bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of crilvastatin, a new cholesterol lowering agent, on unesterified LDL‐cholesterol metabolism into bile salts by rat isolated hepatocytes
Authors: Clerc Thierry , Sbarra Véronique , Diaconescu Nicolas , Lafont Huguette , Jadot Guy , Laruelle Claude , Chanusso Françoise
Published: July 2012

The aim of these experiments was to determine the effect of crilvastatin, a new cholesterol lowering agent, on the metabolism of unesterified low density lipoprotein (LDL)‐cholesterol by rat freshly isolated hepatocytes. This preclinical model was developed as an alternative to in vivo experiments, to mimic the metabolic effects of a molecule on its target cells and to define optimal conditions for future experimentation on human hepatocytes.

Cells were obtained from normolipidaemic or hypercholesterolaemic rats, hypercholesterolaemia was nutritionally induced. Incubations were performed in a medium containing 600 μm taurocholate and 50 μm or 300 μm crilvastatin.

This molecule was shown in vitro to be carried by physiological transporters, i.e., albumin‐bile salt micellar associations and LDL. Crilvastatin induced a significance increase in the synthesis and secretion by hepatocytes of bile salts resulting from the metabolism of unesterified LDL‐cholesterol in both normolipidaemic and hypercholesterolaemic rats. Stimulation involved non‐conjugated as well as tauro‐ and glyco‐conjugated bile salts. These findings corroborate preliminary studies showing in vivo that crilvastatin enhances the secretion of bile acids by stimulating the uptake and incorporation of LDL‐cholesterol by the liver.
Influence of age on the signal transduction pathway of non‐adrenergic non‐cholinergic neurotransmitters in the rat gastric fundus
Authors: Smits Geert J.M. , Lefebvre Romain A.
Published: July 2012

The influence of aging on the relaxant response and the change in cyclic nucleotide content induced by vasoactive intestinal polypeptide (VIP), nitric oxide (NO), electrical field stimulation of the non‐adrenergic non‐cholinergic neurones and substances acting at different levels of the cyclic AMP and cyclic GMP transduction pathways was studied in longitudinal muscle strips of the rat gastric fundus.

The relaxant responses to VIP, sustained electrical stimulation, forskolin and 3‐isobutyl‐1‐methylxanthine were reduced with age, while the responses to dibutyryl cyclic AMP were not. The increase in cyclic AMP content induced by sustained electrical stimulation and forskolin was lower in old rats.

The relaxant responses to NO and to short train electrical stimulation were similar in the three age groups. The inhibitory effect of NG‐nitro‐1‐arginine methyl ester on relaxations induced by short train electrical stimulation was more pronounced in old rats. The relaxant responses to sodium nitroprusside (SNP), 8‐bromo‐cyclic GMP and zaprinast were reduced with age. SNP induced a similar elevation of the cyclic GMP content in the three age groups.

These results suggest that aging differentially affects the cyclic AMP and cyclic GMP pathway for relaxation by VIP and NO in the rat gastric fundus, as the defect seems to occur at the level of the adenylate cyclase and cyclic GMP‐dependent protein kinase respectively.
Relaxation and decrease in [Ca2+]i by hydrochlorothiazide in guinea‐pig isolated mesenteric arteries
Authors: Pickkers Peter , Hughes Alun D.
Published: July 2012

We examined the effect of the thiazide diuretic, hydrochlorothiazide, on intracellular calcium concentration ([Ca2+]i) and tone in guinea‐pig mesentery arteries. Vessels were mounted on a microvascular myograph and loaded with the Ca2+‐sensitive fluorescent dye, Fura‐2.

Hydrochlorothiazide caused relaxation of noradrenaline‐precontracted arteries associated with a fall in [Ca2+]i. Preincubation of arteries with hydrochlorothiazide inhibited both contraction and rise in [Ca2+]i in response to noradrenaline. Hydrochlorothiazide did not affect tone and [Ca2+]i when this was elevated by a combination of depolarizing potassium solution and noradrenaline.

Hydrochlorothiazide‐induced vasorelaxation and decrease of [Ca2+]i was abolished by charybdotoxin, a blocker of large conductance Ca2+‐activated K channels.

The rise in [Ca2+]i elicited by caffeine in Ca2+‐free physiological salt solution, and presumably reflecting Ca2+ release from intracellular stores, was not altered by preincubation with hydrochlorothiazide.

Under depolarizing conditions hydrochlorothiazide did not alter the relationship between the extracellular concentration of Ca2+ and [Ca2+]i; however, hydrochlorothiazide caused a small reduction in the contraction produced for a given rise in [Ca2+]i suggesting hydrochlorothiazide may cause a slight desensitization of the contractile machinery.

These findings suggest that hydrochlorothiazide opens Ca2+‐activated K channels leading to hyperpolarization and consequent closing of voltage‐operated calcium channels. The result of this is an impaired influx of extracellular Ca2+, a decrease in [Ca2+]i and vasorelaxation.
Functional identification of rat atypical β‐adrenoceptors by the first β3‐selective antagonists, aryloxypropanolaminotetralins
Authors: Manara Luciano , Badone Domenico , Baroni Marco , Boccardi Giovanni , Cecchi Roberto , Croci Tiziano , Giudice Antonina , Guzzi Umberto , Landi Marco , Le Fur Gérard
Published: July 2012

We have assessed the relative abilities of compounds belonging to the new aryloxypropanolaminotetralin (APAT) class and of the reference β‐adrenoceptor‐blocking agent, alprenolol, to antagonize functional responses in vitro and in vivo involving atypical (β3) or conventional (β1 and β2) β‐adrenoceptors.

The range of pA2 values for three representative APATs against inhibition of spontaneous motility in the rat isolated colon by the selective β3‐adrenoceptor agonist, SR 58611A (8.1–8.8), was well above similarly calculated values for non‐competitive antagonism of guinea‐pig trachea relaxation by salbutamol (β2, 6.5–6.9) and the atrial chronotropic response by isoprenaline (β1, 6.7‐7.3). Alprenolol, however, was substantially more potent in antagonizing atrial (pA2, 8.2) and tracheal (pA2, 8.9) responses than SR 58611A mediated inhibition of colonic motility (pA2, 6.8).

