bluuebunny/crossref_metadata_2025 huggingface dataset:
The action of SDZ 205,557 at 5‐hydroxytryptamine (5‐HT3 and 5‐HT4) receptors
Authors: Eglen R.M. , Alvarez R. , Johnson L.G. , Leung E. , Wong E.H.F.
Published: July 2012

The interaction of the novel antagonist, SDZ 205,557 (2‐methoxy‐4‐amino‐5‐chloro benzoic acid 2‐(diethylamino) ethyl ester), at 5‐HT3 and 5‐HT4 receptors has been assessed in vitro and in vivo.

In guinea‐pig hippocampus and in the presence of 0.4 μm 5‐carboxamidotryptamine, 5‐HT4‐mediated stimulation of adenylyl cyclase was competitively antagonized by SDZ 205,557, with a pA2 value of 7.5, and a Schild slope of 0.81. In rat carbachol‐contracted oesophagus, 5‐HT4‐receptor mediated relaxations were surmountably antagonized by SDZ 205,557 with a similar pA2 value (7.3). This value was agonist‐independent with the exception of (R)‐zacopride, against which a significantly lower value (6.4) was observed.

In functional studies of 5‐HT3 receptors, SDZ 205,557 exhibited an affinity of 6.2 in guinea‐pig ileum compared with 6.9 at binding sites labelled by [3H]‐quipazine in NG108‐15 cells. In the anaesthetized, vagotomized micropig, SDZ 205,557 produced only a transient blockade of 5‐HT4‐mediated tachycardia. This contrasted with tropisetron, which was active for over 60 min after administration. The half‐lives for the inhibitory responses of SDZ 205,557 and tropisetron were 23 and 116 min, respectively.

In conclusion, SDZ 205,557 has similar affinity for 5‐HT3 and 5‐HT4 receptors. The apparent selectivity observed in guinea‐pig is due to the atypical nature of the 5‐HT3 receptor in this species. The short duration of action of this novel antagonist may complicate its use in vivo. SDZ 205,557 should, therefore, be used with appropriate caution in studies defining the 5‐HT4 receptor.
Effect of opioid receptor subtype‐selective agonists on purinergic and adrenergic components of neurogenic contractions of mouse vas deferens
Authors: Driessen Bernd , Bültmann Ralph , von Kügelgen Ivar , Starke Klaus
Published: July 2012

Effects of opioid agonists on the purinergic and adrenergic components of neurogenic contractions and in some experiments on transmitter overflow were studied in the mouse isolated vas deferens.

When the vas deferens was stimulated every 2 min by pairs of pulses 2 s apart in the presence of prazosin 0.3 μm (to isolate the purinergic component) or α,β‐methylene‐ATP 3 μm (to isolate the adrenergic component), each pulse elicited a separate twitch. The opioid agonists [d‐Ala2,N‐Me‐Phe4,Gly5‐ol]enkephalin (DAMGO, μ‐receptor‐selective), [d‐Pen2,d‐Pen5]enkephalin (DPDPE, δ‐selective) and trans‐(±)‐3,4‐dichloro‐N‐methyl‐N‐[2‐(1‐pyrrolidinyl)‐cyclohexyl]‐benzeneacetamide (U‐50488, κ‐selective) concentration‐dependently reduced both purinergic and adrenergic contractions. For each agonist, maximal effects and concentrations causing half‐maximal effects were very similar for inhibition of the purinergic component on the one hand and for inhibition of the adrenergic component on the other hand, although the adrenergic component was inhibited with a slight preference. Moreover, effects on contractions elicited by the first and the second pulse of the pairs were very similar.

When vasa deferentia preincubated with [3H]‐noradrenaline were stimulated with trains of 100 pulses delivered at 20 Hz, morphine 10 μm reduced significantly both evoked tritium overflow and evoked contractions. Its effect was antagonized by naloxone.

It is concluded that, in contrast to drugs acting at some other presynaptic receptors, opioid μ‐, δ‐and κ‐agonists inhibit purinergic and adrenergic neurogenic contractions of the mouse vas deferens in a similar manner. In contrast to a previous report, no enhancement by morphine of the release of noradrenaline elicited by high frequency pulse trains was observed.
A novel ETA‐receptor antagonist, FR 139317, inhibits endothelin‐induced contractions of guinea‐pig pulmonary arteries, but not trachea
Authors: Cardell Lars Olaf , Uddman Rolf , Edvinsson Lars
Published: July 2012

The effects of a proposed endothelin‐receptor antagonist, FR 139317, on the contraction induced by endothelin‐1, endothelin‐2 and endothelin‐3, were analysed on isolated circular segments of pulmonary arteries and rings of trachea from the guinea‐pig.

The pharmacological profiles of endothelin‐1 and endothelin‐2 were almost identical in the guinea‐pig pulmonary artery, whereas endothelin‐3 demonstrated a weaker and less potent contractile effect. The contractions induced by endothelin‐1 and endothelin‐2 were competitively antagonized by FR 139317. Schild plot analysis revealed a straight line with a slope that did not differ from unity. The pA2 value was 6.65. In contrast, the endothelin‐3 induced contractile response was unaffected by FR 139317.

In tracheal segments endothelin‐1, endothelin‐2 and endothelin‐3 evoked contractions of similar magnitude and sensitivity. FR 139317 had no effect on the endothelin‐induced contractions in tracheal segments.

In ring segments of pulmonary artery and trachea, potassium, noradrenaline and histamine caused concentration‐dependent contractile effects. These contractions were not modified by FR 139317 in the concentration range 10−7 to 3 × 10−6 m.

