bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of bronchoconstriction by pituitary adenylate cyclase activating polypeptide (PACAP 1–27) in guinea‐pigs in vivo
Authors: Lindén Anders , Yoshihara Shigemi , Chan Brendan , Nadel Jay A.
Published: July 2012

We studied the inhibitory effect of pituitary adenylate cyclase activating polypeptide (PACAP 1–27) on the increase in total pulmonary resistance (RL) caused either by allergen or histamine in anaesthetized, ventilated guinea‐pigs.

PACAP 1–27 given via i.v. infusion (0.045‐4.5 nmol kg−1 min−1) dose‐dependently reduced the increase in RL caused by inhaled ovalbumin and histamine. At the highest dose, PACAP 1–27 prevented the increase in RL caused by ovalbumin and histamine completely. Infusion of PACAP 1–27 and the β2‐adrenoceptor agonist, salbutamol (0.045‐4.5 nmol kg‐1 min−1) inhibited the increase in RL similarly, but salbutamol increased the heart rate more than PACAP 1–27.

PACAP 1–27 and salbutamol given via inhaled aerosol (0.1 mM, 20 breaths) significantly reduced the increase in RL caused by histamine infused i.v., whereas aerosolised sterile saline did not. Both PACAP 1–27 and salbutamol caused bronchodilator effects within 1 min of drug inhalation and these effects remained throughout the 20 min of study.

Because PACAP 1–27 produced significant bronchodilatation and rapid onset of sustained action in vivo and without pronounced cardiovascular side effects, we conclude that this peptide may have therapeutic potential as a bronchodilator.
Attenuation of adrenomedullin‐induced renal vasodilatation by NG‐nitro L‐arginine but not glibenclamide
Authors: Miura Katsuyuki , Ebara Tsuneyuki , Okumura Michiaki , Matsuura Takeshi , Kim Shokei , Yukimura Tokihito , Iwao Hiroshi
Published: July 2012

The present study was conducted in order to elucidate the in vivo contribution of nitric oxide (NO) and the glibenclamide‐sensitive potassium channel in the renal action of adrenomedullin in anaesthetized dogs.

Intrarenal arterial infusion of adrenomedullin (20 ng kg−1 min−1) elicited a pronounced increase in renal blood flow with no changes in systemic blood pressure. The renal vasodilator action of adrenomedullin was markedly attenuated by pretreatment with NG–nitro L‐arginine (L‐NOARG), but this was reversed by continuous infusion of L‐arginine.

Pretreatment with glibenclamide almost completely blocked the renal vasodilatation induced by lemakalim, but had no effect on the renal vasodilator and diuretic action of adrenomedullin.

Intrarenal arterial infusion of adrenomedullin induced diuresis and natriuresis. Diuretic and natriuretic action of adrenomedullin was also attenuated by L‐NOARG. L‐Arginine partly reversed the effect of L‐NOARG and adrenomedullin‐induced diuresis and natriuresis.

These data indicate that the in vivo renal vasodilator action of adrenomedullin is mediated by the release of NO. The glibenclamide‐sensitive potassium channel is not involved in the renal action of adrenomedullin, at least, not in anaesthetized dogs. Since the inhibition of L‐NOARG of adrenomedullin‐induced diuresis occurred concomitantly with the attenuation of the renal vasodilator action of adrenomedullin, direct involvement of NO in adrenomedullin‐induced diuresis remains to be established.
Endogenous modulation of excitatory amino acid responsiveness by tachykinin NK1 and NK2 receptors in the rat spinal cord
Authors: Chizh Boris A. , Cumberbatch Michael J. , Birch Philip J. , MaxHeadley P.
Published: July 2012

The effects of selective tachykinin (neurokinin, NK) NK1 and NK2 receptor antagonists have been examined on spinal neurones in α‐chloralose anaesthetized, spinalized rats. They were tested for effects on responses both to excitatory amino acids (EAA) and to noxious heat stimuli. They were also tested for their ability to reverse the actions of selective NK agonists.

The NK1selective antagonists GR82334 (peptide) and CP‐99,994 (non‐peptide), when applied by microiontophoresis, both reduced responses to kainate >AMPA> NMDA. Intravenous CP‐99,994 (3 mg kg−1) also reduced responses to kainate but had inconsistent effects on nociceptive responses.

GR82334, applied microiontophoretically, reduced the enhancement by the selective NK1 agonist, GR73632 of both responses to EAAs and background activity. Systemic CP‐99,994 (≤10 mg kg−1) failed to reverse the effects of GR73632.

The selective peptide NK2 antagonist, GR103537, had no consistent effects on responses to EAAs when applied by iontophoresis. In contrast, the non‐peptide NK2 antagonist, GR159897, administered systemically (0.5‐2 mg kg−1, i.v.) enhanced responses to kainate (but not NMDA); responses to noxious heat were enhanced only weakly.

Iontophoretically‐administered GR 103537 attenuated the effects of the NK2 agonist GR64349, which selectively reduced responses to kainate compared to those to NMDA. Systemically administered GR159897 (≤2 mg kg−1, i.v.) caused little antagonism of the effects of GR64349.

The data indicate that under these conditions the non‐peptide antagonists are not reliable at reversing the actions of selective NK agonists.

These results suggest that there is a tonic release of endogenous tachykinins that can modulate glutamatergic neurotransmission in the spinal cord. They provide further support for the hypothesis that release of endogenous NKs acting on NK1 and NK2 receptors can promote NMDA receptor mediated glutamatergic transmission.
[3H]‐lifarizine, a high affinity probe for inactivated sodium channels
Authors: MacKinnon A.C. , Wyatt K.M. , McGivern J.G. , Sheridan R.D. , Brown C.M.
Published: July 2012

[3H]‐lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (Kd= 10.7 ± 2.9 nM; Bmax = 5.10±1.43 pmol mg−1 protein).

The binding of [3H]‐lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (α‐scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 μm had no effect on [3H]‐saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [3H]‐batrachotoxinin‐A 20‐α‐benzoate (BTX) binding with moderate affinity (pIC50 7.31±0.24) indicating an interaction with toxin site 2. However, lifarizine accelerated the dissociation of [3H]‐BTX and decreased both the affinity and density of sites labelled by [3H]‐BTX, suggesting an allosteric interaction with toxin site 2.

