bluuebunny/crossref_metadata_2025 huggingface dataset:
The influence of bumetanide on the membrane potential of mouse skeletal muscle cells in isotonic and hypertonic media
Authors: Van Mil H G J , Geukes Foppen R J , Siegenbeek van Heukelom J
Published: August 2006

Increasing the medium osmolality, with a non‐ionic osmoticant, from control (289 mOsm) to 319 mOsm or 344 mOsm in the lumbrical muscle cell of the mouse, resulted in a depolarization of the membrane potential (Vm) of 5.9 mV and 10.9 mV, respectively.

In control medium, the blockers of chloride related cotransport bumetanide and furosemide, induced a hyperpolarization of −3.6 and −3.0 mV and prevented the depolarization due to hypertonicity. When bumetanide was added in hypertonic media Vm fully repolarized to control values.

In a medium of 266 mOsm, the hyperpolarization by bumetanide was absent.

At 344 mOsm the half‐maximal effective concentration (IC50) was 0.5 μm for bumetanide and 21 μm for furosemide.

In solutions containing 1.25 mm sodium the depolarization by hypertonicity was reduced to 2.3 mV.

Reducing chloride permeability, by anthracene 9 carboxylic acid (9‐AC) in 289 mOsm, induced a small but significant hyperpolarization of −2.6 mV. Increasing medium osmolality to 344 mOsm enlarged this hyperpolarization significantly to −7.6 mV.

In a solution of 344 mOsm containing 100 μm ouabain, the bumetanide‐induced hyperpolarization of Vm was absent.

The results indicate that a Na‐K‐2Cl cotransporter is present in mouse lumbrical muscle fibre and that its contribution to Vm is dependent on medium osmolality.

British Journal of Pharmacology (1997) 120, 39–44; doi:10.1038/sj.bjp.0700887
Pharmacology of two novel mixed ETA/ETB receptor antagonists, BQ‐928 and 238, in the carotid and pulmonary arteries and the perfused kidney of the rabbit
Authors: Maurice Marie‐Claude , Gratton Jean‐Philippe , D'Orléans‐Juste Pedro
Published: August 2006

In the present study, we have pharmacologically characterized two novel mixed endothelin ETA/ETB receptor antagonists, namely BQ‐928 and BQ‐238, in ETA and ETB preparations, the rabbit carotid artery (RbCA) and the rabbit pulmonary artery (RbPA), respectively. These two antagonists were compared to established ETA (BQ‐123 and BMS 182874), ETB (BQ‐788) and mixed ETA/ETB (SB 209670) receptor antagonists.

In the RbCA, the ETA monoreceptor preparation, BQ‐238 and BQ‐928 had apparent affinities (pA2) of 7.42±0.22 and 7.22±0.18, respectively, BQ‐788 being inactive in this preparation. In the ETB monoreceptor preparation, the RbPA (when IRL‐1620 was used as an ETB receptor agonist), the pA2 for BQ‐238 was 7.05±0.14 and for BQ‐928 was 8.43±0.04. BQ‐123 and BMS 182874 were inactive in this preparation. Similar to SB 209670, BQ‐238 but not BQ‐928 had a higher affinity for the ETA than the ETB receptor.

All of the antagonists were tested for their ability to block and reverse endothelin‐1‐induced vasoconstrictions in the rabbit perfused kidney. In this preparation endothelin‐1‐induced increases in vascular resistance have been shown to be mediated solely by ETA receptors. All compounds (except BQ‐788) blocked the pressor effects of endothelin within the kidney; the calculated IC50 values for BQ‐123, BMS 182874, SB 209670, BQ‐928 and BQ‐238 were 0.4 μm, 2 μm, 0.01 μm, 0.4 μm and 0.09 μm, respectively.

In all experiments in the rabbit perfused kidney, endothelin‐1 was readministered for a third time, 60 min following cessation of infusion of the above‐mentioned antagonists. The response to the third infusion of endothelin‐1 following cessation of infusion of BQ‐123, BMS 182874 and SB 209670 was not significantly different from that to the third infusion of endothelin in control conditions. However, the response to endothelin‐1 was significantly higher than control in tissues pre‐infused with BQ‐788 or BQ‐928 (56±9 and 41.6±15%, respectively, n=8 each, P<0.05).

Our results suggest that in a system where ETA receptor activation is responsible for vasoconstriction and ETB‐receptor activation for vasodilatation, ETA receptor selective antagonists or mixed ETA/ETB receptor antagonists which possess high affinity for ETA receptors do not induce hyperresponsiveness to endothelin‐1. In contrast, ETB selective antagonists or mixed antagonists possessing a high affinity for ETB receptors (such as BQ‐928) interfere with the ETB‐receptor‐dependent physiological antagonism of endothelin‐1‐induced pressor responses in these same tissues.

British Journal of Pharmacology (1997) 120, 319–325; doi:10.1038/sj.bjp.0700895
Potentiation of ATP‐responses at a recombinant P2X2 receptor by neurotransmitters and related substances
Authors: Wildman S S , King B F , Burnstock G
Published: August 2006

The modulator effects of a series of neurotransmitters and related substances were tested on responses to adenosine 5′‐triphosphate (ATP) at a recombinant P2X2 receptor expressed in defolliculated Xenopus oocytes.

Nicotine, 5‐hydroxytryptamine (5‐HT), noradrenaline, adenosine, bradykinin and histamine (all 100 μm) potentiated the responses to ATP (3 μm), an effect found due to acidification of the bathing solution by these drugs.

Arachidonic acid, met‐enkephalin, substance P, calcitonin gene‐related peptide (CGRP) (all 100 μm) and nerve growth factor (NGF; 50 ng ml−1) potentiated the responses to ATP (3 μm) through a largely or wholly pH‐independent effect.

Small acidic and alkaline shifts, as little as 0.03 pH‐units, enhanced or diminished the responses to ATP, respectively. A linear relationship existed between the degree of potentiation of the ATP‐induced responses caused by nicotine, 5‐HT, noradrenaline, adenosine, bradykinin and histamine and the potentiation of these responses induced by the addition of acid to the superfusate.

Since P2X receptors on sensory neurones include P2X2 subunits, the attendant acidosis and ATP‐release associated with tissue injury may play a role in sensitizing sensory nerve fibres.

