bluuebunny/crossref_metadata_2025 huggingface dataset:
β‐Adrenoceptor subtypes and the opening of plasmalemmal K+‐channels in bovine trachealis muscle: studies of mechanical activity and ion fluxes
Authors: Chiu P. , Cook S.J. , Small R.C. , Berry J.L. , Carpenter J.R. , Downing S.J. , Foster R.W. , Miller A.J. , Small A.M.
Published: July 2012

Studies of mechanical activity and 86Rb+ efflux have been made in bovine isolated trachealis with the objectives of (a) identifying which of the β‐adrenoceptor subtypes mediates the opening of plasmalemmal K+‐channels, (b) gaining further insight into the properties of the novel, long‐acting β2‐adrenoceptor agonist, salmeterol and (c) clarifying the role of K+‐channel opening in mediating the mechano‐inhibitory actions of agonists at β‐adrenoceptors.

In bovine trachealis muscle strips precontracted with histamine (460 μm), isoprenaline (0.1 nm– 1 μm), procaterol (0.1–10 nm) and salmeterol (0.1–10 nm) each caused concentration‐dependent relaxation.

ICI 118551 (10 nm–1 μm) antagonized isoprenaline, procaterol and salmeterol in suppressing histamine‐induced tone of the isolated trachealis muscle. The antagonism was concentration‐dependent. In contrast, CGP 20712A (10 nm–1 μm) failed to antagonize isoprenaline, procaterol or salmeterol.

Salmeterol (1–10 μm) antagonized isoprenaline in relaxing strips of bovine trachea which had been precontracted with carbachol (1 μm).

Cromakalim (10 μm), isoprenaline (100 nm–10 μm), procaterol (10 nm–1 μm) and salbutamol (100 nm–10 μm) each promoted the efflux of 86Rb+ from strips of bovine trachealis muscle preloaded with the radiotracer. In contrast, salmeterol (100 nm–10 μm) failed to promote 86Rb+ efflux.

CGP 201712A (1 μm), ICI 118551 (100 nm) and salmeterol (1 μm) did not themselves modify 86Rb+ efflux from trachealis muscle strips, nor did they affect the promotion of 86Rb+ efflux induced by cromakalim (10 μm). In contrast, CGP 20712A (1 μm) and ICI 118551 (100 nm) were each able to inhibit the promotion of 86Rb+ efflux induced by isoprenaline (1 μm) or procaterol (100 nm). Furthermore, salmeterol (10 μm) inhibited isoprenaline (1 μm)‐induced promotion of 86Rb+ efflux.

It is concluded that, in bovine trachealis, activation of either β1‐ or β2‐adrenoceptors can promote the opening of 86Rb+‐permeable K+‐channels in the plasmalemma. The failure of salmeterol to promote plasmalemmal K+‐channel opening may reflect, not its selectivity in activating β2‐ as opposed to β1‐adrenoceptors, but rather its low intrinsic efficacy at β2‐adrenoceptors. The opening of plasmalemmal K+‐channels plays a supportive rather than a crucial role in mediating the mechano‐inhibitory effects of agonists at β‐adrenoceptors acting on trachealis muscle.
Transmitter‐mediated local contracture of the endplate region of the focally innervated mouse diaphragm treated with anticholinesterase
Authors: Hong S.J. , Chang C.C.
Published: July 2012

Local contraction of the endplate region in response to nerve stimulation was studied in isolated mouse diaphragms. In normal preparations, muscle contractions involved the whole length of the muscle fibre with rise and decay times in the order of tens of ms whether evoked with a single or train of pulses.

When acetylcholinesterase was inhibited with neostigmine, tetanic stimulation produced a twitch‐like phasic contraction and a delayed tonic contracture. A brief train of pulse (10 ms, 300 Hz) was enough to trigger a full size tonic contracture which reached an amplitude about one tenth that of control tetanus and had a duration of about 4 s.

Tetanic stimulation evoked a non‐propagating prolonged depolarization at the endplate region lasting for about 1 s following a few muscle action potentials.

μ‐Conotoxin, a specific inhibitor of muscle Na+ channel, selectively abolished the phasic contraction and the muscle action potentials leaving the tonic contracture and the prolonged depolarization unaffected.

Both the tonic contracture and the prolonged depolarization were highly sensitive to blockade by tubocurarine (IC50 0.05–0.1 μm) and vesamicol (1 μm, an inhibitor of packaging acetylcholine into synaptic vesicles), were attenuated by increasing Ca2+ concentration and were prolonged by decreasing Ca2+.

The results suggest that prolonged activation of endplate nicotinic receptors by endogenously released transmitter can produce substantial contractions of the endplate region when acetylcholinesterases are inhibited. The source of Ca2+ for the contraction seems to come mainly from intracellular stores.
Alfuzosin, a selective α1‐adrenoceptor antagonist in the lower urinary tract
Authors: Lefèvre‐Borg F. , O'Connor S.E. , Schoemaker H. , Hicks P.E. , Lechaire J. , Gautier E. , Pierre F. , Pimoule C. , Manoury P. , Langer S.Z.
Published: July 2012

Phenylephrine‐induced contractions of rabbit isolated trigone and urethra were antagonized in a competitive manner by alfuzosin (pA2 7.44 and 7.30, respectively) and prazosin.

The characteristics of [3H]‐prazosin binding to human prostatic adenoma tissue were evaluated. [3H]‐prazosin was potently displaced by α1‐adrenoceptor specific agents including alfuzosin, its (+)‐ and (−)‐enantiomers and prazosin (IC50 0.035, 0.023, 0.019 and 0.004 μm, respectively), but only weakly by α2‐adrenoceptor selective agents, for example, yohimbine (IC50 = 6.0 μm).

In the pithed rat, alfuzosin (0.03–0.3 mg kg−1, i.v.) markedly inhibited pressor responses produced by the α1‐selective agonist, cirazoline but inhibited only slightly responses to the α2‐selective agonist, UK 14,304. Alfuzosin (1 mg kg−1, i.v.) had minimal effects against responses mediated by stimulation of prejunctional α2‐receptors (UK 14,304‐induced inhibition of sympathetic tachycardia).

