence of these mediators such as nitric oxide (NO) or endothelin‐1 (ET‐1) on blood rheology remains elusive. In the present study the influence of nitrovasodilators and ET‐1 on red blood cell (RBC) shape and whole blood viscosity were investigated.

Incubation of whole blood with sodium‐nitroprusside (SNP, 10−5–10−2 M), glyceryl trinitrate (GTN, 0.0001–0.1 mg mL−1), S‐nitroso‐N‐acetylpenicillamine (SNAP, 10−6–10−3 M), and the active metabolite of molsidomine (SIN‐1, 10−6–10−3 M), but not molsidomine (10−6–10−3 M), resulted in significantly increased amounts of methaemoglobin, indicating a relevant interaction with RBCs. Treatment with SNP at 10−2 M induced a marked echinocytosis (morphological index: 2.23±0.98 vs −0.17±0.10; P<0.001) and increased blood viscosity (haematocrit 45%) at a high shear rate of 94.5 s−1 (6.46±0.60 vs 5.07±0.35 mPa·s; P<0.01) and a low shear rate of 0.1 s−1 (88.6±36.8 vs 42.1±11.7 mPa·s; P<0.01). Echinocytosis was probably due to cyanide accumulation. SIN‐1 at 10−3 M slightly decreased high shear viscosity (4.88±0.28 vs 4.95±0.30 mPa·s; P<0.05). SNAP at 10−3 M slightly increased both high (5.14±0.23 vs 5.05±0.24 mPa·s; P<0.01) and low shear (53.9±7.2 vs 51.2±5.9 mPa·s; P<0.05) viscosity. Molsidomine and GTN failed to influence whole blood viscosity. ET‐1 (10−9–10−6 M) had no effect on RBC shape and viscosity.

We conclude that the most important modulators of vascular tone, NO and ET‐1, do not affect RBC shape and blood viscosity, which is important from both a physiological and a pharmacological point of view.

British Journal of Pharmacology (1999) 128, 744–750; doi:10.1038/sj.bjp.0702817
Direct block of native and cloned (Kir2.1) inward rectifier K+ channels by chloroethylclonidine
Authors: Barrett‐Jolley R , Dart C , Standen N B
Published: January 2005

We have investigated the inhibition of inwardly rectifying potassium channels by the α‐adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two‐electrode voltage‐clamp of rat isolated flexor digitorum brevis muscle and whole‐cell patch‐clamp of cell lines transfected with Kir2.1 (IRK1).

In skeletal muscle and at a membrane potential of −50 mV, chloroethylclonidine (CEC), an agonist at α2‐adrenergic receptors and an antagonist at α1x‐receptors, was found to inhibit the inward rectifier current with a Ki of 30 μM.

The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive to high concentrations of α1‐(prazosin) or α2‐(rauwolscine) antagonists.

The degree of current inhibition by CEC was found to vary with the membrane potential (approximately 70% block at −50 mV c.f. ∼10% block at −190 mV). The kinetics of this voltage dependence were further investigated using recombinant inward rectifier K+ channels (Kir2.1) expressed in the MEL cell line. Using a two pulse protocol, we calculated the time constant for block to be ∼8 s at 0 mV, and the rate of unblock was described by the relationship τ=exp((Vm+149)/22) s.

This block was effective when CEC was applied to either the inside or the outside of patch clamped cells, but ineffective when a polyamine binding site (aspartate 172) was mutated to asparagine.

The data suggest that the clonidine‐like imidazoline compound, CEC, inhibits inward rectifier K+ channels independently of α‐receptors by directly blocking the channel pore, possibly at an intracellular polyamine binding site.

British Journal of Pharmacology (1999) 128, 760–766; doi:10.1038/sj.bjp.0702819
Differential effects of bepridil on functional properties of troponin C in slow and fast skeletal muscles
Authors: Kischel P , Stevens L , Mounier Y
Published: January 2005

Bepridil (BPD) is a pharmacological compound able to bind to the Ca2+ sensor protein troponin C (TnC), which triggers skeletal muscle contraction upon Ca2+‐binding. BPD can thereby modulate the Ca2+‐affinity of this protein.

The Ca2+‐sensitizing action of bepridil was investigated on slow and fast isoforms of TnC from skinned slow and fast skeletal muscle fibres, activated by either Ca2+ or Sr2+ ions.

