bluuebunny/crossref_metadata_2025 huggingface dataset:
Desensitization of the P2‐purinoceptors on the rat colon muscularis mucosae
Authors: Hourani S.M.O. , Johnson C.R. , Bailey S.J.
Published: July 2012

Adenosine 5′‐triphosphate (ATP) and adenosine have been shown to contract the rat colon muscularis mucosae, and the receptors at which they act have been classified as P2Y and A1 respectively. Uridine 5′‐triphosphate (UTP) also contracts this tissue, and desensitization was used to investigate the receptors by which it acts, in the light of recent suggestions that specific pyrimidinoceptors may exist for UTP, or that nucleotide receptors may exist which are responsive to both ATP and UTP but not to some ATP analogues such as 2‐methylthioadenosine 5′‐triphosphate (2‐MeSATP).

ATP, UTP and adenosine each contracted the rat colon muscularis mucosae in a concentration‐dependent manner over the concentration range 0.3–300 μm, although maximal responses to ATP and UTP were not obtained. ATP was approximately 4 times as potent as UTP and approximately equipotent with adenosine although the maximal response to adenosine appeared to be less than that to ATP or UTP.

Desensitization of the tissue with ATP (200 μm) given immediately before each concentration of the agonists reduced subsequent contractions induced by ATP itself and also by UTP, but did not reduce contractions induced by adenosine. Desensitization of the tissues with UTP (200 μm) also reduced contractions induced by ATP and UTP but not by adenosine, whereas desensitization with adenosine (200 μm) reduced contractions induced by adenosine itself but not by ATP or UTP.

Desensitization of the tissue with 2‐MeSATP (200 μm), which is a more potent agonist than ATP at P2Y‐purinoceptors, greatly reduced the responses to ATP and to UTP, but had no effect on responses induced by adenosine. Attempts to desensitize the tissue with adenosine 5′‐(α,β‐methylene)triphosphonate (AMPCPP), which is a more potent agonist than ATP at P2X‐purinoceptors but is less potent at P2Y‐purinoceptors, were unsuccessful.

These results show that cross desensitization to ATP and UTP occurred and was specific for these agonists rather than being due to a general decrease in the ability of the muscle to contract. This implies that ATP and UTP act at the same receptor, which does not support the existence of specific pyrimidinoceptors but which could be taken as evidence for the existence of a nucleotide receptor on this tissue. However, the ability of 2‐MeSATP, which is inactive at the proposed nucleotide receptors, also selectively to desensitize this receptor indicates instead that ATP and UTP are both acting at a purinoceptor of the P2Y type in this tissue.
Evidence for the involvement of a 5‐HT4 receptor in the secretory response of human small intestine to 5‐HT
Authors: Borman R.A. , Burleigh D.E.
Published: July 2012

5‐Hydroxytryptamine increases transmucosal short‐circuit current across human isolated small intestinal mucosa. The competitive 5‐HT4 antagonist, DAU 6285 evoked a concentration‐dependent, dextral and parallel shift of the concentration‐response curve to 5‐HT, with no alteration of the maximum response. Schild analysis of this antagonism produced a Schild regression with a slope of 1.00 and an apparent pA2 estimate of 6.17. It appears that a 5‐HT4 receptor may mediate the short‐circuit current response of human small intestinal mucosa to 5‐HT.
Thrombolytic effect of a plasminogen‐plasminogen activator chimera in a photochemically induced thrombosis (PIT) model
Authors: Matsuno Hiroyuki , Uematsu Toshihiko , Nakashima Mitsuyoshi
Published: July 2012

The thrombolytic effects of the plasminogen/plasminogen activator chimera (SUN9216), comprising the fibrin‐binding kringle 1 domain of plasminogen and two kringle and the serine protease domain of the wild‐type tissue plasminogen activator (t‐PA) including a modification of the mannose glycosylation on the kringle 1 of t‐PA (PK1ΔFE1X), was compared with that of t‐PA by use of a photochemically induced thrombus (PIT) in the rat femoral artery. When SUN9216 was administered either as an i.v. infusion (1.0 mg kg−1) or as a single bolus i.v. injection (1.0 mg kg−1), all parameters were markedly improved compared to t‐PA administered as an i.v. infusion (3.0 mg kg−1). A higher concentration of plasminogen activator (PA) activity in plasma was observed after administration of SUN9216 which persisted for longer than that after t‐PA. It is concluded that the thrombolytic effect of SUN9216 is markedly greater than that of t‐PA.
Endotoxin‐induced arterial endothelial barrier dysfunction assessed by an in vitro model
Authors: Berman Rodney S. , Frew Joyce D. , Martin William
Published: July 2012

Using an in vitro model in which albumin transfer across monolayers of bovine aortic endothelial cells (BAEC) was measured, we have shown that lipopolysaccharide (LPS) induces a concentration‐dependent increase in endothelial permeability. This increase was biphasic, having an early peak at 2 h and rising again by 24 h. Both peaks were abolished by polymixin B (PMB) but were unaffected by Nω‐monomethyl‐l‐arginine, Nω‐nitro‐l‐arginine methyl ester or dexamethasone. Furthermore, LPS did not stimulate nitric oxide production by BAEC following 24 h exposure. Thus, the LPS‐induced increase in permeability may account for the vascular leakage of septic shock, but the l‐arginine‐nitric oxide system does not appear to be involved.
Effects of two β3‐adrenoceptor agonists, SR 58611A and BRL 37344, and of salbutamol on cholinergic and NANC neural contraction in guinea‐pig main bronchi in vitro
Authors: Martin Corinne A.E. , Naline Emmanuel , Manara Luciano , Advenier Charles
Published: July 2012

The aim of the present study was to investigate the type of adrenoceptor which modulates constriction of the guinea‐pig isolated main bronchus in response to electrical field stimulation (EFS). Drugs used were salbutamol and two agonists reportedly selective for the putative β3‐adrenoceptor: BRL 37344 and SR 58611A.