In the anaesthetized cat, alfuzosin (0.001–1 mg kg−1, i.v.) and prazosin (0.001–0.3 mg kg−1, i.v.) produced dose‐related inhibition of the increases in urethral pressure caused by stimulation of sympathetic hypogastric nerves. Prazosin was approximately 5 fold more potent than alfuzosin. When phenylephrine was employed to induce urethral and vascular α1‐mediated tone simultaneously, prazosin inhibited both stimuli with similar potency whereas alfusozin was 3–5 fold more potent against elevated urethral pressure. This functional uroselectivity of alfuzosin was more evident by the intraduodenal route, since doses of 0.03 and 0.1 mg kg−1 alfuzosin inhibited urethral pressure with minimal effects on arterial blood pressure.

Alfuzosin is a potent selective α1‐adrenoceptor antagonist in tissues of the lower urinary tract including the human prostate. This provides a pharmacological basis for its use in the treatment of benign prostatic hypertrophy.
Characteristics of the bradykinin‐induced changes in intracellular calcium ion concentration of single bovine tracheal smooth muscle cells
Authors: Marsh Katrina A. , Hill Stephen J.
Published: July 2012

Single bovine tracheal smooth muscle (BTSM) cells were cultured and used to measure bradykinin‐induced changes in [Ca2+]i by dynamic video imaging.

Bradykinin (10 pm‐10 μm)‐induced an increase in [Ca2+]i over basal levels (69 ± 2 nm; n = 353) which was concentration‐dependent (log EC50 = −8.7 m) in the presence of extracellular calcium ions (2 mm). The bradykinin B2 receptor antagonist, d‐Arg[Hyp3,Thi5,8,d‐Phe7]‐bradykinin, produced a parallel shift to the right of the bradykinin concentration‐response curve (log EC50 = −7.1 m and −5.8 m in the presence of 1 μm and 10 μm antagonist respectively) yielding an apparent KD of 26 nm.

In the absence of extracellular calcium ions (with 0.1 mm EGTA), bradykinin (10 pm– 10 μm) produced a uniform increase in [Ca2+]i from a basal level of 33 ± 2 nm (n = 140) to approximately 180 nm in BTSM cells indicating an ‘all‐or‐nothing’ release of intracellular calcium ions. In the presence of 10 μm d‐Arg[Hyp3,Thi5,8,d‐Phe7]‐bradykinin no responses could be induced by bradykinin at concentrations below 100 nm. However, at 100 nm and 1 μm bradykinin there was no change in the uniform increase in [Ca2+]i in these cells previously observed.

In both the absence or presence of d‐Arg[Hyp3,Thi5,8,d‐Phe7]‐bradykinin, there was a concentration‐dependent increase in the percentage of cells responding to bradykinin (frequency) under calcium‐rich or calcium‐free conditions. Individual cells also demonstrated a difference in the sensitivity to any particular concentration of bradykinin.

A latency in the response of cells to bradykinin was observed both in calcium‐containing and calcium‐free conditions.

We conclude that bradykinin B2 receptors are expressed by BTSM cells and are involved in the bradykinin‐induced increase in [Ca2+]i. It appears that the increase in [Ca2+]i can be mediated via a graded influx of calcium ions from the extracellular space or an ‘all‐or‐nothing’ release from intracellular stores.
Glycine stimulates striatal dopamine release in conscious rats
Authors: Yadid Gal , Pacak Karel , Golomb Eliahu , Harvey‐White Judith D. , Lieberman Daniel M. , Kopin Irwin J. , Goldstein David S.
Published: July 2012

Glycine is an inhibitory neurotransmitter in the spinal cord and brainstem. The mechanism of this inhibition is via binding of glycine to specific receptors, increasing transmembrane Cl− conductance and hyperpolarizing neurones. Strychnine selectively antagonizes these effects. The role of glycinergic neurones in supraspinal regions is poorly understood.

Effects of glycine on release of catecholamines in the striatum were examined by microdialysis in freely‐moving rats. Transcription of the genes encoding strychnine‐sensitive glycine receptors was assessed in the striatum and substantia nigra, by use of reverse transcription followed by the polymerase chain reaction.

Glycine administered via the microdialysis probe dose‐dependently increased concentrations of dopamine and its metabolites, dihydroxyphenylacetic acid and homovanillic acid, in the perfusate, indicating increased local release and metabolism of dopamine. Strychnine markedly attenuated these responses. Whereas striatal tissue did not contain mRNA for either the adult or neonatal form of strychnine‐sensitive glycine receptor, nigral tissue contained a message for the adult form.

The results suggest that dopaminergic cells in the substantia nigra synthesize strychnine‐sensitive glycine receptors and transport the receptors to terminals in the striatum. Occupation of the glycine receptors then exerts a net stimulatory effect on striatal dopamine release in vivo.
Cloning and expression of a fish α2‐adrenoceptor
Authors: Svensson Samuel P.S. , Bailey Thomas J. , Pepperl Dave J. , Grundström Nils , Ala‐Uotila Sari , Scheinin Mika , Karlsson Jan Olof G. , Regan John W.
Published: July 2012

Pigment granule aggregation in specialized cells (melanophores) from the skin of teleost fishes has been shown to be mediated by receptors with an α2‐adrenoceptor pharmacology. We now report the cloning of the α2‐F, a fish skin α2‐receptor from the cuckoo wrasse (Labrus ossifagus).

Degenerate oligonucleotides corresponding to conserved regions of the human α2‐adrenoceptor subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated from the skin of the cuckoo wrasse. An 876 base pair (bp) product was obtained that was homologous with that of the human α2‐adrenoceptor and was used to screen a genomic library from the cuckoo wrasse.

A clone (pTB17BS) consisting of ∼5 kb of genomic DNA was obtained which contained the nucleotide sequence of the initial PCR product. In addition, it contained an open reading frame that encoded a protein of 432 amino acids and ∼2 kb of 5′‐untranslated sequence. The deduced amino acid sequence of this protein showed 47–57% identity with the human α2‐adrenoceptors and thus appeared to encode a fish α2‐adrenoceptor.

