bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of homocysteine on acetylcholine‐ and adenosine‐induced vasodilatation of pancreatic vascular bed in rats
Authors: Quéré I. , Hillaire‐Buys D. , Brunschwig C. , Chapal J. , Janbon C. , Blayac J. P. , Petit P. , Loubatières‐Mariani M. M.
Published: January 2005

Epidemiological and experimental data have shown that homocysteine may provoke vascular lesions and that moderate homocysteinaemia may constitute an independent risk factor for vascular disease. It is now documented that homocysteine damages human endothelial cells in culture, possibly by producing hydrogen peroxide in an oxygen‐dependent reaction.

In this study, we have examined the direct effect of this sulphur amino acid on pancreatic vascular resistance. Experiments were performed on the vascular bed of the rat isolated pancreas perfused at constant pressure; thus, any change in pancreatic vascular resistance resulted in a change in the flow rate. D,L‐Homocysteine perfused for one hour at three different concentrations (200 μM, 2 mM, 20 mM) did not induce any significant change in the flow rate per se. Homocysteine infusion for 30 min at a concentration of 200 μM or 2 mM abolished the endothelium‐dependent vasodilatation induced by acetylcholine (0.05 μM), but did not modify adenosine (1.5 μM)‐induced vasodilatation.

The effect of D,L‐homocysteine (200 μM or 2 mM) cannot be ascribed to a direct antimuscarinic effect since 30 min pretreatment of rat ileum with these concentrations did not significantly change the contractile effect of increasing concentrations of acetylcholine (0.015–15 μM).

Preincubation of human umbilical vein endothelial cells with D,L‐homocysteine (0.2–5.0 mM) had no significant effect on overall cell number or viability during 18 h of incubation; the endothelial cells exposed to concentrations up to 5 mM exhibited a spindle‐shaped, whirled pattern. This pattern was reversed 48 h after the removal of homocysteine. A cytotoxic effect was seen after 18 h incubation in 10 mM D,L‐homocysteine.

In conclusion, an acute infusion of homocysteine altered acetylcholine endothelium‐induced vasodilatation, whereas the adenosine vasodilatator effect was insensitive to the deleterious action of homocysteine in vitro.

British Journal of Pharmacology (1997) 122, 351–357; doi:10.1038/sj.bjp.0701358
Demonstration of the existence of nitric oxide‐independent as well as nitric oxide‐dependent vasodilator mechanisms in the in situ renal circulation in near term pregnant rats
Authors: Chu Z. M. , Beilin L. J.
Published: January 2005

We have investigated the role of endogenous nitric oxide on renal vascular reactivity in late pregnancy in in situ blood perfused kidneys of α‐chloralose anaesthetized Wistar‐Kyoto rats. Nitric oxide synthesis inhibition was achieved by intravenous administration of NG‐nitro‐L‐arginine or NG‐nitro‐L‐arginine methyl ester.

Intra‐arterial mean blood pressure was lower in pregnancy compared with nonpregnant controls. Following nitric oxide synthesis inhibition mean blood pressure increased in both pregnant and nonpregnant groups, but remained lower in pregnant animals.

Basal renal perfusion pressure was similar in pregnant and nonpregnant groups. Intravenous administration of NG‐nitro‐L‐arginine resulted in dose‐dependent increases in renal perfusion pressure but responses were substantially depressed in pregnancy.

Renal vasoconstrictor responses to regional angiotensin II (AII) were decreased in pregnancy, whereas those to noradrenaline (NA) did not differ from nonpregnant controls. NG‐nitro‐L‐arginine (5 mg kg−1) potentiated renal responses to regional AII and NA in both groups, but AII responses remained lower in pregnancy. Blunted renal AII responses in pregnancy were still evident following large doses of NG‐nitro‐L‐arginine methyl ester (100 mg kg−1).

The results demonstrate that nitric oxide synthesis inhibition increases renal perfusion pressure to a lesser extent in pregnant compared with nonpregnant rats, and that reduced renal pressor responses to AII are still evident in pregnancy after nitric oxide synthesis inhibition.

These results suggest that although endogenous nitric oxide synthesis modulates renal vasoconstrictor responses in both pregnant and nonpregnant animals, this mechanism does not fully account for the blunted renal vasoconstrictor responses to regional AII or nitric oxide inhibitors in near term pregnant rats. The nature of this important physiological vasodilator mechanism in pregnancy remains to be elucidated.

British Journal of Pharmacology (1997) 122, 307–315; doi:10.1038/sj.bjp.0701363
Central benzodiazepine receptor autoradiography in hippocampal sclerosis
Authors: Hand Kieran S. P. , Baird Virginia H. , Van Paesschen Wim , Koepp Matthias J. , Revesz Tamas , Thom Maria , Harkness William F. J. , Duncan John S. , Bowery Norman G.
Published: January 2005

The γ‐aminobutyric acid (GABA)A/central benzodiazepine receptor (cBZR) complex is a major inhibitory receptor in the vertebrate CNS. Binding of [11C]‐flumazenil to this complex in vivo is reduced in hippocampal sclerosis (HS). It has been uncertain whether reduced cBZR binding is entirely due to neuronal loss in HS.

The objective of this study was to characterize abnormalities of the cBZR in HS with a correlative autoradiographic and quantitative neuropathological study.

Saturation autoradiographic studies were performed with [†H]‐flumazenil to investigate relationships between neuronal density and receptor availability (Bmax) and affinity (Kd) in HS. Hippocampal tissue was obtained at surgery from 8 patients with intractable temporal lobe epilepsy (TLE) due to HS and autopsies of 6 neurologically normal controls. Neuronal densities were obtained by means of a 3‐D counting method.