The binding of [3H]‐lifarizine was voltage‐sensitive, binding to membranes with higher affinity than to synaptosomes (pIC50 for cold lifarizine = 7.99± 0.09 in membranes and 6.68 ± 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KC1 increased the affinity of lifarizine almost 10 fold (pIC50 = 7.86± 0.25). This suggests that lifarizine binds selectively to inactivated sodium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation.

There was negligible [3H]‐lifarizine and [3H]‐BTX binding to solubilized sodium channels, although [3H]‐STX binding was retained under these conditions.

The potencies of a series of compounds in displacing [3H]‐lifarizine from rat cerebrocortical membranes correlated well with their affinities for inactivated sodium channels estimated from whole‐cell voltage clamp studies in the mouse neuroblastoma cell line, N1E‐115 (r = 0.96).

These results show that [3H]‐lifarizine is a high affinity ligand for neuronal sodium channels which potently and selectively labels a site, allosterically linked to toxin binding site 2, associated with inactivated sodium channels.
Pharmacological profile of the novel P2T‐purinoceptor antagonist, FPL 67085 in vitro and in the anaesthetized rat in vivo
Authors: Humphries R.G. , Tomlinson W. , Clegg J.A. , Ingall A.H. , Kindon N.D. , Leff P.
Published: July 2012

The role of endogenous ADP in platelet aggregation in vivo remains unclear due to the lack of suitable P2T‐antagonist probes. This paper describes the potency, selectivity and specificity of the novel P2T‐purinoceptor antagonist, FPL 67085 (2‐propylthio‐D‐β,γ‐dichloromethylene ATP) both in vitro and in the anaesthetized rat in vivo.

FPL 67085 (3–30 nM) produced concentration‐dependent rightward displacement of the concentration‐effect (E/[A]) curve for ADP‐induced aggregation of human washed platelets with no effect on ADP‐independent aggregation at ≤ 10 μm.

Logistic fitting of ADP E/[A] data indicated that the antagonist effect of FPL 67085 did not consistently accord with simple competition: in some preparations depression of the asymptote was seen. Schild analysis of data combined from all preparations, regardless of the antagonist profile observed, gave an apparent pKB of 8.9 (slope parameter 0.90).

The potency of FPL 67085 was unaffected by the P1purinoceptor antagonist, 8‐sulphophenyltheo‐phylline, was similar (IC50 0.6‐3.8 nM) in human and rat washed platelets or whole blood and, in rat blood, did not change following 2–30 min incubation at 37°C.

FPL 67085 was a weak (pA50∼4.2) partial agonist in tissues containing P2x‐ or P2y‐purinoceptors, indicating some 30,000 fold selectivity for the P2T‐subtype.

In anaesthetized rats, intravenous infusion of FPL 67085 produced rapidly‐reversible, dose‐related inhibition of ADP‐induced platelet aggregation measured ex vivo (ID50 1.3μg kg−1 min−1) with no significant effect on haemodynamics or circulating cell counts.

Thus, FPL 67085 is a potent, specific and selective inhibitor of ADP‐induced platelet aggregation both in vitro and in vivo. As such, it represents a novel pharmacological tool to define the role of endogenous ADP in thrombosis and the potential of P2T‐purinoceptor antagonists as a novel class of infusible anti‐thrombotic agents for acute use in man.
Involvement of endothelin in the pressor response following injection of NMDA to the periaqueductal gray area of rats
Authors: D'Amico Michele , Warner Timothy D.
Published: July 2012

Microinjection of N‐methyl‐D‐aspartate (NMDA) (0.068 to 6.8 nmol) into the periaqueductal gray area (PAG) of anaesthetized rats caused dose‐dependent increases in blood pressure. Preinjection (10 min before) of FR 139317 (an ETA receptor selective antagonist; 5 nmol) or SB 209670 (an ETa/ETb receptor non‐selective antagonist; 5 nmol) to the PAG reduced the pressor response to NMDA whereas BQ‐788 (an ETB receptor selective antagonist; 5 nmol) did not affect the NMDA‐induced hypertension. Pretreatment with DL‐2‐amino‐5‐phosphono valeric acid (2‐APV) (an NMDA receptor selective antagonist, 5 nmol) also abolished the pressor response induced by NMDA. Dose‐dependent increases in blood pressure induced by injection of angiotensin II (0.1–10 nmol) to the PAG were unaffected by FR 139317 or SB 209670. Thus, our data indicate that endogenous ET‐1, via an action on ETA receptors, contributes to the pressor effects of NMDA within the brain.
Blockade of acetylcholine release at the motor endplate by a polypeptide from the venom of Phoneutria nigriventer
Authors: Souccar Caden , Gonçalo Maria do Carmo , Lapa Antonio José , Troncone Lanfranco Ranieri Paolo , Lebrun Ivo , Magnoli Fabio
Published: July 2012

The mechanisms underlying the muscle relaxation effect of a fraction (PF3) isolated from the Phoneutria nigriventer spider venom were assessed on mouse diaphragm and chick biventer cervicis muscle preparations.

PF3 (0.25‐4 μg ml−1) produced a concentration‐dependent blockade of the nerve‐elicited muscle twitch of the mouse diaphragm (IC50=0.8 μg ml−1) without affecting the directly induced muscle twitch. In similar preparations, the crude venom (1–10 μg ml−1) produced muscle contracture and blocked both the direct and indirectly induced muscle twitches.

In the chick biventer cervicis muscle, PF3 (1–5 μg ml−1) blocked the nerve stimulated muscle twitch (IC50=1.26 μg ml−1), but did not alter the postjunctional response to exogenous acetylcholine (ACh, 10 μm‐10mm).

PF3 (2–8 μg ml−1) reduced the frequency of miniature endplate potentials (m.e.p.ps) recorded intracellularly from the mouse diaphragm muscle fibres by 58 to 64%, and diminished the amplitude of m.e.p.ps by 20 to 40% of control. The relationship between log m.e.p.p. frequency and log [Ca2+]o was shifted rightwards in the presence of 4 μg ml−1 PF3.