British Journal of Pharmacology (1997) 120, 221–224; doi:10.1038/sj.bjp.0700903
The effects of the peptide KPNFIRFamide (PF4) on the somatic muscle cells of the parasitic nematode Ascaris suum
Authors: Holden‐Dye L. , Brownlee D.J.A. , Walker R.J.
Published: August 2006

Commonly used anthelmintic agents act on the muscle cells of parasitic nematodes to cause paralysis of the parasite and its expulsion from the host.

The motonervous system of nematodes contains neuropeptides, many of which are myoactive and elicit prolonged worm paralysis. Here we describe the actions of a novel peptide, KPNFIRFamide (Lys‐Pro‐Asn‐Phe‐Ileu‐Arg‐Phe‐amide; PF4), which mediates relaxation of the somatic muscle of the parasitic nematode Ascaris suum. Its mechanism of action is compared to that of the inhibitory neuromuscular junction transmitter, γ‐aminobutyric acid (GABA), which gates a chloride channel on Ascaris muscle.

Both PF4 and GABA hyperpolarized the muscle cells (EC50 values 98 nM and 59 μM, respectively; n = 6) and this was accompanied by an increase in input conductance.

The increase in input conductance elicited by PF4 and a supramaximal concentration of GABA were additive (10 μm PF4, 7.78 ± 1.88 μS; 10 mM GABA, 4.68 ± 1.39μS; 10 mM GABA and 10μm PF4 12.05±2.6 μS, n = 6, P<0.02 with respect to PF4 alone; P<0.01 with respect to GABA alone).

The membrane potential response to 10 μm PF4 initially consisted of a fast hyperpolarization that occurred within 1 min of PF4 application. The reversal potential for this early response to PF4 (PF4‐early) was determined at different extracellular chloride concentrations. Linear regression analysis of the natural logarithm of the extracellular chloride concentration against the reversal potential for PF4‐early yielded a straight line with a slope of −29.6±2.4 (‐34.4 to −24.9, 95% confidence limits; r2 = 0.82). This is close to the slope of −26.5 for a chloride‐dependent event, as predicted by the Nernst equation. There was a significant correlation between the reversal potential for this event and the reversal potential for GABA (r = 0.94; P<0.001; n = 12).

The late response to PF4 (PF4‐late) appeared after 1 min and consisted of a slow reduction in the hyperpolarization to a plateau level, before the return of the membrane potential to the resting value. PF4‐late is not likely to be a chloride‐dependent event as during the hyperpolarization caused by a supramaximal concentration of GABA the muscle cells depolarized when a supramaximal concentration of PF4 was added to the perfusate. The membrane potential in the presence of 1 mM GABA was −61.8±4.8 mV and in the presence of 1 mM GABA with 10 μm PF4 was −47.5± 1.5 mV (P<0.02; n = 6).

The conductance increase elicited by 30 μm GABA was blocked by 10 μm ivermectin (before ivermectin 0.97 ±0.2 μS, after ivermectin 0.33 ±0.12 μS; n = 5; P<0.05; Student's paired t test) but the conductance increase elicited by 1 μm PF4 was not (before ivermectin 0.96±0.14 μS, after ivermectin 1.07±0.19 μS; n = 0.34; Student's paired t test).

These data indicate that PF4 elicits a potent, inhibition of Ascaris muscle cells which is partially mediated by chloride and which is independent of the inhibitory GABA receptor.
Functional GABAAreceptors on rat vagal afferent neurones
Authors: Ashworth‐Preece Mark , Krstew Elena , Jarrott Bevyn , Lawrence Andrew J.
Published: August 2006

In the present study,in vitroelectrophysiology and receptor autoradiography were used to determine whether rat vagal afferent neurones possess γ‐aminobutyric acid (GABA)Areceptors.

GABA (1‐100 μM) and isoguvacine (3–100 μm) caused a concentration‐dependent depolarization of the rat isolated nodose ganglion preparation at room temperature. When applied to the tissue 20 min before the agonist, SR95531 (3 μm) and bicuculline (3μm) caused a parallel shift to the right of the GABA and isoguvacine concentration‐response curves, yielding shifts of 81 fold and 117 fold for SR95531 and 4 fold and 12 fold for bicuculline, respectively.

Baclofen (10 nM‐100 μm) was unable to elicit a depolarization of the rat isolated nodose ganglion preparation at either room temperature or at 36°C, whilst 5‐aminovaleric acid (10 μm), a GABABreceptor antagonist, was unable to antagonize significantly the GABA‐induced depolarization at either room temperature or at 36°C.

[3H]‐SR95531 (7.2 nM), a GABAAreceptor‐selective antagonist, bound topographically to sections of rat brainstem. Specific binding was highest in the medial nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMVN). Binding was also observed in certain medullary reticular nuclei, in particular the parvocellular reticular nucleus.

Unilateral nodose ganglionectomy caused a reduction in GABAAbinding site density in the medial NTS from 93 ± 7 to 68 ± 6 d.p.m./mm2. This procedure also caused a reduction in GABAAbinding site density in the side of the NTS contralateral to the lesion, from 151 ± 12 to 93 ± 7 d.p.m./mm2. Sham surgery had no effect on the binding of [3H]‐SR95531 in rat brainstem.

The present data provide evidence for the presence of GABAAreceptors located on the soma and central terminals of rat vagal afferent neurones. Additionally, a population of GABAAreceptors is evidenced postsynaptically in the rat NTS with respect to vagal afferent terminals. These data are discussed in relation to the functional pharmacology of GABA in this region of the NTS.
Preliminary characterization of the role of protein serine/ threonine phosphatases in the regulation of human lung mast cell function
Authors: Peirce Matthew J , Cox Sarah E , Munday Michael R , Peachell Peter T
Published: August 2006

Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE‐dependent release of mediators from human lung mast cells (HLMC). The concentration of okadaic acid required to inhibit by 50% (IC50) the IgE‐dependent release of histamine was 0.2 μm. Okadaic acid also inhibited the IgE‐mediated generation of prostaglandin D2(PGD2) and sulphopeptidoleukotrienes (sLT) with IC50values of 0.2 μmand 0.6 μmrespectively.

