bluuebunny/crossref_metadata_2025 huggingface dataset:
Preservation of vascular function in rat mesenteric resistance arteries following cold storage, studied by small vessel myography
Authors: McIntyre C. A. , Williams B. C. , Lindsay R. M. , McKnight J. A. , Hadoke P. W. F.
Published: January 2005

The use of isolated blood vessels to investigate the physiological and pharmacological control of the vasculature is limited by the requirement to use freshly isolated vessels. Hence, the aim of this study was to determine whether vascular smooth muscle and endothelial cell function could be preserved in resistance arteries by storing them in physiological salt solution (PSS) at 4°C.

Third order mesenteric resistance arteries (mean internal diameter 237±6 μm) were dissected from the mesenteric bed of male Cob‐Wistar rats. The vessel segments were mounted in a small vessel myograph for measurement of isometric tension, and equilibrated at their optimum resting force. Contractile responses to noradrenaline (NA; 1×10−9–3×10−5 M), phenylephrine (PE, 1×10−9–3×10−5 M), potassium chloride (KCl; 2.5–140 mM) and endothelin (ET‐1, 1×10−11–3×10−7 M) and relaxant responses to acetylcholine (ACh; 1×10−9–3×10−5 M) and 3‐morpholinosydnonimine (SIN‐1; 1×10−9–1×10−4 M) were obtained in arteries, immediately after dissection (day 0) and following one to four days storage (day 1–day 4).

All arteries produced concentration‐dependent contractions in response to each of the vasoconstrictors. There were no significant differences in the magnitude or sensitivity (pD2) of the vasoconstrictor responses between fresh and stored vessels.

Arteries precontracted with NA to approximately 80% of the maximum response, relaxed in a concentration‐dependent manner in response to ACh and SIN‐1. Vessel storage for up to three days resulted in no change in response to ACh or SIN‐1.

Vessels analysed after four days of storage demonstrated a significant increase in sensitivity to ACh and SIN‐1 (−logIC50 (M) values; ACh; day 0, 7.46±0.13 vs day 4, 7.97±0.11, P<0.01 and SIN‐1; day 0, 4.87±0.10 vs day 4, 5.52±0.08, P<0.01). There was also a significant increase in the maximum relaxant response to ACh after four days of storage (% relaxation; day 0, 92.65±2.84 vs day 4, 100.36±0.36, P<0.05).

These results demonstrate that small resistance arteries remain viable if stored in PSS at 4°C for up to four days, with no loss in endothelial cell function. The altered sensitivity to the vasodilators on day 4 suggests that vessels should only be stored for up to three days following dissection for analysis of functional responses.

British Journal of Pharmacology (1998) 123, 1555–1560; doi:10.1038/sj.bjp.0701768
Characterization of 5‐HT receptors mediating constriction of porcine carotid arteriovenous anastomoses; involvement of 5‐HT1B/1D and novel receptors
Authors: De Vries Peter , Villalón Carlos M. , Heiligers Jan P. C. , Saxena Pramod R.
Published: January 2005

It was previously shown that porcine cranial arteriovenous anastomoses (AVAs) constrict to 5‐hydroxytryptamine (5‐HT), ergotamine, dihydroergotamine, as well as sumatriptan and that sumatriptan acts exclusively via 5‐HT1B/1D receptors. The present study was devoted to establish the contribution of 5‐HT1B/1D receptors in the constriction of AVAs elicited by 5‐HT (in presence of 0.5 mg kg−1 ketanserin), ergotamine and dihydroergotamine in anaesthetized pigs.

Intracarotid infusion of 5‐HT (2 μg kg−1 min−1) and intravenous doses of ergotamine (2.5–20 μg kg−1) and dihydroergotamine (3–100 μg kg−1) reduced AVA and increased nutrient blood flows and vascular conductances. The vasodilator response to 5‐HT, observed mainly in the skin and ear, was much more prominent than that of the ergot alkaloids.

Treatment with the 5‐HT1B/1D receptor antagonist GR127935 (0.5 mg kg−1, i.v.) significantly attenuated both ergot‐induced AVA constriction and arteriolar dilatation, whereas GR127935 only slightly affected the carotid vascular effects of 5‐HT.

The results suggest that 5‐HT constricts carotid AVAs primarily via receptors, which seem to differ from those (5‐HT1B/1D) stimulated by sumatriptan. The ergot alkaloids produce AVA constriction for a substantial part via 5‐HT1B/1D receptors, but also stimulate unidentified receptors. Both these non‐5‐HT1B/1D receptors may be targets for the development of novel antimigraine drugs.

The moderate vasodilator response to the ergot derivatives seems to be mediated, at least in part, by 5‐HT1B/1D receptors, whereas the arteriolar dilatation caused by 5‐HT may be mediated by other, possibly 5‐HT7 receptors.

British Journal of Pharmacology (1998) 123, 1561–1570; doi:10.1038/sj.bjp.0701770
Dependence of P2‐nucleotide receptor agonist‐mediated endothelium‐independent relaxation on ectonucleotidase activity and A2A‐receptors in rat portal vein
Authors: Guibert Christelle , Loirand Gervaise , Vigne Paul , Savineau Jean‐Pierre , Pacaud Pierre
Published: January 2005

The mechanism of action of P2 nucleotide receptor agonists that produce endothelium‐independent relaxation and the influence of ecto‐ATPase activity on this relaxing effect have been investigated in rat portal vein smooth muscle.

At 25°C, ATP, 2‐methylthioATP (2‐MeSATP) and 2‐chloroATP (2‐ClATP), dose‐dependently inhibited spontaneous contractile activity of endothelium‐denuded muscular strips from rat portal vein. The rank order of agonist potency defined from the half‐inhibitory concentrations was 2‐ClATP (2.7±0.5 μM, n=7)>ATP (12.9±1.1 μM, n=9)2‐MeSATP (21.9±4.8 μM, n=4). In the presence of αβ‐methylene ATP (αβ‐MeATP, 200 μM) which itself produced a transient contractile effect, the relaxing action of ATP and 2‐MeSATP was completely abolished and that of 2‐ClATP strongly inhibited.