Raising the frequency of m.e.p.ps with high K+ medium or theophylline (3 mm) did not prevent the toxin‐induced depression of spontaneous ACh release.

The quantal content of e.p.ps (m), determined in cut‐diaphragm muscle fibres, was reduced by 53% and 77% of control by 1 and 4 μg ml−1 PF3, respectively. At 1 μg ml−1 the toxin shifted the relationship between log m and log [Ca2+]o towards higher values without apparent change of the slope.

E.p.p. trains elicited at 10 to 50 Hz in the presence of PF3 (1 μg ml−1) exhibited irregular amplitudes and facilitation related to the frequency of nerve stimulation.

It is concluded that PF3 blocks neuromuscular transmission by acting prejunctionally and reducing the nerve‐evoked transmitter release. The effect was related to a diminished Ca2+ entry into the nerve terminal associated with inhibition of exocytosis.
Selective blockade of M2 and M3 muscarinic receptors by hexahydrobenzyl‐ fourdapine and a comparison with zamifenacin
Authors: Barlow R.B. , Bond Susan M. , Branthwaite A.G. , Jackson O. , McQueen D.S. , Smith Kirsten M. , Smith Paula J.
Published: July 2012

4‐Diphenylacetoxy‐N‐cyclohexylmethyl‐piperidine HC1 (hexahydro‐benz‐4DAP) is more active as an antagonist of carbachol at receptors in guinea‐pig isolated ileum, log K (pA2) = 6.64±0.14 (s.e. 7 results), than at receptors in guinea‐pig isolated atria, log K= 5.43 ±0.14 (7). In the presence of neostigmine bromide (0.2 μm) the value for atria was 5.62 ±0.19 (4), so the lower activity on atria cannot be attributed to hydrolysis of the compound by cholinesterases present in this tissue.

The limit of solubility of the free base in Krebs solution (pH 7.6) is about 50 μm for both hexahydro‐benz‐4DAP and benzyl‐fourdapine (benz‐4DAP).

In experiments on guinea‐pig isolated ileum with hexahydro‐benz‐4DAP given together with 4‐DAMP methobromide, the combined dose‐ratio was consistent with competition: similar results were obtained with benz‐4DAP.

In rats anaesthetized with pentobarbitone, hexahydro‐benz‐4DAP antagonized the effects of bethanechol on blood‐pressure in doses that had little effect on heart rate or airflow. There was a limit to the effect which could be obtained, however, and the slopes of the Schild plots were less than one. The effects on rat blood‐pressure had a half‐life of at least 30 min.

In similar experiments with zamifenacin the slopes of the Schild plots were close to 1 and the compound was 10 to 20 times as active on blood‐pressure at it was on heart‐rate.

The limited solubility of the base probably accounts for the flat Schild plots obtained with hexahydro‐benz‐4DAP, which had about 10 fold selectivity for effects on blood‐pressure and was more active than expected from tests on isolated ileum.
Cellular localization of the inhibitory action of relaxin against uterine spasm
Authors: Hughes Sarah J. , Hollingsworth Michael
Published: July 2012

The aim of this study was to determine whether the site of action of relaxin as a relaxant of rat myometrium is at the cell membrane or at an intracellular site. Therefore, the potency of relaxin was determined against spasms reliant predominantly upon either extracellular Ca2+ or intracellular Ca2+. Uterine spasms dependent upon extracellular Ca2+ were elicited by (i) oxytocin (0.2 nM) (ii) Bay K 8644 (1 μm) in 10 mM K+‐rich PSS and (iii) KC1 (80 mm). Uterine spasm dependent upon intracellular Ca2+ was elicited by oxytocin (20 nM) in the presence of nifedipine (500 nM). The effects of relaxin against these spasmogens were compared with those of levcromakalim, nifedipine and salbutamol.

Relaxin (0.2–6.3 nM), levcromakalim (25–800 nM), salbutamol (1–63 nM) and nifedipine (1–250 nM) caused concentration‐dependent inhibition of the spasm evoked by oxytocin (0.2 nM) and relaxin was the most potent relaxant.

Relaxin and nifedipine were slightly less potent against the spasm induced by Bay K 8644 (1 μm) than against spasm induced by oxytocin (0.2 nM) (15 fold and 13 fold respectively). Levcromakalim and salbutamol were equipotent against the spasm evoked by Bay K 8644 (1 μm) and that evoked by oxytocin (0.2 nM).

Relaxin induced only 47 ± 7% inhibition of the KC1 (80 mm)‐evoked spasm at a concentration of 0.8 μm. Levcromakalim was much less potent (427 fold) against the spasm evoked by KC1 (80 mm) than against the spasm evoked by oxytocin (0.2 nM). The potency of salbutamol against the spasm evoked by KC1 (80 mm) was modestly reduced (14 fold) compared to that against the spasm evoked by oxytocin (0.2 nM). The potency of nifedipine against the KC1 (80 mm)‐evoked spasm was not different from that against the oxytocin (0.2 nM)‐evoked spasm.

The potencies of relaxin and levcromakalim against the spasm evoked by oxytocin (20 nM) + nifedipine (500 nM) were greatly reduced (74 fold and 234 fold respectively) compared to their potencies against the spasm evoked by oxytocin (0.2 nM). The potency of salbutamol against these two spasmogens was not different.

Relaxin was much less potent against the spasm dependent upon intracellular Ca2+ (that induced by oxytocin (20 nM) + nifedipine (500 nM)) than against the spasms dependent upon extracellular Ca2+, those induced by oxytocin (0.2 nM) and Bay K 8644 (1 μm). In this regard, relaxin resembled levcromakalim and nifedipine rather than salbutamol. Therefore, the major site of action of relaxin appears to be located at the plasma membrane rather than at an intracellular level. The observation that relaxin was less effective against the KC1 (80 mm)‐induced spasm than against the oxytocin (0.2 nM)‐evoked spasm may indicate that relaxin has a minor action involving K+‐channel opening.

