bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrophysiological evidence for different release mechanism of ATP and NO as inhibitory NANC transmitters in guinea‐pig colon
Authors: Zagorodnyuk Vladimir , Maggi Carlo Alberto
Published: July 2012

The effect of the P2‐purinoceptor antagonist, suramin, the specific N‐type voltage‐dependent calcium channel blocker, ω‐conotoxin GVIA (ω‐CgTx) and the δ‐opioid receptor agonist [d‐Pen2, d‐Pen5] enkephalin (DPDPE) on the apamin‐sensitive and apamin‐resistant inhibitory junction potentials (i.j.ps) produced by electrical field stimulation (EFS) were investigated by means of a sucrose‐gap technique in the circular muscle of the guinea‐pig colon.

After incubation of muscle strips in either atropine (1 μm), guanethidine (3 μm) and NG‐nitro‐l‐arginine (l‐NOARG, 30 μm) or atropine, guanethidine and apamin (0.3 μm), the addition of the NK1 receptor antagonist, SR 140,333 (1 μm) abolished the non‐adrenergic, non‐cholinergic (NANC) excitatory junction potential (e.j.p.) and unmasked a pure apamin‐sensitive i.j.p. (in the presence of l‐NOARG) or a pure apamin‐resistant i.j.p. (in the presence of apamin). Both types of i.j.p. were abolished by tetrodotoxin.

Suramin (30–300 μm) concentration‐dependently inhibited the apamin‐sensitive i.j.p., while the apamin‐resistant i.j.p. was not significantly affected by suramin (up to 300 μm). l‐NOARG (30 μm) markedly reduced the apamin‐resistant i.j.p.

The δ‐opioid receptor agonist, DPDPE (0.03–3 μm) concentration‐dependently reduced the apamin‐sensitive i.j.p., while leaving the apamin‐resistant i.j.p. unaffected. Naloxone (1 μm) prevented the i.j.p. inhibition evoked by DPDPE (0.3 μm).

ω‐CgTx (0.3 μm) markedly reduced the apamin‐sensitive but not the apamin‐resistant i.j.p. The application of DPDPE (3 μm), after development of a steady state inhibitory effect by ω‐CgTx, evoked further inhibition of the apamin‐sensitive i.j.p., similar to the effect produced by DPDPE alone. The L‐type calcium channel blocker, nifedipine (1 μm) did not significantly affect either the apamin‐sensitive or the apamin‐resistant i.j.ps.

These findings support the purinergic origin of the fast, apamin‐sensitive i.j.p. produced by EFS in the circular muscle of the guinea‐pig colon and strongly suggest that the apamin‐sensitive and the apamin‐resistant components of the evoked i.j.p. utilize different mechanisms for the secretion of the NANC transmitters, ATP and NO, respectively.
Investigation of the role of 5‐HT3 and 5‐HT4 receptors in ascending and descending reflexes to the circular muscle of guinea‐pig small intestine
Authors: Yuan S.Y. , Bornstein J.C. , Furness J.B.
Published: July 2012

The present study was undertaken to ascertain whether 5‐hydroxytryptamine (5‐HT) acting at either 5‐HT3 or 5‐HT4 receptors plays a significant role in motility reflexes in the guinea‐pig small intestine.

An isolated segment of small intestine was opened along its mesenteric border and pinned, mucosa uppermost, in a three chambered organ bath so that the oral, middle and anal regions of a single preparation could be separately superfused.

Conventional intracellular recording methods were used to monitor the responses of the circular muscle in the oral or the anal end chambers when distension was applied in either of the other two chambers or the mucosal villi were compressed in the middle chamber. Drugs were added to the middle chamber.

5‐HT3 receptor antagonists (tropisetron, 0.1–10 μm; granisetron, 1 μm and BRL 46470, 1 μm) depressed the ascending excitatory reflex evoked by these stimuli but had no effect on the descending inhibitory reflex. The depression of the excitatory reflex was observed whether the reflex was evoked from the chamber containing the drug or was simply conducted, via interneurones, through this chamber.

The 5‐HT4 receptor antagonist, SDZ 205–557 (1 μm), had no significant effect on either the ascending or descending reflex pathways. However, 5‐HT4 receptors were present as cisapride (0.1 μm) significantly enhanced the ascending excitation without affecting the descending inhibition. This effect of cisapride was converted to a significant depression of the ascending reflex by SDZ 205–557.

The results suggest that 5‐HT3, but not 5‐HT4, receptors play an important role in the ascending excitatory reflex and that these receptors may be on interneurones in the reflex pathway.
Role of cyclic AMP‐induced Cl conductance in aqueous humour formation by the dog ciliary epithelium
Authors: Chen Shan , Inoue Ryuji , Inomata Hajime , Ito Yushi
Published: July 2012

The effects of isoprenaline, a forskolin derivative NKH‐477, and dibutyryl cyclic AMP (db cyclic AMP) on the membrane potential, conductance and cell volume of the dog non‐pigmented ciliary epithelium (NPE) were investigated by intracellular potential recording, nystatin‐perforated patch clamp technique and videomicroscopic cytometry.

The resting membrane potential of NPE was about − 70 mV in physiological saline and was depolarized by isoprenaline in a dose‐dependent manner with an ED50 of about 3 nM. This depolarization was competitively antagonized by the β‐adrenoceptor antagonist, timolol (pA2 = ca. 9) and almost completely blocked by the Cl transport blocker, DIDS.

In single dissociated NPE cells, 10 μm isoprenaline induced an inward current and caused a concomitant decrease in cell volume. The reversal potential measurement indicated that this inward current was carried mainly by Cl ion. DIDS (10 μm) abolished both the current and cell volume decrease.

NKH‐477 (10 μm) or db cyclic AMP (1 mM) also induced an inward current together with a cell volume decrease, the properties of which were similar to those caused by isoprenaline.