Bmax values for [†H]‐flumazenil binding in the subiculum, CA1, CA2, CA3, hilus and dentate gyrus were all found to be significantly reduced in HS compared with controls and significant increases in affinity were observed in the subiculum, hilus and dentate gyrus. In HS, cBZR density in the CA1 region was significantly reduced (P<0.05) to a greater extent than could be attributable to neurone loss. In other regions, Bmax was reduced in parallel with neuronal density.

In HS, there is a loss of cBZR in CA1 over and above loss of neurones. This finding and increases in affinity for flumazenil in subiculum, hilus and dentate gyrus imply a functional abnormality of the GABAA/cBZR complex that may have a role in the pathophysiology of epileptogenicity in HS.

British Journal of Pharmacology (1997) 122, 358–364; doi:10.1038/sj.bjp.0701365
Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells
Authors: Kiriyama Michitaka , Ushikubi Fumitaka , Kobayashi Takuya , Hirata Masakazu , Sugimoto Yukihiko , Narumiya Shuh
Published: January 2005

Eight types and subtypes of the mouse prostanoid receptor, the prostaglandin D (DP) receptor, the prostaglandin F (FP) receptor, the prostaglandin I (IP) receptor, the thromboxane A (TP) receptor and the EP1, EP2, EP3 and EP4 subtypes of the prostaglandin E receptor, were stably expressed in Chinese hamster ovary cells. Their ligand binding characteristics were examined with thirty two prostanoids and their analogues by determining the Ki values from the displacement curves of radioligand binding to the respective receptors.

The DP, IP and TP receptors showed high ligand binding specificity and only bound their own putative ligands with high affinity such as PGD2, BW245C and BW868C for DP, cicaprost, iloprost and isocabacyclin for IP, and S‐145, I‐BOP and GR 32191 for TP.

The FP receptor bound PGF2α and fluprostenol with Ki values of 3–4 nM. In addition, PGD2, 17‐phenyl‐PGE2, STA2, I‐BOP, PGE2 and M&B̀‐28767 bound to this receptor with Ki values less than 100 nM.

The EP1 receptor bound 17‐phenyl‐PGE2, sulprostone and iloprost in addition to PGE2 and PGE1, with Ki values of 14–36 nM. 16,16‐dimethyl‐PGE2 and two putative EP1 antagonists, AH6809 and SC‐19220, did not show any significant binding to this receptor. M&B‐28767, a putative EP3 agonist, and misoprostol, a putative EP2/EP3 agonist, also bound to this receptor with Ki values of 120 nM.

The EP2 and EP4 receptors showed similar binding profiles. They bound 16,16‐dimethyl PGE2 and 11‐deoxy‐PGE1 in addition to PGE2 and PGE1. The two receptors were discriminated by butaprost, AH‐13205 and AH‐6809 that bound to the EP2 receptor but not to the EP4 receptor, and by 1‐OH‐PGE1 that bound to the EP4 but not to the EP2 receptor.

The EP3 receptor showed the broadest binding profile, and bound sulprostone, M&B‐28767, GR63799X, 11‐deoxy‐PGE1, 16,16‐dimethyl‐PGE2 and 17‐phenyl‐PGE2, in addition to PGE2 and PGE1, with Ki values of 0.6–3.7 nM. In addition, three IP ligands, iloprost, carbacyclin and isocarbacyclin, and one TP ligand, STA2, bound to this receptor with Ki values comparable to the Ki values of these compounds for the IP and TP receptors, respectively.

8‐Epi‐PGF2α showed only weak binding to the IP, TP, FP, EP2 and EP3 receptor at 10 μM concentration.

British Journal of Pharmacology (1997) 122, 217–224; doi:10.1038/sj.bjp.0701367
The antiarrhythmic agent bertosamil induces inactivation of the sustained outward K+ current in human atrial myocytes
Authors: Tessier Sophie , Rücker‐Martin Catherine , Macé Loïc , Coraboeuf Edouard , Mercadier Jean‐Jacques , Hatem Stéphane N.
Published: January 2005

In whole‐cell patch‐clamped human atrial myocytes, the antiarrhythmic agent bertosamil (10 μM) inhibited the sustained component, Isus (38.6±3.1%), and enhanced the inactivating component, It (9.1±6.1%), of the outward K+ current elicited by 750 ms test pulses from −60 mV to +50 mV. Higher concentrations of bertosamil (>10 μM) inhibited both It and Isus.

Suppression of Isus and stimulation of It by 10 μM bertosamil was observed on renewed stimulation following a 2 min rest period during which the drug was applied and persisted after washout, indicating a rest‐dependent effect of bertosamil on the outward K+ current.

Cell dialysis with an internal solution containing 10 μM bertosamil increased both It (78.0±14.7%) and Itotal (26.7±8.4%) and inhibited Isus (28.9±6.3%, n=6). In the presence of intracellular bertosamil, external application of the drug inhibited It and Isus in a concentration‐dependent and use‐dependent manner.

Following the suppression of Isus by 200 μM 4‐aminopyridine (4‐AP), bertosamil (10 μM) inhibited It. Washout of 4‐AP was associated with a larger It amplitude than that observed in control conditions. In myocytes characterized by a prominent Isus and lack of It, bertosamil (10 μM) induced a rapid and partial inactivation of the current, together with inward rectification of the current measured at the end of the test pulse.

In the presence of bertosamil the activation/voltage relationships, steady‐state inactivation and recovery from inactivation of It were markedly modified, pointing to changes in the conductance underlying It.

We conclude that bertosamil induces rapid inactivation of sustained outward current which leads to an apparent increase in It and decrease in Isus. This effect, which was distinct from the use‐dependent inhibition of the outward K+ current, could represent a new antiarrhythmic mechanism.