High concentrations of relaxin (up to 1 μm) induced significant inhibition of the spasm dependent upon intracellular Ca2+. Thus at high concentrations relaxin also appears to have an additional intracellular action.
Promotion by SR 48692 of gastric emptying and defaecation in rats suggesting a role of endogenous neurotensin
Authors: Croci Tiziano , Giudice Antonina , Manara Luciano , Gully Danielle , Fur Gérard Le
Published: July 2012

We investigated the influence of the nonpeptide neurotensin receptor antagonist, SR 48692, administered orally, on gastric emptying and on acute defaecation. SR 48692 dose‐dependently (ED50 ∼0.7 μg kg−1) increased gastric emptying of a food suspension, but it had no effect on that of a non‐caloric meal. SR 48692 also dose‐dependently promoted defaecation and increased faecal water content. We suggest that antagonism of endogenous neurotensin accounts for the gastric emptying and defaecation promoting action of SR 48692.
Biphasic inhibition of stimulated endogenous dopamine release by 7‐OH‐DPAT in slices of rat nucleus accumbens
Authors: Patel Jyoti , Trout Stephen J. , Palij Peter , Whelpton Robin , Kruk Zygmunt L.
Published: July 2012

Fast cyclic voltammetry was used to investigate the effect of 7‐OH‐DPAT (7‐hydroxy‐N,N‐di‐n‐propyl‐2‐aminotetralin), a putative D3 receptor agonist, on electrically stimulated endogenous dopamine release in slices of rat nucleus accumbens.

7‐OH‐DPAT inhibited single pulse stimulated dopamine release in a concentration‐dependent manner with a maximum inhibition of 95.5%. Analysis of concentration‐response curves to 7‐OH‐DPAT showed that they were biphasic, with the high affinity component contributing 18.0% to the total inhibition and the low affinity component 77.5%. 7‐OH‐DPAT exhibited a 560 fold selectivity between the high and low affinity components (0.015 nm compared to 8.4 nM).

Concentration‐response curves to the nonselective D2/D3 agonist, apomorphine, were monophasic. The maximum inhibition was 93.1% and the EC50 value 82 nM.

The selective D2 antagonist, haloperidol (30 nM), antagonized the low affinity component of the concentration‐response cuve to 7‐OH‐DPAT whilst the high affinity component was essentially unaffected. The pKB values calculated for the high and low affinity components were 7.89 and 9.45 respectively.

In conclusion, these results demonstrate that 7‐OH‐DPAT inhibits stimulated dopamine release by acting at two different sites. Furthermore, the results are consistent with the hypothesis that the high and low affinity components of the concentration‐response curve to 7‐OH‐DPAT may reflect activation of functional D3 and D2 release‐regulating autoreceptors respectively. However, the possibility that the biphasic nature of the curve may reflect different subtypes of the D2 receptor cannot be excluded.
Effect of methylguanidine on rat blood pressure: role of endothelial nitric oxide synthase
Authors: Sorrentino Raffaella , Pinto Aldo
Published: July 2012

The effect of acute i.v. administration of methylguanidine (MG) on mean arterial blood pressure (MABP) was investigated in anaesthetized male Wistar rats.

MG (1–30 mg kg−l i.v.) produced an increase in MABP in a dose‐dependent manner both in normal and in hexamethonium (5 mg kg−1, i.v)‐treated rats.

L‐Arginine (30 or 150 mg kg−1, i.v.), but not its enantiomer D‐arginine (30 or 150 mg kg−1, i.v.), reversed the effect of MG on MABP in both normal and hexamethonium‐treated rats.

L‐Arginine (150 mg kg−1, i.v.) administered 2min before MG (30 mg kg−1, i.v.) prevented the increase in MABP caused by MG in either normal or hexamethonium‐treated rats. This effect was not observed with D‐arginine (150 mg kg−1, i.v.).

Thus, the rise in MABP caused by MG in the anaesthetized rat is due to inhibition of endothelial NO‐synthase activity. We speculate that the rise in the plasma concentration of endogenous MG associated with uraemia may contribute to the hypertension seen in patients with chronic renal failure.
All‐or‐none augmentation of Ca2+ sensitivity in c‐toxin‐permeabilized single smooth muscle cells from guinea‐pig taenia caecum
Authors: Mita Mitsuo , Hashimoto Takao
Published: July 2012

Isolated smooth muscle cells from guinea‐pig taenia caecum were permeabilized by use of Staphylococcus aureus α‐toxin, and the sarcoplasmic reticulum Ca2+ store was depleted by exposure to 0.1 μM A23187.

Shortening of α‐toxin‐permeabilized single smooth muscle cells was induced by increasing free Ca2+ but was not induced by 0.2 μM free Ca2+.

Shortening of the permeabilized cells was caused by application of acetylcholine (ACh) with free Ca2+ concentration held at 0.2 μM. Permeabilized smooth muscle cells responded to 0.3 μM or 1 μM ACh with 0.2 μM Ca2+ with maximal shortening. The concentration‐response relationship to ACh had a very steep slope and the cell shortening appeared to be an all‐or‐none response rather than a graded response, as was the shortening of intact cells to ACh.

The shortening of permeabilized cells was also induced by application of guanosine 5′‐triphosphate (GTP) with 0.2 μM free Ca2+, showing an all‐or‐none response. The threshold concentration of GTP that induced an all‐or‐none response was between 10 μM and 30 μM.

These results suggest that Ca2+ sensitivity is augmented by stimulation of the muscarinic receptor or GTP‐binding protein(s) in an all‐or‐none manner. It seems probable that this contributes to the all‐or‐none response to ACh in intact smooth muscle cells.
Determination β‐adrenoceptor subtype on rat isolated ventricular myocytes by use of highly selective β‐antagonists
Authors: Kitagawa Yoshihiro , Adachi‐Akahane Satomi , Nagao Taku
Published: July 2012

The relative proportions of β1 and β2‐adrenoceptors were determined by radioligand binding studies in three different rat myocardial preparations: membranes prepared from rat ventricle (ventricular membranes), membranes prepared from rat isolated ventricular myocytes (myocyte membranes), and myocytes isolated from rat ventricle (myocytes).

Competition experiments using CGP 20712A or ICI 118,551 with [125I]‐iodocyanopindolol ([1251]‐ ICYP) revealed high‐ and low‐affinity binding sites in ventricular membranes. The concentration at which each β‐antagonist occupied 100% of its high‐affinity binding sites was 300 nM for CGP 20712A (β1‐adrenoceptor) and 50 μM for ICI 118,551 (β2‐adrenoceptor).

