bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for an external location of the dihydropyridine agonist receptor site on smooth muscle and skeletal muscle calcium channels
Authors: Strübing C. , Hering S. , Glossmann H.
Published: July 2012

The location of the binding domain for agonist dihydropyridines (DHP) has been studied by comparing the action of (+)‐202 791 and (−)‐Bay K 8644 on Ba2+ currents (IBa) in whole cell patch clamp experiments. Drug effects were examined upon internal and external (extracellular) application in A7r5 smooth muscle cells and BC3H1 cells, a cell line expressing Ca channels of the skeletal muscle type.

Efficiency of internal drug application in the whole cell studies was demonstrated by inhibition of potassium currents and barium currents (IBa) upon internal perfusion with tetraethylammonium (TEA+) (10 mm) and the permanently charged phenylalkylamine, D 890 (100 μm) respectively. The uncharged DHP, (−)‐STBODIPY‐DHP (2 μm) was used to estimate the time course of internal perfusion by monitoring its fluorescence.

Intracellular application of (+)‐202 791 and (−)‐Bay K 8644 (5 μm) in patch clamp experiments was ineffective in stimulating Ca2+ channel currents in both cell lines. In contrast a 50 fold lower agonist concentration (0.1 μm (−)‐Bay K8644) applied to the external face of the membrane induced typical changes in tail currents and a current increase under conditions when up to 10 μm of the agonist was present in the intracellular perfusion solution.

In cell‐attached patches in A7r5 cells, (−)‐Bay K 8644 increased and (+)‐PN 200 110 inhibited single channel activity when applied via the bath solution. This suggests partitioning and lateral diffusion of the DHPs in the lipid of the plasma membrane.

We conclude that the binding site for agonist DHPs on Ca2+ channels in A7r5 and BC3H1 cells is located close to the external surface of the membrane. The DHP binding domain can be reached by agonists and antagonists from the extracellular but not from the intracellular face of the membrane.
The effects of Nω‐nitro‐L‐arginine methyl ester, sodium nitroprusside and noradrenaline on venous return in the anaesthetized cat
Authors: Bower E.A. , Law A.C.K.
Published: July 2012

The vascular actions of Nω‐nitro‐l‐arginine methyl ester (l‐NAME), sodium nitroprusside and noradrenaline were investigated in cats under chloralose anaesthesia with controlled vascular tone and ventilation. Cardiac output, heart rate, vascular pressures and mean circulatory filling pressure (MCFP) were measured. Total peripheral resistance (TPR) and resistance to venous return (Rvr) were calculated from steady‐state readings.

l‐NAME (37 μmol kg−1, i.v.) administered to ten cats receiving noradrenaline (6 nmol kg−1 min−1, i.v.) increased aortic pressure by 47.5 ± 7.1 mmHg from 106 mmHg, and MCFP by 1.56 ± 0.36 mmHg from 10.0 mmHg (means ± s.e.means). Mean changes in portal venous pressure, RAP and heart rate were not significant. Cardiac output fell by 29.7 ± 3.3% from 130 ml min−1 kg−1. TPR rose by 108 ± 7.2% from 796 mmHg l−1 min kg and Rvr by 58.4 ± 4.5% from 64 mmHg l−1 min kg.

Infusion of sodium nitroprusside into cats receiving noradrenaline evoked dose‐related falls in aortic pressure, MCFP, TPR and Rvr. Changes in portal venous pressure, RAP and heart rate were not significant, and cardiac output fell slightly. After l‐NAME, sensitivity to nitroprusside was increased by 139 ± 34% for MCFP, 176 ± 19% for TPR and 351 ± 39% for Rvr, and cardiac output rose slightly. The nitroprusside infusion required to restore TPR after l‐NAME was estimated to be 5.8 × 10±0.41 nmol kg−1 min−1, which was approximately three times more than that required to restore MCFP.

Infusion of noradrenaline evoked dose‐related increases in aortic and portal venous pressures, heart rate, cardiac output, MCFP, TPR and Rvr. After l‐NAME and nitroprusside (4.4 nmol kg−1 min−1, i.v.), TPR and Rvr were not significantly different, but MCFP was reduced by 1.76 ± 0.24 mmHg, and cardiac output by 22 ± 1.9%. After subsequent expansion of the circulating blood volume (5–7.5 ml kg−1 dextran‐saline), mean values for all parameters were restored to their previous levels. Sensitivity to noradrenaline was not significantly altered for heart rate, TPR and Rvr, but was reduced by 31.8 ± 12% for MCFP and by 66.5 ± 18% for cardiac output.

The depression of cardiac output by l‐NAME is attributed to the increase in Rvr, partly compensated by the rise in MCFP. For a given rise in MCFP, the increase in Rvr was seven times greater after l‐NAME than after noradrenaline, and the difference in the relative actions of the two drugs on resistance and capacitance vessels largely accounts for their contrasting effects on venous return. A procedure is suggested for replacement of vascular nitric oxide by nitroprusside infusion and blood volume expansion.
Relaxing actions of corticotropin‐releasing factor on rat resistance arteries
Authors: Lei S. , Richter R. , Bienert M. , Mulvany M.J.
Published: July 2012

Although it well established that corticotropin‐releasing factor (CRF) injected i.v. can cause hypotension and vasodilatation, there is no in vitro evidence that CRF acts as a vasodilator. We have therefore tested the hypothesis that the hypotensive effect of i.v. CRF is due to a direct vasodilator action by carrying out experiments in vitro on rat resistance arteries (i.d. 150–300 μm).

Initial in vivo experiments confirmed that CRF (1.5 nmol kg−1) injected i.v. caused hypotension in rats, this being partially antagonized by the CRF analogue CRF9–41.

For the in vitro experiments, vessels were taken from the mesenteric, cerebral and femoral vascular beds, and mounted as ring preparations in an isometric myograph. The vessels were pre‐contracted with one of 3 agonists (prostaglandin F2α, arginine vasopressin or noradrenaline) or with a high‐potassium solution (K+).

