bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto‐ATPase
Authors: Crack B.E. , Pollard C.E. , Beukers M.W. , Roberts S.M. , Hunt S.F. , Ingall A.H. , McKechnie K.C.W. , IJzerman A.P. , Leff P. 
Published: July 2012

FPL 67156 (6‐N,N‐diethyl‐β,γ‐dibromomethylene‐d‐ATP), is a newly synthesized analogue of ATP.
In a rabbit isolated tracheal epithelium preparation, measuring P2U‐purinoceptor‐dependent chloride secretion, FPL 67156 was discovered to potentiate the responses to UTP but not those to ATP‐γ‐S. UTP agonist‐concentration effect (E/[A]) curves were shifted to the left by 5‐fold in the presence of 100 μm FPL 67156. The differential effect of FPL 67156 on UTP and ATP‐γ‐S was hypothesized to be due to the greater susceptibility of UTP to enzymatic dephosphorylation and the ability of FPL 67156 to inhibit this process.
FPL 67156 was tested as an ecto‐ATPase inhibitor in a human blood cell assay, measuring [γ32P]‐ATP dephosphorylation. The compound inhibited [γ32P]‐ATP degradation with a pIC50 of 4.6.
FPL 67156 was then tested for its effects on ATP and α,β‐methylene‐ATP responses at P2X‐purinoceptors in the rabbit isolated ear artery. In the concentration range 30 μm‐1 mm, the compound potentiated the contractile effects of ATP but not those of α,β‐methylene‐ATP. At 1 mm, FPL 67156 produced a 34‐fold leftward shift of ATP E/[A] curves.
The effects of FPL 67156 on ATP E/[A] curves in the rabbit ear artery were analysed using a theoretical model (Furchgott, 1972) describing the action of an enzyme inhibitor on the effects of a metabolically unstable agonist. This analysis provided an estimate of the pK1 for FPL 67156 as an ecto‐ATPase inhibitor of 5.2.
Using appropriate assays, FPL 67156 was shown to have weak antagonist effects at P2X‐ and P2T‐purinoceptors (pA2 ≅ 3.3 and 3.5 respectively), and weak agonist effects at P2U‐purinoceptors (p[A50] ≅ 3.5).
The degree of potentiation of ATP and UTP effects elicited by FPL 67156 confirms previous results concerning the influence that ecto‐ATPase has on the position of E/[A] curves for metabolically unstable agonists. The magnitude of this influence is predicted to have a major effect on the agonist potency orders currently used to designate purinoceptors.
This study indicates FPL 67156 to be a potentially valuable probe in studies on the action of nucleotides and in the classification of purinoceptors.
Platelet‐activating factor: the effector of protein‐rich plasma extravasation and nitric oxide synthase induction in rat immune complex peritonitis
Authors: Steil Ana Angelica , Rodriguez Maria del Carmen Garcia , Alonso Andrés , Crespo Mariano Sanchez , Bosca Lisardo 
Published: July 2012

The involvement of platelet‐activating factor (PAF) in immune complex‐induced/polymorphonuclear‐mediated tissue injury was studied by use of a reverse passive Arthus (RPA) model in the peritoneal cavity of rats.
Extravasation of protein‐rich plasma, accumulation of polymorphonuclear leukocytes (PMN), and the production of nitric oxide (NO) by resident peritoneal mononuclear phagocytes were assayed.
Treatment of rats with either UR‐12460 or BB‐823, two compounds which possess different chemical structures, but elicit the same antagonistic effect on the PAF receptor, abrogated protein‐rich plasma extravasation. In contrast, they did not show any effect on the accumulation of PMN.
Inhibition of NO production with both NG‐mono methyl‐l‐arginine and NG‐nitro‐l‐arginine failed to prevent protein‐rich plasma extravasation.
The production of NO by peritoneal adherent cells following RPA was measured in cells maintained for 2 to 28 h in culture, and it was significantly increased in cells removed as early as 15 min after RPA induction, as compared to controls.
Addition of 10 nm PAF to the culture medium reduced the generation of NO by peritoneal cells from RPA rats, whereas this mediator enhanced NO production in cells from naive control animals.
Treatment with either UR‐12460 or BB‐823 prior to the induction of RPA produced an almost complete inhibition of NO production.
Assay of nitric oxide synthase activity in cell homogenates from peritoneal cells showed that the activity was due to the inducible form of the enzyme.
Study by Northen blotting of mRNA coding for the inducible NO synthase (iNOS) showed transcription at 6 and 18 h after the induction of RPA, which was inhibited in UR‐12460‐treated rats.
These data indicate that PAF is the main mediator of the early plasma leakage observed in RPA, and also that PAF is implicated in the triggering of long‐term changes via induction of specific genes, as judged from its ability to promote the expression of iNOS.
Investigation of the subtype of α2‐adrenoceptor mediating prejunctional inhibition of cardioacceleration in the pithed rat heart
Authors: Smith Karen , Gavin Katherine , Docherty James R. 
Published: July 2012