The density of high‐affinity (β1‐adrenoceptor) and low‐affinity (β2‐adrenoceptor) binding sites for CGP 20712A was measured by a saturation experiment using [125I]‐ICYP in the presence and absence of 300 nM CGP 20712A. In ventricular membranes, the proportions of high‐affinity and low‐affinity binding sites for CGP 20712A were 73% and 27%, respectively, whereas in myocyte membranes, the corresponding figures were 90% and 10%, respectively. The density of low‐affinity binding sites for CGP 20712A in ventricular membranes, defined as [125I]‐ICYP‐specific binding in the presence of 300 nM CGP 20712A, was decreased by addition of 50 nM ICI 118,551, whereas that in myocyte membranes was not affected.

In myocytes, specific binding of [125I]‐ICYP and [3H]‐CGP 12177 was not detected by saturation experiments performed in the presence of 300 nM CGP 20712A.

In myocytes, the activation of adenylate cyclase caused by β2‐adrenoceptors was not detected in the presence of 10 nM, 100 nM or 1000 nM CGP 20712A, which selectively antagonized β1‐adrenoceptors. Furthermore, the concentration‐response curve for isoprenaline‐stimulated cyclic AMP accumulation was not shifted by 10 nM or 100 nM ICI 118,551, which selectively antagonized β2‐adrenoceptors, but was shifted to the right by 1000 nM ICI 118,551.

These results indicate that β2‐adrenoceptors are not present on rat ventricular myocytes and that β2‐adrenoceptor stimulation does not cause any detectable production of cyclic AMP. We conclude that only β1‐adrenoceptors exist on rat ventricular myocytes.
Accelerated desensitization of nicotinic receptor channels and its dependence on extracellular calcium in isolated skeletal muscles of streptozotocin‐diabetic mice
Authors: Nojima Hiroshi , Tsuneki Hiroshi , Kimura Ikuko , Kimura Masayasu
Published: July 2012

To elucidate the influence of the diabetic state on desensitization of nicotinic acetylcholine (ACh) receptor channels, we investigated the time course of the decrease in amplitude of ACh potentials elicited by iontophoretic application to isolated diaphragm muscle of streptozotocin‐diabetic mice. We also investigated time‐ and extracellular Ca2+‐dependent changes in the channel opening frequency of ACh‐activated channel currents and the involvement of protein kinases by use of the cell‐attached patch clamp technique in single skeletal muscle cells.

When ACh potentials were evoked at 10 Hz, the decline in trains of ACh potentials was accelerated in the diabetic state.

The time‐dependent decrease in the channel opening frequency of diabetic muscle cells was greatly accelerated compared with normal cells in 2.5 mM Ca2+ medium.

This accelerated decrease in channel opening frequency was restored by pretreatment with a protein kinase C inhibitor, staurosporine (10 nM) but neither a protein kinase A inhibitor, H‐89 (3 μM) nor a calmodulin kinase II inhibitor, KN‐62 (5 μM) were able to restore the fall in opening frequency.

These results demonstrate that in the diabetic state the desensitization of nicotinic ACh receptor channels may be greatly accelerated by activating protein kinase C, which is caused by an increase in the amount of available intracellular Ca2+.
Characterization of the prostanoid receptors mediating constriction and relaxation of human isolated uterine artery
Authors: Baxter G.S. , Clayton J.K. , Coleman R.A. , Marshall K. , Sangha R. , Senior J.
Published: July 2012

This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating constriction and relaxation of human isolated uterine artery.

U‐46619 was a potent constrictor agonist on human uterine artery (EC50 [95% CL] = 3.5 [1.8‐6.7] μM). Prostaglandin E2 (PGE2α), PGF2, PGD2 and PGI2 only weakly constricted the uterine artery, being at least 100 times less potent than U‐46619. The PGE2 and PGI2 constrictor effects may be modified by the potent dilator effects of these compounds. A number of agonists which show selectivity for FP‐, DP‐ and EP‐receptors including ICI 81008, BW 245C, sulprostone, rioprostil and butaprost, failed to cause any constriction at concentrations up to 30 μM.

Constrictor responses induced by all agonists tested were reduced or abolished by the TP‐receptor blocking drugs, GR 32191 and EP 092. pA2 estimates for both antagonists versus U‐46619 were 8.50, values which are consistent with their affinities at TP‐receptors.

In preparations pre‐constricted with phenylephrine (1 μM) both PGI2 and PGE2 were potent relaxant agonists. The selective IP‐receptor agonists, cicaprost and iloprost, also dilated human uterine artery and were approximately 10 fold more potent than PGI2. The EP2‐receptor agonists, butaprost and rioprostil and the selective DP‐agonist, BW 245C, were at least 100 fold weaker than PGI2 and PGE2 suggesting that neither DP‐ nor EP2 receptors were involved.

We conclude that TP‐receptors mediate constriction, whereas IP‐ and possibly EP4‐receptors mediate relaxation of human uterine artery.
Inhibition of carrageenin‐induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2
Authors: Landucci Elen C.T. , Antunes Edson , Donato Jose L. , Faro Renato , Hyslop Stephen , Marangoni Sérgio , Oliveira Benedito , Cirino Giuseppe , de Nucci Gilberto
Published: July 2012

The effect of purified crotapotin, a non‐toxic non‐enzymatic chaperon protein normally complexed to a phospholipase A2 (PLA2) in South America rattlesnake venom, was studied in the acute inflammatory response induced by carrageenin (1 mg/paw), compound 48/80 (3 μg/paw) and 5‐hydroxytryptamine (5‐HT) (3 μg/paw) in the rat hind‐paw. The effects of crotapotin on platelet aggregation, mast cell degranulation and eicosanoid release from guinea‐pig isolated lung were also investigated.