The IgE‐mediated generation of histamine, PGD2and sLT was inhibited by okadaic acid and two analogues of okadaic acid, okadaol and okadaone, with the following rank order of activity; okadaic acid>okadaol>okadaone. This order of activity for the inhibition of mediator release parallels the activity of these compounds as inhibitors of isolated PPs.

Extracts of HLMC liberated32P from radiolabelled glycogen phosphorylase and this PP activity was inhibited by the PP inhibitors (all at 3 μm), okadaic acid (73±4% inhibition,P<0.0005), okadaol (26±7% inhibition,P<0.05) and okadaone (8±7% inhibition,P=0.52). The rank order of activity of okadaic acid>okadaol>okadaone parallels the activity of these compounds as inhibitors of isolated PPs.

Dephosphorylation of radiolabelled glycogen phosphorylase by extracts of HLMC was inhibited by 15±3% (P<0.001) by a low (2 nm) concentration of okadaic acid and by 88±4% (P<0.0005) by a higher (5 μm) concentration of okadaic acid. Because 2 nmokadaic acid may act selectively to inhibit PP2A whereas 5 μmokadaic acid inhibits both PP1 and PP2A, these data suggest that both PP1 and PP2A are present in HLMC.

Inhibitor 2, a PP1‐selective inhibitor, attenuated (71±3% inhibition,P<0.05) PP activity in extracts of HLMC suggesting that HLMC contain PP1 and that it may constitute 71% of the phosphorylase PP activity in extracts of HLMC.

Radiolabelled casein, a PP2A‐restricted substrate, was dephosphorylated by extracts of purified HLMC and this activity was inhibited (81±8% inhibition,P<0.005) by 2 nmokadaic acid suggesting that PP2A is resident in HLMC.

Collectively, these data suggest that both PP1 and PP2A are resident in HLMC. However, although the data suggest that okadaic acid regulates responses in HLMC by interacting with PPs, it has not been possible to determine whether either PP1 or PP2A or both PPs are involved in the okadaic acid‐induced inhibition of mediator release from HLMC.

British Journal of Pharmacology(1997)120, 239–246; doi:10.1038/sj.bjp.0700915
Evidence that both nitric oxide (NO) and a non‐NO hyperpolarizing factor elicit NANC nerve‐mediated relaxation in the rat isolated anococcygeus
Authors: Selemidis S. , Cocks T.M.
Published: August 2006

Responses to electrical field stimulation (EFS; 0.5–10 Hz, 0.2 ms duration, supramaximal voltage for 20 s) of non‐adrenergic, non‐cholinergic, (NANC) nerves were obtained in preparations of rat anococcygeus pre‐contracted with titrated concentrations of phenylephrine (0.1–1 μM) to ∼40% of their maximum contraction to phenylephrine (Fmax) regardless of drug treatment.

With this set level of active force, NANC nerve stimulation resulted in relaxations that were maximal (peak relaxation) at 0.5–1 Hz, abolished by tetrodotoxin (1 μM) but only minimally blocked by the nitric oxide synthase (NOS) inhibitor, NG‐nitro‐L‐arginine, (L‐NOARG; 100 μM). Furthermore, the nitric oxide (NO) scavenger, oxyhaemoglobin (HbO; 30 μM) gave no further block alone or in combination with L‐NOARG (100 μM). By comparison, in preparations contracted with phenylephrine to ∼70% Fmax, relaxations to NANC nerve stimulation were markedly reduced or abolished by combined treatment with L‐NOARG (100 μM) and HbO (30 μM).

Nifedipine (0.3 μM) significantly inhibited NANC nerve‐mediated relaxations, which became frequency‐dependent and abolished those resistant to L‐NOARG (100 μM) and HbO (30 μM).

These data suggest that a non‐NO, hyperpolarizing factor and NO both contribute to NANC nerve‐mediated inhibitory responses in the rat anococcygeus. However, responses to the non‐NO factor were observed only in preparations contracted sub‐maximally by a nifedipine‐sensitive mechanism.
Characterization of the P2 receptors in rabbit pulmonary artery
Authors: Qasabian R.A. , Schyvens C. , Owe‐Young R. , Killen J.P. , Macdonald P.S. , Conigrave A.D. , Williamson D.
Published: August 2006

We have identified the P2 receptors mediating vasomotor responses in the rabbit pulmonary artery.

Neither ATP nor UTP contracted intact or endothelium‐denuded rings. However, both relaxed intact rings of rabbit pulmonary artery that had been preconstricted with phenylephrine (pD2 5.2 and 5.6, respectively).

The vasodilator effect of UTP was endothelium‐dependent and abolished by the nitric oxide synthase inhibitor NG‐nitro‐L‐arginine (L‐NOARG).

The vasodilator effect of ATP was only partially inhibited by removal of endothelium or addition of L‐NOARG, suggesting an additional direct effect on vascular smooth muscle.

The endothelium‐dependent vasodilator responses to UTP and ATP were competitively antagonized by suramin.

Preconstricted, endothelium‐denuded rings were also relaxed by 2‐methylthio ATP (pD2 6.6), a P2Y receptor agonist.

Ca2+‐mobilizing P2U receptors were identified on smooth muscle cells on the basis of single cell responses to ATP (pD2 7.8) and UTP (pD2 7.9; 6.7 in the presence of 100 μm suramin).

There was no evidence of a Ca2+‐mobilizing P2Y receptor in these cultured cells.

The data suggest the presence of (i) a suramin‐sensitive P2U receptor on endothelial cells that induces vasorelaxation through NO release, (ii) a suramin‐sensitive P2U receptor on cultured smooth muscle cells that mobilizes Ca2+ but is not coupled to vasomotor responses and (iii) a putative P2Y receptor on vascular smooth muscle cells that induces relaxation via a Ca2+‐independent signal transduction pathway.
Inhibitory effects of TAK‐044 on endothelin induced vasoconstriction in various canine arteries and porcine coronary arteries: a comparison with selective ETA and ETB receptor antagonists
Authors: Awane‐Igata Yumiko , Ikeda Shota , Watanabe Toshifumi
Published: August 2006

The inhibitory effects of the endothelin (ET) receptor antagonist, TAK‐044, on ET‐induced vasoconstriction in various canine arteries and porcine coronary arteries were studied and were compared to those of selective ETA and ETB receptor antagonists.

