bluuebunny/crossref_metadata_2025 huggingface dataset:
The influence of acetazolamide and amlodipine on the intracellular sodium content of rat proximal tubular cells
Authors: Wong Patrick S.K. , Barclay Paul L. , Newman Michael J. , Johns Edward J.
Published: July 2012

This investigation set out to use 23Na n.m.r. spectroscopy to measure changes in intracellular levels of sodium in isolated suspensions of rat proximal tubules. The effects of temperature, an inhibitor of the sodium pump and known natriuretic drugs on intracellular sodium content of such tubular preparations were measured and compared with calcium channel antagonists where action at this level is unclear.

Rat kidneys were perfused with collagenase, roughly chopped, subjected to mechanical dispersion and washed to remove all traces of the enzyme. The proximal tubules were then purified and concentrated by Percoll density gradient centrifugation and then resuspended in buffer containing dysprosium tripolyphosphate shift reagent.

Distinct peaks corresponding to intracellular and extracellular sodium signals were observed when the tubules were placed into the n.m.r. spectrometer. As the temperature of the suspension rose to 37°C, there was an exponential decrease in sodium content, with a decay constant of 0.15 ± 0.02 min−1, which reached a stable level within 20 to 25 min. Addition of ouabain, 10−3 m, resulted in a significant (P < 0.01) 30% increase in intracellular sodium content within 5 min which peaked at 70% 20 min later. Although acetazolamide (10−3 m) significantly (P < 0.01) increased intracellular sodium content by 45%, amlodipine (10−4m) had no effect.

These data show that changes in the activity of the Na+/K+/ATPase have a considerable influence on the intracellular levels of sodium in proximal tubule cells. Inhibition of carbonic anhydrase activity resulted in a rise in intracellular sodium content which is compatible with its action to reduce the turnover rate of the Na+/(HCO3−)3 symporter. The lack of effect of amlodipine was consistent with the suggestion that it does not have a direct action on the sodium handling processes at the level of the proximal tubule.
Effects of selective ETB‐receptor stimulation on arterial, venous and capillary functions in cat skeletal muscle
Authors: Ekelund Ulf , Adner Mikael , Edvinsson Lars , Mellander Stefan
Published: July 2012

This paper describes, in quantitative terms, the in vivo effects of two selective ETB‐receptor agonists (IRL 1620 and BQ 3020) on vascular resistance (tone) in the following consecutive sections of the vascular bed of sympathectomized cat skeletal muscle: large‐bore arterial resistance vessels (> 25 μm), small arterioles (< 25 μm) and the veins. The effects on capillary pressure and transcapillary fluid exchange were also recorded.

Both IRL 1620 and BQ 3020, infused i.a. to the muscle preparation, evoked an initial transient dilator response followed by a moderate dose‐dependent constrictor response, both being preferentially confined to the small arterioles. The dilator response was associated with a transient increase, and the constrictor response with a sustained decrease, in capillary pressure, the latter causing net transcapillary fluid absorption. The capillary filtration coefficient decreased during the constrictor response, indicating constriction of terminal arterioles/precapillary sphincters.

The vascular responses to the ETB‐receptor agonists were unaffected by blockade of endothelium‐derived nitric oxide (NG‐nitro‐l‐arginine methyl ester) and by selective ETA‐receptor blockade (FR139317). However, blockade of prostacyclin production with indomethacin decreased the amplitude of the dilator response, and decreased the time required to reach a steady‐state vasoconstrictor response to the ETB‐receptor agonists.

The effect of ETB‐receptor stimulation on vascular tone was also evaluated in vitro on the cat femoral artery and vein. IRL 1620 had no effect on the femoral artery but caused a weak dose‐dependent relaxation in the femoral vein. This large vein relaxation response seemed to be mediated by endothelium‐derived nitric oxide and not by prostacyclin.

It may be concluded that ETB‐receptor stimulation is responsible for the dilator response, and can contribute to the constrictor response, elicited by endothelins in cat skeletal muscle in vivo.
Endothelin‐1‐induced myocardial ischaemia and oedema in the rat: involvement of the ETA receptor, platelet‐activating factor and thromboxane A2
Authors: Filep János G. , Fournier Alain , Földes‐Filep Éva
Published: July 2012

The objectives of the present experiments were to assess the role of ETA receptors in mediating endothelin‐1 (ET‐1)‐induced myocardial ischaemia and oedema and to study the involvement of platelet‐activating factor (PAF) and thromboxane A2 (TxA2) in these actions of ET‐1 in rats.

Intravenous bolus injection of ET‐1 (0.1–2 nmol kg−1) into anaesthetized rats induced ST segment elevation of the electrocardiogram in a dose‐dependent manner without causing arrhythmias. ST segment elevation developed within 20–90 s and persisted for at least 10–20 min following administration of ET‐1.

Pretreatment of the animals with the selective endothelin ETA receptor antagonist, FR 139317 (2.5 mg kg−1, i.v.) inhibited by 86% the ST segment elevation elicited by ET‐1 (1 nmol kg−1). Pretreatment with intravenous administration of BM 13505 (1 mg kg−1), a TxA2 receptor antagonist, OKY‐046 (10 mg kg−1), a thromboxane synthase inhibitor or the specific PAF receptor antagonist, WEB 2086 (1 mg kg−1) or BN 52021 (10 mg kg−1) markedly suppressed ST segment elevation in response to ET‐1. Infusion of indomethacin (3 mg kg−1 bolus plus 2 mg kg−1 h−1) did not significantly affect ET‐1‐induced ST segment elevation.

Bolus injection of ET‐1 (1 nmol kg−1, i.v.) to conscious rats resulted in a prolonged pressor effect preceded by a transient depressor response. Corresponding to changes in blood pressure, a small transient tachycardia was followed by a sustained bradycardia. ET‐1 enhanced albumin leakage by 87 and 120% in the left ventricle and right atrium, respectively, as measured by the extravasation of Evans blue dye.

The selective ETA receptor antagonist, FR 139317 (2.5 mg kg−1) significantly blunted the pressor action of ET‐1 and the accompanying bradycardia without affecting the depressor response. Furthermore, FR 139317 almost completely abolished the permeability effect of ET‐1 in both vascular beds studied.