British Journal of Pharmacology (1997) 122, 291–301; doi:10.1038/sj.bjp.0701369
Cyclic AMP‐mediated regulation of vascular smooth muscle cell cyclic AMP phosphodiesterase activity
Authors: Rose R. J. , Liu H. , Palmer D. , Maurice D. H.
Published: January 2005

Rat cultured aortic vascular smooth muscle cells (VSMC) express both cyclic GMP‐inhibited cyclic AMP phosphodiesterase (PDE3) and Ro 20–1724‐inhibited cyclic AMP phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3‐selective inhibitor, or Ro 20–1724, a PDE4‐selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC cyclic AMP phosphodiesterase (PDE) activity.

Treatment of VSMC with either forskolin or 8‐bromo‐cyclic AMP caused significant concentration‐ and time‐dependent increases in total cellular cyclic AMP PDE activity. Using cilostamide or Ro 20–1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8‐bromo‐cyclic AMP treatment, with a relatively larger effect observed on PDE3 activity. The increase in cyclic AMP PDE activity induced by forskolin or 8‐bromo‐cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9‐dideoxyforskolin) or an analogue of cyclic GMP (8‐bromo‐cyclic GMP) did not affect total cyclic AMP PDE activity.

Incubation of VSMC with 8‐bromo‐cyclic AMP for 16 h caused a marked rightward shift in the concentration‐response curves for both isoprenaline‐ and forskolin‐mediated activation of adenylyl cyclase. A role for up‐regulated cyclic AMP PDE activity in this reduced potency is supported by our observation that cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20–1724) partially normalized the effects of isoprenaline or forskolin in treated cells to those in untreated cells.

We conclude that VSMC cyclic AMP PDE activity is increased following long‐term elevation of cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this effect. Furthermore, we conclude that increases in cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long‐term use of cyclic AMP PDE inhibitors as therapeutic agents.

British Journal of Pharmacology (1997) 122, 233–240; doi:10.1038/sj.bjp.0701376
Neurokinin A‐LI release after antigen challenge in guinea‐pig bronchial tubes: influence of histamine and bradykinin
Authors: Lindström Eva G. , Andersson Rolf G. G.
Published: January 2005

Our aim was to determine if antigen challenge stimulates sensory nerves and provokes the release of tachykinins. The involvement of histamine and bradykinin was studied by using specific receptor antagonists. Capsaicin‐induced responses were also examined. Experiments were performed in vitro on tracheal and bronchial preparations from ovalbumin‐sensitized guinea‐pigs.

Characterization of ovalbumin‐induced contraction, with regard to histamine and bradykinin, was carried out on airway ring preparations in the presence of phosphoramidon. The histamine H1 receptor antagonist pyrilamine reduced allergen‐induced bronchial contractions by about 30%, whereas the bradykinin B2 receptor antagonist icatibant (Hoe 140) did not significantly affect the response. Combined treatment with pyrilamine (1 μM) and icatibant (0.1 μM) reduced the contractions by about 80%, indicating a synergistic inhibitory action. Tracheal preparations were not significantly affected by treatments, neither were capsaicin‐induced contractions.

To study the outflow of tachykinins, we used a perfused bronchial‐tube preparation, allowing simultaneous measurement of smooth muscle tension and mediator release. Neurokinin A‐like immunoreactivity (NKA‐LI) and substance P‐like immunoreactivity (SP‐LI) were determined by radioimmunoassay.

The results of the perfusion study showed an increased outflow of NKA‐LI into the perfusate in response to ovalbumin (127% of basal) challenge. SP‐LI determined in some of the samples showed a much lower amount (40 to 70 times lower) of SP‐LI than NKA‐LI. Treatment with icatibant and pyrilamine, separately and in combination, significantly reduced the ovalbumin‐induced NKA‐LI outflow by 38%, 26% and 22%, respectively.

Capsaicin‐induced outflow (124% of basal) was not significantly affected by treatments (icatibant 121%, pyrilamine 107% and combined treatment 111% of basal). However, when pyrilamine was present the increased outflow was not statistically significant.

In conclusion, we found that allergen provocation of guinea‐pig bronchi caused an increased outflow of NKA‐LI that was reduced by treatment with both pyrilamine and icatibant. These findings demonstrate that the allergen‐induced release of histamine and bradykinin stimulate sensory nerves and thereby increase outflow of tachykinins that contribute to the allergic reaction.

British Journal of Pharmacology (1997) 122, 417–422; doi:10.1038/sj.bjp.0701382
Effects of VA‐045 on learning and memory deficits in traumatic brain injury (TBI)‐induced retrograde and anterograde amnesic mice
Authors: Tang Ya‐Ping , Noda Yukihiro , Hasegawa Takaaki , Nabeshima Toshitaka
Published: January 2005

No specific regimen has been developed to treat post‐traumatic amnesia in man. In the present study, we examined the effects of (+)‐eburnamenine‐14‐carboxylic acid (2‐nitroxyethyl) ester (VA‐045), a novel derivative of apovincaminic acid, on learning and memory deficits associated with a mild traumatic brain injury (TBI) in mice.

Two kinds of amnesia, TBI‐induced retrograde amnesia (TRA) and anterograde amnesia (TAA), were produced by means of post‐ and pre‐acquisition head injury, respectively, by a simple weight‐drop device. A novel procedure of water‐finding task was used to assess learning and memory functions.

Both TRA and TAA mice were dramatically impaired in the task performance, with prolonged latencies for finding and drinking in either retention test or retest, indicating that retention was impaired in TRA mice while learning and retention were impaired in TAA mice.

VA‐045 administered 30 min post‐trauma in TRA mice dramatically shortened the prolonged latencies for finding and drinking in both retention test and retest, indicating that VA‐045 significantly improved the retention deficit observed in TRA mice.

VA‐045 administered 30 min post‐trauma in TAA mice dramatically attenuated the prolonged latencies for finding and drinking in both retention test and retest, indicating that VA‐045 significantly improved the learning and retention deficits observed in TAA mice.