ET‐1 (0.1 nM‐0.3 μm) caused vasoconstriction in canine coronary, femoral, renal, mesenteric and basilar arteries, and the strongest responses were obtained in coronary and basilar arteries. TAK‐044 (10 nM, 100 nM) inhibited this ET‐1‐induced vasoconstriction except in the case of mesenteric arteries. The strongest inhibitory effects were obtained in coronary arteries; an EC50 value for ET‐1 was 5.2 ± 0.77 nM (n = 12) in the control and 24 ± 3.8 nM (n = 4) in the presence of TAK‐044 at 10 nM. BQ‐123 (1 μm) inhibited the vasoconstriction in coronary and femoral arteries but did not in renal, mesenteric or basilar arteries.

TAK‐044 (10–100 nM) inhibited the ET‐1‐induced vasoconstriction in porcine coronary arteries to a degree similar to that in canine coronary arteries. In contrast, BQ‐123 (10 μm) did not inhibit the contraction completely, and a BQ‐123‐insensitive component was identified. Although BQ‐788 (1 μm) did not modify the concentration‐response curve at all, it abolished the BQ‐123‐insensitive component when applied together with BQ‐123 (10 μM).

Sarafotoxin S6c (10 pM‐30 nM) caused vasoconstriction in porcine coronary arteries with the maximum amplitude of the contraction being 39% of that with ET‐1. Both TAK‐044 (10 nM, 100 nM) and BQ‐788 (1 μM) inhibited this vasoconstriction, while BQ‐123 (3 μM, 10 μm) did not.

Vasoconstriction induced by ET‐3 (0.1 nM‐0.3 μm) in porcine coronary arteries showed a concentration‐response curve with two distinct phases in contrast to that seen with sarafotoxin S6c. TAK‐044 (0.3 nM‐10 nM) inhibited both phases in a concentration‐dependent manner. BQ‐123 (1 μM, 3 μm) inhibited only the second phase, while BQ‐788 (1 μm) inhibited the first phase.

We concluded that the inhibitory effects of TAK‐044 on ET‐1‐induced vasoconstriction were the strongest in coronary arteries among the canine arteries examined. In addition, we showed that both ETA and ETB receptors mediate vasoconstriction in porcine coronary arteries and TAK‐044 inhibits the vasoconstriction mediated by both of these receptors.
The effect of the protein phosphatases inhibitor cantharidin on β‐adrenoceptor‐mediated vasorelaxation
Authors: Knapp Jörg , Bokník Peter , Linck Bettina , Luss Hartmut , Müller Frank U. , Neumann Peter Nacke Joachim , Vahlensieck Ute , Schmitz Wilhelm
Published: August 2006

Cantharidin, an inhibitor of protein phosphatase types 1 (PP1) and 2A (PP2A), increased basal tone of bovine isolated coronary artery rings (CARs) with and without endothelium in a time‐and concentration‐dependent manner with pEC50 values of about 5.1 and 5.2, respectively, for both preparations.

β‐Adrenoceptor stimulation with isoprenaline (Iso; 0.03–100 μm) or inhibition of phosphodiesterase activity by 3‐isobutyl‐l‐methylxanthine (IBMX; 10–1000 μm), respectively, relaxed CARs precontracted with KC1 (75 mm). CARs with and without endothelium showed no difference in the relaxing response to Iso and IBMX, respectively.

Cantharidin (3 μm) attenuated vasorelaxation induced by Iso (0.03–100 μm) in CARs with and without endothelium in a time‐dependent manner, whereas vasorelaxation induced by IBMX (10–1000 μm) was not attenuated by 3 μM cantharidin.

Cantharidin (3 μm) did not affect cyclic AMP content in bovine cultured vascular cells, i.e. coronary artery smooth muscle cells (BCs), aortic endothelial cells (BAECs) and aortic smooth muscle cells (BASMCs), either under basal conditions, after β‐adrenoceptor stimulation (Iso) or inhibition of phosphodiesterase activity (IBMX), respectively.

Cantharidin inhibited protein phosphatase activity in homogenates from bovine coronary artery rings with a pIC50 of about 6.0. In homogenates of bovine cultured vascular cells pIC50 values of cantharidin amounted to about 6.5 for BCs, 6.7 for BAECs and 6.7 for BASMCs, respectively.

It was concluded that cantharidin differently affects vasorelaxation due to stimulation of [3‐adrenoceptors (Iso) or inhibition of phosphodiesterase activity (IBMX), respectively. The attenuation of β‐adrenoceptor‐mediated vasorelaxation by phosphatase inhibition is not due to diminished adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) generation but could be evidence for different subcellular compartments of cyclic AMP.
Activation and inhibition of rat neuronal nicotinic receptors by ABT‐418
Authors: Papke Roger L. , Thinschmidt Jeffrey S. , Moulton Becky A. , Meyer Edwin M. , Poirier Amy
Published: August 2006

ABT‐418 appeared to function as a relatively broad spectrum activator of neuronal nicotinic receptors, expressed in Xenopus oocytes, with little cross reactivity to the mammalian muscle receptor subtype. However, the relative potencies of ABT‐418 at the various subtypes differed from those acetylcholine (ACh). For example, ACh was most potent at α3β2 (EC50 ∼30 μM) and least potent at α2β2 (EC50∼500 μM). ABT‐418 was most potent at α4β2 and α2β2 (EC50∼6 μM and 11 μM, respectively) and least potent at α3β4 (EC50 ∼188 μM).

In addition to activating neuronal receptors, ABT‐418 exhibited complex properties, including the inhibition of ACh responses.

The current responses elicited by relatively high concentrations of ABT‐418 on the α4β2 receptor subtype were protracted beyond the application interval. The coapplication of ABT‐418 with either of the use‐dependent inhibitors bis(1,2,2,6,6‐tetramethyl‐4‐pipendimyl)sebacate (BTMPS) or tetramethyl‐pipenidine (TMP) eliminated the late protracted phase of the currents with only small effects on the initial activation phase. When the reversible inhibitor TMP was washed from the bath, the previously inhibited late current reappeared, suggesting that the observed mixed agonist‐antagonist effects of ABT‐418 and (±)‐epibatidine on α4β2 were due to a concentration‐dependent noncompetitive inhibition, an effect similar to that obtained for (−)‐nicotine.

