bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelium‐dependent and ‐independent effects of exogenous ATP, adenosine, GTP and guanosine on vascular tone and cyclic nucleotide accumulation of rat mesenteric artery
Authors: Vuorinen Pauli , Pörsti Ilkka , Metsä‐Ketelä Timo , Manninen Vesa , Vapaatalo Heikki , Laustiola Kai E.
Published: July 2012

. The effects of exogenous guanosine 5′‐triphosphate (GTP) and guanosine on vascular tone and cyclic nucleotide accumulation of noradrenaline‐precontracted endothelium‐intact and endothelium‐denuded rat mesenteric artery rings were compared with the effects of the known purinoceptor agonists adenosine 5′‐triphosphate (ATP) and adenosine.

. GTP (10 μm−1 mm) dose‐dependently relaxed endothelium‐intact mesenteric artery rings by producing a rapid initial response followed by sustained relaxation resembling the relaxant response to acetylcholine. GTP also slightly relaxed endothelium‐denuded artery rings. The acetylcholine‐ and GTP‐induced relaxations of endothelium‐intact rings were attenuated by NG‐nitro l‐arginine methyl ester (l‐NAME, 330 μm) which attenuation was reversed with l‐arginine (1 mm).

. Guanosine (10 μm−1 mm) relaxed both endothelium‐intact and ‐denuded artery rings in a dose‐dependent manner. The relaxations were more pronounced in endothelium‐intact preparations and were only slightly attenuated by l‐NAME (330 μm).

. ATP (1 μm−1 mm) and adenosine (10 μm−1 mm) dose‐dependently relaxed endothelium‐intact and ‐denuded artery rings. The responses were more pronounced in endothelium‐intact vascular preparations.

. GTP (100 μm) and guanosine (100 μm) increased guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) accumulation in both endothelium‐intact and ‐denuded artery rings corresponding to the relaxations observed. The concentrations of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) were not affected.

. ATP (100 μm) increased cyclic GMP concentration of endothelium‐intact artery rings. The concentrations of cyclic AMP were not affected by ATP (100 μm) and adenosine (100 μm) in endothelium‐intact and ‐denuded vascular preparations.

. These results provide evidence that exogenous GTP and guanosine relax precontracted endothelium‐intact and ‐denuded rat mesenteric artery rings by increasing cyclic GMP accumulation. The response to GTP of endothelium‐intact rings can mainly be explained by the release of endothelium‐derived relaxing factor (EDRF), but that of guanosine is only partly due to EDRF, and is a combination of endothelium‐dependent and ‐independent effects. The endothelium‐independent response of GTP and guanosine is a direct, unknown effect on smooth muscle and guanylate cyclase.
Decreased inhibition by gravidin of arachidonate release from transformed compared to nontransformed cells
Authors: Wilson Theresa , Ralph Raymond K. , Ganendren Ranjini
Published: July 2012

. Gravidin (a phospholipase A2 inhibitor) reduced the release of arachidonic acid from human lymphocytes by 51% at 10−8 m.

. Under normal culture conditions, nanomolar gravidin caused a significant reduction in the release of free arachidonic acid from human lymphocytes or nontransformed fibroblasts but in transformed cells, nanomolar gravidin was ineffective.

. Inhibition of arachidonate release appeared to be related to rate of growth as inhibitory effects of gravidin on Jurkat cells and HL‐29 cells could be observed if the cells were cultured under conditions where DNA synthesis was low.

. The reported disparate effects of lipocortin on cell phospholipase A2 activity may be reconciled if DNA synthesis is investigated.
Characterization of the P1‐purinoceptors mediating contraction of the rat colon muscularis mucosae
Authors: Bailey S.J. , Hickman D. , Hourani S.M.O.
Published: July 2012

Previous studies had shown that adenosine and adenine nucleotides including adenosine 5′‐triphosphate (ATP) caused contraction of the rat colon muscularis mucosae via P1 and P2Y‐purinoceptors respectively, and that the stable ATP analogue adenylyl 5′‐(β, γ‐methylene)diphosphonate (AMPPCP) had an unexpected direct action on the P1‐purinoceptors. The P1‐purinoceptors have now therefore been further characterized by use of the adenosine analogues 5′‐N‐ethylcarboxamidoadenosine (NECA) and N6‐cyclopropyladenosine (CPA) and the antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX), which is selective for the A1 subtype. The P2‐purinoceptor antagonist suramin was also used, to investigate the selectivity of the P2 agonists.

The order of potency of P1 agonists for contraction was CPA > NECA > AMPPCP ≥ adenosine, and DPCPX (1 nm) caused greater than two fold shifts to the right of the log concentration‐response curves for each of these agonists, although the shifts were not always parallel and Schild analysis of the inhibition of the effect of adenosine resulted in a plot with a slope greater than unity. These results indicate that the P1‐purinoceptor mediating contraction is of the A1 subtype, as has been found in other tissues in which adenosine causes contraction.

The P2‐purinoceptor antagonist suramin (300 μm) had no effect on the responses to adenosine or to AMPPCP, but abolished contractions induced by the related stable ATP analogue adenosine 5′‐(α,β‐methylene)triphosphonate (AMPCPP). Contractions induced by ATP, which were not affected by DPCPX (10 nm) alone, were only partially inhibited by suramin (300 μm), revealing an A1 component to its action which could be blocked by DPCPX (10 nm).

In conclusion, these results show that the rat colon muscularis mucosae possesses contractile A1 receptors in addition to the previously characterized P2Y receptors, and confirms our finding that the stable ATP analogue, AMPPCP, has an unexpected direct action on these A1 receptors.
Evidence that nitric oxide does not mediate the hyperpolarization and relaxation to acetylcholine in the rat small mesenteric artery
Authors: Garland Christopher J. , McPherson Grant A.
Published: July 2012

Acetylcholine caused a concentration‐dependent smooth muscle hyperpolarization and relaxation in rat small mesenteric arteries (diameter at 100 mmHg 250–450 mm) stimulated with noradrenaline (3 μm).