Administration of VA‐045 30 min pre‐trauma in normal mice markedly attenuated the delay of latencies for finding and drinking after trauma in both retention test and retest, which shows that VA‐045 significantly prevented learning and retention deficits after TBI.

Motor activities were not significantly affected by either the TBI or the chemical treatment at the time of task examination in either experimental model.

It is concluded that VA‐045 may have potential effects on learning and memory deficits observed in either TBI‐induced retrograde or anterograde amnesia.

British Journal of Pharmacology (1997) 122, 257–264; doi:10.1038/sj.bjp.0701385
Anti‐inflammatory activity of cationic lipids
Authors: Filion Mario C. , Phillips Nigel C.
Published: January 2005

The effect of liposome phospholipid composition has been assumed to be relatively unimportant because of the presumed inert nature of phospholipids.

We have previously shown that cationic liposome formulations used for gene therapy inhibit, through their cationic component, the synthesis by activated macrophages of the pro‐inflammatory mediators nitric oxide (NO) and tumour necrosis factor‐α (TNF‐α).

In this study, we have evaluated the ability of different cationic lipids to reduce footpad inflammation induced by carrageenan and by sheep red blood cell challenge.

Parenteral (i.p. or s.c) or local injection of the positively charged lipids dimethyldioctadecylammomium bromide (DDAB), dioleyoltrimethylammonium propane (DOTAP), dimyristoyltrimethylammonium propane (DMTAP) or dimethylaminoethanecarbamoyl cholesterol (DC‐Chol) significantly reduced the inflammation observed in both models in a dose‐dependent manner (maximum inhibition: 70–95%).

Cationic lipids associated with dioleyol‐ or dipalmitoyl‐phosphatidylethanolamine retained their anti‐inflammatory activity while cationic lipids associated with dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidylglycerol (DMPG) showed no anti‐inflammatory activity, indicating that the release of cationic lipids into the macrophage cytoplasm is a necessary step for anti‐inflammatory activity. The anti‐inflammatory activity of cationic lipids was abrogated by the addition of dipalmitoylphosphatidylethanolamine‐poly(ethylene)glycol‐2000 (DPPE‐PEG2000) which blocks the interaction of cationic lipids with macrophages.

Because of the significant role of protein kinase C (PKC) in the inflammatory process we have determined whether the cationic lipids used in this study inhibit PKC activity. The cationic lipids significantly inhibited the activity of PKC but not the activity of a non‐related protein kinase, PKA. The synthesis of interleukin‐6 (IL‐6), which is not dependent on PKC activity for its induction in macrophages, was not modified in vitro or in situ by cationic lipids. The synthesis of NO and TNF‐α in macrophages, both of which are PKC‐dependent, was downregulated by cationic lipids.

These results demonstrate that cationic lipids can be considered as novel anti‐inflammatory agents. The downregulation of pro‐inflammatory mediators through interaction of cationic lipids with the PKC pathway may explain this anti‐inflammatory activity. Furthermore, since cationic lipids have intrinsic anti‐inflammatory activity, cationic liposomes should be used with caution to deliver nucleic acids for gene therapy in vivo.

British Journal of Pharmacology (1997) 122, 551–557; doi:10.1038/sj.bjp.0701396
Presynaptic inhibition of synaptic transmission in the rat hippocampus by activation of muscarinic receptors: involvement of presynaptic calcium influx
Authors: Qian Jing , Saggau Peter
Published: January 2005

Modulation of presynaptic voltage‐dependent calcium channels (VDCCs) by muscarinic receptors at the CA3–CA1 synapse of rat hippocampal slices was investigated by using the calcium indicator fura‐2. Stimulation‐evoked presynaptic calcium transients ([Capre]t) and field excitatory postsynaptic potentials (fe.p.s.ps) were simultaneously recorded. The relationship between presynaptic calcium influx and synaptic transmission was studied.

Activation of muscarinic receptors inhibited [Capre]t, thereby reducing synaptic transmission. Carbachol (CCh, 10 μM) inhibited [Capre]t by 35% and reduced fe.p.s.p. by 85%. The inhibition was completely antagonized by 1 μM atropine. An approximate 4th power relationship was found between presynaptic calcium influx and postsynaptic responses.

Application of the N‐type VDCC‐blocking peptide toxin ω‐conotoxin GVIA (ω‐CTx GVIA, 1 μM) inhibited [Capre]t and fe.p.s.ps by 21% and 49%, respectively, while the P/Q‐type VDCC blocker ω‐agatoxin IVA (ω‐Aga IVA, 1 μM) reduced [Capre]t and fe.p.s.ps by 35% and 85%, respectively.

Muscarinic receptor activation differentially inhibited distinct presynaptic VDCCs. ω‐CTx GVIA‐sensitive calcium channels were inhibited by muscarinic receptors, while ω‐Aga IVA‐sensitive channels were not. The percentage inhibition of ω‐CTx GVIA‐sensitive [Capre]t was about 63%.

Muscarinic receptors inhibited presynaptic VDCCs in a way similar to adenosine (Ad) receptors. The percentage inhibition of ω‐CTx GVIA‐sensitive [Capre]t by Ad (100 μM) was about 59%. There was no significant inhibition of ω‐Aga IVA‐sensitive channels by Ad. The inhibitions of [Capre]t by CCh and Ad were mutually occlusive.

These results indicate that inhibition of synaptic transmission by muscarinic receptors is mainly the consequence of a reduction of the [Capre]t due to inhibition of presynaptic VDCCs.

British Journal of Pharmacology (1997) 122, 511–519; doi:10.1038/sj.bjp.0701400
An endogenous A2B adenosine receptor coupled to cyclic AMP generation in human embryonic kidney (HEK 293) cells
Authors: Cooper Jacqui , Hill Stephen J. , Alexander Stephen P. H.
Published: January 2005

Cyclic AMP generation by adenosine analogues was examined in human embryonic kidney (HEK 293) cells by use of a [3H]‐adenine pre‐labelling methodology.