In the 5′‐untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the α2‐F might be regulated by cyclic AMP, calcium and/or steroids.

The α2‐F was expressed in COS‐7 cells and radioligand binding studies were performed with [3H]‐rauwolscine. The binding was of high affinity and it was saturable with a KD of 0.8 ± 0.1 nm and a Bmax of 5.7 ± 1.0 pmol mg−1 of protein.

Competition curves for the displacement of specific [3H]‐rauwolscine binding showed the following order of potency: for agonists, medetomidine > clonidine> p‐aminoclonidine > B‐HT 920 > (−)‐noradrenaline; for antagonists, rauwolscine > atipamezole > yohimbine > phentolamine > prazosin.

These results show that α2‐F has characteristics of both the human α2‐C10 and α2‐C4 and that it might represent an ancestral α2‐adrenoceptor subtype.
Mediation by B1 and B2 receptors of vasodepressor responses to intravenously administered kinins in anaesthetized dogs
Authors: Nakhostine Nabil , Ribuot Christophe , Lamontagne Daniel , Nadeau Réginald , Couture Réjean
Published: July 2012

Vasodepressor responses to intravenous (i.v.) injection of bradykinin (BK) and des‐Arg9‐BK, a selective B1 kinin receptor agonist, were characterized following i.v. pretreatment with selective B1 ([Leu8]‐des‐Arg9‐BK) and B2 (Hoe 140) kinin receptor antagonists in anaesthetized dogs.

Des‐Arg9‐BK (0.05–3.3 nmol kg−1) produced dose‐dependent decreases in mean arterial blood pressure with a ED50 0.4 nmol kg−1. The vasodepressor effects evoked by des‐Arg9‐BK (0.6 nmol kg−1) and BK (0.2 nmol kg−1) were greater after i.v. and i.a. injections, respectively.

The vasodepressor response to BK (0.6 nmol kg−1) but not to des‐Arg9‐BK (0.6 nmol kg−1) was significantly (P < 0.001) blocked by pretreatment with the B2 receptor antagonist, Hoe 140.

The vasodepressor response to des‐Arg9‐BK (0.6 nmol kg−1) but not to BK (0.6 nmol kg−1) was significantly (P < 0.001) reduced by pretreatment with the selective B1 receptor antagonist, [Leu8]‐des‐Arg9‐BK. Although both B1 and B2 receptor antagonists caused a transient fall in blood pressure, their inhibitory action was unlikely to be related to a desensitization mechanism.

Inhibition of prostaglandin synthesis with indomethacin prevented the vasodepressor response induced by arachidonic acid (1 mg kg−1, i.v.) but not that to BK or des‐Arg9‐BK (0.6 nmol kg−1).

These results suggest, firstly, that the vasodepressor responses to i.v. BK and des‐Arg9‐BK are mediated by the activation of B2 and B1 receptors, respectively; secondly, that prostaglandins are not involved in the vasodepressor responses to kinins. These findings provide pharmacological evidence for the existence of functionally active B1 receptors in canine cardiovascular homeostasis.
The positive inotropic effect of compound II, a novel analogue of sotalol, on guinea‐pig papillary muscles and single ventricular myocytes
Authors: White E. , Connors S.P. , Gill E.W. , Terrar D.A.
Published: July 2012

Compound II is a novel analogue of sotalol which has been reported to be free of β‐adrenoceptor and L‐type calcium channel blocking actions. The effects of compound II on the contraction of guinea‐pig papillary muscles (at 2 μm) and single ventricular myocytes (at 100 nm) were investigated.

Exposure to compound II caused a significant increase in the contraction of both preparations.

Compound II prolonged the action potential of the single myocytes and increased the magnitude of the Ca‐activated current which was used as a qualitative indicator of the intracellular calcium transient.

The ratio of first/steady state Ca‐activated currents evoked by short action potentials was not modified. This may indicate that compound II does not influence the normal functioning of the sarcoplasmic reticulum stores.

The observations are consistent with the hypothesis that action potential prolongation by compound II reduces Ca2+ extrusion via the Na‐Ca exchange. This in turn allows increased uptake of calcium into the sarcoplasmic reticulum stores so that more calcium is available for release by subsequent action potentials, leading to an increase in intracellular calcium transients and contractions.
Characterization of P2‐purinoceptor mediated cyclic AMP formation in mouse C2C12 myotubes
Authors: Henning Robert H. , Duin Marry , den Hertog Adriaan , Nelemans Adriaan
Published: July 2012

The formation of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2C12 myotubes.

ATP (100 μm) and ATP7S (100 μm) caused a sustained increase in cyclic AMP content of the cells, reaching a maximum after 10 min. The cyclic AMP content reached a maximum in the presence of 100 μm ATP, followed by a decline at higher ATP concentrations. ATP‐induced cyclic AMP formation was inhibited by the P2‐purinoceptor antagonist, suramin.

Myotubes hydrolysed ATP to ADP at a rate of 9.7 ± 1.0 nmol mg−1 protein min−1. However, further hydrolysis of ADP to AMP and adenosine was negligible.

The cyclic AMP formation induced by ADP (10 μm–1 mm) showed similar characteristics to that induced by ATP, but a less pronounced decline was observed than with ATP. ADP‐induced cyclic AMP formation was blocked by suramin, while cyclic AMP formation elicited by adenosine (10 μm–1 mm) was insensitive to suramin.

The ATP analogue, α,β‐methylene‐ATP also induced a suramin‐sensitive cyclic AMP formation, while 2‐methylthio‐ATP and the pyrimidine, UTP, did not affect cyclic AMP levels.

Stimulation of the myotubes with ATP or UTP (10 μm–1 mm) caused a concentration‐dependent increase in the Ins(1,4,5)P3 content of the cells. ADP (100 μm–1 mm) was less effective. Adenosine did not affect Ins(1,4,5)P3 levels.

Incubation of the cells with UTP (30 μm–1 mm) inhibited the ATP‐ and ADP‐induced cyclic AMP formation, suggesting that stimulation of the ‘nucleotide’ type P2‐receptor inhibits P2‐purinoceptor mediated cyclic AMP formation in C2C12 myotubes. In contrast, UTP (30 μm–1 mm) enhanced adenosine‐induced cyclic AMP formation.