With maximal concentrations of the agonists, CRF caused relaxation of mesenteric and cerebral vessels with 10 nm, and near complete relaxation with 100 nm. Femoral vessels preconstricted with agonists and all vessels preconstricted with K+ were less affected by CRF. In the mesenteric vessels, with sub‐maximal levels of pre‐constriction, CRF caused substantial relaxation at 1 nm and could cause complete relaxation at 10 nm.

The relaxant effect of CRF on contractions of mesenteric vessels was antagonized by 100 nm CRF9–41. Neither tetraethyl ammonium (30 mm) nor glibenclamide (3 μm) antagonized the relaxant effect of CRF.

The relaxant effect of CRF on mesenteric small arteries was found to be unaffected by removal of the endothelium.

The results indicate that CRF causes an endothelial‐independent vasodilatation of rat resistance arteries under in vitro conditions at concentrations which are consistent with this being an important cause of the hypotension observed with i.v. injection of CRF.
Characterization of the inhibitory prostanoid receptors on human neutrophils
Authors: Wheeldon Alan , Vardey Christopher J.
Published: July 2012

We have evaluated the effects of various prostanoid agonists on the release of leukotriene B4 (LTB4) and superoxide anions (O2−) from human neutrophils stimulated with opsonized zymosan (OZ) and formyl‐methionyl‐leucyl‐phenylalanine (FMLP), respectively.

Prostaglandin E2 (PGE2) and PGD2 inhibited both OZ‐induced LTB4 release (EC50 0.72 μm and 0.91 μm respectively), and FMLP‐induced O2− release (EC50 0.42 μm and 0.50 μm respectively). PGF2α, the TP‐receptor agonist, U46619, and the IP‐receptor agonist, iloprost, were also active, but were all at least an order of magnitude less potent than PGE2 and PGD2.

The EP2/EP3‐receptor agonist, misoprostol, and the selective EP2‐agonist, AH13205, were both effective inhibitors of LTB4 release, being approximately equipotent with and 16‐times less potent than PGE2, respectively. In contrast, the EP1/EP3‐receptor agonist, sulprostone, had no inhibitory activity at concentrations of up to 10 μm.

The selective DP‐receptor agonist, BW245C, inhibited LTB4 release, (EC50 0.006 μm) being approximately 50 times more potent than PGD2. BW245C also inhibited O2− release, and this inhibition was antagonized competitively by the DP‐receptor blocking drug, AH6809 (pA2 6.6).

These data indicate the presence of both inhibitory EP‐ and DP‐receptors on the human neutrophil. The rank order of potency of EP‐receptor agonists suggest that the EP‐receptors are of the EP2‐subtype.;
Single channel and whole‐cell K‐currents evoked by levcromakalim in smooth muscle cells from the rabbit portal vein
Authors: Beech D.J. , Zhang H. , Nakao K. , Bolton T.B.
Published: July 2012

Single channel and whole‐cell current recordings were made from single smooth muscle cells isolated from the rabbit portal vein.

Application of 10 μm levcromakalim ((−)‐Ckm) to single cells held with pipettes containing 1 mm GDP induced a K‐current (IK(Ckm)) which occurred in addition to the current caused by GDP alone (IK(GDP)) and averaged 135 pA at − 37 mV. We have investigated whether the same K channels underlie the GDP‐ and Ckm‐induced K‐currents.

If 1 mm GDP was in the pipette but Mg ions were omitted the effect of GDP was absent and IK(Ckm) averaged only 10 pA, suggesting that the action of (−)‐Ckm was Mg‐dependent.

Intracellular ATP was not observed to have much effect on IK(‐Ckm). Loading of cells with 10 mm ATP from the recording pipette had no significant effect and flash photolysis of caged‐ATP loaded into cells from the pipette, estimated to release about 1 mm free ATP, also had no effect on IK(‐Ckm).

Bath‐applied glibenclamide inhibited IK(‐Ckm) with an IC50 of 200 nm, a value 8 times higher than that found for inhibition of IK(GDP). The delayed rectifier K‐current (IK(DR)) was also inhibited by glibenclamide but at higher concentrations (IC50 100 μm). Bath‐applied tetraethylammonium ions (TEA) inhibited IK(‐Ckm) and IK(GDP) to the same extent (IC50 about 7 mm).

In inside‐out patch recordings (−)‐Ckm (10 μm) applied to the intracellular surface of the membrane potentiated the opening of K channels already stimulated by 1 mm GDP and all of the channel activity was abolished by 10 μm glibenclamide. The unitary conductance of the channels was 24 pS in a 60 mm: 130 mm K‐gradient.

We suggest that (−)‐Ckm may hyperpolarize and relax smooth muscle cells by opening KNDP, a class of small conductance K channels that are related to the ATP‐sensitive K channels seen in other tissues.
Effect of capsazepine on the release of calcitonin gene‐related peptide‐like immunoreactivity (CGRP‐LI) induced by low pH, capsaicin and potassium in rat soleus muscle
Authors: Santicioli Paolo , Del Bianco Elena , Figini Michela , Bevan Stuart , Maggi Carlo Alberto
Published: July 2012

We have determined the effect of the competitive antagonist capsazepine at the capsaicin receptor on the release of calcitonin gene‐related peptide‐like immunoreactivity (CGRP‐LI) from rat isolated soleus muscle induced by capsaicin (1 μm), by superfusion with low pH medium (pH 5) or by KCl (80 mm).

Each one of the three stimuli tested produced a marked CGRP‐LI release. Total evoked release (fmol g−1) was 482 ± 69, 169 ± 20 and 253 ± 43 for capsaicin, low pH medium and KCl, respectively.

Prior application of capsaicin (10 μm for 30 min followed by 30 min of washout) to produce capsaicin desensitization in vitro abolished CGRP‐LI release induced by the three stimuli.

