bluuebunny/crossref_metadata_2025 huggingface dataset:
α2‐Adrenoceptor subtypes and imidazoline‐like binding sites in the rat brain
Authors: Brown C.M. , MacKinnon A.C. , McGrath J.C. , Spedding M. , Kilpatrick A.T.
Published: July 2012

The binding of [3H]‐yohimbine and [3H]‐idazoxan to rat cortex and hippocampus is rapid, reversible and of high affinity. Saturation data indicate that a single population of binding sites exist for [3H]‐yohimbine in the cortex (Bmax 121 ± 10 fmol mg−1, protein; Kd 5.2 ± 0.9 nm) and hippocampus (Bmax 72 ± 6 fmol mg−1 protein; Kd 5.8 ± 0.7 nm). [3H]‐idazoxan labels one site in the cortex (Bmax 87 ± 8 fmol mg−1 protein; Kd 4.1± 0.9 nm) and hippocampus (Bmax 30 ± 6 fmol mg−1 protein; Kd 3.5 + 0.5 nm), when 3 μm phentolamine is used to define non‐specific binding. A second distinct [3H]‐idazoxan binding site (Bmax 110 ± 21fmolmg_1 protein; Kd 3.6 ± 0.07 nm) is identified in rat cortex if 0.3 μm cirazoline is used to define non‐specific binding and 3 μm yohimbine is included to prevent binding to α2‐adrenoceptors.

Displacement studies indicate that the α1‐adrenoceptor antagonist prazosin and the 5‐HT1 ligands 8‐OH‐DPAT, RU 24969 and methysergide differentiate [3H]‐yohimbine binding into two components; a high and low affinity site. In contrast the displacement of [3H]‐idazoxan by each ligand was monophasic.

The affinities of 8‐OH‐DPAT, RU 24969 and methysergide determined against [3H]‐idazoxan binding to the cortex and hippocampus correlate significantly with the binding site displaying low affinity for prazosin and previously designated α2A. In contrast, a poor correlation exists for the high affinity site for prazosin designated α2B.

[3H]‐idazoxan, in the presence of 3 μm yohimbine, labels a site that displays high affinity towards cirazoline, naphazoline and guanabenz, but low affinity towards clonidine, p‐aminoclonidine, adrenaline, noradrenaline and the α2‐adrenoceptor antagonists yohimbine, rauwolscine, WY 26703 and BDF 6143.

The results of this study indicate that [3H]‐yohimbine labels two sites; the α2A‐ and α2B‐adrenoceptors whereas [3H]‐idazoxan labels an α2‐adrenoceptor with a profile consistent with the α2A‐adrenoceptor subtype. In addition, [3H]‐idazoxan labels an imidazoline binding site in the rat cortex that is pharmacologically distinct from α2‐adrenoceptors. The low affinity of clonidine and p‐aminoclonidine indicates that the imidazoline‐like binding site in rat cortex is different from the site labelled by [3H]‐clonidine and [3H]‐p‐aminoclonidine in human, rat and bovine brain stem, providing evidence of potential heterogeneity within this class of binding sites.
Selective chiral inhibitors of 5‐lipoxygenase with anti‐inflammatory activity
Authors: McMillan R.M. , Girodeau J.‐M. , Foster S.J.
Published: July 2012

The studies described here, using enantiomers of an optically‐active methoxy alkyl thiazole ICI216800 (1‐methoxy‐6‐(naphth‐2‐yl‐methoxy)‐1‐(thiazol‐2‐yl)indane), provide unequivocal evidence for a specific, chiral interaction with 5‐lipoxygenase. In accordance with their biochemical efficacy these compounds also demonstrate enantio‐specific anti‐inflammatory activity in a leukotriene‐mediated model of inflammation. This is the first class of compounds for which 5‐lipoxygenase inhibition and anti‐inflammatory activity have been shown to be mediated via a specific chiral interaction.
The pharmacological characterization of 5‐HT3 receptors in three isolated preparations derived from guinea‐pig tissues
Authors: Butler A. , Elswood C.J. , Burridge J. , Ireland S.J. , Bunce K.T. , Kilpatrick G.J. , Tyers M.B.
Published: July 2012

The pharmacological characterization of the 5‐HT3 receptors in guinea‐pig isolated tissues is described. The tissues used were ileum (longitudinal muscle‐myenteric plexus), colon and vagus nerve. The guinea‐pig isolated colon is a novel preparation.

In the guinea‐pig isolated ileum, 5‐hydroxytryptamine (5‐HT, 1 × 10−8−3 × 10−5 m) and the selective 5‐HT3 receptor agonist 2‐methyl‐5‐HT (3 × 10−7−1 × 10−4 m) caused concentration‐related contractions. The 5‐HT concentration‐response curve was biphasic whilst the 2‐methyl‐5‐HT curve was monophasic. The EC50 value for the low potency portion of the 5‐HT curve was 4.1 × 10−6 m. The EC50 for 2‐methyl‐5‐HT was 1.23 × 10−5 m. Selective 5‐HT3 receptor antagonists caused rightward shifts of the 2‐methyl‐5‐HT curve and the lower potency portion of the 5‐HT curve. Neither ketanserin (1 × 10−6 m) nor methysergide (1 × 10−5 m) antagonized the responses to 5‐HT or 2‐methyl‐5‐HT.

In the guinea‐pig isolated colon, 5‐HT (3 × 10−7−3 × 10−5 m; EC50 2.4 × 10−6 m) caused contractions which were mimicked by 2‐methyl‐5‐HT (1 × 10−6−1 × 10−4 m; EC50 7.2 × 10−6 m). Selective 5‐HT3 receptor antagonists caused rightward displacements of the 5‐HT concentration‐response curves. Neither ketanserin (1 × 10−6 m) nor methysergide (1 × 10−5 m) had any effect on responses to 5‐HT or 2‐methyl‐5‐HT.

