bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelin‐1 (ET‐1)‐induced contraction in rat isolated trachea: involvement of ETa and ETB receptors and multiple signal transduction systems
Authors: Henry Peter J.
Published: July 2012

Quantitative autoradiographic, biochemical and functional studies were performed to investigate the endothelin receptor subtypes and signal transduction systems that mediate endothelin‐1 (ET‐1)‐induced contraction in rat isolated tracheal smooth muscle.

Specific binding of 0.5 nm [125I]‐ET‐1 to tracheal smooth muscle was inhibited by at least 40% in the presence of either the ETA receptor selective ligand BQ‐123 (1 μm) or the ETB receptor‐selective ligand sarafotoxin S6c (30 nm), indicating the presence of both ETA and ETB receptors in this tissue.

ET‐1 and sarafotoxin S6c were both potent spasmogens of rat isolated tracheal smooth muscle preparations. Sarafotoxin S6c‐induced contractions were unaffected in the presence of the ETA receptor antagonist BQ‐123 (10 μm), but were markedly attenuated in tissue previously exposed to 100 nm sarafotoxin S6c to induce ETB receptor desensitization. ET‐1‐induced contractions were, at most, only partially attenuated either by blocking the ETA receptor‐effector system (with 10 μm BQ‐123) or by desensitizing the ETB receptor‐effector system with sarafotoxin S6c. However, ET‐1‐induced contractions were markedly attenuated by blocking both receptor‐effector systems simultaneously. These findings suggest that ET‐1 could induce contraction by stimulating either ETA or ETB receptors.

ET‐1 (10 μm) induced a 7 fold increase in intracellular [3H]‐inositol phosphate accumulation over basal levels in rat isolated tracheal smooth muscle. In contrast, sarafotoxin S6c (2.5 μm) increased intracellular [3H]‐inositol phosphate accumulation by only 2 fold. ET‐1‐induced accumulation of [3H]‐inositol phosphates was abolished by 10 μm BQ‐123.

In Ca2+‐free Krebs bicarbonate solution, 100 nm ET‐1 induced a significantly larger contraction than that induced by 100 nm sarafotoxin S6c (46.6 ± 5.6% Cmax versus 8.8 ± 2.8% Cmax, n = 5–7). This presumed intracellular Ca2+‐dependent phase of contraction induced by ET‐1 was significantly inhibited by 10 μm BQ‐123 (7.5 ± 1.0% Cmax). Subsequent addition of 2.5 mm Ca2+ induced a second phase of contraction. The extracellular Ca2+‐dependent phase of contraction induced by ET‐1 was similar in magnitude to that induced by sarafotoxin S6c (63.6 ± 4.5% Cmax versus 58.0 ± 3.7% Cmax) and was not inhibited by BQ‐123. Sarafotoxin S6c‐induced contractions were not inhibited by the L‐type Ca2+‐channel antagonists, nicardipine or verapamil.

In summary, ETA and ETB receptors coexist in rat isolated tracheal smooth muscle and stimulation of both receptor subtypes contributes to ET‐1‐induced contraction in this tissue. However, stimulation of these receptor subtypes appears to induce contraction by activating different second messenger pathways; ETA receptor stimulation induces phosphoinositide turnover and subsequent release of intracellular Ca2+ whereas stimulation of ETB receptors facilitates the influx of extracellular Ca2+.
Endothelium‐dependent noradrenaline‐induced relaxation of rat isolated cerebral arteries: pharmacological characterization of receptor subtypes involved
Authors: Hempelmann Ralf G. , Ziegler Albrecht
Published: July 2012

The endothelium‐dependence of catecholamine‐induced relaxation of rat cerebral arteries was investigated in vitro.

In the basilar artery (BA), the maximal relaxant response was most pronounced with noradrenaline (NA), less with isoprenaline (Iso), and only very little with terbutaline. Methoxamine and the α2‐adrenoceptor selective agonists BHT 933 and clonidine, had no relaxant effect.

In BA, the relaxation by NA or Iso was markedly attenuated by Nω‐nitro‐l‐arginine (l‐NOARG) 10−4 m. Short term perfusion of the vessels by Triton X 100 (1:1000) suppressed the NA‐induced relaxation.

The relaxation induced by NA or Iso was markedly reduced in presence of l‐NOARG in the posterior, medial and anterior cerebral artery.

In BA, NA‐induced relaxation was non‐competitively inhibited by propranolol, atenolol, and the β1‐ and β2‐adrenoceptor selective antagonists, CGP 20712 A and ICI 118551.

The relaxant NA‐effect was not affected by prazosin but was non‐competitively blocked by phentolamine.

The Iso‐induced relaxation was competitively blocked by propranolol, whereas atenolol, CGP 20712 A and ICI 118551 caused a non‐competitive inhibition.

The experiments indicate that the catecholamine‐induced relaxation in rat isolated cerebral arteries depends upon the endothelium. They suggest that the NA‐induced relaxation of BA is mediated by different α‐ and β‐adrenoceptors and that the Iso‐induced relaxation is mediated by different β‐receptors. The findings would also be compatible with the idea of a receptor type which cannot be characterized by the pharmacological tools that we have used.
Ca2+ dependency of the release of nitric oxide from non‐adrenergic non‐cholinergic nerves
Authors: Boeckxstaens Guy E. , De Man Joris G. , Pelckmans Paul A. , Cromheeke Kristel M. , Herman Arnold G. , Van Maercke Yvan M.
Published: July 2012

The role of Ca2+ in nitrergic neurotransmission was studied in the canine ileocolonic junction.

The specific N‐type voltage‐sensitive Ca2+ channel blocker ω‐conotoxin GVIA (CTX, 10–100 nm) significantly reduced the electrically‐evoked (2–16 Hz, 1–2 ms pulse width) non‐adrenergic non‐cholinergic (NANC) relaxations, preferentially affecting those to low frequency stimulation, in circular muscle strips of the ileocolonic junction. In contrast, the nerve‐mediated NANC‐relaxations in response to acetylcholine (30 μm), γ‐aminobutyric acid (100 μm) and adenosine 5′‐triphosphate (100 μm), as well as the relaxations to nitric oxide (NO) (3–10 μm) and nitroglycerin (1 μm), remained unaffected.