Capsazepine (1–100 μm, 45 min preincubation) inhibited the evoked CGRP‐LI release. Capsaicin‐induced release was significantly inhibited by 77, 92 and 96% with 10, 30 and 100 μm capsazepine, respectively. Low pH‐induced release was inhibited by 78, 84, 88 and 93% with 3, 10, 30 and 100 μm capsazepine, respectively. KCl‐induced release was significantly inhibited by 55 and 93% with 30 and 100 μm (but not with 10 μm) capsazepine, respectively.

These findings demonstrate that capsazepine prevents low pH‐ and capsaicin‐induced CGRP‐LI release from rat soleus muscle at concentrations which do not affect the release evoked by KCl. These findings imply a relationship between the action of low pH and activation of the capsaicin receptor. At high concentrations, capsazepine produces a nonspecific inhibitory effect on CGRP‐LI release from peripheral endings of the capsaicin‐sensitive primary afferent neurone.
Comparison of the anti‐inflammatory properties of formoterol, salbutamol and salmeterol in guinea‐pig skin and lung
Authors: Whelan C.J. , Johnson M. , Vardey C.J.
Published: July 2012

We have compared some anti‐inflammatory properties of formoterol, salbutamol and salmeterol in guinea‐pig skin and lung.

Intradermal formoterol (1 × 10−10 to 1 × 10−8 mol/site), salbutamol (1 × 10−8 and 1 × 10−7 mol/site) and salmeterol (1 × 10−8 and 1 × 10−7 mol/site) inhibited bradykinin‐induced plasma protein extravasation (PPE) in guinea‐pig skin. A maximally effective dose of formoterol (1 × 10−9 mol/site) and salbutamol (1 × 10−8 mol/site) inhibited PPE in guinea‐pig skin for 2–4 h and 1–2 h respectively, whereas salmeterol (1 × 10−8 mol/site) was effective for >6 h.

Inhaled formoterol (nebuliser concentration 0.1 to 100 μg ml−1) inhibited histamine‐induced plasma protein extravasation (PPE) in guinea‐pig lung, with significant inhibition being observed at 10 and 100 μg ml−1. Formoterol (100 μg ml−1) inhibited PPE in guinea‐pig lung for 2–4 h, a duration of action intermediate between that previously obtained for salbutamol (1 h) and salmeterol (>6 h).

Formoterol, like salbutamol, had no effect on neutrophil accumulation or granulocyte‐dependent PPE (zymosan‐induced) in guinea‐pig skin. Formoterol inhibited neutrophil accumulation (lipopolysaccharide‐induced) in guinea‐pig lung but at doses greater than those required to inhibit granulocyte‐independent PPE (histamine‐induced). In contrast, salmeterol inhibited neutrophil accumulation and granulocyte‐dependent PPE in guinea‐pig skin and inhibited neutrophil accumulation in guinea‐pig lung at doses which inhibit granulocyte‐independent PPE.

Inhaled formoterol (nebuliser concentration 100 μg ml−1) and salmeterol (100 μg ml−1) both inhibited PAF‐induced eosinophil accumulation in guinea‐pig lung. However, unlike salmeterol, this effect of formoterol was observed only at suprabronchodilator doses.

We conclude that to inhibit neutrophil accumulation, at doses which inhibit granulocyte‐independent PPE, agonists acting at β‐adrenoceptors on vascular endothelium require a duration of action greater than that of salbutamol and formoterol. However, we speculate that the mechanism of inhibition of eosinophil accumulation in guinea‐pig lung by β2‐adrenoceptor agonists may involve an action on β2‐adrenoceptors on a cell type other than the endothelial cell.
Partial agonist effect of the platelet‐activating factor receptor antagonists, WEB 2086 and WEB 2170, in the rat perfused heart
Authors: Hu Weimin , McNicholl Ila K. , Choy Patrick C. , Man Ricky Y.K.
Published: July 2012

WEB 2086 and WEB 2170 are potent platelet‐activating factor (PAF) receptor antagonists and have been used widely as pharmacological tools to investigate the actions of PAF in a variety of biological systems.

Low concentrations of WEB 2086 and WEB 2170 blocked the vasoconstrictor action of PAF in the rat perfused heart. In this study, we observed that moderate concentrations of WEB 2086 and WEB 2170 increased the perfusion pressure in rat isolated hearts under constant flow perfusion. The vasoconstrictor actions of WEB 2086 and WEB 2170 were not observed with a structurally different PAF receptor antagonist, FR‐900452.

To determine whether this vasoconstrictor action of WEB 2086 involved non‐specific effects or was via the activation of PAF receptors, hearts were pretreated with 1000 pmol PAF or 50 μm FR‐900452. These pretreatments attenuated the vasoconstrictor action of 1 μm WEB 2086, suggesting that the action of WEB 2086 may be mediated via PAF receptors. Pretreatment with the leukotriene receptor antagonist (L‐649,923, 5 μm) and the leukotriene synthesis inhibitor (MK‐886, 10 μm) that are known to block the vasoconstrictor action of PAF receptor activation also attenuated the vasoconstrictor action of WEB 2086. Pretreatment with PAF or MK‐886 attenuated the vasoconstrictor action of 0.5 μm WEB 2170.

When PAF receptors were activated by PAF in the perfused heart, significant amounts of leukotriene C4 and leukotriene C4/D4/E4 were detected in the coronary effluent. However, no significant amount of these leukotrienes was detected in the coronary effluent when hearts were perfused with 1 μm WEB 2086 or 0.5 μm WEB 2170.

In summary, our results indicate that WEB 2086 and WEB 2170 possess partial agonist effects in the rat perfused heart where they produced vasoconstriction via the activation of PAF receptor. This action could be attenuated by PAF pretreatment or a PAF receptor antagonist. The vasoconstrictor action of WEB 2086 and WEB 2170 involved the production of leukotrienes. But unlike the vasoconstrictor action of PAF, no significant amount of leukotrienes was detected in the effluent suggesting that the vasoconstrictor action of WEB 2086 and WEB 2170 may be explained on the basis of intracellularly or locally produced leukotrienes.
Characterization of the effects of lithium on phosphatidylinositol (PI) cycle activity in human muscarinic ml receptor‐transfected CHO cells
Authors: Atack John R. , Prior Angela M. , Griffith Douglas , Ragan C. Ian
Published: July 2012

The effects of lithium on [3H]‐inositol and [3H]‐cytidine incorporation into [3H]‐inositol monophosphates ([3H]‐InsP1) and [3H]‐cytidine monophosphorylphosphatidate ([3H]‐CMP‐PA), respectively, and inositol 1,4,5‐trisphosphate (InsP3) and inositol 1,3,4,5‐tetrakisphosphate (InsP4) mass were studied in carbachol‐stimulated human ml muscarinic receptor‐transfected Chinese hamster ovary cells (ml CHO cells).