The inhibition of α4β2 receptors by ABT‐418 was voltage‐dependent. When high concentrations of ABT‐418 were applied under depolarizing conditions, additional late currents could be observed under conditions which suggested that a build up of ABT‐418 in an unstirred layer over the surface of the oocyte was occurring. This may have been due to the dissociation of the drug from channel blocking sites on the receptors themselves, or alternatively, from the plasma membrane of the cells.
Evidence against a role of cytochrome P450‐derived arachidonic acid metabolites in endothelium‐dependent hyperpolarization by acetylcholine in rat isolated mesenteric artery
Authors: Fukao Mitsuhiro , Hattori Yuichi , Kanno Morio , Sakuma Ichiro , Kitabatake Akira
Published: August 2006

In rat mesenteric artery, acetylcholine (ACh) causes endothelium‐dependent hyperpolarization by releasing endothelium‐derived hyerpolarizing factor (EDHF). Recent evidence suggests that EDHF may be a cytochrome P450‐derived arachidonic acid metabolite. The aim of the present study was to investigate whether such a metabolite is indeed contributing to ACh‐induced hyperpolarization observed in rat mesenteric artery.

The phospholipase A2 inhibitor quinacrine (30 μm) nearly completely eliminated ACh‐induced hyperpolarization. However, the hyperpolarizing effect of pinacidil was also abolished in the presence of quinacrine.

The imidazole antimycotic agents ketoconazole (50 μm), clotrimazole (30 μm) and miconazole (10 μm), which bind to the heme moiety of cytochrome P450, eliminated not only ACh‐induced hyperpolarizations but also those induced by pinacidil. SKF525A (30 μm), a prototype inhibitor of the enzyme, also abolished the hyperpolarizing responses to both agents. In contrast, neither 17‐octadecynoic acid (10 μm), a mechanism‐based inhibitor of cytochrome P450 metabolism of fatty acids, nor eicosatetraynoic acid (20 μm), an inhibitor of all arachidonic acid metabolic pathways, altered ACh‐induced hyperpolarization. Furthermore, the hyperpolarization was unaffected by the preferential inhibitors of specific cytochrome P450 isozymes, α‐naphtofiavone (1 μm), diedthyldithiocarbamate (50 μm), metyrapone (20 μm) and troleandomycin (10 μm).

Pretreatment of rats with lipopolysaccharide (2mgkg−1) and exposure to nitroprusside (10 μm), both of which are expected to inhibit cytochrome P450 activity due to nitric oxide overproduction, were without effect on ACh‐induced hyperpolarization. Pretreatment of rats for 3 days with pentobarbitone (80 mg kg−1 day−1), a cytochrome P450 inducer, also did not affect the hyperpolarizing response to ACh.

Arachidonic acid in concentrations up to 100 μM had no detectable effect on smooth muscle membrane potential. 11,12‐Epoxyeicosatrienoic acid (EET, 10 μm), one of cytochrome P450‐derived epoxygenase metabolites of arachidonic acid, elicited a small endothelium‐independent membrane hyperpolarization. The hyperpolarizing response to EET was blocked by glibenclamide (30 μm), in contrast to the response to ACh.

These results suggest that the contribution of a cytochrome P450‐derived metabolite of arachidonic acid to ACh‐induced hyperpolarization via EDHF release is minimal or absent in rat mesenteric artery.
Responses of rat substantia nigra dopamine‐containing neurones to (−)‐HA‐966 in vitro
Authors: Grobaski Kenneth C. , Ping Hanxian , Da Silva Helena M.A. , Bowery Norman G. , Connelly Stephen T. , Shepard Paul D.
Published: August 2006

Extracellular single unit recording techniques were used to compare the effects of (−)‐3‐amino‐l‐hydroxypyrrolidin‐2‐one ((—)‐HA‐966) and (±)‐baclofen on the activity of dopamine‐containing neurones in 300 μm slices of rat substantia nigra. Electrophysiological data were compared with the outcome of in vitro binding experiments designed to assess the affinity of (—)‐HA‐966 for γ‐aminobutyric acid (GABAB) receptors.

Bath application of (—)‐HA‐966 produced a concentration‐dependent inhibition of dopaminergic neuronal firing (EC50 = 444.0 μM; 95% confidence interval: 277.6 μm‐710.1 μM, n = 27) which was fully reversible upon washout from the recording chamber. Although similar effects were observed in response to (±)‐baclofen, the direct‐acting GABAB receptor agonist proved to be considerably more potent than (−)‐HA‐966 (EC50 = 0.54 μM; 95% confidence interval: 0.44 μm‐0.66 μM, n = 29) in vitro.

Low concentrations of chloral hydrate (10 μM) were without effect on the basal firing rate of nigral dopaminergic neurones but significantly increased the inhibitory effects produced by concomitant application of (—)‐HA‐966.

The inhibitory effects of (—)‐HA‐966 were completely reversed in the presence of the GABAB receptor antagonists, CGP‐35348 (100 μM) and 2‐hydroxysaclofen (500 μM). Bath application of CGP‐35348 alone increased basal firing rate. However, the magnitude of the excitation (9.2 ± 0.3%) was not sufficient to account for the ability of the antagonist to reverse fully the inhibitory effects of (—)‐HA‐966.

(—)‐HA‐966 (0.1–1.0 mM) produced a concentration‐dependent displacement of [3H]‐GABA from synaptic membranes in the presence of isoguvacine (40 μM). However, the affinity of the drug for GABAB binding sites was significantly less than that of GABA (0.0005 potency ratio) and showed no apparent stereoselectivity.

These results indicate that while (—)‐HA‐966 appears to act as a direct GABAB receptor agonist in vitro, its affinity for this receptor site is substantially less than that of GABA or baclofen and unlikely to account for the depressant actions of this drug which occur at levels approximately ten fold lower in vivo.
Bidirectional modulatory effect of orphanin FQ on morphine‐induced analgesia: antagonism in brain and potentiation in spinal cord of the rat
Authors: Tian Jin‐Hua , Xu Wei , Fang Yuan , Mogil Jeffrey S. , Grisel Judith E. , Grandy David K. , Han Ji‐Sheng
Published: August 2006

The present study was designed to investigate further the effects of the newly discovered orphanin FQ (OFQ)‐the endogenous ligand for the orphan opioid receptor (called, e.g., ORL1 and LC132)‐on pain modulation in the rat. We used the tail‐flick assay as a nociceptive index.