Nitric oxide (NO), generated from either NO‐gas or from acidified sodium nitrite, also induced smooth muscle hyperpolarization but only in the absence of active force. However, unlike the hyperpolarizations to acetylcholine, those to NO were abolished either by prior smooth muscle depolarization caused by noradrenaline, or by the K+ channel blocker, glibenclamide (3 μm).

Hyperpolarization and relaxation to acetylcholine were unaffected by prior exposure of the mesenteric artery to either the cyclo‐oxygenase inhibitor, indomethacin (10 μm), or the nitric oxide synthase inhibitor, NG‐nitro‐l‐arginine (l‐NNA, 100 μm).

Haemoglobin (1.5 μm), which binds and inactivates NO, blocked the hyperpolarizing and vasorelaxant response to NO, but did not alter either response to acetylcholine.

These data show that, in the rat small mesenteric artery, membrane hyperpolarizations to NO and acetylcholine are mediated by different mechanisms, and that the hyperpolarization induced by NO is not involved in the responses to acetylcholine. In addition, they provide evidence that the acetylcholine responses in this artery, which are endothelium‐dependent, are not mediated by the release of NO.
Effects of the P2‐purinoceptor antagonist, suramin, on human platelet aggregation induced by adenosine 5′‐diphosphate
Authors: Hourani S.M.O. , Hall D.A. , Nieman C.J.
Published: July 2012

The effects of suramin, a trypanocidal drug which has been reported to be a P2‐purinoceptor antagonist on smooth muscle, were investigated in human platelets, where adenosine 5′‐diphosphate (ADP) induces aggregation by acting on a subtype of purinoceptors which has been called P2T.

Suramin (100 μm) had no inhibitory effect on ADP‐induced platelet aggregation in plasma, even after 40 min incubation in the presence of bacitracin, a peptidase inhibitor, and did not affect the ability of adenosine 5′‐triphosphate (ATP) (40 μm) to inhibit competitively ADP‐induced aggregation. This lack of effect of suramin on platelets in plasma is probably due to its extensive binding to plasma proteins.

In washed platelets, suramin (50–400 μm) acted as an apparently competitive antagonist, causing parallel shifts to the right of the log concentration‐response curve to ADP. No depression of the maximal response to ADP was observed at concentrations of suramin (50–150 μm) for which full log concentration‐response curves to ADP could be obtained, but the slope of the Schild plot was around 2, indicating that this antagonism was not simply competitive. The apparent pA2 value for suramin, taken from this Schild plot, was 4.6.

Suramin (200–400 μm) also noncompetitively inhibited aggregation induced by U46619 (a thromboxane receptor agonist) or by 5‐hydroxytryptamine in the presence of adrenaline (100 μm), and caused a depression of the maximal response to these agonists. This nonspecific effect of suramin may explain the high Schild plot slope obtained against ADP.

These results provide evidence that the ADP receptor on human platelets is indeed similar to the P2‐purinoceptors responding to adenine nucleotides on smooth muscle and other tissues, and show that suramin cannot distinguish between the proposed subtypes of the P2‐purinoceptors.
4‐Aminopyridine and low Ca2+ differentiate presynaptic inhibition mediated by neuropeptide Y, baclofen and 2‐chloroadenosine in rat hippocampal CA1 in vitro
Authors: Klapstein Gloria J. , Colmers William F.
Published: July 2012

Presynaptic inhibition is mediated by several receptors at the stratum radiatum‐CA1 synapse of rat hippocampus. We tested whether the same mechanism is activated by neuropeptide Y (NPY), baclofen and 2‐chloroadenosine (2‐CA), reasoning that if the receptors all activated the same process, then they should all respond to indirect manipulations of transmitter release in the same manner.

The effects on presynaptic inhibition by the potassium channel blocker, 4‐aminopyridine (4‐AP) and low extracellular concentrations of Ca2+ in the presence of 4‐AP were compared using evoked population excitatory postsynaptic potentials (p.e.p.s.p.) responses in the rat hippocampal slice in vitro.

Log concentration‐effect relationships for the inhibition of excitatory transmission were constructed for all 3 drugs in normal saline, and in the presence of 30 and 100 μm 4‐AP. 4‐AP reduced the inhibition mediated by all three substances, 100 μm 4‐AP was only slightly more effective than 30 μm.

Lowering extracellular Ca2+ from 1.5 to 0.75 mm in the presence of 30 μm 4‐AP restored the presynaptic inhibition caused by all effective concentrations of NPY and baclofen. By contrast, inhibition caused by 2‐CA was not restored by lowering Ca2+, except at concentrations of 2‐CA greater than 10 μm.

The results are consistent with the hypothesis that presynaptic NPY Y2 and GABAB receptors both inhibit transmitter release by the inhibition of voltage‐dependent Ca2+ influx, but that the A1 adenosine receptor may activate a different presynaptic mechanism.
Interaction of pinaverium (a quaternary ammonium compound) with 1,4‐dihydropyridine binding sites in rat ileum smooth muscle
Authors: Feron Olivier , Wibo Maurice , Christen Marie‐Odile , Godfraind Théophile
Published: July 2012

The interaction of pinaverium bromide, a quaternary ammonium compound, with binding sites for (L‐type) calcium channel blockers was investigated in rat ileum smooth muscle.

Pinaverium inhibited [3H]‐(+)‐PN200–110 ([3H]‐(+)‐isradipine) specific binding to tissue homogenates incompletely (Ki 0.38 μm; maximal inhibition 80%). In contrast, binding to single cell preparations (obtained by collagenase treatment) and to saponin‐treated homogenates was completely inhibited. These data are compatible with the view that, in untreated homogenates, 20% of [3H]‐(+)‐isradipine binding sites are not accessible to pinaverium because it is associated with sealed inside‐out vesicles.