Pretreatment of the animals with BM 13505 (1 mg kg−1), OKY‐046 (10 mg kg−1), WEB 2086 (1 mg kg−1) or BN 52021 (10 mg kg−1) significantly reduced ET‐1 (1 nmol kg−1)‐induced albumin extravasation both in the left ventricle and right atrium. The PAF receptor antagonists, WEB 2086 and BN 52021 were equally potent inhibitors in the left ventricle, whereas BN 52021 appeared to be a more potent inhibitor than WEB 2086 in the right atrium. Pretreatment with indomethacin (3 mg kg−1 plus 2 mg kg−1 h−1) did not modify the permeability response to ET‐1. None of these compounds affected significantly ET‐1‐induced changes in mean arterial blood pressure and heart rate.

These results indicate that intravenous administration of ET‐1 provokes ST segment elevation and myocardial oedema and suggest that these events are mediated, in part, through release of secondary mediators, such as PAF and TxA2 via the activation of ETA receptors.
Action of niflumic acid on evoked and spontaneous calcium‐activated chloride and potassium currents in smooth muscle cells from rabbit portal vein
Authors: Hogg R.C. , Wang Q. , Large W.A.
Published: July 2012

The action of niflumic acid was studied on spontaneous and evoked calcium‐activated chloride (ICl(Ca)) and potassium (IK(Ca)) currents in rabbit isolated portal vein cells.

With the nystatin perforated patch technique in potassium‐containing solutions at a holding potential of – 77 mV (the potassium equilibrium potential), niflumic acid produced a concentration‐dependent inhibition of spontaneous transient inward current (STIC, calcium‐activated chloride current) amplitude. The concentration to reduce the STIC amplitude by 50% (IC50) was 3.6 × 10−6 m.

At – 77 mV holding potential, niflumic acid converted the STIC decay from a single exponential to 2 exponential components. In niflumic acid the fast component of decay was faster, and the slow component was slower than the control decay time constant. Increasing the concentration of niflumic acid enhanced the decay rate of the fast component and reduced the decay rate of the slow component.

The effect of niflumic acid on STIC amplitude was voltage‐dependent and at – 50 and + 50 mV the IC50 values were 2.3 × 10−6 m and 1.1 × 10−6 m respectively (cf. 3.6 × 10−6 m at −77 mV).

In K‐free solutions at potentials of – 50 mV and + 50 mV, niflumic acid did not induce a dual exponential STIC decay but just increased the decay time constant at both potentials in a concentration‐dependent manner.

Niflumic acid, in concentrations up to 5 × 10−5 m, had no effect on spontaneous calcium‐activated potassium currents.

Niflumic acid inhibited noradrenaline‐ and caffeine‐evoked ICl(Ca) with an IC50 of 6.6 × 10−6 m, i.e. was less potent against evoked currents compared to spontaneous currents. In contrast niflumic acid (2 × 10−6 m–5 × 10−5 m) increased noradrenaline‐ and caffeine‐induced IK(Ca).

The results are discussed with respect to the mechanism of block of ICl(Ca) by niflumic acid and its suitability as a pharmacological tool for assessing the role of ICl(Ca) in physiological mechanisms.
Biochemical machinery involved in the release of ATP from sympathetic nerve terminals
Authors: Wardell Claire F. , Cunnane Thomas C.
Published: July 2012

The biochemical mechanisms underlying the process of facilitation in sympathetic nerve terminals of the guinea‐pig vas deferens have been investigated by intracellular and focal extracellular recording techniques. Activation of protein kinase C by the phorbol ester, phorbol 12,13‐dibutyrate (PDBu) greatly increased the amplitude of excitatory junction potentials (e.j.ps) from the first stimulus in a train and altered the pattern of facilitation. The configuration of the extracellularly recorded nerve terminal impulse and the sensitivity of the postjunctional membrane to released ATP were unaffected. The specific protein kinase C inhibitor, Ro‐31,8220, abolished facilitation and antagonized the effects of PDBu. These results suggest that protein kinase C plays a fundamental role in ATP release from sympathetic nerves and in particular in the mechanisms underlying facilitation.
The effects of chronic imidazoline drug treatment on glial fibrillary acidic protein concentrations in rat brain
Authors: Olmos Gabriel , Alemany Regina , Escriba Pablo V. , García‐Sevilla Jesús A.
Published: July 2012

The concentration of the astrocytic marker, glial fibrillary acidic protein (GFAP) was quantitated by immunoblotting (western blotting) in the rat brain after treatment with various imidazoline drugs and other agents.

Chronic (7 days) but not acute (1 day) treatment with the imidazoline drugs, cirazoline (1 mg kg−1, i.p.) and idazoxan (10 mg kg−1, i.p.), but not with the structurally related α2‐adrenoceptor antagonists, RX821002 (2‐methoxy idazoxan) (10 mg kg−1, i.p.) and efaroxan (10 mg kg−1, i.p.), markedly increased (45%) GFAP immunoreactivity in the rat cerebral cortex. Chronic treatment (7 days) with yohimbine (10 mg kg−1, i.p.), a non‐imidazoline α2‐adrenoceptor antagonist, did not significantly modify GFAP immunoreactivity in the cerebral cortex.

Chronic treatment (7 days) with cirazoline and idazoxan did not alter the density of brain monoamine oxidase (MAO)‐B sites labelled by [3H]‐Ro 19–6327 (lazabemide), another relevant astroglial marker. Moreover, these imidazoline drug treatments did not modify the levels of α‐tubulin in the cerebral cortex. These negative results reinforced the specificity of the effects of imidazoline drugs on GFAP.

Irreversible inactivation of brain α2‐adrenoceptors (and other neurotransmitters receptors) after treatment with an optimal dose of the peptide‐coupling agent EEDQ (1.6 mg kg−1, i.p., for 6–24 h) did not alter GFAP immunoreactivity in the cerebral cortex. These results further disproved the involvement of these receptors on astroglial cells in the tonic control of GFAP levels.

The binding of [3H]‐idazoxan in the presence of 10−6 m (–)‐adrenaline was used to quantitate in parallel I2‐imidazoline preferring sites in the rat brain after the same treatments. Chronic treatment (7 days) with cirazoline and idazoxan, but not with RX821002, efaroxan or yohimbine, significantly increased (25%) the density of I2‐sites in the cerebral cortex. The up‐regulation of I2‐sites induced by cirazoline was not observed in the liver, a tissue that also expresses I2‐sites but lacks glial cells.