Adenosine analogues showed the following rank order of potency (pD2 value): 5′‐N‐ethylcarboxamidoadenosine (NECA, 5.24)>2‐chloroadenosine (4.41) adenosine (4.19)=N6‐(2‐(4‐aminophenyl)‐ethylamino)adenosine (APNEA, 4.11). The A2A‐selective agonist CGS21680 failed to elicit a significant stimulation of cyclic AMP generation at concentrations below 30 μM.

Of these agents, NECA was observed to exhibit the greatest intrinsic activity, while in comparison maximal responses to adenosine (76±8% NECA response), 2‐chloroadenosine (70±6%) and APNEA (40±3%) were significantly reduced.

Antagonists of the NECA‐evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA2 value): CGS 15943 (7.79)=XAC (7.74)>DPCPX (7.01)=PD115199 (6.93)=8FB‐PTP (6.80)>KF 17837 (5.98)>3‐propylxanthine (5.13).

Agarose gel electrophoresis of the products of the polymerase chain reaction, with cDNA generated from HEK 293 cell total RNA showed virtually identical patterns and nucleotide sizes in comparison with the vector for the full length human brain A2B adenosine receptor.

We concluded that HEK 293 cells express an endogenous adenosine receptor coupled to cyclic AMP generation which is of the A2B subtype.

British Journal of Pharmacology (1997) 122, 546–550; doi:10.1038/sj.bjp.0701401
Acute gentamicin‐induced hypercalciuria and hypermagnesiuria in the rat: dose‐response relationship and role of renal tubular injury
Authors: Parsons P. P. , Garland H. O. , Harpur E. S. , Old S.
Published: January 2005

Standard renal clearance techniques were used to assess the dose‐response relationship between acute gentamicin infusion and the magnitude of hypercalciuria and hypermagnesiuria in the anaesthetized Sprague‐Dawley rat. Also investigated were whether these effects occurred independently of renal tubular cell injury.

Acute gentamicin infusion was associated with a significant hypercalciuria and hypermagnesiuria evident within 30 min of drug infusion. The magnitude of these responses was related to the dose of drug infused (0.14–1.12 mg kg−1 min−1). Increased urinary electrolyte losses resulted from a decreased tubular reabsorption of calcium and magnesium.

A rapid dose‐related increase in urinary N‐acetyl‐β‐D‐glucosaminidase (NAG) excretion was also observed in response to gentamicin infusion. However, there was no evidence of renal tubular cell injury and no myeloid bodies were observed within the lysosomes of the proximal tubular cells. Gentamicin may thus interfere with the mechanisms for cellular uptake and intracellular processing of NAG causing increased NAG release into the tubular lumen.

The absence of changes in renal cellular morphology indicates that the excessive renal losses of calcium and magnesium were an effect of gentamicin per se and not the result of underlying renal tubular injury. The renal effects described in this paper were apparent after administration of relatively low total drug doses, and with plasma concentrations calculated to be within the clinical range. These findings suggest that disturbances of plasma electrolyte homeostasis could occur in the absence of overt renal injury in patients receiving aminoglycoside antibiotics.

British Journal of Pharmacology (1997) 122, 570–576; doi:10.1038/sj.bjp.0701403
Characterization of action potential‐triggered [Ca2+]i transients in single smooth muscle cells of guinea‐pig ileum
Authors: Kohda M. , Komori S. , Unno T. , Ohashi H.
Published: January 2005

To characterize increases in cytosolic free Ca2+ concentration ([Ca2+]i) associated with discharge of action potentials, membrane potential and [Ca2+]i were simultaneously recorded from single smooth muscle cells of guinea‐pig ileum by use of a combination of nystatin‐perforated patch clamp and fura‐2 fluorimetry techniques.

A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca2+]i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca2+]i transients with an almost constant amplitude.

Nicardipine (1 μM) and La3+ (10 μM), blockers of voltage‐dependent Ca2+ channels (VDCCs), abolished both the action potential discharge and the [Ca2+]i transient.

Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca2+‐activated K+ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca2+]i transients.

Thapsigargin (1 μM), an inhibitor of SR Ca2+‐ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential‐triggered [Ca2+]i transients without affecting action potentials. In addition, in voltage‐clamped cells, the drug had little effect on the voltage step‐evoked Ca2+ current but exerted a similar effect on its concomitant rise in [Ca2+]i to that on the action potential‐triggered [Ca2+]i transient.

Similar action potential‐triggered [Ca2+]i transients were induced by brief exposures to high‐K+ solution. They were not decreased, but rather increased, after depletion of intracellular Ca2+ stores by a combination of ryanodine (30 μM) and caffeine (10 mM) through an open‐lock of Ca2+‐induced Ca2+ release (CICR)‐related channels.

The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca2+]i. Intracellular Ca2+ stores have a role, especially in accelerating the falling phase of the action potential‐triggered [Ca2+]i transients by replenishing cytosolic Ca2+. No evidence was provided for the involvement of CICR in the action potential‐triggered [Ca2+]i transient.

British Journal of Pharmacology (1997) 122, 477–486; doi:10.1038/sj.bjp.0701407
FcεRI‐mediated chloride uptake by rat mast cells: modulation by chloride transport inhibitors in relation to histamine secretion
Authors: Redrup A. C. , Foreman J. C. , Hayes N. A. , Pearce F. L.
Published: January 2005

We have examined the role of extracellular chloride in the mast cell secretion process. The immunologically‐directed ligand, antibody to IgE (anti‐IgE) required extracellular chloride ions for optimum secretion from rat peritoneal mast cells. In contrast, replacement of extracellular chloride did not alter the mast cell secretory response to compound 48/80, calcium ionophore A23187 or substance P.