Pinaverium bromide increased the apparent KD of [3H]‐(+)‐isradipine binding to saponin‐treated homogenates but did not significantly affect the Bmax value. Moreover, the dissociation rate constant of [3H]‐(+)‐isradipine binding was not changed by pinaverium. These data suggest that pinaverium interacts with the dihydropyridine binding site in a competitive manner. However, in contrast to uncharged dihydropyridine calcium antagonists, pinaverium inhibited, rather than stimulated, [3H]‐diltiazem binding to rat brain membranes (at 30–37°C).

Although Bmax values of [3H]‐(+)‐isradipine were similar in homogenates prepared from tissue and cells (collagenase‐treated), the KD value was significantly higher in cell homogenates (166 vs 95 pm). Similarly, the Ki value of pinaverium was higher in cell preparations than in tissue homogenates (0.77 vs 0.38 μm). Thus, collagenase can significantly modify the dihydropyridine recognition site.

The competitive interaction of pinaverium, a permanently charged drug, with [3H]‐(+)‐isradipine bound to intact cells and its absence of interaction with [3H]‐(+)‐isradipine bound to sealed inside‐out vesicles imply that the dihydropyridine receptor lies near the external surface of the plasma membrane.
Effects of peptidase inhibition on angiotensin receptor agonist and antagonist potency in rabbit isolated thoracic aorta
Authors: Robertson Mark J. , Cunoosamy Mootoosamy P. , Clark Kenneth L.
Published: July 2012

Experiments were performed with peptidase inhibitors on rabbit aortic strip preparations, to determine whether endogenous peptidase activity can influence the potency estimates for angiotensin receptor agonists and antagonists in this tissue.

Angiotensin II (A II) and angiotensin III (A III) both induced concentration‐related contractions of rabbit aortic strip preparations. A III was approximately 38 fold less potent than A II, and the gradient of the A III concentration‐response curve (1.00 ±0.04) was significantly more shallow than that (1.76 ± 0.05) of the A II curve.

Neither the aminopeptidase‐A and ‐M inhibitor, amastatin, nor the aminopeptidase‐B and ‐M inhibitor, bestatin, affected the potency of, or the maximum response to, A II. In contrast, the potency of A III was increased by both amastatin and bestatin. Amastatin had the most marked effect and at 10 μm caused approximately a 12 fold increase in the potency of A III (EC50 values, 102 nm and 8.6 nm in the absence and presence of amastatin, respectively), and also significantly steepened the gradient of the A III concentration‐response curve. Amastatin did not affect the position or shape of the concentration‐response curve to the α1‐adrenoceptor agonist, phenylephrine. Finally, the carboxypeptidase‐N inhibitor, d‐l‐mercaptomethyl‐3‐guanidine‐ethylpropanoic acid (MERGETPA) did not change the position or shape of the concentration‐response curves to either A II or A III.

In the presence of amastatin, the potency of the peptide angiotensin receptor antagonist, Ile7‐A III (100 nm − 1μm), was increased approximately 13 fold (pA2, with A II as the agonist, 7.0 ± 0.1 and 8.1 ± 0.1, in the absence and presence of amastatin, respectively). However, the potency of the non‐peptide angiotensin receptor antagonist, DuP 753 (30–300 nm), was little affected by amastatin (pA2, 8.2 ± 0.1 and 8.1 ± 0.1 in the absence and presence of amastatin, respectively).

The results of this study suggest that endogenous aminopeptidase activity in the rabbit thoracic aorta can profoundly affect estimates of the potency of peptide angiotensin receptor agonists and antagonists. A suitable aminopeptidase inhibitor should therefore be included in studies, using this tissue, which aim to classify angiotensin receptor subtype(s) based on the rank order of peptide angiotensin receptor agonist and/or antagonist potencies.
Actions of arginine polyamine on voltage and ligand‐activated whole cell currents recorded from cultured neurones
Authors: Scott Roderick H. , Sweeney Marva I. , Kobrinsky Evgeny M. , Pearson Hugh A. , Timms Graham H. , Pullar Ian A. , Wedley Susan , Dolphin Annette C.
Published: July 2012

Toxins from invertebrates have proved useful tools for investigation of the properties of ion channels. In this study we describe the actions of arginine polyamine which is believed to be a close analogue of FTX, a polyamine isolated from the American funnel web spider, Agelenopsis aperta.

Voltage‐activated Ca2+ currents and Ca2+‐dependent Cl− currents recorded from rat cultured dorsal root ganglion neurones were reversibly inhibited by arginine polyamine (AP; 0.001 to 100 μm). Low voltage‐activated T‐type Ca2+ currents were significantly more sensitive to AP than high voltage‐activated Ca2+ currents. The IC50 values for the actions of AP on low and high voltage‐activated Ca2+ currents were 10 nm and 3 μm respectively. AP was equally effective in inhibiting high voltage‐activated currents carried by Ba2+, Sr2+ or Ca2+. However, AP‐induced inhibition of Ca2+ currents was attenuated by increasing the extracellular Ca2+ concentration from 2 mm to 10 mm.

The actions of AP on a Ca2+‐independent K+ current were more complex, 1 μm AP enhanced this current but 10 μm AP had a dual action, initially enhancing but then inhibiting the K+ current.

γ‐Aminobutyric acid‐activated Cl− currents were also reversibly inhibited by 1 to 10 μm AP. In contrast N‐methyl‐d‐aspartate currents recorded from rat cultured cerebellar neurones were greatly enhanced by 10 μm AP.