We have investigated the subtype of α2‐adrenoceptor mediating prejunctional inhibition of cardioacceleration in the pithed rat heart in comparison with α2‐adrenoceptor ligand binding sites.
In pithed rats, prejunctional α2‐adrenoceptors were investigated in terms of the ability of α2‐adrenoceptor antagonists to shift the inhibitory potency of the α2‐adrenoceptor agonist, xylazine, against the tachycardia to a single electrical stimulus given via the pithing rod.
Antagonist potency at prejunctional α2‐adrenoceptors in pithed rat heart was correlated with antagonist affinity at α2‐adrenoceptor ligand binding sites in membranes of rat kidney and submandibular gland labelled with [3H]‐yohimbine.
The correlation with the prejunctional α2‐adrenoceptor in pithed rat heart was best for the α2D‐adrenoceptor ligand binding site of rat submandibular gland (r = 0.98, n = 10, P < 0.0001), as compared to correlations with the α2A‐adrenoceptor ligand binding site of human platelet (r = 0.90, n = 9, P<0.001), the x2B‐adrenoceptor ligand binding site of rat kidney (r = 0.82, n = 10, P<0.01) and with published results for the α2C‐adrenoceptor ligand binding site (r = 0.48, n = 6, NS).
It is concluded that the functional prejunctional α2‐adrenoceptor of pithed rat heart closely resembles the α2D‐adrenoceptor ligand binding site of rat submandibular gland.
Kallikrein rK10‐induced kinin‐independent, direct activation of NO‐formation and relaxation of rat isolated aortic rings
Authors: Wassdal Irene , Hull Robert , Gerskowitch V. Paul , Berg Torill 
Published: July 2012

rK10, a weak T‐kininogenase isolated from the rat submandibular gland, is a protein belonging to the rat kallikrein family. In the present work, we have studied the biological effects of rK10 with respect to its ability to alter vascular resistance, either directly like rK9, i.e., another kallikrein‐like protein, trypsin and thrombin, or through the release of kinins like tissue kallikrein (rK1). The direct effect was studied by its vasomotor activity on rat isolated aortic rings since this preparation was insensitive to the action of kinins. Its ability to induce altered vascular resistance through kinin‐generation was investigated by blood pressure studies in whole animals. The studies were performed in comparison to rK1.
Unlike rK1, which induces hypotension when administered intravenously to rats (ΔBP = − 56 ± 5 mmHg, 5 μg kg−1), rK10 did not have any: effect on systemic blood pressure (ΔBP= −3 ± 1, 5 μg kg−1, i.v.)
rK10 was without effect on uncontracted aortic rings, but showed a concentration‐dependent (10−8−10−6 m) relaxant effect on tissue precontracted with phenylephrine (10−6m). After removal of endothelial cells, no relaxation was observed. The relaxant response to rK10 was transient. rK1 (with and without endothelium), bradykinin and T‐kinin (with endothelium) had no effect on contracted or uncontracted aortic rings.
The relaxant effect of rK10 was dependent on its enzymatic activity since preincubation with aprotinin (1.02 mm) significantly reduced vasorelaxation from 74 ± 4% to 24 ±3%.
The relaxant effect was not inhibited by the kinin antagonist Hoe 140 (10−7 m; 34 ± 4% without, versus 30 ± 2% with Hoe 140), but was totally inhibited by the NO‐synthase inhibitor N.nitro‐L‐arginine methyl ester (l‐NAME) (2.5 × 10−4m; 27 ± 3% without and 2± 1% with l‐NAME).
These results show that rK10 has the ability to induce vascular relaxation by a specific, direct effect on endothelial cell NO‐synthesis, dependent on rK10 proteolytic activity, but independent of its ability to generate kinin. This effect, or its T‐kininogenase activity in blood, was not sufficient for rK10 to have an effect on peripheral vascular resistance since intravenous injections of rK10, unlike rK1, did not induce hypotension. Thus, rK10 does not seem to play a role in blood pressure homeostasis but may have a local effect on vascular resistance.
Thapsigargin‐ and cyclopiazonic acid‐induced endothelium‐dependent hyperpolarization in rat mesenteric artery
Authors: Fukao Mitsuhiro , Hattori Yuichi , Kanno Morio , Sakuma Ichiro , Kitabatake Akira 
Published: July 2012