In the guinea‐pig isolated vagus nerve, 5‐HT (1 × 10−6−3 × 10−4 m) and 2‐methyl‐5‐HT (1 × 10−5−1 × 10−3 m; EC50 7.6 × 10−5 m) caused depolarizations; at higher concentrations there were afterhyperpolarizations. The maximum response to 2‐methyl‐5‐HT was less than half that to 5‐HT. Selective 5‐HT3 receptor antagonists caused rightward displacements of the 5‐HT concentration‐response curves. Antagonists at other 5‐HT receptors (ketanserin, 1 × 10−5 m and methysergide, 1 × 10−6 m) had no effect.

The estimated affinity values of 5‐HT3 receptor antagonists correlated well between the three models. Phenylbiguanide was inactive as an agonist or antagonist (up to 1 × 10−4 m) in each preparation.

Comparisons with antagonist affinity values obtained in the rat isolated vagus nerve revealed marked differences. Antagonists were generally more potent on the rat isolated vagus nerve, although the differences varied considerably between antagonists.

The results are discussed in terms of species‐related receptor differences.
Pharmacological and biochemical comparison of thyrotropin releasing hormone (TRH) and di‐methyl proline‐TRH on pituitary GH3 cells
Authors: McDermott Alison M. , Wilkin Graham P. , Dickinson Stephen L.
Published: July 2012

The binding of [3H]‐thyrotropin releasing hormone ([3H]‐TRH) and [3H]‐RX77368 (di‐methyl proline TRH) and the ability of these peptides to stimulate phosphoinositide hydrolysis were investigated in the GH3 pituitary cell line.

For both peptides binding was found to be saturable with a single component (Hill slopes were, for TRH, 0.98 and for RX77368, 1.13). TRH bound with greater affinity than RX77368 Kd values were 16 nm and 144 nm respectively. Bmax values were 227 fmol mg−1 protein for TRH and 123 fmol mg−1 protein for RX77368.

The rank order of potency of a series of TRH analogues to inhibit binding was the same versus each peptide. However, unlike with saturation analysis, Hill slopes of all displacing ligands were less than 1.0 against both TRH and RX77368 suggeting either multiple binding sites, alteration of affinity state, negative co‐operativity or some allosteric interaction.

Both peptides stimulated phosphoinositide hydrolysis in a dose‐dependent fashion. TRH was more potent than RX77368, EC50 values were 7.9 ± 1 nm and 96.3 ± 3 nm respectively.

These in vitro data suggest that the greater in vivo potency of RX77368 is not the result of enhanced receptor affinity but is more probably due to its greater metabolic stability.
Characterization of P2x‐receptors in rabbit isolated ear artery
Authors: O'Connor S.E. , Wood B.E. , Leff P.
Published: July 2012

The isolated central ear artery of the rabbit contracts in response to adenosine 5′‐triphosphate (ATP) and analogues, effects proposed to be mediated by stimulation of P2x‐receptors. We have extended the characterization of the purinoceptor in this tissue by examining the effects of a series of receptor agonists. The study was designed in such a way as to avoid factors which normally limit attempts to classify receptors on the basis of agonist potency orders.

d‐α,β‐methylene ATP (d‐α,β‐meATP), d‐β,γ‐methylene ATP (d‐β,γ‐meATP), l‐β,γ‐methylene ATP (l‐β,γ‐meATP), 2‐methylthio‐d‐ATP (2‐MeSATP) and ATP produced concentration‐related contractions of the ear artery with similar maximum responses, suggesting that they were full agonists. Selective desensitization of P2x‐receptors abolished or greatly reduced responses to d‐α,β‐meATP, l‐β,γ‐meATP, d‐β,γ‐meATP and 2‐MeSATP. Responses to ATP were inhibited by desensitization but a significant resistant component was still apparent.

d‐α,β‐meATP was the most potent agonist tested (pA50 6.47 ± 0.04) being 2138 times more potent than ATP and approximately 9 times more potent than l‐β,γ‐meATP. The agonist potency order was: d‐α,β‐meATP > l‐β,γ‐meATP > d‐β,γ‐meATP ≥ 2‐MeSATP > ATP. This is generally consistent with the order proposed for P2x‐receptors. The relative potencies of P2x‐agonists in the rabbit ear artery show both similarities to and differences from data obtained in other smooth muscle preparations.
Haemodynamic profile of an inhibitor of phosphodiesterase III, adibendan (BM 14.478): comparison with nitroprusside and dobutamine in conscious dogs
Authors: Dorszewski A. , Müller‐Beckmann B. , Kling L. , Sponer G.
Published: July 2012

This study was performed to investigate whether cardiac positive inotropic as well as peripheral vasodilator properties of adibendan contribute to its overall haemodynamic profile in conscious dogs.

Haemodynamic measurements were carried out in conscious chronically instrumented dogs after administration of adibendan, sodium nitroprusside or dobutamine.

The cardiovascular changes induced by adibendan (0.01 and 0.03 mg kg−1) resembled those of dobutamine (1.0–4.0 μg kg−1 min−1): left ventricular dP/dt60 (LV dP/dt60), stroke volume (SV) and cardiac output (CO) increased to a similar extent, but mean arterial pressure (MAP) and heart rate (HR) remained unchanged.

In contrast to dobutamine, higher doses of adibendan (0.1–1.0 mg kg−1) decreased MAP and LVEDP. These effects were of a similar magnitude to those observed following nitroprusside administration (0.5–12.5 μg kg−1 min−1). In contrast to nitroprusside, adibendan still showed additional effects on LV dP/dt60 and CO.

From these results, it is concluded that both the peripheral vasodilator and the cardiac positive inotropic action of adibendan contribute to its overall haemodynamic profile.
Interleukin‐1 potentiates histamine‐induced release of prostacyclin from human endothelial cells
Authors: Bull Helen A. , Dowd Pauline M.
Published: July 2012

In human cultured umbilical vein endothelial cells, interleukin‐1 potentiated histamine‐induced release of prostacyclin in a time‐ and concentration‐dependent manner.

