bluuebunny/crossref_metadata_2025 huggingface dataset:
The use of receptor desensitization to analyse CCKA and CCKB/gastrin receptors coupled to contraction in guinea‐pig stomach muscle
Authors: Bishop L.A. , Gerskowitch V.P. , Hull R.A.D. , Shankley N.P. , Black J.W.
Published: July 2012

The results of previous studies have been in conflict with respect to the involvement of specific cholecystokinin (CCKA) and CCKB/gastrin receptors in guinea‐pig gastric muscle. Here, in an in vitro, guinea‐pig gastric muscle assay, pentagastrin (PG) and tetragastrin (TG) behaved as high potency agonists and produced symmetrical concentration‐effect curves. In contrast, cholecystokinin‐octapeptide (CCK‐8), while also behaving as a high potency agonist, produced flat asymmetrical curves. Unlike recent data reported using this tissue (Boyle et al., 1993), the CCKA receptor‐selective antagonist, devazepide (3, 10, 30 nm) produced a rightward shift of the upper region of the CCK‐8 curve rendering it biphasic. The lower phase was abolished by the CCKB/gastrin receptor‐selective antagonist, L‐365260 (300 nm) indicating that the contractile effects of CCK‐8 in this tissue are mediated by both receptor types.

L‐365260 produced a concentration‐dependent, parallel rightward displacement of PG concentration‐effect curves. However, a flat Schild plot slope parameter (0.77 ± 0.06) was obtained. Therefore, an empirical pA2 value of 8.64 ± 0.21 was estimated from the smallest dose ratio. This value is consistent with published values characteristic of an interaction at CCKB/gastrin receptors.

TG (1 μm) was used to densensitize selectively the CCKB/gastrin receptors in the gastric muscle assay and thereby expose a population of receptors capable of responding to subsequent stimulation by CCK‐8 but not by PG. The selectivity of TG for CCKB/gastrin‐ over CCKA receptors was demonstrated by its low efficacy compared to CCK‐8 in the guinea‐pig gallbladder assay, a tissue shown previously to contain a homogeneous population of CCKA receptors. In TG‐desensitized gastric muscle, CCK‐8 concentration‐effect curves were symmetrical and could be displaced in a simple parallel fashion by devazepide at nanomolar concentrations consistent with an interaction at CCKA receptors (pKB ∼ 10).

These results indicate that the guinea‐pig gastric muscle contains both CCKA‐ and CCKB/gastrin receptors and the effects of CCK‐8 are mediated via both of these receptors. Notwithstanding the complexity of the behaviour of L‐365260, it was possible to obtain a reasonable description of the system using a simple 2‐receptor model in which the effects of individual receptor activation were assumed to be additive. The absence of a simple competitive interaction of PG with L‐365260 may indicate, for example, non‐homogeneity of CCKB/gastrin receptors or lack of concentration equilibrium between the bath and the receptor biophase.
Mediation by 5‐hydroxytryptamine2B receptors of endothelium‐dependent relaxation in rat jugular vein
Authors: Ellis Elizabeth S. , Byrne Clare , Murphy Olive E. , Tilford Nicholas S. , Baxter Gordon S.
Published: July 2012

An ‘atypical’ 5‐HT2 receptor which is located on the endothelium of rat jugular vein has been described. In the present study we have further defined the nature of the 5‐HT2 receptor subtype present in this preparation.

In experiments conducted in the presence of ketanserin to preclude involvement of 5‐HT2 receptors, the mixed 5‐HT2B/2C antagonist, SB 200646, acted as an antagonist of 5‐HT at the endothelial 5‐HT receptor (pA2 = 7.2). Yohimbine, which exhibits negligible affinity for rat 5‐HT2C receptors but has high 5‐HT2B receptor affinity, acted as a potent but non‐surmountable antagonist (pA2 ≥ 7.3) in rat jugular vein. Neither yohimbine nor SB 200646 affected endothelium‐dependent relaxations induced by carbachol.

Mianserin also acted as a surmountable antagonist (pA2 = 7.3) and the 5‐HT2B agonist, BW 723C86, acted as a potent partial agonist (pEC50 [95% C L], intrinsic activity ± s.e.mean = 7.9 [7.6–8.3], 0.84 ± 0.04). Responses to BW 723C86 were antagonized by SB 200646 (0.3 μm) yielding an ‘apparent’ pA2 [95% CL] of 7.03 [6.76–7.32].

These data are consistent with the presence of 5‐HT2B receptors mediating endothelium‐dependent relaxation of rat jugular vein.
Species‐related differences in inotropic effects of angiotensin II in mammalian ventricular muscle: receptors, subtypes and phosphoinositide hydrolysis
Authors: Ishihata Akira , Endoh Masao
Published: July 2012

Experiments were carried out to clarify the mechanisms responsible for variations in the positive inotropic effect (PIE) of angiotensin II (AII) on ventricular muscles from various mammals. We examined the density of All receptors, the relative proportions of receptor subtypes and the acceleration of the hydrolysis of phosphoinositide that was induced by AII, as well as the PIE of AII in ventricular muscles from the rabbit, dog, rat and ferret.

In the rabbit, AII (1 μm) in the presence of bupranolol (0.3 μm) and prazosin (0.1 μm) elicited a concentration‐dependent PIE, which was antagonized by a selective AT1 subtype antagonist, losartan, but not by an AT2 antagonist, PD123319. AII did not have any inotropic effects in ventricular muscles from the dog, rat and ferret.

Specific high‐affinity binding of [125I]‐AII, with a similar Kd value in each case (1–2 nm), was observed with membrane fractions derived from ventricular muscle of all four species tested.

In the rabbit, losartan and PD123319 each displaced approximately 50% of [125I]‐AII specific binding having high affinity for the receptors, and indicating that AT1 and AT2 subtypes were present in equal numbers. In the other species the AT1 subtype of receptors was predominant.