Several APAT isomers with different configurations at the chiral carbons, when tested on isolated organs, presented stringent stereochemical requirements for β3‐selectivity, including high antagonist potency‐ratios between active and inactive enantiomers.

In vivo, the inhibition of colonic motility and the thermogenic response of brown adipose tissue elicited in rats by the selective β3‐adrenoceptor agonists SR 58611A and BRL 37344 respectively were substantially diminished by the representative APAT, SR 59230A, at oral doses (≤5 mg kg−1) well below those half maximally effective (ID50) for preventing β1‐(isoprenaline tachycardia ≥80 mg kg−1) or β2‐(salbutamol bronchodilatation, 44 mg kg−1) mediated responses. Alprenolol, as expected, was a less potent and nonselective antagonist of the putative β3‐responses.

These findings support APATs as the first potent, orally effective selective antagonists at β3‐adrenoceptors, and provide final unambiguous evidence that β3‐adrenoceptors underlie inhibition of colonic motility and brown adipose tissue thermogenesis in rats
Characterization of Ca2+‐activated 86Rb+ fluxes in rat C6 glioma cells: a system for identifying novel IKCa‐channel toxins
Authors: de‐Allie Frank A. , Bolsover Steven R. , Nowicky Alex V. , Strong Peter N.
Published: July 2012

The pharmacological characteristics of a putative Ca2+ activated K+ channel (IKCa channel) in rat glioma C6 cells were studied in the presence of the Ca2+ ionophore, ionomycin and various K+ channel blockers, 86Rb+ being used as a radioisotopic tracer for K+.

The resting 86Rb+ influx into C6 cells was 318±20 pmol s−1. The threshold for ionomycin activation of 86Rb+ influx was approx. 100 nM. At ionomycin concentrations above the activation threshold, the initial rate of 86Rb+ influx was proportional to ionophore concentration. Ionomycin‐activated 86Rb+ flux was saturable (EC50 = 0.62±0.03 μm) and was not inhibited by ouabain.

Intracellular Ca2+ increased within 30 s from a basal level of 42±2 nM to 233±17 nM, after addition of 2 μm ionomycin. During this period, intracellular pH fell from 7.03±0.04 to 6.87±0.03 and the cell hyperpolarized from −34±10 mV to −76±2 mV.

Single channel conductance measurements on inside‐out patches in physiological K+ solutions identified a 14±3 pS Ca2+ ‐activated K+ current between −25 mV and +50 mV. In symmetrical (100 mM) K+, the single channel conductance was 26 pS.

Externally applied quinine (IC50 = 0.12±0.34 mM) and tetraethylammonium chloride (IC50 = 10±1.9 mM) inhibited 86Rb+ influx into C6 cells in a concentration‐dependent manner. Charybdotoxin (IC50 = 0.5±0.02 nM) and iberiotoxin (IC50 = 800±150 nM), as well as the crude venoms from the scorpions Leiurus quinquestriatus and Mesobuthus tamulus, also inhibited 86Rb+ influx. In contrast, apamin and toxin I had no inhibitory effects on 86Rb+ flux. A screen of fractions from cation exchange h.p.l.c. of Mesob. tamulus venom revealed the presence of at least four charybdotoxin‐like peptides. One of these was iberiotoxin; the other three are novel toxins.

The ionomycin‐activated 86Rb+ influx into rat C6 glioma cells has proved to be a valuable pharmacological assay for the screening of toxins and crude venoms which modify intermediate conductance, Ca2+ activated K+ channel activity.
Role of opioid receptors in the spinal antinociceptive effects of neuropeptide FF analogues
Authors: Gouardères Christine , Jhamandas Khem , Sutak Maaja , Zajac Jean‐Marie
Published: July 2012

Neuropeptide FF (NPFF) has been shown to produce antinociceptive effects and enhance morphine‐induced antinociception after intrathecal (i.t.) injection. In this study, the spinal effects of two NPFF analogues, [D‐Tyr1, (NMe)Phe3]NPFF (1DMe) and [D‐Tyr1, D‐Leu2, D‐Phe3]NPFF (3D), which are resistant to degradation and exhibit a high affinity for NPFF binding sites, were examined in tests of thermal and mechanical nociception.

1DMe and 3D produced potent dose‐dependent spinal antinociception in the tail‐flick test. On a molar basis, 1DMe was 20 and 50 times more potent than 3D and morphine, respectively, and high doses of 1DMe and 3D produced a sustained antinociceptive effect without visible signs of motor impairment.

Spinal antinociceptive effects produced by 1DMe (0.86 nmol) or 3D (8.6 nmol) were significantly reduced by i.t. co‐administration of naloxone (11 nmol) or i.t. pre‐administration of D‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP, 9.25 nmol) or β‐funaltrexamine (β‐FNA, 2 nmol) or naltrindole (2.2 nmol). The doses of the μ‐antagonists (CTOP and β‐FNA) or the δ‐antagonist (naltrindole) used in 1DMe and 3D experiments blocked the antinociceptive effects of μ‐or δ‐receptor‐selective agonists.

When administered in combination with antinociceptive doses of the μ‐receptor agonist, morphine (13.2 nmol) or the δ‐receptor agonist, [D‐Ala2]deltorphin I (20 nmol), sub‐effective dose of 1DMe or 3D (0.009 nmol) enhanced and prolonged the spinal effects of these opioid agonists.

The results of this study show that spinal μ‐and δ‐opioid receptors play a role in antinociception produced by NPFF analogues. These results also suggest a role for NPFF in modulation of nociceptive signals at the spinal level.
Modulating effect of the nootropic drug, piracetam on stress‐ and subsequent morphine‐induced prolactin secretion in male rats
Authors: Matton A. , Engelborghs S. , Bollengier F. , Finne E. , Vanhaeist L.
Published: July 2012

The effect of the nootropic drug, piracetam on stress‐ and subsequent morphine‐induced prolactin (PRL) secretion was investigated in vivo in male rats, by use of a stress‐free blood sampling and drug administration method by means of a permanent indwelling catheter in the right jugular vein.