FR 139317 seems to be a selective ETA‐receptor antagonist which competitively antagonizes the endothelin‐1‐ and endothelin‐2‐induced contractions of guinea‐pig isolated pulmonary arteries. Thus, the guinea‐pig pulmonary artery appears to be endowed with one receptor type (ETA) which is antagonized by FR 139317 and with another endothelin‐receptor subtype which responds to endothelin‐3, but is not antagonized by FR 139317. In the trachea, all three peptides act on a homogeneous population of receptors which is unaffected by FR 139317. This suggests an ETA‐receptor in the guinea‐pig pulmonary artery and another receptor, probably of ETB‐type, in the guinea‐pig trachea.
The affinity of betaxolol, a β1‐adrenoceptor‐selective blocking agent, for β‐adrenoceptors in the bovine trachea and heart
Authors: Satoh Eisaku , Narimatsu Akihiro , Hosohata Yoshiaki , Tsuchihashi Hiroshi , Nagatomo Takafumi
Published: July 2012

The specificity of betaxolol, a β‐adrenoceptor antagonist, for β1 and β2‐adrenoceptors was compared with that of other β‐antagonists, atenolol, ICI‐118551, butoxamine and (±)‐propranolol, in the bovine trachea and heart by competitive interaction with [3H]‐CGP12177 as a radioligand.

The radioligand Kd values were 0.75 ±0.12 and 1.60 ± 0.11 nm in the trachea and heart, respectively, and the Bmax values were 34.00 ± 4.41 and 21.54 ± 2.94 fmol mg−1 protein, respectively.

Using ICI‐118551, we determined the ratio of β1:β2‐adrenoceptors in the trachea and heart to be approximately 29:71 and 56:44, respectively.

In the trachea, a β2‐predominant tissue, betaxolol and atenolol were more selective for β1‐adrenoceptor binding sites than β2‐adrenoceptor binding sites, whereas ICI‐118551 and butoxamine were more selective for β2‐adrenoceptor binding sites.

The β1‐selectivity of betaxolol was 2.2 and 2.7 fold higher than that of atenolol in the bovine trachea and heart. These findings suggest that betaxolol may be useful in the treatment of hypertension, cardiac arrhythmia and angina pectoris.
Quantitative analysis of the agonist and antagonist actions of some ATP analogues at P2X‐purinoceptors in the rabbit ear artery
Authors: Leff P. , Wood B.E. , O'Connor S.E. , McKechnie K.
Published: July 2012

The agonist and antagonist effects of a series of β,γ‐methylene dihalo‐ and 2‐methylthio‐substituted analogues of ATP at P2x‐purinoceptors have been analysed on the rabbit isolated ear artery preparation. Cumulative and sequential dosing experimental protocols were employed in the construction of agonist concentration‐effect curves in order to address the possible influence of acute receptor desensitization on subsequent analyses.

Using the cumulative curve design the following results were obtained: d‐AMP‐PCBr2P, 2‐methylthio‐d‐AMP‐PCCl2P, l‐AMP‐PCF2P, l‐AMP‐PCCl2P and LAMP‐PCBr2P each behaved as partial agonists. d‐AMP‐CPP was used as a reference full agonist and these analogues were analysed by the comparative method of Barlow et al. (1967), to provide estimates of affinity and efficacy. 2‐Methylthio‐l‐AMP‐PCBr2P was virtually silent as an agonist and was analysed as a competitive antagonist by Schild analysis.

Two agonists, l‐AMP‐PCCl2P and l‐AMP‐PCBr2P, were analysed by the sequential curve design, and the antagonist effects of one of the agonists, l‐AMP‐PCBr2P were also analysed using this protocol. The resulting estimates of affinity and efficacy, while similar to those obtained with the cumulative design, indicated that acute desensitization may affect curve definition and estimation of these quantities.

The following structure‐activity trends emerged: d‐analogues tended to have higher efficacy but lower affinity than l‐analogues; efficacy varied markedly and inversely with the atomic weight of the halogen while affinity was only minimally affected; 2‐methylthio‐ substitution also reduced efficacy with minimal effect on affinity.

The results of this analysis are discussed in terms of the utility of affinity and efficacy information in the classification of purinoceptors and the design of chemical probes for them.
Interference of a neutrophil recruitment inhibitory factor upon the accumulation of inflammatory cells and airway hyperreactivity in sensitized guinea‐pigs after intranasal antigen challenge
Authors: Tavares‐Murta B.M. , Lefort J. , Cunha F.Q. , Ferreira S.H. , Vargaftig B.B.
Published: July 2012

A neutrophil recruitment inhibitory factor (NRIF) recovered from the crude supernatant of lipopolysaccharide (LPS)‐stimulated macrophages inhibited neutrophil migration following both intratracheal and intravenous administration of LPS, but did not alter the pattern of leukopenia/leucocytosis induced by intravenous LPS.

The correlation between airway infiltration by inflammatory cells and hyperreactivity in lungs from actively sensitized and challenged guinea‐pigs was investigated by use of NRIF.

Increased eosinophil counts were found in the bronchoalveolar lavage fluid from guinea‐pigs sensitized with 10 μg ovalbumin and challenged at day 14 by the intranasal administration of the antigen. The increase was evident 5 h after challenge and persisted at 24 h. Neutrophil numbers were also increased at this time. Pretreatment with NRIF suppressed the leucocyte increase in the bronchoalveolar lavage fluid.

Bronchoconstriction and histamine release induced by 3 ng PAF injected into the isolated lungs were increased in challenged guinea‐pigs as compared to sensitized but unchallenged controls. Pretreatment of the animals with NRIF did not interfere with this response, but significantly reduced the bronchoconstriction induced by ovalbumin injection.

Even though the increased number of inflammatory cells in bronchoalveolar lavage and airway hyperresponsiveness were concomitant, NRIF inhibited cellular infiltration but failed to alter airway hyperreactivity to PAF, demonstrating that these events may occur independently. Conversely, the inhibition of antigen‐induced bronchoconstriction by NRIF suggests that this response is dependent upon the emigration of granulocytes.
Role of the endothelium in the response to cholinoceptor stimulation of rabbit ear and femoral arteries during cooling
Authors: Monge L. , García‐Villalón A.L. , Montoya J.J. , García J.L. , Fernández N. , Gómez B. , Diéguez G.
Published: July 2012

The role of the endothelium in the effects of cooling on the response to cholinoceptor stimulation of the rabbit central ear (cutaneous) and femoral (non‐cutaneous) arteries was studied using 2 mm long cylindrical segments.

Concentration‐response curves for acetylcholine (10−9‐10−5 m), methacholine (10−9‐10−5 m) and sodium nitroprusside (10−9‐10−4 m) were isometrically recorded in arteries under conditions, with and without endothelium or following pretreatment with the nitric oxide‐synthesis inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME, 10−6‐3 x 10−4 m) at 37°C and at 24°C (cooling).