Bepridil did not modify the Ca2+ maximal tension of slow and fast fibres, suggesting that binding of the drug to TnC did not induce a change in the number of cross‐bridges involved in maximal tension.

Sr2+ ions induced lower maximal tension than Ca2+ ions. However, in fast fibres, these lower Sr2+ maximal tensions could be reinforced by bepridil, suggesting an effect of bepridil on the function of site I of fast TnC.

Under submaximal tension, bepridil induced an increase in Ca2+ affinity of TnC in both slow and fast fibres. However, slow fibres were more drug reactive than fast fibres, and the increase in tension appeared to be modulated by the Ca2+ concentration.

Thus, bepridil exerted a differential effect on slow and fast fibres. Moreover, the results suggest that bepridil was more effective when activation conditions were unfavourable.

British Journal of Pharmacology (1999) 128, 767–773; doi:10.1038/sj.bjp.0702820
Evidence for a role for central 5‐HT2B as well as 5‐HT2A receptors in cardiovascular regulation in anaesthetized rats
Authors: Knowles Ian D , Ramage Andrew G
Published: January 2005

The effects of injections i.c.v. of quipazine, (2 μmol kg−1) and 1‐(2,5‐di‐methoxy‐4‐iodophenyl)‐2‐aminopropane (DOI; 2 μmol kg−1) on renal sympathetic and phrenic nerve activity, mean arterial blood pressure (MAP) and heart rate were investigated in α‐chloralose anaesthetized rats pretreated with a peripherally acting 5‐HT2 receptor antagonist.

Quipazine or DOI caused a rise in MAP which was associated with a tachycardia and renal sympathoinhibition in rats pretreated (i.c.v.) with the antagonist vehicle 10% PEG. These effects of quipazine were completely blocked by pretreatment with cinanserin (a 5‐HT2 receptor antagonist) and attenuated by spiperone (a 5‐HT2A receptor antagonist). However, pretreatment with SB200646A (a 5‐HT2B/2C receptor antagonist) only blocked the sympathoinhibition, while pretreatment with SB204741 (a 5‐HT2B receptor antagonist) reversed the sympathoinhibition to excitation as it also did for DOI. Quipazine also caused renal sympathoexcitation in the presence (i.v.) of a vasopressin V1 receptor antagonist.

Injection (i.v.) of the V1 receptor antagonist at the peak pressor response evoked by quipazine alone and in the presence of SB204741 caused an immediate fall in MAP. For quipazine alone the renal sympathoinhibition was slowly reversed to an excitation, while the renal sympathoexcitation observed in the presence of SB204741 was potentiated. In both, the quipazine‐evoked tachycardia was unaffected.

The data indicate that cardiovascular responses caused by i.c.v. quipazine and DOI are primarily due to activation of central 5‐HT2A receptors, which causes the release of vasopressin and a tachycardia. This released vasopressin appears to suppress a 5‐HT2A receptor‐evoked central increase in sympathetic outflow, which involves the activation of central 5‐HT2B receptors indirectly by the released vasopressin.

British Journal of Pharmacology (1999) 128, 530–542; doi:10.1038/sj.bjp.0702822
Beneficial effects of Mn(III)tetrakis (4‐benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic, in zymosan‐induced shock
Authors: Cuzzocrea Salvatore , Costantino Giuseppina , Mazzon Emanuela , De Sarro Angela , Caputi Achille P
Published: January 2005

The therapeutic efficacy of Mn(III)tetrakis (4‐benzoic acid) porphyrin (MnTBAP), a novel superoxide dismutase mimetic which scavenges peroxynitrite, was investigated in rats subjected to shock induced by peritoneal injection of zymosan.

Our data show that MnTBAP (given at 1, 3 and 10 mg kg−1 intraperitoneally, 1 and 6 h after zymosan injection) significantly reduce in dose dependent manner the development of peritonitis (peritoneal exudation, high nitrate/nitrite and peroxynitrite plasma levels, leukocyte infiltration and histological examination).

Furthermore, our data suggest that there is a reduction in the lung, small intestine and liver myeloperoxidase (MPO) activity and lipid peroxidation activity from MnTBAP‐treated rats.

MnTBAP also reduced the appearance of nitrotyrosine immunoreactivity in the inflamed tissues.