Four doses of piracetam were tested (20, 100, 200 and 400 mg kg−1), being given intraperitoneally 1 h before blood sampling; control rats received saline instead. After a first blood sample, rats were subjected to immobilization stress and received morphine, 6 mg kg−1, 90 min later.

Piracetam had no effect on basal plasma PRL concentration.

While in the non‐piracetam‐treated rats, stress produced a significant rise in plasma PRL concentration, in the piracetam‐pretreated rats PRL peaks were attenuated, especially in the group given 100 mg kg−1 piracetam, where plasma PRL concentration was not significantly different from basal values. The dose‐response relationship showed a U‐shaped curve; the smallest dose had a minor inhibitory effect and the highest dose had no further effect on the PRL rise.

In unrestrained rats, morphine led to a significant elevation of plasma PRL concentration. After the application of immobilization stress it lost its ability to raise plasma PRL concentration in the control rats, but not in the piracetam‐treated rats. This tolerance was overcome by piracetam in a significant manner but with a reversed dose‐response curve; i.e. the smaller the dose of piracetam, the higher the subsequent morphine‐induced PRL peak.

There is no simple explanation for the mechanism by which piracetam induces these contradictory effects. Interference with the excitatory amino acid system, which is also involved in opiate action, is proposed speculatively as a possible mediator of the effects of piracetam.
Inhibition of prostanoid formation in intact cells by 2,5‐di‐(tert‐butyl)‐1,4‐benzohydroquinone, a blocker of Ca2+‐ATPases
Authors: Leis Hans J. , Zach Doris , Huber Evelyn , Ziermann Laszlo , Gleispach Helmut , Windischhofer W.
Published: July 2012

The blocker of endoplasmic reticulum Ca2+‐ATPase, 2,5‐di‐(tert‐butyl)‐1,4‐benzohydroquinone (BHQ) was shown to inhibit formation of prostaglandin E2 and prostacyclin in the osteoblast‐like cell lines, MC3T3‐E1 and ROS 17/2.8, respectively, in a dose‐dependent manner with an IC50 of 0.5‐1 μm. Inhibition was observed with various stimuli (arachidonic acid, bradykinin, melittin and calcium ionophore, A23187).

This effect was also observed in human platelets, where BHQ dose‐dependently blocked thromboxane biosynthesis and formation of 12‐hydroxy‐heptadecatrienoic acid after stimulation with arachidonic acid, but not formation of 12‐hydroxy‐eicosatetraenoic acid.

Inhibition of prostaglandin E2 formation in MC3T3‐E1 cells was not observed with thapsigargin after stimulation with arachidonic acid, A23187 or melittin, whereas bradykinin‐induced prostaglandin E2 biosynthesis was blocked.

Taken together, the results suggest a direct inhibitory action of BHQ on the cyclo‐oxygenase in these three cell systems.
Comparison of the profiles of agonists as stimulants of the β3‐adrenoceptor in vitro with their gastroprotective effects in the conscious rat
Authors: Bahl A.K. , Clayton N.M. , Coates J. , Martin D.P. , Oakley I.G. , Strong P. , Trevethick M.A.
Published: July 2012

This paper compares the activity of a range of agonists as stimulants of the β3‐adrenoceptor in rat isolated oesophagus with their ability to afford protection against indomethacin‐induced gastric damage in the conscious rat.

The β3‐adrenoceptor agonists, CL 316243 and BRL 37344, the non‐selective β‐adrenoceptor agonist, isoprenaline and the selective β2‐adrenoceptor agonist, salmeterol, all evoked concentration‐dependent relaxation of precontracted muscularis mucosa from rat oesophagus. The rank order of agonist potency was BRL 37344 > CL 316243 > isoprenaline > > salmeterol. The selective β1‐adrenoceptor agonist, denopamine, did not relax the preparation.

The relaxant responses to all agonists were resistant to blockade by atenolol (10 μm), and ICI 118551 (1 μm) thus suggesting that they were not mediated by either β1‐ or β2‐adrenoceptor stimulation. In contrast, cyanopindolol and propranolol did inhibit responses to BRL 37344, CL 316243 and isoprenaline, giving pA2 values or pKB estimates which were consistent with an interaction at β3‐adrenoceptors (i.e. approximately 8.0 and 6.5 respectively). However, responses to salmeterol were resistant to blockade by all the antagonists tested, which suggests that the high (> 1 μm) concentrations of salmeterol used exerted non‐specific relaxant effects.

The agonist effects of CL 316243 and BRL 37344 on β1‐ and β2‐adrenoceptors were assessed on guinea‐pig right atrium and precontracted trachea respectively. Both agonists had minimal activity as stimulants of heart rate, but did relax trachea, being 380 (CL 316243) and 21 (BRL 37344) fold less potent than isoprenaline.

CL 316243 and BRL 37344 were potent inhibitors of indomethacin‐induced gastric antral ulceration in the conscious rat (ED50 values = 0.24 and 0.09 μmol kg−1, p.o.) Salmeterol was approximately 100 times less potent than BRL 37344 as a gastroprotective agent and denopamine was without effect.

The gastroprotective effects of CL 316243 and BRL 37344 were resistant to blockade by ICI 118551 (10 mg kg−1, p.o.) and propranolol (10 mg kg−1, p.o.). In contrast, both antagonists caused dose‐related inhibition of the protective action of salmeterol (10 mg kg−1, p.o.). Cyanopindolol was not assessed as an antagonist in vivo because preliminary experiments revealed that it exacerbated indomethacin‐induced gastric damage in its own right.