Subplantar co‐injection of crotapotin (1 and 10 μg/paw) with carrageenin or injection of crotapotin (10 μg/paw) into the contralateral paw significantly inhibited the carrageenin‐induced oedema. This inhibition was also observed when crotapotin (10–30 μg/paw) was administered either intraperitoneally or orally. Subplantar injection of heated crotapotin (15 min at 60°C) failed to inhibit carrageenin‐induced oedema. Subplantar injection of crotapotin (10 μg/paw) also significantly inhibited the rat paw oedema induced by compound 48/80, but it did not affect 5‐HT‐induced oedema.

In adrenalectomized animals, subplantar injection of crotapotin markedly inhibited the oedema induced by carrageenin. The inhibitory effect of crotapotin was also observed in rats depleted of histamine and 5‐HT stores.

Crotapotin (30 μg/paw) had no effect on either the histamine release induced by compound 48/80 in vitro or on the platelet aggregation induced by both arachidonic acid (1 mm) and platelet activating factor (1 μm) in human platelet‐rich plasma. The platelet aggregation and thromboxane B2 (TXB2) release induced by thrombin (100 mu ml−1) in washed human platelets were also not affected by crotapotin. In addition, crotapotin (10 μg/paw) did not affect the release of 6‐oxo‐prostaglandin F1α and TXB2 induced by ovalbumin in sensitized guinea‐pig isolated lungs.

Our results indicate that the anti‐inflammatory activity of crotapotin is not due to endogenous corticosteroid release or inhibition of cyclo‐oxygenase activity. It is possible that crotapotin may interact with extracellular PLA2 generated during the inflammatory process thereby reducing its hydrolytic activity.
Regional haemodynamic effects of human and rat adrenomedullin in conscious rats
Authors: Gardiner S.M. , Kemp P.A. , March J.E. , Bennett T.
Published: July 2012

Male, Long Evans rats were chronically instrumented with pulsed Doppler flow probes and intravascular catheters to permit assessment of the regional haemodynamic responses to human and rat adrenomedullin, to compare the responses to human adrenomedullin to those of human α‐CGRP in the absence and presence of the CGRP1‐receptor antagonist, human α‐CGRP [8–37], and to determine the involvement of nitric oxide (NO)‐mediated mechanisms in the responses to human adrenomedullin, relative to human α‐CGRP.

Human and rat adrenomedullin (0.3, 1, and 3 nmol kg−1, i.v.) caused dose‐dependent hypotension and tachycardia, accompanied by increases in renal, mesenteric and hindquarters flows and vascular conductances. At the lowest dose only, the hypotensive and mesenteric vasodilator effects of rat adrenomedullin were significantly greater than those of human adrenomedullin.

Human α‐CGRP at a dose of 1 nmol kg−1 caused hypotension, tachycardia and increases in hindquarters flow and vascular conductance, but reductions in renal and mesenteric flows, and only transient vasodilatations in these vascular beds. These effects were substantially inhibited by human α‐CGRP [8–37] (100 nmol kg−1 min−1), but those of human adrenomedullin (1 nmol kg−1) were not; indeed, the mesenteric haemodynamic effects of the latter peptide were enhanced by the CGRP1‐receptor antagonist.

In the presence of the NO synthase inhibitor, NG‐nitro‐1‐arginine methyl ester (1‐NAME, 183 nmol kg−1 min−1), there was only a slight, but significant, inhibition of the hindquarters hyperaemic vasodilator effect of human adrenomedullin, but not that of human α‐CGRP.

These results indicate that the marked regional vasodilator effects of human (and rat) adrenomedullin are largely independent of NO and, in vivo, do not involve CGRP1‐receptors.
Different pressor and bronchoconstrictor properties of human big‐endothelin‐1, 2 (1–38) and 3 in ketamine/xylazine‐anaesthetized guinea‐pigs
Authors: Gratton Jean‐Philippe , Rae Giles A. , Claing Audrey , Télémaque Sabine , D'Orléans‐Juste Pedro
Published: July 2012

In the present study, the precursors of endothelin‐1, endothelin‐2 and endothelin‐3 were tested for their pressor and bronchoconstrictor properties in the anaesthetized guinea‐pig. In addition, the effects of big‐endothelin‐1 and endothelin‐1 were assessed under urethane or ketamine/xylazine anaesthesia.

When compared to ketamine/xylazine, urethane markedly depressed the pressor and bronchoconstrictor properties of endothelin‐1 and big‐endothelin‐1.

Under ketamine/xylazine anaesthesia, the three endothelins induced a biphasic increase of mean arterial blood pressure. In contrast, big‐endothelin‐1, as well as big‐endothelin‐2 (1–38), induced only sustained increase in blood pressure whereas big‐endothelin‐3 was inactive at doses up to 25 nmol kg−1.

Big‐endothelin‐1, but not big‐endothelin‐2, induced a significant increase in airway resistance. Yet, endothelin‐1, endothelin‐2 and endothelin‐3 were equipotent as bronchoconstrictor agents.

Big‐endothelin‐1, endothelin‐1 and endothelin‐2, but not big‐endothelin‐2, triggered a marked release of prostacyclin and thromboxane A2 from the guinea‐pig perfused lung.

Our results suggest the presence of a phosphoramidon‐sensitive endothelin‐converting enzyme (ECE) which is responsible for the conversion of big‐endothelin‐1 and big‐endothelin‐2 to their active moieties, endothelin‐1 and 2. However, the lack of bronchoconstrictor and eicosanoid‐releasing properties of big‐endothelin‐2, as opposed to endothelin‐2 or big‐endothelin‐1, suggests the presence of two distinct phosphoramidon‐sensitive ECEs in the guinea‐pig. The ECE responsible for the systemic conversion of big‐endothelins possesses the same affinity for big‐endothelin‐1 and 2 but not big‐endothelin‐3. In contrast, in the pulmonary vasculature is localized in the vicinity of the sites responsible for eicosanoid release, an ECE which converts more readily big‐endothelin‐1 than big‐endothelin‐2.
Involvement of NK2 receptors rather than NK1 receptors in bronchial hyperresponsiveness induced by allergic reaction in guinea‐pigs
Authors: Mizuguchi Masayuki , Fujimura Masaki , Amemiya Tokunao , Nishi Kouichi , Ohka Takio , Matsuda Tamotsu
Published: July 2012

In this study, the role of neuropeptides in antigen‐induced bronchoconstriction and bronchial responsiveness in guinea‐pigs was evaluated by use of phosphoramidon, the inhibitor of neutral endopeptidases (NEP), the NK1 receptor antagonist, FK888, and the dual NK1/NK2 receptor antagonist, FK224. The role of endogenous tachykinins in bronchial hyperresponsiveness induced by inhaled capsaicin was also observed with FK888 and FK224.