Ear and femoral arteries showed endothelium‐dependent relaxation to acetylcholine and methacholine at 37°C and 24°C. The extent of relaxation of the control ear arteries, but not of the control femoral arteries, to acetylcholine and methacholine increased during cooling.

l‐NAME (10−6−3 × 10−4 m) reduced in a concentration‐dependent way the response of ear arteries to acetylcholine at both 37°C and 24°C, this reduction being more potent at 37°C. l‐Arginine (10−5‐10−3 m) reversed in a concentration‐dependent manner the inhibitor effects of 10−5 m l‐NAME at both temperatures.

Sodium nitroprusside caused a concentration‐dependent relaxation in both arteries that was endothelium‐independent. However, the extent of relaxation to this nitrovasodilator in ear and femoral arteries was lower at 24°C.

These results suggest that cooling augments the reactivity of cutaneous (ear) arteries, but not that of non‐cutaneous (femoral) arteries to cholinoceptor stimulation by endothelium‐mediated mechanisms. Cooling could therefore facilitate the stimulated release of endothelial nitric oxide in cutaneous vessels.
Sensory nerve‐mediated relaxation of guinea‐pig isolated pulmonary artery: prejunctional modulation by α2‐adrenoceptor agonists but not sumatriptan
Authors: Butler A. , Worton S.P. , O'Shaughnessy C.T. , Connor H.E.
Published: July 2012

Effects of the α2‐adrenoceptor agonists, UK14304 and clonidine, the 5‐HT1 receptor agonist, sumatriptan and the κ‐opioid receptor agonist, GR103545, on sensory neurotransmission in histamine‐contracted guinea‐pig isolated pulmonary artery (GPPA) have been studied.

Electrical field stimulation (EFS) induced frequency‐dependent relaxations of histamine‐contracted GPPA, which were attenuated by tetrodotoxin and capsaicin pretreatment but not by the nitric oxide synthase inhibitor, Nω‐nitro‐l‐arginine methyl ester (l‐NAME).

Substance P (0.3 μm) induced relaxations which were subject to rapid tachyphylaxis. Neither the NK1 receptor antagonist, (±)‐CP 96,345, nor desensitization to substance P had any effect of EFS‐induced relaxations of histamine‐contracted GPPA.

Calcitonin gene‐related peptide (CGRP; 3 and 30 nm) induced concentration‐dependent relaxations of histamine‐contracted GPPA. The putative CGRP receptor antagonist, CGRP8–37 (1 μm), markedly attenuated EFS‐induced relaxations as well as relaxations induced by a low concentration of CGRP.

Sumatriptan (0.1 and 1 μm) and the selective K‐opioid receptor agonist, GR103545 (10 and 100 nm) had no effect on EFS‐induced relaxations of histamine‐contracted GPPA. In contrast, the α2‐adrenoceptor agonists UK14304 (1–100 nm) and clonidine (300 nm) attenuated responses to EFS, the attenuation of UK14304 (100 nm) being reversed by yohimbine (300 nm).

It is concluded that in GPPA, where a presynaptic inhibition of sensory neurotransmission by α2‐adrenoceptor activation could be shown, there was no evidence for such modulation by either sumatriptan‐sensitive 5‐HT1 receptors or κ‐opioid receptors.
Modulation by histamine of the delayed outward potassium current in guinea‐pig ventricular myocytes
Authors: Yazawa Kazuto , Abiko Yasushi
Published: July 2012

Histamine receptor‐mediated modulation of the delayed outward potassium current (IK) was investigated in guinea‐pig single ventricular cells by the whole‐cell voltage clamp.

Histamine increased IK in a dose‐ dependent manner with a half‐maximum dose of 3.8 × 10−8 M. Histamine (10−6 m) increased IK by a factor of 3.02 without a significant change in the current kinetics. The threshold dose of histamine for increasing IK was 10−9 m and this value was similar to that for calcium current.

Cimetidine decreased IK in the presence of histamine, by shifting the dose‐response curve to histamine to the right. The pA2 value of cimetidine against histamine was 6.38.

Forskolin did not increase IK after application of 10−6 m histamine, and histamine scarcely increased IK in the presence of a heat‐stable inhibitor of cyclic AMP‐dependent protein kinase (PKI).

We conclude that stimulation by histamine of IK is mainly by way of the H2‐receptor, and is mediated by cyclic AMP‐dependent phosphorylation.
Stimulation of insulin secretion and improvement of glucose tolerance in rat and dog by the P2y‐purinoceptor agonist, adenosine‐5′‐O‐(2‐thiodiphosphate)
Authors: Hillaire‐Buys D. , Bertrand G. , Chapal J. , Puech R. , Ribes G. , Loubatières‐Mariani M.M.
Published: July 2012

In vivo effect of a P2y‐purinoceptor agonist, adenosine‐5′‐O‐(2‐thiodiphosphate) (ADPβS), on insulin secretion and glycaemia were studied both in rats and dogs.

In anaesthetized rats, i.v. administered ADPβS (0.2 mg kg−1) produced an insulin response dependent on the nutritional state of the animals, since we observed only a transient increase in overnight‐fasted rats and a sustained insulin secretion followed by a reduction in plasma glucose levels in fed rats. During an i.v. glucose tolerance test, ADPβS enhanced insulin release and thus increased the glucose disappearance rate.

In anaesthetized fasted dogs, i.v. administered ADPβS (0.1 mg kg−1) increased pancreaticoduodenal insulin output and slightly decreased blood glucose levels.

In conscious fasted dogs, orally administered ADPβS (0.1 mg kg−1) transiently increased insulinemia and punctually reduced glycaemia. Furthermore, during an oral glucose tolerance test, orally administered ADPβS at the same dose markedly enhanced insulin secretion and consequently reduced the hyperglycaemia.