The non‐selective P2‐receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS, 100 μM) did not affect the relaxation induced by ATP, 2‐MeSATP, and 2‐ClATP.

The A2A‐adenosine receptor antagonist ZM 241385 inhibited the ATP‐induced relaxation in a concentration‐dependent manner (1–100 nM). In the presence of 100 nM ZM 241385, the relaxing effects of 2‐MeSATP and 2‐ClATP were also inhibited.

ADP, AMP and adenosine also produced concentration‐dependent inhibition of spontaneous contractions. The relaxing effects of AMP and adenosine were insensitive to αβ‐MeATP (200 μM) but were inhibited by ZM 241385 (100 nM).

Simultaneous measurements of contraction and ecto‐ATPase activity estimated by the degradation of [γ‐32P]‐ATP showed that muscular strips rapidly (10–60 s) hydrolyzed ATP. This ecto‐ATPase activity was abolished in the presence of EDTA and was inhibited by 57±11% (n=3) by 200 μM αβ‐MeATP.

These results suggest that ATP and other P2‐receptor agonists are relaxant in rat portal vein smooth muscle, because ectonucleotidase activity leads to the formation of adenosine which activates A2A‐receptors.

British Journal of Pharmacology (1998) 123, 1732–1740; doi:10.1038/sj.bjp.0701773
Involvement of protein kinases in the induction of NO synthase II in human DLD‐1 cells
Authors: Kleinert Hartmut , Euchenhofer Christian , Fritz Gerhard , Ihrig‐Biedert Irmgard , Förstermann Ulrich
Published: January 2005

Protein phosphorylation is involved in the induction of nitric oxide synthase II (NOS II, iNOS) in several types of animal cells. Here we have investigated the possible involvement of major protein kinases in the induction of NOS II expression in human DLD‐1 cells.

In DLD‐1 cells, interferon‐γ alone induced a submaximal NOS II expression; a cytokine mixture consisting of interferon‐γ, tumour necrosis factor‐α and interleukin‐1β produced maximal NOS II induction.

Activators of protein kinase A (forskolin, 8‐dibutyryl‐cyclic AMP), of protein kinase C (tetradecanoylphorbol‐13‐acetate), and of protein kinase G (8‐bromo cyclic GMP) did not induce NOS II mRNA by themselves, nor did they alter NOS II mRNA induction in response to cytokines.

Inhibitors of protein kinase A (compound H89), of protein kinase C (bisindolylmaleimide, chelerythrine or staurosporine), of phosphatidylinositol 3‐kinase (wortmannin), of p38 mitogen‐activated protein kinase (compound SB 203580) and of extracellular signal‐regulated kinase (compound PD 98059) also had no influence on basal or cytokine‐induced NOS II mRNA expression.

Immunoprecipitation kinase assays showed no activation of extracellular signal‐regulated kinase or p38 mitogen‐activated protein kinase in cytokine‐incubated DLD‐1 cells. The c‐Jun NH2‐terminal kinase was activated by cytokines, but the most efficacious cytokine was tumour necrosis factor‐α which did not induce NOS II by itself.

In contrast, the protein tyrosine kinase inhibitor tyrphostin B42 (a specific inhibitor of interferon‐γ‐activated janus kinase 2) and the protein tyrosine kinase inhibitor tyrphostin A25 both reduced CM‐induced NOS II mRNA expression in a concentration‐dependent manner.

These results suggest that activation of NOS II expression in DLD‐1 cells is independent of the activities of protein kinases A, C and G, phosphatidylinositol 3‐kinase, extracellular signal regulated kinase and p38 mitogen‐activated protein kinase, but seems to require protein tyrosine kinase activity, especially the interferon‐γ‐activated janus kinase 2.

British Journal of Pharmacology (1998) 123, 1716–1722; doi:10.1038/sj.bjp.0701782
Further insights into the anti‐aggregating activity of NMDA in human platelets
Authors: Franconi Flavia , Miceli Mauro , Alberti Luisa , Seghieri Giuseppe , De Montis M Graziella , Tagliamonte Alessandro
Published: January 2005

In the present study the effect of N‐methyl‐D‐aspartate (NMDA) on thromboxane B2 synthesis and on [Ca2+]i was studied in human platelets.

NMDA (10−7M) completely inhibited the synthesis of thromboxane B2 from exogenous arachidonic acid (AA), while it did not interfere with the aggregating effect of the thromboxane A2 receptor agonist U‐46619.

NMDA (0.1 μM–10 μM) dose‐dependently increased intracellular calcium in washed platelets pre‐loaded with fura 2 AM, and this effect was not additive with that of AA.

NMDA shifted the dose‐response curve of AA to the right. At the highest AA concentrations platelet aggregation was not inhibited.

The antiaggregating effect of NMDA was not antagonized by NG‐monomethyl‐L‐arginine (L‐NMMA), a nitric oxide synthase (NOS) inhibitor.

Finally, NMDA (0.01 nM–100 nM) associated with either aspirin or indomethacin significantly potentiated the antiaggregating activity of both cyclo‐oxygenase inhibitors.

It was concluded that NMDA is a potent inhibitor of platelet aggregation and thromboxane B2 synthesis in human platelet rich plasma (PRP).
Pharmacological characterization of muscarinic receptors in the uterus of oestrogen‐primed and pregnant rats
Authors: Munns Melinda , Pennefather Jocelyn N.
Published: January 2005

Radioligand binding and contractility studies were undertaken to determine the subtype/s of muscarinic receptors present in uteri of oestrogen‐treated and late pregnant rats.