In conclusion, the β3‐adrenoceptor agonists CL 316243 and BRL 37344 were potent inhibitors of indomethacin‐induced gastric antral ulceration in the rat. These data suggest that an agonist which is potent and selective for the human β3‐adrenoceptor may confer mucosal protection in man.
Allosteric interactions between cyclothiazide and AMPA/kainate receptor antagonists
Authors: Yamada Kelvin A. , Turetsky Dorothy M.
Published: July 2012

Cyclothiazide blocks α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor desensitization and potentiates AMPA receptor gated currents. Interactions between cyclothiazide, and the non‐competitive antagonist GYKI52466 (GYKI) and competitive antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulphamoyl‐benzo (F) quinoxaline (NBQX) were studied at native and recombinant AMPA/kainate receptors using whole‐cell recording in order to characterize the modulation by cyclothiazide of these two antagonist sites.

GYKI 100 μm, which is sufficient to eliminate virtually hippocampal kainate (100 μm) currents, failed to prevent access of cyclothiazide to its site of potentiation, and was unable to enhance removal of cyclothiazide potentiation. However, cyclothiazide reduced GYKI (30 μm) block from 84±8.3% to 38±12%, and slowed the onset of the block with a time course much faster than the time course for onset and offset of potentiation induced by cyclothiazide. Cyclothiazide had qualitatively similar effects upon antagonism by NBQX 1 μm.

Kainate activated desensitizing currents in dorsal root ganglion (DRG) neurones, which were unaffected by cyclothiazide. GYKI blocked these kainate currents with lower affinity (IC50>120 μm) than for hippocampal neurones (IC50<30 μm), and cyclothiazide did not affect GYKI antagonism.

Steady‐state AMPA currents from homomeric GluRA‐Dflip receptors in HEK 293 cells were dramatically potentiated (up to 216 fold) by cyclothiazide via reduction of desensitization. In contrast, kainate‐gated currents in HEK 293 cells expressing GluR6R receptors exhibited pronounced desensitization that was unaffected by cyclothiazide. GYKI retains its inhibition at both recombinant AMPA and kainate receptors.

These results indicate that cyclothiazide allosterically influences two important antagonist sites on AMPA receptors. In addition, AMPA/kainate receptor subunit composition influences the affinity of GYKI for the receptor.
Ouabain‐induced increases in resting tone of human hyperplastic prostate following repeated noradrenaline and electrical field stimulation
Authors: Guh Jih‐Hwa , Ko Feng‐Nien , Chueh Shih‐Chieh , Lai Ming‐Kuen , Teng Che‐Ming
Published: July 2012

The effect of ouabain on contractions to repeated noradrenaline stimulation and electrical field stimulation of human hyperplastic prostate was examined. Ouabain (1 μm) did not induce contractile response per se but progressively increased the resting tone (i.e., the tone between one noradrenaline stimulation, or electrical field stimulation, and the following) of human hyperplastic prostate.

The increased tone by ouabain following repeated noradrenaline stimulations or electrical field stimulation was fully relaxed by the removal of external calcium, and recovered following restoration of calcium.

The effect of noradrenaline on Na+ uptake was measured. Noradrenaline (10 μm) significantly increased the rate of Na+ accumulation in the presence of ouabain (1 μm); this stimulatory effect was almost completely blocked by prazosin (0.1 μm) and ethylisopropylamiloride (100 μm). In contrast, tetrodotoxin (1 μm) had no effect on noradrenaline‐stimulated Na+ transport in human hyperplastic prostate.

Intracellular Na+ loading by noradrenaline (10 μm) in the presence of ouabain (1 μm) significantly increased the transmembrane Ca2+ uptake as compared with the absence of ouabain; however, nifedipine (1 μm) was ineffective on Ca2+ uptake under this condition.

Transmembrane Ca2+ efflux was stimulated by noradrenaline (10 μm) in human hyperplastic prostate; this effect was significantly decreased in the presence of ouabain (1 μm).

It is suggested that the increased tone of human hyperplastic prostate following repeated excitation in the presence of ouabain is due to increased Ca2+ entry and reduced efflux of Ca2+ through the Na+/Ca+ exchange system as a consequence of Na+ pump inhibition by ouabain.
Metabolism of the macrolide immunosuppressant, tacrolimus, by the pig gut mucosa in the Ussing chamber
Authors: Lampen Alfonso , Christians Uwe , Gonschior Ann‐Katrin , Bader Augustinus , Hackbarth Ingelore , von Engelhardt Wolfgang , Sewing Karl‐Friedrich
Published: July 2012

The macrolide tacrolimus (FK506), used as an immunosuppressant, is a cytochrome P450 (CYP) 3A substrate in the liver. The metabolism of tacrolimus and the transport of its metabolites in the pig gut was studied in the Ussing chamber. Tacrolimus and its metabolites were quantified by h.p.l.c./mass spectrometry.

In the Ussing chamber, demethyl, didemethyl, hydroxy and hydroxy‐demethyl tacrolimus were generated. Their formation was concentration‐ and time‐dependent. The metabolite pattern was not different from that after incubation of tacrolimus with human small intestinal microsomes.

The metabolite formation was highest in the duodenum and declined in the order duodenum >

Since tacrolimus metabolism was inhibited by the specific CYP3A inhibitors, troleandomycin and ketoconazole, we concluded that these enzymes are involved in intestinal metabolism of tacrolimus.

Tacrolimus metabolites re‐entered the mucosa chamber (>90%) and passed through the small intestinal preparation into the serosa chamber.

It is concluded that tacrolimus is metabolized in the intestine, that the metabolites are able to re‐enter the gut lumen and also enter into the portal vein and that small intestinal metabolism and transport is at least in part responsible for the low oral bioavailability of tacrolimus
Antiarrhythmic action of rilmenidine on adrenaline‐induced arrhythmia via central imidazoline receptors in halothane‐anaesthetized dogs
Authors: Mammoto Tadanori , Kamibayashi Takahiko , Hayashi Yukio , Yamatodani Atsushi , Takada Koji , Yoshiya Ikuto
Published: July 2012

To elucidate the role of central imidazoline receptors in the genesis of adrenaline‐induced arrhythmias under halothane anaesthesia, we investigated the effects of rilmenidine, a selective agonist at imidazoline receptors, on this type of arrhythmia in dogs. Rilmenidine (1, 3, 10 μg kg−1, i.v.) did not affect basal haemodynamic parameters (heart rate and blood pressure), but dose‐dependently inhibited adrenaline‐induced arrhythmias under halothane anaesthesia.