These results suggest that β‐adrenoceptor stimulation in NPE leads to an increased rate of aqueous humour production by increasing Cl− efflux via an elevation of cyclic AMP and this effect is efficiently blocked by timolol.
Pharmacokinetics, plasma protein binding and urinary excretion of Nω‐nitro‐L‐arginine in rats
Authors: Tabrizi‐Fard Mohammad A. , Fung Ho‐Leung
Published: July 2012

The in vivo disposition of Nω‐nitro‐l‐arginine (l‐NOARG) has been examined in rats. Plasma concentrations of l‐NOARG following an intravenous dose of 10 mg kg−1 were determined by high‐performance liquid chromatography. Plasma l‐NOARG concentrations declined biexponentially, with average half‐lives of 11 min and 20 h. l‐NOARG clearance did not appear to exhibit concentration‐dependency below a plasma concentration of 4.56 × 10−4 m (100 mg 1−1). Ultrafiltration studies revealed insignificant binding of l‐NOARG to rat plasma proteins. Urinary excretion of unchanged l‐NOARG was minimal. These pharmacokinetic information may be useful in the design of in vivo experiments involving l‐NOARG, as well as in the interpretation of the pharmacodynamics of this important nitric oxide synthase inhibitor.
Effects of bradykinin B2 receptor antagonism on the hypotensive effects of ACE inhibition
Authors: Bouaziz Herve , Joulin Yves , Safar Michel , Benetos Athanase
Published: July 2012

The aim of this study was to determine the participation of endogenous bradykinin (BK) in the antihypertensive effects of the angiotensin converting enzyme inhibitor (ACEI), perindoprilat, in the spontaneously hypertensive rat (SHR) on different salt diets.

Conscious SHRs receiving either a low or a high NaCl diet were used in order to evaluate the respective roles of angiotensin II suppression and bradykinin stimulation in the acute hypotensive effects of perindoprilat. Two different B2 receptor antagonists (B 4146 and Hoe 140) were used after bolus administration of 7 mg kg−1 of the ACEI, perindoprilat. In separate animals, Hoe 140 was administered before the injection of perindoprilat. In other experiments, the effects of Hoe 140 on the hypotensive effects of the calcium antagonist, nicardipine, were tested.

The different NaCl diets had no effect on baseline blood pressure. Hoe 140 injection before ACE inhibition did not modify blood pressure. Perindoprilat caused more marked hypotension in the low salt‐fed rats than in the high salt animals (P < 0.01). Administration of Hoe 140 or B4146 after perindoprilat significantly reduced the antihypertensive effects of perindoprilat in the different groups, but this effect was more pronounced in high salt‐fed rats. However, in SHRs receiving Hoe 140 before perindoprilat, the antihypertensive effect of perindoprilat was completely abolished in both high or low salt diet rats. In separate experiments we confirmed that Hoe 140 did not affect the hypotensive efficacy of the calcium antagonist, nicardipine.

Our study shows that inhibition of endogenous bradykinin degradation participates in the acute antihypertensive effects of perindoprilat in SHRs. The role of bradykinin is more pronounced following exposure to a high salt diet i.e., when the renin‐angiotensin system is suppressed. Blockade of bradykinin B2 receptors by Hoe 140 before administration of perindoprilat completely abolished the hypotensive effect of perindoprilat suggesting an increased role of bradykinin in the onset of hypotensive action of ACE inhibitors. However, the exact mechanism of this interaction remains unclear.
Role of interleukin‐5 in enhanced migration of eosinophils from airways of immunized guinea‐pigs
Authors: Coéffier Eliane , Joseph Danielle , Vargaftig B. Boris
Published: July 2012

Platelet activating factor (PAF), leukotriene B4 (LTB4) and interleukin‐5 (IL‐5) are potent chemoatt‐ractants for guinea‐pig eosinophils, which may be involved in eosinophil recruitment and up‐regulation in allergic diseases. Eosinophils from the bronchoalveolar lavage fluid (BALF) of ovalbumin‐sensitized guinea‐pigs were collected 24 h after antigen provocation and migration induced by PAF, LTB4 and rhIL‐5 was studied.

Total BALF content and distribution of eosinophils were greater in immunized, ovalbumin‐challenged guinea‐pigs (5.0 ± 0.8 × 106/guinea‐pig; 12 ± 1%) than in immunized, saline‐challenged animals (3.0 ± 0.7 × 106/guinea‐pig; 7 ± 1%).

The chemoattraction of eosinophils isolated on a metrizamide gradient was studied in micro‐Boyden chambers, results being expressed as the number of migrating cells (mean ± s.e.mean). PAF and LTB4‐induced migration of eosinophils from immunized and OA‐challenged guinea‐pigs were significantly enhanced, as compared to immunized and saline‐challenged animals (170 ± 36 vs 35 ± 9 migrating eosinophils for 10 nm PAF; 271 ± 60 vs 110 ± 19 for 1 nm LTB4).

The IL‐5 antibody TRFK‐5, in vivo, reduced eosinophil recruitment in BALF of antigen‐challenged immunized animals as well as the enhanced responsiveness of eosinophils from the challenged animals, suggesting a role for IL‐5 in the priming of eosinophils in vivo.

In contrast to TRFK‐5, nedocromil sodium reduced to a similar extent eosinophil, macrophage and lymphocyte recruitment into the BALF of antigen‐challenged, but failed to down‐regulate the enhanced responsiveness of eosinophils from the challenged animals.

The increased eosinophil content in lungs from antigen‐challenged guinea‐pigs is thus selectively reduced by the anti‐IL‐5 antibody, which also attenuates the concomitant enhancement of the eosinophil responsiveness, supporting the concept that IL‐5 is essential for recruitment and priming of eosinophils in vivo. In contrast, nedocromil sodium reduced non‐selectively the total cell recruitment to the airways, but failed to attenuate the enhanced responsiveness of those eosinophils which migrated, indicating that its effects involve a different target.
Dual effects of tetrandrine on cytosolic calcium in human leukaemic HL‐60 cells: intracellular calcium release and calcium entry blockade
Authors: Leung Yuk‐Man , Kwan Chiu‐Yin , Loh Tatt‐Tuck
Published: July 2012

Tetrandrine (TET, a Ca2+ antagonist of Chinese herbal origin) and thapsigargin (TSG, an endoplasmic reticulum Ca2+ pump inhibitor) concentration‐dependently mobilized Ca2+ from intracellular stores of HL‐60 cells, with EC50 values of 20 μm and 0.8 nm, respectively. After intracellular Ca2+ release by 30 nm TSG, there was no more discharge of Ca2+ by TET (100 μm), and vice versa.