In all four species AII caused a concentration‐dependent acceleration of the hydrolysis of phosphoinositide in ventricular slices that had been prelabelled with myo‐[3H]‐inositol.

The results indicate that the signal‐transduction process distal to acceleration of the hydrolysis of phosphoinositide may be responsible for the wide range of species variations in the inotropic action of AII on mammalian ventricular myocardium.
Electrophysiological effects of SD‐3212, a new antiarrhythmic agent with vasodilator action, on guinea‐pig ventricular cells
Authors: Kodama Itsuo , Suzuki Ryoko , Maruyama Kazuyasu , Toyama Junji
Published: July 2012

The effects of SD‐3212 on transmembrane action potentials were examined in right ventricular papillary muscles and in single ventricular myocytes isolated from guinea‐pig hearts.

In papillary muscles, SD‐3212 ≥ 3 μm caused a significant decrease in the maximum upstroke velocity () of action potential without affecting resting membrane potential. The inhibition of was enhanced at higher stimulation frequencies.

In the presence of SD‐3212, trains of stimuli at rates ≥ 0.5 Hz led to a use‐dependent inhibition of . The time constant for the recovery of from the use‐dependent block was 1.3 s.

Voltage‐dependence of inhibition by SD‐3212 was investigated in single myocytes. The curves relating membrane potential and were shifted by SD‐3212 (10 μm) in a hyperpolarizing direction by 6.2 mV.

In myocytes treated with SD‐3212 (10 μm), the of test action potentials preceded by conditioning clamp to 0 mV was decreased progressively as the clamp pulse duration was prolonged. of test action potentials following a long (1 s) 0 mV clamp recovered at a time constant ranging from 1.01 to 1.22 s, being shorter at the more negative potential within a range from −70 to −90 mV.

These findings suggest that the primary electrophysiological effect of SD‐3212 is a use‐ and voltage‐dependent inhibition of sodium channels. From the onset and offset kinetics of the use‐dependent block, SD‐3212 is located between fast and intermediate kinetic Class‐I drugs. From the state‐dependence of sodium channel block, SD‐3212 belongs to inactivated channel blockers.
A reassessment of the modulatory role of cyclic AMP in catecholamine secretion by chromaffin cells
Authors: Parramón M. , González M.P. , Oset‐Gasque M.J.
Published: July 2012

The role of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) in the regulation of catecholamine (CA) secretion in chromaffin cells remains equivocal from previous studies.

In the present study the effect of this cyclic nucleotide on basal CA secretion, as well as on intracellular calcium and membrane potential has been examined.

Forskolin and the permeable cyclic AMP analogue, 8‐(4‐chlorphenylthio)‐adenosine‐3′‐5′ monophosphate cyclic (pClpcAMP), increased basal CA secretion in a dose‐dependent manner. The EC50S were 0.43 ± 0.10 μm for forskolin and 39 ± 9 μm for pClpcAMP. Other agonists with adenylate cyclase activity such as stimulants of adenosine receptors, β‐adrenoceptors, GABAB receptors and intestinal vasoactive peptide (VIP), also increased basal CA secretion in a highly significant manner. However, when they were added together with forskolin, CA secretion was not affected although an additive increase in cyclic AMP levels was produced.

Statistical analysis of the correlation between cyclic AMP levels and CA secretion evoked by these cyclic AMP increasing compounds showed that a significant direct correlation between both parameters existed only when low levels of cyclic AMP were produced by secretogogue stimulation. When the increase in intracellular cyclic AMP concentrations exceeded approximately 8 times the basal cyclic AMP levels the correlation was not significant. These results indicate a dual dose‐dependent effect of cyclic AMP on basal CA secretion.

The stimulatory effect of low cyclic AMP on basal CA secretion was accompanied by an increase in membrane potential and in intracellular calcium concentrations ([Ca2+]i), the latter mainly being due to an increase in intracellular Ca2+ entry through L‐type voltage‐dependent Ca2+ channels.

The possible mechanisms involved in these cyclic AMP effects are discussed.
Different effects of endothelin‐3 on the Ca2+ discharge induced by agonists and Ca2+‐ATPase inhibitors in human platelets
Authors: Astarie‐Dequeker Catherine , Korichneva Irina , Devynck Marie‐Aude
Published: July 2012

The present study demonstrates that endothelin‐3 (ET‐3), previously shown to attenuate thrombin‐evoked aggregation of human platelets, delayed the dose‐dependent aggregatory response to thapsigargin (Tg). As this Ca2+‐ATPase inhibitor induces platelet activation in part through the depletion of internal Ca2+‐stores, we examined the influence of ET‐3 on Ca2+ discharge from internal pools.

Cytosolic Ca2+ concentration was evaluated with Fura‐2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 times 10−7 m ET‐3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane‐mimetic. However, ET‐3 did not affect Ca2+ movements induced by 1 μm ADP. Addition of Tg (0.5 to 5 μm) to resting platelets induced a cytosolic [Ca2+] rise with concentration‐dependent increase of the initial rate and decrease of the time to reach the peak. ET‐3 slowed down these dose‐dependent effects with a more marked influence on the responses induced by low concentrations of Tg.

ET‐3 did not modify the Ca2+ response to another Ca2+‐ATPase inhibitor, 2,5‐di‐(tert‐butyl)‐1,4‐benzohydroquinone(tBuBHQ). The thromboxane A2 receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 μm Tg, which became similar to that induced by 15 μm tBuBHQ. Under these conditions, the ET‐3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET‐3.