Furthermore, a significant reduction of suppression of mitochondrial respiration, DNA strand breakage and reduction of cellular levels of NAD+ was observed in ex vivo macrophages harvested from the peritoneal cavity of zymosan‐treated rat.

In vivo treatment with MnTBAP significantly reduced in a dose‐dependent manner peroxynitrite formation and prevented the appearance of DNA damage, the decrease in mitochondrial respiration and the loss of cellular levels of NAD+.

In conclusion our results showed that MnTBAP was effective in preventing the development of zymosan‐induced shock.

British Journal of Pharmacology (1999) 128, 1241–1251; doi:10.1038/sj.bjp.0702826
Regulation of induction of nitric oxide synthase and the inhibitory actions of dexamethasone in the human intestinal epithelial cell line, Caco‐2: influence of cell differentiation
Authors: Cavicchi M , Whittle B J R
Published: January 2005

The inducible isoform of nitric oxide synthase (iNOS) may be involved in the pathogenesis of inflammatory bowel disease. Using the human intestinal epithelial cell line, Caco‐2, iNOS expression, regulation and sensitivity to the glucocorticoid, dexamethasone after cytokine exposure and its relationship to the degree of differentiation has been studied.

NOS activity, assessed by NO2− and NO3− release, was time‐dependently increased after exposure to interferon γ alone or in combination with interleukin‐1β and tumour necrosis factor α.

Cytokine‐induced iNOS activity was increased with days in culture over 20 days and number of passages, suggesting iNOS up‐regulation during enterocyte‐like differentiation. This activity was inhibited by the selective iNOS inhibitor 1400 W (0.1–100 μM). In addition, iNOS protein induction was confirmed by Western blot.

Actinomycin D (5 μg ml−1) inhibited cytokine‐induced iNOS activity, protein expression and mRNA level. Pyrrolidine dithiocarbamate (PDTC: 10–200 μM) and 3,4 dichloroisocoumarin (0.1–100 μM) reduced cytokine‐induced iNOS activity and protein expression at both day 10 and 15 after confluence. PDTC also decreased iNOS mRNA levels, suggesting NF‐κB involvement in its transcription at these times.

The tyrphostins A25 and B42 reduced cytokine‐induced iNOS activity at both day 10 and 15 after confluence, indicating the JAK‐2 kinase is also involved at these times. The tyrphostins also reduced the iNOS protein expression.

Dexamethasone (0.1–10 μM, for 24 h) reduced cytokine‐induced iNOS activity at day 15 and 20 after cell confluence, but not at day 5 or 10.

Dexamethasone (5 μM) decreased cytokine‐induced iNOS protein expression at day 10 as well as at day 15 after confluence.

These findings indicate that iNOS induction and its inhibition by dexamethasone in this human intestinal epithelial cell line is dependent on the degree of differentiation.

British Journal of Pharmacology (1999) 128, 705–715; doi:10.1038/sj.bjp.0702827
Structural determinants of the partial agonist‐inverse agonist properties of 6′‐azidohex‐2′‐yne‐Δ8‐tetrahydrocannabinol at cannabinoid receptors
Authors: Ross Ruth A , Gibson T Michael , Stevenson Lesley A , Saha Bijali , Crocker Peter , Razdan Raj K , Pertwee Roger G
Published: January 2005

We have extended previous investigations of four analogues of Δ8‐tetrahydrocannabinol (Δ8‐THC): 6′‐azidohex‐2′‐yne‐Δ8‐THC (O‐1184), 6′‐azidohex‐cis‐2′‐ene‐Δ8‐THC (O‐1238) and octyl‐2′‐yne‐Δ8‐THC (O‐584) and its 1‐deoxy‐analogue (O‐1315).

O‐1184, O‐1238 and O‐584 displaced [3H]‐CP55940 from specific binding sites on Chinese hamster ovary (CHO) cell membranes expressing CB1 or CB2 cannabinoid receptors, with pKi values of 8.28 to 8.45 (CB1) and 8.03 to 8.13 (CB2). The pKi values of O‐1315 were significantly less, 7.63 (CB1) and 7.01 (CB2).

All the analogues inhibited forskolin‐stimulated cyclic AMP production by CB1‐transfected CHO cells (pEC50=9.16 to 9.72). Only O‐1238 behaved as a full agonist in this cell line.