A strong correlation (r = 0.97) was found when the mean percentage changes in GFAP immunoreactivity were related to the mean percentage changes in I2 imidazoline sites after the various drug treatments.

Together the results suggest a direct physiological function of glial I2‐imidazoline preferring sites in the regulation of GFAP levels.
Routes of delivery of muscarinic acetylcholine receptors to the plasma membrane in NG108‐15 cells
Authors: Koenig Jennifer A. , Edwardson J. Michael
Published: July 2012

We have examined the routes of delivery of muscarinic acetylcholine receptors to the plasma membrane in unstimulated and agonist‐stimulated NG108‐15 cells. Delivery of receptors to the plasma membrane was measured by irreversibly alkylating receptors already at the cell surface with propyl‐<***>benzilylcholine mustard (PrBCM) and then following the recovery of binding of the polar radioligand [3H]‐N‐methylscopolamine ([3H]‐NMS) in intact cells.

In unstimulated cells, recovery of [3H]‐NMS binding after 2 h of incubation at 37°C was 30% of binding in control cells. Binding affinity of [3H]‐NMS was unchanged. In cells that had been pre‐exposed to carbachol (0.5 mm) for 30 min, initial [3H]‐NMS binding was reduced by 38% but recovery of binding was increased from 30% to 43% of control binding.

When the cells were pre‐incubated for 1 h with the protein synthesis inhibitor cycloheximide (20 μg ml−1), recovery of [3H]‐NMS binding was reduced by similar extents in unstimulated (30% to 9%) and carbachol‐stimulated (43% to 19%) cells. Incubation of the cells at 20°C instead of 37°C reduced recovery of [3H]‐NMS binding from 30% to 9% in unstimulated cells and from 43% to 23% in carbachol‐stimulated cells.

Depletion of cellular ATP by addition of antimycin (50 nm) and deoxyglucose (50 mm), reduced recovery of binding to 12% in unstimulated cells and to 6% in carbachol‐stimulated cells.

These results indicate that in unstimulated NG108‐15 cells, delivery of muscarinic receptors to the plasma membrane is almost exclusively through the synthetic pathway. In agonist‐stimulated cells, receptor sequestration into an intracellular compartment (probably endosomes) occurs. Under these circumstances, delivery of receptors to the plasma membrane along the synthetic route is unaffected but an additional route of delivery (recycling) now operates.
Induction of kinin B1 receptor‐dependent vasoconstriction following balloon catheter injury to the rabbit carotid artery
Authors: Pruneau D. , Luccarini J.M. , Robert C. , Bélichard P.
Published: July 2012

Balloon catheter injury to the rabbit carotid artery damaged the endothelium and induced neointima formation over 7 days. The area of intima, expressed as a percentage of the media, was 16.2 ± 4.2% and 8.2 ± 0.1% in balloon catheter‐injured and sham‐operated arteries.

Seven days after arterial injury, carotid arteries were isolated and set up as ring preparations in organ baths for isometric tension measurements. Balloon catheter‐injured arteries first contracted with noradrenaline (0.01–0.1 μm), contracted further in a concentration‐dependent manner to bradykinin (BK; pD2, 5.98 ± 0.22; Emax, 41.3 ± 5.2% of KCl) and to des‐Arg9‐BK (pD2, 7.12 ± 0.36; Emax, 46.0 ± 9.9% of KCl). In contrast, vessel segments with endothelium either intact or acutely removed were unresponsive to both BK receptor agonists.

The concentration‐contraction curves for BK and for des‐Arg9‐BK were shifted to the right by the B1 receptor antagonist, [Leu8]des‐Arg9‐BK (3 μm), but not by the selective B2 receptor antagonist, Hoe 140 (1 μm).

Thus, BK and its metabolite, des‐Arg9‐BK act as vasoconstrictor agents following balloon catheter injury. These effects appear to be mediated by activation of B1 receptors.
Stimulation of phospholipase C in cultured microvascular endothelial cells from human frontal lobe by histamine, endothelin and purinoceptor agonists
Authors: Purkiss John R. , West David , Wilkes Leslie C. , Scott Caroline , Yarrow Peter , Wilkinson Graeme F. , Boarder Michael R.
Published: July 2012

Cultures of endothelial cells derived from the microvasculature of human frontal lobe have been investigated for phospholipase C (PLC) responses to histamine, endothelins and purinoceptor agonists.

Using cells prelabelled with [3H]‐inositol and measuring total [3H]‐inositol (poly)phosphates, histamine acting at H1 receptors stimulated a substantial response with an EC50 of about 10 μm.

Endothelin‐1 also gave a clear stimulation of phosphoinositide‐specific phospholipase C. Both concentration‐response curves and binding curves showed effective responses and binding in the rank order of endothelin‐1>sarafotoxin S6b>endothelin‐3, suggesting an ETA receptor.

Assay of total [3H]‐inositol (poly)phosphates showed no response to the purinoceptor agonists, 2‐methylthioadenosine 5′‐trisphosphate (2MeSATP), adenosine 5′‐O‐(3‐thiotrisphosphate) (ATPγS) or β,γ‐methylene ATP. Both ATP and UTP gave a small PLC response.

Similarly, when formation of [32P]‐phosphatidic acid from cells prelabelled with 32Pi was used as an index of both PLC and phospholipase D, a small response to ATP and UTP was seen but there was no response to the other purinoceptor agonists tested.

Study by mass assay of stimulation by ATP of inositol (1,4,5) trisphosphate accumulation revealed a transient response in the first few seconds, a decline to basal, followed by a small sustained response.

These results show that human brain endothelial cells in culture are responsive to histamine and endothelins in a manner which may regulate brain capillary permeability. Purines exert a lesser influence.
Effect of subthreshold ouabain on the tone of guinea‐pig aortic strips following repeated noradrenaline stimulation
Authors: Bova Sergio , Rossi GianPaolo , Luciani Sisto , Debetto Patrizia , Pessina Achille C. , Cargnelli Gabriella
Published: July 2012

The effect of ouabain at a concentration (0.8 μm) that does not induce contractile response in guinea‐pig aortic strips has been studied on endothelium‐denuded strips repeatedly stimulated with 1 μm noradrenaline or 60 mm K+applied for 5 min every 30 min.