ET‐3 attenuates Ca2+ mobilization from an internal pool dependent on the stimulation of thrombin and thromboxane A2 receptors and insensitive to the direct effect of Ca2+‐ATPase inhibitors. The small but significant inhibitory effect of ET‐3 leads us to propose that endothelin‐3 acts as a modulator of platelet activation.
Differences between 5‐HT3 receptor antagonists in modulation of visceral hypersensitivity
Authors: Banner S.E. , Sanger G.J.
Published: July 2012

Noxious colo‐rectal distension was applied in conscious rats by acute balloon inflation and the effects observed as abdominal muscle contraction with the threshold typically between 10–40 mmHg. The effects of 5‐HT3 receptor antagonists on responses to noxious colo‐rectal distension were then studied in both normal rats and those pretreated with 5‐hydroxytryptophan (5‐HTP).

Granisetron and ondansetron (10 μg kg−1 and 1 mg kg−1, s.c.) had no effect on visceromotor thresholds to colo‐rectal distension in normal rats.

Hypersensitivity of the colo‐rectum was achieved by systemic administration of a low dose of 5‐HTP (10 mg kg−1, s.c.) which lowered the distension pressure required to induce the visceromotor reflex; analysis of variance showed a highly significant treatment effect (F1,11 = 84.26, P<0.001).

Granisetron, zatosetron, bemesetron and renzapride equi‐potently increased the threshold values at which distension evoked a visceromotor reflex after dosing with 5‐HTP, with a maximal response 3.6 to 4.2 fold above saline controls, at 10 μg kg−1, s.c. Metoclopramide (10 μg kg−1) also raised the level of distension required to elicit a response. By comparison, tropisetron caused a small, non‐significant increase in visceromotor threshold values and only at high doses (1 mg kg−1), whilst ondansetron and BRL 46470 had no signficant effects at doses up to 10 mg kg−1.

The response to granisetron (10 μg kg−1, s.c.) in 5‐HTP‐treated rats was unaltered by pre‐administration of naloxone (5 mg kg−1, s.c).

These results suggest that a 5‐HT3‐like receptor modulates 5‐HTP‐ evoked visceral hypersensitivity. However, the rank order of antagonist potency does not correlate with their order of potency against the classically defined 5‐HT3 receptor.
A new class of furoxan derivatives as NO donors: mechanism of action and biological activity
Authors: Ferioli R. , Folco G.C. , Ferretti C. , Gasco A.M. , Medana C. , Fruttero R. , Civelli M. , Gasco A.
Published: July 2012

The mechanism of action and biological activity of a series of R‐substituted and di‐R‐substituted phenylfuroxans is reported.

Maximal potency as vasodilators on rabbit aortic rings, precontracted with noradrenaline (1 μm), was shown by phenyl‐cyano isomers and by the 3,4‐dicyanofuroxan, characterized by a potency ratio 3–10 fold higher than glyceryl trinitrate (GTN). This effect was reduced upon coincubation with methylene blue or oxyhaemoglobin (10 μm).

The furoxan derivatives showing maximal potency as vasodilators were also able to inhibit collagen‐induced platelet aggregation, with IC50 values in the sub‐micromolar range.

The furoxan derivatives were able to stimulate partially purified, rat lung soluble guanylate cyclase; among the most active compounds, the 3‐R‐substituted isomers displayed a higher level of stimulatory effect than the 4‐R analogues.

Solutions (0.1 mm) of all the tested furoxans, prepared using 50 mm phosphate buffer, pH 7.4, (diluting 10 mm DMSO stock solutions) did not release nitric oxide (NO) spontaneously; however in presence of 5 mm l‐cysteine, a significant NO‐releasing capacity was observed, which correlated significantly with their stimulation of the guanylate cyclase activity.
Pharmacological characterization of RS 25259‐197, a novel and selective 5‐HT3 receptor antagonist, in vivo
Authors: Eglen R.M. , Lee C.‐H. , Smith W.L. , Johnson L.G. , Clark R. , Whiting R.L. , Hegde S.S.
Published: July 2012

The pharmacological effects in vivo, of RS 25259‐197, a selective 5‐HT3 receptor antagonist, have been investigated.

In anaesthetized rats, RS 25259‐197, administered by the intravenous, intraduodenal or transdermal route, dose‐dependently inhibited the von Bezold‐Jarisch reflex induced by 2‐methyl 5‐HT (ID50 = 0.04 μg kg−1, i.v., 3.2 μg kg−1, i.d. and 32.8 μg per chamber, respectively). In this regard, when administered intraduodenally, RS 25259‐197 was more potent and exhibited a longer duration of action than either ondansetron or granisetron.

In conscious ferrets, RS 25259‐197, administered intravenously or orally, dose‐dependently inhibited emesis induced by cisplatin. The ID50 estimates of RS 25259‐197 were 1.1 μg kg−1, i.v. and 3.2 μg kg−1, p.o. In this respect, RS 25259‐197 was more potent than ondansetron and equipotent with granisetron.

In conscious dogs, RS 25259‐197, administered intravenously or orally, dose‐dependently inhibited emesis induced by cisplatin (ID50 = 1.9 μg kg−1, i.v. and 8.5 μg kg−1, p.o.), dacarbazine (ID50 = 4.1 μg kg−1, i.v. and 9.7 μg kg−1, p.o.), actinomycin D (ID50 = 4.9 μg kg−1, i.v. and 2.5 μg kg−1, p.o.) and mechlorethamine (ID50 = 4.4 μg kg−1, i.v. and 3.0 μg kg−1, p.o.). Against each of the emetogenic agents, RS 25259‐197 was very much more potent than ondansetron. When tested at equi‐effective intravenous doses against cisplatin‐induced emesis in dogs, RS 25259‐197 had a longer duration of anti‐emetic activity (7 h) than ondansetron (4 h). At doses up to and including 1000 μg kg−1, p.o., neither RS 25259‐197 nor ondansetron was capable of inhibiting apomorphine‐induced emesis.