A NO‐related substance (NO‐R), released from the ileocolonic junction in response to NANC nerve stimulation (4 and 16 Hz, 2 ms pulse width), was assayed with a superfusion bioassay cascade. CTX (50 nm) reduced the release of NO‐R induced by electrical impulses (4 Hz: from 18 ± 4% to 6 ± 4%; 16 Hz: from 33 ± 2% to 14 ± 4%, n = 5), but not that in response to the nicotinic receptor agonist, 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP, 0.3 mm). In Ca2+‐free medium, the release of NO‐R evoked by electrical impulses or DMPP was inhibited. The L‐type Ca2+ channel blockers verapamil (1–3 μm) and nifedipine (1 μm) had no effect.

From these results we conclude that the release of NO‐R in response to NANC nerve stimulation is Ca2+‐dependent. The electrically‐evoked release of NO‐R results from Ca2+ entry through CTX‐sensitive N‐type voltage‐sensitive Ca2+ channels, whereas that induced by nicotinic receptor activation involves CTX‐insensitive Ca2+ channels, different from the L‐ or N‐type.
Nasal administration of an ACTH(4–9) peptide analogue with dimethyl‐β‐cyclodextrin as an absorption enhancer: pharmacokinetics and dynamics
Authors: Schipper Nicolaas G.M. , Verhoef J. Coos , De Lannoy Larissa M. , Romeijn Stefan G. , Brakkee Jan H. , Wiegant Victor M. , Gispen Willem Hendrik , Merkus Frans W.H.M.
Published: July 2012

The systemic absorption and the neurotrophic effect of the metabolically stabilized ACTH (4–9) analogue, Org2766, were investigated following intranasal (i.n.) administration.

Without additives the nasal bioavailability of the peptide was in the order of 15 and 10% in rats and rabbits, respectively. The absorption could be improved by addition of a variety of absorption enhancers to the nasal preparation. The β‐cyclodextrin derivative, dimethyl‐β‐cyclodextrin (DMβCD) at a concentration of 5% (w/v) improved the absorption in rats about 5 fold from 13 ± 4% (mean ± s.d.) for administration of the peptide alone to 65 ± 21%, and in rabbits 1 to 2 fold, from 10 ± 6% to 17 ± 8%.

The increased permeability of the rat nasal mucosa for Org2766 caused by DMβCD in rats reversed substantially within 1 h. However, the nasal absorption had not yet completely returned to the level without enhancer.

S.c. administered Org2766 accelerated the functional recovery from peripheral nerve damage in rats. However, the peptide did not facilitate nerve repair following i.n. administration with DMβCD, in spite of the fact that Org2766 was well absorbed. I.v. injection of Org2766 was also ineffective.
5‐HT3 antagonists reduce morphine self‐administration in rats
Authors: Hui Siu‐Chun G. , Sevilla Elenita L. , Ogle Clive W.
Published: July 2012

The effects of the 5‐HT3 receptor antagonists, ondansetron and tropisetron, on morphine consumption were studied in naive and morphine‐dependent rats.

The administration of ondansetron (1 μg kg−1, i.p. twice daily) 7 days prior to, and during a 21‐day period of, morphine availability (increasing concentration from 0.1 to 0.4 mg ml−1) in 5% sucrose solution reduced opiate intake from the 9th day of morphine treatment.

The administration of ondansetron (0.1 μg kg−1, i.p. twice daily) or tropisetron (0.1 μg kg−1, i.p. twice daily) on the 14th day of the 21‐day period of morphine treatment failed to reduce opiate consumption. Administration of the larger doses of tropisetron (1 μg kg−1) or ondansetron (1 μg kg−1) reduced morphine consumption.

After receiving 21 days of treatment with morphine alone or with the ondansetron or tropisetron regimens identified above, the sucrose solutions were substituted with tap water for 7 days. These detoxified rats were then allowed a free choice of sucrose or morphine for 10 days. Animals that had received concomitant treatment with ondansetron or tropisetron showed reduced morphine intake when compared with the controls treated with morphine only or with vehicle‐treated controls.

The administration of cyproheptadine (100 or 250 μg kg−1, i.p. twice daily) on the 14th day of 21‐day morphine treatment failed to modify morphine intake and also failed to influence the subsequent intake of the opiate in the free choice situation.

It is concluded that ondansetron and tropisetron can reduce morphine intake in both naive and morphine‐dependent rats.
Binding and structure‐activity‐relation of benzo[f]isoquinoline‐ and norcodeinone‐derivatives at μ‐opioid receptors in the rat cerebral cortex
Authors: Freissmuth Michael , Beindl Wolfgang , Kratzel Martin
Published: July 2012

We have probed the ligand binding site of the μ‐opioid receptor using a series of isoquinoline‐ and norcodeinone‐derivatives; in these morphine‐ and codeine‐analogues, the position of the piperidine‐nitrogen as well as its mobility is altered relative to that found in morphine.

The μ‐receptor in rat cortical membranes was labelled with [3H]‐naloxone and competition experiments were carried out in the absence and presence of Gpp(NH)p and NaCl: conditions, which are associated with affinity shifts for agonists whilst antagonist affinity remains unaffected. Moving the piperidine‐nitrogen closer to the phenolic ring or reducing its mobility by incorporation into an additional ring drastically decreases the affinity.

In contrast, we find that the piperidine‐nitrogen in a distal position is well tolerated provided that additional structural criteria, in particular a phenolic hydroxyl‐group and a 6 carbon ring corresponding to ring C in morphine, are met. This assumption was verified by the synthesis of WB4/PH (4aR, 10bS, 11R)‐10, 11‐epoxy‐1, 2, 3, 4, 5, 6‐hexahydro‐9‐hydroxy‐3‐methyl‐4a,10b‐butano‐benzo[f]isochinolin‐12‐on(10). This compound is an agonist with an affinity comparable to that of morphine.