Lithium alone (10 mm) had no appreciable effects on any of the four parameters measured; it was only in carbachol‐stimulated cells that the effects of lithium became apparent.

In the presence of carbachol (1 mm), lithium (10 mm) caused a relatively rapid (within 5 min) accumulation of [3H]‐InsP1 and [3H]‐CMP‐PA which continued up to about 20–30 min, after which accumulation slowed down. On the other hand, the elevation in InsP3 and InsP4 levels produced by carbachol was not altered by lithium in the short‐term and only at later times (> 20–30 min) was the response attenuated, with InsP3 and InsP4 levels approaching basal.

The effects of lithium on carbachol‐stimulated [3H]‐InsP1 and [3H]‐CMP‐PA accumulation and the attenuation of the carbachol‐induced elevation of InsP3 and InsP4 were all dose‐dependent, with EC50s in the region of 1 mm.

The lithium‐induced effects on [3H]‐CMP‐PA and InsP3 and InsP4 in carbachol‐stimulated cells could be reversed, in a dose‐dependent manner, by preincubation with exogenous myo‐inositol (EC50 = 2–3 mm) but not by the inactive analogue scyllo‐inositol, indicating that these effects occur as a consequence of depletion of inositol.

The temporal effects of lithium are consistent with lithium inhibiting inositol monophosphatase, causing accumulation of InsP1, resulting in lower free inositol levels. This leads to accumulation of CMP‐PA and reduced PI synthesis which, once agonist‐linked membrane inositol phospholipids are depleted, produces attenuated InsP3 and InsP4 responses.

These results in ml CHO cells support the hypothesis that lithium affects the PI cycle cell signalling pathway by depletion of inositol due to inhibition of inositol monophosphatase.
Evidence that depletion of internal calcium stores sensitive to noradrenaline elicits a contractile response dependent on extracellular calcium in rat aorta
Authors: Noguera M. Antonia , D'Ocon M. Pilar
Published: July 2012

Noradrenaline 1 μm induced a contractile response in rat isolated aorta in the presence or in the absence of extracellular Ca2+ with depletion of intracellular Ca2+ stores. Thereafter, during incubation in the presence of Ca2+, an increase in the resting tone was observed. Such a contractile response did not occur after exposure to caffeine or 5‐hydroxytryptamine.

This increase in tension was inhibited in a concentration‐dependent manner by α‐adrenoceptor antagonists (prazosin, phentolamine and yohimbine), the non‐specific relaxing compound, papaverine and by the Ca2+‐entry blocker, nifedipine. Therefore, this contractile process is related to depletion of Ca2+ stores sensitive to noradrenaline and is linked to Ca2+ entry through voltage‐operated Ca2+ channels and α‐adrenoceptors.

Phentolamine and yohimbine did not block the Ca2+ refill pathway; prazosin and nifedipine inhibited the reuptake of Ca2+ by an internal store sensitive only to noradrenaline; papaverine inhibited the refilling of caffeine‐ and noradrenaline‐sensitive Ca2+‐stores.
Evidence that M1 muscarinic receptors enhance noradrenaline release in mouse atria by activating protein kinase C
Authors: Costa Mary , Barrington Michelle , Majewski Henryk
Published: July 2012

The M1 selective muscarinic agonist, McNeil A 343, enhanced the electrically evoked release of noradrenaline from postganglionic sympathetic nerves in mouse atria. This has been found previously to be due to activation of muscarinic receptors of the M1 subtype, probably located on sympathetic nerve terminals. The present study investigated the signal transduction mechanisms involved in the release‐enhancing effects of McNeil A 343. The release of noradrenaline from mouse atria was assessed by measuring the electrically‐induced (3 Hz, 60 s) outflow of radioactivity from atria which had been pre‐incubated with [3H]‐noradrenaline.

8‐Bromo cyclic AMP in the presence of IBMX was used to enhance maximally S‐I noradrenaline release through cyclic AMP‐dependent mechanisms. However, the facilitatory effect of McNeil A 343 (10 μm) was not different from the effect in the absence of these drugs, suggesting that McNeil A 343 enhances noradrenaline release independently of the cyclic AMP system. Furthermore, the release‐enhancing effect of McNeil A 343 (10 μm) on noradrenaline release was also not altered by the 5‐lipoxygenase inhibitor, BW A4C.

The facilitatory effect of McNeil A 343 was not altered in the presence of drugs (trifluoperazine, W7, and calmidazolium) which inhibit calmodulin‐dependent processes, suggesting that the mechanisms of action of McNeil A 343 does not depend on calmodulin.

It was considered likely that the facilitatory effect of McNeil A 343 on noradrenaline release may be due to activation of protein kinase C, since activators of protein kinase C enhance noradrenaline release. The facilitatory effect of McNeil A 343 was abolished by the non‐selective protein kinase C inhibitor, K‐252a. To investigate further the involvement of protein kinase C, mouse atria were chronically incubated (9–10 h) with the protein kinase C activator, 4β‐phorbol dibutyrate (1.0 μm) in order to down‐regulate protein kinase C activity. In protein kinase C‐down‐regulated atria, the facilitatory effect of McNeil A 343 (30 μm) was abolished. Incubation with 4α‐phorbol dibutyrate which does not affect protein kinase C did not reduce the facilitatory effect of McNeil A 343. This provides evidence that activation of protein kinase C is involved in the signal transduction process of McNeil A 343.
Pharmacology of high‐threshold calcium currents in GH4C1 pituitary cells and their regulation by activation of human D2 and D4 dopamine receptors
Authors: Seabrook G.R. , Knowles M. , Brown N. , Myers J. , Sinclair H. , Patel S. , Freedman S.B. , McAllister G.
Published: July 2012

The objective of this study was to characterize the pharmacology of calcium currents in GH4C1 pituitary cells and determine whether activation of heterologously expressed human dopamine receptors can regulate their function. Human D2(short), D3 and D4,2 receptor cDNA's were separately transfected into GH4C1 cells and whole cell calcium currents were recorded by use of nystatin‐perforated patch clamp techniques.