In cells incubated with interleukin‐1 for 24 h, maximal potentiation was observed when cells were pre‐incubated with 0.5 u ml−1 interleukin‐1 before stimulation with histamine (1 μm‐1 mm).

In cells incubated with 0.5 u ml−1 interleukin‐1, 20 min pre‐incubation was sufficient to induce a statistically significant potentiation of prostacyclin release induced by 1 μm histamine (P > 0.05).

Nifedipine but not cycloheximide, significantly (P > 0.05) inhibited histamine‐induced release of prostacyclin and interleukin‐1 potentiation of histamine‐induced release of prostacyclin (P > 0.05).

Incubation with 1 u ml−1 interleukin‐1 induced a two fold increase in cellular prostaglandin synthetase activity within 30 min. The enzyme activity increased up to 6 h and was maintained up to 24 h. In cells co‐incubated with cycloheximide and 1 u ml−1 interleukin 1, prostaglandin synthetase activity at 24 h was the same as that in unstimulated cells. Prostacyclin release was not significantly inhibited in cells co‐incubated with cycloheximide and interleukin 1.

These results suggest that interleukin‐1 potentiates histamine‐induced release of prostacyclin by rapid up‐regulation of prostaglandin synthetase activity as well as by inducing synthesis of enzyme protein. These mechanisms may act to potentiate/regulate vascular endothelial responses in inflammatory reactions.
Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo
Authors: Rees D.D. , Palmer R.M.J. , Schulz R. , Hodson H.F. , Moncada S.
Published: July 2012

Three analogues of l‐arginine were characterized as inhibitors of endothelial nitric oxide (NO) synthase by measuring their effect on the endothelial NO synthase from porcine aortae, on the vascular tone of rings of rat aorta and on the blood pressure of the anaesthetized rat.

NG‐monomethyl‐l‐arginine (l‐NMMA), N‐iminoethyl‐l‐ornithine (l‐NIO) and NG‐nitro‐l‐arginine methyl ester (l‐NAME; all at 0.1–100 μm) caused concentration‐dependent inhibition of the Ca2+‐dependent endothelial NO synthase from porcine aortae.

l‐NMMA, l‐NIO and l‐NAME caused an endothelium‐dependent contraction and an inhibition of the endothelium‐dependent relaxation induced by acetylcholine (ACh) in aortic rings.

l‐NMMA, l‐NIO and l‐NAME (0.03–300 mg kg−1, i.v.) induced a dose‐dependent increase in mean systemic arterial blood pressure accompanied by bradycardia.

l‐NMMA, l‐NIO and l‐NAME (100 mg kg−1, i.v.) inhibited significantly the hypotensive responses to ACh and bradykinin.

The increase in blood pressure and bradycardia produced by these compounds were reversed by l‐arginine (30–100 mg kg−1 i.v.) in a dose‐dependent manner.

All of these effects were enantiomer specific.

These results indicate that l‐NMMA, l‐NIO and l‐NAME are inhibitors of NO synthase in the vascular endothelium and confirm the important role of NO synthesis in the maintenance of vascular tone and blood pressure.
Effects of inhibitors of protein kinase C and Na +/H + exchange on α1‐adrenoceptor‐mediated inotropic responses in the rat left ventricular papillary muscle
Authors: Otani Hitomi , Otani Hajime , Uriu Toshiko , Hara Mitsuyoshi , Inoue Masafumi , Omori Kyoko , Cragoe Edward J. , Inagaki Chiyoko
Published: July 2012

α1‐Adrenoceptor stimulation of rat left ventricular papillary muscle produced a triphasic inotropic response: an initial transient positive inotropic effect (PIE) followed by a transient negative inotropic effect (NIE) and a sustained PIE.

The protein kinase C inhibitor, staurosporine, at concentrations ranging from 30 nm to 100 nm inhibited the sustained PIE, but had no significant effect on the transient PIE and NIE.

H‐7, 1‐(5‐isoquinoline sulphonyl)‐2‐methylpiperazine, a less specific inhibitor of protein kinase C than staurosporine, at a concentration of 100 μm inhibited both the transient NIE and the sustained PIE without affecting the transient PIE.

Amiloride, an inhibitor of Na+/H+ exchange, at concentrations ranging from 0.1 mm to 1 mm inhibited the sustained PIE and, at higher concentrations, also inhibited the transient NIE.

An amiloride analogue, 5‐(N‐methyl‐N‐isobutyl)amiloride (MIBA), inhibited only the sustained PIE with an IC50 of 0.3 μm which is approximately two orders of magnitude lower than amiloride.

The receptor‐linked stimulation of Na+/H+ exchange through protein kinase C activation may be a mechanism for α1‐adrenoceptor‐mediated sustained PIE.
Local formation of angiotensin II in the rat aorta: effect of endothelium
Authors: Eglème Cécile , Cressier Floriane , Wood Jeanette M.
Published: July 2012

The local formation of angiotensin II (AII) from its precursors, angiotensin I (AI) and tetradecapeptide (TDP) renin substrate, was studied in intact (with endothelium) and rubbed (without endothelium) aortic rings of the rat.

AI and TDP renin substrate maximally contracted intact tissues in a similar way to AII. The same observations were made in rubbed tissues.

The maximal response and the sensitivity of the aorta to these agonists were greater in rubbed than in intact tissues.

In intact preparations, methylene blue increased the contractile response to AII and TDP to the same extent as endothelium removal.

In intact preparations, AII receptor blockade completely suppressed all contractile responses, converting enzyme inhibition completely blocked the responses to AI and TDP, and renin inhibition partially blocked the responses to TDP.

In rubbed preparations, AII receptor blockade completely inhibited all contractile responses, converting enzyme inhibition completely suppressed the responses to AI but only partially inhibited those to TDP, and renin inhibition partially blocked the responses to TDP.