We therefore conclude that both the mobility of the piperidine nitrogen of the ligand and of its counterpart anionic site in the ligand binding pocket of the μ‐opioid receptor (presumably aspartic acid) are important determinants for fruitful interaction. The mobility of the anionic site is restricted in one direction but is sufficient to bridge the 2Å distance that exists between the position of the nitrogen in morphine and WB4/PH.
Effects of selective antagonism of β‐adrenoceptor sub‐types on responses to isoprenaline in rat distal colon in vitro
Authors: MacDonald Allan , Lamont Michelle
Published: July 2012

The effects of the β1‐ and β2‐adrenoceptor selective antagonists, CGP 20712A and ICI 118551 respectively, on responses to isoprenaline‐induced relaxation of rat distal colon were investigated in order to determine the contributions of these subtypes to relaxation. In addition, the properties of ICI D7114, a novel putative stimulant of atypical β‐adrenoceptors, were investigated. Our preliminary experiments with ICI D7114 showed that this compound lacked agonist activity in rat distal colon and in fact antagonized responses to isoprenaline. We therefore studied the antagonism of isoprenaline by ICI D7114 in more detail.

Longitudinal segments of rat distal colon were suspended in Krebs solution at 37°C for isometric recording. The Krebs solution contained EDTA (23 μm) and prazosin (0.1 μm) and was gassed with 95/5% O2/CO2. After an initial equilibration period, reproducible contractions to a submaximal concentration of methacholine (1 μm) were obtained before carrying out a concentration‐response curve (CRC) to isoprenaline in a non‐cumulative manner. Four consecutive CRCs to isoprenaline were carried out in each tissue with a 1 h interval between each curve. Antagonists were present in increasing concentrations during the intervals between CRCs. Control tissues received no antagonists to allow estimation of the magnitude of time‐dependent changes.

Isoprenaline produced a concentration‐dependent inhibition of methacholine‐induced contractions. CRCs to isoprenaline were reproducible with no significant time‐dependent changes. Propranolol produced no shift of the isoprenaline CRC at 0.01 μm and a 5 fold shift at 0.1 μm. No further shift was observed with 1 μm. CGP 20712A had no effect on the CRC to isoprenaline at 0.1, 1 and 3 μm. ICI 118551 produced little or no shift at 0.1 μm and a six fold shift with 1 μm. No further shift was observed with 3 μm. ICI D7114 produced a concentration‐dependent parallel rightward shift of the CRC to isoprenaline. Schild analysis gave a slope close to unity and a mean pA2 value of 7.29 for ICI D7114.

The results with propranolol and β1‐ and β2‐adrenoceptor antagonists confirm the mainly atypical nature of β‐adrenoceptors in rat distal colon. There may also be a small contribution from β2‐adrenoceptors in the response to isoprenaline but β1‐adrenoceptors are absent. ICI D7114 has no agonist activity and behaves as a relatively high affinity reversible competitive antagonist of atypical β‐adrenoceptors in this preparation.
Electrophysiological and antiarrhythmic actions of the κ agonist PD 129290, and its R,R (+)‐enantiomer, PD 129289
Authors: Pugsley M.K. , Saint D.A. , Penz M.P. , Walker M.J.A.
Published: July 2012

The S,S (−)‐enantiomer PD 129290, a κ agonist, and its corresponding inactive R,R (+)‐enantiomer (PD 129289) were studied in rat isolated hearts and in intact rats for cardiovascular and antiarrhythmic actions. The electrophysiological actions of PD 129290 were also studied in rat isolated cardiac myocytes using voltage clamp.

Ventricular pressure, heart rate and ECG were studied in isolated hearts while blood pressure, heart rate and ECG were studied in pentobarbitone‐anaesthetized rats. In the latter, responses to electrical stimulation and coronary occlusion were also investigated.

In isolated hearts both enantiomers, over the concentration range 2–16 μm, dose‐dependently reduced systolic ventricular pressure and heart rate while prolonging the P–R and QRS intervals of the ECG.

At doses of 1–32 μmol kg−1 both enantiomers reduced blood pressure and heart rate in anaesthetized rats. In addition, both enantiomers increased the size of the RSh and increased P–R, QRS, and Q–T intervals of the ECG. The thresholds for premature beats and ventricular fibrillation were dose‐dependently increased by PD 129289. At lower doses PD 129290 decreased thresholds. These decreases were blocked by naloxone to reveal underlying increases similar to those seen with PD 129289. Both enantiomers increased refractory periods.

Naloxone (8 μmol kg−1) did not alter any of the actions of PD 129290, except to abolish the initial decreases in thresholds in intact rats seen with lower doses of PD 129290.

Both enantiomers (2 and 8 μmol kg−1) equally reduced arrhythmias in anaesthetized rats subject to occlusion of a coronary artery.

In rat isolated cardiac myocytes 20 μm PD 129290, in the presence and absence of naloxone decreased the amplitude of the transient sodium current by about 50% without affecting the voltage‐dependence of activation or inactivation of this current.

The antiarrhythmic actions of both enantiomers appear to primarily result from their Class I (sodium channel blockade) properties which are independent of κ agonism.
Electrophysiological effects of S 16257, a novel sino‐atrial node modulator, on rabbit and guinea‐pig cardiac preparations: comparison with UL‐FS 49
Authors: Thollon Catherine , Cambarrat Christine , Vian Joël , Prost Jean‐François , Peglion Jean Louis , Vilaine Jean Paul
Published: July 2012

S 16257 is a new bradycardic agent. Its electropharmacological profile has been compared to that of the known bradycardic compound UL‐FS 49 (Zatebradine). Intracellular recordings of action potentials (APs) were performed with conventional glass microelectrodes.

In the rabbit isolated sino‐atrial node (SAN) tissue, S 16257 and UL‐FS 49 (1 μm, 3 μm and 10 μm) were equipotent in slowing spontaneous APs firing predominantly by decreasing the rate of diastolic depolarization (at 3 μm, – 23.8 ± 3.9% and – 27.9 ± 2.6%, respectively). For the two compounds a maximal effect was obtained at 3 μm. In these preparations, action potential duration at 50% of total repolarization (APD50) was more affected by UL‐FS 49 than S 16257 at any concentration tested (at 3 μm, + 8.9 ± 2.9% and + 29.1 ± 3.7% for S 16257 and UL‐FS 49, respectively; P ≤ 0.01).