High‐threshold calcium currents were antagonized in a biphasic manner by the dihydropyridine, nisoldipine. The half‐maximally effective concentration for each site was 0.2 nm (pIC50 = 9.78 ± 0.21, n = 4) and 339 nm (pIC50 = 6.47 ± 0.12, n = 4). The component of current inhibited by 10 nm nisoldipine was also blocked by ω‐conotoxin GVIA (30 ± 9% at 30 nm, n = 6) or by ω‐agatoxin IVA (34 ± 7% at 100 nm, n = 4).

Activation of either D2 or D4 receptors by dopamine (10 μm) or quinpirole (0.1 to 10 μm) reduced the peak calcium current by ca. 20% in the majority of cells studied. No inhibition was observed in control or D3 transfected GH4C1 cell lines.

The mobilisation of intracellular calcium by thyrotropin releasing hormone in hD4‐GH4C1 cells was also studied using Fura‐2 AM microspectrofluorimetry. Thyrotropin releasing hormone caused a concentration‐dependent increase in calcium mobilisation with an EC50 of 7 nm. D4 receptor activation had no effect upon either basal or hormone‐induced [Ca2+]i transients.

These results demonstrate that GH4C1 pituitary cells have at least two types of dihydropyridine‐sensitive high‐threshold calcium currents and that like D2 receptors, human D4 receptors can also regulate calcium channel function.
Moguisteine: a novel peripheral non‐narcotic antitussive drug
Authors: Gallico L. , Borghi A. , Rosa C. Dalla , Ceserani R. , Tognella S.
Published: July 2012

The antitussive effects of moguisteine have been compared with codeine in several experimental models of cough in guinea‐pigs and dogs.

Moguisteine and codeine dose‐dependently (respective ED50 values are given in parentheses) inhibited cough induced in guinea‐pigs by 7.5% citric acid aerosol (25.2 and 29.2 mg kg−1, p.o.), by 30 μm capsaicin aerosol (19.3 and 15.2 mg kg−1, p.o.), by mechanical stimulation (22.9 and 26.4 mg kg−1, p.o.) and by tracheal electrical stimulation (12.5 and 13.9 mg kg−1, p.o.).

Moguisteine was effective against cough induced by tracheal electrical stimulation in dogs (ED50 17.2 mg kg−1, p.o.); codeine was not tested because of its emetic effect.

After repeated dosing (12–15 days), moguisteine did not induce tolerance in either guinea‐pigs or dogs.

Moguisteine did not interact with opiate receptors, since it did not show affinity for [3H]‐naloxone binding sites and furthermore naloxone (5mg kg−1, s.c.) did not antagonize its antitussive effects.

Moguisteine had no antitussive effect after i.c.v. administration (20 μg), whilst codeine (2–10 μg) and dextromethorphan (2.5–20 μg) were highly effective.

Our findings demonstrate that moguisteine is a novel peripherally acting non‐narcotic antitussive agent, the mode of action of which remains to be elucidated fully.
Covariation of α2‐adrenoceptor density and function following irreversible antagonism with EEDQ
Authors: Durcan Michael J. , Morgan Philip F. , Van Etten Michelle L. , Linnoila Markku
Published: July 2012

Administration of the irreversible antagonist, N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline, (EEDQ, 2 mg kg−1, i.p.) to mice reduced binding of [3H]‐RX 821002 (2‐methoxy‐idazoxan) to α2‐adrenoceptors in whole mouse brain by 75% 24 h later. The receptor binding returned over time only being reduced by 25% by 16 days post administration; the time taken for binding to return to 50% of control levels was estimated to be 5.25 days.

EEDQ administration also resulted in the loss of the sedative effect of the α2‐adrenoceptor agonist, medetomidine, measured by the holeboard test of directed exploration and locomotor activity. Agonistinduced sedation returned to control values by 8 days post EEDQ administration.

EEDQ administration also resulted in the loss of the hypothermic response to medetomidine (0.1 mg kg−1, i.p.). Medetomidine‐induced hypothermia returned to control values by 12 days post EEDQ administration.

Pretreatment with the selective α2‐adrenoceptor antagonist, RX 821002 (0.1–3.0 mg kg−1, i.p.) 45 min before EEDQ prevented the loss of α2‐adrenoceptors as well as the blockade of medetomide‐induced sedation and hypothermia by EEDQ.

The results of these experiments indicate that there is significant receptor reserve for α2‐adrenoceptor‐mediated behavioural and physiological responses.
Mediation of most atypical effects by species homologues of the β3‐adrenoceptor
Authors: Blin Nathalie , Nahmias Clara , Drumare Marie F. , Strosberg A. Donny
Published: July 2012

A wide panel of compounds acting on β‐adrenoceptors active either in mammalian heart or in rodent digestive tract and adipose tissues, were investigated for their effects on Chinese hamster ovary cells transfected with the human or murine β3‐adrenoceptor gene.

The β3‐agonists, bucindolol, CGP 12177A and pindolol exhibited the highest binding affinities; BRL 37344, LY 79771, ICI 201651 and SR 58611A presented high potencies in stimulating adenylyl cyclase; bupranolol appeared as the most efficient β3‐antagonist.