Competition binding studies with uterine membrane preparations and [3H]‐QNB (quinuclidinyl benzilate) provided negative log dissociation constants (pKi) for each antagonist as follows; oestrogen‐treated – atropine (7.98)himbacine (7.83)>methoctramine (7.52)hexahydrosiladiphenidol (HHSiD; 7.32)5,11‐dihydro‐11‐[[[2‐[2 ‐ [(dipropylamino)methyl] ‐ 1piperidinyl]ethyl]amino] ‐ carbonyl] ‐ 6H‐pyrido‐ [2,3 ‐ b][1,4] ‐ benzodiazepin ‐ 6‐one (AF ‐ DX 384; 7.10)>11 ‐ [[2 ‐ [(diethylamino)methyl]‐1‐piperidinyl]‐ acetyl]5,11‐dihydro‐6H‐pyridol]2,3,‐b][1,4]benzodiazepin‐6‐one (AF‐DX 116, 6.77)>pirenzepine (6.17); late pregnant – atropine (8.05)methoctramine (7.95)himbacine (7.71)HHSiD (7.52)AF‐DX 384 (7.34)>AF‐DX 116 (6.72)>pirenzepine (6.18).

The potency of carbachol in causing uterine contraction was similar in preparations from pregnant and non‐pregnant animals (pD2=5.57 and 5.46, respectively). Each muscarinic antagonist caused parallel, rightward shifts of carbachol concentration‐response curves. The pA2 estimates were: oestrogen‐treated – atropine (9.42)>himbacine (8.73)HHSiD (8.68)methoctramine (8.49)AF‐DX 384 (7.91)AF‐DX 116 (7.36)pirenzepine (7.26); late pregnant – atropine (9.48)>himbacine (8.37)HHSiD (8.22)methoctramine (8.01)AF‐DX 116 (7.73)AF‐DX 384 (7.44)pirenzepine (6.92).

The relative pKi estimates for antagonists obtained in membrane preparations from oestrogen‐treated rats suggest the presence of muscarinic M2 subtypes. In functional studies pA2 values indicated the additional presence of muscarinic M3 receptor or, possibly an atypical receptor subtype. The similarity between pKi and pA2 estimates obtained in uteri from oestrogen‐treated and pregnant animals, respectively, indicates that pregnancy does not affect myometrial muscarinic receptors in the rat.

British Journal of Pharmacology (1998) 123, 1639–1644; doi:10.1038/sj.bjp.0701794
Comparative, general pharmacology of SDZ NKT 343, a novel, selective NK1 receptor antagonist
Authors: Walpole C S J , Brown M C S , James I F , Campbell E A , McIntyre P , Docherty R , Ko S , Hedley L , Ewan S , Buchheit K‐H , Urban L A
Published: January 2005

The in vitro and in vivo pharmacology of SDZ NKT 343 (2‐nitrophenyl‐carbamoyl‐(S)‐prolyl‐(S)‐3‐(2‐naphthyl)alanyl‐N‐benzyl‐N‐methylamide), a novel tachykinin NK1 receptor antagonist was investigated.

SDZ NKT 343 inhibited [3H]‐substance P binding to the human NK1 receptor in transfected Cos‐7 cell membranes (IC50=0.62±0.11 nM). In comparison, in the same assay Ki values for FK888, CP 99,994, SR 140,333 and RPR 100,893 were 2.13±0.04 nM, 0.96±0.20 nM, 0.15±0.06 nM and 1.77±0.41 nM, respectively. SDZ NKT 343 showed a markedly lower affinity at rat NK1 receptors in whole forebrain membranes (IC50=451±139 nM).

SDZ NKT 343 caused an increase in EC50 as well as reduction in the number of binding sites (Bmax) determined for [3H]‐substance P, suggesting a non‐competitive interaction at the human NK1 receptor. SDZ NKT 343 also caused a reduction in the maximum elevation of [Ca2+]i evoked by substance P (SP) in human U373MG cells and depressed the maximum [Sar9]SP sulphone‐induced contraction of the guinea‐pig isolated ileum. The antagonism of SP effects on U373MG cells by SDZ NKT 343 was reversible.

SDZ NKT 343 showed weak affinity to human NK2 and NK3 receptors in transfected Cos‐7 cells (Ki of 0.52±0.04 μM and 3.4±1.2 μM, respectively). SDZ NKT 343 was inactive in a broad array of binding assays including the bradykinin B2 receptor the histamine H1 receptor, opiate receptors and adrenoceptors. SDZ NKT 343 only weakly inhibited the voltage‐activated Ca2+ and Na+currents in guinea‐pig dorsal root ganglion neurones. The enantiomer of SDZ NKT 343, (R,R)‐SDZ NKT 343 was about 1000 times less active at human NK1 receptors expressed in Cos‐7 cell membranes.

Contractions of the guinea‐pig ileum by [Sar9]SP sulphone were inhibited by SDZ NKT 343 in a concentration‐dependent manner, with an IC50=1.60±0.94 nM, while the enantiomer (R,R)‐SDZ NKT 343 was 100 times less active (IC50=162±26 nM). In comparison, in the same assay IC50 values for other NK1 receptor antagonists CP 99,994, SR 140,333, RPR 100,893 and FK 888 were 2.90±07 nM, 0.14±0.02 nM, 11.4±2.9 nM and 2.4±0.83 nM, respectively.

In anaesthetized guinea‐pigs i.v. administered SDZ NKT 343 antagonized [Sar9]SP sulphone‐evoked bronchoconstriction (70% reduction at 0.4 mg kg−1, i.v.). Basal airway resistance, mean arterial blood pressure and heart rate were not affected.

In conclusion, SDZ NKT 343 is a highly selective NK1 receptor antagonist with high potency at the human and guinea‐pig receptors. SDZ NKT 343 may be used as a potential novel therapeutic agent in human diseases where NK1 receptor hyperfunction is involved.
Molecular cloning, expression and characterization of cDNA encoding a mouse α1a‐adrenoceptor
Authors: Xiao Lei , Scofield Margaret A , Jeffries William B
Published: January 2005

In this study, we have cloned, expressed, and characterized an α1a‐adrenoceptor gene from the mouse. We designed oligonucleotide PCR primers complementary to regions of the rat α1a‐adrenoceptor sequence and amplified cDNA fragments from total RNA of mouse cerebral cortex, liver and kidney by reverse transcription‐polymerase chain reaction (RT–PCR).