Although, rilmenidine has a weak affinity for α2‐adrenoceptors, pretreatment with idazoxan (10 μg kg−1, intracistenally i.c.), an imidazoline receptor antagonist which has also α2‐adrenoceptor blocking potency, blocked the antiarrhythmic effect of rilmenidine (10 μg kg−1, i.v.). In contrast, pretreatment with rauwolscine (20 μg kg−1, i.c.), a classical α2‐adrenoceptor antagonist with little affinity for imidazoline receptors, did not affect the effect of rilmenidine (10 μg kg−1, i.v.). Furthermore, bilateral vagotomy completely blocked the antiarrhythmic action of rilmenidine (10 μg kg−1, i.v.).

It is suggested that the antiarrhythmic action of rilmenidine is due to the activation of central imidazoline receptors and that vagal tone is critical for this action of rilmenidine.
Block of pancreatic ATP‐sensitive K+ channels and insulinotrophic action by the antiarrhythmic agent, cibenzoline
Authors: Ishida‐Takahashi Ayako , Horie Minoru , Tsuura Yoshiyuki , Ishida Hitoshi , Ai Tomohiko , Sasayama Shigetake
Published: July 2012

We investigated the effect of cibenzoline (a class Ia antiarrhythmic drug) on basal insulin secretory activity of rat pancreatic islets and ATP‐sensitive K+ channels (KATP) in single pancreatic β cells of the same species, using radioimmunoassay and patch clamp techniques.

Micromolar cibenzoline had a dose‐dependent insulinotrophic action with an EC50 of 94.2±46.4 μm;. The compound inhibited the activity of the KATP channel recorded from a single β‐cell in a concentration‐dependent manner. The IC50 was 0.4 μm in the inside‐out mode and 5.2 μm in the cell‐ attached mode, at pH 7.4.

In the cell‐attached mode, alkalinization of extracellular solution increased the inhibitory action of cibenzoline and the IC50 was reduced from 26.8 μm at pH 6.2 to 0.9 μm at pH 8.4. On the other hand, the action of cibenzoline in the excised inside‐out mode was acute in onset with a small IC50, indicating that the drug attains its binding site from the cytoplasmic side of the cell membrane.

In the inside‐out mode, micromolar ADP reactivated the cibenzoline‐blocked KATP channels in a manner similar to that by which ADP restored ATP‐dependent block of the channel.

The binding of [3H]‐glibenclamide to pancreatic islets was inhibited by glibenclamide but not by cibenzoline. In contrast, the [3H]‐cibenzoline binding was displaced by unlabelled cibenzoline but not by glibenclamide. It is concluded that cibenzoline blocks pancreatic KATP channels via a binding site distinct from the sulphonylurea receptor.
Investigation of the involvement of the N‐methyl‐D‐aspartate receptor macrocomplex in the development of spermine‐induced CNS excitation in vivo
Authors: Doyle K.M. , Shaw G.G.
Published: July 2012

The involvement of the N‐methyl‐D‐asparate (NMDA) receptor macrocomplex in the development of spermine‐induced CNS excitation in vivo was investigated.

Injection of 100 μg of spermine into the left lateral cerebral ventricle of female Laca mice (20–25 g) resulted in the development of two distinct phases of CNS excitatory effects which were quantified by a scoring system.

The first phase effects occurred within minutes of injection and generally lasted for about 1 h. Most mice showed scratching of the upper body, frequent face washing and some mice developed clonic convulsions. By about 2 h after injection, the second phase of effects began to develop in the form of body tremor which worsened with time and culminated in fatal tonic convulsions, generally within 8 h of injection.

Pretreatment of the mice with dizocilpine (0.3 mg kg−1, i.p.) resulted in antagonism of the first phase of spermine‐induced effects, but a higher dose (0.3 mg kg−1, (x2), i.p.) was necessary to inhibit the second phase effects.

Whereas the glutamate antagonist, 3‐((R)‐2‐carboxypiperazin‐4‐yl) propyl‐1‐phosphonic acid (D‐CPP) (10, 20 mg kg−1, i.p.), the glycine antagonist 7‐chlorokynurenate (10, 30, 50 nmol, i.c.v.), or the polyamine antagonist ifenprodil (30, 60 mg kg−1, i.p.) antagonized the first phase of effects produced by spermine, these agents given as monotherapy, were ineffective against the development of the second phase of effects.

Co‐administration of ifenprodil with either D‐CPP or 7‐chlorokynurenate resulted in a dose‐dependent antagonism of the development of the second phase of spermine‐induced effects.

It is concluded that the development of the two temporally distinct phases of spermine‐induced effects may be mediated by pharmacologically distinct mechanisms, although the results suggest that the NMDA receptor macrocomplex may be involved in both phases of effects. Furthermore, a moderate dose of D‐CPP or 7‐chlorokynurenate appears to enhance the inhibitory potential of ifenprodil in vivo.
Effects of ranolazine on L‐type calcium channel currents in guinea‐pig single ventricular myocytes
Authors: Allen T.J.A. , Chapman R.A.
Published: July 2012

Ranolazine has protective effects against ischaemia as exemplified by a reduction of the associated enzyme release and an attenuation of the fall of ATP and other metabolic changes. It has been suggested that ranolazine may affect GTP‐binding proteins involved in the β‐adrenergic protein kinase A (PKA) cascade by interacting with Gs. Calcium channel currents are stimulated by this cascade but the effect of ranolazine upon them is not known. The whole cell patch clamp technique was used to examine the action of ranolazine on basal calcium channel currents and those stimulated by activation at various steps in the PKA cascade.