At basal tone, all three drugs induced relaxation, however, SR 58611A and BRL 37344 (10−9 to 10−6 m) relaxed guinea‐pig isolated main bronchus more weakly than salbutamol (10−9 to 10−6 m). The effects observed at 10−6 m were 43% ± 9%, 63% ± 4% and 98% ± 1% of the maximal effect induced by theophylline (3 × 10−3 m) for SR 58611A, BRL 37344 and salbutamol, respectively.

SR 58611A and BRL 37344 (10−8 to 10−6 m) did not significantly modify the cholinergic component of the response to EFS, but caused a concentration‐dependent reduction of the nonadrenergic non‐cholinergic (NANC) excitatory component (41.8% ± 10.1% and 56.8% ± 7.4% respectively at 10−6 m, n = 6–7). Salbutamol (10−9 to 10−7 m) strongly inhibited both components, with 91.1% ± 4.2% of inhibition for the NANC contraction and 62.0% ± 5.2% of inhibition for the cholinergic contraction (10−7 m, n = 7).

Whereas the inhibitory effects of salbutamol were strongly inhibited by both propranolol (10−6 m) and ICI 118,551 (10−6 m), those of BRL 37344 were only slightly, albeit significantly reduced by both propranolol and ICI 118,551, and those of SR 58611A were unaffected by treatment with either β‐adrenoceptor antagonist. An α2‐adrenoceptor antagonist, yohimbine, did not influence the inhibitory effects of any of the β‐adrenoceptor agonists tested.

Concentration‐response curves to acetylcholine (10−8 to 10−3 m), [Nle10]NKA(4–10) (10−10 to 10−6 m) and substance P (10−10 to 3 × 10−6 m) were also significantly shifted to the right by salbutamol (10−6 m), whereas SR 58611A and BRL 37344 (10−6 m) had no effect.

These results suggest that the stimulation of putative β3‐adrenoceptors exerts a specific prejunctional inhibitory action on NANC excitatory response induced by EFS of the isolated main bronchus of the guinea‐pig. They also suggest that a β2‐adrenoceptor agonistic component may be involved in the effects of BRL 37344.
Cardiovascular effects of intrathecally administered bradykinin in the rat: characterization of receptors with antagonists
Authors: Lopes Paulo , Regoli Domenico , Couture Réjean
Published: July 2012

The effects of intrathecal (i.t.) pretreatment with selective B1 or B2 kinin receptor antagonists were studied on the cardiovascular response to i.t. injection of bradykinin (BK) in conscious freely moving rats.

BK (81 pmol) produced an increase in mean arterial pressure (MAP: 9–13 mmHg) and decrease in heart rate (HR: 20–30 beats min−1) that reached a maximum 2 min after injection.

The BK‐induced cardiovascular responses were dose‐dependently and reversibly reduced by four antagonists with the following rank order of potency: Tyr, d‐Arg[Hyp3,d‐Phe7,Leu8]‐BK = d‐Arg[Tyr3,d‐Phe7,Leu8]‐BK = d‐Arg[Hyp3,d‐Phe7,Leu8]‐BK > d‐Arg[Hyp3,Thi5,d‐Tic7,Oic8]‐BK (Hoe 140). These compounds failed to alter the cardiovascular response to i.t. injection of 8.1 nmol of substance P.

Other compounds acting on the B2 receptor, namely d‐Arg[Hyp3,Gly6,Leu8]‐BK, d‐Arg[Hyp3,d‐Phe7]‐BK, d‐Arg[Hyp2,Thi5,8,d‐Phe7]‐BK and d‐Arg[Hyp3,Gly6,d‐Phe7,Leu8]‐BK or on the B1 receptor, [Leu8]‐desArg9‐BK, did not influence the cardiovascular responses to BK at doses devoid of intrinsic activity on MAP and HR.

None of the kinin receptor antagonists caused motor impairment, respiratory arrest or persisting cardiovascular changes.

These results confirm that the cardiovascular effects induced by i.t. BK are mediated by the activation of a B2 receptor in the rat spinal cord. However, the rank order of potency of antagonists does not conform to the classical B2 functional site characterized in peripheral tissues.
Cardiotonic actions of selective phosphodiesterase inhibitors in rat isolated ventricular cardiomyocytes
Authors: Kelso Elizabeth J. , McDermott Barbara J. , Silke Bernard
Published: July 2012

The contractile effects of the novel cardiotonic agent HN‐10200 (2‐[3‐methoxy‐5‐methylsulphinyl‐2‐thienyl]‐1H‐imidazo‐[4,5‐c]‐pyridine hydrochloride), were examined and comparisons made with the responses obtained to a structurally similar compound, sulmazole, and to a number of other compounds which are known to inhibit phosphodiesterase (PDE) isoenzymes with differing selectivities; namely, enoximone (PDE III inhibitor), Ro 20–1724 (PDE IV inhibitor) and 3‐isobutyl‐1‐methylxanthine (non‐selective PDE inhibitor).

Contractile function, as measured by mechanical shortening, and biochemical systems involving cyclic AMP were investigated in ventricular cardiomyocytes isolated from adult Sprague‐Dawley rats (200–250 g).

HN‐10200 exerted a concentration‐dependent (10−8 m − 10−4 m) positive contractile effect, which was independent of α‐ or β‐adrenoceptor, or histamine receptor stimulation.

The efficacies of the contractile responses to the PDE inhibitors were of the order: HN‐10200 > IBMX > sulmazole > enoximone and maximum stimulations, which were obtained at concentrations of 10−4 m, were 54 ± 4%, 41 ± 7%, 38 ± 7% and 26 ± 5% (mean ± s.e.) greater than basal levels, respectively (n = 6); the basal value of contractile amplitude (dL), in the absence of PDE inhibitors was 7.39 ± 0.18% (mean ± s.e.). Ro 20–1724 did not have any effect on contractile activity.

Due to low basal levels of cyclic nucleotides in isolated cells, accumulation of cyclic AMP due to the presence of the PDE inhibitors was detected only when the levels of cyclic nucleotide were enhanced with forskolin (10 μm).