In conclusion, the P2y‐agonist, ADPβS, is a potent insulin secretagogue in vivo, improves glucose tolerance and is effective after oral administration. Thus, the P2y‐purinoceptors of the β cell may be a target for new antidiabetic drugs.
Pharmacology of GAB A ρ1 and GAB A α/β receptors expressed in Xenopus oocytes and COS cells
Authors: Kusama T. , Spivak C.E. , Whiting P. , Dawson V.L. , Schaeffer J.C. , Uhl G.R.
Published: July 2012

The ρ1 protein, which we previously cloned from retina, assembles as a homooligomer that transduces the binding of γ‐aminobutyric acid (GABA) into robust chloride currents. However, its insensitivity to bicuculline, pentobarbitone and benzodiazepines, all potent agents at typical GABAA receptors, suggested that it may react atypically to other GABA agonists and antagonists.

cDNAs for the ρ1 and the α5β1 receptors for GABA were expressed as homo‐ and heterooligomers, respectively, in Xenopus oocytes. The selectivities of the respective receptors for various agonists were investigated using concentration‐response experiments in voltage clamped cells.

The most potent agonists at the ρ1 receptor were trans‐4‐aminocrotonic acid (TACA) > GABA > muscimol; at the α5β1 receptor the rank order was muscimol > GABA > 4,5,6,7‐tetrahydroisoxazole[4,5‐c]pyridine‐3‐ol (THIP). The most specific agonists were cis‐(2‐(aminomethyl)‐cyclopropyl‐carboxylic acid (CAMP) and THIP for the ρ1 and the α5β1 receptors, respectively.

Comparing GABA, TACA and cis‐aminocrotonic acid (CACA) at ρ1 receptors expressed in COS cells gave results almost indistinguishable from those found at oocytes; the pharmacology of ρ1 seems independent of the expression system.

Agonists THIP, piperidine‐4‐sulphonic acid (P4S), and isoguvacine, whose C‐C‐C‐N chains are constrained by rings into a folded conformation and were potent at the α5β1 receptor, were among the weakest at the ρ1 receptor. However CACA and CAMP, which align better with the extended than the folded conformation, were weakest at the α5β1 receptor but moderately potent at the ρ1 receptor. These findings suggest that the ρ1 receptor recognizes agonists in the extended conformation, in contrast to GABAA receptors, which are believed to recognize agonists in the partially folded conformation.

In contrast to the α5β1 receptor, gradations in maximum responses were apparent in the ρ1 receptor, suggesting various degrees of partial agonism. In particular, imidazole‐4‐acetic acid (I4AA), whose maximum response was only 3% of GABA's maximum, had an apparent Kd for activating the ρ1 receptor of 16 μm; but it had an apparent Kd for competitively blocking the receptor of 0.64 μm. This difference suggests that steric constraints in the activated (open channel) receptor are tighter than in the resting receptor.

Hill coefficients approached 2 at the ρ1 receptor, but were closer to unity at the α5β1 receptor. Thus, the ρ1 receptor displayed higher cooperativity.

Unlike typical GABAA receptors, the ρ1 receptor was insensitive to the competitive antagonists bicuculline, SR95531, securinine, and (+)‐tubocurarine.
Novel regulation of muscarinic receptors and their coupling with G proteins in smooth muscle: transient resensitization during desensitizing process
Authors: Hishinuma Shigeru , Matsumoto Yukio , Uchida Masaatsu K. , Kurokawa Masanori
Published: July 2012

Muscarinic stimulation of the smooth muscle of guinea‐pig taenia caeci was produced with 10−4 m carbachol for 15 s, 30 s, 1 min, 2 min and 30 min, and the time course of developing desensitization was studied by measuring the muscle contractility and the binding characteristics of muscarinic receptors.

The contractile response to carbachol was analyzed using dose‐response curves. The response to 10−7 m carbachol was reduced by treatment for 15 s with 10−4 m carbachol (fast desensitization), but recovered partially after 30 s treatment and completely after 1 min treatment (resensitization). Contractility was reduced again after 2 min and 30 min treatment (re‐desensitization).

The affinity of carbachol for muscarinic receptors was changed by the carbachol treatment in a manner similar to the contractility. Thus, the affinity was reduced at 15 s, restored slightly at 30 s and completely at 1 and 2 min, and was reduced again at 30 min.

5′‐Guanylylimidodiphosphate (GppNHp), a non‐hydrolysable analogue of guanosine triphosphate (GTP) reduced the affinity of muscarinic receptors for carbachol via guanine nucleotide‐binding regulatory proteins (G proteins). A similar effect was observed in tissues desensitized by 15 s carbachol treatment. This effect disappeared after 30 s, recovered completely after 1 and 2 min, and disappeared again after 30 min carbachol treatment.

Neither the dissociation constant (Kd value) nor the maximal binding (Bmax) of [3H]‐quinuclidinyl benzilate ([3H]‐QNB) to muscarinic receptors were changed by the carbachol treatment.

These results indicate that the whole process of desensitization, resensitization and re‐desensitization are related to changes in the binding ability of muscarinic receptors, in their coupling with G proteins and in the post‐receptor steps of the signal transduction. We emphasize that the desensitizing process involves an early transient phase of resensitization that could be caused by restoration of both the affinity of carbachol for muscarinic receptors and their coupling with G proteins. This novel resensitization mechanism may have some physiological significance for cellular homoeostasis by modulating cellular responsiveness transiently or even in an oscillatory manner during the process of desensitization.
The superficial buffer barrier in venous smooth muscle: sarcoplasmic reticulum refilling and unloading
Authors: Chen Qian , van Breemen Cornelis
Published: July 2012

The interaction of Ca2+ transport in the plasmalemma and the sarcoplasmic reticulum (SR) was investigated in smooth muscle of the rabbit inferior vena cava. We tested the possibility of direct refilling of the SR with extracellular Ca2+ and of the existence of a vectorial Ca2+ extrusion pathway from the SR lumen to the extracellular space suggested by earlier results.

After depletion with caffeine the SR was loaded with Ca2+ to increasing levels by incubation in a high potassium 1.5 mm Ca2+ solution and a 10 mm Ca2+ zero Na+ solution, respectively. Thapsigargin, 2 μm, (a specific SR Ca2+‐ATPase blocker) completely blocked refilling of the SR in either of the above solutions, indicating that the SR Ca2+‐ATPase is essential for this process.