In mouse vasa deferentia, O‐1238 inhibited electrically‐evoked contractions (pEC50=10.18 and Emax=70.5%). Corresponding values for O‐1184 were 9.08 and 21.1% respectively. At 1 nM, O‐1184 produced surmountable antagonism of the cannabinoid receptor agonist, CP55940. However, at 0.1 nM, O‐1184 did not attenuate CP55940‐induced inhibition of cyclic AMP production by CB1‐transfected CHO cells.

In CB2‐transfected CHO cells, cyclic AMP production was inhibited by CP55940 (pEC50=8.59), enhanced by O‐1184 and O‐584 (pEC50=8.20 and 6.86 respectively) and not significantly affected by O‐1238 or O‐1315.

At 100 nM, O‐1184 and O‐1238 produced surmountable antagonism of CP55940 in CB2 cells, decreasing the pEC50 of CP55940 from 8.61 to 7.42 (O‐1184) or from 8.54 to 7.44 (O‐1238).

These data support the hypothesis that increasing the degree of unsaturation of the aliphatic side‐chain of Δ8‐THC analogues has little effect on CB1 or CB2 receptor affinity but can reduce CB1 receptor efficacy and reverse the direction of responses elicited at CB2 receptors.

British Journal of Pharmacology (1999) 128, 735–743; doi:10.1038/sj.bjp.0702836
5‐hydroxytryptamine receptors mediating contraction in human small muscular pulmonary arteries: importance of the 5‐HT1B receptor
Authors: Morecroft Ian , Heeley Robert P , Prentice Howard M , Kirk Alan , MacLean Margaret R
Published: January 2005

The 5‐hydroxytryptamine (5‐HT) receptors mediating vasoconstriction in isolated human small muscular pulmonary arteries (SMPAs) were determined using techniques of wire myography and reverse transcription‐polymerase chain reaction (RT–PCR).

The agonists 5‐HT, 5‐carboxamidotryptamine (5‐CT, unselective for 5‐HT1 receptors) and sumatriptan (selective for 5‐HT1B/D receptors) all caused contraction and were equipotent (pEC50s: 7.0±0.2, 7.1±0.3 and 6.7±0.1, respectively) suggesting the presence of a 5‐HT1 receptor.

Ketanserin (5‐HT2A‐selective antagonist, 0.1 μM) inhibited 5‐HT‐induced contractions only at non‐physiological/pathological concentrations of 5‐HT (>0.1 μM) whilst GR55562 (5‐HT1B/1D‐selective antagonist, 1 μM) inhibited 5‐HT‐induced contractions at all concentrations of 5‐HT (estimated pKB=7.7±0.2). SB‐224289 (5‐HT1B‐selective antagonist, 0.2 μM) inhibited sumatriptan‐induced contractions (estimated pKB=8.4±0.1) whilst these were unaffected by the 5‐HT1D‐selective antagonist BRL15572 (0.5 μM) suggesting that the 5‐HT1B receptor mediates vasoconstriction in this vessel.

RT–PCR confirmed the presence of substantial amounts of mRNA for the 5‐HT2A and 5‐HT1B receptor subtypes in these arteries whilst only trace amounts of 5‐HT1D receptor message were evident.

These findings suggest that a heterogeneous population of 5‐HT2A and 5‐HT1B receptors co‐exist in human small muscular pulmonary arteries but that the 5‐HT1B receptor mediates 5‐HT‐induced vasoconstriction at physiological and pathophysiological concentrations of 5‐HT. These results have important implications for the treatment of pulmonary hypertension in which the 5‐HT1B receptor may provide a novel and potentially important therapeutic target.

British Journal of Pharmacology (1999) 128, 730–734; doi:10.1038/sj.bjp.0702841
Cannabinoid CB1 receptors fail to cause relaxation, but couple via Gi/Go to the inhibition of adenylyl cyclase in carotid artery smooth muscle
Authors: Holland Michael , Challiss R A John , Standen Nicholas B , Boyle John P
Published: January 2005

The aim of the current study was to characterize which cannabinoid receptors, if any, are present on rat carotid artery smooth muscle. Additionally, the effects of cannabinoids on carotid artery tone, on cyclic AMP accumulation and on forskolin‐induced relaxation were examined in the same tissue.