The PDE inhibitors increased levels of cyclic AMP only at concentrations > 10−4 m. HN‐10200 and sulmazole had similar concentration‐dependent profiles for the accumulation of cyclic AMP; their potencies were lower than that of IBMX (concentrations of forskolin required to increase cyclic AMP by 4 pmol mg−1 protein, in the presence of maximum concentrations of the PDE inhibitors, were 13 ± 3 μm, 14 ± 3 μm and 3 ± 0.6 μm [mean ± s.e.], respectively).

These results indicate that a similar mechanism, probably through a weak inhibition of the cyclic AMP‐specific PDE isoenzymes, is responsible for the increase in levels of cyclic AMP by HN‐10200 and sulmazole. However, cyclic AMP is only partially responsible for the positive contractile effect of HN‐10200 and, similarly, sulmazole and IBMX. The lack of apparent increase in levels of cyclic AMP by enoximone, highlights its degree of selectivity for the PDE III isoenzyme, such that the PDE IV isoform is still present in sufficient quantity to degrade cyclic AMP within the cell. On the other hand, the potent action of Ro 20–1724 on accumulation of cyclic AMP, in addition to the lack of effect on contractile function, is in agreement with the selectivity of this compound for the PDE IV isoenzyme and compartmentalization of cyclic AMP in rat isolated ventricular cardiomyocytes.
Selective inhibition by dexamethasone of induction of NO synthase, but not of induction of l‐arginine transport, in activated murine macrophage J774 cells
Authors: Baydoun Anwar R. , Bogle Richard G. , Pearson Jeremy D. , Mann Giovanni E.
Published: July 2012

Effects of dexamethasone on induction of nitric oxide (NO) synathase and l‐arginine transport by lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line J774. Metabolism of l‐arginine to l‐citrulline and subsequent changes in intracellular amino acids pools were correlated with changes in nitrite production.

Despite a high intracellular concentration of arginine in activated J774 cells, LPS (1 μg ml−1, 8 h) induced a 2.4 fold increase in arginine transport. Treatment of cells with cycloheximide (1 μg ml−1) inhibited the time‐dependent (1–8 h) induction of NO synthase and arginine transport mediated by LPS.

Induction of NO synthase by LPS (1 μg ml−1, 24 h) alone was accompanied by a marked increase in arginine utilisation leading to decreased intracellular arginine levels and elevated intracellular and extracellular l‐citrulline levels. These changes were further enhanced in the presence of interferon‐γ (IFN‐γ, 100 units ml−1, 24 h).

Dexamethasone (1 μm) abolished the increases in both nitrite and citrulline production induced by LPS alone but only partially reversed the combined effects of LPS and IFN‐γ. In contrast, treatment of cells with dexamethasone (10 μm) had no effect on the LPS‐mediated induction of arginine transport or the decrease in intracellular arginine concentration.

We conclude that induction of arginine transporter activity in LPS‐stimulated J774 cells involves de novo synthesis of carrier proteins, which increases transport of exogenous arginine during enhanced NO production. Moreover, the intracellular signalling pathways mediating induction of arginine transport and of NO synthase by LPS in activated macrophages diverge, since only the latter is sensitive to dexamethasone.
Induction of bradykinin B1 receptors in vivo in a model of ultra‐violet irradiation‐induced thermal hyperalgesia in the rat
Authors: Perkins M.N. , Kelly D.
Published: July 2012

The role of bradykinin B1 receptors in the thermal hyperalgesia following unilateral ultra‐violet (u.v.) irradiation of the hindpaw of rats has been investigated.

In non‐irradiated (naive) animals the B1 receptor agonist des‐Arg9‐bradykinin and bradykinin (BK) (up to 1 μmol kg−1 i.v.) had no effect on withdrawal latency to a noxious heat stimulus when administered 60 min before testing.

Following exposure of one hindpaw to strong u.v. irradiation the withdrawal latency of the u.v.‐treated paw to radiant noxious heat fell by a maximum of 50% after 48 h. There was no reduction in latency in the contralateral paw.

des‐Arg9‐BK (1–100 nmol kg−1 i.v.) administered 24 h after u.v. exposure caused a further dose‐dependent fall (50 ± 4% reduction from saline injected animals at 100 nmol kg−1 i.v.) in withdrawal latency in the u.v.‐treated paw when measured 60 min after injection. The withdrawal latency of the contralateral paw was also reduced but to a lesser extent following des‐Arg9‐BK (100 nmol kg−1 i.v.) with a maximum reduction of 19 ± 3%.

Bradykinin also induced a further reduction in withdrawal latency (33 ± 5% reduction at 1 μmol kg−1) although it was not as effective as des‐Arg9‐BK. Bradykinin did not reduce the withdrawal latency in the contralateral paw.

The hyperalgesic action of both des‐Arg9‐BK (10 nmol kg−1 i.v.) and bradykinin (100 nmol kg−1 i.v.) were antagonized by the B1 receptor antagonist, des‐Arg9,Leu8‐BK (200 nmol kg−1 i.v.) but not by the B2 receptor antagonist, HOE 140 (0.5 μmol kg−1 i.v.).

The results suggest that in conditions of inflammatory hyperalgesia bradykinin B1 receptors are induced both locally and distant to the inflamed area, activation of which leads to further thermal hyperalgesia. In addition, in these conditions bradykinin appears to act predominantly via B1 receptors, presumably after degradation to des‐Arg9‐BK.
Homologous and heterologous desensitization of histamine H1‐and ATP‐receptors in the smooth muscle cell line, DDT1MF‐2: the role of protein kinase C
Authors: Dickenson John M. , Hill Stephen J.
Published: July 2012

The possible role of protein kinase C (PKC) in homologous and heterologous desensitization of histamine H1‐ and ATP‐receptors has been studied in monolayers of cultured vas deferens smooth muscle cells (DDT1MF‐2). Cells were loaded with the calcium‐sensitive fluorescent dye fura‐2 and increases in intracellular free Ca2+ concentration ([Ca2+]i) monitored in response to histamine H1‐ or ATP‐receptor activation.