When injected into a cerebral ventricle, OFQ (4 fmol‐10 nmol) has no effect on basal tail‐flick latency by itself at any dose, but dose‐dependently antagonizes systemic morphine analgesia (400 fmol‐50 nmol).

Injected intrathecally, OFQ (3 and 10 nmol) displayed an analgesic effect without producing motor dysfunction, and potentiated morphine analgesia (1 and 10 nmol).

The anti‐opioid effect of OFQ in rat brain and the high level of expression of LC132/ORL1 receptor in the locus coeruleus indicated a possible role of OFQ in the precipitation of opiate withdrawal symptoms. However, no such precipitation was observed by OFQ in morphine‐dependent rats.
Inhibition of glycine currents by dextromethorphan in neurones dissociated from the guinea‐pig nucleus tractus solitarii
Authors: Takahama Kazuo , Fukushima Hidenao , Isohama Yoichiro , Kai Hirofumi , Miyata Takeshi
Published: August 2006

The effect of dextromethorphan (DM) on the current induced by glycine in acutely dissociated nucleus tractus solitarii (NTS) neurones of guinea‐pigs was studied by use of the whole‐cell patch clamp technique. The effect of DM on γ‐aminobutyric acid (GABA)‐induced currents (Igaba) was also examined.

DM inhibited 30 μm glycine‐induced current (IGly), without affecting the current caused by 30 μm GABA. The action of DM was concentration‐dependent, with a maximum effect at 100 μm, and reversible. The half‐maximum inhibitory concentration (IC50) of DM was 3.3 μm, about 85 times higher than that of strychnine.

DM 3 μm shifted the concentration‐response curve for glycine to the right without affecting the maximum value. DM 10 μm shifted the curve even more to the right, although it was not a parallel shift. Strychnine at a concentration of 0.1 μm shifted the curve for glycine in a nearly parallel fashion.

The effect of 10 μm DM was slightly weak voltage‐dependency, but the lower concentration of DM, 3 μm, inhibited IGly equally at −50 mV and + 50 mV. The effect of 3 μm DM on IGly showed no use‐dependence. Blockade by strychnine 0.1 μm showed no voltage‐or use‐dependence.

The results indicate that DM inhibits IGly in single neurones of NTS, and further suggest that DM at a low concentration may act on the glycine receptor‐ionophore complex, but not on the Cl− channel of the complex. However, a relatively high concentration of DM may at least partly affect the Cl− channel of the complex.
Characteristics of L‐type calcium channel blockade by lacidipine in guinea‐pig ventricular myocytes
Authors: Cerbai Elisabetta , Giotti Alberto , Mugelli Alessandro
Published: August 2006

The Ca2+‐antagonistic properties of lacidipine were investigated in patch‐clamp guinea‐pig ventricular myocytes.

In basal conditions, 0.1 μm lacidipine reduced the action potential duration, associated with a decrease in the L‐type calcium current (Ica,L) to 66 ± 4% of the control value, without a change in the current‐voltage relationship. Sodium current and background potassium currents were not affected. All the effects reached a steady state within 2 min.

The Ca2+‐antagonistic effect of lacidipine was voltage‐dependent: a marked negative shift (about 20 mV) of the steady‐state inactivation curve was observed with long (10 s) conditioning prepulses, but not with short (350 ms) prepulses.

The onset of and recovery from the voltage‐dependent effect caused by 0.1 μm lacidipine were significantly slower when compared to those of equiactive concentrations of nimodipine (0.5 μm) and nisoldipine (0.1 μm). ICa,L measured after prepulses at —40 mV lasting 500 ms or less was unchanged (95 ± 5% of maximum current value) while it was reduced to 49±10% by nimodipine and 43 ± 9% by nisoldipine (P <0.05 vs lacidipine for both).

Similarly, the recovery from block in the presence of lacidipine was slower than with nimodipine and nisoldipine. After a prepulse of 1 s at —80 mV, ICa,L recovered up to 54 ± 2% of the maximum current value in the presence of lacidipine, and up to 91 ±3% and 93 ± 5% in the presence of nimodipine and nisoldipine, respectively (P<0.05 vs lacidipine).

Blockade of ICa,L by lacidipine was use‐dependent. After ten 200 ms long pulses (1 Hz) from −80 mV, ICa,L was reduced to 55 ± 7% of the current measured at the first pulse. In the presence of nimodipine and nisoldipine, ICa,L elicited by the tenth pulse amounted to 93 ± 3% and 80 ± 6% of the first pulse value, respectively (P <0.05 vs lacidipine). Lacidipine did not cause use‐dependent blockade of Ica,L in cells stimulated with 10 ms long pulses.

These results demonstrate that lacidipine selectively inhibits ICa,L in isolated cardiomyocytes and suggest that this effect occurs mainly through binding to the inactivated Ca2+ channels.
Involvement of a glibenclamide‐sensitive mechanism in the nitrergic neurotransmission of the pig intravesical ureter
Authors: Hernández Medardo , Prieto Dolores , Orensanz Luis M. , Barahona María Victoria , Jiménez‐cidre Miguel , Rivera Luis , García‐sacristán Albino , Simonsen Ulf
Published: August 2006

The present study was designed to investigate whether potassium (K+) channels are involved in the relaxations to nitric oxide (NO) of pig intravesical ureteral preparations suspended in organ baths for isometric tension recordings. In ureteral strips treated with guanethidine (10−5 m) and atropine (10−7 m) to block adrenergic neurotransmission and muscarinic receptors, respectively, NO was either released from nitrergic nerves by electrical field stimulation (EFS, 0.5–10 Hz, 1 ms duration, 20 s trains), or exogenously‐applied as an acidified solution of sodium nitrite (NaNO2, 10−6‐10−3 m).

Incubation with an inhibitor of guanylate cyclase activation by NO, methylene blue (10−5 m) did not change the basal tension of intravesical ureteral strips but inhibited the relaxation induced by EFS or exogenous NO on ureteral preparations contracted with the thromboxane analogue U46619 (10−7 m).