In conclusion, the formation of AII from TDP and its blockade by a converting enzyme inhibitor and a renin inhibitor shows that converting enzyme and a renin‐like aspartic proteinase are present in the aortic wall. Furthermore, the results show that the endothelium is not essential for the conversion of the TDP to AII, but modulates the responses to locally formed AII through the release of endothelium‐derived relaxing factor (EDRF).
2‐Chloroadenosine induction of vagally‐mediated and atropine‐resistant bronchomotor responses in anaesthetized guinea‐pigs
Authors: Manzini Stefano , Ballati Lido
Published: July 2012

The bronchoconstrictor effects of intravenous administration of adenosine derivatives in anaesthetized non‐curarized guinea‐pigs have been studied.

2‐Chloroadenosine (2‐Cl‐Ade), 5′‐N‐ethylcarboxamideadenosine (NECA) and l‐N6‐phenylisopropyl‐adenosine (l‐PIA) all produced dose‐dependent, transient increases in tracheal insufflation pressure, with an order of potency (NECA ≤ 2‐Cl‐Ade ≫ l‐PIA) typical of A2‐receptor mediated biological responses.

2‐Chloroadenosine‐induced bronchoconstrictor responses disappeared after vagotomy or topical application of tetrodotoxin (TTX) on cervical vagal trunks.

2‐Chloradenosine‐induced bronchospasm was unaffected by atropine (1 mg kg−1 i.v.), physostigmine (50 μg kg−1 i.v.) and hexamethonium (30 mg kg−1 i.v.) but was significantly reduced by theophylline (25 mg kg−1 i.v.).

The magnitude of 2‐Cl‐Ade‐induced bronchospasm was significantly reduced by acute (10 μg kg−1 i.v.) or chronic (55 mg kg−1 s.c. four days before the experiment) pretreatment with capsaicin.

Guanethidine (20 mg kg−1 s.c. on two consecutive days), prazosin (10 μg kg−1 i.v.), diphenhydramine (1 mg kg−1 i.v.) and indomethacin (1 mg kg−1 i.v.) failed to block the bronchomotor response to 2‐Cl‐Ade. In contrast, cyproheptadine (1–5 mg kg−1 i.v.) markedly reduced, but did not abolish the bronchospasm elicited by the purine derivative.

We conclude that in anaesthetized non‐curarized guinea‐pigs, a transient vagally‐mediated bronchospasm can be induced by stimulation of A2‐purinoceptors. This effect is complex and involves, at least in part, stimulation of capsaicin‐sensitive sensory nerves and 5‐hydroxytryptamine release. This experimental model might be useful for the further study of the potential role of adenosine in asthma, and for the evaluation of new antiasthma drugs.
Role of adrenoceptors in vitro and in vivo in the effects of lithium on blood glucose levels and insulin secretion in the rat
Authors: Fontela T. , Hermida O. García , Gómez‐Acebo J.
Published: July 2012

Pretreatment of rats with the non‐selective α‐adrenoceptor antagonist dihydroergotamine counteracts the inhibition of glucose‐induced insulin secretion caused by lithium both in vitro and in vivo. The present study was therefore carried out to specify further which type of adrenoceptor is involved in lithium‐induced hyperglycaemia and inhibition of insulin secretion.

The lithium‐induced effects were reversibly blocked by pretreatment of rats with the α2‐adrenoceptor antagonist yohimbine or a combination of yohimbine and the non‐selective β‐receptor antagonist propranolol, whereas the α1‐receptor antagonist prazosin and propranolol alone were ineffective in blocking these effects.

These findings suggest that the effects of lithium on plasma glucose and insulin levels are mediated mainly by the stimulation of α2‐adrenoceptors.
FPL 63012AR: a potent D1‐receptor agonist
Authors: Smith G.W. , Farmer J.B. , Ince F. , Matu K. , Mitchell P.D. , Naya I. , Springthorpe B.
Published: July 2012

FPL 63012AR is a D1‐receptor agonist in the dog kidney, 10 times as potent as dopamine, reducing renal vascular resistance by 20% with an intra‐arterial dose of 0.42 nmol kg−1.

No prejunctional inhibitory D2‐receptor agonist activity was detected in either the isolated ear artery of the rabbit or in the conscious dog as D2‐receptor‐mediated emesis.

Unlike dopamine, FPL 63012AR had no significant agonist activity at α1‐, α2‐, β1‐ or β2‐adrenoceptors.

FPL 63012AR is a potent inhibitor of [3H]‐noradrenaline uptake (Uptake1) into brain synaptosomes, with an IC50 of 29.5 nm, i.e. 9.2 times more potent than dopamine.

The ability to block Uptake1 in the anaesthetised dog was confirmed by inhibition of the tyramine‐induced pressor and inotropic responses.

Intravenous infusion of FPL 63012AR in anaesthetized and conscious dogs (0.3 to 3 nmol kg−1 min−1) reduced vascular resistance and increased blood flow to the kidney which was accompanied by hypotension and tachycardia.

It is concluded that FPL 63012AR is an example of a novel class of potent agonists at the D1‐receptor. Such compounds may have the potential for use clinically in improving renal perfusion and reducing afterload.
On the blockade of acetylcholine release at mouse motor nerve terminals by β‐bungarotoxin and crotoxin
Authors: Rowan E.G. , Pemberton K.E. , Harvey A.L.
Published: July 2012

β‐Bungarotoxin and crotoxin are phospholipose A2 neurotoxins, which block irreversibly the evoked release of acetylcholine from motor nerve terminals of mouse triangularis sterni preparations.

Extracellular recording of nerve terminal action potentials reveal that inhibition of transmitter release is not associated with failure of the action potential to invade nerve terminals.

When evoked transmitter release (measured as intracellularly recorded endplate potentials) was blocked by β‐bungarotoxin, spontaneous acetylcholine release was stimulated as in control experiments by K+‐induced depolarization and by the Ca2+‐ionophore A23187.