At doses up to 1000 μg kg−1, i.v., RS 25259‐197 produced no meaningful haemodynamic changes in anaesthetized dogs.

In summary, RS 25259‐197 is a novel, highly potent and orally active 5‐HT3 receptor antagonist in vivo. With respect to its anti‐emetic activity, RS 25259‐197 appears to be a significant improvement over ondansetron in terms of potency and duration of action.
Group B Streptococcus and E. coli LPS‐induced NO‐dependent hyporesponsiveness to noradrenaline in isolated intrapulmonary arteries of neonatal piglets
Authors: Villamor Eduardo , Pérez‐Vizcaíno Francisco , Ruiz Teresa , Leza Juan C. , Moro Manuel , Tamargo Juan
Published: July 2012

The effects of endotoxin (E. coli lipopolysaccharide, LPS) and heat inactivated group B Streptococcus (GBS) were studied on the contractile responses to noradrenaline (NA) in isolated pulmonary arteries and on the activity of the constitutive and inducible nitric oxide synthase (NOS) in lung fragments of neonatal piglets.

Short‐term (≤ 5 h) incubation with LPS (1 μg ml−1) or GBS (3 × 107 colonies forming units ml−1) did not modify the vascular responsiveness to NA (10−8 M‐10−4 m) in isolated intrapulmonary arteries. However, long‐term incubation (20 h) with LPS or GBS produced a significant reduction in the maximal contractile responses and shifted the concentration‐response curve for NA downwards.

Endothelium removal or the cyclo‐oxygenase inhibitor meclofenamate (10−5 m) did not affect the GBS‐ and LPS‐induced hyporesponsiveness to NA.

The presence of the nitric oxide (NO) precursor, l‐arginine (10−5 m), 30 min prior to the contractility challenge increased the LPS‐ and GBS‐induced pulmonary vascular hyporesponsiveness to NA. In contrast, the addition, prior to the challenge with NA, of the NOS inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME, 10−4m) or coincubation with dexamethasone (3 × 10−6m), a potent inhibitor of the induction of NOS, or with the protein synthesis inhibitor cycloheximide (10−5 m) completely restored the reactivity to NA in LPS‐ and GBS‐treated pulmonary arteries.

The incubation for 20 h of lung fragments with LPS and GBS produced a significant increase in the Ca2+‐independent (inducible) NOS activity determined by the conversion of radiolabelled l‐arginine to citrulline, but did not modify the constitutive NOS activity. This NOS induction was abolished by coincubation with dexamethasone (3 × 10−6 m).

These results demonstrated that prolonged incubation with GBS and LPS causes an induction of NOS activity which results in a reduced vascular responsiveness to NA in pulmonary arteries of neonatal piglets. Thus, induction of NOS seems to be responsible for the delayed pulmonary vascular hyporesponsiveness induced by GBS (a Gram‐positive) and E. coli (a Gram‐negative), the most common causal agents of neonatal sepsis.
Actions of general anaesthetics on a neuronal nicotinic acetylcholine receptor in isolated identified neurones of Lymnaea stagnalis
Authors: McKenzie D. , Franks N.P. , Lieb W.R.
Published: July 2012

Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were studied under two‐electrode voltage‐clamp. Neuronal nicotinic acetylcholine receptor currents were studied at low acetylcholine (ACh) concentrations (≤ 200 nM). At these levels, control currents were non‐desensitizing and proportional to the square of the ACh concentration.

IC50 concentrations were determined for the steady‐state inhibition of the ACh‐activated current by 31 general anaesthetics plus the non‐anaesthetic alcohol n‐tridecanol. The general anaesthetics included inhalational agents, n‐alcohols, n‐alkane‐(α,ω)‐diols, cycloalcohols and an n‐alkane.

Anaesthetic inhibition was independent of voltage and consistent with two anaesthetic‐binding sites on the receptor.

IC50 concentrations for inhibiting the neuronal nicotinic ACh receptor correlated well (r = 0.97) with EC50 concentrations for general anaesthesia. The maximum deviation from the line of identity was less than fourfold. The inhalational agents tended to be more potent as inhibitors of the ACh receptor than as general anaesthetics, while the alcohols and diols were less potent.

The inhibition of the ACh‐induced current by the homologous series of n‐alcohols exhibited a cutoff at the same position (just after dodecanol) as found for the induction of general anaesthesia in tadpoles.

Polarity profile maps of the anaesthetic‐binding sites on the neuronal nicotinic ACh receptor were calculated from IC50 concentrations for the homologous series of n‐alcohols and n‐alkane‐(α,ω)‐diols. They reveal amphiphilic sites with apolar regions capable of accommodating the hydrocarbon chains of n‐alcohols as large as decanol. A striking resemblance was found to profiles previously calculated from data for tadpole general anaesthesia.
Quinidine‐induced open channel block of K+ current in rat ventricle
Authors: Clark R.B. , Sanchez‐Chapula J. , Salinas‐Stefanon E. , Duff H.J. , Giles W.R.
Published: July 2012

The effects of quinidine on calcium‐independent outward K+ currents in rat ventricular myocytes were studied using whole‐cell patch clamp techniques.

Quinidine sulphate (6 μm) significantly prolonged repolarization of the ventricular action potential. This effect was larger during early repolarization (25% level) than at later times (90% level).

Quinidine reduced the amplitude of a transient outward current, and accelerated its rate of decay by approximately 4 fold at membrane potentials between 0 to +50mV. Quinidine also reduced the amplitude of a slowly inactivating, tetraethylammonium‐sensitive ‘pedestal’ component of the outward current.

