bluuebunny/crossref_metadata_2025_split huggingface dataset:
Binding and effects of KATP channel openers in the vascular smooth muscle cell line, A10
Authors: Russ Ulrich , Metzger Friedrich , Kickenweiz Elisabeth , Hambrock Annette , Krippeit‐Drews Peter , Quast Ulrich 
Published: January 2005

The ATP‐sensitive K+ channel (KATP channel) in A10 cells, a cell line derived from rat thoracic aorta, was characterized by binding studies with the tritiated KATP channel opener, [3H]‐P1075, and by electrophysiological techniques.
Saturation binding experiments gave a KD value of 9.2±5.2 nM and a binding capacity (BMax) of 140±40 fmol mg−1 protein for [3H]‐P1075 binding to A10 cells; from the BMax value a density of binding sites of 5–10 per μm2 plasmalemma was estimated.
KATP channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [3H]‐P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [3H]‐P1075 binding by 85%, minoxidil sulphate and glibenclamide by 70%. The inhibition constants were similar to those determined in strips of rat aorta.
Resting membrane potential, recorded with microelectrodes, was −51±1 mV. P1075 and levcromakalim produced a concentration‐dependent hyperpolarization by up to −25 mV with EC50 values of 170±40 nM and 870±190 nM, respectively. The hyperpolarization induced by levcromakalim (3 μM) was completely reversed by glibenclamide with an IC50 value of 86±17 nM.
Voltage clamp experiments were performed in the whole cell configuration under a physiological K+ gradient. Levcromakalim (10 μM) induced a current which reversed around −80 mV; the current‐voltage relationship showed considerable outward rectification. Glibenclamide (3 μM) abolished the effect of levcromakalim.
Analysis of the noise of the levcromakalim (10 μM)‐induced current at −40 and −20 mV yielded estimates of the channel density, the single channel conductance and the probability of the channel to be open of 0.14 μm−2, 8.8 pS and 0.39, respectively.
The experiments showed that A10 cells are endowed with functional KATP channels which resemble those in vascular tissue; hence, these cells provide an easily accessible source of channels for biochemical and pharmacological studies. The density of binding sites for [3H]‐P1075 was estimated to be one order of magnitude higher than the density of functional KATP channels; assuming a plasmalemmal localization of the binding sites this suggests a large receptor reserve for the openers in A10 cells.

British Journal of Pharmacology (1997) 122, 1119–1126; doi:10.1038/sj.bjp.0701514
Imaging of intracellular calcium during desensitization of nicotinic acetylcholine receptors of rat chromaffin cells
Authors: Khiroug L. , Giniatullin R. , Sokolova Elena , Talantova Maria , Nistri A. 
Published: January 2005

The possible role of intracellular Ca2+ levels ([Ca2+]i) in desensitization of nicotinic acetylcholine receptors (AChRs) was investigated in rat cultured chromaffin cells by use of combined whole‐cell patch clamping and confocal laser scanning microscopy with the fluorescent dye fluo‐3.
On cells held at −70 mV, pressure‐application of nicotine elicited inward currents with associated [Ca2+]i rises mainly due to influx through nicotinic AChRs. These responses were blocked by (+)‐tubocurarine (10 μM) but were insensitive to α‐bungarotoxin (1 μM) or Cd2+ (0.1 mM).
Pressure applications of 1 mM nicotine for 2 s (conditioning pulse) evoked inward currents which faded biexponentially to a steady state level due to receptor desensitization and were accompanied by a sustained increase in [Ca2+]i. Inward currents evoked by subsequent application of brief test pulses of nicotine were depressed but recovered with a time course reciprocal to the decay of the [Ca2+]i transient induced by the conditioning pulse.
Omission of intracellular Ca2+ chelators or use of high extracellular Ca2+ solution (10 mM) lengthened recovery of nicotinic AChRs from desensitization while adding BAPTA or EGTA intracellularly had the opposite effect. When the patch pipette contained fluo‐3 or no chelators, after establishing whole cell conditions the rate of recovery became progressively longer presumably due to dialysis of endogenous Ca2+ buffers. None of these manipulations of external or internal Ca2+ had any effect on onset or steady state level of desensitization.
High spatial resolution imaging of [Ca2+]i in intact cells (in the presence of 0.1 mM Cd2+) showed that its level in the immediate submembrane area decayed at the same rate as in the rest of the cell, indicating that Ca2+ was in a strategic location to modulate (directly or indirectly) AChR desensitization.
The present data suggest that desensitized nicotinic AChRs are stabilized in their conformation by raised [Ca2+]i and that this phenomenon retards their recovery to full activity.
Influence of flupirtine on a G‐protein coupled inwardly rectifying potassium current in hippocampal neurones
Authors: Jakob Regina , Krieglstein Josef 
Published: January 2005