The site of action of the toxins remains to be elucidated but would appear to be associated with the coupling of action potential induced‐depolarization to the release mechanism, rather than with the release mechanism itself.
Effects of platelet activating factor on contractile force and 45Ca fluxes in guinea‐pig isolated atria
Authors: Diez J. , Delpón E. , Tamargo J.
Published: July 2012

The effects of platelet activating factor (PAF) were studied on the electromechanical properties and 45Ca2+ fluxes of guinea‐pig isolated atria.

Both in spontaneously beating and electrically driven atria, PAF (10−12‐10−7m) increased atrial rate but produced a biphasic effect on contractile force. At low concentrations (up to 10−10m) it produced a positive inotropic effect, while at higher concentrations PAF exerted a negative inotropic effect. A similar biphasic effect was observed in the slow contractions elicited by isoprenaline in K+‐depolarized atrial fibres.

The positive inotropic effect of PAF was prevented by verapamil, whereas pretreatment of atria with propranolol, phentolamine, indomethacin or atropine did not modify its positive and negative inotropic actions. BN 52021, a specific PAF antagonist, abolished both the positive and negative inotropic effects.

PAF had no effect on the characteristics of the action potentials recorded in either normally polarized or K+‐depolarized (slow action potential) atrial fibres.

At concentrations at which it increased contractile force, PAF potentiated the contractile responses to Ca2+ (0.9‐9 mm), whereas at negative inotropic concentrations it inhibited them. The negative inotropic effect of PAF was partially reversed in 70% Na+ medium.

At 10−11 m, PAF increased 45Ca2+ uptake and reduced the rate coefficient (kcm for the 45Ca2+ efflux. This increase in 45Ca2+ uptake was abolished in atria pretreated with verapamil or BN 52021. However, 10−7 μ PAF modified neither 45Ca2+ uptake nor efflux in atrial muscle.

These results suggest that in guinea‐pig atria the biphasic inotropic effects of PAF cannot be explained through modifications in the slow inward Ca2+ current or in Na+‐Ca2+ exchange, but may be related to changes in trans‐sarcolemmal Ca2+ entry mediated by specific PAF receptors.
Membrane hyperpolarization, cyclic nucleotide levels and relaxation in the guinea‐pig internal anal sphincter
Authors: Baird A. Anne , Muir T.C.
Published: July 2012

Changes in membrane potential (measured with an intracellular microelectrode) and in cyclic nucleotide (adenosine 3′:5′‐cyclic monophosphate, cyclic AMP and guanosine 3′:5′‐cyclic monophosphate, cyclic GMP) levels (measured by radioimmunoassay) in response to inhibitory non‐adrenergic non‐cholinergic (NANC) field stimulation and drugs were investigated in the guinea‐pig internal anal sphincter (gpIAS) in the presence of phentolamine and atropine (each 10−6 m).

Inhibitory NANC nerve stimulation (single pulse, 5 pulses at 5, 10 and 20 Hz, 0.5 ms supramaximal voltage) and adenosine triphosphate (ATP, 10−7‐10−3 m) inhibited spike discharge, hyperpolarized the membrane and relaxed the sphincter. The effects of inhibitory nerve stimulation were blocked by tetrodotoxin (TTX, 10−6m) and, with those of ATP, were blocked by apamin (5 χ 10−6m).

Isoprenaline (10−9‐10−4m), cromakalim (10−9‐10−5m), sodium nitroprusside (NaNP 10−5m), M&B 22948 (10−4m) and 8‐bromocyclic GMP (8‐Br‐cyclic GMP, 10−4m) also inhibited spike discharge, hyperpolarized the membrane and relaxed the sphincter. The effects of isoprenaline were blocked by propranolol (10−6m). However, forskolin (10−9‐10−7m), M&B 22948 (10−9‐10−5m) and lower concentrations of NaNP (10−9‐10−6m) relaxed the sphincter without affecting the membrane potential.

The characteristics of the membrane potential changes in response to different inhibitory stimuli in the gpIAS differed. Hyperpolarization produced by inhibitory NANC nerve stimulation and ATP were rapid in onset, of brief duration and of comparable amplitude. Isoprenaline and direct electrical stimulation also hyperpolarized the membrane and relaxed the muscle although the extent of the relaxation in these two cases was much less than that with nerve stimulation and ATP. In each case, the membrane potential change preceded relaxation and probably accounted for it.

Both inhibitory NANC nerve stimulation (80 pulses 8 Hz supramaximal voltage 0.5 ms) and ATP (10−4m) raised levels of cyclic GMP significantly and to a comparable degree and relaxed the sphincter. The effect of nerve stimulation was prevented by TTX (10−6m) but not by apamin (5 χ 10−6 m). Isoprenaline (10−4 m), cromakalim (10−5 m) and forskolin (10−5 m) were ineffective.

Inhibitory NANC nerve stimulation (80 pulses 8 Hz 0.5 ms supramaximal voltage) and ATP (10−4m) raised levels of cyclic AMP significantly to a comparable degree and relaxed the sphincter. The increase produced by nerve stimulation was abolished by TTX (10−6m) and apamin (5 × 10−6 m). Isoprenaline (10−4 m), cromakalim (10−5 m) and forskolin (10−5 m) raised levels of this nucleotide significantly.
Effect of diadenosine polyphosphates on catecholamine secretion from isolated chromaffin cells
Authors: Castro E. , Torres M. , Miras‐Portugal M.T. , Gonzalez M.P.
Published: July 2012

The action of several diadenosine polyphosphates (AP3A, AP4A and AP5A) on basal, and on nicotine‐and high K+‐evoked, catecholamine (CA) release has been investigated. Each of the three diadenosine polyphosphates weakly but significantly increased basal CA secretion. This enhancement represented about 10% of the response evoked by 2 μm nicotine.

The evoked secretory response to diadenosine polyphosphates had an absolute requirement for extracellular Ca2+.

In contrast, these compounds had an inhibitory action on nicotine‐evoked release. This response was concentration‐dependent, EC50 values being 3.2 ± 0.4 μm, 4.0 ± 1.6 μm and 19.3 ± 4.0 μm for AP3A, AP4A, and AP5A, respectively. The lower the concentration of nicotine used to evoke secretion, the higher the inhibitory power of these compounds.