Stimulation of carotid arteries with forskolin (10 μM) significantly increased cyclic AMP accumulation, an effect that was inhibited in a concentration‐dependent manner by the cannabinoid receptor agonist, methanandamide.

Similar inhibition was seen with the CB1 agonist HU‐210 but this inhibition was not mimicked by the CB2 agonist, WIN 55,2212‐2.

The inhibitory effect of methanandamide on cyclic AMP accumulation was prevented by incubation of the arteries with pertussis toxin and was significantly reduced by LY320135, a selective CB1 antagonist, but not by SR 144528, a CB2‐selective antagonist.

Methanandamide failed to relax carotid arteries pre‐contracted with phenylephrine, but inhibited forskolin‐induced relaxation of these arteries. This functional inhibition of relaxation by methanandamide was inhibited by CB1‐selective (LY320135 and SR 141716A), but not a CB2‐selective antagonist (SR 144528).

These data demonstrate the presence of functional G protein‐linked cannabinoid receptors of the CB1 subtype in the rat carotid artery, but show that these receptors inhibit cyclic AMP accumulation rather than cause relaxation.

British Journal of Pharmacology (1999) 128, 597–604; doi:10.1038/sj.bjp.0702842
Response‐selective C5a agonists: differential effects on neutropenia and hypotension in the rat
Authors: Short Anna J , Paczkowski Natalii J , Vogen Shawn M , Sanderson Sam D , Taylor Stephen M
Published: January 2005

Some in vivo activities of two complement C5a agonist analogues have been evaluated by measuring changes in blood pressure and neutropenia in the rat and comparing the results with their receptor affinities in peritoneal macrophages and polymorphonuclear leucocytes (PMNs). In vitro C5a receptor (C5aR) binding experiments showed that YSFKPMPLaR and YSFKD(NMeNle)PlaR had similar affinities for the macrophage C5aR (IC50 0.2, 0.1 μM respectively). In PMNs, the affinity of YSFKPMPLaR (IC50 0.1 μM) was similar to that in macrophages, whereas the affinity of YSFKD(NMeNle)PLaR for the PMN C5aR was >100 μM. Given i.v., YSFKD(NMeNle)PLaR had similar activity to YSFKPMPLaR on blood pressure but did not cause neutropenia. These results demonstrate selectivity of a new C5a agonist in vitro, which is paralleled in vivo. The results suggest the possibility of developing selective agonists of C5a for in vivo use in humans.

British Journal of Pharmacology (1999) 128, 511–514; doi:10.1038/sj.bjp.0702847
Receptor density as a factor governing the efficacy of the dopamine D4 receptor ligands, L‐745,870 and U‐101958 at human recombinant D4.4 receptors expressed in CHO cells
Authors: Gazi L , Bobirnac I , Danzeisen M , Schüpbach E , Langenegger D , Sommer B , Hoyer D , Tricklebank M , Schoeffter P
Published: January 2005

The relationships between the density of dopamine D4.4 receptors and the agonist efficacies of L‐745,870 (3‐(4‐[4‐chlorophhenyl]piperazin‐1‐yl)‐methyl‐1H‐pyrrolo [2,3‐b]pyridine) and U‐101958 ((1‐benzyl‐piperidin‐4‐yl)‐(3‐isopropoxy‐pyridin‐2‐yl)‐methyl‐amine) were investigated in Chinese hamster ovary (CHO) cells, after treatment with the gene expression enhancer, sodium butyrate.

In CHO cells expressing D4.4 receptors (CHO/D4 cells), dopamine inhibited forskolin‐stimulated cyclic AMP accumulation (Emax 56±1% inhibition, pEC50 7.4±0.1, n=10). U‐101958 behaved as a partial agonist (39±7% the efficacy of dopamine, pEC50 8.1±0.3, n=4), whereas L‐745,870 had no detectable agonist effect.

Receptor density, as estimated by [3H]‐spiperone saturation binding was 240±30 fmol mg−1 protein (n=8) in CHO/D4 cell homogenates. It reached 560±150 (n=6), 1000±190 (n=4) and 840±120 (n=4) fmol mg−1 protein after treatment with sodium butyrate (5 mM) for 6, 18 and 48 h, respectively.