Both the nucleotide and deduced peptide sequences of the cDNA showed high sequence identity with those of cloned α1a‐adrenoceptors from other species. The cDNA clone had an open reading frame of 1398 nucleotides encoding a 466 amino acid peptide which had 97%, 92% and 90% identity with the deduced amino acid sequences of the rat, human and bovine α1a‐adrenoceptor, respectively.

The amplified mouse cDNA was inserted into a mammalian expression vector pcDNA3.1(+) and expressed in COS‐1 cells. The pharmacological properties of the mouse cDNA clone were examined in radioligand binding studies and functional assays. The expressed mouse protein had a high affinity for [3H]‐prazosin (Kd=0.48 nM) and pattern of affinity for antagonists in competition studies that is similar to that of the rat α1a‐adrenoceptor. Chloroethylclonidine (CEC) could slowly alkylate the expressed protein, with a rate similar to that of the rat α1a‐adrenoceptor.

The expressed receptors were able to mediate noradrenaline (NA) stimulation of the production of inositol phosphates in COS‐1 cells, consistent with coupling to phospholipase C. This response to NA could be reversed by pretreatment of the transfected cells with prazosin.

Based on the above evidence, we concluded that the cloned cDNA is that of the mouse α1a‐adrenoceptor.
Enhancement of opioid inhibition of GABAergic synaptic transmission by cyclo‐oxygenase inhibitors in rat periaqueductal grey neurones
Authors: Vaughan C. W.
Published: January 2005

Cyclo‐oxygenase (COX) inhibitors potentiate opioid inhibition of GABAergic synaptic transmission in rat periaqueductal grey (PAG) (Vaughan et al., 1997). In the present study, the relative contribution of cyclo‐oxygenase‐1 (COX‐1) and COX‐2 inhibition to this phenomenon was examined by use of whole‐cell patch clamp recordings in brain slices. The μ‐receptor partial agonist morphine (10 μM) had little effect on GABAergic synaptic transmission. Morphine reduced the frequency of spontaneous miniature inhibitory postsynaptic currents (m.i.p.s.cs) by 13%. The nonselective COX inhibitor, indomethacin, produced a dose‐dependent potentiation of the morpine inhibition of m.i.p.s.c. frequency (maximum inhibition 42%, IC50=6 nM). More selective COX‐2 inhibitors produced a similar potentiation of the morphine inhibition of m.i.p.s.c. frequency; however, at greater concentrations (IC50=57 nM piroxicam, 1.7 μM DFU). Maintaining slices in the protein synthesis inhibitor cycloheximide (1 μM), to prevent COX‐2 induction, had no effect on the potentiation action of DFU (10 μM). These results demonstrate that the potentiation of opioid inhibition of GABAergic synaptic transmission in PAG is largely a result of inhibition of COX‐1 activity. These findings suggest that COX‐1, rather than COX‐2 inhibition, mediates the synergistic analgesic actions of opioids and non‐steroidal anti‐inflammatory drugs (NSAIDs) in the midbrain PAG.

British Journal of Pharmacology (1998) 123, 1479–1481; doi:10.1038/sj.bjp.0701818
Respiratory and cardiovascular effects of the μ‐opioid receptor agonist [Lys7]dermorphin in awake rats
Authors: Negri Lucia , Lattanzi Roberta , Tabacco Fabio , Melchiorri Pietro
Published: January 2005

Changes in respiratory variables, arterial blood pressure and heart rate were studied in awake rats after injection of the opioid peptide [Lys7]dermorphin and its main metabolites, [1‐5]dermorphin and [1‐4]dermorphin.

Fifteen minutes after injection, doses of [Lys7]dermorphin producing antinociception (i.c.v., 36–120 nmol; s.c., 0.12–4.7 μmol kg−1) significantly increased respiratory frequency and minute volume of rats breathing air or hypoxic inspirates. This respiratory stimulation was reversed to depression by the 5‐HT receptor antagonist ritanserin (2 mg kg−1, s.c.), was blocked by naloxone (0.1 mg kg−1, s.c.), significantly reduced by the μ1 opioid receptor antagonist naloxonazine (10 mg kg−1, s.c., 24 h before) but unaffected by peripherally acting opioid antagonist naloxone methyl bromide (3 mg kg−1, s.c.). Forty five minutes after injection, doses of the peptide producing catalepsy (s.c., 8.3–14.2 μmol kg−1, i.c.v., 360 nmol) significantly reduced respiratory frequency and volume of rats breathing air and blocked the hypercapnic ventilator response of rats breathing from 4% to 10% CO2. I.c.v. administration of [1‐5]dermorphin and [1‐4]dermorphin (from 36 to 360 nmol) never stimulated respiration but significantly reduced basal and CO2‐stimulated ventilation. Opioid respiratory depression was only antagonized by naloxone.

In awake rats, [Lys7]dermorphin (0.1–1 mg kg−1, s.c.) decreased blood pressure. This hypotensive response was abolished by naloxone, reduced by naloxone methyl bromide and unaffected by naloxonazine.

In conclusion, the present study indicates that analgesic doses of [Lys7]dermorphin stimulate respiration by activating central μ1 opioid receptors and this respiratory stimulation involves a forebrain 5‐hydroxytryptaminergic excitatory pathway.
Outward current produced by somatostatin (SRIF) in rat anterior cingulate pyramidal cells in vitro
Authors: Hicks G A , Feniuk W , Humphrey P P A
Published: January 2005

A high density of receptors for somatostatin (SRIF) exists in the anterior cingulate cortex but their function is unknown. Whole‐cell patch clamp recordings were made from visualized deep layer pyramidal cells of the rat anterior cingulate cortex contained in isolated brain slices to investigate the putative effects of SRIF and to identify the receptor subtype(s) involved.

SRIF (1–1000 nM) produced a concentration‐dependent outward current which was associated with an increased membrane conductance, was sensitive to Ba2+ (300 μM–1 mM), and was absent in the presence of a maximal concentration of the GABAB receptor agonist, baclofen (100 μM). These observations suggest the outward current was carried by K+ ions.