The resting tone (i.e. the tone between one noradrenaline stimulation and the following) of the aortic strips exposed to ouabain increased progressively, whereas the control strips (no ouabain) completely relaxed on washout of the agonist. In the aortic strips stimulated by 60 mm K+, the resting tone did not increase.

The calcium antagonist, verapamil, did not affect the increase in tone, that was nevertheless strictly dependent on external calcium, since the contracted strips completely relaxed on calcium removal and promptly contracted again on calcium readdition. This finding indicates a mechanism independent of voltage‐gated calcium channels.

Caffeine‐induced contractions, taken as a measure of sarcoplasmic reticulum calcium content, were amplified by the presence of ouabain in aortic strips either stimulated by noradrenaline or unstimulated, with a larger increase in the former.

These results suggest that the repeated stimulation of guinea‐pig aortic strips by noradrenaline in the presence of ouabain, by raising both intracellular Na+and Ca2+, decreases the ouabain threshold concentration required for contraction, thus increasing the responsiveness of vascular smooth muscle to the glycoside.
Effect of the putative cognitive enhancer, linopirdine (DuP 996), on quantal parameters of acetylcholine release at the frog neuromuscular junction
Authors: Provan Spencer D. , Miyamoto Michael D.
Published: July 2012

The subcellular mechanism and site of action of linopirdine or DuP 996 (3,3‐bis(4‐pyridinylmethyl)‐1‐phenylindolin‐2‐one) was investigated at the frog neuromuscular junction, using miniature endplate potential (m.e.p.p.) counts and a new method for obtaining unbiased estimates of n (number of functional release sites), p (probability of release), and varsp (spatial variance in p).

DuP 996 produced an increase in m (no. of quanta released), which was due to an increase in n and p. The increase in m was concentration‐dependent over a range of 0.1–100 μm and completely reversible with 15 min of wash. There was a saturation in the increase in p, but not in the increase in m and n, for [DuP 996] >10 μm. By contrast, there was no major change in varsp.

Block of presynaptic Na+‐ and Ca2+‐channels with 3 μm tetrodotoxin and 1.8 mm Co2+prevented the m.e.p.p. frequency increase to DuP 996, and this effect was completely reversed by washing.

Application of the neuronal Ca2+‐channel blocker, ω‐conotoxin GVIA (1 μm) brought about a rapid and profound decrease in the m.e.p.p. frequency increase produced by DuP 996. The effect of the toxin was not reversed by prolonged washing.

Block of voltage‐gated K+‐channels with 100 μm 4‐aminopyridine (4‐AP) resulted in only a small (28%) increase in m. The combination of 4‐AP (100 μm) and DuP 996 (10 μm) produced an increase in m (189%) which was much greater than the sum of the responses to each agent alone. This increase in m was due solely to an increase in n, as p and varsp were unchanged.

For [DuP 996] up to 100 μm, there was no apparent change in the mean size, amplitude distribution, or time course of m.e.p.ps, signifying that it had no anticholinesterase activity.

It is concluded that DuP 996 increases the release of quantal transmitter but not the postsynaptic response to the quanta. This appears to involve an effect at the nerve terminal membrane, most likely an increase in Ca2+‐conductance, and not an action to block K+‐conductance or to release Ca2+from intraterminal organelles.
Mechanical and biochemical responses to endothelin‐1 and endothelin‐3 in bovine bronchial smooth muscle
Authors: Nally Jane E. , McCall Raymond , Young Louise C. , Wakelam Michael J.O. , Thomson Neil C. , McGrath John C.
Published: July 2012

In this study, mechanical responses to endothelin‐1 and endothelin‐3 were examined in bovine bronchial smooth muscle. In addition, the involvement of phosphatidylinositol 4,5‐bisphosphate hydrolysis (PIP2) in the responses to these peptides was assessed by measurement of inositol (1,4,5) trisphosphate (I(1,4,5)P3) production using a specific mass assay.

ET‐1 evoked contractions of bovine bronchi which were concentration‐dependent and initiated at between 10−9m and 10−8m. ET‐1‐evoked responses were unaffected by slight elevation of tone with potassium chloride (3 × 10−2m), methacholine (10−6m) or U46619 (10−7m).

Contractions to ET‐1 were not altered by pre‐incubation with atropine (10−5m), indomethacin (10−5m), nifedipine (10−5m), phosphoramidon (3.67 × 10−5m) or by removal of the epithelium.

ET‐3 evoked small contractions which were not concentration‐dependent. In the presence of phosphoramidon (3.67 × 10−5m) however, concentration‐dependent contractions were obtained to ET‐3 which were unaffected by atropine (10−5m) or by removal of the epithelium, but were significantly attenuated by indomethacin (10−5m). Nifedipine (10−5m) virtually abolished this response.

Both ET‐1 and ET‐3 (in the presence of phosphoramidon)‐evoked contractions were significantly enhanced by the presence of the phorbol ester phorbol 12,13‐dibutyrate (10−8m). Neither ET‐1‐, nor ET‐3‐mediated responses were antagonized by the protein kinase C (PKC) inhibitor, Ro 31–8220 (3 × 10−9–3 × 10−8m).

ET‐1 (3 × 10−7m) evoked a biphasic rise in levels of I(1,4,5)P3 which was unaltered by pre‐incubation with atropine, whilst ET‐3 (10−10–3 × 10−7m) failed to alter levels of I(1,4,5)P3 at any time point examined, even in the presence of phosphoramidon (3.67 × 10−5m).