The quinidine‐induced block of the transient outward current was dependent on time and membrane potential. Maximal block occurred with depolarizations of about 100 ms duration, and longer depolarizations (up to 1.5 s) produced little additional block. The membrane potential dependence of quinidine‐induced block was very similar to the membrane potential dependence of activation of the transient outward current. The membrane potential dependence of steady‐state inactivation of the transient outward current was not significantly affected by quinidine.

These results show that quinidine blocks outward K+ currents in rat ventricular cells. The time and potential dependence of this block suggests that quinidine blocks the transient outward K+ current by acting primarily on the open state of these channels.
Effect of nitric oxide on integrity, blood flow and cyclic GMP levels in the rat gastric mucosa: modulation by sialoadenectomy
Authors: Tripp M.A. , Tepperman B.L.
Published: July 2012

The effects of the nitrosothiol, S‐nitroso N‐acetylpenicillamine (SNAP) which liberates nitric oxide (NO), on ethanol‐mediated gastric damage, blood flow and cyclic GMP levels in siaoloadenectomized (SALX) rats have been investigated.

Intraluminal instillation of ethanol (5–50% w/v) dose‐dependently induced haemorrhagic damage and decreased NO synthase activity in the gastric mucosa. Both the extent of mucosal damage and inhibition of NO synthase activity were exacerbated in SALX rats.

Epidermal growth factor administration (5 and 10 μg kg−1, s.c.) reduced mucosal damage but did not restore NO synthase activity in ethanol‐treated SALX rats.

SNAP infusion (0.01‐1.0 μg kg−1 min−1, i.v.) attenuated haemorrhagic damage in ethanol‐treated rats. The reduction in mucosal damage was significantly greater in SALX rats.

SNAP administration also caused an increase in gastric mucosal blood flow and cyclic GMP levels in control rats and both responses were augmented in SALX animals.

These data suggest that SALX is associated with increases in mucosal susceptibility to ethanol‐mediated damage and reduces mucosal NO synthase activity. Epidermal growth factor does not appear to influence mucosal NO synthase in ethanol‐treated rats. Furthermore, SALX augments the responsiveness of the gastric mucosa to NO administration. Therefore, factors from the salivary glands influence gastric NO formation and mucosal responsiveness to a NO donor.
Pharmacological activity of the C‐terminal and N‐terminal domains of secretory leukoprotease inhibitor in vitro
Authors: Masuda Kenichi , Kamimura Takashi , Watanabe Kunihito , Suga Tetsuya , Kanesaki Masaharu , Takeuchi Akiko , Imaizumi Atsushi , Suzuki Yoji
Published: July 2012

In order to characterize the physiological functions of the domain structure of secretory leukoprotease inhibitor (SLPI), the biological capacities of half‐length SLPIs, (Serl‐Pro54)SLPI and (Asn55‐Ala107)SLPI, were investigated and compared with those of full‐length SLPI.

The activities of these inhibitors against several serine proteases were determined using synthetic chromogenic substrates. The inhibitory capacity of the C‐terminal domain, (Asn55‐Ala107)SLPI, was as strong as that of full‐length SLPI against human neutrophil elastase (NE), cathepsin G and chymotrypsin. It possessed less trypsin inhibitory activity than intact SLPI. For the N‐terminal domain of SLPI, (Serl‐Pro54)SLPI, no inhibitory activity could be detected against the serine proteases tested in this study.

The inhibitory activity of (Asn55‐Ala107)SLPI against the proteolysis of the natural substrates elastin and collagen by NE was comparable with that of full‐SLPI (elastin, IC50 = 907±31 nM for SLPI, 767±33nM for (Asn55‐Ala107)SLPI; collagen, IC50 = 862 ± 36 nM for SLPI, 727 ±47 nM for (Asn55‐Ala107)SLPI).

The binding affinities of full‐ and half‐length SLPIs for heparin were measured by affinity column chromatography. Full‐length SLPI showed high affinity for heparin while the binding capacities of both half‐length SLPIs were lower. (Concentration of NaCl for elution, 0.45 m for SLPI, 0.24 m for (Serl‐Pro54)SLPI, 0.27 m for (Asn55‐Ala107)SLPI).

The effects of full‐SLPI and (Asn55‐Ala107)SLPI on blood coagulation were measured using the activated partial thromboplastin time (APTT). Full‐length SLPI prolonged clotting time dose‐dependently (1.25, 2.5 and 5.0 um), whereas (Asn55‐Ala107)SLPI had no effect even at the highest concentration.

In conclusion, the C‐terminal domain of SLPI is a promising candidate for the treatment of inflammatory diseases in which participation of neutrophil proteases has been suggested.
A novel receptor for diadenosine polyphosphates coupled to calcium increase in rat midbrain synaptosomes
Authors: Pintor Jesús , Miras‐Portugal M. Teresa
Published: July 2012

Diadenosine polyphosphates, AP4A and Ap5A, as well as ATP, α,β‐MeATP and ADP‐β‐S, were able to elicit variable intrasynaptosomal Ca2+ increases in rat midbrain synaptic terminals. The origin of the Ca2+ increment was the extrasynaptosomal space since the elimination of extracellular Ca2+ abolished the effect of all the agonists.

The P2‐purinoceptor antagonist, suramin, did not affect the Ca2+–increase evoked by diadenosine polyphosphates but dramatically blocked the Ca2+ entry induced by ATP and its synthetic analogues.

The actions of Ap5A and ATP on the intrasynaptosomal Ca2+ increase did not cross‐desensitize.

Concentration‐response studies for diadenosine polyphosphates showed pD2 values of 54.5 ±4.2 μm and 55.6 ±3.8 μm for AP4A and Ap5A, respectively.

The entry of calcium induced by diadenosine polyphosphates could be separated into two components. The first represented a selective voltage‐independent Ca2+ entry; the second, a sustained phase which was voltage‐dependent.