The increase in receptor density was associated with a gradual enhancement of the agonist effects (increased Emax and pEC50 values) of dopamine. The efficacy of U‐101958 (relative to dopamine) doubled and L‐745,870 was turned into a partial agonist (efficacy 49% relative to dopamine, pEC50 8.6±0.2, n=6, after 48 h treatment with sodium butyrate). These agonist effects of U‐101958 and L‐745,870 could be antagonized by spiperone (0.1 μM) but not by raclopride (10 μM).

The results show that U‐101958 and L‐745,870 are partial agonists at human dopamine D4.4 receptors expressed in CHO cells. Their efficacy is governed by receptor density. Agonist effects of these two compounds in vivo cannot be excluded under circumstances of increased receptor levels.

British Journal of Pharmacology (1999) 128, 613–620; doi:10.1038/sj.bjp.0702849
Preweanling naltrindole administration differentially affects clonidine induced antinociception and plasma adrenaline levels in male and female neonatal rats
Authors: Alberti Israel , Fernández Beatriz , Alguacil Luis Fernando , Aguilar Antonio , Caamaño Manuel , Romero Eva M , Viveros M Paz
Published: January 2005

The influence of a chronic treatment with the δ‐selective opioid antagonist naltrindole (1 mg kg−1) during the preweanling period (daily injections from birth to postnatal day 19), on the antinociceptive and sympatholytic effects of the α2‐adrenergic agonist clonidine in male and female rats of 20 and 25 days of age was investigated.

Nociception was assessed using the tail immersion test (water at 50°C) and plasma levels of adrenaline were measured by high‐performance liquid chromatography.

The dose of clonidine (1.5 mg kg−1) and the time point at which nociceptive responses were recorded (30 min after the administration of the drug) were chosen on the basis of dose‐response (0.5, 1, 1.5 and 2 mg kg−1) and time‐response (5, 10, 15, 30 and 60 min) curves which were previously carried out in naive control neonatal rats.

In females, the functional blockade of the δ‐receptor by neonatal naltrindole treatment did not modify the sympatholytic effect of clonidine but prevented clonidine induced antinociception. Conversely, in males naltrindole treatment allowed the appearance of clonidine antinociception and the sympatholytic effect of clonidine.

The results indicate that the δ‐receptor is involved in the modulation of antinociceptive and sympatholytic responses to clonidine in neonatal rats and suggest the existence of sex differences in the interactions between δ‐opioid and α2‐adrenergic receptors.

British Journal of Pharmacology (1999) 128, 953–960; doi:10.1038/sj.bjp.0702852
Acetylcholine‐induced arteriolar dilation is reduced in streptozotocin‐induced diabetic rats with motor nerve dysfunction
Authors: Terata K , Coppey L J , Davidson E P , Dunlap J A , Gutterman D D , Yorek M A
Published: January 2005

Diabetes mellitus produces marked abnormalities in motor nerve conduction, but the mechanism is not clear. In the present study we hypothesized that in the streptozotocin (STZ)‐induced diabetic rat impaired vasodilator function is associated with reduced endoneural blood flow (EBF) which may contribute to nerve dysfunction.

We examined whether diabetes‐induced reductions in sciatic nerve conduction velocity and EBF were associated with impaired endothelium‐dependent dilation in adjacent arterioles. We measured motor nerve conduction velocity (MNCV) in the sciatic nerve using a non‐invasive procedure, and sciatic nerve nutritive blood flow using microelectrode polarography and hydrogen clearance. In vitro videomicroscopy was used to quantify arteriolar diameter responses to dilator agonists in arterioles overlying the sciatic nerve.

MNCV and EBF in 4‐week‐STZ‐induced diabetic rats were decreased by 22% and 49% respectively. Arterioles were constricted with U46619 and dilation to acetylcholine (ACh), aprikalim, or sodium nitroprusside (SNP) examined. All agonists elicited dose‐dependent dilation in control and diabetic rats, although ACh‐induced dilation was significantly reduced in diabetic rats. Treating vessels from normal or diabetic rats with indomethacin (INDO) alone did not significantly affect ACh‐induced relaxation. However, ACh‐induced vasodilation was significantly reduced by treatment with KCl or Nω‐nitro‐L‐arginine (LNNA) alone. Combining LNNA and KCl further reduced ACh‐induced dilation in these vessels.