Previous studies have shown that flupirtine, a centrally acting, non‐opioid analgesic agent, also exhibits neuroprotective activity in focal cerebral ischaemia in mice and reduces apoptosis induced by NMDA, gp 120 of HIV, prior protein fragment or lead acetate as well as necrosis induced by glutamate or NMDA in cell culture. To study the potential mechanism of the neuroprotective action of flupirtine, we investigated whether flupirtine is able to modulate potassium or NMDA‐induced currents in rat cultured hippocampal neurones by use of the whole‐cell configuration of the patch‐clamp technique.
We demonstrated that 1 μM flupirtine activated an inwardly rectifying potassium current (Kir) in hippocampal neurones (ΔI=−39±18 pA at −130 mV; n=10). This effect was dose‐dependent (EC50=0.6 μM). The reversal potential for Kir was in agreement with the potassium equilibrium potential predicted from the Nernst equation showing that Kir was predominantly carried by K+. Furthermore, the induced current was blocked completely by Ba2+ (1 mM), an effect typical for Kir.
The activation of Kir by flupirtine was largely prevented by pretreatment of the cells with pertussis toxin (PTX) indicating the involvement of a PTX‐sensitive G‐protein in the transduction mechanism (ΔI=−3±6 pA at −130 mV; n=8). Inclusion of cyclic AMP in the intracellular solution completely abolished the activation of Kir (n=7).
The selective α2‐adrenoceptor antagonist SKF‐86466 (10 μM), the selective 5‐HT1A antagonist NAN 190 as well as the selective GABAB antagonist 2‐hydroxysaclofen (10 μM) failed to block the flupirtine effect on the inward rectifier.
Flupirtine (1 μM) could not change the current induced by 50 μM NMDA.
These results show that in cultured hippocampal neurones flupirtine activates an inwardly rectifying potassium current and that a PTX‐sensitive G‐protein is involved in the transduction mechanism.
Assessment of the role of α2‐adrenoceptor subtypes in the antinociceptive, sedative and hypothermic action of dexmedetomidine in transgenic mice
Authors: Hunter J. C. , Fontana D. J. , Hedley L. R. , Jasper J. R. , Lewis R. , Link R. E. , Secchi R. , Sutton J. , Eglen R. M. 
Published: January 2005