Histamine and ATP stimulated the release of Ca2+ from intracellular Ca2+ stores and Ca2+ influx across the plasma membrane. Activation of PKC with the phorbol ester β‐phorbol‐12,13 dibutyrate (PDBu; 1 μm) attenuated histamine (100 μm) and ATP (10 μm)‐induced release of intracellular Ca2+ and Ca2+ influx.

The selective PKC inhibitor, Ro 31–8220 (10 μm), reversed the PDBu‐induced attenuation of histamine (100 μm)‐stimulated Ca2+ responses.

Histamine H1‐ and ATP‐receptors are readily susceptible to homologous desensitization since short‐term exposure to histamine or ATP (450 s) attenuated the Ca2+ responses elicited by a second application of the same agonist. Furthermore, H1‐receptor activation‐induced heterologous desensitization of ATP stimulated Ca2+ responses and vice versa.

Homologous and heterologous desensitization of histamine and ATP Ca2+ responses still occurred in the presence of the PKC inhibitor, Ro 31–8220 (10 μm).

These data suggest that PKC activation can attenuate histamine H1‐ and ATP‐receptor mediated Ca2+ responses. However, based on our experimental data, PKC‐independent mechanisms appear to be involved in the homologous and heterologous desensitization of histamine H1‐ and ATP‐receptor mediated Ca2+ responses in DDT1MF‐2 cells.
EDRF release from canine coronary artery by lectins
Authors: Kleha Joseph F. , Devesly Pierre , Johns Anthony
Published: July 2012

The effect of wheat germ agglutinin (WGA), concanavalin A (Con A) and lentil lectin agglutinin (LCA) were investigated on pre‐contracted canine coronary artery rings in vitro.

In endothelium‐intact canine coronary artery, contracted with the thromboxane A2‐analogue, U46619, WGA relaxed the tissue in a concentration‐dependent manner, with an inhibitory concentration (IC50) of 112 ± 17 nm (n = 6). In the absence of an endothelium, WGA did not cause any relaxation of the tissue.

In endothelium‐intact canine coronary artery, contracted with the thromboxane A2‐analogue, U46619, LCA relaxed the tissue in a concentration‐dependent manner, with an inhibitory concentration (IC50) of 423.1 ± 41 nm (n = 6). In the absence of an endothelium, LCA produced a 20.1 ± 1.1% (n = 6) relaxation at the highest concentration tested (3 μm).

Concanavalin A (Con A) relaxed canine coronary artery in a partial endothelium‐dependent manner with an IC50 of 104 ± 19 nm on endothelium‐intact coronary artery and an IC50 of 1.3 ± 0.3 μm (n = 6) on endothelium‐denuded tissues.

The relaxation effects of WGA were attenuated by 1 mm NG‐monomethyl l‐arginine (l‐NMMA) and completely inhibited by haemoglobin (3 μm), methylene blue (10 μm) and LY 83583 (30 μm). Ibuprofen had no effect on WGA‐induced relaxation.

The relaxant effects of WGA were reversed by addition of 20 mm N‐acetyl‐d‐glucosamine (GlcNAc) and N‐acetyl‐d‐galactosamine (Ga1NAc) but not by α‐mannose, d‐(+)‐galactose, and β‐lactose, whereas the endothelin‐dependent relaxations to LCA and Con A were unaffected.

The endothelium‐dependent relaxation induced by the lectins was unaffected by pretreatment of the tissue with 1 μm atropine.

In the absence of extracellular calcium, WGA was also able to release EDRF suggesting that WGA acts through a second messenger system to release intracellular calcium.

We suggest that WGA acts as an agonist to release EDRF from endothelial cells possibly by binding to a sugar moiety, specific receptor or adhesion molecules on the endothelial cell surface.
ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro
Authors: Brownlie R.P. , Brownrigg N.J. , Butcher H.M. , Garcia R. , Jessup R. , Lee V.J. , Tunstall S. , Wayne M.G.
Published: July 2012

The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)‐receptor blocking action of ZD1542 (4(Z)‐6‐[2S,4S,5R)‐2‐[1‐methyl‐1‐(2‐nitro‐4‐tolyloxy)ethyl]‐4‐(3‐pyridyl)‐1,3‐dioxan‐5‐yl]hex‐4‐enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro.

ZD1542 caused concentration‐dependent inhibition of human platelet microsomal thromboxane B2 (TXB2) production in vitro (IC50 = 0.016 μm); this inhibition was associated with an increase in prostaglandin E2 (PGE2) and PGF2α formation.

ZD1542 also inhibited collagen‐stimulated TXS in human, rat and dog whole blood giving IC50 values of 0.018, 0.009 and 0.049 μm respectively. The drug did not modify platelet cyclo‐oxygenase activity as inhibition of TXB2 formation was associated with a concomitant increase in the levels of PGD2, PGE2 and PGF2α. ZD1542 had little if any effect against cultured human umbilical vein endothelial cell (HUVEC) cyclo‐oxygenase (IC50 > 100 μm) and prostacyclin (PGI2) synthase (IC50 = 18.0 ± 8.6 μm).

ZD1542 caused concentration‐dependent inhibition of U46619‐induced aggregation responses of human, rat and dog platelets yielding apparent pA2 values of 8.3, 8.5 and 9.1 respectively. The drug was selective as, at concentrations up to 100 μm, it did not modify 5‐hydroxytryptamine (5‐HT) or the primary phases of adenosine diphosphate (ADP) and adrenaline‐induced aggregation. Furthermore, ZD1542 (100 μm) modified only weakly the platelet inhibitory effects of PGD2, PGE1 and PGI2.

ZD1542 also caused concentration‐dependent inhibition of U46619‐mediated contractions of rat thoracic aorta, guinea‐pig trachea and lung parenchyma preparations giving apparent pA2 values of 8.6, 8.3 and 8.5 respectively. At concentrations approaching three orders of magnitude greater than those required to block U46619‐mediated contractions, the drug did not affect the actions of non‐prostanoid agonists or exhibit agonist activity in any of the smooth muscle preparations employed; neither did it interact at EP‐ or FP‐receptors.