This pharmacological analysis further established that the β3‐adrenoceptor is the prototype of the adipose tissue atypical β‐adrenoceptor, since these receptors share a number of pharmacological properties which differ strikingly from those of β1‐ and β2‐adrenoceptors: low affinities for conventional β‐adrenoceptor agonists and antagonists, high potencies for novel compounds active in adipose tissues, partial agonistic activites for several β1/β2‐antagonists.

Although the pharmacological profiles of the human and murine β3‐receptor were very similar, some quantitative or even qualitative differences were observed for particular compounds such as propranolol, which exhibited weak and partial agonistic effects at the human β3‐receptors and antagonistic effects at the murine β3‐receptors. These differences may result from key amino‐acid substitutions between the human and the murine β3‐receptor sequences, which may alter the binding site or signal processing.

Compounds active on atypical β‐sites of other tissues such as heart and digestive tract were also potent on the β3‐adrenoceptor expressed in Chinese hamster ovary cells, suggesting that this receptor mediates most of the atypical properties described in various tissues, and that differences in ligand effects may result from tissue‐related specificities.
Blockade by oral or parenteral RPR 100893 (a non‐peptide NK1 receptor antagonist) of neurogenic plasma protein extravasation within guinea‐pig dura mater and conjunctiva
Authors: Lee Won S. , Moussaoui Saliha M. , Moskowitz Michael A.
Published: July 2012

The ability of an NK1 receptor antagonist, RPR 100893, and its enantiomer, RPR 103253 to block neurogenic plasma protein extravasation in guinea‐pig dura mater and conjunctiva was assessed following 125I‐labelled bovine serum albumin ([125I]‐BSA, 50 μCi kg−1, i.v.) and unilateral electrical stimulation of the trigeminal ganglion (0.6 mA, 5 ms, 5 Hz, 5 min) or capsaicin administration (150 μg kg−1, i.v.).

When administered p.o. 60 min prior to electrical stimulation, RPR 100893 (≥ 0.1 μg kg−1) decreased plasma protein extravasation in dura mater in a dose‐dependent manner, whereas the enantiomer (10 or 100 μg kg−1, p.o.) was inactive.

When given i.v. 30 min prior to electrical stimulation, RPR 100893 (≥ 0.5 ng kg−1) significantly inhibited plasma protein extravasation in the dura mater evoked by electrical stimulation in a dose‐dependent manner.

RPR 100893 (100 μg kg−1, p.o.) also reduced the leakage when given 45 min before the guinea‐pigs were killed and 10, 40 and 80 min after electrical trigeminal stimulation.

RPR 100893 given p.o. dose‐dependently inhibited capsaicin‐induced plasma protein extravasation with ID50s of 7.4 μg kg−1 and 82 μg kg−1 for dura mater and conjunctiva, respectively.

These results are consistent with the contention that NK1 receptors mediate neurogenic plasma protein leakage following trigeminal stimulation, and suggest that NK1 receptor antagonists of the perhydroisoindolone series may be useful for treating migraine and cluster headaches.
Des‐Arg9‐bradykinin‐induced increases in intracellular calcium ion concentration in single bovine tracheal smooth muscle cells
Authors: Marsh Katrina A. , Hill Stephen J.
Published: July 2012

Dynamic video imaging was used to measure the des‐Arg9‐bradykinin‐induced changes in the intracellular free calcium ion concentration ([Ca2+]i) of single bovine tracheal smooth muscle (BTSM) cells.

In the presence of extracellular calcium ions, des‐Arg9‐bradykinin (1 nm–10 μm) produced a concentration‐dependent increase in the [Ca2+]i over basal levels yielding an EC50 value of 316 nm. The percentage of cells responding to each concentration of des‐Arg9‐bradykinin also increased in a concentration‐dependent manner (from 9% to 100%).

The bradykinin B2 receptor antagonist, d‐Arg[Hyp3,Thi5,8,d‐Phe7]‐bradykinin (10 μm), was without effect on the calcium response of the cells when added 2 min prior to des‐Arg9‐bradykinin (100 nm). However, the B1 receptor antagonist, des‐Arg9Leu8‐bradykinin (10 μm), completely abolished the des‐Arg9‐bradykinin‐induced response.

Under calcium‐free conditions, des‐Arg9‐bradykinin induced an increase in [Ca2+]i at concentrations of 1 μm and 10 μm. The response to 10 μm des‐Arg9‐bradykinin was reduced by preincubation of either d‐Arg[Hyp3,Thi5,8,d‐Phe7]‐bradykinin (10 μm) or des‐Arg9Leu8‐bradykinin (10 μm).

We conclude that bradykinin B1 receptors are expressed by cultured BTSM cells and mediate the des‐Arg9‐bradykinin‐induced influx of calcium ions at low agonist concentrations (< 1 μm). At higher concentrations, des‐Arg9‐bradykinin (1 μm and 10 μm) can stimulate both B1 and B2 receptors to effect intracellular calcium release under calcium‐free conditions.
The P2Z‐purinoceptor of human lymphocytes: actions of nucleotide agonists and irreversible inhibition by oxidized ATP
Authors: Wiley James S. , Chen Joan R. , Snook Marie B. , Jamieson Gary P.
Published: July 2012

Extracellular adenosine triphosphate (ATP) is known to open a receptor‐operated ion channel (P2Z class) in human lymphocytes which conducts a range of cationic permeants. The activity of a range of different agonists and inhibitors towards the P2Z‐purinoceptor was investigated by measuring the agonist‐induced influx of Ba2+ into fura‐2 loaded lymphocytes.

The most potent agonist was 2′ & 3′‐0‐(4‐benzoylbenzoyl)‐ATP (benzoylbenzoic ATP) which gave 2 fold greater maximum Ba2+ influx and had a 10 fold lower EC50 than for ATP. The rank order of agonist potency in K+‐media was benzoylbenzoic ATP>>ATP = 2‐methylthio ATP = 2‐chloro ATP>ATP‐γ‐S. ADP, UTP and α,β‐methylene ATP were unable to stimulate Ba2+ influx.