Three different agents, caffeine, ryanodine and thapsigargin, which inhibit Ca2+ accumulation by the SR, increased the steady state intracellular Ca2+ concentration in the rabbit inferior vena cava.

Measurements of Mn2+ induced quenching of the intracellular fura‐2 signal during pharmacological manipulation of the SR content showed that these three agents did not stimulate divalent cation entry.

On the other hand, stimulation with noradrenaline caused a marked increase in Mn2+ influx, which was blocked by 2 mm Ni2+. Mn2+ entry stimulated by high K+ solution was blocked by 1 μm diltiazem.

We conclude that the SR refilling has to be mediated by the SR Ca2+‐ATPase. Inhibition of Ca2+ accumulation by the SR causes an increase in the steady state intracellular Ca2+ concentration. This observation cannot be explained by an increase in Ca2+ influx into the smooth muscle cells of the rabbit inferior vena cava. Alternatively these results suggest the existence of a continuous vectorial release of Ca2+ from the SR lumen to the extracellular space.
Sodium modulation of 3H‐agonist and 3H‐antagonist binding to α2‐adrenoceptor subtypes
Authors: MacKinnon A.C. , Spedding M. , Brown C.M.
Published: July 2012

The α2‐adrenoceptors on human platelets and neonatal rat lung were characterized with the agonist and antagonist ligands [3H]‐adrenaline and [3H]‐RS‐15385‐197 respectively. A correlation of affinities for 3H‐antagonist binding showed the receptors to be of the α2A‐(platelet) and α2B‐(neonatal rat lung) adrenoceptor subtypes, whereas a correlation of affinities for 3H‐agonist binding showed the receptors to have similar characteristics (r = 0.88).

NaCl (100 mm) had no effect on [3H]‐RS‐15385‐197 binding in the human platelet, but increased the density of sites labelled with [3H]‐RS‐15385‐197 in neonatal rat lung by 52%. NaCl increased the density of sites labelled by [3H]‐adrenaline in neonatal rat lung, but there was a consequent 3.5 fold decrease in affinity. In the human platelet, no specific [3H]‐adrenaline binding was observed in the presence of 100 mm NaCl.

In the neonatal rat lung, NaCl had no significant effect on the affinity of prazosin for [3H]‐RS‐15385‐197 binding; however, imiloxan affinity was increased 13 fold. The affinity of the catecholamines, adrenaline and noradrenaline was significantly decreased, whereas the imidazolines, oxymetazoline and UK‐14,304 were much less affected. The affinity of prazosin and imiloxan for [3H]‐adrenaline binding was significantly increased in the presence of 10 and 100 mm NaCl. Conversely, the affinity of adrenaline and noradrenaline was decreased in the presence of NaCl, and there was no change in the affinity of the imidazoline agonists.

In the human platelet, NaCl had no effect on the affinity of prazosin for [3H]‐RS‐15385‐197 binding but the affinity of imiloxan was significantly increased. NaCl significantly decreased the affinity of the catecholamines adrenaline and noradrenaline, whereas the affinity of UK‐14,304 and oxymatazoline was much less affected. Competition experiments with [3H]‐adrenaline in the presence of NaCl in platelets were difficult to characterize as there was no specific binding under these conditions.

The results show that both the α2A‐ and α2B‐adrenoceptor subtypes are allosterically regulated by Na+, but only the α2B‐subtype showed a significant increase in density. Interestingly, there is a differential regulation of imidazoline (unchanged) and catecholamine (decreased affinity) agonist interactions with these subtypes. Na+ may therefore critically affect receptor subtype selectivity of drugs. The implications for receptor subclassification are discussed.
Caffeine and related compounds block inhibitory amino acid‐gated Cl− currents in freshly dissociated rat hippocampal neurones
Authors: Uneyama Hisayuki , Harata Nobutoshi , Akaike Norio
Published: July 2012

The effects of caffeine and related compounds on responses mediated by inhibitory amino acids were investigated in freshly dissociated rat hippocampal pyramidal neurones by conventional and nystatin perforated patch‐clamp techniques.

Glycine and γ‐aminobutyric acid (GABA) evoked Cl− currents in hippocampal neurones. The half‐maximum effective concentrations (EC50) of glycine and GABA were 8.5 × 10−5 and 5 × 10−6 m, respectively.

Caffeine reversibly inhibited both 10−4 m glycine‐ and 10−5 m GABA‐induced Cl− currents in a concentration‐dependent manner. The half‐maximum inhibitory concentrations (IC50) of caffeine were 4.5 × 10−4 m for the glycine response and 3.6 × 10−3 m for the GABA response.

Caffeine shifted the concentration‐response curve of IGly to the right without affecting the maximum response.

The inhibitory action of caffeine did not show voltage‐dependency.

The blocking action of caffeine was not affected by intracellular perfusion with 5 mm BAPTA or by pretreatment with the protein kinase A inhibitor, H‐8. This excludes the participation of Ca2+ or cyclic AMP in the inhibitory action of caffeine.

Caffeine failed to inhibit the augmentations of aspartate‐ and N‐methyl‐d‐aspartate (NMDA) ‐gated current by glycine, suggesting that caffeine has no effect on the allosteric glycine binding site on the NMDA receptor.

The inhibitory effects of some xanthine derivatives on IGly were compared. The inhibitory potency of those compounds on IGly was in the order of pentoxifylline > theophylline ≥ caffeine > paraxanthine > IBMX ≥ theobromine > dyphylline. Xanthine had no effect.

The results indicate that methylxanthines including caffeine may act directly on the glycine receptor Cl− channel complex in rat hippocampal pyramidal neurones. The blockade of the inhibitory amino acid response by methylxanthines may be involved in the excitatory side effects of methylxanthines in the mammalian central nervous system.
Activation by calcium alone of chloride secretion in T84 epithelial cells
Authors: Kachintorn Udom , Vajanaphanich Mana , Traynor‐Kaplan Alexis E. , Dharmsathaphorn Kiertisin , Barrett Kim E.
Published: July 2012

The goal of this study was to determine if an increase in cytoplasmic calcium concentration ([Ca2+]i), in the absence of additional second messengers derived from membrane phospholipid turnover, is a sufficient signal to induce chloride secretion across monolayers of the human colonic epithelial line, T84.