The present study was designed to determine whether putative, selective inhibitors of the Ca2+–pump ATPase of endoplasmic reticulum, thapsigargin (TSG) and cyclopiazonic acid (CPA), induce endothelium‐dependent hyperpolarization in the rat isolated mesenteric artery. The membrane potentials of smooth muscle cells of main superior mesenteric arteries were measured by the microelectrode technique.
In tissues with endothelium, TSG (10−8–10−5m) caused sustained hyperpolarization in a concentration‐dependent manner. In tissues without endothelium, TSG did not cause any change in membrane potential. CPA (10−5 m) also hyperpolarized the smooth muscle membrane, an effect that was endothelium‐dependent and long‐lasting.
The hyperpolarizing responses to these agents were not affected by indomethacin or NG–nitro‐L‐arginine (L‐NOARG).
In Ca2+–free medium, neither TSG nor CPA elicited hyperpolarization, in contrast to acetylcholine which generated a transient hyperpolarizing response.
In rings of mesenteric artery precontracted with phenylephrine, TSG and CPA produced endothelium‐dependent relaxations. L‐NOARG significantly inhibited the relaxations to these agents, but about 40–60% of the total relaxation was resistant to L‐NOARG. The L‐NOARG‐resistant relaxations were abolished by potassium depolarization.
These results indicate that TSG and CPA can cause endothelium‐dependent hyperpolarization in rat mesenteric artery possibly by releasing endothelium‐derived hyperpolarizing factor and that membrane hyperpolarization can contribute to the endothelium‐dependent relaxations to these agents. The mechanism of hyperpolarization may be related to increased Ca2+ influx into endothelial cells triggered by depletion of intracellular Ca2+ stores due to inhibition of endoplasmic reticulum Ca2+–pump ATPase activity.
Potentiation by viral respiratory infection of ovalbumin‐induced guinea‐pig tracheal hyperresponsiveness: role for tachykinins
Authors: Ladenius A. Rudolf C. , Folkerts Gert , van der Linde Henk J. , Nijkamp Frans P. 
Published: July 2012

We investigated whether virus‐induced airway hyperresponsiveness in guinea‐pigs could be modulated by pretreatment with capsaicin and whether viral respiratory infections could potentiate ovalbumin‐aerosol‐induced tracheal hyperresponsiveness.
Animals were inoculated intratracheally with bovine parainfluenza‐3 virus or control medium 7 days after treatment with capsaicin (50 mg kg−1, s.c). Four days after inoculation, tracheal contractions were measured to increasing concentrations of substance P, histamine and the cholinoceptor agonist, arecoline.
In tracheae from virus‐infected guinea‐pigs, contractions in response to substance P, histamine and arecoline were significantly enhanced (P<0.01) by 144%, 46% and 77%, respectively. Capsaicin pretreatment inhibited the hyperresponsiveness to substance P partly (62%) and to histamine and arecoline completely.
In another series of experiments animals were first sensitized with ovalbumin (20 mg kg−1, i.p.). After 14 days animals were exposed to either saline or ovalbumin aerosols for 8 days. After 4 aerosol exposures (4 days) animals were inoculated with either parainfluenza‐3 virus or control medium. One day after the last ovalbumin aerosol, tracheal contraction in response to increasing concentrations of substance P, histamine and arecoline was measured.
Tracheae from ovalbumin‐aerosol‐exposed control inoculated animals showed a similar degree of airway hyperresponsiveness to saline‐aerosol‐exposed virus‐treated guinea‐pigs. Virus inoculation of ovalbumin‐treated animals significantly potentiated the tracheal contractions to substance P compared to either of the treatments alone. The contractions in response to histamine and arecoline were only slightly enhanced.
In conclusion, sensory nerves and/or tachykinins are involved in virus‐induced airway hyperresponsiveness in guinea‐pigs and viral respiratory infections can potentiate the increase in tracheal responsiveness to bronchoconstrictor agonists after ovalbumin exposure.
Inhibition by cromoglycate and some flavonoids of nucleoside diphosphate kinase and of exocytosis from permeabilized mast cells
Authors: Martin Matthew W. , O'Sullivan Antony J. , Gomperts Bastien D. 
Published: July 2012