Adenosine‐sensitive P1‐purinoceptors activating cyclic AMP formation were found in C2C12 myotubes. Further, a novel P2‐purinoceptor is postulated, sensitive to ATP, ADP and ATPγS, which also activates the formation of cyclic AMP in C2C12 myotubes.
Partial inhibition by epithelium of tracheal smooth muscle relaxation induced by the potassium channel activator, BRL 38227
Authors: Pavlovic Dragan , Brione Eveline , De Vernejoul Dominique , Aubier Michel
Published: July 2012

A method is described whereby either the serosal (Out) or epithelial (In) sides of rat isolated tracheae were selectively perfused. Perfusion with BRL 38227 (10−8–5 × 10−6 m; In/Out) of preparations with intact epithelium (+ EP) precontracted with carbachol (10−6 m; Out/In) produced complete relaxation. Perfusion with aminophylline (10−5–10−3 m; In) of + EP preparations precontracted with carbachol (10−6 m; Out) also produced complete relaxation.

In preparations precontracted with carbachol (10−6 m) epithelium removal (− EP) increased the sensitivity to the relaxant effect of BRL 38227 (In), but not BRL 38227 (Out) [− log EC50, + EP/ − EP; carbachol (In), BRL 38227 (Out): 6.76 ± 0.11 vs 6.67 ± 0.15; carbachol (Out), BRL 38227 (In): 5.93 ± 0.06 vs 6.25 ± 0.07]. Removal of the epithelium increased also the sensitivity to BRL 38227 (In) of preparations precontracted with a lower concentration (5 × 10−7 m) of carbachol (Out). [− log EC50, + EP/ − EP, carbachol (Out), BRL 38227 (In): 6.19 ± 0.14 vs 6.58 ± 0.17].

Removal of the epithelium did not affect the sensitivity to BRL 38227 (In) of preparations precontracted with a higher concentration (5 × 10−6 m) of carbachol (Out).

In both + EP and − EP preparations precontracted with carbachol (10−6 M; Out), BRL 38227 (In) had a more potent relaxant effect than aminophylline (In) (EC50, BRL 38227 vs aminophylline, + EP/ − EP: 5.93 ± 0.06 vs 3.66 ± 0.11/6.25 ± 0.07 vs 3.77 ± 0.11).

In preparations precontracted with carbachol (10−6 m; Out), removal of the epithelium did not affect the sensitivity to aminophylline (In) but increased the degree of precontraction (Tmax) following epithelial but not serosal stimulation with carbachol.

We conclude that BRL 38227, a K+ channel activator, is a potent relaxant of rat tracheal smooth muscle precontracted with carbachol, and that the effect can be partially inhibited by the presence of an intact tracheal epithelium, whereas the relaxant effect of aminophylline is not.
Involvement of nitric oxide in the endothelium‐dependent relaxation induced by hydrogen peroxide in the rabbit aorta
Authors: Zembowicz Artur , Hatchett Richard J. , Jakubowski Andrzej M. , Gryglewski Richard J.
Published: July 2012

The effects of hydrogen peroxide (H2O2, 0.1–1 mm) on the tone of the rings of rabbit aorta precontracted with phenylephrine (0.2–0.3 μm) were studied.

H2O2 induced a concentration‐dependent relaxation of both the intact and endothelium‐denuded rings. However, in the presence of intact endothelium, H2O2‐induced responses were 2–3 fold larger than in its absence, demonstrating the existence of endothelium‐independent and endothelium‐dependent components of the vasorelaxant action of H2O2.

The endothelium‐dependent component of H2O2‐induced relaxation was prevented by NG‐nitro‐l‐arginine methyl ester (l‐NAME, 30 μm) or NG‐monomethyl‐l‐arginine (300 μm), inhibitors of nitric oxide synthase (NOS), in a manner that was reversible by l‐, but not by d‐arginine (2 mm). The inhibitors of NOS did not affect the responses of denuded rings.

Methylene blue (10 μm), an inhibitor of soluble guanylate cyclase, blocked H2O2‐induced relaxation of both the intact and denuded rings.

H2O2 (1 mm) enhanced the efflux of cyclic GMP from both the endothelium‐intact and denuded rings. The effect of H2O2 was 4 fold greater in the presence of intact endothelium and this endothelium‐dependent component was abolished after the inhibition of NOS by l‐NAME (30 μm).

In contrast to the effects of H2O2, the vasorelaxant action of stable organic peroxides, tert‐butyl hydroperoxide or cumene hydroperoxide, did not have an endothelium‐dependent component. Moreover, they did not potentiate the efflux of cyclic GMP from the rings of rabbit aorta.

Exogenous donors of NO, specifically, 3‐morpholinosydnonimine (SIN‐1), glyceryl trinitrate or sodium nitroprusside were used to decrease the tone of denuded rings to the level induced by endogenous NO released from intact endothelium. This procedure did not influence the vasorelaxant activity of H2O2, showing that H2O2 does not potentiate the vasorelaxant action of NO within the smooth muscle.

Thus, H2O2‐induced relaxation in the rabbit aorta has both endothelium‐dependent and independent components. The endothelium‐dependent component of the relaxant action of H2O2 is due to enhanced endothelial synthesis of NO.
Blockade by 2,2′,2″‐tripyridine of the nicotinic acetylcholine receptor channels in embryonic Xenopus muscle cells
Authors: Hsu K.S. , Fu W.M. , Lin‐Shiau S.Y.
Published: July 2012

The effects of 2,2′,2″‐tripyridine on the nicotinic acetylcholine (ACh) receptor channels were studied in the cultured myocytes of 1‐day‐old Xenopus embryos.

2,2′,2″‐Tripyridine depressed the amplitude of iontophoretic ACh‐induced current at a low frequency of 0.7 Hz stimulation and it not only decreased the initial responses but also enhanced the run‐down of ACh‐induced current at higher frequency stimulation of 7 Hz and 30 Hz.

Single ACh channel recordings showed that 2,2′,2″‐tripyridine decreased the channel conductance, the opening frequency and mean open time of both types of low‐ and high‐conductance channels.