The role of α2‐adrenoceptor (AR) subtypes in the modulation of acute nociception, motor behaviour and body temperature, has been investigated by determining the activity of the α2AR selective agonist dexmedetomidine (Dex) in mice devoid of individual α2AR subtypes through either a point (α2A) or null (α2B/α2C) mutation (‘knock‐out’).
In a rodent model of acute thermal nociception, the mouse tail immersion test, Dex, in wild type (WT) control animals, produced a dose‐dependent increase in the threshold for tail withdrawal from a 52°C water bath with mean ED50 values of 99.9±14.5 (α2A), 94.6±17.8 (α2B) and 116.0±17.1 (α2C) μg kg−1, i.p.
In comparison to the WT controls, Dex (100–1000 μg kg−1, i.p.), was completely ineffective as an antinociceptive agent in the tail immersion test in the α2AAR D79N mutant animals. Conversely, in the α2BAR and α2CAR knock‐outs, Dex produced a dose‐dependent antinociceptive effect that was not significantly different from that observed in WT controls, with ED50 values of 85.9±15.0 (P>0.05 vs WT control) and 226.0±62.7 (P>0.05 vs WT control) μg kg−1 i.p., respectively.
Dex (10–300 μg kg−1, i.p.) produced a dose‐dependent reduction in spontaneous locomotor activity in the α2A, α2B and α2CAR WT control animals with ED50 values of 30.1±9.0, 23.5±7.1 and 32.3±4.6 μg kg−1, i.p., respectively. Again, Dex (100–1000 μg kg−1, i.p.) was ineffective at modulating motor behaviour in the α2AAR D79N mutants. In the α2BAR and α2CAR knock‐out mice, Dex produced a dose‐dependent reduction in spontaneous locomotor activity with ED50 values of 29.1±6.4 (P>0.05 vs WT control) and 57.5±11.3 (P>0.05 vs WT control) μg kg−1, respectively.
Dex was also found to produce a dose‐dependent reduction in body temperature in the α2A, α2B and α2CAR WT control mice with ED50 values of 60.6±11.0, 16.2±2.5 and 47.2±9.1 μg kg−1, i.p., respectively. In the α2AAR D79N mutants, Dex had no effect on body temperature at a dose (100 μg kg−1, i.p.) that produced a significant reduction (−6.2±0.5°C; P<0.01 vs vehicle) in temperature in WT controls. However, higher doses of Dex (300 and 1000 μg kg−1, i.p) produced a small, but statistically significant decrease in temperature corresponding to −1.7±0.4°C and −2.4±0.3°C (both P<0.01 vs vehicle), respectively. In the α2BAR and α2CAR knock‐out mice, Dex produced a dose‐dependent reduction in body temperature with ED50 values of 28.4±4.8 (P>0.05 vs WT control) and 54.1±8.0 (P>0.05 vs WT control) μg kg−1, respectively.
In conclusion, the data are consistent with the α2AAR being the predominant subtype involved in the mediation of the antinociceptive, sedative and hypothermic actions of Dex. This profile would appear to indicate that an α2AAR subtype selective analgesic will have a narrow therapeutic window, particularly following systemic administration.
Influence of interleukin 1α on superoxide anion, platelet activating factor release and phospholipase A2 activity of naive and sensitized guinea‐pig alveolar macrophages
Authors: Mugnai Sabrina , Ciuffi Mario , Maurizi Manuela , Bindi Daniela , Franchi‐Micheli Sergio , Zilletti Lucilla 
Published: January 2005

We studied the effect exerted by hr‐interleukin‐1α (IL‐1α) on responsiveness of alveolar macrophages (AM) from naive and sensitized guinea‐pigs, through O2‐. production (by ferricytochrome C reduction), platelet‐activating factor (PAF) release (by platelet aggregation), prostaglandin E2 (PGE2) release (by a radioimmunoassay), and cytosolic phospholipase A2 (cPLA2) activity (by hydrolysis of radioactive substrate).
In naive guinea‐pig AM, 0.06 nM hr‐IL‐1α pretreatment decreased by 65% O2‐. release stimulated with 10 nM fMLP. In contrast, O2‐. production was not affected in sensitized guinea‐pig AM.
O2‐. release elicited by fMLP stimulation in both cell groups was affected by PLA2 inhibitors (10 μM bromophenacyl bromide, BPB or 10 μM methylprednisolone, MP). In contrast, 10 μM arachidonyl trifluoromethyl ketone (AACOCF3), a cPLA2 inhibitor, was ineffective.
In naive AM, PAF release was elicited by hr‐IL‐1α pretreatment and by separate fMLP‐stimulation, but when the stimulus was added to hr‐IL‐1α‐pretreated cells inhibition of PAF release was observed. In sensitized AM, PAF release was lower than that found in naive guinea‐pig AM in both hr‐IL‐1α‐pretreated and fMLP‐stimulated cells.
PGE2 release was unaffected by hr‐IL‐1α pretreatment and it was decreased by fMLP in both naive and sensitized AMs. The latter released less PGE2 than naive cells in basal conditions and after fMLP treatment.
Sensitized AM showed a greater cPLA2 activity in all experimental conditions in comparison to naive cells. cPLA2 activity assayed in the cytosolic fraction was found to be enhanced by hr‐IL‐1α pretreatment and by fMLP stimulation in naive but not in sensitized AM. However, when the stimulus was added to hr‐IL‐1α‐pretreated cells we observed a decrease in cPLA2 activity in the cytosol and an increase in the membranes, thus suggesting a translocation of enzymatic activity.
In conclusion, hr‐IL‐1α can modulate the responsiveness of AM from naive and sensitized guinea‐pigs, as suggested by changes found in the release of PAF and O2− and in cPLA2 activity; therefore, sensitization itself may affect cellular responsiveness.
Lipolytic effects of conventional β3‐adrenoceptor agonists and of CGP 12,177 in rat and human fat cells: preliminary pharmacological evidence for a putative β4‐adrenoceptor
Authors: Galitzky Jean , Langin Dominique , Verwaerde Patrick , Montastruc Jean‐Louis , Lafontan Max , Berlan Michel 
Published: January 2005