Diabetes causes vasodilator dysfunction in a microvascular bed that provides circulation to the sciatic nerve. These studies imply that ACh‐induced dilation in these vessels is mediated by multiple mechanisms that may include the endothelial‐dependent production of nitric oxide and endothelial‐derived hyperpolarizing factor. This impaired vascular response is associated with neural dysfunction.

British Journal of Pharmacology (1999) 128, 837–843; doi:10.1038/sj.bjp.0702856
Characterization of the binding of [3H]‐clobenpropit to histamine H3‐receptors in guinea‐pig cerebral cortex membranes
Authors: Harper E A , Shankley N P , Black J W
Published: January 2005

We have investigated the binding of a novel histamine H3‐receptor antagonist radioligand, [3H]‐ clobenpropit ([3H]‐VUF9153), to guinea‐pig cerebral cortex membranes.

Saturation isotherms for [3H]‐clobenpropit appeared biphasic. Scatchard plots were curvilinear and Hill plot slopes were significantly less than unity (0.63±0.03; n=12±s.e.mean). The radioligand appeared to label two sites in guinea‐pig cerebral cortex membranes with apparent affinities (pKD′) of 10.91±0.12 (Bmax=5.34±0.85 fmol mg−1 original wet weight) and 9.17±0.16 (Bmax=23.20± 6.70 fmol mg−1).

In the presence of metyrapone (3 mM) or sodium chloride (100 mM), [3H]‐clobenpropit appeared to label a homogeneous receptor population (Bmax=3.41±0.46 fmol mg−1 and 3.49±0.44 fmol mg−1, pKD′ =10.59±0.17 and 10.77±0.02, respectively). Scatchard plots were linear and Hill slopes were not significantly different from unity (0.91±0.04 and 0.99±0.02, respectively). Granisetron (1 μM), rilmenidine (3 μM), idazoxan (0.3 μM), pentazocine (3 μM) and 1,3‐di‐(2‐tolyl)guanidine (0.3 μM) had no effect on the binding of [3H]‐clobenpropit.

The specific binding of [3H]‐clobenpropit appeared to reach equilibrium after 25 min at 21±3°C and remained constant for >180 min. The estimated pKD′ (10.27±0.27; n=3±s.e.mean) was not significantly different from that estimated by saturation analysis in the presence of metyrapone.

A series of histamine H3‐receptor ligands expressed affinity values for sites labelled with [3H]‐clobenpropit which were not significantly different from those estimated when [3H]‐R‐α‐MH was used to label histamine H3‐receptors in guinea‐pig cerebral cortex membranes.

British Journal of Pharmacology (1999) 128, 881–890; doi:10.1038/sj.bjp.0702860
Block of human aorta Kir6.1 by the vascular KATP channel inhibitor U37883A
Authors: Surah‐Narwal S , Xu S Z , McHugh D , McDonald R L , Hough E , Cheong A , Partridge C , Sivaprasadarao A , Beech D J
Published: January 2005

A human aorta cDNA library was screened at low stringency with a rat pancreatic Kir6.1 cDNA probe and a homologue of Kir6.1 (hKir6.1) was isolated and sequenced.

Metabolic poisoning of Xenopus laevis oocytes with sodium azide and application of the K+ channel opener drug diazoxide induced K+ channel currents in oocytes co‐injected with cRNA for hKir6.1 and hamster sulphonylurea receptor (SUR1), but not in oocytes injected with water or cRNA for hKir6.1 or SUR1 alone.

K+ channel currents due to hKir6.1+SUR1 or mouse Kir6.2+SUR1 were strongly inhibited by 1 μM glibenclamide. K+‐current carried by hKir6.1+SUR1 was inhibited by the putative vascular‐selective KATP channel inhibitor U37883A (IC50 32 μM) whereas current carried by Kir6.2+SUR1 or Shaker K+ channels was unaffected.

The data support the hypothesis that hKir6.1 is a component of the vascular KATP channel, although the lower sensitivity of hKir6.1+SUR1 to U37883A compared with native vascular tissues suggests the need for another factor or subunit. Furthermore, the data suggest that pharmacology of KATP channels can be determined by the pore‐forming subunit as well as the sulphonylurea receptor and point to a molecular basis for the pharmacological distinction between vascular and pancreatic/cardiac KATP channels.

British Journal of Pharmacology (1999) 128, 667–672; doi:10.1038/sj.bjp.0702862