Anti‐IgE‐stimulation of mast cells evoked a significant uptake of chloride ions compared to non‐stimulated cells. The magnitude of chloride uptake correlated with the magnitude of stimulated histamine secretion.

Compound 48/80, substance P and A23187 did not alter the rate of chloride ion uptake, although these agents caused significant histamine secretion.

The Na+/K+/2Cl− cotransport inhibitor, furosemide, reduced the rate of anti‐IgE‐stimulated chloride uptake at a relatively high concentration (700 μM). However, the more potent Na+/K+/2Cl− cotransport inhibitors, bumetanide (100 μM) and piretanide (100 μM) had no effect on the stimulated chloride uptake.

Furosemide inhibited anti‐IgE‐induced histamine secretion, bumetanide potentiated the response and piretanide had no effect. This suggests that their respective action on histamine secretion are unrelated to inhibition of the Na+/K+/2Cl− carrier.

The chloride channel blocker, 5‐nitro‐2‐((3‐phenylpropyl)‐amino)‐benzoic acid (NPPB), reduced both anti‐IgE‐stimulated chloride uptake and the corresponding histamine secretion in a dose‐dependent manner. The magnitude of the inhibitory action of the drug on these two cellular processes was comparable, implying that chloride channel activity is related to the mechanism of histamine secretion.

It is concluded that chloride uptake has a role in the control of FcεRI‐mediated histamine secretion from rodent mast cells.

British Journal of Pharmacology (1997) 122, 1188–1194; doi:10.1038/sj.bjp.0701418
Inhibition of nociceptin‐induced allodynia in conscious mice by prostaglandin D2
Authors: Minami Toshiaki , Okuda‐Ashitaka Emiko , Nishizawa Mikio , Mori Hidemaro , Ito Seiji
Published: January 2005

We recently showed that intrathecal administration of nociceptin induced allodynia by innocuous tactile stimuli and hyperalgesia by noxious thermal stimuli in conscious mice. In the present study, we examined the effect of prostaglandins on nociceptin‐induced allodynia and hyperalgesia.

Prostaglandin D2 (PGD2) blocked the allodynia induced by nociceptin in a dose‐dependent manner with an IC50 of 26 ng kg−1, but did not affect the nociceptin‐induced hyperalgesia at doses up to 500 ng kg−1. BW 245C (an agonist for PGD (DP) receptor) blocked the allodynia with an IC50 of 83 ng kg−1.

The blockade of nociceptin‐induced allodynia by PGD2 was reversed by the potent and selective DP‐receptor antagonist BW A868C in a dose‐dependent manner with an ED50 of 42.8 ng kg−1.

Glycine (500 ng kg−1) almost completely blocked the nociceptin‐induced allodynia. A synergistic effect on the inhibition of nociceptin‐evoked allodynia was observed between glycine and PGD2 at below effective doses.

Dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, blocked the nociceptin‐induced allodynia with an IC50 of 2.9 μg kg−1.

PGE2, PGF2α, butaprost (an EP2 agonist) and cicaprost (a PGI receptor agonist) did not affect the nociceptin‐induced allodynia.

These results demonstrate that PGD2 inhibits the nociceptin‐evoked allodynia through DP receptors in the spinal cord and that glycine may be involved in this inhibition.

British Journal of Pharmacology (1997) 122, 605–610; doi:10.1038/sj.bjp.0701421
Local neurogenic regulation of rat hindlimb circulation: role of calcitonin gene‐related peptide in vasodilatation after skeletal muscle contraction
Authors: Yamada Masami , Ishikawa Tomohisa , Fujimori Akira , Goto Katsutoshi
Published: January 2005

The mechanism of neurogenic regulation of skeletal muscle circulation was studied in the hindlimb of anaesthetized rats in vivo. Regional blood flow (RBF) of the hindlimb was recorded with a pulsed Doppler flow probe positioned in the iliac artery.

A short period (1 min) of sciatic nerve stimulation at 10 Hz caused a sustained increase in RBF (from 2.0±0.2 to 3.7±0.2 kHz at the peak), but no appreciable change in either MBP or HR, suggesting that the nerve stimulation produced local vasodilatation of the peripheral vasculature. The hyperaemic response reached a peak within 15 s and characteristically remained above the basal level for more than 5 min after the cessation of nerve stimulation. The response was regarded as a secondary response brought about by the contraction of skeletal muscles since (+)‐tubocurarine (0.73 μmol kg−1, i.a.) almost abolished it.

Lignocaine (43 μmol kg−1, i.a.) and capsaicin (0.33 μmol kg−1, i.a.) significantly suppressed the hyperaemic response to skeletal muscle contraction, suggesting that capsaicin‐sensitive sensory nerves contribute to the hyperaemia. In contrast, an inhibitor of NO synthase, Nω‐nitro‐L‐arginine methyl ester (1 μmol kg−1 min−1, i.v.), did not affect the hyperaemic response.

Serum levels of calcitonin gene‐related peptide (CGRP) in iliac venous effluent significantly increased from 51±4 to 77±5 fmol ml−1 during the hyperaemic response to skeletal muscle contraction. A bolus injection of CGRP (300 pmol kg−1, i.a.) induced a long‐lasting increase in RBF of the hindlimb. Moreover, CGRP(8–37) (100 nmol kg−1 min−1, i.v.), a specific CGRP1 receptor antagonist, significantly suppressed the hyperaemic response, especially the sustained phase of the response which was almost abolished by this antagonist.

These results suggest that CGRP, which is released from peripheral endings of capsaicin‐sensitive sensory nerves, partly mediates the hyperaemia evoked by skeletal muscle contraction of the rat hindlimb.