Ranolazine had only a small effect on the basal calcium current (100 μm caused 11.3% inhibition), but markedly attenuated the β‐adrenoceptor stimulated current (20 nM isoprenaline increased current by 2.3 fold, 10 μm ranolazine inhibited this increase by 47.6%). When the PKA cascade was activated downstream to the receptor by either G‐protein activation with Gpp[NH]p or adenylate cyclase activation with forskolin, the calcium current showed a sensitivity to ranolazine similar to the basal current. Activation of the PKA cascade via H2 receptors gave rise to currents which showed an intermediate sensitivity to ranolazine. Ranolazine inhibition of ICa persisted during muscarinic attenuation of β‐adrenoceptor activation.

The results indicate that ranolazine, at concentrations which have significantly beneficial effects during ischaemic episodes, only greatly affects whole cell calcium current when facilitated by β‐adrenoceptor or histamine receptor activation. Ranolazine would appear to act at the receptor level, rather than at the GTP‐binding or Gs/adenylate cyclase level. An additional smaller effect is also present, which may be mediated by a direct effect on the channel, or components closely associated with it.
Ability of angiotensin II to modulate striatal dopamine release via the AT1 receptor in vitro and in vivo
Authors: Brown D. Charles , Steward Lucinda J. , Ge Jian , Barnes Nicholas M.
Published: July 2012

The ability of angiotensin II to modulate dopamine release from rat striatal slices in vitro and in the intact rat striatum in vivo was assessed by the microdialysis technique.

In slices of rat striatum, angiotensin II (0.1‐1.0 μm) induced a concentration‐related increase in endogenous dopamine release which was maximal (approximately 250% above basal levels) within the first 2–4 min of agonist application and subsequently declined to near basal values. The angiotensin II‐induced increase in dopamine release was Ca2+‐dependent and was completely antagonized by the selective AT1 receptor antagonist, losartan (1.0 μm). In contrast, the AT2 receptor antagonist, PD123177 (1.0 μm) failed to modify the angiotensin II‐induced response. Neither antagonist alone modified basal dopamine release from striatal slices.

In freely moving rats, angiotensin II (1.0–10 μm; administered via the microdialysis probe) induced a concentration‐related increase in extracellular levels of dopamine which was maximal (approximately 150% above basal levels) within 20–40 min of agonist application and subsequently declined. The angiotensin II (10 μm)‐induced increase in extracellular levels of dopamine was completely antagonized by the AT1 receptor antagonist, losartan (0.1‐1.0 μm; administered via the microdialysis probe) but not by the AT2 receptor antagonist, PD123177 (1.0 μm; administered via the microdialysis probe). Neither antagonist alone modified basal extracellular levels of dopamine.

Homogenate radioligand binding studies with [125I]‐angiotensin II (0.1 nM) identified relatively low levels of specific binding sites in rat striatal homogenates compared to homogenates of pyriform cortex (51.3±9.2 and 651.3±55.1 fmol g−1 wet weight, respectively, mean ± s.e. mean, n = 3; non‐specific binding defined by unlabelled angiotensin II). The majority of the specific [125I]‐angiotensin II (0.1 nM) binding in the striatal and pyriform cortex homogenates was sensitive to the selective AT1 receptor antagonist, losartan (1.0 μm).

In conclusion, the present study provides direct evidence that angiotensin II acting via the AT1 receptor subtype facilitates the release of dopamine in the rat striatum in vitro and in vivo. This receptor‐mediated response may account for the modulation of dopamine‐mediated behavioural responses by antagonists of the AT1 receptor and inhibitors of angiotensin converting enzyme.
Discrimination by PPADS between endothelial P2Y‐ and P2U‐ purinoceptors in the rat isolated mesenteric arterial bed
Authors: Ralevic Vera , Burnstock Geoffrey
Published: July 2012

The main aim of this study was to characterize the antagonistic effects of pyridoxalphosphate‐6‐ azophenyl‐2′,4′‐disulphonic acid (PPADS) at coexisting endothelial P2Y‐ and P2U‐purinoceptors. Studies were conducted in Krebs‐perfused mesenteric arterial preparations isolated from the rat, with tone raised by methoxamine (5–50 μm).

Purine and pyrimidine compounds elicited vasodilatation with a rank order of potency of 2‐methylthio ATP (2‐MeSATP) = ADP>ATP = UTP>P1, P3‐diadenosine triphosphate (Ap3A)>P1, P2‐ diadenosine pyrophosphate (Ap2A)>NADP> adenosine. 8‐para‐Sulphophenyltheophylline (8‐PSPT; 3 μm) had no effect on vasodilator responses to 2MeSATP, ADP, ATP, UTP, Ap3A or NADP, but blocked responses to adenosine and the maximal response to Ap2A.

PPADS (3–100 μm) attenuated vasodilator responses to the P2Y‐selective agonists 2MeSATP and ADP, shifting the dose‐response curves to the right. The pA2 values for PPADS at 2MeSATP and ADP were 5.97 ± 0.69 and 5.98 ± 0.86 respectively. In contrast, PPADS had no effect on vasodilator responses mediated by the P2U‐selective agonist, UTP, or on vasodilator responses mediated by ATP.

PPADS (10 μm) was used to characterize responses mediated by the adenine dinucleotides; dose‐response curves for vasodilator responses to Ap3A and NADP, but not those to Ap2A, were shifted to the right by PPADS. The estimated pA2 values for the effect of PPADS on Ap3A and NADP were 6.38 and 6.26 respectively.

Indomethacin (10 μm) had no effect on vasodilator responses to 2MeSATP, ADP, ATP or UTP.