Thapsigargin was used to increase [Ca2+]i by inhibiting the endomembrane Ca2+‐ATPase. [Ca2+]i was monitored in monolayers by fura‐2 fluorescence spectroscopy, chloride secretion by measuring changes in short circuit current (Isc) in modified Ussing chambers, and inositol phosphates were measured by radio‐h.p.l.c. of extracts of cells prelabelled with [3H]‐inositol.

Thapsigargin increased [Ca2+]i and Isc in parallel, without increasing any inositol phosphates. The effect of thapsigargin on Isc was abolished by the intracellular calcium chelator, bis‐(o‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA).

Increasing [Ca2+]i with thapsigargin did not prevent a subsequent calcium response to carbachol or histamine if extracellular calcium was available. In the absence of extracellular calcium, only one such release of calcium to hormonal stimulation occurred when cells were pretreated with thapsigargin, and a second response to either carbachol or histamine was essentially abolished.

Addition of carbachol or histamine to thapsigargin‐treated cells mounted in Ussing chambers caused a transient further increase in Isc followed by termination of the response, even though [Ca2+]i continued to rise.

We conclude that an elevation in [Ca2+]i is a sufficient signal to induce chloride secretion in T84 cells. Rather than being required to stimulate secretory responses, additional second messengers induced by hormonal secretagogues (such as inositol phosphates) may in fact serve to limit the secretory response.
Mediation of endothelin‐1‐induced inhibition of platelet aggregation via the ETB receptor
Authors: McMurdo Lorraine , Lidbury Paul S. , Thiemermann Christoph , Vane John R.
Published: July 2012

The effects of FR139317 (ETA antagonist) or PD145065 (non‐selective ETA/ETB antagonist) on endothelin‐1 (ET‐1)‐induced changes in blood pressure and inhibition of ex vivo platelet aggregation were investigated in the anaesthetized rabbit.

ET‐1 (1 nmol kg−1, i.a. bolus) caused a sustained increase in mean arterial pressure (MAP) (peak increase 47 ± 5 mmHg, n = 8). Intravenous infusion of FR139317 at 0.2 (n = 4) or 0.6 mg kg−1 min−1 (n = 4) inhibited the ET‐1 pressor response by 83 or 89%, respectively. Infusion of PD 145065 at 0.2 (n = 4) or 0.6 mg kg−1 min−1 (n = 4) inhibited the ET‐1‐induced increase in MAP by 79 or 75%, respectively.

The transient depressor response (−16 ± 3 mmHg) which preceded the rise in blood pressure induced by ET‐1 (1 nmol kg−1, i.a., n = 8) was enhanced by an intravenous infusion of FR139317 (0.6 mg kg−1 min−1) to −35 ± 5 mmHg (P < 0.05, n = 4). This enhancement was abolished by indomethacin (5 mg kg−1, i.v.) pretreatment (−17 ± 1 mmHg, n = 4). PD145065 (0.2 mg kg−1 min−1, i.v.) attenuated the ET‐1‐induced fall in blood pressure to −9 ± 1 mmHg (n = 4), while a higher dose of this antagonist (0.6 mg kg−1 min−1, i.v.) completely abolished the ET‐1‐mediated depressor response.

ET‐1 (1 nmol kg−1, n = 8) inhibited ex vivo platelet aggregation by 96% at 5 min after injection of the peptide. FR139317 (0.2 or 0.6 mg kg−1 min−1, i.v.) or PD145065 (0.2 mg kg−1 min−1, i.v.) did not affect the inhibition of ex vivo platelet aggregation in response to ET‐1. In contrast, intravenous infusion of PD145065 (0.6 mg kg−1 min−1) abolished the anti‐aggregatory effects of ET‐1.

Thus, FR139317 inhibits the pressor, but not the depressor actions of ET‐1 and has no effect on the ET‐1‐induced inhibition of ex vivo platelet aggregation. In contrast, PD145065 antagonizes the pressor and depressor responses to ET‐1 and abolishes the anti‐aggregatory effects of the peptide.

These results strongly suggest that ET‐1‐induced vasoconstriction in the anaesthetized rabbit is primarily mediated via the ETA receptor while the depressor and antiaggregatory actions of ET‐1 are due to activation of the ETB receptor.
Oestradiol inhibits smooth muscle cell proliferation of pig coronary artery
Authors: Vargas Roberto , Wroblewska Barbara , Rego Alfredo , Hatch Joshua , Ramwell Peter W.
Published: July 2012

The effect of oestradiol 17β on vascular smooth muscle proliferation was examined in segments of the pig left anterior descending coronary artery (LAD). It was established by cytochemical techniques that out‐growth from the segments was composed of vascular smooth muscle cells.

[3H]‐thymidine uptake by pig LAD segments was used as an index of vascular smooth muscle cell proliferation. Nitroprusside and forskolin significantly inhibited [3H]‐thymidine uptake and were used as positive controls.

Oestradiol 17β (180–360 nm) inhibited thymidine uptake by pig LAD segments (P < 0.05). The inhibition was observed only in the absence of phenol red, which is a weak oestrogen receptor agonist. The anti‐oestrogens tamoxifen and its more potent metabolite 4‐hydroxytamoxifen, both of which are partial oestrogen receptor agonists, also significantly inhibited thymidine uptake. However, pretreatment with either tamoxifen or 4‐hydroxytamoxifen did not signficantly block oestradiol 17β‐induced inhibition of thymidine uptake.

The LAD segments bound [3H]‐oestradiol 17β in a time‐dependent manner and about 20 to 30% was displaced by an excess of unlabelled oestradiol 17β. Autoradiography showed [3H]‐oestradiol 17β was evenly distributed in the cytosol and nuclei of cells in the three layers of the vessel wall.