British Journal of Pharmacology (1997) 122, 703–709; doi:10.1038/sj.bjp.0701422
Augmentation of human neutrophil and alveolar macrophage LTB4 production by N‐acetylcysteine: role of hydrogen peroxide
Authors: Dent Gordon , Rabe Klaus F. , Magnussen Helgo
Published: January 2005

The actions of N‐acetylcysteine (NAC) on hydrogen peroxide (H2O2) and leukotriene B4 (LTB4) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated.

At a concentration of 100 μM, NAC significantly (P<0.01) suppressed the accumulation of H2O2 in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml−1)‐ or N‐formylmethionyl‐leucyl‐phenylalanine (fMLP; 1 μM)‐stimulated neutrophils and of resting and OZ‐stimulated macrophages. At concentrations of 10 μM and above, NAC augmented significantly the level of LTB4 in the supernatants of OZ‐ and fMLP‐stimulated neutrophils (P<0.01 and P<0.05, respectively) and OZ‐stimulated macrophages (P<0.05 at 10 μM, P<0.01 at 100 μM NAC).

NAC (100 μM) caused a significant (P<0.01) reduction in the quantity of measurable H2O2 when incubated with exogenous H2O2 concentrations equivalent to those released from OZ‐stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB4 when incubated with exogenous LTB4.

Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H2O2 had no significant effect on LTB4 production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H2O2 to H2O and O2, caused a pronounced, statistically significant (P<0.01) increase in the levels of LTB4 measured in the supernatants of OZ‐ and fMLP‐stimulated neutrophils.

H2O2 (12.5 μM and 25 μM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB4 (P=0.051 and P<0.05 at 12.5 μM and 25 μM, respectively) that was inhibited by NAC (100 μM) but not by catalase (400 u ml−1).

In conclusion, the anti‐oxidant drug, NAC, increases LTB4 production by human neutrophils and alveolar macrophages, probably through the elimination of cell‐derived H2O2. LTB4 undergoes a H2O2‐dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB4, suggesting that NAC may increase LTB4 production by blocking the H2O2‐dependent inhibition of a synthetic enzyme, such as 5‐lipoxygenase.

British Journal of Pharmacology (1997) 122, 758–764; doi:10.1038/sj.bjp.0701428
Stimulation of intracellular chloride accumulation by noradrenaline and hence potentiation of its depolarization of rat arterial smooth muscle in vitro
Authors: Davis Julian P. L. , Harper Alexander A. , Chipperfield Alan R.
Published: January 2005

Double‐barrelled ion‐selective microelectrodes were used to examine the effects of exogenous noradrenaline upon the membrane potential (Em) and intracellular chloride concentration ([Cl]i) of arterial smooth muscle from the saphenous branch of the femoral artery of the rat.

After treatment with 0.6 mM 6‐hydroxydopamine (to functionally denervate the tissue), exogenous noradrenaline (5 nM) caused repeatable depolarization of Em from −63.7±2.4 mV (s.d., n=18) to −53.8±3.4 mV (P<0.0001) and increases in [Cl]i from 31.0±0.5 mM to 42.5±2.2 mM (P<0.0001).

In the presence of 10 μM bumetanide (an inhibitor of (Na‐K‐Cl) cotransport), 5 nM noradrenaline caused a depolarization of Em of 3.0±3.2 mV, and a rise in [Cl]i of 4.5±2.5 mM.

In the presence of bumetanide and 1 mM acetazolamide (used as an inhibitor of a Na‐independent inward Cl pump), noradrenaline had no effect on Em or [Cl]i.

In the absence of extracellular chloride, the rise in apparent [Cl]i in response to 5 nM noradrenaline was abolished but there was a depolarization of 2.0±3.9 mV.

These results are consistent with the stimulation of (Na‐K‐Cl) cotransport and a Na‐independent Cl pump by exogenous noradrenaline and with the consequent increase in [Cl]i and shift in ECl potentiating the depolarization caused by noradrenaline. The possibility that modulation of [Cl]i may be a general mechanism of Em regulation is discussed.

British Journal of Pharmacology (1997) 122, 639–642; doi:10.1038/sj.bjp.0701431
Chemokine production by human vascular smooth muscle cells: modulation by IL‐13
Authors: Jordan Nicola J. , Watson Malcolm L. , Williams Robert J. , Roach Alan G. , Yoshimura Teizo , Westwick John
Published: January 2005

The production of chemokines by vascular smooth muscle cells (SMC) is implicated in the pathogenesis of atherosclerosis, although the factors regulating chemokine production by these cells are incompletely characterized.

We describe the differential stimulation of interleukin‐(IL)‐8, monocyte chemoattractant protein (MCP)‐1 and regulated on activation normal T‐cell expressed and secreted (RANTES) synthesis following treatment of human vascular SMC with IL‐1α or tumour necrosis factor α (TNFα). Under basal conditions, cultured SMC release very low amounts of IL‐8, MCP‐1 and RANTES as assessed by specific ELISA. Concentration‐response studies with IL‐1α or TNFα revealed that each stimulus induced a similar amount of MCP‐1. In contrast approximately three fold more IL‐8 was induced by IL‐1α than by TNFα whereas significant RANTES production was induced only by TNFα. These findings point to a divergence in the regulation of synthesis of the different chemokines in response to IL‐1α or TNFα stimulation.

The T‐cell derived cytokines IL‐10 and IL‐13 were also found to have differential effects on chemokine production by SMC. IL‐13, but not IL‐10, significantly enhanced IL‐8 and MCP‐1 release in response to IL‐1α or TNFα. This increase in chemokine release appeared to be accounted for by increased mRNA expression.

These findings provide support for the concept that smooth muscle cells can have an active role in a local immune response via the production of chemokines which can be selectively modulated by T‐cell derived cytokines.