In conclusion, these results show that PPADS is an antagonist at endothelial P2Y‐ but not P2U‐ purinoceptors in rat mesenteric arteries. These receptors cannot be discriminated by inhibition of prostaglandin synthesis; P2Y‐purinoceptors are, however, sensitive to ADP. Selective antagonism by use of PPADS showed that ATP acts at P2U‐ and not P2Y‐purinoceptors. Ap3A and NADP mediate vasodilatation via P2Y‐purinoceptors, whereas vasodilatation to Ap2A is mediated partly via P1 and possibly via P2U‐purinoceptors.
Chloride channel blockers decrease intracellular pH in cultured renal epithelial LLC‐PK1 cells
Authors: Brown Colin D.A. , Dudley Adam J.
Published: July 2012

The effects of chloride channel blockers upon intracellular pH (pHi) were examined in renal epithelial monolayers of LLC‐PK1 cells. A significant intracellular acidification was found with addition of 100 μm 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB), niflumic acid, flufenamate and diphenylamine‐2‐carboxylate (DPC) but not with 4,4′‐diisothiocyanatostilbene‐2‐2′disulphonic acid (DIDS). The effects of these agents upon pHi was dose‐dependent with apparent K0.5 values of: 16.7±0.3 μm, 34.2±0.9 μm and 740±13 μm for niflumic acid, flufenamate and DPC respectively. The results indicate that at concentrations commonly used to block channel activity these chloride channel blockers have profound effects upon pHi.
Dissociation of P2 purinoceptor‐mediated increase in intracellular Ca2+ level from myosin light chain phosphorylation and contraction in rat aorta
Authors: Kitajima Satoshi , Harada Ken‐Ichi , Hori Masatoshi , Ozaki Hiroshi , Karaki Hideaki
Published: July 2012

The effects of P2 agonists, adenosine‐5′‐triphosphate (ATP), α, β‐methylene‐adenosine‐5′‐triphosphate (α, β‐me‐ATP) and adenosine‐5‐O‐(3‐thiotriphosphate) (ATPγS), on the intracellular free Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction were examined in vascular smooth muscle of rat aorta.

ATP (0.1 μm‐1 mM), α, β‐me‐ATP (0.1–100 μm) and ATPγS (1–100 μm) induced transient increases followed by sustained increase in [Ca2+]i. The effects of these agonists were concentration‐dependent. Compared with the effects of a high concentration of KCl (17.5–72.4 mM), the contractions induced by these P2 purinoceptor agonists were smaller at a given [Ca2+]i.

In the absence of extracellular Ca2+ (with 0.5 mM EGTA), ATPγS (10 μm) induced large transient increase in [Ca2+]i with only small contraction in Ca2+‐free solution. In contrast, α, β‐me‐ATP (10 μm) induced only a very small increase in [Ca2+]i and contraction.

ATP (1 mM), α, β‐me‐ATP (10 μm) and ATPγS (10 μm), added during stimulation with 0.1 μm noradrenaline, induced additional and transient increases in [Ca2+]i which were also not associated with contraction.

High K+ (72.4 mM) increased MLC phosphorylation with a similar time course to that of the increase in [Ca2+]i (peak phosphorylation was 56% when [Ca2+]i increased to 100%). In contrast, the time course of the increase in MLC phosphorylation due to ATP (1 mM) did not coincide with that of the large increases in [Ca2+]i; MLC phosphorylation increased to only 31% when [Ca2+]i increased to 163%. The MLC phosphorylation due to α, β‐me‐ATP (10 μm) and ATPγS (10 μm), measured at peak [Ca2+]i, were only 19% and 14%, respectively, irrespective of a large increase in [Ca2+]i (138% and 188%, respectively).

The absence of a clear relationship between P2‐purinoceptor‐mediated increase in [Ca2+]i (either by Ca2+ influx or Ca2+ release) and MLC phosphorylation or force generation appears to imply that elevation in [Ca2+]i does not contribute to these responses.
Effects of an AT1 receptor antagonist, an ACE inhibitor and a calcium channel antagonist on cardiac gene expressions in hypertensive rats
Authors: Kim Shokei , Ohta Kensuke , Hamaguchi Akinori , Yukimura Tokihito , Miura Katsuyuki , Iwao Hiroshi
Published: July 2012

This study was undertaken to determine whether the AT1 receptor directly contributes to hypertension‐induced cardiac hypertrophy and gene expressions.

Stroke‐prone spontaneously hypertensive rats (SHRSP) were given orally an AT1 receptor antagonist (losartan, 30 mg kg−1 day−1), an angiotensin converting enzyme inhibitor (enalapril 10 mg kg−1 day−1), a dihydropyridine calcium channel antagonist (amlodipine, 5 mg kg−1 day−1), or vehicle (control), for 8 weeks (from 16 to 24 weeks of age). The effects of each drug were compared on ventricular weight and mRNA levels for myocardial phenotype‐ and fibrosis‐related genes.

Left ventricular hypertrophy of SHRSP was accompanied by the increase in mRNA levels for two foetal phenotypes of contractile proteins (skeletal α‐actin and β‐myosin heavy chain (β‐MHC)), atrial natriuretic polypeptide (ANP), transforming growth factor‐β‐1 (TGF‐β1) and collagen, and a decrease in mRNA levels for an adult phenotype of contractile protein (α‐MHC). Thus, the left ventricle of SHRSP was characterized by myocardial transition from an adult to a foetal phenotype and interstitial fibrosis at the molecular level.

Although losartan, enalapril and amlodipine lowered blood pressure of SHRSP to a comparable degree throughout the treatment, losartan caused regression of left ventricular hypertrophy of SHRSP to a greater extent than amlodipine (P < 0.01).

Losartan significantly decreased mRNA levels for skeletal α‐actin, ANP, TGF‐β1 and collagen types I, III and IV and increased α‐MHC mRNA in the left ventricle of SHRSP. Amlodipine did not alter left ventricular ANP, α‐MHC and collagen types I and IV mRNA levels of SHRSP.

The effects of enalapril on left ventricular hypertrophy and gene expressions of SHRSP were similar to those of losartan, except for the lack of inhibition of collagen type I expression by enalapril.

Unlike the hypertrophied left ventricle, there was no significant difference between losartan and amlodipine in the effects on non‐hypertrophied right ventricular gene expressions of SHRSP.