The data suggest that oestradiol 17β inhibits smooth muscle cell proliferation in porcine LAD segments, possibly through an oestrogen receptor mechanism. This in vitro effect suggests an in vivo role for oestradiol 17β in directly protecting coronary arteries against myointimal proliferation in premenopausal women.
[3H]‐idazoxan binding to rabbit cerebral cortex recognises multiple imidazoline I2‐type receptors: pharmacological characterization and relationship to monoamine oxidase
Authors: Renouard Anne , Widdowson Peter S. , Cordi Alex
Published: July 2012

In rabbit cerebral cortical homogenates, saturation analysis of [3H]‐idazoxan, an α2‐adrenoceptor antagonist, revealed high affinity binding to a single site with high density. Competition experiments demonstrated that the [3H]‐idazoxan recognition site was insensitive to the catecholamines, adrenaline and noradrenaline and possessed a low affinity for the α2‐ and α1‐adrenoceptor antagonists, rauwolscine, yohimbine and prazosin, suggesting that the site was not an adrenoceptor. Mapping [3H]‐idazoxan binding sites in the forebrain of rabbits by autoradiography, showed high densities of I2 sites in the medial preoptic area and in the stria terminalis. Moderate binding was found in caudate nucleus, putamen, cerebral cortex and hippocampus.

The imidazolines cirazoline, naphazoline, guanabenz and BRL44408 along with amiloride, which is structurally related to the imidazolines, all had high affinity for the [3H]‐idazoxan site, suggesting that the site was related to the I2 imidazoline‐recognition site described by other groups. However, the imidazolines, clonidine and UK‐14,304 and the structurally related rilmenidine all had a low affinity for the binding site, showing that [3H]‐idazoxan was not binding to the I1 imidazoline‐recognition site found in rat, bovine and human medulla oblongata.

Naphazoline, guanabenz, clonidine and amiloride competition studies had Hill slopes which were significantly different from unity (P < 0.01) and computer analysis showed that the [3H]‐idazoxan binding data could be best fitted to a model which considers binding to two sites (P < 0.01). One site has a high affinity for idazoxan, cirazoline, naphazoline, guanabenz and amiloride and a moderate affinity for BRL44408 and clonidine (70% of binding) and the second site (30% of binding) has a high affinity for idazoxan and cirazoline, but a lower affinity for naphazoline, guanabenz, amiloride, BRL44408 and clonidine.

Experiments using [3H]‐RX821002, in contrast to [3H]‐idazoxan, clearly demonstrated the presence of a single type of α2‐adrenoceptor in rabbit cortex with a pharmacological profile which is similar to the α2A‐adrenoceptor possessing a high affinity for yohimbine, rauwolscine, BRL44408 and oxymetazoline, but a lower affinity for prazosin.

The monoamine oxidase inhibitors, clorgyline, pargyline and deprenyl had at least a ten fold lower affinity at the rabbirt cortex I2 site as compared to their known affinity at monoamine oxidase suggesting that the I2 site is not related to the active site of the enzyme, monoamine oxidase. In addition, the peripheral benzodiazepine ligands, PK‐11195 or Ro 5–4864 both had very low affinities at the I2 site in rabbit cortex suggesting that the [3H]‐idazoxan binding was not to the peripheral benzodiazepine binding site.
Dual effects of chlorobutanol on secretory response and intracellular Ca2+ dynamics in isolated pancreatic acini of the rat
Authors: Habara Y. , Kanno T.
Published: July 2012

The effects of chlorobutanol, a widely used drug preservative, on exocrine response and intracellular Ca2+ dynamics were examined in isolated pancreatic acini of the rat.

Chlorobutanol (1 mg ml−1) markedly inhibited the secretory response to cholecystokinin octapeptide (CCK‐8), carbamylcholine chloride (carbachol), or sodium fluoride, a direct G‐protein activator. However, chlorobutanol itself induced a maximal release of amylase when the dose was increased to 4 mg ml−1.

An oscillatory fluctuation of cytoplasmic Ca2+ concentration, [Ca2+]c, induced by 5 pm CCK‐8 or 0.3 μm carbachol was totally abolished in the presence of 1 mg ml−1 chlorobutanol.

A biphasic change in [Ca2+]c induced by 100 pm CCK‐8, a rapid rise followed by a gradual decay, was transformed to an oscillatory fluctuation by the preservative.

Chlorobutanol inhibited 13 pm [125I]‐CCK‐8 or 0.5 nm [3H]‐methylscopolamine chloride binding to the acinar cells in a dose‐dependent manner.

These results indicate that chlorobutanol produces discernible pharmacological effects on the secretory response in rat pancreatic acinar cells through changes in the Ca2+ dynamics. Possible sites of action could be at a binding process of secretagogues to their receptors, at an activation process of a G‐protein located in the plasma membrane, or at the processes following G‐protein activation. However, the possibility that the preservative may distort the Ca2+‐transport function of the plasma membrane or the membrane of intracellular organella, especially Ca2+‐sequestering pools, cannot be excluded.
Pharmacological characterization of muscarinic receptor‐activated cation channels in guinea‐pig ileum
Authors: Chen Shan , Inoue Ryuji , Ito Yushi
Published: July 2012

The pharmacological properties of cationic currents activated by acetylcholine (ACh) (Icat) in guinea‐pig ileal smooth muscle cells were investigated, with conventional single patch electrode or nystatin‐perforated whole‐cell recording. Cs‐aspartate was used as the internal solution to allow selective measurement of Icat.

Well‐known K channel blockers, tetraethylammonium (TEA), 4‐aminopyridine (4‐AP), procaine and quinine as well as a Ca releasing agent, caffeine, all produced concentration‐dependent inhibition of Icat with rapid onset (time constant ∼100 ms), when applied externally. The recovery from the inhibition on washout also occurred rapidly in the order of 100 ms except in the case of quinine. Approximate values of the half inhibitory concentrations (IC50) were 10 mm for TEA and caffeine, 1–5 mm for 4‐AP and procaine, and 1 μm for quinine. The mode of inhibition was voltage‐dependent, i.e., depolarization relieved the inhibition with no change in reversal potential.

Externally applied diphenylamine‐2‐carboxylate (DPC) derivatives, DCDPC and flufenamic acid, produced potent inhibition of Icat at micromolar concentrations (IC50s were < 30 μm for DCDPC and 32 μm for flufenamic acid). The onset of and recovery from inhibition occurred slowly and the degree of inhibition depended on the membrane potential only weakly, without any discernible change in the reversal potential.