We conclude that at a concentration of 10 nm, AP is a selective inhibitor of low threshold T‐type voltage‐activated Ca2+ currents. However, at higher concentrations 1–10 μm AP interacts with ion channels or other membrane constituents to produce a variety of actions on both voltage and ligand gated ion channels.
Vasoconstriction of guinea‐pig submucosal arterioles following sympathetic nerve stimulation is mediated by the release of ATP
Authors: Evans R.J. , Surprenant A.
Published: July 2012

The nature of the transmitter mediating vasoconstriction of guinea‐pig submucosal arterioles following sympathetic nerve stimulation was studied.

Prazosin (0.1 μm) abolished the response to exogenously applied phenylephrine (1 μm) but had no effect on constrictions of submucosal arterioles evoked by nerve stimulation (100 pulses at 10 Hz).

Vasoconstrictions and excitatory junction potentials elicited by nerve stimulation were potentiated by idazoxan (0.1 μm).

Following reserpine treatment, catecholamine fluorescence was absent in submucosal arterioles but nerve‐evoked vasoconstrictions were unaltered.

Vasoconstrictions and excitatory junction potentials recorded in response to sympathetic nerve stimulation, as well as constrictions evoked by exogenously applied ATP (3 μm), were abolished by the P2‐purinoceptor antagonist, suramin (100 μm). Suramin had no effect on the vasoconstriction in response to noradrenaline (3 μm), or the nicotinic excitatory postsynaptic potentials (e.p.s.ps) and noradrenergic inhibitory postsynaptic potentials (i.p.s.ps) recorded from submucosal neurones.

We conclude that postjunctional responses of submucosal arterioles following sympathetic nerve stimulation are mediated solely through the activation of P2X‐purinoceptors by ATP or a related purine nucelotide. The function of neurally released noradrenaline is to act through prejunctional α2‐adrenoceptors to depress transmitter release.
The contribution of α‐adrenoceptors to neurally‐mediated contractions of the rabbit urethral smooth muscle
Authors: Chen Hong‐I , Brading Alison F.
Published: July 2012

The nature of the nerve‐mediated contractions in the urethral smooth muscle from the rabbit was studied in vitro. Field stimulation caused smaller contractile responses than in the detrusor of the rabbit.

There was no significant difference in response to field stimulation or exogenous agents acting on adrenoceptors between longitudinal and circular strips from the rabbit urethra. Histological studies showed that the urethral muscle is arranged in three layers, which run circularly and longitudinally.

Atropine had very little effect on the response to field stimulation, phentolamine almost abolished the contractile response to nerve stimulation and sometimes unmasked a relaxation.

The α1‐adrenoceptor blocking agent, prazosin, blocked both the contractile response to the α1‐receptor agonist phenylephrine and that to intrinsic nerve stimulation, with similar potencies. The α2‐blocking agent yohimbine shifted the dose‐response curve of the contractile response to the α2‐agonist, clonidine, in a dose‐dependent manner, 10−7 m causing a 10 fold shift. This concentration had no effect on the response to intrinsic nerve stimulation, suggesting that α2‐receptors are not involved in the response. Higher concentrations of yohimbine caused a suppression of the nerve‐evoked response which is assumed to be non‐specific.

Noradrenaline, phenylephrine, and clonidine caused dose‐dependent contractile responses in the rabbit urethral strips. The contractions induced by clonidine developed more slowly than those induced by noradrenaline and phenylephrine.

These results demonstrate that the rabbit urethral smooth muscle contains both α1‐ and α2‐adrenoceptors, and the nerve‐mediated contraction of the rabbit urethra is adrenergic in nature and mediated mainly via α1‐adrenoceptors.
Cholinoceptor regulation of cyclic AMP levels in bovine adrenal medullary cells
Authors: Anderson Karen , Robinson Phillip J. , Marley Philip D.
Published: July 2012

The regulation of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) levels by cholinoceptors has been studied in cultured bovine adrenal medullary cells.

Acetylcholine (100 μm), nicotine (10 μm) and dimethylphenylpiperazinium (20 μm) each increased cellular cyclic AMP levels 2 to 4 fold over 5 min in the absence of phosphodiesterase inhibitors. The muscarinic agonist acetyl‐β‐methylcholine (100 μm) had no effect either on its own or on the response to nicotine. The responses to acetylcholine and nicotine were unaffected by atropine (1 μm) but were abolished by mecamylamine (5 μm).

Cellular cyclic AMP increased transiently during continuous exposure to nicotine (1–20 μm), with the largest response seen after 5 min, a smaller response after 20 min, and no change in cyclic AMP levels seen after 90 or 180 min. The maximal response after 5 min stimulation was seen with 5‐ 10 μm nicotine and the EC50 was about 2 μm. In contrast, extracellular cyclic AMP levels did not change after 5 or 20 min stimulation with nicotine, but increased slightly after 90 min and further after 180 min.

The cellular cyclic AMP response to nicotine (10 μm) was unchanged or weakly enhanced in the presence of the unselective phosphodiesterase inhibitor, isobutylmethylxanthine, and was unchanged in the presence of rolipram. Nicotine did not interact synergistically with low concentrations of forskolin. The response was however completely abolished in the absence of extracellular Ca2+.

The nicotinic cyclic AMP response was almost abolished by sphingosine (30 μm), which did not inhibit the cyclic AMP response to phorbol‐12,13‐dibutyrate (PDB). The nicotinic response was reduced by 55% by the calmodulin antagonist W7 at 3 μm, and by >90% at 10 μm W7. At these concentrations, W7 had no effect on the cyclic AMP response to 100 nm PDB. The nicotinic response was also selectively abolished by another calmodulin antagonist, trifluoperazine (10 μm).