To estimate the direct effects on AP duration, driven cardiac preparations were exposed to these agents. In guinea‐pig papillary muscles, paced at a frequency of 1 Hz, increasing concentrations of S 16257 or UL‐FS 49 (0.1 to 10 μm, 30 min exposure for each concentration) slightly prolonged AP repolarization. This prolongation was more marked for UL‐FS 49 (at 1 μm, + 6.1 ± 0.6% and + 11.2 ± 1.3% elevation of APD50, for S 16257 and UL‐FS 49, respectively).

Application of UL‐FS 49 (3 μm) to rabbit Purkinje fibres, triggered at a frequency of 0.25 Hz, induced a marked prolongation of APD50 and APD90 (+ 149.4 ± 51.2% and + 86.0 ± 15.4%, respectively). S 16257 (3 μm) induced only a weak prolongation of AP (+ 14.1 ± 5.0% and + 14.8 ± 3.3% for APD50 and APD90, respectively) significantly smaller than in the case of UL‐FS 49.

These results show that S 16257 slows the rate of spontaneous AP firing in isolated SAN mainly by a reduction of the diastolic depolarization of the cells, which suggests an inhibition of the pace‐maker current (If). S 16257 and UL‐FS 49 are equipotent in their bradycardic effect but S 16257 is more specific as it induces less increase in myocardial repolarization time.
Metabolism of methylarginines by human vasculature; implications for the regulation of nitric oxide synthesis
Authors: MacAllister Raymond J. , Fickling Sara A. , Whitley Guy St. J. , Vallance Patrick
Published: July 2012

The metabolism of methylarginines by human cultured endothelial cells and human saphenous vein was studied in vitro. The human endothelial cell line (SGHEC‐7), primary cultures of human umbilical vein endothelial cells (HUVEC) and human saphenous vein were incubated with [14C]‐monomethyl‐l‐arginine ([14C]‐l‐NMMA) and the cytosolic extract analysed by high performance liquid chromatogaphy (h.p.l.c.) with on‐line radioisotope detection.

SGHEC‐7, HUVEC and human saphenous vein metabolized [14C]‐l‐NMMA to a compound which co‐eluted with [14C]‐citrulline. A second metabolite which co‐eluted with [14C]‐arginine was evident on the radiochromatograms of HUVEC cytosol and saphenous vein extracts.

The intracellular levels of [14C]‐l‐NMMA and [14C]‐citrulline in SGHEC‐7 cells incubated with [14C]‐l‐NMMA (0.5 μCi ml−1: 8.9 μm) for 1 h were 113 ± 22 and 67.6 ± 6.2 pmol mg−1 cell protein respectively (n = 7). Co‐incubation with NGNGdimethyl‐l‐arginine (ADMA; 100 μm) but not NGN′Gdimethyl‐l‐arginine (SDMA; 100 μm) reduced the intracellular level of [14C]‐citrulline to 26.3 ± 3.7 pmol mg−1 cell protein (P < 0.01; n = 3) without reducing the intracellular level of [14C]‐l‐NMMA.

The intracellular levels of [14C]‐citrulline in SGHEC‐7 cells incubated with [14C]‐l‐NMMA for 1 h were reduced following co‐incubation with NGnitro‐l‐arginine methylester (l‐NAME; 1 mm), NGnitro‐l‐arginine (l‐NOARG; 1 mm) and l‐canavanine (1 mm) to 47.1 ± 6.2, 24.7 ± 3.6 and 12.5 ± 2.8% of control levels (P < 0.001; n = 9). ADMA (1 mm; n = 3) reduced intracellular [14C]‐citrulline levels to 4 ± 4% of control (P < 0.01) but SDMA (1 mm; n = 3) had no effect.

The accumulation of endogenously synthesized ADMA in the culture supernatant of SGHEC‐7 cells was increased by co‐incubation with l‐NMMA (1 mm) from 1.98 ± 0.08 to 2.74 ± 0.36 nmol mg−1 cell protein, an increase of 40%.

These results demonstrate that human vasculature possesses an enzyme which has similar properties to dimethylarginase; human endothelial cells and human saphenous vein metabolize l‐NMMA to citrulline via a process inhibited by ADMA but not SDMA. The increase in endothelium‐derived ADMA following co‐incubation with l‐NMMA is consistent with competition between ADMA and l‐NMMA for dimethylarginase. Inhibition of this enzyme might increase the intracellular concentration of ADMA, an endogenously produced compound that inhibits nitric oxide synthesis.
Modulation of vasodilatation to levcromakalim by adenosine analogues in the rabbit ear: an explanation for hypoxic augmentation
Authors: Randall Michael D. , Ujiie Hiroshi , Griffith Tudor M.
Published: July 2012

We have used a rabbit isolated ear, buffered‐perfused preparation to investigate the effects of adenosine analogues on the vasodilatation to the potassium channel opener, levcromakalim (the active (−)‐enantiomer of cromakalim). We have examined the effects of 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), a selective adenosine A1 antagonist, on vasodilatation to levcromakalim under hypoxic conditions and also following inhibition of nitric oxide synthesis.

Levcromakalim relaxed preconstricted preparations with an EC50 = 369 ± 48 nm and maximum relaxation of tone (Rmax) = 81.0 ± 3.2%. In the presence of 1 μm N6‐cyclohexyladenosine (CHA) a selective adenosine A1 agonist, there was a significant (P < 0.01) leftward shift in the concentration‐response curve with an EC50 = 194 ± 54 nm and Rmax = 93.2 ± 2.0%. Conversely, the presence of CHA did not influence vasodilatation to either pinacidil or sodium nitroprusside.

Hypoxia also significantly (P < 0.001) increased the vasodilator potency of levcromakalim (EC50 = 134 ± 22 nm), and this enhancement was completely reversed (EC50 = 380 ± 107nm, P<0.01) by pretreatment of the preparations with 5 μm DPCPX, a selective A1 adenosine antagonist. However, under normoxic conditions DPCPX did not influence vasodilatation to levcromakalin.