These results suggest that the blocking actions of 2,2′,2″‐tripyridine on ACh receptor channels in the skeletal muscle may contribute to the depression of the nerve‐evoked contraction of the mouse diaphragm as reported previously.
Estimation of partial agonist affinity by interaction with a full agonist: a direct operational model‐fitting approach
Authors: Leff P. , Dougall I.G. , Harper D.
Published: July 2012

The operational model of agonism (Black & Leff, 1983) has been extended to describe the interaction between a partial agonist and a full agonist at the same receptor. The derived equation explicitly describes the interaction and allows the affinity (and efficacy) of the partial agonist to be estimated by direct fitting of raw experimental agonist concentration‐effect (E/[A]) curve data.

The model was used to analyse experimental E/[A] curve data generated for the interaction between pilocarpine (partial agonist) and carbachol (full agonist) at the M3‐muscarinic receptor mediating contraction of the guinea‐pig isolated trachea. Pilocarpine affinity estimates obtained by operational model‐fitting were compared with those obtained by use of the null method (Stephenson, 1956). These analyses demonstrated that the two methods gave comparable results (mean pKB estimates were 5.79 and 5.86 for the operational model and null method respectively).

When multiple concentrations of partial agonist are used, simultaneous operational model‐fitting of all the E/[A] curve data allows the competitive nature of the interaction to be studied.

We conclude that operational model‐fitting is a valid and analytically simple alternative to the conventional null method of analysing full/partial agonist interactions.
Effects of morphine metabolites on micturition in normal, unanaesthetized rats
Authors: Igawa Yasuhiko , Westerling Dagmar , Mattiasson Anders , Andersson Karl‐Erik
Published: July 2012

By means of continuous cystometry in normal, unanaesthetized rats, the effects on micturition of intrathecally (i.t.) administered morphine‐3‐glucuronide (M3G) and morphine‐6‐glucuronide (M6G), the two main metabolites of morphine, were studied and compared with those of i.t. morphine.

Both M6G (0.01, 0.1, and 0.5 μg) and M3G (5 μg) were found to have significant effects on micturition. Like morphine (0.1, 0.5, and 10 μg), M6G was able to inhibit the micturition reflex, and produce urinary retention and dribbling incontinence in a dose‐dependent manner. The potency of M6G for inhibiting micturition was approximately 10 times higher than that of morphine, and the duration of its effect was longer. All effects of M6G could be reversed by naloxone.

M3G (5 μg) facilitated the micturition reflex, resulting in decreases in bladder capacity and micturition volume, and an increase in spontaneous contractile activity. Pretreatment with naloxone (10 μg), which by itself had no effect on micturition, enhanced the facilitatory effects of M3G. In addition, M3G tended to counteract the inhibitory effects of both morphine and M6G on micturition. M3G (5 μg) also produced an excitatory behavioural syndrome.

It is concluded that in rats, i.t. M3G has excitatory effects on micturition and behaviour, probably not mediated via opioid receptors. I.t M6G has a potent inhibitory effect on micturition mediated by stimulation of opioid receptors. It may have effects on somatosensory afferent input in lower doses than those required for effects on micturition.
Inhibitory actions of ZENECA ZD7288 on whole‐cell hyperpolarization activated inward current (If) in guinea‐pig dissociated sinoatrial node cells
Authors: BoSmith Robert E. , Briggs Ian , Sturgess Nicholas C.
Published: July 2012

ZENECA ZD7288 (4‐(N‐ethyl‐N‐phenylamino)‐1,2‐dimethyl‐6‐(methylamino) pyrimidinium chloride) is a sinoatrial node (SAN) modulating agent which produces a selective slowing of the heart rate. Its effects have been studied in single, freshly dissociated guinea‐pig SAN cells, by standard patch clamp procedures.

Whole‐cell inward currents were evoked by hyperpolarizing voltage clamp steps from a holding potential of −40 mV. ZD7288 inhibited the hyperpolarization activated cationic current (If) in a concentration‐dependent manner. The ‘selective bradycardic agents' alinidine and UL‐FS 49 (zatebradine) both also inhibited If.

The activation of If was investigated by measuring tail current amplitudes at +20 mV after hyperpolarizing steps to different potentials to activate the current. The reduction in If resulted from both a shift in the If current activation curve in the negative direction on the voltage axis, and also a reduction in the activation curve amplitude.

ZD7288 did not affect the ion selectivity of the If channel, since the tail current reversal potential was unchanged in the presence of the drug.

With ZD7288 the inhibition of If was not use‐dependent, whereas UL‐FS 49 displayed use‐dependence in the block of the If current.

Whereas ZD7288 had no significant effect on the delayed rectifier current (Ik) in these cells, both alinidine and UL‐FS 49 significantly reduced Ik at the same concentrations which reduced If.

The data show that ZD7288 reduces If by affecting the activation characteristics of the If current; this inhibition may account for this agent's selective bradycardic properties.
Nitric oxide‐dependent and ‐independent hyperaemia due to calcitonin gene‐related peptide in the rat stomach
Authors: Holzer P. , Lippe I.Th. , Jocic̆ M. , Wachter Ch. , Erb R. , Heinemann A.
Published: July 2012

Calcitonin gene‐related peptide (CGRP) potently enhances mucosal blood flow in the rat stomach. The aim of this study was to examine whether CGRP also dilates extramural arteries supplying the stomach and whether the vasodilator action of CGRP involves nitric oxide (NO).

Rat CGRP‐α (0.03–1 nmol kg−1, i.v.) produced a dose‐dependent increase in blood flow through the left gastric artery (LGA) as determined by an ultrasonic transit time technique in urethane‐anaesthetized rats. Blockade of NO synthesis by NG‐nitro‐l‐arginine methyl ester (l‐NAME, 20 and 60 μmol kg−1, i.v.) significantly reduced basal blood flow (BF) in the LGA and attenuated the hyperaemic activity of CGRP by a factor of 2.8–4. d‐NAME tended to enhance basal BF in the LGA but had no influence on the dilator activity of CGRP. The ability of vasoactive intestinal polypeptide to increase left gastric arterial blood flow remained unaltered by l‐NAME.

l‐NAME (20 and 60 μmol kg−1, i.v.) evoked a prompt and sustained rise of mean arterial blood pressure (MAP) and caused a slight decrease in the hypotensive activity of CGRP. In contrast, d‐NAME induced a delayed and moderate increase in MAP and did not influence the hypotensive activity of CGRP.