In conclusion, the present study demonstrates that ZD1542 is a drug that exhibits both potent, selective TXS inhibition and TXA2 receptor antagonism.
Antinociceptive activity of NK1 receptor antagonists: non‐specific effects of racemic RP67580
Authors: Rupniak N.M.J. , Boyce S. , Williams A.R. , Cook G. , Longmore J. , Seabrook G.R. , Caeser M. , Iversen S.D. , Hill R.G.
Published: July 2012

Release of substance P in the dorsal horn is considered a primary event in the perception of pain. The profile of racemic RP67580, a non‐peptide antagonist at the NK1 (substance P) receptor, was examined in a range of antinociception tests on rodents.

At doses up to 30 mg kg−1, s.c. racemic RP67580 exhibited antinociceptive activity in writhing and formalin paw tests in mice and gerbils. Acetic acid induced writhing and the licking response to formalin were reduced to 40–50% of the level observed in vehicle‐treated animals (P < 0.05). However, this agent was not active in mouse tail flick, rat paw pressure or rat and guinea‐pig formalin paw tests.

Like racemic RP67580, the calcium channel blockers nifedipine (30 mg kg−1, i.p.) and verapamil (10 or 20 mg kg−1, s.c.) inhibited the response to formalin by approximately 60% in gerbils (P < 0.05 compared with vehicle‐treated animals).

Evidence for calcium channel antagonist activity of RP67580 was obtained in vitro. Racemic RP67580 inhibited calcium entry into depolarized strips of guinea‐pig ileum longitudinal muscle myenteric plexus (apparent KB = 587 ± 115 nm), inhibited [3H]‐diltiazem binding to rabbit skeletal membranes (IC50 = 298 nm) and depressed high threshold calcium currents in neurones cultured from rat cortex (10% inhibition at 10 μm).

These findings indicate that the acute antinociceptive effects of RP67580 may not be attributable to a specific interaction with NK1 receptors and may be mediated via calcium channel blockade.
Effects of P1 and P2Y purinoceptor antagonists on endothelium‐dependent and ‐independent relaxations of rat mesenteric artery to GTP and guanosine
Authors: Vuorinen Pauli , Wu Xiumin , Arvola Pertti , Vapaatalo Heikki , Pörsti Ilkka
Published: July 2012

Guanosine 5′‐triphosphate (GTP) and guanosine can relax both endothelium‐intact and ‐denuded arterial preparations. In the present work the P1 and P2Y purinoceptor antagonists, 8‐phenyltheophylline and reactive blue 2, respectively, were used to study the mechanisms of relaxation responses induced by GTP, guanosine, adenosine 5′‐triphosphate (ATP) and adenosine in noradrenaline‐precontracted rat mesenteric artery rings.

GTP (10 μm – 1 mm) dose‐dependently relaxed endothelium‐intact mesenteric artery rings and also induced moderate relaxation responses in endothelium‐denuded preparations. Pretreatment of the rings with 8‐phenyltheophylline (10 μm) or reactive blue 2 (10 μm) did not attenuate the relaxant effect of GTP.

Guanosine (10 μm – 1 mm) relaxed both endothelium‐intact and ‐denuded artery rings in a dose‐dependent manner. The presence of 8‐phenyltheophylline or reactive blue 2 had no effects on guanosine‐induced relaxations.

ATP‐induced (0.1 μm – 0.1 mm) relaxation of endothelium‐intact artery rings was attenuated by reactive blue 2 while 8‐phenyltheophylline was ineffective. ATP also relaxed endothelium‐denuded artery rings and this relaxation was inhibited by 8‐phenyltheophylline, but not by reactive blue 2.

Adenosine‐induced (10 μm–1 mm) relaxation of endothelium‐intact and ‐denuded artery rings was attenuated by the presence of 8‐phenyltheophylline, but not of reactive blue 2.

In conclusion, the endothelium‐dependent and ‐independent relaxations of rat mesenteric arteries to GTP and guanosine are not mediated via P1 and P2Y purinoceptors. Therefore, these results support our previous suggestion on the presence of a novel guanine nucleotide‐specific receptor, a putative PG receptor, on both endothelial and smooth muscle cells, which may participate in the regulation of arterial tone.
Characterization of the prostaglandin E2 sensitive (EP)‐receptor in the rat isolated trachea
Authors: Lydford S.J. , McKechnie K.
Published: July 2012

Using a range of natural and synthetic prostanoid receptor agonists and antagonists, we have shown that the rat isolated trachea contains a heterogeneous population of prostaglandin receptor sub‐types mediating both relaxation and contraction of the smooth muscle. Prostaglandin E2 (PGE2) elicits smooth muscle relaxation of pre‐contracted preparations, the responses being well defined, with a mean potency (p[A50]) of 7.81 ± 0.05.

11‐deoxy PGE1 16,16‐dimethyl PGE2 and misoprostol were all full agonists at this receptor, whilst AH13205 was a low potency agonist, and sulprostone was inactive.

The EP1 receptor antagonist, AH6809 (5 μm), and the selective DP receptor antagonist, BW A868C (0.1 μm), had no significant effect on the concentration‐effect (E/[A]) curves to PGE2.

The putative EP4‐receptor antagonist, AH23848B, produced non‐competitive antagonism of the PGE2 response curves; pA2 values of 5.07 ± 0.15 and 5.24 ± 0.19 were obtained at concentrations of 30 μm and 100 μm respectively.

The synthetic thromboxane A2 mimetic, U46619, caused smooth muscle contractions, with a mean p[A50] of 6.90 ± 0.11. This response was antagonized by the TP receptor antagonist, GR32191B, yielding a mean pA2 of 8.31.

At concentrations of 1 μm and above, prostaglandin D2 (PGD2) and the IP‐receptor agonist, cicaprost, generally elicited concentration‐dependent relaxations of the rat trachea. Prostaglandin F2α (PGF2α) was without affinity or efficacy.