The results indicate that nicotinic cholinoceptors can increase cyclic AMP production in chromaffin cells by a mechanism that does not directly involve Gs, that is dependent on extracellular Ca2+ and that is sensitive to the calmodulin antagonists W7 and trifluoperazine. We propose that a Ca2+/calmodulin‐sensitive adenylate cyclase may mediate the nicotinic cyclic AMP response in bovine chromaffin cells.
Effect of lignocaine on arginine‐vasopressin plasma levels: baseline or induced by frusemide
Authors: Gariépy Louise , Larose Patrice , Bailey Benoit , du Souich Patrick
Published: July 2012

To assess whether or not lignocaine influences baseline and frusemide‐induced (5 mg kg−1) plasma concentrations of arginine‐vasopressin (AVP), 2 groups of rabbits received an infusion of lignocaine (130 μg min−1 kg−1) for 6 h. Lignocaine‐induced changes in AVP plasma concentrations were substantiated by measurement of diuresis and natriuresis and hepatic plasma flow, by means of an infusion of indocyanine green (ICG) (249 μg min−1 kg−1).

Baseline plasma AVP levels were 4.9 ± 0.9 pg ml−1 (± s.e.), and following lignocaine, these values were reduced to 0.7 ± 0.1 pg ml−1 (P < 0.01). Frusemide increased AVP levels to 134.1 ± 73.6 pg ml−1 (P < 0.05) and lignocaine totally prevented this increase, e.g. mean AVP levels of 2.7 pg ml−1.

Lignocaine enhanced baseline diuresis secondary to an increase in free water clearance; none of the experimental conditions affected the diuresis and natriuresis induced by frusemide.

Frusemide reduced the hepatic plasma flow and this decrease was not reversed by the infusion of lignocaine.

It is concluded that in healthy rabbits lignocaine reduces baseline secretion of AVP and its antidiuretic effect; in addition, lignocaine prevents the rise in AVP induced by frusemide.
Effect of the β‐adrenoceptor agonist clenbuterol and phytohaemagglutinin on growth, protein synthesis and polyamine metabolism of tissues of the rat
Authors: Bardocz S. , Brown D.S. , Grant G. , Pusztai A. , Stewart J.C. , Palmer R.M.
Published: July 2012

The kidney bean lectin, phytohaemagglutinin (PHA), induced a marked atrophy of skeletal muscle which was evident from the changes in tissue composition (protein, RNA, DNA and polyamine content) and from the reduction in weight and protein synthesis of hind leg muscles of rats fed on kidney bean‐diets for four days. The β‐adrenoceptor agonist, clenbuterol, induced skeletal muscle hypertrophy by transiently stimulating protein synthesis. As a consequence, the muscle loss caused by a short exposure to PHA was, in part, ameliorated by clenbuterol treatment.

Cardiac muscle was affected to a lesser extent than skeletal muscle by both clenbuterol and the lectin. However, there was evidence that protein synthesis in heart was reduced by PHA.

PHA had opposite effects on the gut, the lectin‐induced hyperplasia of the jejunum was accompanied by a large increase in protein synthesis. Clenbuterol alone had no effect on the jejunum whereas a combination of PHA and clenbuterol appeared to exacerbate the effect of the lectin on gut.

Both the lectin‐induced gut growth and the hypertrophy of skeletal muscle caused by clenbuterol were preceded by the accumulation of polyamines in the respective tissues. Of particular note was the observation that a significant increase in the proportion of the intraperitoneally injected 14C‐labelled spermidine or putrescine taken up by the growing tissues could be detected by the second day. Therefore, the measurement of uptake of labelled polyamines may be used as a sensitive indicator of early alterations in tissue metabolism.
(S)‐homoquisqualate: a potent agonist at the glutamate metabotropic receptor
Authors: Porter Richard H.P. , Roberts Peter J. , Jane David E. , Watkins Jeffrey C.
Published: July 2012

The synthetic quisqualate analogue, (S)‐homoquisqualate was examined for activity at the glutamate metabotropic receptor, in relation to its ability to stimulate phosphoinositide hydrolysis in rat pup cerebro‐cortical slices. The compound produced a robust increase in hydrolysis (EC50 = 50.2 ± 1.6 μm), which, in common with responses to quisqualate and (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylate ((1S,3R)‐ACPD), was antagonized uncompetitively by l‐2‐amino‐3‐phosphonopropionate (l‐AP3). In contrast to quisqualate which exhibits low efficacy, (S)‐homoquisqualate behaves as a full agonist at the metabotropic receptor.
Pathological events in experimental acute pancreatitis prevented by the bradykinin antagonist, Hoe 140
Authors: Griesbacher Thomas , Tiran Beate , Lembeck Fred
Published: July 2012

In a previous investigation, Hoe 140, a specific and potent bradykinin B2 receptor antagonist, prevented the pancreatic oedema and the hypotension observed during acute experimental pancreatitis; however, it augmented the associated rises in the serum activities of pancreatic enzymes. Therefore, we have now investigated the consequences of the pancreatic oedema for the fate of activated enzymes released into the tissue during the course of acute pancreatitis.

Acute oedematous pancreatitis was induced in rats, pretreated with captopril (50 μmol kg−1, i.p.), by hyperstimulation of the exocrine function of the pancreas with the cholecystokinin analogue, caerulein (4 nmol kg−1 h−1, i.v.), for up to 120 min.

Pancreatic oedema began to develop 10 min after the start of the caerulein infusion, reached a maximum within about 45 min, and then declined slightly. The development of the oedema parallelled the second phase of the caerulein‐induced fall in blood pressure found in earlier experiments. No further extravasation of plasma proteins occurred during the 2nd hour of the caerulein infusion. The oedema formation was completely blocked in animals pretreated with the bradykinin receptor antagonist, Hoe 140 (100 nmol kg−1, s.c.). Pretreatment with aprotinin or soy bean trypsin inhibitor did not result in a significant inhibition of the oedema.

The haematocrit of animals with experimental pancreatitis showed a pronounced increase which started 10 min after the start of the caerulein infusion and reached maximal values at 60 min. The changes in haematocrit showed a reduction in total blood volume of 28% due to a 48% loss of plasma. This effect was completely blocked by Hoe 140.