These results suggest that, in bovine bronchial smooth muscle, ET‐1 does not evoke contraction via cyclo‐oxygenase metabolites, does not evoke release of the neurotransmitter substance acetylcholine, or require calcium influx via dihydropyridine‐sensitive channels. ET‐1 evokes I(1,4,5)P3 production, but stimulation of protein kinase C may not be critical for the associated contraction. In contrast, ET‐3‐evoked contractions are partly mediated by cyclo‐oxygenase metabolites. ET‐3 does not stimulate PIP2 hydrolysis, nor activate PKC, but may, either directly or as a requirement of intermediates released in response to ET‐3, rely upon extracellular calcium.
Influence of S‐nitrosothiols and nitrate tolerance in the rat gastric fundus
Authors: Barbier A.J.M. , Lefebvre R.A.
Published: July 2012

The relaxant responses of S‐nitroso‐l‐cysteine (CysNO), S‐nitroso‐N‐acetyl‐d,l‐penicillamine (SNAP), S‐nitroso‐N‐acetyl‐l‐cysteine (SNAC) and S‐nitrosoglutathione (GSNO) in the rat gastric fundus (forestomach) were studied and compared to the relaxant responses obtained in response to nitric oxide (NO) and electrical field stimulation (EFS, 10 s trains) of non‐adrenergic non‐cholinergic (NANC) nerves.

CysNO (10−7–3 × 10−4m) caused transient relaxation of the precontracted rat gastric fundus, comparable to the response to NO (10−4–10−4m) and EFS. SNAP, SNAC and GSNO elicited more sustained relaxations.

The cyclic GMP‐specific phosphodiesterase inhibitor, zaprinast (3 × 10−5m), increased the relaxant effect of CysNO, SNAP and GSNO while the NO‐synthase inhibitor, NG‐nitro‐l‐arginine (l‐NOARG, 3 × 10−4m) had no influence.

In the presence of LY 83583 (10−5m), which releases superoxide anions, the relaxant response to NO and CysNO was decreased, whereas that to all other stimuli was unaltered. The inhibitory effect of LY 83583 on CysNO‐induced relaxations was prevented by superoxide dismutase (SOD, 1000 u ml−1).

Tissues incubated for 1 h with 5.5 × 10−4m nitroglycerin (GTN) became tolerant to GTN. In this condition, the relaxant response to 10−5m NO was maintained, while the relaxations by EFS (8 Hz) and 3 × 10−5m SNAP were significantly decreased. The reduction of the response to the other S‐nitrosothiols was not significant.

The combination of nitrate tolerance and 10−5m LY 83583 caused a significantly larger inhibition of the relaxant response to EFS (8 Hz) than nitrate tolerance alone. The combination of LY 83583 and GTN tolerance reduced the relaxant effect of 10−5m NO to a similar extent to LY 83583 alone, while the relaxant response to 10−4m GTN was reduced to the same extent as after 1 h exposure to 5.5 × 10−4m GTN alone.

It is concluded that S‐nitrosothiols potently relax the rat gastric fundus, possibly by a cyclic GMP‐dependent mechanism and S‐nitrosothiols such as SNAC and GSNO may be involved in NANC neurotransmission.
Prevention by NMDA receptor antagonists of the centrally‐evoked increases of cardiac inotropic responses in rabbits
Authors: Monassier Laurent , Tibiriça Eduardo , Roegel Jean‐Christophe , Mettauer Bertrand , Feldman Josiane , Bousquet Pascal
Published: July 2012

The purpose of this study was to investigate further the role of the excitatory amino acid (EAA) system of neurotransmission, particularly of the NMDA receptor, in the central regulation of cardiac function.

Electrical stimulation of the paraventricular nucleus of the hypothalamus (PVN) in pentobarbitone anaesthetized rabbits induced a cardiovascular response mainly characterized by a positive inotropic effect, hypertension and a marked increase in the myocardial oxygen demand index.

The intracerebroventricular (i.c.v.) or intravenous (i.v.) injection of different EAA antagonists acting on different sites of the NMDA receptor/channel complex dose‐dependently blunted the excitatory cardiovascular effects of PVN stimulation.

5,7 Dichlorokynurenic acid was used as a specific glycine site antagonist and 2‐amino‐5‐phosphonovaleric acid was used to block the agonist recognition site; ketamine was used as a channel blocker site antagonist and ifenprodil as a blocker of the polyamine binding site.

5,7 Dichlorokynurenic acid (125 and 250 μg kg−1, i.c.v.) virtually abolished the cardiovascular responses, inducing only haemodynamic depression at the highest dose used. 2‐Amino‐5‐phosphonovaleric acid (0.1 to l.0 mg kg−1, i.c.v.) elicited a reduction of the peak values observed during PVN stimulation which was accompanied by a decrease of the basal cardiovascular parameters. Ketamine (2.5 and 10 mg kg−1) and ifenprodil (1 mg kg−1), injected intravenously, blocked the haemodynamic response induced by PVN stimulation without marked reduction of the basal haemodynamics.

It is concluded that glutamate neurotransmission is not only involved in vasomotor tone control but also in the central control of cardiac function and can therefore modulate the myocardial oxygen demand.
Adenosine‐induced hyperpolarization of the membrane voltage in rat mesangial cells in primary culture
Authors: Pavenstädt Hermann , Ruh Joachim , Greger Rainer , Schollmeyer Peter
Published: July 2012

The effect of adenosine on membrane voltage and ion currents was studied in rat mesangial cells in primary culture. Membrane voltage was measured with the patch clamp technique in the slow‐ or fast whole cell configuration. The resting membrane voltage of mesangial cells was −48±0.5mV. Adenosine (10−8−10−3 m) induced a sustained and concentration‐dependent hyperpolarization of membrane voltage (ED50≅6 × 10−7m). Adenosine (10−5m) hyperpolarized the membrane voltage by 14 ± 0.5 mV. During the hyperpolarization ion currents were monitored simultaneously. An increase of the outward current by 51 ± 11% was observed.

An increase of the extracellular K+ concentration (from 3.6 to 18.6 m) caused a depolarization of membrane voltage to −34 ± 2 mV. In the presence of increased K+ the hyperpolarization of membrane voltage induced by adenosine was significantly attenuated by 61 ± 5%. The K+‐channel blocker, Ba2+ (5 × 10−3m) depolarized membrane voltage to −24 ± 2mV. In the presence of Ba2+ the adenosine‐induced hyperpolarization was significantly inhibited by 72 ± 8%.