SRIF analogues also elicited outward currents with a rank potency order of (EC50, nM): octreotide (1.8)>BIM‐23027 (3.7)>SRIF (20)=L‐362,855 (20). BIM‐23056 was without agonist or antagonist activity. Responses to L‐362,855 were unlike those to the other agonists since they were sustained for the duration of the application.

The sst2 receptor antagonist, L‐Tyr8Cyanamid 154806 (1 μM), had no effect alone but partially reversed responses to submaximal concentrations of SRIF (100 nM, 44±6% reversal) and L‐362,855 (100 nM, 70±6% reversal) and fully reversed the response to BIM‐23027 (10 nM). In contrast, L‐Tyr8Cyanamid 154806 did not antagonize the response to baclofen (10 μM).

We conclude that SRIF activates a K+ conductance in anterior cingulate pyramidal neurones via an action predominantly at sst2 receptors.
Modulation of striatal quinolinate neurotoxicity by elevation of endogenous brain kynurenic acid
Authors: Harris C A , Miranda A F , Tanguay J J , Boegman R J , Beninger R J , Jhamandas K
Published: January 2005

Nicotinylalanine, an inhibitor of kynurenine metabolism, has been shown to elevate brain levels of endogenous kynurenic acid, an excitatory amino acid receptor antagonist. This study examined the potential of nicotinylalanine to influence excitotoxic damage to striatal NADPH diaphorase (NADPH‐d) and γ‐aminobutyric acid (GABA)ergic neurones that are selectively lost in Huntington's disease.

A unilateral injection of the N‐methyl‐D‐aspartate (NMDA) receptor agonist, quinolinic acid, into the rat striatum produced an 88% depletion of NADPH‐d neurones. Intrastriatal infusion of quinolinic acid also produced a dose‐dependent reduction in striatal GABA content.

Nicotinylalanine (2.3, 3.2, 4.6, 6.4 nmol 5 μl−1, i.c.v.) administered with L‐kynurenine (450 mg kg−1), a precursor of kynurenic acid, and probenecid (200 mg kg−1), an inhibitor of organic acid transport, 3 h before the injection of quinolinic acid (15 nmol) produced a dose‐related attenuation of the quinolinic acid‐induced loss of NADPH‐d neurones. Nicotinylalanine (5.6 nmol 5 μl−1) in combination with L‐kynurenine and probenecid also attenuated quinolinic acid‐induced reductions in striatal GABA content.

Nicotinylalanine (4.6 nmol, i.c.v.), L‐kynurenine alone or L‐kynurenine administered with probenecid did not attenuate quinolinic acid‐induced depletion of striatal NADPH‐d neurones. However, combined administration of kynurenine and probenecid did prevent quinolinic acid‐induced reductions in ipsilateral striatal GABA content.

Injection of nicotinylalanine, at doses (4.6 nmol and 5.6 nmol i.c.v.) which attenuated quinolinic acid‐induced striatal neurotoxicity, when combined with L‐kynurenine and probenecid produced increases in both whole brain and striatal kynurenic acid levels. Administration of L‐kynurenine and probenecid without nicotinylalanine also elevated kynurenic acid, but to a lesser extent.

The results of this study demonstrate that nicotinylalanine has the potential to attenuate quinolinic acid‐induced striatal neurotoxicity. It is suggested that nicotinylalanine exerts its effect by increasing levels of endogenous kynurenic acid in the brain. The results of this study suggest that agents which influence levels of endogenous excitatory amino acid antagonists such as kynurenic acid may be useful in preventing excitotoxic damage to neurones in the CNS.
Modulation of angiotensin‐converting enzyme by nitric oxide
Authors: Ackermann A , Fernández‐Alfonso M S , Sánchez de Rojas R , Ortega T , Paul M , González C
Published: January 2005

The aim of the present study was to determine the effect of nitric oxide (NO) on angiotensin‐converting enzyme (ACE) activity.

A biochemical study was performed in order to analyse the effect of the NO‐donors, SIN‐1 and diethylamine/NO (DEA/NO), and of an aqueous solution of nitric oxide on the ACE activity in plasma from 3‐month old male Sprague‐Dawley rats and on ACE purified from rabbit lung. SIN‐1 significantly inhibited the activity of both enzymes in a concentration‐dependent way between 1 and 100 μM. DEA/NO inhibited the activity of purified ACE from 0.1 μM to 10 μM and plasma ACE, with a lower potency, between 1 and 100 μM. An aqueous solution of NO (100 and 150 μM) also inhibited significantly the activity of both enzymes. Lineweaver‐Burk plots indicated an apparent competitive inhibition of Hip‐His‐Leu hydrolysis by NO‐donors.

Modulation of ACE activity by NO was also assessed in the rat carotid artery by comparing contractions elicited by angiotensin I (AI) and AII. Concentration‐response curves to both peptides were performed in arteries with endothelium in the presence of the guanylyl cyclase inhibitor, ODQ (10 μM), and the inhibitor of NO formation, L‐NAME (0.1 mM). NO, which is still released from endothelium in the presence of 10 μM ODQ, elicited a significant inhibition of AI contractions at low concentrations (1 and 5 nM). In the absence of endothelium, 1 μM SIN‐1 plus 10 μM ODQ, as well as 10 μM DEA/NO plus 10 μM ODQ induced a significant inhibition on AI‐induced contractions at 1 and 5 nM and at 1–100 nM, respectively.