These data suggest that the rat isolated trachea contains EP‐receptors, TP‐receptors, and few, if any, DP, IP or FP‐receptors. The inactivity of sulprostone (EP3/EP1 receptor selective) and the low potency of AH13205 (EP2‐receptor selective) suggest that the rat trachea contains an atypical EP‐receptor that does not conform to the current classification system.
Block of ATP‐sensitive K+ channels in isolated mouse pancreatic β‐cells by 2,3‐butanedione monoxime
Authors: Smith Paul A. , Williams Beatrice A. , Ashcroft Frances M.
Published: July 2012

The patch‐clamp technique has been used to examine the action of the chemical phosphatase 2,3‐butanedione monoxime (BDM) on ATP‐sensitive K+ channels (KATP‐channels) from mouse isolated pancreatic β‐cells in the absence of ATP and Mg2+.

BDM reversibly inhibited whole‐cell KATP‐currents with a concentration for half maximal inhibition (Ki) of 15 ± 1 mm and a Hill coefficient (n) of 2.5 ± 0.2 (n = 4).

In outside‐out patches, external BDM reversibly reduced the activity of single KATP‐channels with an affinity similar to that observed in whole‐cell recordings (Ki = 11 ± 3 mm, n = 2.0 ± 0.3, n = 7). In inside‐out patches, internally applied BDM also reversibly blocked the activity of KATP‐channels (Ki = 31 ± 2 mm, n = 2.2 ± 0.4, n = 8). In both excised patch configurations, BDM decreased the mean open life‐time and the burst duration, thereby producing a decrease in the channel open probability. The drug had no effect on the short intraburst closed times.

BDM had no effect on the single‐channel current amplitude.

The results suggest that BDM blocks the KATP‐channel directly, by mechanisms independent of channel dephosphorylation.
Stimulation of chloride secretion by P1 purinoceptor agonists in cystic fibrosis phenotype airway epithelial cell line CFPEo‐
Authors: Chao Anthony C. , Zifferblatt Jonathan B. , Wagner John A. , Dong Y.‐J. , Gruenert Dieter C. , Gardner Phyllis
Published: July 2012

P1 purinoceptor agonists like adenosine have been shown to stimulate Cl−1 transport in secretory epithelia. In the present study, we investigated whether P1 agonist‐induced Cl−1 secretion is preserved in cystic fibrosis airway epithelium and which signalling mechanism is involved. The effects of purinoceptor agonists on Cl−1 secretion were examined in a transformed cystic fibrosis airway phenotype epithelial cell line, CFPEo‐.

Addition of adenosine (ADO; 0.1 – 1 mm) markedly increased 125I efflux rate. The rank order of potency of purinoceptor agonists in stimulating 125I efflux was ADO > AMP > ADP ≃ ATP. A similar order of potency was seen in transformed cystic fibrosis nasal polyp cells, CFNPEo‐ (ADO > ATP > AMP > ADP). These results are consistent with the activation of Cl−1 secretion via a P1 purinoceptor.

The P1 agonists tested (at 0.01 and 0.1 mm) revealed a rank order of potency of 5′‐N‐ethylcarboxamine adenosine (NECA) > 2‐chloro‐adenosine (2‐Cl‐ADO) > R‐phenylisopropyl adenosine (R‐PIA).

The known potent A2 adenosine receptor (A2AR) agonist, 5′‐(N‐cyclopropyl) carboxamidoadenosine (CPCA, 2 μm) but not the A1 adenosine receptor agonist, N6‐phenyl adenosine (N6‐phenyl ADO, 10 μm) markedly increased 125I efflux rate (baseline, 5.9 ± 2.0% min−1, + CPCA, 10.9 ± 0.6% min−1; P < 0.01). The stimulant effect of CPCA (10 μm) was abolished by addition of the A2AR antagonist 3,7‐dimethyl‐1‐propargylxanthine (DMPX) (100 μm; reported K1 = 11 ± 3 μm). These results favour the involvement of A2AR.

ADO (0.1 – 1 mm) and CPCA (2 μm) both induced a marked increase in intracellular [Ca2+] ([Ca2+]i); the effect of the latter was again abolished by pretreatment of the cells with DMPX. By contrast, N6‐phenyl ADO did not affect [Ca2+]i.

In patch‐clamp experiments, ADO (1 mm) induced an outwardly‐rectified whole‐cell Cl−1 current (baseline, 2.5 ± 0.8 pA pF−1, + ADO, 78.4 ± 23.8 pA pF−1; P < 0.02), which was largely inhibited in cells internally perfused with a selective inhibitory peptide of the multifunctional Ca2+/calmodulin‐dependent protein kinase, CaMK [273–302] (20 μm), as compared to a control peptide, CaMK [284–302]. Addition of BAPTA (10 mm), a Ca2+ chelator, to the perfusion pipette also abolished the ADO‐elicited Cl−1 current.

In conclusion, our results suggest that A2AR participates in regulation of airway Cl−1 secretion via a Ca2+‐dependent signalling pathway, which involves CaMK and appears to be at least partially conserved in cystic fibrosis airway epithelial cells.
Differences in the effects of NK1‐receptor antagonists, (±)‐CP 96,345 and CP 99,994, on agonist‐induced responses in guinea‐pig trachea
Authors: Longmore J. , Razzaque Z. , Shaw D. , Hill R.G.
Published: July 2012

The effects of the NK1‐receptor antagonists, (±)‐CP 96,345 and CP 99,994, on NK1‐agonist evoked contractions were compared in isolated rings of guinea‐pig tracheal smooth muscle.

(±)‐CP 96,345 and CP 99,994 were similarly effective in antagonizing responses evoked by septide, whereas CP 99,994 was more effective than (±)‐CP 96,345 in inhibiting responses evoked by [Sar9Met11(O2)] substance P.

These results suggest that responses to septide and [Sar9Met11(O2)] substance P may be operated via different populations of NK1‐receptors.
Attenuation by chlormethiazole administration of the rise in extracellular amino acids following focal ischaemia in the cerebral cortex of the rat
Authors: Baldwin H.A. , Williams J.L. , Snares M. , Ferreira T. , Cross A.J. , Green A.R.
Published: July 2012

In vivo microdialysis has been used to investigate the concentration of various amino acids and lactate in the extracellular fluid of the rat cortex following focal ischaemia, the probe being placed in the core of the infarct area.