Preincubation of the adenosine antagonist, 8‐phenyltheophylline (10−4m) significantly inhibited the adenosine (10−5 m) mediated membrane voltage response by 67 ± 8%. The adenosine agonists 5‐N‐ethylcarboxamidoadenosine (NECA), R‐(−)N6‐(2‐phenylisopropyl)adenosine (R‐(−)−PIA), S‐(+)‐N6‐(2‐phenylisopropyl)adenosine (S‐(+)‐PIA), N6‐[2‐(3,5‐dimethoxyphenyl)‐2‐(2‐methylphenyl)‐ethyl]adenosine (DPMA), and 2‐chloroadenosine (2‐CA) also hyperpolarized membrane voltage of mesangial cells. The rank order of potency of the agonists at 10−5 m was NECA > adenosine = > R‐(−)−PIA = DPMA = 2‐CA > S‐(+)‐PIA.

Stimulation of cyclic AMP by forskolin induced a concentration‐dependent hyperpolarization of membrane voltage (ED50≅2 × 10−7m). Application of forskolin (10−5m) in the presence of adenosine (10−4m) had no additive hyperpolarizing effect on the membrane voltage.

Activation of protein kinase C by phorbol 12,13 dibutyrate (PDBu) induced a sustained depolarization of membrane voltage (ED50≅5 × 10−9m). In the presence of PDBu, adenosine (10−5m) still hyperpolarized membrane voltage of mesangial cells.

The data indicate that adenosine activates K+‐conductance via an A2 receptor in mesangial cells; the activation of the K+‐conductance, which is probably mediated by cyclic AMP led to a hyperpolarization of membrane voltage.
The 5‐HT1‐like receptor mediating the increase in canine external carotid blood flow: close resemblance to the 5‐HT1D subtype
Authors: Villalón Carlos M. , Terrón José A.
Published: July 2012

It has recently been shown that the increase in external carotid blood flow induced by 5‐hydroxytryptamine (5‐HT) in the anaesthetized dog, being mimicked by 5‐carboxamidotryptamine (5‐CT), inhibited by methiothepin, vagosympathectomy and sympatho‐inhibitory drugs, and resistant to blockade by ritanserin and MDL 72222, is mediated by stimulation of prejunctional 5‐HT1‐like receptors leading to an inhibitory action on carotid sympathetic nerves; these 5‐HT1‐like receptors are unrelated to either the 5‐HT1A, 5‐HT1B or 5‐HT1C (now 5‐HT2C) receptor subtypes. Inasmuch as 5‐CT, 5‐methoxytryptamine, sumatriptan and metergoline display high affinity, amongst other 5‐HT binding sites, for the 5‐HT1D subtype, in the present study we have used these drugs in an attempt to determine whether the above inhibitory prejunctional 5‐HT1‐like receptors correlate with the 5‐HT1D subtype.

One‐minute intracarotid (i.c.) infusions of 5‐HT (0.3, 1, 3 and 10 μg), 5‐CT (0.01, 0.03, 0.1 and 0.3 μg), 5‐methoxytryptamine (1, 3, 10 and 30 μg) and sumatriptan (1, 3, 10, 30 and 100 μg) resulted in dose‐dependent increases in external carotid blood flow (without changes in mean arterial blood pressure or heart rate) with the following rank order of agonist potency: 5‐CT> >5‐HT > 5‐methoxytryptamine ≤ sumatriptan. Interestingly, sumatriptan‐induced vasodilatation was followed by a more pronounced vasoconstriction.

The external carotid vasodilator effects of 5‐HT, 5‐CT, 5‐methoxytryptamine and sumatriptan were dose‐dependently and specifically antagonized by metergoline (10, 30 and/or 100 μg kg−1, i.v.). In addition, 5‐methoxytryptamine‐ and sumatriptan‐induced vasodilator effects were, respectively, markedly inhibited or abolished after vagosympathectomy, as previously shown for 5‐CT and 5‐HT.

Sumatriptan showed tachyphylaxis in its vasodilator component and antagonized 5‐HT‐induced external carotid vasodilatation in a specific manner, suggesting that a common site of action may be involved.

Taken together, the above results support our contention that 5‐HT, 5‐CT, 5‐methoxytryptamine and sumatriptan produce external carotid vasodilatation in the dog by an action that might primarily involve a prejunctional inhibition on carotid sympathetic nerves; a secondary component of this vasodilator response may be postsynaptic (endothelium‐dependent and/or even directly on the vasculature). Based on the rank order of agonist potency, inhibition by vagosympathectomy and blockade by metergoline, we suggest that the inhibitory prejunctional 5‐HT1‐like receptors mediating external carotid vasodilatation in the dog closely resemble the 5‐HT1D receptor subtype. The pharmacological profile of these receptors is similar (sympathetic nerves of the rat kidney and human saphenous vein, as well as porcine coronary endothelium) to other putative 5‐HT1D receptors mediating vascular responses.
Investigation of the endogenous chemoattractants involved in 111In‐eosinophil accumulation in passive cutaneous anaphylactic reactions in the guinea‐pig
Authors: Weg Vivian B. , Watson Malcolm L. , Faccioli Lucia H. , Williams Timothy J.
Published: July 2012

Eosinophil accumulation and plasma extravasation are features of type I allergic responses. In an attempt to characterize the mediators of these responses, we have examined the local accumulation of 111In‐eosinophils and leakage of 125I‐human serum albumin (125I‐HSA) during passive cutaneous anaphylaxis (PCA) reactions and in response to defined inflammatory mediators in the guinea‐pig. Animals were passively sensitized by intradermal injection of anti‐bovine gamma globulin antibody (50 μl, 1/50 dilution). After 20–24 h, animals were injected intravenously with 111In‐eosinophils and 125I‐HSA for the measurement of cell accumulation and plasma leakage, respectively.

When injected into sensitized sites, antigen caused a dose‐related increase in the accumulation of 111In‐eosinophils and plasma leakage in guinea‐pig skin. Time course experiments over 24 h revealed that the maximal rate of 111In‐eosinophil accumulation occurred over the first 90 min, with little accumulation at later time points. Plasma leakage was completed within the first 30 min after challenge. Responses to the mast cell degranulator, compound 48/80, exhibited very similar responses to the PCA reaction.