All of the above‐tested drugs exhibited comparable inhibitory actions on the voltage‐dependent Ca current in the concentration ranges effective at inhibiting Icat. However, amongst them, quinine and flufenamic acid seemed to have several‐fold better selectivity for the Icat channel than for the voltage‐dependent Ca channel.

Internally dialysed GTPγS (100 μm) induced inward cationic currents. The effects of drugs on these currents were similar to their effects on the Icat current.

These results clearly indicate that many drugs used as pharmacological tools in smooth muscle research exert considerable nonspecific effects on various types of channels. The mechanism of inhibition and the relevance to use of these drugs as blockers for the Icat channel are discussed.
Involvement of nitric oxide synthase in the delayed vasodilator response to ultraviolet light irradiation of rat skin in vivo
Authors: Warren J.B. , Loi R.K. , Coughlan M.L.
Published: July 2012

The role of nitric oxide synthase and cyclo‐oxygenase in the skin blood flow response to ultraviolet light B (u.v.B) irradiation was investigated in the rat in vivo.

Local skin blood flow changes were measured in the shaved dorsal skin of anaesthetized male Sprague‐Dawley rats with a laser Doppler flow probe.

u.v.B irradiation caused delayed onset vasodilatation and by 18 h basal blood flow increased by 125 ± 25% (P < 0.05, n = 12 rats, mean ± s.e.mean).

Indomethacin, 3 nmol per site, NG‐nitro‐l‐arginine methyl ester (l‐NAME) 100 nmol per site, but not d‐NAME 100 nmol per site, injected locally 17.5 h after u.v.B irradiation abolished the 18 h increase in blood flow.

The nitric oxide synthase inhibitor l‐NAME, NG‐monomethyl‐l‐arginine (l‐NMMA) and canavanine, 10 and 100 nmol per site injected at 17.5 h, suppressed significantly the u.v.B 18 h response in a dose‐dependent manner. The order of potency was l‐NAME > canavanine = l‐NMMA. The effect of l‐NAME was reversed partially by the co‐injection of an excess of l‐arginine.

Topical application of the corticosteroid, clobetasol 17‐propionate, immediately after irradiation inhibited the 18 h u.v.B response in a dose‐dependent manner.

The delayed onset microcirculatory vasodilatation induced by u.v.B involves both nitric oxide synthase and cyclo‐oxygenase in this in vivo model. Topical corticosteroids may attenuate the response by inhibiting both prostaglandin and nitric oxide synthesis pathways.
Effects of intracerebroventricular injection of histamine and related compounds on corticosterone release in rats
Authors: Tsujimoto Susumu , Okumura Yasushi , Kamei Chiaki , Tasaka Kenji
Published: July 2012

The effects of intracerebroventricular (i.e.v.) injection of histamine and related compounds on plasma adrenocorticotrophic hormone (ACTH) and corticosterone concentrations were studied in conscious rats.

Histamine at doses of 5–20 μg kg−1 rapidly increased plasma ACTH and corticosterone concentrations almost simultaneously, and subsequent courses were also similar to each other. However, in the case of CRF‐41 (i.v.), the plasma ACTH concentration first increased followed by an increase in plasma corticosterone concentration. Even in hypophysectomized rats, a significant increase in plasma corticosterone concentration was induced by histamine at doses of 20 and 50 μg kg−1.

Histamine at doses of 10 and 20 μg kg−1 elicited an increase in the amplitude of adrenal nerve activity, and electrical stimulation to the adrenal nerves resulted in an increase in plasma corticosterone concentration.

Both H1‐agonist (2‐methylhistamine) and H2‐agonists (4‐methylhistamine and impromidine) also induced similar effects to those of histamine. Pretreatment with pyrilamine caused an inhibition of histamine‐induced increase in plasma ACTH and corticosterone concentrations, while both cimetidine and ranitidine failed to inhibit this effect. However, both H2‐blockers were effective in inhibiting the 4‐methylhistamine‐induced elevation of plasma ACTH and corticosterone concentrations.

Neither (R)‐α‐methylhistamine nor thioperamide had a significant effect, indicating that the H3‐receptor is not involved in the histamine‐induced increase in plasma ACTH and corticosterone concentrations.

From these findings, it was concluded that (1) electrical signals transmitted from the brain to the adrenal gland through the neurones may be involved in the rapid corticosterone release induced by histamine, and (2) not only H1‐ but also H2‐receptors are implicated in histamine‐induced hormone secretions in rats, though the contribution of the H2‐receptor is less important than that of the H1‐receptor.
Antiarrhythmic drugs, clofilium and cibenzoline are potent inhibitors of glibenclamide‐sensitive K+ currents in Xenopus oocytes
Authors: Sakuta Hidenari , Okamoto Koichi , Watanabe Yasuhiro
Published: July 2012

The novel K+ channel opener, Y‐26763 induced outward K+ currents in voltage‐clamped follicle‐enclosed Xenopus oocytes in a concentration‐dependent manner with an EC50 value of 58 μm.

The Y‐26763‐induced K+ current was completely and reversibly blocked by glibenclamide (an ATP‐sensitive K+ channel blocker) in a concentration‐dependent manner (IC50 140 nm). Effects of several antiarrhythmic drugs on Y‐26763‐induced glibenclamide‐sensitive K+ currents were investigated.

(±)‐Cibenzoline, RS‐2135, pirmenol, lorcainide and KW‐3407 (class I antiarrhythmic drugs, Na+ channel blockers) suppressed Y‐26763 responses in a concentration‐dependent manner with IC50 values (in μm) of 6.6, 54, 68, 71 and 370, respectively.

Clofilium, E‐4031, MS‐551 and bretylium (class III antiarrhythmic drugs which increase the action potential duration) also suppressed Y‐26763 responses concentration‐dependently, IC50 values (in μm) were 3.3, 660, 980 and ≥ 2000, respectively. N‐acetylprocainamide (class III antiarrhythmic drug) scarcely suppressed Y‐26763 responses.

The glibenclamide‐sensitive K+ currents elicted by KRN2391 were also suppressed by all these antiarrhythmic drugs.

The antiarrhythmic drugs, clofilium and (±)‐cibenzoline block glibenclamide‐sensitive K+ channels in Xenopus oocytes at concentrations comparable to their therapeutic plasma levels.