Inhibition of nitric oxide synthesis with 100 μm NG‐nitro‐l‐arginine methyl ester (l‐NAME) caused a significant (P < 0.001) leftward shift in the concentration‐response curve to levcromakalim (EC50 = 73.0 ± 7.6 nm). Pretreatment of preparations with DPCPX partially reversed the increase in potency found in the absence of nitric oxide synthesis (EC50 = 153 ± 18 nm, P < 0.001).

We have shown that an adenosine A1 agonist may increase the potency of levcromakalim indicating that adenosine receptor activation may augment the vasodilator activity of levcromakalim. That responses to levcromakalim but not those to pinacidil were affected by CHA points to further differences in the pharmacology of these potassium channel openers. The reversal by the adenosine A1 antagonist of the hypoxic‐potentiation of vasodilatation to levcromakalim, and also augmentation following inhibition of nitric oxide synthesis, suggests that under these conditions there is an endogenous release of adenosine which may enhance responses to levcromakalim. The findings of this study suggest that levcromakalim may selectively dilate vessels where there is elevated adenosine release.
Prevention by specific chemical classes of α1‐adrenoceptor antagonists of veratrine‐contractures in rat left atria independently of α1‐adrenoceptor blockade
Authors: Grand Bruno Le , Marty Aline , Vié Bruno , Patoiseau Jean‐François , John Gareth W.
Published: July 2012

The putative direct protective effects of a series of chemically diverse α1‐adrenoceptor antagonists against veratrine alkaloid‐induced tetanic contractures in rat isolated left atria have been investigated.

Atria were mounted in organ baths containing normal, oxygenated physiological salt solution (20 ml, pH 7.4), for isometric tension recording. Atria were electrically driven at 4 Hz and were maintained at 34°C. Veratrine (100 μg ml−1) was applied to the atria to elicit tetanic (diastolic) contracture.

Concentration‐dependent protective effects against veratrine‐contractures, in the absence of negative inotropic responses, were observed with the quinazoline congeners, prazosin and doxazosin and with the benzodioxane‐related compounds, WB 4101 and its thio analogue, benoxathian. IC50 concentrations and apparent Hill coefficients of all four drugs ranged from 0.27 to 0.93 μm, and from 0.86 to 1.09, respectively, and are consistent with interaction at a single site.

In contrast, no protective activity versus veratrine‐contractures was observed with corynanthine, 5‐methyl‐urapidil, phenoxybenzamine, phentolamine or chloroethylclonidine (10 μm).

Contractures were prevented by prazosin at concentrations 2–3 log units higher than those which antagonized methoxamine‐evoked inotropic responses. In addition, concommitant α1‐adrenoceptor occupancy by high concentrations of methoxamine (100 μm), phentolamine (10 μm, inactive per se in preventing contracture), or both drugs together, failed, in each case, to modify significantly the protective effects of prazosin or WB 4101 against veratrine‐contractures.

Our findings demonstrate that α1‐adrenoceptor antagonists which prevent veratrine‐contractures belong to specific chemical classes of the quinazoline‐ and benzodioxane‐type. The mechanism by which these drugs afford protection is apparently independent of an interaction with defined α1‐adrenoceptors.
Attenuated renal response to moxonidine and rilmenidine in one kidney‐one clip hypertensive rats
Authors: Li Ping , Penner S. Brian , Smyth Donald D.
Published: July 2012

I1 non‐adrenoceptor, imidazoline receptor agonists, such as moxonidine, increase urine flow rate and sodium excretion following infusion into the renal artery. The functions of these agonists in genetic and acquired models of hypertension have not been determined.

We therefore studied the renal effects of two known non‐adrenoceptor, imidazoline receptor agonists, rilmenidine and moxonidine, in 1K‐1C hypertensive and 1K‐sham normotensive rats. Rilmenidine (0, 3, 10, 30 nmol kg−1 min−1) or moxonidine (0, 1, 3, 10 nmol kg−1 min−1) was infused directly into the renal artery (30 gauge needle) of 1K‐sham normotensive and 1K‐1C hypertensive rats.

In 1K‐sham normotensive rats, rilmenidine and moxonidine produced dose related increases in urine flow rate, sodium excretion and osmolar clearance. Both rilmenidine and moxonidine failed to increase urine flow rate, sodium excretion and osmolar clearance in 1K‐1C hypertensive rats to the same extent as in 1K‐sham animals. At comparable doses, rilmenidine had no effect, while moxonidine (3 and 10 nmol kg−1 min−1) did result in a small increase in urine volume and osmolar clearance which was less than that observed in the 1K sham control animals.

These studies indicate that the renal effects of non‐adrenoceptor, imidazoline receptor stimulation are diminished in 1K‐1C hypertensive rats compared with 1K‐sham normotensive rats. Whether this decrease in activity of the natriuretic non‐adrenoceptor, imidazoline receptors contributes to the increase in blood pressure in the 1K‐1C acquired model of hypertension remains to be determined.
Dual effects of mastoparan on intracellular free Ca2+ concentrations in human astrocytoma cells
Authors: Nakahata Norimichi , Ishimoto Hiromi , Mizuno Kohichi , Ohizumi Yasushi , Nakanishi Hironori
Published: July 2012

The effect of mastoparan, a wasp venom toxin, on intracellular free Ca2+ concentration ([Ca2+]i) was examined in human astrocytoma cells. Mastoparan inhibited [Ca2+]i induced by carbachol (100 μm) in a concentration‐dependent manner in the absence of extracellular Ca2+, consistent with our previous results showing that mastoparan inhibits phosphoinositide hydrolysis in human astrocytoma cells.

In contrast, mastoparan itself increased [Ca2+]i and augmented carbachol‐induced increase in the [Ca2+]i in the presence of extracellular Ca2+, suggesting that mastoparan elicited Ca2+ influx from the extracellular medium. The increase appeared to be maximum at extracellular Ca2+ concentrations of 0.1–0.2 mm. The higher concentrations of extracellular Ca2+ depressed the influx.