Pretreatments with 100 nm rauwolscine (α2‐adrenoceptor antagonist), 100 nm prazosin (α1‐adreno‐ceptor antagonist), 10 nm phorbol myristate acetate (PMA, a protein kinase C activator) or 100 nm staurosporine (a protein kinase C inhibitor) had no effect on 100 μm TET‐induced intracellular Ca2+ release.

After intracellular Ca2+ release by 30 nm TSG in Ca2+‐free medium, readmission of Ca2+ caused a substantial and sustained extracellular Ca2+ entry. The latter was almost completely inhibited by 100 μm TET (IC50 of 20 μm) added just before Ca2+ readmission. In Ca2+‐containing medium, 30 nm TSG caused a sustained phase of cytosolic Ca2+ elevation, which could be abolished by 100 μm TET. TET was also demonstrated to retard basal entry of extracellular Mn2+ and completely inhibit TSG‐stimulated extracellular Mn2+ entry.

TSG‐induced extracellular Ca2+ entry was insensitive to the L‐type Ca2+ channel blocker, nifedipine (1 μm), but was completely inhibited by the non‐selective Ca2+ channel blocker La3+ (300 μm). Depolarization with 100 mm KCl did not raise the cytosolic Ca2+ level.

These data suggest that (a) TET and TSG mobilized the same Ca2+ pool and TET‐induced intracellular Ca2+ release was independent of protein kinase C activity and a‐adrenoceptor activation, and (b) TET blocked the voltage‐insensitive Ca2+ entry pathway activated by TSG. These dual effects on HL‐60 cells were also observed with hernandezine (HER), a TET‐like compound and in another cell type, murine B lymphoma M12.4 cells.
Antagonism by (R)‐ and (S)‐trihexyphenidyl of muscarinic stimulation of adenylyl cyclase in rat olfactory bulb and inhibition in striatum and heart
Authors: Onali Pierluigi , Aasen Arne J. , Olianas Maria C.
Published: July 2012

Activation of muscarinic receptors in rat olfactory bulb stimulates adenylyl cyclase activity. This response was competitively antagonized by the (R)‐ and (S)‐enantiomers of trihexyphenidyl with pA2 values of 8.84 and 6.09, respectively.

Similarly, in rat striatal homogenates, muscarinic inhibition of adenylyl cyclase activity was antagonized by the (R)‐ and (S)‐enantiomers with pA2 values of 8.75 and 6.12, respectively.

In contrast, in rat myocardium the muscarinic inhibition of the adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) formation was more weakly antagonized by trihexyphenidyl, with a particularly marked loss (15 fold) in activity of the (R)‐enantiomer. The (R)‐ and (S)‐enantiomers had pA2 values of 7.64 and 5.72, respectively.

Each muscarinic response was completely antagonized by increasing concentrations of (R)‐trihexyphenidyl with a Hill coefficient not significantly different from unity.

The present study shows that the muscarinic receptors coupled to stimulation of adenylyl cyclase in the olfactory bulb display high stereoselectivity for the enantiomers of trihexyphenidyl. The affinities of these receptors for the antagonists are similar to those shown by the striatal receptors. This finding supports the hypothesis that both the muscarinic stimulation of adenylyl cyclase in the olfactory bulb and the muscarinic inhibition of the enzyme in striatum are mediated by activation of a receptor subtype pharmacologically equivalent to the m4 gene product. On the other hand, the weaker affinities and the lower stereoselectivity for the trihexyphenidyl enantiomers exhibited by the muscarinic inhibition of adenylyl cyclase in the heart are consistent with the involvement of M2 receptors in this response.
Agonist‐induced desensitization of histamine Hi receptor‐mediated inositol phospholipid hydrolysis in human umbilical vein endothelial cells
Authors: McCreath G. , Hall LP. , Hill S.J.
Published: July 2012

The regulation of histamine‐induced [3H]‐inositol phosphate formation was studied in human cultured umbilical vein endothelial cells (HUVEC).

Histamine (EC50 4.8 μm) produced a 12.7 fold increase in [3H]‐inositol phosphate formation over basal levels. Prior exposure to 0.1 mm histamine (2h) produced a 78% reduction in the response to subsequent histamine (0.1 μm) challenge. The IC50 for this histamine‐induced desensitization was 0.9 μm.

The inositol phosphate response to histamine (0.1 μm) was inhibited by phorbol dibutyrate (IC50 40 nm; maximal reduction 64%). This effect was antagonized by both staurosporine (100 nm) and Ro 31–8220 (10 μm). However, the histamine‐induced desensitization of the H1‐receptor‐mediated inositol phosphate response was insensitive to the protein kinase inhibitors, staurosporine, Ro 31–8220, K252a and KN62.

Prior exposure to sodium nitroprusside (100 μm), forskolin (10 μm) or dibutyryl cyclic AMP (1 μm) had no effect upon histamine‐induced [3H]‐inositol phosphate formation.

NaF (20 μm) and thrombin (EC50 0.4 u ml−1) also induced inositol phosphate formation in HUVEC. Histamine pretreatment (0.1 mm, 10‐120min) failed to modify the inositol phosphate response to a subsequent NaF or thrombin challenge.

We conclude that the desensitization of histamine Hrreceptor‐mediated [3H]‐inositol phosphate formation occurs at the level of the receptor and involves a mechanism independent of activation of protein kinase A, G, or C, or calcium calmodulin‐dependent protein kinase II.
Contribution of intercellular adhesion molecule 1 (ICAM‐1) to the pathogenesis of splanchnic artery occlusion shock in the rat
Authors: Squadrito Francesco , Altavilla Domenica , Canale Patrizia , Ioculano MariaPatrizia , Campo Giuseppe M. , Ammendolia Letteria , Squadrito Giovanni , Saitta Antonino , Calapai Gioacchino , Caputi Achille P.
Published: July 2012

It has been suggested that leukocytes play a key role in the pathogenesis of splanchnic artery occlusion shock. Intercellular adhesion molecule 1 (ICAM‐1) is an adhesion molecule of crucial importance in the phenomenon of leukocyte accumulation.