The nature of rat and human fat cell β3‐adrenoceptors was investigated by studying the effects of the new β3‐adrenoceptor selective antagonist, SR 59,230A, on lipolysis induced by the conventional β3‐adrenoceptor agonists, CL 316,243 and SR 58,611A, and by the non‐conventional partial β3‐adrenoceptor agonist CGP 12,177 (a potent β1‐ and β2‐adrenoceptor antagonist with partial β3‐adrenoceptor agonist property).
In rat fat cells, the rank order of potency of agonists was: CL 316,243>isoprenaline>SR 58,611A>CGP 12,177. The three former agents were full agonists whereas CGP 12,177 was a partial agonist (intrinsic activity of 0.70). In human fat cells, the lipolytic effect of CGP 12,177 reached 25 % of isoprenaline effect. CL 316,243 was a poor inducer of lipolysis and SR 58,611A was ineffective.
In rat fat cells, lipolysis induced by CL 316,243 and SR 58,611A was competitively antagonized by SR 59,230A. Schild plots were linear with pA2 values of 6.89 and 6.37, respectively. Conversely, 0.1, 0.5 and 1 μM SR 59,230A did not modify the concentration‐response curve of CGP 12,177. A rightward shift of the curve was however observed with 10 and 100 μM of SR 59,230A. The apparent pA2 value was 5.65. The non‐selective β‐adrenergic antagonist, bupranolol, competitively displaced the concentration‐response curve of CGP 12,177 and CL 316,243. Schild plots were linear with pA2 values of 6.70 and 7.59, respectively. CL316,243‐mediated lipolytic effect was not antagonized by CGP 20,712A. In human fat cells, CGP 12,177‐mediated lipolytic effect was antagonized by bupranolol and CGP 20,712A. SR 59,230A (0.1, 1 and 10 μM) did not modify the concentration‐response curve of CGP 12,177. A rightward shift was however observed at 100 μM leading to an apparent pA2 value of 4.32.
The results suggest that the non‐conventional partial agonist CGP 12,177 can activate lipolysis in fat cells through the interaction with a β‐adrenoceptor pharmacologically distinct from the β3‐adrenoceptor, i.e. through a putative β4‐adrenoceptor. They suggest that the two subtypes coexist in rat fat cells whereas only the putative β4‐adrenoceptor mediates lipolytic effect of CGP12,177 in human fat cells.