Incubation with charybdotoxin (3 × 10−8 m) and apamin (5 × 10−7 m), which are inhibitors of large and small conductance calcium (Ca2+)‐activated K+ channels, respectively, did not modify basal tension or the relaxations induced by EFS and exogenous NO. Treatment with charybdotoxin or apamin plus methylene blue (10−5 m) significantly reduced the relaxations to EFS and exogenous NO. However, in both cases the reductions were similar to the inhibition evoked by methylene blue alone. The combined addition of charybdotoxin plus apamin did not change the relaxations to EFS or exogenously added NO of the porcine intravesical ureter.

Cromakalim (10−8‐3 × 10−6 m), an opener of ATP‐sensitive K+ channels, evoked a dose‐dependent relaxation with a pD2of 7.3 + 0.2 and maximum relaxant effect of a 71.8 + 4.2% of the contraction induced by U46619 in the pig intravesical ureter. The blocker of ATP‐sensitive K+ channels, glibenclamide (10−6 m), inhibited markedly the relaxations to cromakalim.

Glibenclamide (10−6 m) had no effect on the basal tone of ureteral preparations but significantly reduced the relaxations induced by both EFS and exogenous NO. Combined treatment with methylene blue (10−5 m) and glibenclamide (10−6 m) did not exert an effect greater than that of methylene blue alone on either EFS‐or NO‐evoked relaxations of the pig ureter.

The present results suggest that NO acts as an inhibitory neurotransmitter in the pig intravesical ureter and relaxes smooth muscle through a guanylate cyclase‐dependent mechanism which seems to favour the opening of glibenclamide‐sensitive K+ channels.
Involvement of adrenoceptors in the ovarian vascular pedicle in the regulation of counter current transfer of steroid hormones to the arterial blood supplying the oviduct and uterus of pigs
Authors: Stefańczyk‐Krzymowska S , Grzegorzewski W , Skipor J , Wasowska B , Krzymowski T
Published: August 2006

On Day 10 of the oestrous cycle in pigs, after laparotomy noradrenaline (NA), methoxamine (α1‐adrenomimetic, M), Prazosin (α1‐adrenolytic, Pr) in total doses of 4 μmol, and saline were infused (10 min) into the superficial layer of mesovarium on both sides of the ovarian pedicle vasculature, close to the ovary.

Blood flow in the ovarian artery, heart rate and progesterone (P4) and androstenedione (A4) secretion from the ovary and their concentrations in the ovarian venous effluent, as well as the concentrations of P4 and A4 in the blood supplying the oviduct and the uterus, were determined.

A significant increase of P4 and A4 secretion after NA and M infusion and increased concentrations of P4 and A4 in the ovarian venous effluent were found, but these changes did not influence the counter current transfer of hormones from the venous effluent into arterial blood supplying the oviduct and the uterus.

Infusion of Pr caused a significant decrease of P4 and A4 secretion and their concentrations in the ovarian venous effluent and significantly increased A4 concentration in the blood supplying the oviduct and uterus.

The results indicate that stimulation of α1‐adrenoceptors in the area of ovarian vasculature did not influence, whereas block of α1‐adrenoceptors affected, the local concentration of steroid hormones in the blood supplying the oviduct and the part of the uterus proximal to the ovary, despite the changes in the concentrations of steroid hormones in the ovarian effluent.

British Journal of Pharmacology (1997) 120, 763–768; doi:10.1038/sj.bjp.0700953
1‐(3‐(9H‐Carbazol‐9‐yl)‐1‐propyl)‐4‐(2‐methoxyphenyl)‐4‐piperidinol, a novel subtype selective inhibitor of the mouse type II GABA‐transporter
Authors: Thomsen Christian , Sørensen Per Olaf , Egebjerg Jan
Published: August 2006

The selectivity of new derivatives of the γ‐aminobutyric acid (GABA)‐uptake inhibitor, tiagabine was characterized at the four cloned mouse GABA transporters (mGAT1 through mGAT4) by measuring [3H]‐GABA uptake into stably transfected baby hamster kidney cells. While tiagabine is a highly selective inhibitor of mGAT1 (Ki=0.11±0.02 μm), these derivatives exhibited low potencies at mGAT1 but differential activities at mGAT2, mGAT3 and mGAT4. In particular, 1‐(3‐(9H‐carbazol‐9‐yl)‐1‐propyl)‐4‐(2‐methoxyphenyl)‐4‐piperidinol (NNC 05‐2090) was a potent inhibitor of mGAT2 (Ki=1.4±0.3 μm) showing at least 10 fold selectivity over mGAT1, mGAT3 and mGAT4. NNC 05‐2090 is the first subtype selective inhibitor of mGAT2 and may represent a novel useful tool for investigating the physiological roles of GAT2 in the brain and periphery.

British Journal of Pharmacology (1997) 120, 983–985; doi:10.1038/sj.bjp.0700957
Role of α2‐adrenoceptors in the regulation of intestinal water transport
Authors: Liu Lu , Coupar Ian M
Published: August 2006

The influence of the sympathetic nervous system on intestinal fluid transport by the jejunum and ileum of the anaesthetized rat was investigated under basal conditions and during active secretion induced by intra‐arterial infusion of vasoactive intestinal peptide (VIP).

Intra‐arterial infusion of noradrenaline (3, 10, 30 nmol min−1, i.a.) and i.v. injection of the selective α2‐adrenoceptor agonist UK 14,304 (1 μmol kg−1, i.v.) increased the rate of basal fluid absorption. The effect of UK 14,304 was blocked by yohimbine (10 μmol kg−1, i.v). However, the selective α1‐adrenoceptor agonist phenylephrine (5 μmol kg−1, i.v.) did not alter either the jejunal or ileal absorption rate.

The α2‐adrenoceptor antagonists yohimbine (0.3, 1.0, 3 and 10 μmol kg−1, i.v.) and rauwolscine (10 μmol kg−1, i.v.) decreased the basal absorption rate, while the α1‐adrenoceptor antagonist prazosin (3 μmol kg−1, i.v.) was without effect. Intracerebroventricular injection of yohimbine (3 μmol kg−1) caused a significant antiabsorptive effect in the jejunum but not ileum.