We investigated the involvement of ICAM‐1 in the pathogenesis of splanchnic artery occlusion shock. Splanchnic artery occlusion (SAO) shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min. Sham‐operated animals were used as controls. Survival time, serum tumour necrosis factor‐α (TNF‐α), white blood cell (WBC) count, mean arterial blood pressure, myeloperoxidase activity (MPO; studied as a quantitative means to assess leukocyte accumulation) and the responsiveness to acetylcholine of aortic rings were investigated. SAO shocked rats had a decreased survival time (90 ± 9.5 min, while sham‐shocked rats survived more than 4 h), reduced mean arterial blood pressure, increased serum levels of TNF‐α (201 ± 10 u ml−1) and MPO activity in the ileum (0.15 ± 0.03 u × 10−3 per g tissue) and in the lung (1.9 ± 0.8 u × 10−3 per g tissue), leukopenia and reduced responsiveness to acetylcholine (ACh, 10nM‐10 μm) of aortic rings.

Administration of monoclonal antibody raised against rat ICAM‐1 significantly increased survival time (225 ± 9 min), reduced leukopenia and MPO activity both in the ileum (0.031 ± 0.003 u × 10−3 per g tissue) and in the lung 0.23 ± 0.03 u × 10−3 per g tissue), improved the cardiovascular changes and restored the responsiveness to ACh of aortic rings.

Our findings are consistent with an involvement of adhesion mechanisms in vivo in the pathogenesis of SAO shock and suggest that specific adhesion mechanisms, which support leukocyte accumulation, may represent potentially important therapeutic targets in circulatory shock.
Blockade by dendrotoxin homologues of voltage‐dependent K+ currents in cultured sensory neurones from neonatal rats
Authors: Hall Adam , Stow John , Sorensen Roger , Dolly J. Oliver , Owen David
Published: July 2012

Homologues of dendrotoxin (Dtx) were isolated from the crude venom of Green and Black Mamba snakes and examined for K+ channel blocking activity in neonatal rat dorsal root ganglion cells (DRGs) by whole‐cell patch clamp recording.

Outward potassium current activated by depolarization was composed of two major components: a slowly inactivating current (SIC, τdecay ≅ 50ms, 200 ms and 2 s), and a non‐inactivating current (NIC, τdecay > 2 min). Tail current analysis revealed two time constants of deactivation of total outward current, 3–12 ms and 50–150 ms (at −80 mV) which corresponded to SIC and NIC, respectively.

All the homologues (α‐, β‐, γ‐ and γ‐Dtx and toxins I and K) blocked outward current activated by depolarization in a dose‐dependent manner. The most potent in blocking total outward current was γ‐Dtx (EC50 of 0.5 ± 0.2 nm), although there were no statistically significant differences in potency between any of the homologues.

Qualitative differences in the nature of the block were noted between homologues. In particular, the block by γ‐Dtx was time‐dependent, whereas that by γ‐Dtx was not.

α‐Dtx was a much better blocker of SIC (EC50 = 1.0 ± 0.4 nm) than was γ‐Dtx (EC50 = 17.6 ± 5.8 nm). Furthermore, γ‐Dtx was selective for NIC (EC50 ± 0.24 ± 0.03 nm) over SIC and reduced the slow component of tail currents (NIC), preferentially. On the other hand, α‐Dtx did not significantly distinguish between SIC and NIC although tail current analysis showed that α‐Dtx preferentially reduced the fast component of tail currents (SIC).

The results confirm, using direct electrophysiological methods, that homologues of dendrotoxins from Mamba snake venom block K+ channels in rat sensory neurones. Furthermore, α‐Dtx and γ‐Dtx distinguish between sub‐types of K+ channels in these cells and may thus be useful pharmacological tools in other neuronal K+ channel studies.
Role of bradykinin in the hyperaemia following acid challenge of the rat gastric mucosa
Authors: Pethö Gábor , Jocič Milana , Holzer Peter
Published: July 2012

This study examined whether the hyperaemia following acid challenge of the rat gastric mucosa involves bradykinin, a peptide formed in response to tissue injury.

Gastric mucosal blood flow in urethane‐anaesthetized rats was assessed by the hydrogen gas clearance method. Infusion of a bradykinin solution (10 μm) into the gastric wall augmented gastric mucosal blood flow by a factor of 2.3, an effect that was prevented by the bradykinin B2 antagonist Hoe‐140 (icatibant; 100 μmol kg−1, i.v.).

I.v. injection of bradykinin (20–60 nmol kg−1) caused a 2.3‐3.5 fold increase in blood flow through the left gastric artery as measured by the ultrasonic transit time shift technique. The hyperaemic effect of bradykinin in this gastric artery was also prevented by Hoe‐140 (100 μ mol kg−1, i.v.).

Gastric acid backdiffusion was evoked by perfusing the stomach with 15% ethanol, to break the gastric mucosal barrier, in the presence of luminal acid. Depending on the concentration of acid (0.05 and 0.15 M HC1), this procedure increased gastric mucosal blood flow by a factor of 1.6‐2.8 and caused formation of gross damage in 1.5‐3% of the glandular mucosa. Hoe‐140 (100 μmol kg−1, i.v.) failed to alter the moderate vasodilatation seen in the presence of 0.05 M HC1 but significantly (P < 0.05) attenuated the marked hyperaemia and enhanced the gross mucosal damage observed in the presence of 0.15 M HC1.

These data show that bradykinin is able to enhance gastric mucosal blood flow via activation of B2 receptors. It appears as if this kinin is formed during severe acid challenge of the rat gastric mucosa and participates in the hyperaemic reaction to gastric acid backdiffusion.
Substrate specificity of the di/tripeptide transporter in human intestinal epithelia (Caco‐2): identification of substrates that undergo H+‐coupled absorption
Authors: Thwaites David T. , Hirst Barry H. , Simmons Nicholas L.
Published: July 2012

pH‐dependent transepithelial transport and intracellular accumulation of the hydrolysis‐resistant dipeptide glycylsarcosine (Gly‐Sar) have been demonstrated in the model human intestinal epithelial cell line, Caco‐2.