The anti‐allergic compound, cromoglycate, is reported to possess affinity for, and to suppress the autophosphorylation of a 72kDa protein having the sequence of nucleoside diphosphate kinase (NDPK).We have tested the ability of cromoglycate, and a panel of ten structurally related flavonoids of plant origin, to inhibit the NDPK reaction and the exocytotic process of permeabilized mast cells. The conditions of permeabilization (use of an isotonic medium based on sodium glutamate) were selected to ensure that NDPK activity would be an essential component in the induction of Ca2+–induced exocytosis in which ATP is required for generation of GTP. For comparison, we also measured the inhibition of exocytosis induced by GTP‐γ‐S; this proceeds in the absence of ATP and bypasses the need for NDPK activity.We found that cromoglycate does not discriminate between Ca2+and GTP‐γ‐S‐induced exocytosis and is a poor inhibitor of NDPK activity. Concentrations in the millimolar range are required for inhibition of all these functions. By comparison, many of the flavonoids are effective at concentrations in the micromolar range.While we were unable to discern any systematic relationships between their ability to inhibit the three functions, two compounds, quercetin and genistein, inhibit Ca2+–induced, but not GTP‐γ‐S‐induced exocytosis. Inhibition of the late stages of the stimulus‐response pathway in mast cells by these compounds is therefore likely to be due to inhibition of NDPK and the consequent failure to generate GTP.
Effect of morphine on the nerve terminal impulse and transmitter release from sympathetic varicosities innervating the mouse vas deferens
Authors: Lavidis Nickolas A. 
Published: July 2012

The effect of morphine on both the propagation of the nerve terminal impulse along the sympathetic varicose axons as well as the evoked and spontaneous transmitter release has been evaluated.
Morphine (1 μm) did not significantly change the shape or the regularity by which the nerve terminal impulse was recorded while evoked transmitter release was greatly reduced.
Morphine induced a uniform decrease in evoked transmitter release irrespective of the release probability of individual varicosities of their position along terminal branches.
Procedures which are thought to increase intracellular calcium concentration such as increasing the extracellular calcium concentration, stimulation of the nerve with trains of impulses and increasing the duration of the action potential with 4‐aminopyridine reduced the ability of morphine to decrease evoked transmitter release.
Morphine had to act directly on the varicosities to induce a decrease in evoked transmitter release.
The decrease in evoked quantal release does not involve an affect on the nerve terminal impulse or the vesicle release process and morphine may affect the dependence of the secretory process on calcium.
Inducible nitric oxide synthase activity and expression in a human colonic epithelial cell line, HT‐29
Authors: Kolios George , Brown Zarin , Robson Rachel L. , Robertson Duncan A.F. , Westwick John 
Published: July 2012

We have determined which cytokines regulate the expression of human inducible nitric oxide synthase (iNOS) mRNA and nitrite generation in the human colonic epithelial cell line HT‐29.
Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin‐lα (IL‐lα), tumour necrosisfactor‐α (TNF‐α), interferon γ (IFN‐γ) or vehicle added alone or in combination. Human iNOS mRNA was determined by Northern blot analysis and nitrite generation by the use of a fluorometric assay.
Unstimulated cells produced a small time‐dependent increase in nitrite generation of 50 ± 4, 75 ± 8, and 103 ±8 nM per 106 cells at 24 h, 48 h, and 72 h respectively. This nitrite generation was unaffected by cycloheximide (5 μg ml−1) pretreatment and iNOS mRNA was not detected.
None of cytokines alone induced either iNOS mRNA expression or an increase in nitrite generation. The combination of IL‐lα/IFN‐γ produced a highly significant (P< 0.001) 4 fold increase in nitrite production at 48 h, compared to basal values, while no other pair of cytokines was effective.
Time course studies with IL‐lα/IFN‐γ combination revealed significant (P< 0.001) increases in nitrite at 24 h (153 ±7), 48 h (306 ±24), and 72 h (384 ±15) compared to basal values of 50 ±4, 75 ±8, and 103 ±8 nM per 106 cells respectively.
Studies with IL‐lα/IFN‐γ combination demonstrated a time dependent expression of iNOS mRNA, first observed at 6 h, peaked at 24 h and was undetectable by 72 h. IL‐lα (0.3–10 ng ml−1) and IFN‐γ (10–300 u ml−1) in combination induced a concentration‐dependent expression of iNOS mRNA at 24 h.
Pretreatment with cycloheximide before IL‐lα/IFN‐γ stimulation reduced nitrite levels to basal values. These data suggest that the IL‐lα/IFN‐γ‐induced nitrite production by HT‐29 cells is dependent on de novo protein synthesis, probably the iNOS enzyme.
The addition of TNF‐α produced a significant (P< 0.001) 3 fold increase of IL‐lα/IFN‐γ‐induced nitrite generation. In marked contrast the presence of TNF‐α had no effect on IL‐lα/IFN‐γ‐induced iNOS mRNA expression by HT‐29 cells. These findings suggest that the up‐regulation by TNF‐α of IL‐lα/IFN‐γ‐induced nitrite generation is at the post‐transcriptional level.
These data suggest that pro‐inflammatory cytokines induce NO production in colonic epithelial cells probably due to the induction of iNOS and these cells may be a major source of NO generation in inflammatory bowel disease.
Nitric oxide is a mediator of tachykinin NK3 receptor‐induced relaxation in rat mesenteric artery
Authors: Mizuta Aki , Takano Yukio , Honda Kenji , Saito Ryo , Matsumoto Takafumi , Kamiya Hiro‐O 
Published: July 2012