The CA secretion evoked by K+‐rich solutions was further enhanced by AP3A and AP5A, whereas AP4A inhibited it. The possible physiological role of these dual actions is discussed.
GABAB receptor‐mediated inhibition of the neurogenic vasopressor response in the pithed rat1
Authors: Kohlenbach Andreas , Schlicker Eberhard
Published: July 2012

The effects of γ‐aminobutyric acid (GABA) and related drugs on the vasopressor response induced by electrical stimulation (single pulse of 30 V and 1 ms) of the preganglionic sympathetic nerve fibres or by injection of noradrenaline 0.3 nmol kg−1 were studied in the pithed rat.

The electrically‐induced increase in diastolic blood pressure was inhibited by GABA and the GABAB‐receptor agonist R‐(−)−baclofen but was not affected by its S‐(+)‐enantiomer and by the GABAA‐receptor agonists muscimol and 3‐aminopropane sulphonic acid.

The dose‐response curve of R‐(−)−baclofen for its inhibitory effect on the electrically‐induced vasopressor response was shifted to the right by the GABAB‐receptor antagonist 2‐hydroxysaclofen, but was not affected by the GABAA‐receptor antagonist bicuculline. 2‐Hydroxysaclofen and bicuculline by themselves did not affect the electrically‐induced vasopressor response.

The increase in diastolic blood pressure induced by exogenous noradrenaline was not affected by the GABA‐related drugs, which also had no (or very slight) effects on the basal diastolic blood pressure.

It is concluded that GABA inhibits catecholamine release in the resistance vessels of the rat via GABAB‐receptors, probably located presynaptically on the postganglionic sympathetic nerve fibres.
Platelet‐leukocyte interaction in adhesion to endothelial cells induced by platelet‐activating factor in vitro
Authors: Hirafuji Masahiko , Shinoda Hisashi
Published: July 2012

Platelet‐activating factor (PAF, 10 nm) did not induce platelet adhesion to endothelial cells cultured in monolayer but it induced their adhesion to protein‐coated plastic. However, PAF induced a marked platelet adhesion to endothelial cells when polymorphonuclear leukocytes (PMNs) were present. Lyso‐PAF had no effect.

Phase‐contrast microscopic examination showed that single platelets rather than their aggregates adhered to the endothelial cell surface around aggregating and adhering PMNs.

Significant platelet adhesion was induced by PAF at concentrations higher than 0.01 nm with the maximal response at 10 nm. Platelet adhesion occurred within minutes after PAF addition, reaching a maximum approximately after 30 min. Platelet adhesion also occurred significantly at a PMN: platelet ratio of 1:800, and linearly up to 1:50.

The PAF‐induced platelet adhesion was suppressed by three structurally unrelated PAF antagonists, WEB 2086, ONO 6240 and BN 52021, in a concentration‐dependent manner.

PAF also increased PMN adhesion to endothelial cell monolayers, which was further augmented by the presence of platelets.

The present study demonstrates that PAF induces platelet adhesion to endothelial cells in vitro when PMNs are present and that there is a close interaction between platelets and PMNs in their adhesion to endothelial cells. The present study further suggests that PMNs could play a central role in platelet adhesion to vascular endothlium in certain pathological conditions.
Regulation of bradykinin receptor level by cholera toxin, pertussis toxin and forskolin in cultured human fibroblasts
Authors: Etscheid Beth G. , Ko Peter H. , Villereal Mitchell L.
Published: July 2012

The effect of bacterial toxins on bradykinin‐triggered release of arachidonic acid was studied in serum‐deprived human foreskin (HSWP) fibroblasts prelabelled with [3H]‐arachidonic acid. An 18‐h exposure of HSWP cells to cholera toxin, pertussis toxin, or forskolin enhanced the bradykinin‐stimulated release of arachidonic acid and metabolites.

Prolonged treatment of HSWP cells with these agents also caused a 3 to 4 fold rise in cell surface [3H]‐bradykinin binding. The rise was inhibited by concurrent incubation with cycloheximide or actinomycin D. In addition, cholera toxin and foreskolin increased [3H]‐bradykinin binding in wildtype PC12 cells, but not in mutant PC12 cells with reduced cyclic AMP‐dependent protein kinase type II activity.

In conclusion, cholera toxin, pertussis toxin and forskolin enhanced arachidonic acid release in response to bradykinin, and increased the number of bradykinin receptors in HSWP fibroblasts. A cyclic AMP‐dependent mechanism appears to mediate the actions of the toxins and forskolin.
The spider toxin, argiotoxin636, binds to a Mg2+ site on the N‐methyl‐d‐aspartate receptor complex
Authors: Reynolds Ian J.
Published: July 2012

The mechanism of action of the arylalkylamine spider toxin, argiotoxin636, on the N‐methyl‐d‐aspartate (NMDA) receptor was investigated by use of [3H]‐dizocilpine binding to well‐washed membranes obtained from rat brain.

Argiotoxin636 decreased [3H]‐dizocilpine binding with an apparent potency of about 3 μm. The inhibition of [3H]‐dizocilpine by argiotoxin636 was insensitive to the concentration of glutamate, glycine and spermidine in the assay.

Argiotoxin636 alone had no effect on the dissociation of [3H]‐dizocilpine. However, argiotoxin636 reversed the actions of Mg2+ on the dissociation of [3H]‐dizocilpine by decreasing the apparent potency of Mg2+. Argiotoxin636 also reversed the action of Ca2+ on the dissociation of [3H]‐dizocilpine.

These results suggest that argiotoxin636 exerts a novel inhibitory effect on the NMDA receptor complex by binding to one of the Mg2+ sites located within the NMDA‐operated ion channel.
Inhibition by galanin and by high K+ of human basophil histamine release triggered by calcium ionophores but not responses induced by anti‐IgE, chemotactic peptide or phorbol ester
Authors: Bergstrand Håkan , Lundquist Britta , Ahrén Bo
Published: July 2012

Galanin concentration‐dependently blocked human leukocyte histamine release triggered by the calcium ionophores A23187 and ionomycin. Almost complete inhibition of release was recorded at 410 nm whereas 41 nm galanin mediated close to 50% inhibition of responses.