Our results show that hypertension causes not only left ventricular hypertrophy but also molecular transition of myocardium to a foetal phenotype and interstitial fibrosis‐related molecular changes. These hypertension‐induced left ventricular molecular changes may be at least in part mediated by the direct action of local angiotensin II via the AT1 receptor.
Bosentan‐improved cardiopulmonary vascular performance and increased plasma levels of endothelin‐1 in porcine endotoxin shock
Authors: Weitzberg Eddie , Hemsén Anette , Rudehill Anders , Modin Agnes , Wanecek Michael , Lundberg Jan M.
Published: July 2012

To evaluate the possible contribution of endothelin‐1 (ET‐1) to the pathophysiology of porcine septic shock, the non‐peptide, mixed ET‐receptor antagonist, bosentan (RO 47–0203) was administered (5 mg kg−1, i.v.) 30 min before infusion of lipopolysaccharide (LPS) (E. coli., serotype 0111:B4) (15 μg kg−1 h−1) and at 3.5 h of endotoxaemia in six anaesthetized and mechanically ventilated pigs. Six other pigs served as controls and received only LPS infusion. Pulmonary and systemic haemodynamics as well as splenic, renal and intestinal blood flows were measured continuously. Release and synthesis of ET‐1 and Big ET‐1 were also measured.

Only three of the six pigs in the control group survived 3 h of LPS infusion while in the bosentan‐treated group all six pigs were alive at that time. A biphasic increase in mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance (PVR) was seen in control pigs. Pretreatment with bosentan did not influence the first peak but markedly attenuated the second, more prolonged increase in MPAP and PVR. The second dose of bosentan completely restored these parameters to pre‐LPS levels. The LPS‐induced changes in mean arterial blood pressure, heart rate and systemic vascular resistance were similar in both groups, while cardiac output (CO) was significantly higher in the bosentan‐treated group. The second bosentan dose increased CO and splenic and intestinal blood flow without further lowering of blood pressure.

Bosentan caused an increase of the basal arterial plasma levels of ET‐1‐like immunoreactivity (LI), from 16.8 ± 1.3 pM to 49.6 ± 10.0 pM (n = 6, P < 0.01). However, the rate of the increase of ET‐1 levels during the LPS infusion was not affected by bosentan. Repeated administration of bosentan during LPS infusion caused an additional increase of ET‐1‐LI levels. Neither the basal levels of Big ET‐LI nor the LPS induced 8 fold increase in Big ET‐LI were changed by bosentan. The level of preproET‐1 mRNA in the lung was increased about 3 fold after 4.5 h of LPS treatment. This elevation was not influenced by bosentan.

From these studies using bosentan, a non‐peptide, selective and mixed ET‐receptor antagonist, we conclude that during LPS‐induced shock bosentan can abolish the late phase pulmonary hypertension and improve cardiac output as well as increase blood flow to the splenic and intestinal vascular beds without causing a further decrease in mean arterial blood pressure. Further investigations in the clinical setting are needed to evaluate the use of ET‐receptor antagonists, such as bosentan, in treatment of septic shock.
The effect of forskolin on 5‐HT1‐like and angiotensin II‐induced vasoconstriction and cyclic AMP content of the rabbit isolated femoral artery
Authors: Randall V.A. , MacLennan S.J. , Martin G.R. , Wilson V.G.
Published: July 2012

A characteristic feature of vasoconstrictor 5‐HT1‐like receptors in vitro is that responses mediated by these receptors are enhanced by other vasoconstrictor agents. In the present study, we have examined the influence of cellular cyclic AMP on vasoconstrictor responses to activation of 5‐HT1‐like receptors in isolated ring segments of the rabbit femoral artery (RbFA), and determined whether modulation of this second messenger underlies the ability of angiotensin II, an endogenous vasoconstrictor, to enhance 5‐HT1‐like responses.

In the presence of 0.1 μm ketanserin (to antagonize 5‐HT2‐receptors) and 0.3 μm prazosin (to antagonize α1‐adrenoceptors), 5‐HT produced a concentration‐related contraction, which was significantly augmented by pre‐contraction of the vessel with 0.1‐0.45 nM ([A30]) angiotensin II. Responses to 5‐HT in the presence of angiotensin II were inhibited by the 5‐HT1‐like/5‐HT2 antagonist, metergoline (1 μm).

The directly‐acting adenylyl cyclase activator, forskolin (1 μm), abolished responses to angiotensin II and caused a rightward shift and concomitant depression of the 5‐HT concentration‐effect (E/[A]) curve. Higher concentrations of forskolin (> 10 μm) abolished responses to 5‐HT and 1 μm sodium nitroprusside abolished responses to 5‐HT and angiotensin II (n = 7).

In the presence of angiotensin II (0.1‐0.45 nM), however, 1 μm forskolin failed to inhibit 5‐HT‐induced contractions; the E/A curve for 5‐HT (in the presence of forskolin and angiotensin II) was not significantly different from that produced in the presence of angiotensin II alone. Similarly, the presence of angiotensin II (0.1‐0.45 nM) was also able to overcome partially the inhibitory effect of 1 μm sodium nitroprusside against 5‐HT‐induced contractions (n = 7). In marked contrast, 5‐HT failed to elicit a contraction in the presence of angiotensin II and 10 μm forskolin (n = 5).

5‐HT (1 μm) significantly reduced basal cyclic AMP accumulation by 35%, whereas angiotensin II (0.45 nM) was without effect. The combination of angiotensin II and 5‐HT failed to alter significantly the reduction in cyclic AMP produced by 5‐HT alone. Forskolin (1 μm) increased cyclic AMP levels 7 fold above basal, but neither 1 μm 5‐HT nor a combination of 1 μm 5‐HT and 0.45 nM angiotensin II produced a significant decrease in cyclic AMP content.

Whilst moderate concentrations of forskolin can inhibit the reponses to either agent, simultaneous activation of angiotensin II and 5‐HT1‐like receptors can overcome the inhibitory effect of elevated levels of cyclic AMP. Since the potentiating effect of angiotensin II, in either the presence or absence of forskolin, occurs without significant alteration of cellular cyclic AMP, it seems likely that a cyclic AMP‐independent pathway is implicated in the synergistic interaction between angiotensin II and vasoconstrictor 5‐HT1‐like receptors.