Studies on the voltage‐dependent Ca2+–channels involved in the effects of the diadenosine polyphosphates, demonstrated that Ω‐conotoxin G‐VI‐A inhibited the sustained Ca2+–entry, suggesting the participation of an N‐type Ca2+–channel. This toxin was unable to abolish the initial cation entry induced by AP4A or Ap5A. Ω‐Agatoxin IV‐A, tetrodotoxin, or nifedipine did not inhibit the effects of the diadenosine polyphosphates.

The effect of ATP on Ca2+–entry was abolished by nifedipine and Ω‐conotoxin G‐VI‐A, suggesting the participation of L‐ and N‐type Ca2+–channels in the response to ATP. 8 These data suggest that AP4A, Ap5A and ATP activate the same intracellular Ca2+ signal through different receptors and different mechanisms. AP4A and Ap5A induce a more selective Ca2+–entry in a voltage‐independent process. This is the first time that a selective action of diadenosine polyphosphate through receptors other than P1 and P2‐purinoceptors has been described.
Mode of action of thrombin in the rabbit aorta
Authors: Godin Denis , Rioux Francis , Marceau François , Drapeau Guy
Published: July 2012

Thrombin is a vasoactive protease that elicits the contraction of the rabbit aorta by activating a G‐protein coupled receptor through cleavage of its N‐terminal extracellular domain. Synthetic peptides corresponding to the newly exposed N‐terminus, following thrombin cleavage, have been shown to reproduce some of the activities of thrombin in the rabbit aorta.

Intracellular pathways involved in the contractile response of the rabbit aorta to thrombin and synthetic peptides were examined by use of a series of inhibitors. A similar method was applied to characterize the mitogenic effect of thrombin on cultured smooth muscle cells (SMCs) derived from the same tissue.

Results from this study indicate that the contractile response of the rabbit aorta to thrombin is dependent on the activation of protein kinase C (PKC) and independent of extracellular calcium. The contractile response to thrombin can be fully reproduced by peptide agonists related to the N‐terminal receptor sequence. However, subtle differences seem to exist between the mechanism of the contractile effect of thrombin and of the synthetic peptides, as both PKC activation and extracellular calcium were found to participate in the contractile effect of the synthetic peptides.

In cultured SMCs, both thrombin and the synthetic peptides increased inositol phosphate turnover; however, only thrombin elicited a mitogenic effect, which occurs at thrombin concentrations well below those needed to increase inositol phosphate turnover significantly. Activation of a tyrosine kinase pathway is involved in the mitogenic effect of thrombin on aortic SMCs.

Altogether these results suggest the existence of subtle differences between the mode of action of thrombin and of synthetic peptides related to the N‐terminal thrombin receptor sequence, in the rabbit aorta.
Characterization of acute homologous desensitization of μ‐opioid receptor‐induced currents in locus coeruleus neurones
Authors: Osborne P.B. , Williams J.T.
Published: July 2012

Acute homologous desensitization of μ‐opioid receptor‐induced currents was pharmacologically characterized in locus coeruleus (LC) neurones by use of intracellular and whole cell recording in superfuised brain slices.

Following desensitization of opioid receptors by perfusion with a high concentration of [Met5] enkephalin (ME) for 5 min, there was a reduction in the maximum response and a rightward shift of the concentration ‐ response curves for ME, [D‐Ala2, N‐MePhe4, Gly ‐ ol] enkephalin (DAMGO) and nor‐morphine.

By simultaneously fitting the operational model to the paired pre‐ and post‐desensitization concentration‐response data for each agonist, estimates of the level of desensitization were obtained. The values obtained for the three agonists (between 88% and 96%) were similar and did not vary according to the efficacy of the agonist used.

Use of whole cell patch recording techniques caused a slow rundown in the amplitude of ME currents (approx. 40% reduction over 60 min) but did not greatly affect the expression of acute desensitization of opioid currents.

When included in the patch recording solution, the phosphatase inhibitors, microcystin (50 nM‐4 μm) and okadaic acid (1 μm) had no effect on the induction of desensitization or the normal ability of opioid or α2‐adrenoceptors to produce currents. Microcystin decreased the rate of recovery of the ME (300 nM) currents following desensitization; however, okadaic acid had little effect on the rate of recovery from desensitization.

Strong calcium buffering with BAPTA (10–20 mM) had no effect on desensitization or the recovery from desensitization.

These results suggest that acute homologous desensitization of μ‐opioid receptors in LC neurones entails a rapid loss of responsiveness that involves a majority of the receptor population. The mechanism by which desensitization is reversed may involve a non‐calcium‐dependent protein phosphatase but the processess that cause desensitization remain unclear.
Ca2+‐dependent and ‐independent mechanism of cyclic‐AMP reduction: mediation by bradykinin B2 receptors
Authors: Sipma Henk , den Hertog Adriaan , Nelemans Adriaan
Published: July 2012

Bradykinin caused a transient reduction of about 25% in the cyclic AMP level in forskolin prestimulated DDT1 MF‐2 smooth muscle cells (IC50: 36.4 ±4.9 nM) and a pronounced, sustained inhibition (40%) of the isoprenaline‐stimulated cyclic AMP level (IC50: 37.5 ±1.1 nM).

The Ca2+ ionophore, ionomycin, mimicked both the bradykinin‐induced transient reduction in the forskolin‐stimulated cyclic AMP level and the sustained reduction in the isoprenaline‐stimulated cyclic AMP level.

The Ca2+‐dependent effect on cyclic AMP induced by bradykinin was mediated solely by Ca2+ release from internal stores, since inhibition of Ca2+ entry with LaCl3 did not reduce the response to bradykinin.

The involvement of calmodulin‐dependent enzyme activities, protein kinase C or an inhibitory GTP binding protein in the bradykinin‐induced responses was excluded since a calmodulin inhibitor, calmidazolium, a PKC inhibitor, staurosporine and pertussis toxin, respectively did not affect the decline in the cyclic AMP level.