Rat CGRP‐α dilated the isolated perfused bed of the rat LGA precontracted with methoxamine and was 3 times more potent in this respect than rat CGRP‐β. The dilator action of rat CGRP‐α in this preparation was not affected by l‐NAME or d‐NAME (40 μm).

L‐NAME (60 μmol kg−1, i.v.) reduced gastric mucosal blood flow as assessed by laser Doppler flowmetry and diminished the hyperaemic activity of rat CGRP‐α in the gastric mucosa by a factor of 4.5, whereas d‐NAME was without effect.

These data show that CGRP is a potent dilator of mucosal and extramural resistance vessels in the rat stomach. Its dilator action involves both NO‐dependent and NO‐independent mechanisms.
E‐type prostaglandins enhance local oedema formation and neutrophil accumulation but suppress eosinophil accumulation in guinea‐pig skin
Authors: Teixeira M.M. , Williams T.J. , Hellewell P.G.
Published: July 2012

Prostaglandins possess both pro‐ and anti‐inflammatory actions depending on their route of administration and the experimental model used. In this study, we have investigated the effect of locally injected prostaglandins on oedema formation, neutrophil accumulation and eosinophil accumulation in inflammatory responses in guinea‐pig skin.

Prostaglandin E1 (PGE1) significantly enhanced local oedema formation induced by zymosan‐activated plasma (ZAP), bradykinin and in a passive cutaneous anaphylatic (PCA) reaction. The accumulation of ZAP‐induced 111In‐labelled neutrophils was also significantly enhanced by PGE1. In addition, the prostacyclin analogue, iloprost, enhanced ZAP‐induced responses.

In contrast PGE1 decreased the accumulation of 111In‐labelled eosinophils in skin sites. This was demonstrated on eosinophil accumulation and local eodema formation induced by PAF, compound 48/80 and in the PCA reaction. PGE2 also suppressed eosinophil accumulation while iloprost had no detectable effect.

Isoprenaline inhibited eosinophil accumulation in a dose‐dependent manner with no effect on local oedema formation, except in the case of responses to ZAP where suppression was observed.

The vasodilator neuropeptide, calcitonin gene‐related peptide (CGRP), enhanced local oedema formation but had no detectable effect on eosinophil accumulation.

In conclusion, the magnitude of a given response to an inflammatory mediator in vivo depends on the net effect of stimulation of several cell types e.g. arteriolar smooth muscle cells, microvascular endothelial cells, mast cells and accumulating leukocytes. In this study, we have demonstrated that different components of the inflammatory response in guinea‐pig skin can be differentially modulated by E‐type prostaglandins and isoprenaline, suggesting that cyclic AMP has an important regulatory role.
Cyclopiazonic acid, an inhibitor of Ca2+‐ATPase in sarcoplasmic reticulum, increases excitability in ileal smooth muscle
Authors: Uyama Yoshiaki , Imaizumi Yuji , Watanabe Minoru
Published: July 2012

Effects of cyclopiazonic acid (CPA), a specific inhibitor of Ca2+‐ATPase in endo‐ and sarcoplasmic reticulum (ER/SR), on contractile responses, cytosolic Ca2+ concentration and spontaneous electrical activity were examined in ileal longitudinal smooth muscle strips.

After intracellular stored Ca2+ in intact ileal strips was depleted by application of 25 mm caffeine in Ca2+‐free solution, Ca2+‐loading was performed in the absence or presence of 10 μm CPA in a standard solution containing 2.2 mm Ca2+. Subsequent application of caffeine in Ca2+‐free solution induced a phasic contraction which was significantly smaller in the strip pretreated with CPA than that in the control.

Spontaneous and 20 mm K+‐induced contractions in the presence of 1 μm atropine were markedly enhanced by 1–30 μm CPA, whereas that induced by 80 mm K+ was not. The magnitude of repetitive transient elevation of cytosolic Ca2+ concentration ([Ca2+]i) and concomitant phasic contractions were markedly enhanced by CPA. The effects were abolished by 10 μm verapamil and restored by 10 μm Bay K 8644.

Application of 10 μm CPA depolarized the cell by about 5 mV, decreased the action potential (AP) afterhyperpolarization and markedly increased the frequency of spontaneous AP. These effects were mimicked by 100 nm charybdotoxin.

The rate of decay of [Ca2+]i and tension after the bathing solution was changed from one containing 140 mm K+ and 2.2 mm Ca2+ to one containing 5.9 mm K+ and 0 mm Ca2+ was significantly slowed when 10 μm CPA was added to the latter solution.

These results indicate that CPA enhances ileal smooth muscle excitability and increases Ca2+‐influx through voltage‐dependent Ca2+ channels. The effect may be consistent with the hypothesis that CPA‐induced decrease in stored Ca due to Ca‐pump inhibition reduces the Ca2+‐dependent K+ current and indirectly enhances Ca2+‐influx through membrane activity resulting from the increased excitability. Direct evidence for the regulation of Ca2+ channel activity by intracellular Ca storage sites was not obtained in the present study.
Protection by oestradiol against the development of cardiovascular changes associated with monocrotaline pulmonary hypertension in rats
Authors: Farhat Michel Y. , Chen Ming‐Fong , Bhatti Tahira , Iqbal Azhar , Cathapermal Seedabarum , Ramwell Peter W.
Published: July 2012

We studied the effects of oestradiol 17β on the development of pulmonary vascular changes and right ventricular (RV) hypertrophy in response to monocrotaline in male Sprague‐Dawley rats.

Rats were treated with either placebo or oestradiol 17β (10 mg) in the form of slow release pellets implanted subcutaneously 48 h before monocrotaline administration. Rats were injected with either saline or a single dose of monocrotaline (60 mg kg−1, i.m.). Pulmonary vascular changes and RV hypertrophy were studied at 4 weeks following monocrotaline administration.