Allergic bronchoconstriction and bronchial responsiveness was evoked by inhalation of ovalbumin (OA), and increasing doses of methacholine were inhaled at 5‐min intervals for 30 min after OA challenge in passively sensitized and artificially ventilated guinea‐pigs. Animals were treated with a 30 s inhalation of phosphoramidon (10−3m) or saline 10 min before the OA challenge. FK888 (1.0 or 10 mg kg−1) or FK224 (1.0 or 10 mg kg−1) was administered intravenously 5 min before the OA challenge.

Treatment with phosphoramidon did not alter the increase in the lateral pressure at the tracheal tube (Pao) caused by OA inhalation or the increase in bronchial response to methacholine following the allergic reaction. Pretreatment with FK224 did not inhibit the increase in Pao after antigen provocation but did significantly inhibit antigen‐induced bronchial hyperresponsiveness in a dose‐dependent manner, while FK888 did not affect either allergic bronchoconstriction or post‐allergic bronchial hyperresponsiveness.

Histamine, 25, 50, 100 or 200 μ ml−1 was inhaled for 20 s at 5‐min intervals in non‐sensitized guinea‐pigs which were pretreated with inhalation of subthreshold dose of capsaicin (10−7 m). FK888 or FK224, each at a dose of 0.1 or 1.0 mg kg−1, or vehicle was given to guinea‐pigs intravenously 3 min before inhalation of capsaicin. The capsaicin inhalation significantly potentiated bronchial responsiveness to histamine, compared with control. The capsaicin‐induced bronchial hyperresponsiveness was completely blocked by FK224 in a dose‐dependent manner but not by FK888.

These results suggest that NK2 receptors rather than NK1 receptors may play an important role in bronchial hyperresponsiveness induced by antigen challenge as well as capsaicin while tachykinins do not play a primary role in the acute bronchospasm elicited by antigen challenge in passively sensitized guinea‐pigs.
Characterization of the endothelin receptor selective agonist, BQ3020 and antagonists BQ123, FR139317, BQ788, 50235, Ro462005 and bosentan in the heart
Authors: Peter Markus G. , Davenport Anthony P.
Published: July 2012

In this study we used ligand binding techniques to determine the affinity and selectivity of endothelin receptor agonists and antagonists in human left ventricle which expresses both ETA and ETB receptors, and compared these results with cardiovascular tissues from rat and porcine hearts.

The linear tripeptide antagonist, FR139317 competed for [125I]‐ET‐1 binding to human left ventricle with over 200,000 fold selectivity for the ETA receptor (KD ETA=1.20±0.28 nM, KDETB = 287±93 μm). The ETA‐selective non‐peptide antagonist, 50235, competed with lower affinity and selectivity (KDETA = 162±61 nM, KDETB= 171±42 μm) in this tissue. BQ123 and FR139317 also showed high selectivity (greater than 20,000 fold) and affinity in rat (BQ123: KDETA = 1.18±0.16 nM, KDETB = 1370±1150 μm; FR139317: KDETA = 2.28±0.30 nM, KDETB = 292±114 μm) and pig heart (BQ123: KDETA = 0.52±0.05 nM, KDETB = 70.4±4.0 μm; FR139317: KDETA = 2.17±0.51 nM, KDETB = 47.1±5.7 μm) (n≥3 individuals±s.e.mean).

Although BQ3020 competed with over 1000 fold selectivity for the ETB subtype in human heart KDETB = 1.38±0.72 nM, KDETA = 2.04±0.21 μm) the peptide inhibited only the binding of [125I]‐ET‐1 at concentrations greater than 100 nM in rat and porcine heart. This is in contrast to the data from the ETA‐selective antagonists which indicated the presence of ETB sites in these tissues from animal hearts.

The peptide antagonist, BQ788, had a low, micromolar affinity (KD = 1.98±0.13 μm) using human left ventricle and no significant selectivity for the human ETB‐subtype in this tissue.

The non‐peptide ET antagonists, Ro462005 (KD = 50.3±9.5 μm) and bosentan (Ro470203; KD = 77.9±7.9 nM) competed monophasically for [125I]‐ET‐1 binding sites in human left ventricle.

The results show that the ETA antagonists, BQ123 and FR139317, are highly selective for ETA receptors in all cardiac tissues tested, whereas BQ788 has a low affinity and no selectivity in this human tissue. Further we showed that there are species differences in the binding of BQ3020 to the ETB receptors in the hearts derived from human, rat and pig.
Electrophysiological effects of monoamine oxidase inhibition on rat midbrain dopaminergic neurones: an in vitro study
Authors: Mercuri Nicola B. , Bonci Antonello , Siniscalchi Antonio , Stefani Alessandro , Calabresi Paolo , Bernardi Giorgio
Published: July 2012

The effects of the inhibition of monoamine oxidase (MAO) type A and B have been evaluated on the spontaneous firing activity of the dopaminergic (principal) neurones of the rat midbrain intracellularly recorded from a slice preparation.

The non‐specific MAO inhibitor, pargyline, superfused at a concentration of 10–100 μm, decreased or abolished the spontaneous firing discharge of the principal neurones in the subtantia nigra pars compacta and ventral tegmental area. This effect had a slow onset and appeared to be sustained.

The administration of the dopamine D2/3 receptor antagonist, sulpiride (100–300 μm), antagonized the pargyline‐induced effect, while the superfusion of the dopamine D1 receptor antagonist, SCH 23390 (1–3 μm) did not counteract the induced inhibition of the firing rate.

The inhibitor for the MAO A, clorgyline (30–100 μm), reduced the firing rate of the dopaminergic neurones. A similar depressant effect was also observed when a MAO B inhibitor, deprenyl (30–100 μm), was applied. Lower concentrations of both drugs (300 nM‐10 μm) did not produce consistent effects on neuronal discharge.