Experiments with BCECF (2′,7′‐bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein)‐loaded Caco‐2 cells demonstrated that dipeptide (Gly‐Sar) transport across the apical membrane is coupled to proton flow into the cell.

A range of postulated substrates for the intestinal di/tripeptide carrier were tested for their abilities to: (a) inhibit pH‐dependent [14C]Gly‐Sar apical‐to‐basal transport and intracellular accumulation and (b) stimulate H+ flow across the apical surface of BCECF‐loaded Caco‐2 cell monolayers.

A range of compounds (including Gly‐Gly, Leu‐Leu, Gly‐Gly‐Gly, cefadroxil and cephalexin) caused marked acidification of intracellular pH when perfused at the apical surface of Caco‐2 cell monolayers. In contrast leucine and D‐Leu‐D‐Leu failed to induce proton flow. The ability to induce proton‐flow across the apical surface by these compounds, in this intestinal epithelium, was directly correlated to the relative inhibitory effects on [14C]‐Gly‐Sar transport and accumulation.

The determination of substrate‐induced intracellular pH change in the Caco‐2 cell system may provide a useful rapid screen for candidate substrates for absorption via H+‐coupled transport mechanisms such as the intestinal di/tripeptide carrier in an appropriate physiological context.
Prevention by dexamethasone of the marked antiarrhythmic effects of preconditioning induced 20 h after rapid cardiac pacing
Authors: Vegh Agnes , Papp Julius Gy , Parratt James R.
Published: July 2012

Dogs were paced, via a pacing electrode in the right ventricle, for four 5 min periods at a rate of 220 beats min−1. On the following day they were reanaesthetized, thoracotomized and the left anterior descending coronary artery occluded for 25 min. Pacing markedly reduced the severity of ischaemia‐induced arrhythmias (e.g. reduction in VF from 45% in unpaced dogs to 10% in paced dogs; P < 0.05), an effect reversed by dexamethasone (4 mg kg−1 i.v., 45 min prior to pacing). This protection may be due to the induction of nitric oxide synthase or cyclo‐oxygenase.
Attenuation by dexamethasone of endotoxin protection against ischaemia‐induced ventricular arrhythmias
Authors: Song Wu , Furman Brian L. , Parratt James R.
Published: July 2012

Ventricular arrhythmias from a 30 min occlusion of the left coronary artery were assessed in Langendorff perfused isolated hearts removed from rats administered either saline, or endotoxin derived from Escherichia coli (2.5mg kg−1 i.p.) given either 2, 4, 8, 24 or 48 h previously. Arrhythmia severity was markedly reduced in those hearts removed from rats administered endotoxin with a maximum protection at 8h; there was a marked reduction in the incidence of ventricular fibrillation (from 54% to 4%) and in the number of ventricular premature beats during the occlusion period (e.g. from 1165 ± 144 to 37 ± 19; P < 0.01). Dexamethasone (3 mg kg−1, given 1 h prior to endotoxin or saline) markedly attenuated the protection afforded by endotoxin.
Smooth muscle relaxing effects of NO, nitrosothiols and a nerve‐induced relaxing factor released in guinea‐pig colon
Authors: Iversen H.H. , Gustafsson L.E. , Leone A.M. , Wiklund N.P.
Published: July 2012

The aim of the present study was to compare the biological activity of S‐nitroso‐L‐cysteine (CYSNO), S‐nitrosoglutathione (GSNO), S‐nitroso‐N‐acetyl‐D, L‐penicillamine (SNAP) and hydrox‐ylamine to that of nitric oxide (NO) and a vascular relaxing factor released by nerve stimulation in the guinea‐pig intestine. The biological activity was examined in a bioassay system with guinea‐pig colon as donor tissue and a series of spiral strips of rabbit aorta without endothelium as detector tissues.

Electrical stimulation of the guinea‐pig colon released a vascular relaxing factor. The half‐life of the relaxing factor down the bioassay cascade was the same as exogenously applied NO. Nω‐nitro‐L‐arginine (L‐NOARG) inhibited the release of bioactivity.

The relaxations of the assay tissues caused by exogenous CYSNO also declined during the passage down the cascade. However, in the presence of L‐cysteine (10−5 m) the half‐life of CYSNO increased and there was no significant breakdown through the cascade. In contrast, the half‐life of applied NO and the vascular relaxing factor released by nerve stimulation was unaffected by the presence of L‐cysteine.

Exogenously applied GSNO (20–50 nm), SNAP (2–4 nm) and hydroxylamine (300–600 nm) caused relaxations that did not decline during the passage down the cascade.

In summary, the relaxation of the bioassay tissues during nerve stimulation was indistinguishable from the relaxation induced by NO, whereas relaxations induced by CYSNO, GSNO, SNAP and hydroxylamine showed different pharmacological profiles. The released bioactivity is thus likely to be NO itself.
Neutral endopeptidase (NEP) inhibition in rats with established pulmonary hypertension secondary to chronic hypoxia
Authors: Thompson J.S. , Sheedy W. , Morice A.H.
Published: July 2012

Atrial natriuretic peptide (ANP) causes vasorelaxation in the pulmonary vasculature. ANP levels are elevated in conditions characterized by pulmonary hypertension and it has been hypothesized that ANP may be autoregulatory in the pulmonary circulation.

One route of ANP metabolism in vivo is by the action of the enzyme neutral endopeptidase (NEP). We have studied the effects of the NEP inhibitor, SCH 42495, in rats with established pulmonary hypertension secondary to chronic hypoxia.

Rats (n = 32) were divided into 4 groups. Normoxic controls were kept in air for 10 days (NC10) and all other animals were placed in a normobaric hypoxic chamber (Fi 02 10%). Chronic hypoxic controls were studied at 10 days (CHC10). After 10 days hypoxia the two remaining groups received oral treatment for a further 10 days, consisting of either SCH 42495 (30 mg kg−1, twice daily CHT20) or methyl cellulose vehicle (0.4%, twice daily, CHV20).

Animals were anaesthetized and blood collected for measurement of plasma ANP. Hearts were dissected and ventricles weighed and the histology of the pulmonary vasculature examined.