British Journal of Pharmacology (1997) 122, 749–757; doi:10.1038/sj.bjp.0701433
The role of the IsK protein in the specific pharmacological properties of the IKs channel complex
Authors: Busch A. E. , Busch G. L. , Ford E. , Suessbrich H. , Lang H.‐J. , Greger R. , Kunzelmann K. , Attali B. , Stühmer W.
Published: January 2005

IKs channels are composed of IsK and KvLQT1 subunits and underly the slowly activating, voltage‐dependent IKs conductance in heart. Although it appears clear that the IsK protein affects both the biophysical properties and regulation of IKs channels, its role in channel pharmacology is unclear. In the present study we demonstrate that KvLQT1 homopolymeric K+ channels are inhibited by the IKs blockers 293B, azimilide and 17‐β‐oestradiol. However, IKs channels induced by the coexpression of IsK and KvLQT1 subunits have a 6–100 fold higher affinity for these blockers. Moreover, the IKs activators mefenamic acid and DIDS had little effect on KvLQT1 homopolymeric channels, although they dramatically enhanced steady‐state currents through heteropolymeric IKs channels by arresting them in an open state. In summary, the IsK protein modulates the effects of both blockers and activators of IKs channels. This finding is important for the action and specificity of these drugs as IsK protein expression in heart and other tissues is regulated during development and by hormones.

British Journal of Pharmacology (1997) 122, 187–189; doi:10.1038/sj.bjp.0701434
Imbalance between the endothelial cell‐derived contracting factors prostacyclin and angiotensin II and nitric oxide/cyclic GMP in human primary varicosis
Authors: Schuller‐Petrovic Sanja , Siedler Susanne , Kern Thomas , Meinhart Johann , Schmidt Kurt , Brunner Friedrich
Published: January 2005

The role of the endothelium in the vasomotor control of human veins in the lower extremity is little understood. We tested the hypothesis that the production of relaxing and contracting factors is altered in endothelial cells from varicose saphenous veins which may predispose to the decreased vessel tone observed in primary varicosis.

We determined the intracellular accumulation of guanosine 3′:5′‐cyclic monophosphate cyclic GMP; a measure of nitric oxide production) and the release of endothelin and prostacyclin (measured as its stable metabolite 6‐keto‐prostaglandin F1α) from cultured cells derived from the long saphenous veins of patients with primary varicosis (Varicose saphena group, n=27) or from patients undergoing coronary artery bypass surgery (Healthy saphena group, n=22). In addition, levels of endothelin, angiotensin II, bradykinin, cyclic GMP and cyclic AMP in plasma from patients with primary varicosis and healthy volunteers (n=8–11 in each group) were determined.

Although basal cyclic GMP levels were similar, more cyclic GMP accumulated in response to histamine (1–100 μmol l−1) in cells from varicose saphenous veins (0.75±0.1 pmol per well) than in cells from veins without varicosis (0.27±0.05 pmol per well). Furthermore, the relaxant potency of nitroprusside (1 nmol l−1–300 μmol l−1) in vitro was higher for varicose veins (mean EC50=5.9 μmol l−1; n=8) than healthy veins (mean EC50=20.0 μmol l−1; n=7).

The production of prostacyclin was significantly less in cells from varicose than healthy saphenous veins (66±8.7 and 121±20.1 nmol g−1 protein), but the production of endothelin was similar in both groups. Prostacyclin (3 nmol l−1–30 μmol l−1) consistently contracted rings of varicose saphenous vein in vitro with a mean EC50 value of 10–20 μmol l−1 (n=7); the maximum tension generated was ∼50% of that of a completely depolarizing solution of K+ (120 mmol l−1).

In plasma from patients with varicose veins, levels of cyclic GMP were higher than in healthy controls (9.2±0.03 and 7.2±0.02 nmol l−1), levels of angiotensin II were lower (81±11.5 and 147±21.7 pmol l−1), and levels of endothelin, cyclic AMP, and bradykinin were not different.

It is concluded that endothelial cells from diseased saphenous veins secrete less constrictor mediators than cells from healthy veins and that in diseased veins the nitric oxide/cyclic GMP system is up‐regulated which may shift the balance of vasoactive factors towards vasodilatation and contribute to the development of primary varicosis.

British Journal of Pharmacology (1997) 122, 772–778; doi:10.1038/sj.bjp.0701437
Muscarinic receptor subtypes controlling the cationic current in guinea‐pig ileal smooth muscle
Authors: Zholos Alexander V. , Bolton Thomas B.
Published: January 2005

The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch‐clamp recording techniques in guinea‐pig single ileal smooth muscle cells.

Ascending concentrations of carbachol (3–300 μM) activated the cationic conductance in a concentration‐dependent manner with conductance at a maximally effective carbachol concentration (Gmax) of 27.4±1.4 nS and a mean −log EC50 of 5.12±0.03 (mean±s.e.mean) (n=114).

Muscarinic antagonists with higher affinity for the M2 receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration‐effect curve to the right in a concentration‐dependent manner with pA2 values of 8.1, 8.0 and 9.1, respectively.

All M3 selective muscarinic antagonists tested, 4‐DAMP, p‐F‐HHSiD and zamifenacin, reduced the maximal response in a concentration‐dependent and non‐competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC50 for carbachol. At higher concentrations M3 antagonists shifted the agonist curve to the right, increasing the EC50, and depressed the maximum conductance response. Atropine, a non‐selective antagonist, produced both reduction in Gmax (M3 effect) and significant increase in the EC50 (M2 effect) in the same concentration range.

The depression of the conductance by 4‐DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPγS to the pipette (without application of carbachol) was unaffected.

The results support the hypothesis that carbachol activates M2 muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M3 receptors. As an increasing fraction of M3 receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of activated M2 receptors are disabled.

British Journal of Pharmacology (1997) 122, 885–893; doi:10.1038/sj.bjp.0701438