Co‐injection of antigen with the PAF antagonist, WEB 2086 (10−7 mol/site) or the 5‐lipoxygenase inhibitor, PF 5901 (10−7 mol/site) did not significantly alter the accumulation of 111In‐eosinophils or plasma leakage, whereas these drug doses abolished responses to exogenous PAF (10−9 mol/site) and arachidonic acid (AA, 3 × 10−8 mol/site), respectively. The H1 receptor antagonist chlorpheniramine (2.5 × 10−8 mol/site) did not reduce antigen‐induced 111In‐eosinophil accumulation. Drug combinations were also injected with antigen into sensitized sites, but were unable to reduce 111In‐eosinophil accumulation.

These results indicate that anaphylactic eosinophil accumulation in this model involves mediators other than histamine, PAF or lipoxygenase products. This is in contrast to plasma leakage in this reaction, which can be abolished by a combination of antagonists blocking these mediators.
Inhibition by KF17837 of adenosine A2A receptor‐mediated modulation of striatal GABA and ACh release
Authors: Kurokawa Masako , Kirk Ian P. , Kirkpatrick Karen A. , Kase Hiroshi , Richardson Peter J.
Published: July 2012

The effect of the A2A adenosine receptor agonist, 2‐p‐(‐2‐carboxyethyl)phenethyl‐amino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) on the potassium evoked release of [3H]‐γ‐aminobutyric acid ([3H]‐GABA) from nerve terminals derived from the caudate‐putamen and the globus pallidus of the rat was compared. In both preparations CGS 21680 (1 nm) inhibited the [3H]‐GABA release evoked by 15 mm KCl but had no effect on that evoked by 30 mm KCl.

The ability of CGS 21680 (1 nm) to inhibit the release of [3H]‐GABA from striatal nerve terminals was unaffected by the presence of the GABA receptor antagonists, bicuculline (10 μm), phaclofen (100 μm) and 2‐hydroxysaclofen (100 μm). Similarly the opioid receptor antagonist, naloxone (10 μm), the adenosine A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 40 nm), and the cholinoceptor antagonists, mecamylamine (10 μm) and atropine (100 nm) had no effect on this inhibition.

The ability of CGS 21680 (0.1 nm) to stimulate the release of [3H]‐acetylcholine ([3H]‐ACh) from striatal nerve terminals was unaffected by the presence of bicuculline (10 μm), 2‐hydroxysaclofen (100 μm), phaclofen (100 μm), naloxone (10 μm) and DPCPX (4 nM).

The novel A2A receptor antagonist, (E)‐8‐(3,4‐dimethoxystyryl)‐1,3‐dipropyl‐7‐methylxanthine (KF 17837), blocked the CGS 21680 (1 nm)‐induced inhibition of [3H]‐GABA efflux with an EC50 of approximately 30 nm and also antagonized the CGS 21680 (0.1 nm)‐induced stimulation of [3H]‐ACh release with an EC50 of approximately 0.3 nm.

It is concluded that the A2A adenosine receptor is present on both GABAergic and cholinergic nerve terminals of the rat striatum and that in both the caudate‐putamen and the globus pallidus this receptor inhibits [3H]‐GABA release. No evidence was seen for a difference in the ligand binding sites of this receptor in the two groups of nerve terminals.
Action of adenosine receptor antagonists on the cardiovascular response to defence area stimulation in the rat
Authors: St Lambert J.H. , Dawid‐Milner M.S. , Silva‐Carvalho L. , Spyer K.M.
Published: July 2012

The action of adenosine in the mediation of the cardiovascular changes associated with the defence reaction has been investigated in the rat using two A1 receptor antagonists.

Cumulative doses of 1,3 dipropyl‐cyclopentylxanthine (DPCPX) (0.3–3 mg kg−1) and ethanol (0.03–0.25 ml) and bolus doses of DPCPX (3 mg kg−1) and 8‐sulphophenyltheophylline (8‐SPT) (20 mg kg−1) were given into α‐chloralose, paralysed and artificially ventilated rats. Recordings were made of arterial blood pressure and heart rate.

Ethanol, the vehicle for DPCPX, failed to modify the magnitude of the defence response; however, cumulative doses of DPCPX produced a dose‐dependent decrease in the HDA (hypothalamic defence area)‐evoked increase in arterial blood pressure, accompanied by a similar fall in the magnitude of the evoked heart rate response.

The evoked rise in arterial blood pressure was reduced significantly by intravenous injection of DPCPX (3 mg kg−1) but not 8‐SPT (20 mg kg−1), a purely peripherally acting adenosine antagonist.

These results suggest that adenosine acting at A1 receptors located in the central nervous system, is involved in the HDA‐evoked pressor response. Whilst the site of action of the A1 receptors is not known, possible locations are discussed.
Hydrolysis of iodine labelled urodilatin and ANP by recombinant neutral endopeptidase EC. 3.4.24.11
Authors: Abassi Zaid A. , Golomb Eliahu , Agbaria Riad , Roller Peter P. , Tate John , Keiser Harry R.
Published: July 2012

Urodilatin is a 32 amino‐acid peptide of similar sequence to atrial natriuretic peptide (ANP), with four additional amino‐acids at the N‐terminus. Although ANP and urodilatin bind to the same receptors with similar affinities, urodilatin is more active than ANP as a natriuretic agent. Previous studies, using neutral endopeptidase EC 3.4.24.11 (NEP) derived from crude membrane preparations, were inconclusive, but suggested that urodilatin was more resistant than ANP to degradation by this enzyme. In the present study, we compared the degradation rates of [125I]‐urodilatin and [125I]‐ANP by pure recombinant NEP (rNEP).

Incubation of radioactively labelled ANP with rNEP resulted in a much more rapid degradation of the peptide than that for labelled urodilatin.

Both phosphoramidon and SQ‐28,603, potent inhibitors of NEP, completely protected both peptides from metabolism by rNEP.

The circular dichroism spectra of the two peptides indicate that they are very similar and exist largely in unordered or flexible conformations.

These results support the relative resistance of urodilatin to NEP, and indicate that urodilatin may be of use as a therapeutic agent, in conditions in which ANP is ineffective.
The electrophysiological effects of antiarrhythmic potential of a secoaporphine, N‐allylsecoboldine
Authors: Wu Mei‐Hwan , Su Ming‐Jai , Lee Shoei‐Sheng , Young Ming‐Long
Published: July 2012

A satisfactory antiarrhythmic potential of N‐allylsecoboldine, a synthetic derivative of secoaporphine, has been documented. Its effects on the ionic currents of cardiac myocytes and the influence on the electrophysiological properties of the conduction system in Langendorff perfused hearts were investigated.