CHC10 rats had significant right ventricular hypertrophy (0.53 ± 0.08) and pulmonary vascular remodelling (29.0 ± 0.01%) and had gained significantly less body weight (33.2 ± 5.5 g) than NC10 rats (0.31 ± 0.04, 10.9 ± 0.01%, and 59.2 ± 11.9 g respectively). CHC10 rats had significantly elevated plasma ANP levels (58.4 ± 9.9 pM) compared with NC10 rats (23.9 ± 32pM). Treatment with SCH 42495 caused a significant reduction in pulmonary vascular remodelling (25.0 ± 0.01%) and right ventricular hypertrophy (0.52 ± 0.09) in CHT20 rats compared with CHV20 controls (33.0 ± 0.02% and 0.61 ± 0.09 respectively). Pulmonary vascular remodelling was also significantly lower in CHT20 rats than CHC10 animals.

Thus, short term inhibition of NEP causes regression of established pulmonary vascular remodelling and may be a useful therapeutic strategy in pulmonary hypertension.
Interactions of constitutive nitric oxide with PAF and thromboxane on rat intestinal vascular integrity in acute endotoxaemia
Authors: László F. , Whittle B.J.R , Moncada S.
Published: July 2012

The involvement of endogenous platelet activating factor (PAF) and thromboxane A2 in the acute microvascular damage in the ileum and colon induced by the nitric oxide (NO) synthase inhibitor, NG‐nitro‐L‐arginine methyl ester (1‐NAME) following endotoxin administration was investigated in the rat over a 1 h period.

Administration of 1‐NAME (1–10 mg kg−1, s.c.) concurrently with E. coli lipopolysaccharide (LPS;

mg kg1, i.v.) dose‐dependently increased vascular permeability in the ileum and colon, as determined by the leakage of radiolabeled albumin, and caused macroscopic mucosal damage in the ileum determined 1 h later. Neither LPS administration nor 1‐NAME (5 mg kg1) alone affected resting vascular permeability.

Infusion of phenylephrine (lOμg kg−1 min−1, i.v. for 1 h) caused an elevation in blood pressure similar to that found following 1‐NAME administration (5 mg kg−1, i.v. or s.c), but did not increase intestinal vascular permeability, when administered with LPS (3 mg kg−1, i.v.).

The increased vascular permeability in the ileum and colon and macroscopic damage in the ileum, induced by 1‐NAME (5 mg kg−1, s.c.) and LPS (3 mg kg−1, i.v.) was dose‐dependently inhibited following s.c. pretreatment (15min before challenge) with the thromboxane synthase inhibitors, OKY 1581 (5–25 mg kg−1) or 1‐benzyl‐imidazole (1–50 mg kg−1), or with the thromboxane receptor antagonist, BM 13177 (0.2‐2 mg kg−1).

Pretreatment with the cyclo‐oxygenase inhibitor, indomethacin (2‐5mg kg−1, s.c, 15min before challenge) reduced the microvascular injury in the ileum and colon and macroscopic lesions in the ileum, observed after the concurrent administration of 1‐NAME and LPS.

Pretreatment (15min) with the PAF‐receptor antagonists, WEB 2086 (0.5‐1 mg kg−1, s.c) or BN 52021 (2.5–10 mg kg−1, s.c.) likewise attenuated this intestinal vascular injury.

Combined administration of low doses of 1‐benzyl‐imidazole (1 mg kg−1) with WEB 2086 (0.5 mg kg−1) 15min before 1‐NAME and LPS challenge, abolished this vascular damage and macroscopic injury.

These results suggest that PAF and thromboxane A2 are released acutely following challenge with a low dose of endotoxin. However, these mediators do not appear to injure the intestinal microvascular bed unless NO synthase is concurrently inhibited. Such findings support the protective role of constitutively‐formed NO, counteracting the injurious vascular actions of cytotoxic mediators released under pathological conditions.
Participation of tumour necrosis factor and nitric oxide in the mediation of vascular dysfunction in splanchnic artery occlusion shock
Authors: Squadrito Francesco , Altavilla Domenica , Canale Patrizia , Ioculano Mariapatrizia , Campo Giuseppe M. , Ammendolia Letteria , Ferlito Marcella , Zingarelli Basilia , Squadrito Giovanni , Saitta Antonino , Caputi Achille P.
Published: July 2012

Splanchnic artery occlusion (SAO) shock is characterized by irreversible circulatory failure. Tumour necrosis factor (TNF‐α) may affect the 1‐arginine/nitric oxide (NO) pathway, thus contributing to the cardiovascular derangements of circulatory shock.

We investigated the contribution of both TNF‐α and the 1‐arginine/nitric oxide pathway to the vascular dysfunction of SAO shock. Anaesthetized rats, subjected to total occlusion of the superior mesenteric artery and the coeliac trunk for 45 min developed a severe shock state (SAO shock) resulting in a fatal outcome within 75–90 min after the release of occlusion. Sham operated animals were used as controls. SAO shocked rats had also a marked hypotension and enhanced macrophage and serum levels of TNF‐α. Furthermore, aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE 1 nm‐10 μm) and reduced responsiveness to acetylcholine (ACh lOnM‐lOμm). Endothelium‐denuded aortic rings had also a marked hyporeactivity to phenylephrine, which was restored to control values by in vitro administration of N° nitro‐1‐arginine‐methyl ester (1‐NAME 10 μm).

In vivo administration of cloricromene (2 mg kg−1, i.v.), an inhibitor of TNF‐α biosynthesis, increased survival, enhanced mean arterial blood pressure and reduced macrophage and serum levels of TNF‐α. Furthermore, aortic rings from shocked rats treated with cloricromene exhibited a greater contractile response to phenylephrine and improved responsiveness to ACh when compared to aortic rings from vehicle‐treated SAO shocked rats.