In rats with caerulein‐induced pancreatitis, there was a time‐dependent increase in the activities of amylase and lipase in blood serum as well as in the pancreas. Pretreatment with Hoe 140 greatly augmented the caerulein‐induced rise in enzyme activities in blood serum but potently attenuated it in the pancreas. The activities of trypsin in both the blood serum and the pancreas were below or near the limit of detection in all experimental groups.

It is concluded that the second phase of hypotension in this model of acute pancreatitis is due to the liberation of kinins which cause a massive loss of blood plasma into the pancreas and into the retroperitoneal space. Activated enzymes are trapped in the pancreas, at least in part, by the oedema of the gland. Treatment with Hoe 140 prevents the oedema formation and greatly facilitates the egress of activated enzymes from the pancreas.
Actions of agonists of metabotropic glutamate receptors on synaptic transmission and transmitter release in the olfactory cortex
Authors: Collins G.G.S.
Published: July 2012

The effects of agonists of metabotropic glutamate receptors on the evoked N‐wave complex in slices of mouse olfactory cortex have been studied: most experiments were carried out using slices perfused with Mg2+‐free solution to which 10 μm of either 6,7‐dinitroquinoxaline‐2,3‐dione or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione was applied.

Following agonist washout, a slowly developing, long lasting potentiation of the complex occurred which was confined to the N‐methyl‐d‐aspartate (NMDA) receptor‐mediated component of the potential. The relative agonist potencies were 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD, 5–250 μm) = quisqualate (5–50 μm) > 1RS,3RS‐cis‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (ACPD, 25–1000 μm) > l‐glutamate (0.25–2.5 mm); NMDA, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate (AMPA) and l‐aspartate were inactive.

Potentiation of the NMDA receptor‐mediated component by 1S,3R‐ACPD (0.1 mm) was non‐competitively antagonised by S‐(+)‐ but not R‐(−)‐2‐amino‐3‐phosphonopropionate (AP3, 0.125 mm), equally by d‐(−) and l‐(+)‐2‐amino‐4‐phosphonobutyrate (0.25 mm) and also by the protein kinase C inhibitors sphingosine, (25 μm), sangivamycin (25 μm) and 5‐(isoquinolinylsulphonyl)‐3‐methylpiperazine (50 μm).

In a series of input‐output experiments, 1S,3R‐ACPD (0.1 mm) reversibly reduced the latency to peak of the NMDA receptor‐mediated component at submaximal stimulus intensities, an effect blocked by S‐(+)‐AP3 (0.125 mm). On agonist washout, there was an increase in the area of the NMDA receptor‐mediated component over all stimulus intensities, an effect blocked by the inhibitors of protein kinase C and by S‐(+)‐AP3 (0.125 mm). 4‐β‐Phorbol‐12,13‐diacetate (2.5 μm) also potentiated the component, an action inhibited by protein kinase C inhibitors but not by S‐(+)‐AP3.

1S,3R‐ACPD (0.1 mm) had no significant effect on postsynaptic responses evoked by NMDA, AMPA and kainate, but significantly reversed a partial antagonism of NMDA responses produced by 7‐chlorokynurenate (2.5 μm).

The K+‐evoked release of glycine was selectively and significantly increased in the presence of 0.1 mm 1S,3R‐ACPD (antagonized by 0.125 mm S‐(+)‐AP3) whereas following agonist washout, release of glycine fell to control levels but there was a significant increase in release of aspartate (antagonized by 25 μm sangivamycin and 0.125 mm S‐(+)‐AP3).

It is concluded that metabotropic glutamate receptors mediate (i) a reduction in the latency of the NMDA receptor‐mediated component of potentials by a mechanism that is independent of protein kinase C but which may depend on increased glycine release and (ii) a long lasting increase in the total area of the potential by increasing transmitter (possibly aspartate) release by a mechanism that is protein kinase C‐dependent.
Effects of capsaicin and 5‐HT3 antagonists on 5‐hydroxytryptamine‐evoked release of calcitonin gene‐related peptide in the guinea‐pig heart
Authors: Tramontana Manuela , Giuliani Sandro , Del Bianco Elena , Lecci Alessandro , Maggi Carlo A. , Evangelista Stefano , Geppetti Pierangelo
Published: July 2012

The effect of 5‐hydroxytryptamine (5‐HT) on the release of calcitonin gene‐related peptide (CGRP) was studied directly in the isolated perfused heart and indirectly in the isolated left atria of guinea‐pig.

5‐HT injection into the guinea‐pig isolated and perfused heart evoked a dose‐dependent (1–100 μm) release of CGRP‐like immunoreactivity (LI) that was abolished by in vitro pretreatment with capsaicin and was not affected by indomethacin.

Chlorophenyldiguanide (CPD, 100 μm), but not 8‐hydroxy‐dipropylaminotetralin (8‐OH‐DPAT, 100 μm), sumatriptan (100 μm) or 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI, 100 μm) evoked a release of CGRP‐LI. Ondansetron (10 μm) or ICS205‐930 (20 μm) completely abolished the 5‐HT (100 μm)‐evoked CGRP‐LI release.

In the isolated electrically driven left atria of the guinea‐pig 5‐HT (1–10 μm) and CPD (3–100 μm) produced a positive inotropic response, which was abolished by capsaicin pretreatment. 8‐OH‐DPAT (10 μm) and DOI (10 μm) were inactive. Ondansetron inhibited the response to 5‐HT with a pA2 of 6.50 (CL 6.08–6.91).