Ionic currents were studied by voltage clamp in the whole cell configuration. N‐allylsecoboldine blocked the Na channel with a leftward‐shift of its half voltage‐dependent inactivation and a slower rate of recovery from the inactivation state. Similarly, calcium inward currents were inhibited but to a much smaller extent.

N‐allylsecoboldine inhibited the 4‐AP‐sensitive transient outward K current. Currents through the K1 channels were also reduced.

As compared with quinidine, N‐allylsecoboldine caused a comparable degree of block on Na and K1 currents but blocked to a lesser extent the Ca and Ito currents.

In the perfused whole‐heart model, N‐allylsecoboldine caused a dose‐dependent prolongation in sinoatrial, atrioventricular and His‐Purkinje system conduction intervals and prolonged the effective refractory periods of the atrium, AV node, His‐Purkinje system and ventricle. However, the basic cycle length was not significantly affected. As compared to quinidine, N‐allylsecoboldine exerted less pronounced effects on both the basic cycle length and the atrial and AV nodal refractory periods.

We conclude that N‐allylsecoboldine predominantly blocks Na and K1 channels and in similar concentrations partly blocks Ca channels and Ito. These effects result in a modification of the electrophysiological properties of the conduction system which provides a satisfactory therapeutic potential for the treatment of cardiac arrhythmias.
Nitric oxide as a mediator of the laxative action of magnesium sulphate
Authors: Izzo Angelo A. , Gaginella Timothy S. , Mascolo Nicola , Capasso Francesco
Published: July 2012

Magnesium sulphate was studied for its effects on diarrhoea, fluid secretion, gastrointestinal transit and nitric oxide (NO) synthase activity in rats.

At a dose of 2 g kg−1 orally magnesium sulphate produced diarrhoea that was delayed in onset and intensity in a dose‐related manner by the NO synthase inhibitor NG‐nitro‐L‐arginine methyl ester (l‐NAME). This was prevented by the NO precursor, l‐arginine and the NO donating compound, isosorbide‐5‐mononitrate (IMN).

Nitric oxide synthase activity was stimulated in gut tissue from rats given magnesium sulphate and this was inhibited by l‐NAME. Dexamethasone (1 mg kg−1, i.p.), an inhibitor of inducible NO synthase, had no effect on magnesium sulphate‐induced diarrhoea.

Magnesium sulphate stimulated fluid and electrolyte accumulation in the intestinal lumen; these effects were prevented by l‐NAME but not d‐NAME.

Gastrointestinal transit of a non‐absorbable marker (charcoal suspension) was increased by oral magnesium sulphate from a mean value of 54.1% to 72.9% (P<0.01), and this was prevented by pretreatment with l‐NAME.

The results demonstrate that oral magnesium sulphate produces diarrhoea in rats by increasing the accumulation of fluid in the intestinal lumen and enhancing flow from the proximal to distal intestine. The mechanism involves release of NO, probably through stimulation of the constitutive form of NO synthase. Whether or not the effects of magnesium sulphate are due to an osmotic action or an intrinsic effect of the magnesium or sulphate ions cannot be determined from these experiments.
Effect of 7‐nitro indazole on neurotransmission in the rat vas deferens: mechanisms unrelated to inhibition of nitric oxide synthase
Authors: Allawi Hala S. , Wallace P. , Pitcher Ann , Gaffen Zoe , Bland‐Ward P.A. , Moore P.K.
Published: July 2012

The effect of the nitric oxide synthase (NOS) inhibitor, 7‐nitro indazole (7‐NI), on sympathetic and purinergic neurotransmission in the rat isolated vas deferens preparation has been studied.

7‐NI (50–200 μm) caused a dose‐ and frequency‐dependent inhibition of the phasic (predominantly purinergic) contractile response of the rat vas deferens to electrical (field) stimulation (100 V, 0.5 ms). Greatest inhibition occurred at lower frequencies of stimulation (0.1–10 Hz). The sustained tonic contractile response (predominantly noradrenergic) was inhibited only at a high frequency of stimulation (60 Hz) and only at the highest concentration of 7‐NI studied (200 μm).

7‐NI (100 μm) significantly reduced the contractile response of the vas deferens to exogenous ATP (20 μm‐5mm) and the stable P2x purinoceptor agonist, α,β‐methylene ATP (2.5 and 25.0 μm) but was without effect on contractions due to noradrenaline (0.1–50 μm) indicating a lack of antagonist effect on post‐junctional α1 adrenoceptors.

The effect of 7‐NI (100 μm) on the phasic contractile response to field stimulation (0.1 and 2.0 Hz) was unaffected by preincubation of preparations with yohimbine (1.0 μm) or propranolol (0.01–10.0 μm) indicating the absence of involvement of α2– or β‐adrenoceptors in this response.

7‐NI (50–600 μm) caused dose‐related inhibition of contractions elicited by addition of a depolarizing concentration of KC1 (64 mm).

The effect of 7‐NI (100 μm) on the phasic contractile response to field stimulation (0.1 and 2.0 Hz) was unaffected by preincubation of preparations with l‐arginine (1 mm). Neither l‐arginine (1 mm) nor NG nitro l‐arginine methyl ester (l‐NAME, 100 μm) affected the response of the vas deferens to field stimulation at 0.1 or 2.0 Hz. Nitric oxide synthase (NOS) enzyme activity, measured as the conversion of [3H]‐l‐arginine to [3H]‐citrulline, was not detectable in rat vas deferens homogenates.

7‐Nr preferentially inhibits the purinergic component of the response of the rat vas deferens to field stimulation. The mechanism of action of 7‐NI is not known but is not related to NOS inhibition. It seems likely that 7‐NI combines an antagonist action at smooth muscle cell P2x‐purinoceptors with the ability to inhibit the cellular influx of calcium ions. Although these hitherto unrecorded effects of 7‐NI occur at relatively high concentrations, the effects described may contribute to the pharmacological effects of this NOS inhibitor.