Bradykinin enhanced the rate of cyclic AMP breakdown in intact cells, which effect was not mimicked by ionomycin. This suggested a Ca2+‐independent activation of phosphodiesterase activity by bradykinin in DDT1 MF‐2 cells.

The bradykinin B1 receptor agonist, desArg9–bradykinin, did not affect cyclic AMP formation in isoprenaline prestimulated cells, while the bradykinin B2 receptor antagonists, Hoe 140 (D‐Arg[Hyp3, Thi5, D‐Tic7, Oic8]‐BK) and D‐Arg[Hyp3, Thi5–8, D‐Phe7]‐BK completely abolished the bradykinin response in both forskolin and isoprenaline prestimulated cells.

Bradykinin caused an increase in intracellular Ca2+, which was antagonized by the bradykinin B2 receptor antagonists, Hoe 140 and D‐Arg[Hyp3, Thi5,8, D‐Phe7]‐BK. The bradykinin B2 receptor agonist, desArg9–bradykinin, did not evoke a rise in cytoplasmic Ca2+.

It is concluded, that stimulation of bradykinin B2 receptors causes a reduction in cellular cyclic AMP in DDT1 MF‐2 cells. This decline in cyclic AMP is partly mediated by a Ca2+/calmodulin independent activation of phosphodiesterase activity. The increase in [Ca2+]i mediated by bradykinin B2 receptors inhibited forskolin‐ and isoprenaline‐activated adenylyl cyclase differently, most likely by interfering with different components of the adenylyl cyclase signalling pathway.
A role for mast cells in adenosine A3 receptor‐mediated hypotension in the rat
Authors: Hannon J.P. , Pfannkuche H.J. , Fozard J.R.
Published: July 2012

The adenosine A3 receptor agonist, N6–2‐(4‐aminophenyl)ethyladenosine (APNEA) induces hypotension in the anaesthetized rat. The present experiments were carried out to explore the role of mast cells in the response.

Intravenous injection of APNEA (1–30 μg kg−1 to rats in which the A3 receptor‐mediated response had been isolated by pretreatment with 8‐(p‐sulphophenyl) theophylline (8‐SPT)), induced dose‐related falls in blood pressure accompanied at higher doses by small falls in heart rate. Responses to the mast cell degranulating agent, compound 48/80 (10–300 μg kg−1, i.v.) were qualitatively similar to those to APNEA.

Pretreatment with sodium cromoglycate (0.25‐20 mg kg−1, i.v.) induced dose‐related, although incomplete, blockade of the hypotensive responses to APNEA. At 20 mg kg−1, sodium cromoglycate also inhibited the cardiovascular response to compound 48/80 but had no effects on those to the selective A1 receptor agonist, N6–cyclopentyladenosine (CPA) or the selective A2A receptor agonist, 2‐[p‐(2‐carboxyethyl)phenylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680). Lodoxamide (0.01–20 mg kg−1) also blocked selectively but incompletely the response to APNEA.

The cardiovascular responses to compound 48/80 (10–300 μg kg−1, i.v.) were markedly suppressed in animals which had received repeated doses of the compound by the intraperitoneal route. Similarly APNEA was essentially devoid of cardiovascular activity in such preparations. In contrast, responses to CPA were similar in animals treated repeatedly with compound 48/80 to those obtained in control animals.

Plasma and serum histamine concentrations were markedly increased associated with the pronounced hypotensive responses induced by intravenous injections of APNEA (30 or 100 μg kg−1) in the presence of 8‐SPT, or compound 48/80 (300 μg kg−1).

Taken together the data implicate the mast cell in a key role in adenosine A3 receptor‐mediated hypotension in the anaesthetized rat.
Potentiation, activation and blockade of GABAA receptors of clonal murine hypothalamic GT1‐7 neurones by propofol
Authors: Adodra Snehal , Hales Tim G.
Published: July 2012

The actions of GABA and the intravenous general anaesthetic propofol (2,6‐düsopropylphenol) on GABAA receptors of self‐replicating GT1‐7 hypothalamic neurones were investigated by the patch clamp technique.

GABA (1 μm‐1 mM) dose‐dependently activated inward currents with an EC50 of 27 μm, recorded from whole cells voltage‐clamped at —60 mV. GABA (100 μm)‐activated currents reversed at the Cl− equilibrium potential.

Propofol (0.1 −100 μm) dose‐dependently potentiated GABA (100 μm)‐evoked currents with an EC50 of 5 μm.

In the absence of GABA, propofol (10 μm‐1 mM) activated small inward currents with a reversal potential similar to the CI− equilibrium potential. The peak current amplitudes activated by propofol were only 31% of those activated by GABA in the same cells.

Like GABA (100 μm)‐activated currents, propofol (100 μm)‐activated currents were inhibited by the GABAA receptor antagonist, bicuculline (10 μm) and were abolished by Zn2+ (100 μm).

Propofol (10, 30 and 100 μm) dose‐dependently activated currents in the absence of GABA. However, the peak amplitude of currents activated by propofol declined with concentrations > 100 μm. The cessation of application of a high dose of propofol (1 mM) was associated with a current ‘surge’.

The surge current, seen after application of propofol (1 mM), had a reversal potential similar to the CI− equilibrium potential. The ratio between peak current amplitude in the presence of propofol (1 mM) and surge current amplitude after propofol application, were not dependent on holding potential. Thus, it is unlikely that the surge current represents reversal of a voltage‐dependent block of GABAA receptors by propofol.

The amplitude of the surge current exceeded the amplitude of the initial propofol (1 mM)‐evoked current following brief applications, but declined after prolonged applications of the drug.