In conclusion, we demonstrated that (i) NO and NO‐releasing compounds inhibit ACE activity in a concentration‐dependent and competitive way and that (ii) NO release from endothelium physiologically reduces conversion of AI to AII.
Competitive and selective antagonism of P2Y1 receptors by N6‐methyl 2′‐deoxyadenosine 3′,5′‐bisphosphate
Authors: Boyer José L , Mohanram Arvind , Camaioni Emidio , Jacobson Kenneth A , Harden T Kendall
Published: January 2005

The antagonist activity of N6‐methyl 2′‐deoxyadenosine 3′,5′‐bisphosphate (N6MABP) has been examined at the phospholipase C‐coupled P2Y1 receptor of turkey erythrocyte membranes. N6MABP antagonized 2MeSATP‐stimulated inositol phosphate hydrolysis with a potency approximately 20 fold greater than the previously studied parent molecule, adenosine 3′,5′‐bisphosphate. The P2Y1 receptor antagonism observed with N6MABP was competitive as revealed by Schild analysis (pKB=6.99±0.13). Whereas N6MABP was an antagonist at the human P2Y1 receptor, no antagonist effect of N6MABP was observed at the human P2Y2, human P2Y4 or rat P2Y6 receptors.
The contribution of nitric oxide to cardiovascular status and responses to vasodilators in conscious, hypertensive, transgenic ((mRen‐2)27) rats
Authors: Gardiner S M , March J E , Kemp P A , Bennett T
Published: January 2005

The aim of the study was to measure the regional haemodynamic responses to vasodilators, and the effects of nitric oxide (NO) synthase inhibition, in conscious, hypertensive, transgenic ((mRen‐2)27) rats (TG rats) and normotensive, Hannover Sprague‐Dawley (SD) rats.

The hypotensive response to acetylcholine was greater in TG than in SD rats, but the renal vasodilator responses were not different.

The responses to bradykinin were similar in the two strains, except that hindquarters vasodilatation occurred only in SD rats.

Salbutamol caused smaller renal and hindquarters vasodilatation in TG rats than in SD rats, and there was mesenteric vasodilatation only in the latter strain.

The hypotensive response to sodium nitroprusside was smaller, but the accompanying mesenteric vasodilatation was greater, in SD than in TG rats.

The contribution of NO to the vasodilator responses was taken as the difference between the responses in the presence of the NO synthase inhibitor, NG‐nitro‐L‐arginine methylester (L‐NAME), compared to those in the presence of a co‐infusion of angiotensin II and vasopressin (to match the haemodynamic effects of L‐NAME).

In TG rats, L‐NAME caused a greater absolute pressor effect, but a smaller mesenteric vasoconstriction, than in SD rats.

L‐NAME affected the vasodilator responses to all the challenges similarly in the two strains.

Collectively, the results provide no direct evidence for impaired NO‐mediated vasodilator mechanisms in TG rats. It is feasible that the reduced hindquarters response to bradykinin and the reduced renal and hindquarters responses to salbutamol, in TG rats are due to abnormal β2‐adrenoceptor‐mediated processes.
The profile of sabcomeline (SB‐202026), a functionally selective M1 receptor partial agonist, in the marmoset
Authors: Harries M H , Samson N A , Cilia J , Hunter A J
Published: January 2005

Sabcomeline (SB‐202026, 0.03 mg kg−1, p.o.), a potent and functionally selective M1 receptor partial agonist, caused a statistically significant improvement in the performance of a visual object discrimination task by marmosets. No such improvement was seen after RS86 (0.1 mg kg−1, p.o.).

Initial learning, which only required an association of object with reward and an appropriate response to be made, was not significantly affected. Reversal learning, which required both the extinction of the previously learned response and the acquisition of a new response strategy, was significantly improved after administration of sabcomeline (0.03 mg kg−1, p.o.).

Sabcomeline (0.03 and 0.1 mg kg−1, p.o.) had no significant effect on mean blood pressure measured for 2 h after administration in the conscious marmoset.

Sabcomeline (0.03 mg kg−1, p.o.) caused none of the overt effects such as emesis or behaviours often seen after the administration of muscarinic agonists, e.g. face rubbing and licking.

This is the first study to demonstrate cognitive enhancement by a functionally selective M1 receptor partial agonist in a normal (i.e. non‐cognitively impaired) non‐human primate and this effect was seen at a dose which did not cause side effects.

Perseverative behaviour and deficient acquisition of new information are seen in patients with Alzheimer's disease (AD). Therefore the data suggest that sabcomeline might be of therapeutic benefit in the treatment of AD.
Superoxide and endothelium‐dependent constriction to flow in porcine small pulmonary arteries
Authors: Liu Qiang , Wiener Charles M , Flavahan Nicholas A
Published: January 2005

The aim of this study was to determine the response of porcine small pulmonary arteries to intralumenal flow and to identify the cellular mechanisms and potential mediators involved in the response.

Porcine small pulmonary arteries were isolated from a branch of the main intrapulmonary artery of the lower lung lobe and studied in a perfusion myograph system that allowed independent control of transmural pressure and intralumenal flow. At a transmural pressure of 20 mmHg, the baseline internal diameter (BID) of the arteries was 251.2±16.1 μm (n=16).

Under quiescent conditions or during constriction with U46619 to ∼60% of BID, intralumenal flow caused reversible constriction in arteries with endothelium (in the presence of U46619, flow decreased diameter from 60.0±2.5% to 49.5±3.0% BID at 10 μl min−1, n=16, P<0.05) but no change in diameter of arteries without endothelium.

In the presence of superoxide dismutase (SOD, 150 u ml−1), the response to flow was converted from constriction to vasodilatation (in presence of U46619 and SOD, flow increased diameter from 54.2±3.4% to 76.7±4.5% BID at 10 μl min−1, n=10, P<0.05). Inhibition of NO synthase with L‐NAME (3×10−5 M) abolished the flow‐induced vasodilatation occurring in the presence of SOD and the flow‐induced constriction occurring in the absence of SOD. In arteries with endothelium, L‐NAME (3×10−5 M) caused significant vasoconstriction, whereas SOD did not alter vasomotor tone.

Acetylcholine (10−8 to 10−6 M) caused endothelium‐dependent relaxation of small pulmonary arteries that was not significantly affected by SOD (150 u ml−1) but was inhibited by L‐NAME (3×10−5 M).

These results suggest that in small, porcine, isolated pulmonary arteries, intralumenal flow increases the production of NO but this is obscured by the generation of superoxide which causes vasoconstriction.
Effect of rolipram and dibutyryl cyclic AMP on resequestration of cytosolic calcium in FMLP‐activated human neutrophils
Authors: Anderson R , Mahomed A Goolam , Theron A J , Ramafi G , Feldman C
Published: January 2005

We have investigated the effects of the selective phosphodiesterase (PDE) type 4 inhibitor, rolipram (0.01–1 μM) on cytosolic Ca2+ fluxes in FMLP‐activated human neutrophils, as well as on superoxide production by, and release of elastase from, these cells.