Peripheral chemical sympathectomy induced by pretreating animals with 6‐hydroxydopamine (150 mg kg−1, i.p., total dose) induced a trend towards impaired absorption in the jejunum and ileum.

The findings provide evidence that the sympathetic nervous system exerts tonic control on intestinal fluid transport and that the effect is mainly through peripheral α2‐adrenoceptors.

The subtype determination of α2‐adrenoceptors in modulating intestinal fluid transport was assessed by determining the effects of α2‐adrenoceptor agents on intestinal fluid secretion induced by i.a. infusion of VIP (0.8 μg min−1).

Intravenous administration of UK 14,304 caused a dose‐dependent reversal of the secretory phase of the VIP‐induced response, but failed to restore fluid transport to the control level of net absorption. EC50 values were 0.17 μmol kg−1 in the jejunum and 0.22 μmol kg−1 in the ileum.

The effect of UK 14,304 was blocked by the selective α2A/D antagonist BRL 44408 and the non‐selective α2 antagonist yohimbine (each 10 μmol kg−1). The selective α2B/C antagonist ARC 239 (10 μmol kg−1) did not affect the antisecretory action of UK 14,304. It is suggested that the α2‐adrenoceptors in the rat intestinal epithelium are the α2D or α2A‐like subtype.

British Journal of Pharmacology (1997) 120, 892–898; doi:10.1038/sj.bjp.0700958
Purinergic facilitation of ATP‐sensitive potassium current in rat ventricular myocytes
Authors: Babenko Andrey P. , Vassort Guy
Published: August 2006

The effects of different purinergic agonists on the cardiac adenosine 5′‐triphosphate (ATP)‐sensitive potassium current (IK(atp)) appearing during dialysis of rat isolated ventricular myocytes with a low‐ATP (100 μm) internal solution under whole‐cell patch‐clamp conditions, were examined in the presence of a P1 purinoceptor antagonist.

The extracellular application of ATP in the micromolar range induced, besides known inward currents through cationic and chloride channels, the facilitation of IK(atp) once IK(atp) had already been partially activated during the low‐ATP dialysis.

Analogues of ATP, α,β‐methyleneadenosine 5′‐triphosphate (α,βmeATP), 2‐methylthioadenosine triphosphate (2MeSATP), adenosine 5′‐O‐3‐thiotriphosphate (ATPγS) similarly facilitated IK(atp). UTP and ADP were very weak agonists while AMP and adenosine had no detectable effect.

The half‐maximal stimulating concentration (C50) of α,βmeATP, an analogue that did not elicite the interfering inward cationic current was 1.5 μm. Similar apparent C50 (1–2 μm) were observed for ATP and analogues tested with somewhat less maximal effect of ATPγS.

Suramin, a nonselective P2‐purinoceptor antagonist, altered IK(atp) at the relatively high concentration required to inhibit purinoceptors. Pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), a supposedly predominantly P2X‐purinoceptor antagonist, at micromolar concentration inhibited the transient inward current but did not block the facilitation of IK(atp).

Our results demonstrate that ATP and its analogues facilitate IK(atp) in rat ventricular myocytes by stimulation of non‐P1‐, non‐P2X‐purinoceptors.
Differential actions of charybdotoxin on central and daughter branch arteries of the rabbit isolated ear
Authors: Berman Rodney S. , Griffith Tudor M.
Published: August 2006

By use of rabbit isolated perfused intact ears and isolated perfused segments of central and first generation daughter branch ear arteries, we investigated the actions of charybdotoxin (ChTX), a blocker of calcium‐activated K+ channels (KCa channels), and Nω‐nitro‐L‐arginine methyl ester (l‐NAME) on pressure‐flow and diameter‐flow relationships.

ChTX (1 nM) induced an upwards shift in the pressure‐flow curve in the rabbit intact isolated ear preconstricted with 5‐hydroxytryptamine (5‐HT; 100 nM) with subsequent administration of L‐NAME (100 μM) inducing a further upwards shift. L‐NAME itself induced an upwards shift in the pressure‐flow curve, but subsequent administration of ChTX was without significant effect.

Microangiographic analysis revealed a tendency of ChTX (1 nM) to decrease vessel diameter in the central ear artery (G0) with little effect on the first two generations of daughter branch arteries (G1 and G2) in the intact ear. Subsequent addition of L‐NAME (100 μM) did not significantly further decrease vessel diameter in G0, but did decrease vessel diameter in G1 and G2. L‐NAME itself showed a tendency to decrease vessel diameter in G0, G1 and G2 vessels with subsequent addition of ChTX being without significant effect.

In an isolated G0 preparation which was preconstricted with 5‐HT (100 nM), ChTX (1 nM) caused an upwards shift in the pressure‐flow curve which was augmented by subsequent addition of L‐NAME (100 μM). L‐NAME (100 μM) itself caused an upwards shift in the pressure‐flow curve but subsequent addition of ChTX (1 nM) had no significant effect.

In comparison, in an isolated G1 preparation which was preconstricted with 5‐HT (100 nM), ChTX (1 nM) had no significant effect on the pressure‐flow curve relative to control, but subsequent addition of L‐NAME (100 μm) caused an upwards shift. L‐NAME (100 μM) itself induced an upwards shift in the pressure‐flow curve with subsequent addition of ChTX (1 nM) being without significant effect.

ChTX (10 pM‐10 nM) caused a concentration‐dependent increase in perfusion pressure in isolated G0 and G1 preparations at fixed flow rates of 2 ml min−1 and 0.5 ml min−1, respectively. These responses were enhanced in the presence of L‐NAME (100 μM) in G1 but not G0 preparations.

We conclude that at 1 nM, ChTX exhibits differential actions on central and daughter branch arteries of the intact ear of the rabbit, which are also apparent in the corresponding arteries when studied in isolation. The action of 1 nM ChTX in G0 vessels may reflect inhibition of either the release or action of nitric oxide as it was blocked in the presence of L‐NAME. At higher concentrations of ChTX, there would appear to be a direct constrictor effect on vascular smooth muscle which is apparent in both G0 and G1 vessels. This observed heterogeneity could reflect different distributions of KCa channels between central and daughter branch arteries at either the endothelial or smooth muscle levels, or both.