It is concluded that 5‐HT causes a release of CGRP in the whole heart and a positive inotropic response in the isolated atria of guinea‐pig. Both these effects are sensitive to capsaicin pretreatment and to 5‐HT3 antagonists.
In vitro characterization of prostanoid receptors on human myometrium at term pregnancy
Authors: Senior J. , Marshall K. , Sangha R. , Clayton J.K.
Published: July 2012

Prostanoid receptors present on the pregnant human myometrium in vitro have been characterized according to the receptor classification proposed by Coleman et al. (1984) using natural prostanoids and synthetic, selective analogues and antagonists where available.

Prostaglandin E2 (PGE2) produced a biphasic effect consisting of an initial excitation followed by a dose‐related inhibition. The EP2/EP3‐receptor agonists, rioprostil and misoprostol, produced similar effects to PGE2, however, the excitatory event of the misoprostol response was related to dose. The EP1/EP3‐receptor agonist, sulprostone, evoked a purely excitatory response which was unaffected by AH6809. The selective EP2‐receptor agonist butaprost produced a long‐lasting dose‐dependent inhibition of activity. The results from these prostanoids indicated that inhibitory EP2‐ and excitatory EP3‐receptors are present on myometrium from pregnant donors at term.

PGF2α and the synthetic FP‐receptor agonist, fluprostenol, caused equipotent excitatory effects, indicating the presence of contractile FP‐receptors.

PGD2 produced a biphasic effect of which the inhibition appeared dose‐related and was antagonized by the selective DP‐receptor antagonist BW A868C. The selective DP‐receptor agonist, BW245C, produced a potent inhibitory effect that was competitively antagonized by BW A868C (pA2 = 8.6).

PGI2 produced a biphasic response qualitatively similar to PGE2. The EP1/IP‐receptor agonist, iloprost, produced an occasional unquantifiable excitation and dose‐related inhibition. The selective IP‐receptor prostanoid, cicaprost, evoked only an inhibitory response.

The stable thromboxane A2 (TXA2)‐mimetic, U46619, produced potent excitation which was competitively antagonized by the TP‐receptor antagonist, GR32191 (pA2 = 7.2).

The prostanoids tested indicate that a heterogeneous population of prostanoid receptors are present on human myometrium from pregnant donors. It may be concluded that excitation is EP3‐, FP‐ and TP‐receptor‐mediated and inhibition is EP2‐, DP‐ and IP‐receptor‐mediated. Comparison of data obtained from non‐pregnant specimens indicates that the lower segment tissue from pregnant donors demonstrated more pronounced responses to EP2 and IP‐receptor activation.
Investigations into factors determining the duration of action of the β2‐adrenoceptor agonist, salmeterol
Authors: Nials Anthony T. , Sumner Michael J. , Johnson Malcolm , Coleman Robert A.
Published: July 2012

This study has explored the mechanism underlying the long duration of action of the β2‐adrenoceptor agonist, salmeterol.

Salmeterol, salbutamol and isoprenaline caused a concentration‐related inhibition of electrically‐induced contractile responses of the guinea‐pig superfused trachea preparation. The effects of both isoprenaline and salbutamol were rapid in onset and rapidly reversed upon removal of the agonist. In contrast, the effects of salmeterol were slower in onset and could not be reversed by superfusion of the tissue with agonist‐free Krebs solution even for periods of up to 10 h.

The effects of salmeterol were, however, readily reversed by a number of β‐adrenoceptor blocking drugs, as was the effect of a continuous infusion of isoprenaline. Upon removal of the antagonist, however, the effects of salmeterol and of the isoprenaline infusion were reasserted at a rate which was inversely related to the lipophilicity of a β‐adrenoceptor blocking drugs.

Salmeterol inhibited the binding of [125I]‐(−)‐iodopindolol (100 pm) to rat lung membranes (pIC50 7.1), with isoprenaline (pIC506.2) and salbutamol (pIC50 5.1) having lower potencies. The inhibition of binding by salmeterol was apparently non‐competitive, whereas that produced by salbutamol and isoprenaline was competitive in nature.

Isoprenaline and salbutamol rapidly dissociated from their binding sites, whereas in marked contrast, the binding of salmeterol showed no dissociation for periods of up to 1 h.

These data are consistent with a mechanism in which salmeterol binds adjacent to the active site of the β2‐adrenoceptor, such that the drug cannot be washed out of the tissue, yet can interact with and activate the receptor. This latter property is susceptible to antagonism by β‐adrenoceptor blocking drugs but is reasserted when the antagonists are removed.
Investigation into the role of phosphodiesterase IV in bronchorelaxation, including studies with human bronchus
Authors: Cortijo J. , Bou J. , Beleta J. , Cardelús I. , Llenas J. , Morcillo E. , Gristwood R.W.
Published: July 2012

We have investigated the role of cyclic nucleotide phosphodiesterase IV (PDEIV) in the relaxation of human bronchus and guinea‐pig trachea in vitro and in guinea‐pigs in vivo.

Functional studies showed that the selective PDE IV inhibitors, rolipram and denbufylline, relaxed human and guinea‐pig preparations in vitro.

Two clinically used xanthine non‐selective PDE inhibitors, theophylline and pentoxifylline, were also effective in these preparations, but were much less potent than the selective agents used.

The rank order of potency for the four PDE inhibitors in both species was similar.

Biochemical studies indicated that PDE IV was the major PDE isoform present in the human bronchial tissue. PDEs I, II and V were also identified.

Theophylline and pentoxifylline were, as expected, non‐selective inhibitors of the human enzymes, but there was a good correlation between PDE IV inhibitory and bronchorelaxation potencies, suggesting that PDE IV inhibition is important for the clinical bronchodilator activities of the two xanthine compounds.

We have confirmed the ability of selective PDE IV inhibitors to cause bronchodilatation in guinea‐pigs in vivo.