The mechanism of vasodilatation induced by tachykinin peptides was studied in isolated mesenteric arteries of rats.
Senktide, a selective NK3 agonist, elicited potent endothelium‐dependent relaxation of arteries precontracted with phenylephrine (10‐5m), but an NK1 agonist did not.
A non‐peptide NK3 antagonist, SR 142801, inhibited senktide‐induced relaxation. However, a non‐peptide NK1 antagonist, CP‐96,345, and a peptide‐based NK2 antagonist, L‐659,877, had no effect on senktide‐induced relaxation.
Nω‐nitro‐L‐arginine (l‐NOARG), a nitric oxide synthesis inhibitor, markedly attenuated the relaxant response to senktide.
These results suggest that the endothelium of rat mesenteric arteries possesses tachykinin NK3 receptors, and that NK3 agonist‐induced vasodilatation is mediated by release of nitric oxide (NO) from the endothelium.
An endogenous protectant effect of cardiac cyclic GMP against reperfusion‐induced ventricular fibrillation in the rat heart
Authors: Pabla Ravinder , Bland‐Ward Philip , Moore Philip K. , Curtis Michael J. 
Published: July 2012

. After a period of myocardial ischaemia, reperfusion of the myocardium can elicit cardiac arrhythmias. Susceptibility to these arrhythmias declines with time, such that a preceding period of more than approximately 40 min ischaemia is associated with few reperfusion‐induced arrhythmias. We have tested the hypothesis that this decline in susceptibility occurs, in part, because of protection by endogenous guanosine 3′:5′‐cyclic monophosphate (cyclic GMP).
Rat isolated hearts were subjected to 60 min left regional ischaemia followed by reperfusion (n= 10 per group). Methylene blue (20 μm), a soluble guanylate cyclase inhibitor, raised the incidence of reperfusion‐induced ventricular fibrillation (VF) from 10% in control hearts to 80% (P<0.05). This effect of methylene blue was abolished by co‐perfusion with zaprinast (100 μm), a phosphodiesterase inhibitor which, in the rat heart, is cyclic GMP‐specific (specific for the type‐V phosphodiesterase isozyme).
Methylene blue reduced cyclic GMP levels in the ischaemic, non‐ischaemic and reperfused myocardium (P<0.05) to 50±10, 52±12 and 70 ± 7 fmol mg−1 tissue wet weight, respectively from control values of 143 ± 38, 147±43 and 156±15 fmol mg−1. Co‐perfusion with zaprinast prevented this effect, and cyclic GMP levels were actually elevated (P<0.05) to 366±102, 396±130 and 293 ± 22 fmol mg−1 in ischaemic, non‐ischaemic and reperfused myocardium, respectively. Zaprinast by itself also elevated cyclic GMP content. Cyclic AMP levels were not affected by zaprinast or methylene blue.
In conclusion, when endogenous cardiac cyclic GMP synthesis is reduced, susceptibility to reperfusion‐induced VF after sustained ischaemia is substantially increased. The effect is prevented by inhibiting cyclic GMP degradation. Therefore cyclic GMP appears to be an endogenous intracellular cardioprotectant, and its actions may account for the low susceptibility to VF normally encountered in hearts reperfused after sustained ischaemia.