Extracellular Na+ inhibited the increment of Ba2+ influx induced by all concentrations of ATP, 2‐methylthio ATP, 2‐chloroATP and ATP‐γ‐S. This inhibitory effect of extracellular Na+ is also reflected in the different EC50s for benzoylbenzoic ATP (8 μm in K+‐media, 18 μm in Na+‐media) but the maximal response to this agonist was the same in the presence or absence of Na+.

Treatment of lymphocytes with 2,3 dialdehyde ATP (oxidized ATP) at 300 μm for 60 min gave total and irreversible inhibition of ATP‐induced Ba2+ influx. 5′‐p‐Fluorosulphonyl benzoyladenosine (FSBA) also was an irreversible inhibitor but the maximal inhibition achieved was 90%.

It is concluded that the P2Z‐purinoceptor of human lymphocytes has a rank order of agonist potency which clearly distinguishes it from other P2‐receptors and that oxidized ATP is a convenient irreversible inhibitor for the P2Z‐purinoceptor.
Production of PGE2 by bovine cultured airway smooth muscle cells and its inhibition by cyclo‐oxygenase inhibitors
Authors: Delamere F. , Holland E. , Patel S. , Bennett J. , Pavord I. , Knox A.
Published: July 2012

Prostaglandin E2 (PGE2) is thought to be an important inhibitory modulator of inflammatory processes in the airway. It inhibits inflammatory cell function and cholinergic neurotransmission in vitro and roles have been postulated in vivo in refractoriness and in the mechanism of action of the diuretic agent, frusemide.

The production of PGE2 by bovine cultured airway smooth muscle cells has been studied under a range of conditions. The effects of cyclo‐oxygenase inhibitors (flurbiprofen, indomethacin, acetyl salicylic acid) on serum‐induced production of PGE2 were assessed over a range of concentrations (10−7–10−4 m).

Serum‐stimulated production of PGE2 in control wells ranged from 350 to 800 ng PGE2 ml−1 in cells from different animals. All three cyclo‐oxygenase inhibitors inhibited PGE2 production with an order of potency, flurbiprofen > indomethacin > acetyl salicylic acid. Log IC50 values were − 6.24 for flurbiprofen, − 5.23 for indomethacin and − 3.50 for acetyl salicylic acid.

PGE2 production was stimulated by arachidonic acid (10−5 m) or addition of the proinflammatory mediator, bradykinin (10−8–10−5 m).

Incubation of cells for 24 h with 5 bromo deoxyuridine (BRDU) (10−4 m) to prevent DNA synthesis did not alter PGE2 production in response to serum, suggesting that it was not a function of proliferation per se.

Our study suggests that airway smooth muscle may be an important source of PGE2. Production of PGE2 may be a novel feedback mechanism whereby airway smooth muscle cells can negatively modulate airways inflammation. The differing potencies of the cyclo‐oxygenase inhibitors may explain the contrasting effect of these drugs in recent studies in asthma.
Characterization of the capsaicin‐sensitive component of cyclophosphamide‐induced inflammation in the rat urinary bladder
Authors: Ahluwalia A. , Maggi C.A. , Santicioli P. , Lecci A. , Giuliani S.
Published: July 2012

Cyclophosphamide (CYP) (150 mg kg−1, i.p. 0.5–48 h before) caused a time‐dependent plasma protein extravasation in the rat urinary bladder with the maximal extravasation occuring at between 2 and 4 h after administration of the drug.

Prior capsaicin desensitization of capsaicin‐sensitive primary afferent neurones (CSPANs) (50 mg kg−l, s.c., 4 days before) resulted in approximately 50% inhibition of the magnitude of the extravasation response at the 2 h time‐point.

Intraperitoneal (i.p.) pretreatment with the tachykinin NK1 receptor antagonist, RP 67,580 (0.44 mg kg−1) or the bradykinin B2 receptor antagonist, Hoe 140 (0.13 mg kg−1) had significant inhibitory effects, giving responses of 56 ± 6% and 39 ± 4% of the control extravasation response to CYP treatment after 2 h. Pretreatment with the tachykinin NK2 receptor antagonist, SR 48,968 (0.3 mg kg−1, i.p.), the histamine H1 receptor blocker, chlorpheniramine (10 mg kg−1, i.p.), the 5‐HT receptor blocker, methysergide (6 mg kg−l, i.p.) or the cyclo‐oxygenase inhibitor indomethacin (5 mg kg−1, i.p.) had no significant effect upon the development of the extravasation response at this same time‐point.

In rat isolated urinary bladder strips, the active metabolite of CYP, acrolein (1–300 μm) produced a concentration‐dependent contraction that was significantly reduced by in vitro capsaicin desensitization (10 μm for 15 min) indicating direct stimulation of CSPANs. CYP was without appreciable effect.

The effect of acrolein in vitro was significantly reduced by pretreatment of the bladder with a combination of tachykinin NK1 and NK2 receptor antagonists, RP 67,580 (3 μm) and SR 48,968 (1 μm). The dose‐response curve to acrolein was also significantly inhibited by treatment with indomethacin (10 μm) and slightly affected by Hoe 140 (1 μm).

These findings demonstrate the contribution of CSPANs to the development of CYP‐induced cystitis. Plasma protein extravasation involves activation of tachykinin NK1 and bradykinin B2 receptors. Activation of CSPANs in the urinary bladder is likely to be due to the conversion of CYP into its active metabolite, acrolein, and not to a direct effect of CYP upon these nerve‐endings.
Functional effects and ligand binding of chimeric galanin‐neuropeptide Y (NPY) peptides on NPY and galanin receptor types
Authors: Kahl Ulrika , Langel Ülo , Bartfai Tamas , Grundemar Lars
Published: July 2012

The effects and binding characteristics of a series of chimeric galanin‐neuropeptide Y (NPY) peptides were examined in various preparations known to contain a predominant population of either Y1 or Y2 receptors for NPY or galanin receptors.