British Journal of Pharmacology (1997) 122, 1244–1250; doi:10.1038/sj.bjp.0701523
Activation of two sites by adenosine receptor agonists to cause relaxation in rat isolated mesenteric artery
Authors: Prentice D. J. , Payne S. L. , Hourani S. M. O. 
Published: January 2005

In this study we have characterized the receptor(s) in the rat mesenteric artery mediating relaxant responses to adenosine and a number of adenosine analogues, N6 ‐R‐phenylisopropyladenosine (R‐PIA), N6‐cyclopentyladenosine (CPA), N6‐(3‐iodo‐benzyl)‐adenosine‐5′‐N‐methyluronamide (IB‐MECA) and 5′‐N‐ethylcarboxamidoadenosine (NECA), by use of the non‐selective antagonist 8‐sulphophenyltheophylline (8‐SPT) and the A2A selective ligands 2‐[p‐(2‐carbonylethyl)‐phenylethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) and 4‐(2‐[7‐amino‐2‐(2‐furyl)[1,2,4]‐triazolo[2,3‐a][1,3,5]‐triazin‐5‐ylamino]ethyl) phenol (ZM 241385). We have also studied the effects of endothelial removal and uptake inhibition by nitrobenzylthioinosine (NBTI) and the effects of the A3 receptor antagonist 1,3‐dipropyl‐8‐(4‐acrylate)phenylxanthine (BWA1433).
Adenosine, NECA, CPA and R‐PIA all elicited relaxant responses in tissues precontracted with phenylephrine (1 μM) with the following potency order: NECA>R‐PIA>adenosine=CPA. However, E/[A] curves to NECA were biphasic. CGS 21680 was inactive at concentrations up to 30 μM and IB‐MECA elicited relaxant responses which were resistant to blockade by 8‐SPT and BWA1433 (100 μM).
Removal of the endothelium produced a small but significant decrease in the asymptote of the high potency phase of E/[A] curves to NECA with no change in p[A]50. E/[A] curves to adenosine were not altered by removal of the endothelium. However, there were small rightward shifts of E/[A] curves to CPA and R‐PIA in the absence of endothelium.
Inhibition of uptake by NBTI (1 μM) had no effect on E/[A] curves to NECA, CPA or R‐PIA, but E/[A] curves to adenosine were significantly left‐shifted in the presence of NBTI.
8‐SPT (10–100 μM) caused significant rightward shifts of the high potency phase of the E/[A] curves to NECA (pA2=5.63±0.26). The second phase of the concentration‐response curve to NECA appeared to be resistant to blockade by 8‐SPT, as were E/[A] curves for adenosine, CPA or R‐PIA. However, in the presence of NBTI (1 μM), 8‐SPT (100 μM) gave significant rightward shifts of E/[A] curves to adenosine.
ZM 241385 (0.1–1 μM) produced significant rightward shifts of the high potency phase of NECA E/[A] curves (pA2=7.65±0.25 in the presence and 7.20±0.12 in the absence of endothelium), while curves to R‐PIA were not significantly shifted by 1 μM ZM 241385. In the presence of NBTI E/[A] curves to adenosine were significantly rightward shifted by ZM 241385 (0.1 μM, pA2=7.50±0.16).
In conclusion, the results suggest activation of A2B receptors located primarily on the smooth muscle by low concentrations of NECA and by adenosine under conditions of uptake blockade, and of another, as yet undefined site which may be intracellular, by higher concentrations of NECA, by CPA, R‐PIA and adenosine under conditions where uptake is operational.
Protective effect of darodipine, a calcium antagonist, on rat cardiomyocytes against oxygen radical‐mediated injury
Authors: Matucci Rosanna , Francesca Ottaviani Maria , Barbieri Mario , Cerbai Elisabetta , Mugelli Alessandro 
Published: January 2005