Cytosolic Ca2+ fluxes were measured by use of fura‐2 spectrofluorimetry in combination with a radiometric procedure that enables distinction between net efflux and influx of the cation. Superoxide production and elastase release were measured by lucigenin‐enhanced chemiluminescence and a colorimetric procedure, respectively.

Pretreatment of neutrophils with rolipram did not affect the FMLP‐activated release of Ca2+ from intracellular stores, but was associated with dose‐related acceleration of the rate of decline in fura‐2 fluorescence and with decreased efflux, as well as store‐operated influx of 45Ca2+, indicative of enhancement of resequestration of the cation by the endo‐membrane Ca2+‐ATPase.

Inhibition of superoxide production and elastase release was observed at concentrations of rolipram which accelerated the clearance of Ca2+ from the cytosol of FMLP‐activated neutrophils.

These effects of rolipram on FMLP‐activated Ca2+ fluxes, superoxide generation and elastase release were mimicked by pretreatment of neutrophils with dibutyryl cyclic AMP (0.5–4 mM), while theophylline (10–150 μM), a non‐specific PDE inhibitor, as well as the β2‐agonist, salbutamol, were less effective.

We conclude that rolipram deactivates FMLP‐stimulated human neutrophils by enhancement of cyclic AMP‐dependent resequestration of cytosolic Ca2+.
The effect of nitric oxide on cytokine‐induced release of PGE2 by human cultured astroglial cells
Authors: Mollace Vincenzo , Colasanti Marco , Muscoli Carolina , Lauro Giuliana M , Iannone Michelangelo , Rotiroti Domenicantonio , Nistico' Giuseppe
Published: January 2005

The role of the L‐arginine‐nitric oxide (NO) pathway on the formation of prostaglandin E2 (PGE2) by human cultured astroglial cells incubated with interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) was investigated.

Incubation of T 67 astroglial cell line with IL‐β (10 ng ml−1) and TNF‐α (500 u ml−1) produced a significant (P<0.05) increase of both nitrite (the breakdown product of NO), cyclic GMP and PGE2 levels in cell supernatants. Nω‐nitro‐L‐arginine methyl ester (L‐NAME; 20–300 μM), an inhibitor of NO synthase (NOS), inhibited the increase of cyclic GMP and nitrite levels found in supernatants of cytokine‐treated astroglial cells and reduced the release of PGE2. The latter effect showed that the enhanced arachidonic acid (AA) metabolism subsequent to stimulation of astroglial cells with IL‐1β and TNF‐α was, at least in part, induced by NO. This occurred also when sodium nitroprusside (SNP; 120 μM), an NO donor, was incubated with astroglial cells, an effect antagonized by oxyhaemoglobin (OxyHb; 10 μM).

The inhibition elicited by L‐NAME on PGE2‐release by cytokine‐treated astroglial cells was reversed by adding AA (40 μM), showing that the effect of NO on cytokine‐dependent PGE2 release occurred at the cyclo‐oxygenase (COX) level. Furthermore, the release of PGE2 in cytokine‐treated astroglial cells was inhibited by indomethacin (10 μM), a COX inhibitor as well as by preincubating cells with dexamethasone (20 μM), an inhibitor of inducible enzymes, showing that the inducible isoform of COX (COX‐2) was involved.

On the other hand, pretreating astroglial cells with methylene blue (MB; 10 μM), an inhibitor of NO biological activity acting at the guanylate cyclase level, failed to affect PGE2 release in cytokine‐treated astroglial cells, leading to the conclusion that cyclic GMP changes related to NO formation are not involved in the generation of AA metabolites.

The present experiments demonstrated that the release of PGE2 by astroglial cells pretreated with IL‐1β and TNF‐α is due to enhanced COX‐2 activity via activation of the L‐arginine‐NO pathway, and this may be relevant to the understanding of the pathophysiological mechanisms underlying neuroimmune disorders.
Inhibition of the ATP‐sensitive potassium channel from mouse pancreatic β‐cells by surfactants
Authors: Smith Paul A , Proks Peter
Published: January 2005

We have used patch‐clamp methods to study the effects of the detergents, Cremophor, Tween 80 and Triton X100 on the KATP channel in the pancreatic β‐cell from mouse.

All three detergents blocked KATP channel activity with the following order of potency: Tween 80 (Ki<∼83 nM)>Triton X100 (Ki=350 nM)>Cremophor. In all cases the block was poorly reversible.

Single‐channel studies suggested that at low doses, the detergents act as slow blockers of the KATP channel.

Unlike the block produced by tolbutamide, that produced by detergent was not affected by intracellular Mg2+‐nucleotide, diazoxide or trypsin treatment, nor did it involve an acceleration of rundown or increase in ATP sensitivity of the chanel.

The detergents could block the pore‐forming subunit, Kir6.2ΔC26, which can be expressed independently of SUR1 (the regulatory subunit of the KATP channel). These data suggest that the detergents act on Kir6.2 and not SUR1.

The detergents had no effect on another member of the inward rectifier family: Kir1.1a (ROMK1).

Voltage‐dependent K‐currents in the β‐cell were reversibly blocked by the detergents with a far lower potency than that found for the KATP channel.

Like other insulin secretagogues that act by blocking the KATP channel, Cremophor elevated intracellular Ca2+ in single β‐cells to levels that would be expected to elicit insulin secretion.

Given the role of the KATP channel in many physiological processes, we conclude that plasma borne detergent may have pharmacological actions mediated through blockage of the KATP channel
No evidence for a significant non‐nitrergic, hyperpolarising factor contribution to field stimulation‐induced relaxation of the mouse anococcygeus
Authors: Fonseca Mario , Uddin Nazim , Gibson Alan
Published: January 2005

The aim of the study was to determine whether a nerve‐derived hyperpolarizing factor (NDHF) might contribute to non‐adrenergic, non‐cholinergic (NANC) relaxations of the mouse anococcygeus when low concentrations of contractile agent are used to raise tone and low frequencies of field stimulation applied; such a non‐nitrergic NDHF has been proposed to contribute to NANC relaxations of the rat anococcygeus and guinea‐pig taenia coli.