NPY suppressed the electrically stimulated twitches of the rat vas deferens (Y2 receptors), while galanin enhanced the electrically stimulated twitches. The galanin‐NPY peptides m 32 (galanin(1–13)‐NPY(25–36)), M69A (galanin(1–13)‐Lys‐[εNH‐Gly‐NPY(4‐1)]NPY(25–36)) and M88 (galanin(1–12)‐Ala‐NPY(25–36)) evoked a concentration‐dependent suppression of the electrically stimulated twitches. These chimeric peptides were about equipotent with NPY, while NPY (13–36) was about five times less potent than NPY itself. Also a stochiometric combination of the N‐ and C‐terminal fragments NPY (1–24)NH2 and NPY (25–36) (each at 1 μm) was inactive in vas deferens. M120 (galanin (1–13)‐NPY(14–36) (1 μm) did not affect the NPY‐mediated suppression of the stimulated twitches.

NPY evoked a concentration‐dependent contraction in the guinea‐pig isolated caval vein (Y1 receptors), while galanin (≤ 1 μm) was inactive. M32, M69A and M88 induced a slight contraction at very high concentrations only (≥ 0.3 m), while M120 was inactive at 1 μm. None of the four chimeric peptides affected the contraction evoked by NPY.

Since the number of NPY receptors in the rat vas deferens and guinea‐pig caval vein were too low, the affinities of the galanin‐NPY peptides for [3H]‐NPY binding sites were examined in membranes from rat brain areas known to contain predominant populations of Y1 receptors (cerebral cortex) and Y2 receptors (hippocampus), respectively. The chimeric peptides M32, M69A, M88, M120 and NPY (13–36) all had higher affinities for hippocampal binding sites than for cerebral cortical binding sites. These peptides were 90–440 times less potent than NPY at cerebral cortical binding sites and 15–125 times less potent than NPY at hippocampal binding sites. The most selective chimeric peptide was M32, which had a 20 fold higher affinity for hippocampal than for cerebral cortical binding sites.

At hypothalamic [125I]‐galanin binding sites M32, M88 and M69A were equipotent with galanin, while M120 was about 10 times less potent than galanin. M32, M88 and M69A, like galanin contracted the rat isolated jejunum.

The N‐terminal portion (1–12) of galanin seems to permit a steric conformation of the attached NPY (25–36) part of the chimeric galanin‐NPY peptides, which results in a facilitated Y2 but not Y1 receptor recognition and activation. None of the galanin‐NPY peptides appeared to act as antagonists at either type of NPY receptor, probably due to their low affinity. Instead, they displayed a very high affinity for hypothalamic galanin receptors and probably act as galanin agonists in the rat jejunum.
The effects of propofol and enflurane on single calcium channel currents of guinea‐pig isolated ventricular myocytes
Authors: Takahashi H. , Puttick R.M. , Terrar D.A.
Published: July 2012

The effects of the anaesthetics, propofol (100 μm) and enflurane (3%, 1.46 mm), on single L type calcium channel currents were investigated in single myocytes isolated from guinea‐pig ventricles. Channel activity was recorded from membrane patches by use of the ‘cell‐attached’ patch‐clamp technique (pipette solution containing 110 mm BaCl2, 5 μm Bay K 8644, 5 μm HEPES, pH 7.4; temperature 36°C).

Channel conductance was calculated from the slope of the relationship between single channel current and membrane potential during step depolarizations to activate the channel over a range of approximately − 20 to + 20 mV. Neither propofol (6 cells) nor enflurane (7 cells) caused any significant reduction in channel conductance.

Both propofol (7 cells) and enflurane (9 cells) decreased the probability of the channel being open during depolarizations to + 10 mV (measured from histograms of the fraction of time spent by the channel at different current levels, taking areas under the Gaussian curves fitted to the open and closed components of the distributions to represent the proportion of time spent in the two states).

A fraction of the current traces showed no detectable channel openings in response to step depolarizations to + 10 mV. Both propofol and enflurane significantly increased the fraction of silent traces.

Transitions across a threshold halfway between the open and closed levels were used to define periods spent in the open and closed states. Both propofol (7 cells) and enflurane (9 cells) reduced the mean open times and increased the mean closed times of the calcium channel.

Histograms were plotted showing the distributions of times spent by the channels in the open and closed states. Two exponentials were fitted to the open and closed time distributions. Both propofol (7 cells) and enflurane (9 cells) shortened both time constants fitted to the open times and lengthened both time constants fitted to the closed times.

It is concluded that both propofol and enflurane appear to alter the kinetics of opening and closing of calcium channels to favour shut channels without altering channel conductance. This effect would be expected to result in a reduction of the macroscopic calcium current and thus contribute to the negative inotropic action of these anaesthetics.
Reduction of endothelial function with age in the mesenteric arterial bed of the normotensive rat
Authors: Atkinson Jeffrey , Tatchum‐Talom Rabelais , Capdeville‐Atkinson Christine
Published: July 2012

Age‐related changes in endothelial vasodilator function were studied in an in vitro preparation of the mesenteric arterial bed removed from male, normotensive, Wistar rats.

Animals were killed at 2, 12 or 22 months of age, the superior mesenteric artery was cannulated and the gut removed. The mesenteric arterial bed was perfused at a constant flow rate of 4 ml min−1and perfusion pressure was taken as an index of arteriolar tone.

The muscarinic agonist, carbachol, antagonized noradrenaline‐induced vasoconstriction in the presence, but not in the absence, of endothelium. This cholinoceptor agonist‐induced release of endothelial‐derived relaxing factor (EDRF) was impaired in 22 month old rats.

Noradrenaline‐induced vasoconstriction increased following removal of endothelium suggesting that agonist‐induced release of EDRF attenuates vasoconstrictor responses to noradrenaline measured in the presence of endothelium.

Removal of endothelium had less effect on noradrenaline‐induced vasoconstriction in old rats suggesting once again that agonist‐induced release of EDRF is impaired in old rats.

The noradrenaline dose‐response curve established in the presence of endothelium was shifted to the left in 22 month old rats.

In conclusion, aging in the rat appears to lead to a reduction in endothelial vasodilator function in a resistance vessel.