We used electrophysiological and electron spin resonance (e.s.r.) techniques to study the mechanism of the protective effect of darodipine on rat isolated cardiomyocytes exposed to an exogenous source of oxygen free radicals (OFR).
The L‐type calcium current (ICa,L), action potential and cell shortening were measured in patch‐clamped cells in the whole‐cell configuration. ICa,L blockade by darodipine was concentration‐dependent, peak current being reduced by 20% with 50 nM and by 58% with 100 nM darodipine. The lowest concentration of darodipine did not affect action potential or twitch profile.
Exposure to OFR‐generating solution (5 mM dihydroxyfumarate, DHF) caused the appearance of electrophysiological alterations and/or spontaneous activity in 73% of cells (n=26) within 5 min; action potential duration (APD) was prolonged (195±16 ms vs 140±6 ms in the control) and maximum diastolic potential (MDP) was reduced (−59.5±2.6 mV vs −69.8±0.8 mV in the control) (P<0.05, n=25).
A 2 min pretreatment with 50 nM darodipine significantly reduced the incidence of these arrhythmogenic events following a 5 min exposure to OFR (36% of cells, n=14; P<0.05 vs nonpretreated cells). Pretreatment with darodipine also prevented APD prolongation caused by OFR (137±12 ms after DHF vs 117±6 ms before DHF n=14, not significant) but not the decrease of MDP (−63.4±2.5 mV after DHF vs −70.9±1.0 mV before DHF, P<0.05).
The e.s.r. spectra obtained from the DHF‐DMPO solution in the absence of darodipine demonstrated the presence of two components corresponding to two DMPO adducts. The addition of darodipine (50–500 nM) led to a concentration‐dependent decrease in intensity of the signals, the intensity of the DMPO‐COO.− adduct being decreased more than that of the DMPO‐OH. adduct.
Our results demonstrate that darodipine dose‐depentently blocks ICa,L in rat isolated cardiomyocytes. Furthermore it exerts protective effects against free‐radical‐induced electrophysiological alterations independently of its calcium antagonistic properties; this effect is possibly due to trapping of specific radical species.
Recording of the saphenous vein compliance by an ultrasonic echo‐tracking device in the dog: effects of S 18149
Authors: Vayssettes‐Courchay Christine , Cordi Alex A. , Lacoste Jean‐Michel , Laubie Michel , Verbeuren Tony J. 
Published: January 2005

Saphenous vein reactivity was recorded in the anaesthetized dog by use of an ultrasonic echo‐tracking device to measure the internal diameter of the vein and to calculate the venous compliance. This method was used to investigate the effects of a new partial α1/α2‐adrenoceptor agonist, S 18149, on the canine saphenous vein in vivo after intravenous (i.v.) or oral administration.
Venoconstrictions induced by i.v. or local administration of compounds were evaluated by continuous recording of the internal diameter of the saphenous vein with the echo‐tracking method. Venous compliance was calculated in two ways: (1) as the slope of the diameter‐pressure curve obtained by increasing the venous pressure with an inflatable cuff and (2) in veins in which pressure was higher than 12 mmHg, pulsatile variations in the venous diameter and venous pressure were detected and used to calculate the pulsatile compliance of the vein.
S 18149 administered i.v. at 0.5 μg kg−1 min−1 for 10 min induced a decrease in the saphenous vein diameter (−15±3%) and blood flow (−72±6%) associated with an increase in saphenous vein resistance; at the dose used, S 18149 did not modify venous pressure and caused only a weak increase in arterial pressure (+7±2 mmHg).
The pulsatile compliance of the saphenous vein averaged 8.65±1.37 mm2×100 mmHg−1 in control dogs and was significantly decreased to 5.13±0.68 mm2×100 mmHg−1 in the same animals after treatment with S 18149 at 100 μg kg−1 per os (n=10). The saphenous vein compliance calculated with the increased external pressure method averaged 24.90±1.49 μm mmHg−1 in control dogs and was significantly reduced in the same animals after treatment with S 18149 at 100 μg kg−1 per os to 9.06±3.42 μm mmHg−1 (n=5). When constrictions of the vein were induced with increasing doses of (−)‐phenylephrine, injected locally at 1, 3 or 6 μg min−1, only the responses obtained with the lower dose of (−)‐phenylephrine were increased in dogs treated with S 18149 100 μg kg−1 per os (−16±4% versus −4±3%, n=5).
These results show that the high resolution echo‐tracking device previously used for arterial compliance measurements, allows the detection of pulsatile changes in the canine saphenous vein and thus permits calculation of both the pulsatile and the static compliance of superficial veins in vivo. Using this technique, we have demonstrated that the novel α‐adrenoceptor agonist S 18149 constricts the canine saphenous vein in vivo and decreases the saphenous vein compliance after oral administration.