Pretreatment of the cells with pertussis toxin did not influence the inhibitory effects of galanin.

Leukocyte responses triggered by anti‐IgE, the chemotactic peptide formyl‐methionyl‐leucyl‐phenylalanine (FMLP) or 4β‐phorbol 12‐myristate 13‐acetate (PMA) were not affected by galanin.

Ionophore‐induced basophil histamine release, but not responses triggered by anti‐IgE, FMLP or PMA, was inhibited when cells were challenged in medium containing high potassium (120 mm).

We conclude that galanin and a depolarizing medium selectively inhibit ionophore‐induced basophil histamine release.
N‐nitro l‐arginine causes coronary vasoconstriction and inhibits endothelium‐dependent vasodilatation in anaesthetized greyhounds
Authors: Woodman O.L. , Dusting G.J.
Published: July 2012

The effect of N‐nitro‐l‐arginine (l‐NNA), an inhibitor of nitric oxide biosynthesis, on large coronary artery diameter and coronary blood flow was examined in anaesthetized greyhounds. The effects of l‐NNA on the coronary vascular responses to acetylcholine (ACh), glyceryl trinitrate (GTN) and 5‐hydroxytryptamine (5‐HT) were also assessed.

l‐NNA (5 mg kg−1), infused into the left circumflex coronary artery, increased systemic mean arterial pressure and decreased the external diameter of the artery. Infusion of l‐NNA decreased coronary blood flow in 5 of the 7 dogs tested and increased mean coronary resistance but neither of these effects was statistically significant. There was no change in heart rate.

Intra‐arterial injection of both ACh (0.01–0.05 μg kg−1) and GTN (0.1–0.5μg kg−1) increased large coronary artery diameter and coronary blood flow. Coronary vascular responses to the endothelium‐dependent vasodilator ACh were significantly reduced by l‐NNA, whereas the responses to the endothelium‐independent vasodilator GTN were not significantly affected.

5‐HT (0.1 μg kg−1, injected into the left circumflex coronary artery) decreased coronary artery diameter but increased coronary blood flow. After the administration of l‐NNA the 5‐HT‐induced dilatation of the coronary resistance vessels was significantly attenuated whereas the constriction of the circumflex coronary artery was increased in 3 out of 3 dogs in which diameter could be measured, although the latter effect was not statistically significant.

These data indicate that l‐NNA causes coronary and systemic vasoconstriction and selectively inhibits endothelium‐dependent vasodilatation in the coronary circulation of the anaesthetized greyhound. Therefore endothelium‐derived NO has an important role in the regulation of coronary vascular tone in the large arteries and the resistance vessels.
Electrophysiological effects of the combination of mexiletine and flecainide in guinea‐pig ventricular fibres
Authors: Delpón Eva , Valenzuela Carmen , Tamargo Juan
Published: July 2012

The effects of flecainide alone, mexiletine alone and their combination at the Na+ channel level were studied in guinea‐pig papillary muscles. The maximum upstroke velocity (Vmax) was used as an indirect index of the magnitude of the fast inward Na+ current (INa).

In muscles driven at 0.02 Hz, neither mexiletine (10−5 m) nor flecainide (10−6 m) nor the combination of both drugs modified the action potential characteristics. Mexiletine, but not flecainide, increased the effective refractory period/action potential duration ratio; this enhancement was greater when flecainide was also present.

Mexiletine or flecainide alone produced a frequency‐dependent Vmax block. Although at 0.5 Hz the blockade induced by the combination of flecainide and mexiletine was similar to that produced by flecainide alone, in muscles driven at 1 and 2 Hz the combination increased the magnitude and the onset rate of the Vmax block.

The time constant of recovery of Vmax block was similar in the presence of flecainide or the combination mexiletine plus flecainide (τre = 16.4 ± 2.3 s and 16.7 ± 2.7 s, respectively), but the combination decreased the magnitude of the slow component of reactivation induced by flecainide (93.8 ± 1.5% versus 68.9 ± 1.7%). Moreover, the combination of both drugs was more effective in inhibiting the Vmax of early test stimuli than either drug alone.

It is concluded that the combination of mexiletine and flecainide is synergistic at driving rates faster than 0.5 Hz without detracting from the characteristics of flecainide.
A comparison of the effects of angiotensin II and Bay K 8644 on responses to noradrenaline mediated via postjunctional α1‐and α2‐adrenoceptors in rabbit isolated blood vessels
Authors: Dunn W.R. , Daly C.J. , McGrath J.C. , Wilson V.G.
Published: July 2012

The effects of angiotensin II (AII) and Bay K 8644 on responses to noradrenaline (NA) mediated via postjunctional α1‐ and/or α2‐adrenoceptors have been compared in three isolated venous preparations from the rabbit, the lateral saphenous vein, the left renal vein and the ear vein.

A similar action of AII and Bay K 8644 was observed only in the lateral saphenous vein; each potentiated responses to NA after isolation of a homogeneous population of postjunctional α2‐ adrenoceptors. However, even in this preparation the mechanism of action for these agents was not identical. The sensitivity of KCl‐induced contraction to changes in extracellular calcium ions (reflecting activation of voltage‐dependent Ca2+ channels) was enhanced by Bay K 8644 but reduced by AII.

AII produced a selective facilitation of responses mediated via postjunctional α2‐adrenoceptors. In the lateral saphenous vein it reduced the effectiveness of prazosin and facilitated responses after isolation of α2‐adrenoceptors with phenoxybenzamine and rauwolscine. It directly enhanced responses to NA in the ear vein, where only α2‐adrenoceptors are involved. In contrast, AII did not influence responses mediated via postjunctional α1‐adrenoceptors in the left renal vein (even after the receptor reserve had been removed with phenoxybenzamine) nor the ‘rauwolscine‐resistant’ component of responses to NA in the saphenous vein.