The observed modulatory actions of propofol may be due to separate potentiation, activation and inhibitory sites for this anaesthetic agent on GT1‐7 cell GABAA receptors.
Application of a model to explore interspecies differences in acetylcholine M‐receptor‐stimulated gastric acid secretion
Authors: Welsh Nicola J. , Shankley Nigel P. , Black James W.
Published: July 2012

Concentration‐effect curves were obtained, in the absence and presence of histamineH2‐receptor blockade, to 5‐methylfurmethide (5‐MeF) and McN‐A 343, high efficacy and low efficacy acetylcholine (ACh) M‐receptor agonists, respectively, in isolated stomach preparations from the mouse and immature rat and guinea‐pig.

In the immature guinea‐pig assay, the responses to 5‐MeF and McN‐A 343 were abolished by histamineH2‐receptor blockade suggesting that the responses were totally dependent upon gastric mucosal histamine. However, in the mouse and immature rat assays, although the histamineH2‐receptor antagonists produced small but significant rightward shifts and, in some cases, depression of the maximum of the agonist concentration‐effect curves, a significant secretory response remained, presumed to be due to direct stimulation of oxyntic cells.

Previously, by assuming that the histamineH2‐receptor blockade alters the mode of agonist‐stimulated acid secretion from mainly an indirect action mediated by histamine release to direct stimulation of the oxyntic cell, we applied an operational model of agonism to similar data obtained in the mouse preparation. In that study we were able to account for the behaviour of 5‐MeF and McN‐A 343 by assuming that the agonists expressed 6 fold higher efficacy, τ in the operational model of agonism, at ACh M‐receptors on the histamine‐releasing cells than on the oxyntic cells. In this study it was possible to account for the variation in the behaviour of the agonists both between and within assays by simply varying the efficacy expressed by the agonists at each of the cells in the model. The efficacy variation could be due to receptor concentration variation.

The data and analysis are discussed in terms of contemporary models for the role of histamine in the regulation of gastric acid secretion.
Accumulation of endogenous inhibitors for nitric oxide synthesis and decreased content of L‐arginine in regenerated endothelial cells
Authors: Azuma Hiroshi , Sato Jun , Hamasaki Hidehisa , Sugimoto Akiko , Isotani Eiji , Obayashi Satoshi
Published: July 2012

We examined regeneration of endothelial cells (ECs), neointima formation, decreased endothelium‐dependent relaxation (EDR) and changes in the contents of L‐arginine, NG–monomethyl‐L‐arginine (l‐NMMA), asymmetrical NG, NG–dimethylarginine (ADMA) and symmetrical NG, NG–dimethylarginine (SDMA) in the regnerated ECs, 6 weeks after balloon denudation of the rabbit carotid artery.

Regeneration of ECs was completed in 6 weeks and a significant neointima formation accompanied by the decreased EDR was observed.

L‐NMMA and ADMA contents in the regenerated ECs (23.5 ± 4.3 and 21.2 ± 2.0 pmol mg−1 DNA, respectively) were significantly (P<0.05 and P<0.01) higher than those in the control ECs (8.8 ± 3.0 and 7.4plusmn;1.9 pmol mg−1 DNA, respectively), whereas L‐arginine was significantly (P< 0.005) decreased in the regenerated ECs (31,470±1,050 pmol mg−1 DNA) as compared to that in the control ECs (47,870±1,890 pmol mg−1 DNA). SDMA content was below the assay limits.

L‐NMMA and ADMA, but not SDMA, inhibited the EDR induced by acetylcholine in a concentration‐dependent manner. The inhibition with L‐NMMA and ADMA was prevented by an addition of L‐arginine, but not by D‐arginine.

These results suggest that the accumulation of endogenous inhibitors for nitric oxide synthesis and decreased L‐arginine content are associated with decreased NO production/release from regenerated ECs and neointima formation.
Modulation of excitatory amino acid responses by tachykinins and selective tachykinin receptor agonists in the rat spinal cord
Authors: Cumberbatch Michael J. , Chizh Boris A. , MaxHeadley P.
Published: July 2012

The effects of tachykinins and agonists selective for the three subtypes of neurokinin (NK) receptor have been tested on spinal neuronal responses both to the excitatory amino acids (EAAs) NMDA, AMPA and kainate, and to noxious heat stimuli. The agonists were applied by microiontophoresis in in vivo experiments in α‐chloralose‐anaesthetized, spinalized rats.

The NK1selective agonist, GR 73632, enhanced responses to all three EAAs similarly, whilst the NK2‐selective agonist, GR64349, reduced responses to AMPA and kainate without affecting those to NMDA, and the NK3 selective agonist, senktide, enhanced responses to AMPA and kainate.

The endogenous ligands substance P (SP) and neurokinin A (NKA) both enhanced responses to NMDA with little effect on responses to kainate, whereas neurokinin B (NKB) selectively enhanced responses to kainate without affecting those to NMDA.

The effects of GR73632 on EAA responses showed some differences between the dorsal and ventral horn, with more selectivity towards enhancement of NMDA responses in the ventral horn, but a smaller maximum effect.

Background activity was significantly enhanced by GR73632, GR64349, SP and NKA but not by senktide or NKB. GR73632 had the greatest effect on background firing, but this action was variable between cells and was related both to the location within the spinal cord and to the degree of spontaneous activity prior to GR73632 administration.

Responses to noxious heat were enhanced consistently only by NKA.

These data show that selective agonists for the tachykinin receptors are capable of modulating EAA responses differentially. SP, NKA and NKB appear to act via more than one receptor type when modulating EAA responses in vivo. This indicates that NK‐EAA interactions can be more specific than suggested hitherto, with the combined actions at NK1 and NK2 receptors biasing EAA responsiveness towards NMDA receptor mediated functions, whereas NK3 receptor activation would have the opposite effect. The physiological role of such interactions is likely to be complex.