An ischaemic infarct was produced in the cortex by use of a photochemical dye (Rose Bengal) and light irradiation. There was a marked increase in lactate concentration (300%) over the next 4 h. Substantial increases were also seen in the concentration of the excitatory (glutamate and aspartate), inhibitory (GABA and taurine) and other amino acids (serine, alanine, asparagine).

Administration of chlormethiazole (200 mg kg−1, i.p.) 5 min after the onset of ischaemia reduced the ischaemia‐induced neurodegeneration by approximately 30%, measured histologically 24 h later.

Chlormethiazole (200 mg kg−1, i.p.) administration also reduced the rise in the concentration of lactate and all the amino acids by between 30–60% during the first 4 h after the onset of ischaemia.

Analysis of the time course of the amino acid changes suggested that chlormethiazole is not neuroprotective because of the inhibition of excitatory amino acid release but rather that the attenuated rise in the concentration of all the amino acids is reflective of neuroprotection and therefore decreased cell death.

This conclusion was supported by the observation that the enhanced efflux of glutamate from slices of cerebral cortex which had been induced by incubation of the slices in an hypoxic medium was unaltered by the presence of a high concentration of chlormethiazole (1 mm) in the medium.

Overall the data strengthen the evidence for the neuroprotective effect of chlormethiazole in this model of focal ischaemia.
Effects of naftidrofuryl oxalate on microsphere embolism‐induced decrease in regional blood flow of rat brain
Authors: Miyake Keiko , Takagi Norio , Takeo Satoshi
Published: July 2012

The purpose of the present study was to determine whether naftidrofuryl oxalate (naftidrofuryl), a vasodilator, is capable of improving brain regional blood flow of animals in sustained ischaemia.

Cerebral ischaemia was induced by injecting 900 microspheres (48 μm in diameter) into the right internal carotid artery of rats. Cerebral blood flow of brain regions was measured by a hydrogen clearance method on the 3rd, 7th and 28th days after the onset of ischaemia. Ischaemic animals were treated with naftidrofuryl, 15 mg kg−1 day−1 i.p., from the first to 28th day.

Microsphere‐embolism caused a sustained decrease in cortical and striatal blood flow over a period of 28 days, whereas hippocampal blood flow was decreased on the 3rd day but not on the 7th or 28th day. On the 3rd day, the striatal and hippocampal but not cortical blood flow of naftidrofuryl‐treated, microsphere‐embolized rats was higher than untreated rats. On the 7th and 28th days, the cortical and striatal blood flow of the treated and untreated animals did not differ.

Brain slices from microsphere‐embolized rats contained areas, which were not stained with triphenyltetrazolium chloride (TTC), to a similar degree on the 3rd, 7th and 28th days, indicating the genesis of cerebral infarction. TTC‐unstained areas of microsphere‐embolized rats that had received naftidrofuryl treatment were smaller than those of untreated rats on the 3rd and 7th days, but not on the 28th day.

The results suggest that naftidrofuryl improves cerebral circulation impaired by microsphere‐induced ischaemia and this higher level of cerebral blood flow of the treated animal may account for the delayed development of cerebral infarction.
Comparative pharmacology of recombinant rat AT1A, AT1B and human AT1 receptors expressed by transfected COS‐M6 cells
Authors: Balmforth Anthony J. , Bryson Susan E. , Alison J. Aylett , Warburton Philip , Ball Stephen G. , Pun Kwok‐Tao , Middlemiss David , Drew G. Michael
Published: July 2012

Currently available antagonists and agonists cannot distinguish between angiotensin AT1 receptor subtypes.

We synthesized a series of compounds selected on the basis of having the most diverse structural features with respect to losartan (DuP753), the prototype non‐peptide AT1 receptor antagonist. Using a radioligand‐receptor binding assay and membranes prepared from COS‐M6 cells transfected with individual AT1 receptor subtypes, we determined whether any of these compounds could distinguish between the receptor subtypes.

The diversity of the structural features of this series of compounds was reflected by the wide range of affinities (pIC50 values) displayed towards competing with [125I]‐Sar1Ile8 angiotensin II for binding to the AT1 receptors.

Direct comparisons of the pIC50 values of individual compounds for rat AT1A, AT1B and human AT1 receptors revealed only minor differences.

It is concluded that compounds based structurally on losartan are unlikely to distinguish between these receptors.
Relative roles of nitric oxide and cyclo‐oxygenase and lipoxygenase products of arachidonic acid in the contractile responses of rat renal arcuate arteries
Authors: Wu X.C. , Richards N.T. , Michael J. , Johns E.
Published: July 2012

We have examined the effects of inhibition of nitric oxide synthase, cyclo‐oxygenase and lipoxygenase on the responses of renal arcuate arteries of Wistar rats, with and without endothelium, to noradrenaline, potassium chloride, endothelin‐1, acetylcholine and sodium nitroprusside.

Noradrenaline, potassium chloride and endothelin‐1 caused concentration‐dependent contraction of the vessels. Indomethacin (14 μm) attenuated the contractile response to noradrenaline and to potassium chloride. The inhibitory effect of indomethacin persisted following endothelial removal.

Acetylcholine produced concentration‐dependent relaxation of the vessels which was potentiated by indomethacin (14 μm).

NG‐nitro‐l‐arginine methyl ester (l‐NAME, 100 μm) did not affect the contractile response to either noradrenaline or potassium chloride but abolished relaxation to acetylcholine. In addition, l‐NAME abolished the affects of indomethacin on acetylcholine‐induced relaxation and noradrenaline‐ and potassium chloride‐induced contraction.

BWC755C attenuated noradrenaline and potassium chloride‐induced contraction. This effect persisted in the presence of indomethacin.

In vessels pretreated with CHAPS, BW755C inhibited both noradrenaline and potassium chloride‐induced contraction. In these vessels BW755C had no additional inhibitory effect to indomethacin on noradrenaline‐ and potassium‐induced contraction.