Our results suggest that TNF‐α alters both endothelial and muscular L‐arginine/nitric oxide pathways which in turn produce vascular dysfunction in SAO shock.
‘Sensory‐efferent’ neural control of mucus secretion: characterization using tachykinin receptor antagonists in ferret trachea in vitro
Authors: Ramnarine Sean I. , Hirayama Yoshitaka , Barnes Peter J. , Rogers Duncan F.
Published: July 2012

We characterized the tachykinin receptor(s) mediating ‘sensory‐efferent’ neural control of release of 35S04‐labelled macromolecules (mucus) from ferret trachea in vitro in Ussing chambers using selective tachykinin antagonists. Secretion was induced by substance P (SP), neurokinin A (NKA), capsaicin, the NK1 tachykinin receptor agonist [Sar9, Met(O2))11] substance P ([Sar9SP), or acetylcholine (ACh), or by electrical stimulation of nerves. Antagonists used were FK888 and L‐668,169, selective for the NK, receptor, SR 48968, selective for the NK2 receptor, and FK224, a dual antagonist at NK1 and NK2 receptors. The selectivity of FK888 and SR 48968 was examined on NKA‐induced contraction of ferret tracheal smooth muscle in vitro.

SP (1 μm) increased mucus secretion by 695% above vehicle controls. FK888 (0.1 μm‐30μm) inhibited SP‐induced secretion in a dose‐dependent manner, with complete inhibition at IOJXM and an IC50 of 1 μm. L‐668,169 (1 μm) also completely inhibited SP‐induced secretion.

NKA (1 μm) significantly increased mucus secretion by 271% above baseline, a response which was completely inhibited by FK888 (10 μm) or L‐668,169 (μm). Secretion induced by ACh (10 μm: 317% above baseline) was not inhibited by FK888 but was inhibited by atropine. Capsaicin (10μm)‐induced secretion (456% above vehicle controls) was significantly inhibited by FK888 and by L‐668,169 (111% and 103% inhibition respectively).

Electrical stimulation (50 V, 10 Hz, 0.5 ms, 5 min) increased mucus output above baseline (increased by 12 to 26 fold), a response blocked by tetrodotoxin (0.1 μm). FK888 (10μm) inhibited the increase in secretion due to electrical stimulation by 47%. Atropine, propranolol and phentolamine in combination (APP) inhibited the response to electrical stimulation by 48%. The remaining NANC response, i.e. in the presence of APP, was further reduced by 66% with FK888. FK224 (10μm) inhibited neurally‐evoked secretion by 73%. SR 48968 (0.1 μm) had no effect on electrically‐stimulated or [Sar9]SP‐induced secretion.

NKA (10μm‐10μm: in the presence of DMSO control vehicle) induced tracheal smooth muscle contraction in a concentration‐dependent manner with a maximal contraction of 30% of the maximal response to ACh (10 μm) and an EC50 of 0.3 JIM. SR 48968 (0.1 μ in DMSO) inhibited the NKA‐induced contraction whereas FK888 did not. Neither antagonist had any inhibitory effect on ACh‐induced contraction.

We conclude that ‘sensory‐efferent’ neurogenic mucus secretion in ferret trachea in vitro is mediated via tachykinin NK1 receptors with no involvement of NK2 receptors. Potent and selective tachykinin antagonists may have therapeutic potential in bronchial diseases such as asthma and chronic bronchitis in which neurogenic mucus hypersecretion may be aetiologically important.
Nitric oxide, and not vasoactive intestinal peptide, as the main neurotransmitter of vagally induced relaxation of the guinea pig stomach
Authors: Desai K.M. , Warner T.D. , Bishop A.E. , Polak J.M. , Vane J.R.
Published: July 2012

Nitric oxide synthase (NOS) was localized in the guinea pig stomach by immunocytochemistry. In vitro experiments were carried out on the isolated stomach of the guinea pig to study any possible links between nitric oxide (NO) and vasoactive intestinal peptide (VIP) in mediating relaxations induced by vagal stimulation.

NOS was localized to nerve cell bodies and nerve fibre varicosities of the myenteric plexus in wholemounts of the longitudinal muscle‐myenteric plexus of the stomach fundus. The NOS‐positive cells had a Dogiel type I morphology characteristic of motor neurones.

The cross‐sections of the stomach wall showed NOS‐positive neurones mainly in the myenteric plexus ganglia and NOS‐positive nerve fibre varicosities in the circular muscle layer.

Relaxations induced by vagal stimulation were almost completely prevented by 1‐NAME with an IC50 value of 5.5 × 10−6m. This inhibition was reversed by L‐arginine (2 μm).

VIP (100 nm) induced reproducible relaxations of the stomach. These were unaffected by tet‐rodotoxin (2μm) or NW‐nitro‐L‐arginine methyl ester (1‐NAME, 100 μm).

Desensitization to the relaxant effect of VIP partially reduced relaxations induced by vagal stimulation, glyceryl trinitrate or sodium nitroprusside but not noradrenaline.

These results show that NO has a neuronal origin in the guinea pig stomach, and support NO, and not VIP, as the major neurotranmitter of vagally induced gastric relaxation in the guinea pig.
β‐Adrenoceptors mediate inhibition of [3H]‐acetylcholine release from the isolated rat and guinea‐pig trachea: role of the airway mucosa and prostaglandins
Authors: Wessler Ignaz , Reinheimer Torsten , Brunn Gernot , Anderson Gary P. , Maclagan Jennifer , Racké Kurt
Published: July 2012

Rat or guinea pig isolated tracheae were labelled with [3H]‐choline to measure evoked tritium outflow, which reflects neuronal release of [3H]‐acetylcholine. Tritium outflow was evoked either by electrical stimulation of the extrinsic vagal nerve (rat tracheae) or by 27 mm potassium (guinea pig tracheae).

In rat tracheae isoprenaline (0.01, 0.1 μm) inhibited evoked [3H]‐acetylcholine release, whereas β‐adrenoceptor‐selective agonists (fenoterol, formoterol, salbutamol) were ineffective.

The inhibitory effect of isoprenaline was abolished under the following conditions: (i) presence of propranolol (1 μm) or of the β‐selective antagonist CGP 20712 A (0.1 μm); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 μm indomethacin.

In rat isolated tracheae prelabelled with [3H]‐arachidonic acid, isoprenaline (0.1 μm) but not formoterol (0.01 μm) enhanced the outflow of [3H]‐prostaglandins (PGD2, PGE2). This effect was blocked by 0.1 μm CGP 20712 A.