Monocrotaline induced a significant increase in the ratio of right ventricle (RV) to left ventricle‐plus‐septum (LV + S) weights. Monocrotaline‐treated rats also showed significant myointimal proliferation in small pulmonary arteries, decrease of arterial numbers and increase in the number of abnormal alveolar macrophages.

Oestradiol 17β attenuated myointimal hyperplasia in pulmonary vessels, decreased the RV/(LV + S) ratio in monocrotaline‐treated rats. Oestradiol 17β had no significant effect on control animals.

Oestradiol treatment prevented the increase in lung wet to dry weight ratio, observed 7 days post monocrotaline administration.

These results suggest that oestradiol 17β protects against the pulmonary vascular remodelling and RV hypertrophy associated with monocrotaline‐induced pulmonary hypertension in the rat. Oestradiol also protects against microvascular leak observed in the early days of lesion.
Pharmacological activity of (−)‐discretamine, a novel vascular α‐adrenoceptor and 5‐hydroxytryptamine receptor antagonist, isolated from Fissistigma glaucescens
Authors: Ko Feng‐Nien , Yu Sheu‐Meei , Su Ming‐Jai , Wu Yang‐Chang , Teng Che‐Ming
Published: July 2012

The pharmacological activity of (−)‐discretamine, isolated from Fissistigma glaucescens, was determined in rat isolated thoracic aorta, cardiac tissues and ventricular myocytes and guinea‐pig isolated trachea.

(−)‐Discretamine was found to be an α1‐adrenoceptor blocking agent in rat thoracic aorta as revealed by its competitive antagonism of noradrenaline (pA2 = 7.20 ± 0.10)‐ or phenylephrine (pA2 = 7.60 ± 0.09)‐induced vasoconstriction. It was as potent as phentolamine (pA2 = 7.51 ± 0.10), but was more potent than yohimbine (pA2 = 6.18 ± 0.06). Removal of endothelium significantly increased the antagonistic potency of (−)‐discretamine on noradrenaline (pA2 = 7.52 ± 0.09)‐ or phenylephrine (pA2 = 7.90 ± 0.09)‐induced vasoconstriction.

(−)‐Discretamine was also an α2‐adrenoceptor blocking agent (pA2 = 6.30 ± 0.15) and a 5‐hydroxytryptamine antagonist (pA2 = 6.87 ± 0.06), both in rat aorta denuded of endothelium.

(−)‐Discretamine protected α‐adrenoceptors from alkylation by the irreversible blocking agent, phenoxybenzamine.

[3H]‐inositol monophosphate formation caused by noradrenaline (3 μm) in rat thoracic aorta was suppressed by (−)‐discretamine (10 and 30 μm) and prazosin (3 μm).

A high concentration of (−)‐discretamine (30 μm) did not affect the contraction induced by the thromboxane receptor agonist U‐46619, prostaglandin F2α (PGF2α), angiotensin II, high K+ or endothelin in rat aorta denuded of endothelium. Neither cyclic AMP nor cyclic GMP content of rat thoracic aorta was changed by (−)‐discretamine.

Contraction of guinea‐pig trachea caused by histamine, leukotriene C4 or carbachol was not affected by (−)‐discretamine (30 μm). (−)‐Discretamine also did not block β1 or β2‐adrenoceptor‐mediated responses induced by isoprenaline in rat right atria and guinea‐pig trachea.

A voltage clamp study in rat ventricular single myocytes revealed that sodium inward current, slow inward Ca2+ current or transient (Ito) and steady state (I800) outward current was not affected by (−)‐discretamine (30 μm).

It is concluded that (−)‐discretamine is a selective α‐adrenoceptor and 5‐HT receptor antagonist in vascular smooth muscle.
Solubilization and characterization of GABABreceptor binding sites from porcine brain synaptic membranes
Authors: Facklam M. , Bowery N.G.
Published: July 2012

The characteristics of membrane bound GABABreceptors in pig brain are similar to those in rat brain as judged byin vitrobinding experiments and sensitivity to GTP. The rank order of affinity of GABABreceptor ligands was CGP 54626 > GABA ∼(−)‐baclofen >> CGP 35348 = CGP 36742 > (+)‐baclofen in membranes from both species.

For solubilization of GABABreceptors from pig brain, washed membranes were preincubated with 5 mmMgSO4and subsequently incubated with various detergents. 3‐[(3‐Cholamidopropyl)dimethyl‐ammoniol]‐1‐propane sulphonate (CHAPS) (0.5%) proved to be the most successful, solubilizing 22.7 ± 4.7% (mean ± s.e.mean,n= 6) of GABABreceptors.

Binding of [3H]‐GABA to GABABreceptors solubilized with 0.5% CHAPS exhibited similar characteristics to the binding at membrane bound receptors since, firstly, theKdandBmaxvalues (around 30 nmand 450 fmol mg−1protein, respectively) were comparable; secondly, stereospecific binding for baclofen was obtained in both forms; thirdly, the affinity for the agonists GABA and (−)‐baclofen and the antagonists CGP 35348, CGP 36742 and CGP 54626 were the same; fourthly, comparable sensitivity to Ca2+(2.5 mm) was observed and finally, a similar sensitivity to GTP was apparent.

Saturation experiments performed with the GABABantagonist, [3H]‐CGP 54626, indicated a higherKdvalue and a lowerBmaxvalue for solubilized (7.7 ± 2.6 nmand 1033 ± 41 fmol mg−1protein, mean ± s.e.mean,n= 3) than for membrane bound receptors (1.35 ± 0.08 nm, 1171 ± 20 fmol mg−1protein,n= 3).
Protection by scoparone against the alterations of plasma lipoproteins, vascular morphology and vascular reactivity in hyperlipidaemic diabetic rabbit
Authors: Huang Huei‐Chen , Weng Yu‐I , Lee Chai‐Rong , Jan Tong‐Rong , Chen Yuh‐Lien , Lee Yuan‐Teh
Published: July 2012

The in vivo pharmacological effects of scoparone (6,7‐dimethoxycoumarin) in a hyperlipidaemic diabetic rabbit model were investigated.