Our data suggest that only the blockade of both types of MAO enzymes favours the inhibitory action of endogenous dopamine on somato‐dendritic D2/3 autoreceptors.
Comparative studies of the angiogenic activity of vasoactive intestinal peptide, endothelins‐1 and −3 and angiotensin II in a rat sponge model
Authors: Hu De‐En , Hiley C. Robin , Fan Tai‐Ping D.
Published: July 2012

The angiogenic activity of four vasoactive peptides with a range of vasodilator and vasoconstrictor properties, i.e. vasoactive intestinal peptide (VIP), endothelin‐1, endothelin‐3 and angiotensin II, were investigated in a rat sponge model. Neovascularization was assessed by the 133Xe clearance technique and confirmed by histological studies.

Daily doses of the vasodilator peptide, VIP (1000 pmol), caused intense neovascularization, but a lower dose (10 pmol) produced no apparent effect. However, the lower dose of VIP, when given with a subthreshold dose of interleukin‐1α (0.3 pmol), produced an angiogenic response similar to that seen with the higher dose of VIP. The neovascular response induced by co‐administration of VIP and interleukin‐1α was inhibited by simultaneous administration of 100 pmol VIP (10–28), a specific VIP receptor antagonist.

In contrast, daily doses of 10, 100 or 1000 pmol endothelin‐3 (a mixed vasoconstrictor and vasodilator with more marked vasodilator activity) or of 100 or 1000 pmol endothelin‐1 (also with mixed activity but with much more pronounced vasoconstrictor response) produced no apparent effect on sponge‐induced angiogenesis.

The vasoconstrictor peptide, angiotensin II, in daily doses of 1000 pmol, caused an intense neovascularization like VIP but lower doses of angiotensin II (10 or 100 pmol) produced no apparent effect. The lowest dose of angiotensin II (10 pmol) when administered with the subthreshold dose of interleukin‐1α (0.3 pmol) had no effect on the basal neovascular response in the sponges. The angiotensin II‐induced neovascular response was inhibited by co‐administration of 100 nmol of the specific AT1 receptor antagonist, losartan, but not by the AT2 receptor antagonist, PD 123319.

These data show that VIP and angiotensin II possess angiogenic activity. However, endothelin‐1 and endothelin‐3 had no activity at the doses used. Thus the angiogenic response is not related to local vasoconstriction or vasodilatation in the sponges. The blockade of VIP‐ and angiotensin II‐induced angiogenesis at the receptor level suggests that receptor modulation could provide a strategy for the management of angiogenic diseases.
The longitudinal muscle of rat ileum as a sensitive monoreceptor assay for bradykinin B1 receptors
Authors: Meini Stefania , Lecci Alessandro , Maggi Carlo Alberto
Published: July 2012

Various bradykinin derivatives, acting preferentially at B1 or B2 receptors, were tested in the isolated longitudinal smooth muscle of rat ileum. Experiments were carried out in the presence of chlorpheniramine and atropine (both 1 μm), guanethidine and indomethacin (both 3 μm) and of the peptidase inhibitors (captopril, bestatin and thiorphan, all 1 μm).

The rank order of potency was (pD2 values±s.e.mean, n = 5 in parentheses, at 5 h from set‐up): [des‐ Arg9]‐BK (8.27±0.11)≥ [des‐Arg10]‐kallidin (7.67±0.24) > bradykinin (6.69±0.25). The B2 receptor selective agonist, [Hyp3, Tyr(Me)8]‐BK, was approximately 10 fold less active than bradykinin. Contractile responses to all agonists increased with time. The maximal response to the B1 receptor agonist, [desArg9]‐BK at 5 h (94±2%) was significantly (P<0.05) greater than that measured at 2 h (74±2%).

The B2 receptor antagonist, D‐Arg[Hyp3, Thi5, D‐Tic7, Oic8]‐BK (Hoe 140, 0.1 μm) did not affect responses to the B1 receptor agonist [des‐Arg9‐BK (0.1 nM‐1 μm) nor those to the B2 receptor agonist, [Hyp3, Tyr(Me)8]‐BK (1 nM‐10 μm). In control experiments performed in the longitudinal smooth muscle of guinea‐pig ileum and rat isolated urinary bladder as bioassays for B2 receptors, the B2 receptor antagonist Hoe 140 (0.1 μm) antagonized bradykinin‐induced contractions.

In the rat isolated ileum the B1 receptor antagonist, D‐Arg[Hyp3, Thi5, D‐Tic7, Oic8, des‐Arg9]‐BK ([des‐Arg10]‐Hoe 140, 0.3–10 μm) competitively antagonized contractile responses to [des‐Arg9]‐BK with an estimated pKB of 6.74±0.08 (Schild plot slope with confidence limits 1.22, (0.70‐1.73) n = 13). In control experiments in the guinea‐pig isolated ileum and rat isolated urinary bladder, [des‐Arg10]‐Hoe 140 (1–10 μm) did not inhibit B2 receptor‐mediated contractile responses.

The putative B1 receptor antagonist, [Leu8, des‐Arg9]‐BK, behaved as a partial agonist when responses were determined 2 h from set‐up (pD2 6.43±0.21, n = 5; Emax 30% of that evoked by [des‐Arg9‐BK); at 5 h from set‐up it behaved as a full agonist (pD2 7.48±0.12, n = 5; Emax 90% of that evoked by [des‐ Arg9]‐BK). At this time the response to [Leu8, des‐Arg9]‐BK was antagonized in a concentration‐dependent manner by [des‐Arg10]‐Hoe 140, which at 1 μm and 10 μm, produced dose‐ratios of 6.33±3.66 (n = 4) and 103±40 (n = 4).

In view of the rank order of potency of agonists, the antagonist activity by [des‐Arg10]‐Hoe 140 and the lack of antagonist activity of Hoe 140, we conclude that the longitudinal smooth muscle of rat ileum, after histamine, acetylcholine, noradrenaline, and prostanoid production blockade, is a sensitive monoreceptor assay for studying the pharmacology of bradykinin B1 receptors. Further the preparation can also be used as a sensitive bioassay to identify partial agonist activity of B1 receptor antagonists such as [Leu8, desArg9]‐BK.