In guinea pig tracheae electrical stimulation of the extrinsic vagal nerve did not cause a constant release of [3H]‐acetylcholine, but 27 mm potassium elicited a reproducible release of [3H]‐acetylcholine. In this species both isoprenaline (0.1 μm) and formoterol (0.01 μm) inhibited evoked [3H]‐acetylcholine release. Inhibition was abolished under the following conditions: (i) presence of propranolol (1 μm) or of the β2‐selective antagonist ICI 118551 (0.3 μm); (ii) removal of the mucosa at the start of the experiments; (iii) blockade of cyclooxygenase activity by 3 μm indomethacin.

In conclusion, the present experiments have demonstrated that activation of β‐adrenoceptors localized in the mucosa mediates inhibition of [3H]‐acetylcholine release from the neuroeffector junctions of the pulmonary, parasympathetic nerves most probably by the liberation of inhibitory prostaglandins from the airway mucosa. The adrenoceptor subtype involved differs in rat (β1 subtype) and guinea pig (β 2subtype) airways.
Potentiation of the anti‐obesity effect of the selective β3‐adrenoceptor agonist BRL 35135 in obese Zucker rats by exercise
Authors: Santti Eriika , Huupponen Risto , Rouru Juha , Hanninen Virve , Pesonen Ullamari , Jhanwar‐Uniyal Meena , Koulu Markku
Published: July 2012

The effects of chronic treatments with a selective β3‐adrenoceptor agonist and a selective α2‐adrenoceptor antagonist and their interactions with physical exercise training were studied in experimental obesity.

BRL 35135 (β3‐agonist, 0.5 mg kg−1 day−1 p.o.), atipamezole (α2‐antagonist, 4.0 mg kg−1 day−1 p.o.) and placebo were given to genetically obese male Zucker rats. Half of the rats were kept sedentary whereas the other half were subjected to moderate treadmill exercise training. Body weight gain, cumulative food intake, the neuropeptide Y content of the hypothalamic paraventricular nucleus, brown adipose tissue thermogenic activity (measured as GDP binding), plasma insulin and glucose levels were measured after 3 weeks' treatment and exercise.

Treatment with BRL 35135 reduced weight gain by 19%, increased brown adipose tissue thermogenic activity 45‐fold and reduced plasma insulin by 50%. Atipamezole slightly increased food intake and neuropeptide Y content in the paraventricular hypothalamic nucleus but had no effect on the other measured parameters. Exercise alone had no effect on weight gain, food intake or thermogenic activity, whereas it reduced plasma insulin and glucose levels.

The effect of BRL 35135 on weight gain and thermogenic activity was significantly potentiated by exercise; the reduction in weight gain was 56% in comparison with 19% in sedentary animals. Food intake was significantly reduced in the BRL 35135‐treated‐exercise‐trained animals, although neither β3‐agonist nor exercise alone affected it.

Based on the present results in genetically obese Zucker rats, combination of β3‐agonist treatment with a moderate physical training may offer a new feasible approach to the therapy of obesity.
Interactions of chloroethylclonidine with rauwolscine‐ and prazosin‐sensitive adrenoceptors in dog saphenous vein
Authors: Low A.M. , Bowdish D.M. , Prashad T.R. , Gaspar V.
Published: July 2012

α1‐Adrenoceptors have been classified pharmacologically into four subtypes (α1A, α1B, â1c and α1D) on the basis of their differential affinity for novel antagonists such as chloroethylclonidine (CEC). While CEC is considered an α1B‐adrenoceptor antagonist, our earlier studies revealed that it also acted like an agonist in the dog saphenous vein (DSV). The present study characterized the contraction induced by CEC in endothelium‐denuded rings from DSV.

Concentration‐response curves for CEC were constructed in the absence (EC50 value of 11.13 ± 3.6 μm, n = 8) and presence of propranolol (β‐adrenoceptor antagonist, 30 nm), rauwolscine (α2‐adrenoceptor antagonist, 30 nm), prazosin (α1‐adrenoceptor antagonist, 30 nm) or methysergide (5HT2 antagonist, 30 nm) or both prazosin and rauwolscine. Pretreatment with methysergide (9.83 ± 5.14μm, n = 4) or propranolol (23.78 ± 12.32μm, n = 4) had no consistent effect. In the presence of rauwolscine, the concentration‐response curve for CEC was significantly shifted to the right with an EC50 value of 48.82 ± 13.2 JAM (n = 8). In the presence of prazosin, the CEC concentration‐response curve had an EC50 value of 29.12 ± 6.42 μm (n = 8). Pretreatment with both prazosin and rauwolscine shifted the concentration‐response curve for CEC to the right with an EC50 value of 72.67 ± 10.69 JAM (n = 8, P < 0.05). Maximum responses were significantly reduced only in tissues that were treated with both prazosin and rauwolscine.

CEC (100 μm) pretreatment abolished prazosin binding sites and reduced the Bmax for rauwolscine by 50% without affecting the IQ value or the Hill slope.

In Ca2+‐free Krebs solution containing 50 /IM EGTA, CEC produced a small transient contraction, suggesting that it can mobilize internally‐stored Ca2+. Pretreatment with rauwolscine abolished the CEC‐induced contraction in Ca2+‐free medium; prazosin pretreatment reduced but did not abolish CEC response in Ca2+“free medium.

Restoring Ca2+ (0.5‐2.5 μm) to the extracellular solution increased CEC contraction in a concentration‐dependent manner, reaching a plateau at around 1.5 mm Ca2+. The contraction was insensitive to nicardipine (1 μm), a voltage‐operated Ca2+channel blocker, but was blocked in a concentration‐dependent manner by the putative receptor‐operated Ca2+channel blockers, SK&F 96365 (1–10 μm) and genistein, also a tyrosine kinase inhibitor (10–100μm).

We conclude that CEC acts on rauwolscine‐ and, to a less extent, prazosin‐sensitive adrenoceptors in DSV to release internally stored Ca2+ and to open receptor‐operated Ca2+ channels. The inhibitory effect on CEC‐induced contraction that depended on external Ca2+ by genistein suggests a role for tyrosine kinase in the regulation of dihydropyridine‐insensitive Ca2+ entry.