Three groups of rabbits were studied: (1) normal, (2) hyperlipidaemic and diabetic‐untreated and (3) hyperlipidaemic and diabetic‐scoparone treated. The hyperlipidaemic diabetic rabbits were fed with 1% cholesterol and treated with alloxan, a diabetogenic agent. The plasma levels of total cholesterol, total triglyceride, very low‐density lipoprotein (VLDL) cholesterol, low density lipoprotein (LDL) cholesterol and high‐density lipoprotein (HDL) cholesterol were markedly increased as soon as the rabbit became diabetic at the second week. Scoparone‐treatment (5 mg kg−1 day−1, s.c.) significantly reduced the plasma lipid and lipoprotein cholesterol levels of the hyperlipidaemic diabetic rabbit to 73.3% of total cholesterol, 48.3% of total triglyceride, 66.0% of VLDL cholesterol, 55.7% of LDL cholesterol and 79.5% of HDL cholesterol.

Six weeks after cholesterol‐feeding, the aortic arch and thoracic aorta were dissected for morphological and functional studies. In vascular rings from the untreated hyperlipidaemic diabetic rabbit, there was intimal thickening with accumulation of fatty streaks, foam cells and migration of smooth muscle cells to the intima. In the rabbits treated with scoparone, there were fewer pathological morphology changes found in vascular segments than in the untreated hyperlipidaemic diabetic rabbits.

In the vascular reactivity experiments, the phenylephrine‐induced contraction and nitroprusside‐induced dilatation did not differ significantly among the three rabbit groups, except that the contraction was enhanced in the thoracic aorta of hyperlipidaemic diabetic rabbits either untreated or treated with scoparone, as compared to the normal group, and the sensitivity to nitroprusside was increased in the thoracic aorta of the scoparone‐treated group as compared to the untreated group.

The endothelium‐dependent dilatation induced by acetylcholine was significantly attenuated in both the aortic arch and thoracic aorta from the hyperlipidaemic diabetic rabbits as compared to the normal rabbits. This attenuation was partially prevented, when scoparone (5 mg kg−1) was administered daily.

These results suggest that scoparone protects against some alterations of plasma lipoproteins, vascular morphology and vascular reactivity in the hyperlipidaemic diabetic rabbit. These protective effects of scoparone may be partly related to its free radical scavenging property.
Different pathways of calcium sensitization activated by receptor agonists and phorbol esters in vascular smooth muscle
Authors: Hori Masatoshi , Sato Koichi , Miyamoto Shigeki , Ozaki Hiroshi , Karaki Hideaki
Published: July 2012

It has been shown that receptor agonists and activators of protein kinase C, phorbol esters, increase Ca2+ sensitivity of contractile elements in vascular smooth muscle. To discover if protein kinase C is involved in the agonist‐mediated Ca2+ sensitization, we examined the effects of receptor agonists in the rat isolated aorta in which protein kinase C activity had been diminished by pretreatment with phorbol 12‐myristate 13‐acetate for 24 h.

In the aorta with protein kinase C activity, a high concentration (1 μm) of 12‐deoxyphorbol 13‐isobutyrate induced contraction and a low concentration (100 nm) potentiated high K+‐induced contraction. In addition, prostaglandin F2α induced greater contractions than high K+ at a given cytosolic Ca2+ level. The maximally effective concentrations of noradrenaline and endothelin‐1 also induced greater contraction than high K+. In the aorta without protein kinase C activity, the contraction induced by 12‐deoxyphorbol 13‐isobutyrate and its potentiation of the high K+‐induced contraction were abolished. However, prostaglandin F2α, noradrenaline and endothelin‐1 still induced a greater contraction than high K+.

In the aorta without protein kinase C activity, noradrenaline, endothelin‐1 and prostaglandin F2α, but not 12‐deoxyphorbol 13‐isobutyrate, induced contractions in the presence of the Ca2+ channel blocker, verapamil, or in the absence of external Ca2+, by increasing Ca2+ sensitivity.

In the permeabilized preparations, inhibition of protein kinase C activity abolished the effect of potentiation of the Ca2+‐induced contraction by 12‐deoxyphorbol 13‐isobutyrate although the potentiation of the contraction by prostaglandin F2α did not change.

These results suggest that there are two pathways for Ca2+ sensitization in rat aorta; a protein kinase C‐dependent pathway which is activated by phorbol esters, and a protein kinase C‐independent pathway which is activated by receptor agonists.
GABAB receptors modulate catecholamine secretion in chromaffin cells by a mechanism involving cyclic AMP formation
Authors: Oset‐Gasque M.J. , Parramón M. , González M.P.
Published: July 2012

The function of γ‐aminobutyric acidB (GABAB) receptors in modulation of catecholamine secretion by chromaffin cells and the possible mechanism involved in this action have been examined.

The GABAB agonists (−)‐baclofen and 3‐aminopropylphosphinic acid (3‐APPA) were found to induce a dose‐dependent increase of basal catecholamine secretion. The EC50s were 151 ± 35 μm and 225 ± 58 μm for baclofen and 3‐APPA, respectively. This stimulatory effect was specific since it could be blocked by 0.5 mm of the specific GABAB antagonist CGP‐35348.

In contrast, preincubation of chromaffin cells with the GABAB agonists was found to inhibit, in a dose‐dependent manner, the catecholamine secretion evoked by 10 μm nicotine and 200 μm muscimol.

The effects of GABAB agonists on both basal and evoked catecholamine secretion were found to be accompanied by parallel changes in intracellular calcium concentration ([Ca2+]i). GABAB agonists produced a dose‐dependent increase in [Ca2+]i which was partially blocked by CGP 35348, but they produced a strong inhibition of the [Ca2+]i increase induced by nicotine and muscimol.

The GABAB agonists also produced a dose‐dependent increase in intracellular cyclic AMP levels, there being a direct correlation between both increase in catecholamine secretion and in intracellular cyclic AMP levels.

The pretreatment of chromaffin cells with pertussis toxin doubled the catecholamine secretion and increased by four times the intracellular cyclic AMP levels evoked by GABAB agonists.

The possible involvement of adenylate cyclase in the mechanism of GABAB receptor modulation of catecholamine secretion is discussed.