Phenylephrine (0.1–100 μM) produced concentration‐related contractions of the mouse isolated anococcygeus muscle; 0.2 μM phenylephrine (EC26) was used to raise tone in subsequent experiments.

Field stimulation (0.5, 1.0 and 5.0 Hz) produced frequency‐dependent relaxations of phenylephrine‐induced tone. In the presence of the nitric oxide synthase inhibitor L‐NG‐nitro‐arginine (L‐NOARG; 100 μM), the soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxodiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 5 μM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01–5 μM) were unaffected by L‐NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8‐Br‐cyclic GMP (10 μM).

Nifedipine (1 μM) reduced the contractile response to 0.2 μM phenylephrine by 38%; however, it had no effect on NANC relaxations.

It is concluded that NANC relaxations of the mouse anococcygeus are purely nitrergic and that there is no significant contribution from a putative NDHF.
Effect of inhibitors of Na+/H+‐exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes
Authors: Ritter M , Schratzberger P , Rossmann H , Wöll E , Seiler K , Seidler U , Reinisch N , Kähler C M , Zwierzina H , Lang H J , Lang F , Paulmichl M , Wiedermann C J
Published: January 2005

Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N‐formyl‐Met‐Leu‐Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs.

Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling.

Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP‐induced cell swelling.

Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP.

H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP‐induced increase in CV.

Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP‐stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited.

The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole.

Western analysis with a monoclonal anti gastric H+/K+ATPase β‐subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs.

The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+‐dependent H+‐extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.
Species differences in the distribution of β‐adrenoceptor subtypes in bladder smooth muscle
Authors: Yamazaki Yoshinobu , Takeda Hiroo , Akahane Masuo , Igawa Yasuhiko , Nishizawa Osamu , Ajisawa Yukiyoshi
Published: January 2005

The β‐adrenoceptor (β‐AR) subtypes mediating relaxation of the rabbit, rat and canine detrusors were subjected to functional investigation using selective β‐AR agonists and antagonists.

In all three species, isoprenaline, noradrenaline and adrenaline each produced a concentration‐dependent relaxation of the detrusor. The rank order for their relaxing potency was isoprenaline> adrenaline>noradrenaline in rabbits and rats, but isoprenaline>noradrenaline>adrenaline in dogs.

Dobutamine did not produce relaxation of the detrusors at concentrations that are selective for β1‐AR. The selective β2‐AR agonist, procaterol, had a more potent relaxing effect on rabbit and rat detrusors than on the canine detrusor. CGP‐12177A, a selective β3‐AR agonist, was more effective in the rabbit than in the other two species. On the other hand, the relaxing effect of another β3‐AR agonist, CL316243, was more pronounced in dogs and rats than in rabbits.

CGP‐20712A (10−9 to 10−7 M), a selective β1‐AR antagonist, caused a slight rightward shift of the concentration‐relaxation response curve for isoprenaline in the canine detrusor (pA2 9.41), but not in the rabbit and rat detrusors. ICI‐118,551, a selective β2‐AR antagonist, antagonized the isoprenaline‐induced relaxation in rabbits (pA2 9.45) and rats (pA2 9.05), but not in dogs. Bupranolol, a non‐selective β‐AR antagonist, caused a rightward shift of the concentration‐relaxation curve for isoprenaline in the rabbit (pA2 9.32) and rat (pA2 8.98). However, higher concentrations (3×10−8 to 10−5 M) were needed to induce a rightward shift of the curve for isoprenaline in the dog (pA2 8.19) than in the other two species.

We have confirmed that the distribution of β‐AR subtypes in the detrusor muscle varies significantly from species to species and we provide here the first evidence of the presence of β3‐AR in the detrusor. It is suggested that the relaxation induced by adrenoceptor agonists in urinary bladder smooth muscle may be mediated mainly via β2‐AR in rabbits, via both β2‐ and β3‐AR in rats, but mainly via β3‐AR in dogs.
Synergistic effects of caspase inhibitors and MK‐801 in brain injury after transient focal cerebral ischaemia in mice
Authors: Ma Jianya , Endres Matthias , Moskowitz Michael A
Published: January 2005

Excitotoxic and apoptotic mechanisms have been implicated in the pathophysiology of cerebral ischaemia. Both MK‐801, an NMDA receptor antagonist, or peptide inhibitors of the caspase family (z‐VAD.FMK and z‐DEVD.FMK), protect mouse brain from ischaemic cell damage. In this study, we examined whether these drugs which act via distinct mechanisms, afford even greater neuroprotection when given in combination following 2 h MCA occlusion (filament model) and 18 h reperfusion.

Given alone as pretreatment, MK‐801 (1, 3 and 5 mg kg−1, but not 0.3 mg kg−1, i.p.) decreased infarct size by 34–75%. When injected 1 h after occlusion and before reperfusion, 3 mg kg−1 reduced injury but not when administered 1 h after reperfusion.

Pretreatment with a subthreshold dose of MK‐801 (0.3 mg kg−1) plus a subthreshold dose of z‐VAD.FMK (27 ng) or z‐DEVD (80 ng) significantly decreased infarct size by 29 and 30%, respectively, and enhanced neurological function.

Administering a subthreshold dose of z‐VAD.FMK (27 ng) or z‐DEVD.FMK (80 ng) as pretreatment extended the time window for MK‐801 (3 mg kg−1) by 2 h from 1 h before reperfusion to at least 1 h after reperfusion.

Pretreating with a subthreshold dose of MK‐801 (0.3 mg kg−1) extended the time window for z‐DEVD.FMK (480 ng) from 1 h after reperfusion to at least 3 h after reperfusion.

We conclude that caspase inhibitors which putatively block apoptotic cell death and inhibit cytokine production and the NMDA antagonist MK‐801 act synergistically and prolong their respective therapeutic windows in cerebral ischaemia.