We conclude that our study has provided further evidence that selective PDE IV inhibitors could act as bronchodilators in the clinic.
Active secretion of the fluoroquinolone ciprofloxacin by human intestinal epithelial Caco‐2 cell layers
Authors: Griffiths Nina M. , Hirst Barry H. , Simmons Nicholas L.
Published: July 2012

The bidirectional transepithelial fluxes of ciprofloxacin, an antibacterial fluoroquinolone, across the human intestinal epithelial Caco‐2 cell‐line show marked asymmetry. Basal‐to‐apical flux of ciprofloxacin (10 μm) exceeds apical‐to‐basal flux indicating net secretion. Net ciprofloxacin secretion is abolished by azide/2‐deoxy‐d‐glucose treatment, displays saturation kinetics (Km = 0.89 ± 0.23 mm, Vmax 44.3 ± 4.9 nmol cm−2.h) and competition by other fluoroquinolones. A specific, active secretion in Caco‐2 epithelia may explain the transintestinal elimination of ciprofloxacin observed in pharmacokinetic studies in man.
Ca2+‐dependent aggregation of rabbit platelets induced by maitotoxin, a potent marine toxin, isolated from a dinoflagellate
Authors: Watanabe Akiko , Ishida Yukisato , Honda Hiromi , Kobayashi Masaki , Ohizumi Yasushi
Published: July 2012

Administration of maitotoxin (MTX), a dinoflagellate toxin, caused aggregation of rabbit washed platelets. The cytosolic Ca2+ concentration ([Ca2+]i), measured by fura‐2 fluorescence technique, was also increased by the presence of MTX. Rates of aggregation response and [Ca2+]i‐increase were dependent on tested concentrations (3–100 ng ml−1) of the toxin.

The MTX‐induced platelet aggregation and [Ca2+]i‐increase were totally abolished in a Ca2+‐free solution. The successive administration of Ca2+ in the presence of MTX elicited the aggregation and increase in [Ca2+]i.

Ba2+ was capable of substituting for Ca2+ in the MTX‐induced platelet aggregation. In the presence of external Ca2+, transition metals, Co2+, Cd2+ and Ni2+, inhibited the aggregation response to MTX.

Organic calcium antagonists (verapamil and nifedipine) as well as a cyclo‐oxygenase‐inhibitor (aspirin) did not apparently inhibit the aggregation response to MTX, except for a high concentration (10−5 m) of verapamil, while procaine (10 mm) reduced the rate of platelet aggregation.

MTX also elicited a release of ATP from platelets, which was abolished in the absence of external Ca2+.

In contrast, thrombin 0.5 unit ml−1 could elicit platelet shape change, [Ca2+]i‐increase and ATP‐release in the absence of external Ca2+.

These results suggest that the MTX‐induced platelet activation is caused by an enhanced Ca2+‐influx presumably through voltage‐independent Ca2+ channels on the plasma membrane.
Effects of 5‐HT‐receptor and α2‐adrenoceptor ligands on the haemodynamic response to acute central hypovolaemia in conscious rabbits
Authors: Evans Roger G. , Haynes John M. , Ludbrook John
Published: July 2012

We set out to elucidate the pharmacological mechanisms by which α2‐adrenoceptor and 5‐HT‐receptor ligands affect the haemodynamic response to acute central hypovolaemia in conscious rabbits.

Acute central hypovolaemia was produced by inflating an inferior vena caval cuff so that cardiac output fell at a constant rate of ∼8.5% of its baseline level per min.

Drugs were administered into the fourth cerebral ventricle in either 154 mm NaCl (saline) or 20% w/v 2‐hydroxypropyl‐β‐cyclodextrin (β‐CDX). After vehicle treatments, the haemodynamic response to acute central hypovolaemia had the usual two phases. During Phase I, systemic vascular conductance fell in proportion to cardiac output so that mean arterial pressure fell by only 8 mmHg. Phase II commenced when cardiac output had fallen to ∼60% of its baseline level, when vascular conductance rose abruptly and arterial pressure fell to ≤ 40 mmHg. The haemodynamic response was not dependent on the vehicle used (saline or β‐CDX).

Methysergide delayed the occurrence of Phase II in a dose‐dependent manner, and prevented it at a dose of 30– 600 nmol (geometric mean = 186 nmol). The effects and potency of methysergide were not dependent on the vehicle used, indicating that β‐CDX can be used as a vehicle for fourth ventricular administration of lipophilic drugs to conscious rabbits. Clonidine (10 nmol) reversed the effects of a critical dose of methysergide.

Phase II was also prevented by 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (5‐HT1A‐selective agonist, geometric mean critical dose (range) = 13.1 (10–30) nmol), sumatriptan (5‐HT1D‐selective agonist, 72.1 (10–300) nmol), mesulergine (5‐HT2/1c‐selective antagonist, 173 (30–1000) nmol), idazoxan (α2‐adrenoceptor‐selective antagonist, 548 (100–3000) nmol), and mianserin (5‐HT2/1c‐selective antagonist, 548 (100– 3000) nmol). It was not affected by MDL 72222 (5‐HT3‐selective antagonist, 300 nmol) or ketanserin (5‐HT2/1c‐selective antagonist, 3000 nmol).

To characterize the nature of α2‐adrenoceptors in rabbit brainstem, we examined the binding of [3H]‐rauwolscine to membrane homogenates of whole brainstem. [3H]‐rauwolscine bound to a population of sites with the characteristics of α2A‐adrenoceptors.

From these results we suggest that activation of 5‐HT1A receptors in the brainstem can prevent Phase II of the response to acute central hypovolaemia in conscious rabbits. Our results do not support the notion of an endogenous 5‐hydroxytryptaminergic mechanism mediating Phase II. The mechanism by which the α2‐adrenoceptor antagonists yohimbine and idazoxan prevent Phase II remains to be elucidated. However, their potency relative to other 5‐HT‐receptor ligands indicates that an agonist action at 5‐HT1A‐receptors is more likely than an antagonist action at α2‐adrenoceptors.