Pertussis toxin did not affect mastoparan‐induced inhibition of [Ca2+]i in the absence of extracellular Ca2+, consistent with the previous results that pertussis toxin did not affect mastoparan‐induced inhibition of phosphoinositide hydrolysis.

Pertussis toxin augmented mastoparan‐induced increase in [Ca2+]i in the presence of extracellular Ca2+, suggesting that pertussis toxin substrate(s) seems to be inhibitory for Ca2+ influx induced by mastoparan.

Verapamil, nifedipine and diltiazem (each 10 μm), L‐type Ca2+ antagonists, did not affect mastoparan‐induced Ca2+ influx. However, verapamil (10 μm) slightly inhibited the increase in [Ca2+]i induced by carbachol in the presence of mastoparan.

The results obtained in the present study indicate that mastoparan has two opposite effects on [Ca2+]i in human astrocytoma cells and possibly has at least two sites of action.
Regulation by hypoxia of endothelin‐1‐stimulated phospholipase D activity in sheep pulmonary artery cultured smooth muscle cells
Authors: Plevin Robin , Kellock Nicola A. , Wakelam Michael J.O. , Wadsworth Roger
Published: July 2012

The aim of the study was to characterize the effects of hypoxia on agonist‐stimulated phospholipase D (PLD) and phospholipase C activity of sheep pulmonary artery cultured smooth muscle cells.

Endothelin‐1 (ET‐1), 5‐hydroxytryptamine (5‐HT) and the protein kinase C (PKC) activator tetradecanoylphorbol acetate (TPA), stimulated a time‐ and concentration‐dependent increase in [3H]‐phosphatidylbutanol accumulation. This was abolished by pretreatment of the cells with the PKC inhibitor, Ro‐318220, suggesting that agonist‐stimulated phospholipase D activity is dependent upon the activation of PKC.

Hypoxia (Po2 20 mmHg for 30 min) stimulated basal [3H]‐phosphatidylbutanol accumulation by approximately 2 fold and this activity was abolished by preincubation of the cells with 10 μm Ro‐318220.

In cells preincubated in low O2 containing medium for 30 min, the subsequent agonist‐stimulated accumulation of [3H]‐phosphatidylbutanol was reduced. However, the decrease in stimulation was greater for ET‐1 and 5‐HT than for TPA.

ET‐1 and TPA stimulated a time‐dependent increase in protein kinase C‐ mediated psuedosubstrate phosphorylation. Following preincubation for 30 min in low O2 containing media, basal pseudosubstrate phosphorylation increased whilst the fold stimulation by TPA and ET‐1 decreased.

In cells preincubated in low O2 containing medium, ET‐1‐stimulated [3H]‐inositol phosphate accumulation was reduced by approximately 30– 40%. This reduction was reversed by preincubation of the cells with Ro‐318220.

These results suggest a role for PKC in the effects of hypoxia on PLD in pulmonary artery smooth muscle cells.
The effect of calcium removal on the suppression by adenosine of epileptiform activity in the hippocampus: demonstration of desensitization
Authors: Hosseinzadeh H. , Stone T.W.
Published: July 2012

Previous work has suggested that presynaptic effects of adenosine may be dependent on divalent cations. The present study was undertaken to determine whether a similar requirement existed at postsynaptic sites.

Extracellular recordings were made in the CA1 pyramidal cell layer of rat hippocampal slices following orthodromic stimulation of Schaffer collateral fibres in stratum radiatum or antidromic stimulation of the alveus. In antidromic stimulation experiments, CaCl2 was omitted (calcium‐free medium) or reduced to 0.24 mm (low calcium medium) and in some experiments MgS04 was increased to 2 mm. Kynurenic acid at concentrations of 1 and 5 mm in calcium‐free medium and 1 mm in low calcium medium had no effect on secondary spike size.

Adenosine and baclofen induced a concentration‐dependent reduction in the amplitude of orthodromic potentials with maximum effects at 20 and 5 μm respectively.

In nominally calcium‐free medium, bursts of multiple population spikes were obtained in response to antidromic stimulation. Adenosine had little effect in reducing the secondary spike amplitude. At high concentration (2 mm) an initial depression was seen which declined within 3–5 min.

Sensitivity to adenosine was restored in low calcium medium or by raising magnesium. Although raising the divalent cation concentration increased the inhibitory effect of adenosine, desensitization was still seen.

2‐Chloroadenosine (100–500 μm) and R‐PIA (50 μm), which are not substrates for either the nucleoside transporters or adenosine deaminase, were inactive in the absence of calcium. S‐(2‐hydroxy‐5 nitrobenzyl)‐6‐thioinosine, an adenosine uptake blocker, at a concentration 100 μm had no effect on secondary potential size and did not restore adenosine sensitivity in calcium‐free medium.

Thapsigargin, which discharges intracellular calcium stores, had no significant effect at 1 μm on the bursts of action potentials and did not change the effect of 0.5 mm adenosine in calcium‐free medium.

Unlike adenosine, baclofen concentration‐dependently reduced the secondary spike size in calcium‐free medium and no sign of recovery was observed during maintained superfusion for up to 45 min. No cross‐desensitization was seen between baclofen and adenosine.

Applications of adenosine locally by pressure to neuronal somata or dendrites still resulted in desensitized responses in calcium‐free medium.

It is concluded that the postsynaptic sensitivity to adenosine is dependent on the concentration of divalent cations in the extracellular space implying an effect of cations on adenosine receptor activation or transduction processes.
Benzodiazepine/cholecystokinin interactions at functional CCK receptors in rat brain
Authors: Boden P.R. , Woodruff G.N.
Published: July 2012

The effects of benzodiazepines on cholecystokinin (CCK) responses produced following activation of CCKB receptors by pentagastrin in the ventromedial hypothalamus (VMH) or CCKA receptors by CCK‐8S in the dorsal raphe of the rat brain in vitro have been investigated.