Inhibition of nitric oxide synthase with l‐NAME (100 μm) attenuated the effect of BW755C on noradrenaline‐ and potassium‐induced contraction.

BW755C alone did not affect endothelium‐dependent relaxation as assessed by the response to acetylcholine. However, in the presence of indomethacin, BW755C inhibited acetylcholine‐induced relaxation.

BW755C did not affect endothelium‐independent relaxation as assessed by the response to sodium nitroprusside in vessels with or without endothelium.

These data support the existence of two vasoconstrictor products of arachidonic acid released during contraction of renal arcuate arteries with noradrenaline and potassium chloride. A cyclo‐oxygenase product which appears to be endothelium‐independent and the other an endothelium‐dependent lipoxygenase product.
The renal handling of dopamine originating from l‐DOPA and γ‐glutamyl‐l‐DOPA
Authors: Pestana M. , Soares‐da‐Silva P.
Published: July 2012

The formation and outflow of dopamine and its deaminated metabolite 3,4‐dihydroxyphenylacetic acid (DOPAC) was studied in cortical fragments of the rat kidney loaded with l‐β‐3,4‐dihydroxyphenylalanine (l‐DOPA) or γ‐glutamyl‐l‐DOPA (GluDOPA). Dopamine and DOPAC in the tissues and in the effluent were assayed by means of h.p.l.c. with electrochemical detection.

In rats given 30 mg kg−1 l‐DOPA, tissue and outflow levels of both dopamine and DOPAC were 3 fold those observed with a lower dose of l‐DOPA (10 mg kg−1). In rats given GluDOPA (16.7 mg kg−1) levels of dopamine in renal tissues and in perifusate samples were found to be higher than those obtained with an equimolar dose of l‐DOPA (10 mg kg−1); however, no significant difference was observed for DOPAC. The outflow of both dopamine and DOPAC in kidney slices of rats injected with l‐DOPA (10 and 30 mg kg−1) or GluDOPA (16.7 mg kg−1) was found to decline monophasically with similar slopes of decline. The rate constants of loss (k, min−1) of DOPAC (10 mg kg−1 l‐DOPA, k = 0.0070; 30 mg kg−1 l‐DOPA, k = 0.0087; 16.7 mg kg−1 GluDOPA, k = 0.0080) were 2 to 3 fold those of dopamine (10 mg kg−1 l‐DOPA, k = 0.0027; 30 mg kg−1 l‐DOPA, k = 0.0034; 16.7 mg kg−1 GluDOPA, k = 0.0030). With both precursors the DOPAC/dopamine ratio in perifusate samples were 2.0 fold those in the tissues.

Tissue and outflow levels of dopamine after incubation of renal tissues with l‐DOPA, 50 and 100 μm were found to be lower than those observed with GluDOPA (50 and 100 μm). DOPAC/dopamine ratios in tissues and perifusate samples of experiments performed with l‐DOPA were significantly higher (P < 0.01) than those observed with GluDOPA. The outflow of both dopamine and DOPAC in renal slices incubated with l‐DOPA (50 and 100 μm) were found to decline with time, but presented a biphasic shape. DOPAC/dopamine ratios in perifusate samples were 3 fold that in the tissues with both precursors.

In conclusion, the present results show that both l‐DOPA and GluDOPA give origin to substantial amounts of dopamine and the newly‐formed amine undergoes considerable deamination to DOPAC. However, dopamine originating from GluDOPA was less deaminated than that resulting from l‐DOPA; it appears that this different behaviour may concern aspects related to the formation of the amine and also those related to its deamination and disposition, namely the processes involved in the access of newly‐formed dopamine to MAO.
Effects of dietary marine oil supplementation on reactivity of human buttock subcutaneous arteries and forearm veins in vitro
Authors: Chin Jaye P.F. , Kaye David M. , Hurlston Robyn M. , Angus James A. , Jennings Garry L. , Dart Anthony M.
Published: July 2012

The vascular reactivity of resistance arteries isolated from gluteal skin biopsies and veins isolated from forearms of subjects fed marine oils were examined.

Twenty seven healthy adult males were randomly allocated to one of two different treatment groups. The first group received maxEPA (eicosapentaenoic acid 0.178 g g−1; docosahexaenoic acid 0.116 g g−1) capsules 10 g per day for 28 days while the second group received an equivalent amount of mixed oil placebo capsules. Biopsies were performed on day 29 (13 for gluteal sections; 14 for forearm vein biopsies). Subcutaneous arteries and veins were mounted in myographs and standard organ baths, respectively.

The internal diameter of the subcutaneous arteries at a calculated transmural pressure of 100 mmHg averaged 183.7 ± 10.3 μm in the maxEPA group and 182.6 ± 19.8 μm in the placebo controls. Arteries from subjects on maxEPA demonstrated increased sensitivity to angiotensin II (maxEPA vs placebo: – log EC50 (m) − 8.36 ± 0.18 vs − 7.91 ± 0.14) but not to noradrenaline or 5‐hydroxytryptamine. Concentration‐response curves to acetylcholine, substance P and sodium nitroprusside obtained for noradrenaline precontracted vessels were unaltered with marine oil treatment as was the concentration‐response curve to calcium in K+‐depolarized vessels.

Vein internal diameter at a calculated transmural pressure of 20 mmHg averaged 3.06 ± 0.23 mm in the maxEPA group and 2.96 ± 0.89 in the placebo group. Responses to noradrenaline, 5‐hydroxytryptamine, angiotensin II and endothelin‐1 were obtained in the absence and presence of indomethacin (1 μm) in veins from both maxEPA and placebo‐treated subjects. Neither dietary supplementation with marine oils nor indomethacin had any effect on the responses obtained to these agonists.

The major finding of the present study was that in general, maxEPA supplementation did not affect responses to various vasoactive substances on isolated subcutaneous arteries or forearm veins. An exception was the observation of an enhanced response to angiotensin II in subcutaneous resistance arteries studied in vitro. This effect was selective for angiotensin II and was not apparent in veins isolated from the forearm.