Bay K 8644 enhanced contractile responses to NA mediated both via α2‐adrenoceptors, in the lateral saphenous vein, and via α1‐adrenoceptors in the left renal vein. Thus, unlike angiotensin II, no preferential effect was apparent.

Bay K 8644 was inactive against responses to NA in the rabbit isolated ear vein. Since the sustained component of responses to NA in this preparation is dependent upon the influx of extracellular Ca2+, these observations suggest that the influx of Ca2+ stimulated by NA is mediated via receptor‐operated (1,4‐dihydropyridine‐resistant) Ca2+ channels.
Postjunctional α‐adrenoceptors in the rabbit isolated distal saphenous artery: indirect sensitivity to prazosin of responses to noradrenaline mediated via postjunctional α2‐adrenoceptors
Authors: Dunn W.R. , McGrath J.C. , Wilson V.G.
Published: July 2012

Under normal experimental conditions, the rabbit isolated distal saphenous artery appears to contain a homogeneous population of postjunctional α1‐adrenoceptors. Prazosin competitively antagonized responses to noradrenaline (NA) with a pA2 value of 8.6, while a relatively high concentration of rauwolscine (1 μm), produced only a 2 fold rightward displacement of the NA cumulative concentration‐response curve (CCRC).

Despite the fact that angiotensin II (AII) was without effect on responses to NA or phenylephrine in this preparation, this peptide made responses to NA less susceptible to the antagonistic action of prazosin. This was particularly evident on the lower portion of the CCRC for NA. These results suggest that in the presence of AII, NA produces contractile responses by an action mediated through a prazosin‐resistant adrenoceptor.

An attempt was made to isolate a homogeneous population of postjunctional α2‐adrenoceptors by use of a receptor protection procedure involving the combination of rauwolscine and phenoxybenzamine. After the protection protocol no responses were observed to the α‐adrenoceptor agonists NA, phenylephrine or UK‐14304. In the presence of angiotensin II however, concentration‐dependent contractions were observed to each of these agonists. Under these conditions the rank order of potency, UK‐14304 > NA > phenylephrine, is consistent with that of an effect mediated through postjunctional α2‐adrenoceptors.

The responses to NA, after the protection protocol, in the presence of AII, were susceptible to the selective α2‐adrenoceptor antagonist, rauwolscine (1 μm), but resistant to the selective α1‐adrenoceptor antagonist prazosin (0.1 μm). Furthermore, the combination of rauwolscine (1 μm) and prazosin (0.1 μm) was no more effective in blocking responses to NA than was rauwolscine (1 μm) alone. These results are consistent with the presence of a homogeneous population of postjunctional α2‐adrenoceptors.

Inducing a small degree of tone with a low concentration of the selective α1‐adrenoceptor agonist, phenylephrine, markedly increased the threshold sensitivity to the selective α2‐adrenoceptor agonist UK‐14304, in a manner analogous to that seen with AII.

The results in the present study indicate that responses mediated via postjunctional α2‐adrenoceptors in the rabbit isolated distal saphenous artery are dependent upon a degree of vascular smooth muscle stimulation by some other receptor system. It is hypothesized that under normal experimental conditions, this function is fulfilled by stimulation of α1‐adrenoceptors, while after α1‐adrenoceptor blockade the necessary positive influence can be provided by stimulation of AII receptors. The implications for such an interaction between postjunctional α‐adrenoceptor subtypes in demonstrating prazosin‐resistant, rauwolscine‐ or yohimbine‐sensitive responses in isolated blood vessels is discussed.
The effects of nifedipine on α2‐adrenoceptor‐mediated contractions in several isolated blood vessels from the rabbit
Authors: Dunn W.R. , Daly C.J. , McGrath J.C. , Wilson V.G.
Published: July 2012

The effects of the dihydropyridine calcium channel blocker, nifedipine, on noradrenaline‐induced contractile responses have been examined in several isolated blood vessels from the rabbit, with particular emphasis on responses mediated via postjunctional α2‐adrenoceptors.

In the isolated renal vein, ear vein, distal saphenous artery, saphenous vein and plantaris vein, 0.1 μm and 1 μm nifedipine reduced responses elicited by 54 mm KCl by more than 70%. The remaining responses were abolished by α‐adrenoceptor blockade, suggesting the involvement of noradrenaline released from neurones activating a dihydropyridine‐resistant mechanism.

In the renal vein (α1), ear vein (predominantly α2‐), distal saphenous artery (αr‐ > α2‐), saphenous vein and plantaris vein (α2‐ > α1‐), 0.01 μm and 0.1 μm nifedipine produced concentration‐related reductions in the maximum response to noradrenaline. However, 1 μm nifedipine was no more effective than 0.1 μm nifedipine and the reduction in the maximum varied from 10–25% of the control response. Thus, a sizeable component of the α‐adrenoceptor‐medaited response in all blood vessels is resistant to dihydropyridine calcium channel blockers and this appears to be unrelated to the α‐adrenoceptor subtype involved.

Following irreversible inactivation of α1‐adrenoceptors and isolation of functional α2‐adrenoceptors in the saphenous vein, plantaris vein and distal saphenous artery (the latter requiring the presence of angiotensin II), the effect of nifedipine on responses to noradrenaline was increased. However, a component of the α2‐adrenoceptor response in each preparation was present even after the concentration of nifedipine was increased to 1 μm.

In the saphenous vein, a preparation in which it has been demonstrated previously that α2‐adrenoceptor‐mediated responses are highly dependent upon the presence of extracellular calcium ions, partial depolarization with 20 mm KCl failed to increase the inhibitory effect of 0.1 μm nifedipine. This suggests the involvement of dihydropyridine‐resistant Ca2+ channels. The possible relationship between these dihydropyridine‐resistant Ca2+ channels, α‐adrenoceptor subtypes and ‘receptor‐operated’ Ca2+ channels is discussed.