The benzodiazepine agonist, flurazepam, at high concentrations, blocked pentagastrin‐induced excitations in the rat VMH yielding an equilibrium constant (Ke) value of 12.5 μm.

In the rat dorsal raphe, where activation of CCKA receptors leads to neuronal depolarization, flurazepam also produced a weak block of the CCK response.

Flurazepam blocked CCK responses but not carbachol‐induced excitations of VMH neurones. The inhibition of CCK responses by flurazepam was not blocked by the benzodiazepine antagonist, flumazenil.

These data suggest that flurazepam is a weak antagonist at central CCKB receptors.

At central CCKA receptors, flurazepam blocked CCK‐8S responses but the inhibition was not competitive, with a reduction in the peak CCK‐8S obtainable in the presence of flurazepam. These results suggest that flurazepam acts at a site other than the CCKA receptor itself to block CCK responses in the dorsal raphe.
Enhancement of noradrenergic constriction of large coronary arteries by inhibition of nitric oxide synthesis in anaesthetized dogs
Authors: Woodman Owen L. , Pannangpetch Patchareewan
Published: July 2012

Coronary vascular responses to bilateral carotid occlusion (BCO) and the intravenous infusion of tyramine (Tyr, 20 μg kg−1 min−1) and noradrenaline (NA, 0.5 μg kg−1 min−1) were examined after bilateral vagotomy and antagonism of β‐adrenoceptors. BCO, Tyr and NA decreased large coronary artery diameter and increased mean coronary resistance and systemic arterial pressure without affecting heart rate.

Inhibition of nitric oxide (NO) synthase with NG‐nitro‐l‐arginine (l‐NNA, 5 and 15 mg kg−1) significantly increased mean arterial pressure and decreased heart rate and large coronary artery diameter. Mean coronary resistance was unaffected by either dose of l‐NNA. l‐NNA significantly reduced depressor and coronary vasodilator responses to the endothelium‐dependent vasodilator acetylcholine (ACh, 10 μg kg−1, i.v.). Systemic and coronary vasodilator responses to sodium nitroprusside (SNP, 5 μg kg−1) were unaffected by l‐NNA with the exception that the dilatation of the large coronary artery was significantly enhanced by the higher dose.

−1‐NNA significantly enhanced constriction of the large coronary arteries caused by BCO, Tyr and NA but did not affect the increases in mean coronary resistance or systemic arterial pressure.

Inhibition of NO synthesis enhances adrenergic constriction of large coronary arteries caused by both neuronally released and exogenous noradrenaline. In contrast, l‐NNA did not affect adrenergic constriction of coronary or systemic resistance vessels. Endothelium‐derived NO may play an important role in the modulation of noradrenergic vasoconstriction in coronary conductance arteries.
Effects of a novel nonpeptide bradykinin B2 receptor antagonist on intestinal and airway smooth muscle: further evidence for the tracheal B3 receptor
Authors: Farmer Stephen G. , DeSiato Mark A.
Published: July 2012

We examined the effects of phosphonium, [[4‐[[2‐[[bis(cyclohexylamino)methylene] amino]‐3‐(2‐naphthalenyl) 1‐oxopropyl]amino]‐phenyl]‐tributyl, chloride, monohydrochloride (WIN 64338), a novel, nonpeptide bradykinin B2 receptor antagonist, on bradykinin‐induced contractions of guinea‐pig isolated ileum, and guinea‐pig and ferret trachea.

WIN 64338 potently and competitively antagonized ileal contractions, in response to bradykinin, exhibiting a pA2 value of 7.97 ± 0.10. The compound was without effect on contractions elicited by methacholine, a muscarinic receptor antagonist. Thus, WIN 64338 is a competitive and selective antagonist of ileal B2 receptors.

In contrast, WIN 64338 was completely without effect on bradykinin‐induced contractions of guinea‐pig or ferret trachea. Thus, even at a concentration of 1 μm, which was sufficient to cause a 100 fold decrease in ileal sensitivity to bradykinin, WIN 64338 failed to shift the bradykinin log concentration‐response curves in trachea isolated from either species.

These data confirm that WIN 64338 represents the first reported nonpeptide antagonist of guinea‐pig ileal B2 receptors. They also provide additional evidence for heterogeneity of bradykinin receptors within the same species (guinea‐pig) and, furthermore, indicate that the tracheal bradykinin receptor (B3?) is different from that in ileal tissue (B2).
Binding and functional properties of hexocyclium and sila‐hexocyclium derivatives to muscarinic receptor subtypes
Authors: Waelbroeck M. , Camus J. , Tastenoy M. , Feifel R. , Mutschler E. , Tacke R. , Strohmann C. , Rafeiner K. , de Miranda J.F. Rodrigues , Lambrecht G.
Published: July 2012

We have compared the binding properties of several hexocyclium and sila‐hexocyclium derivatives to muscarinic M1 receptors (in rat brain, human neuroblastoma (NB‐OK 1) cells and calf superior cervical ganglia), rat heart M2 receptors, rat pancreas M3 receptors and M4 receptors in rat striatum, with their functional antimuscarinic properties in rabbit vas deferens (M1/M4‐like), guinea‐pig atria (M2), and guinea‐pig ileum (M3) muscarinic receptors.

Sila‐substitution (C/Si exchange) of hexocyclium (→ sila‐hexocyclium) and demethyl‐hexocyclium (→ demethyl‐sila‐hexocyclium) did not significantly affect their affinities for muscarinic receptors. By contrast, sila‐substitution of o‐methoxy‐hexocyclium increased its affinity 2 to 3 fold for all the muscarinic receptor subtypes studied.

The p‐fluoro‐ and p‐chloro‐derivatives of sila‐hexocyclium had lower affinities than the parent compound at the four receptor subtypes, in binding and pharmacological studies.

In binding studies, o‐methoxy‐sila‐hexocyclium (M1 = M4 ≥ M3 ≥ M2) had a much lower affinity than sila‐hexocyclium for the four receptor subtypes, and discriminated the receptor subtypes more poorly than sila‐hexocyclium (M1 = M3 > M4 > M2). This is in marked contrast with the very clear selectivity of o‐methoxy‐sila‐hexocyclium for the prejunctional M1/M4‐like heteroreceptors in rabbit vas deferens.

The tertiary amines demethyl‐hexocyclium, demethyl‐sila‐hexocyclium and demethyl‐o‐methoxy‐sila‐hexocyclium had 10 to 30 fold lower affinities than the corresponding quaternary ammonium derivatives.
Mediation by M3‐muscarinic receptors of both endothelium‐dependent contraction and relaxation to acetylcholine in the aorta of the spontaneously hypertensive rat
Authors: Boulanger Chantal M. , Morrison Keith J. , Vanhoutte Paul M.
Published: July 2012

Experiments were designed to characterize the subtype(s) of endothelial muscarinic receptor that mediate(s) endothelium‐dependent relaxation and contraction in the aorta of spontaneously hypertensive rats (SHR).

Rings of SHR aorta with endothelium were suspended in organ baths for the measurement of isometric force. Ecothiopate (an inhibitor of acetylcholinesterase) was present throughout the experiments. Endothelium‐dependent contraction to acetylcholine was studied in quiescent aortic rings in the presence of NG‐nitro‐l‐arginine (to prevent the formation of nitric oxide). Endothelium‐dependent relaxation to acetylcholine was obtained during contraction to phenylephrine and in the presence of indomethacin (to inhibit cyclo‐oxygenase activity). Responses to acetylcholine were assessed against the non‐preferential muscarinic receptor antagonist, atropine, and the preferential antagonists pirenzepine (M1), methoctramine (M2) and 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP; M3).

The potency of acetylcholine in inducing endothelium‐dependent contraction was 6.54 ± 0.07 (EC50). Atropine, pirenzepine, methoctramine and 4‐DAMP displayed competitive antagonism towards the endothelium‐dependent contraction to acetylcholine. The pA2 values for these muscarinic receptor antagonists were estimated from Arunlakshana‐Schild plots to be (– log m) 9.48 ± 0.07, 6.74 ± 0.22, 6.30 ± 0.20 and 9.39 ± 0.22 respectively. The potency of acetylcholine in inducing endothelium‐dependent relaxation was 7.82 ± 0.09 (IC50). Atropine, pirenzepine and 4‐DAMP displayed competitive antagonism towards the endothelium‐dependent relaxation to acetylcholine but methoctramine had no effect. The pA2 values for atropine and 4‐DAMP for the relaxation to acetylcholine were estimated from Arunlakshana‐Schild plots to be (– log m) 9.15 ± 0.23 and 9.63 ± 0.28, respectively. These results suggest that the muscarinic M3 receptor subtype mediates both endothelium‐dependent relaxation and contraction to acetylcholine in SHR aorta.
Pilocarpine‐induced relaxation of rat tail artery by a non‐cholinergic mechanism and in the absence of an intact endothelium
Authors: Tonta M.A. , Partington Helena C. , Tare M. , Coleman H.A.
Published: July 2012

The partial muscarinic agonist, pilocarpine, evoked concentration‐dependent relaxation with an EC50 of 2.4 × 10−3 m in isolated segments of rat tail artery that were constricted with phenylephrine (10−8 to 2 × 10−7 m). Acetylcholine also evoked concentration‐dependent relaxation but was more potent than pilocarpine (EC50, 6.5 × 10−7 m).

The concentration‐relaxation curves for pilocarpine were not affected by the muscarinic antagonists, atropine (10−9 m) or pirenzepine (5 × 10−7 m), while the concentration‐relaxation curves for acetylcholine‐evoked relaxation of the same tissues were shifted some 10 fold to the right by these concentrations of atropine and pirenzepine.

Acetylcholine failed to evoke relaxation following removal of the endothelium. The smooth muscle of the rat tail artery was some 10 fold more sensitive to the relaxing action of pilocarpine following denudation of the endothelium.

The effects of pilocarpine and acetylcholine on membrane potential were studied in tissues that were depolarized to − 39 ± 1 mV with phenylephrine (5 × 10−8 to 2 × 10−7 m). In intact tissues, pilocarpine caused hyperpolarization, an effect that persisted in the presence of muscarinic antagonists. Acetylcholine also evoked hyperpolarization.

Following removal of the endothelium, pilocarpine (10−5 to 10−3 m) evoked hyperpolarization in 6 of 15 preparations and a decrease in the frequency of action potentials in the remainder. Both of these responses were associated with relaxation.

The effects of pilocarpine were compared with other agents that evoke endothelium‐independent relaxation. The concentration‐relaxation curves in response to pilocarpine and nitroprusside were shifted to the right by ferricyanide (10−5 m) and methylene blue (10−5 m). Glibenclamide (10−6 m) was without effect on the hyperpolarization and relaxation evoked by pilocarpine (10−4 to 10−3 m).

Thus, pilocarpine evokes relaxation of rat tail artery independently of the cholinergic system and it is suggested that this is achieved by decreasing the frequency of action potentials in the smooth muscle.
Relationship between plasma lipids and palmitoyl‐CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates
Authors: Alegret M. , Ferrando R. , Vázquez M. , Adzet T. , Merlos M. , Laguna J.C.
Published: July 2012

The time‐course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl‐CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague‐Dawley rats.

The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB.

Drug treatment produced gross hepatomegaly and increase in peroxisomal β‐oxidation, and these parameters were strongly correlated. The order of potency was BFB > CFB ≥ GFB.

Both plasma cholesterol (BFB ∼ CFB > GFB) and triglyceride (BFB ∼ GFB > CFB) levels were reduced in treated animals. There was an inverse correlation between these parameters and peroxisomal β‐oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed.

Cytosolic and microsomal (but not mitochondrial) palmitoyl‐CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro. The increased hydrolase activities were directly and strongly correlated with peroxisomal β‐oxidation.

Palmitoyl‐CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB >GFB), probably as a consequence of the enhancement of hydrolase activities.

Some of the effects of fibrate treatment can be explained, at least in part, in terms of peroxisomal induction and caution should be exercised in the extrapolation of these results to species, such as man, that are insensitive to peroxisomal proliferation.