bluuebunny/crossref_metadata_2025 huggingface dataset:
[Ca2+]i oscillations induced by muscarinic stimulation in airway smooth muscle cells: receptor subtypes and correlation with the mechanical activity
Authors: Roux Etienne , Guibert Christelle , Savineau Jean‐Pierre , Marthan Roger
Published: August 2006

Cytosolic calcium concentration ([Ca2+]i) by indo 1 microspectrofluorimetry in freshly isolated cells and isometric contraction of isolated rings were measured in response to muscarinic cholinoceptor stimulation in rat tracheal smooth muscle.

In isolated myocytes, acetylcholine (ACh, 0.031 μm) caused a rapid and graded increase in [Ca2+]i up to a net amplitude of 492±26 nm (n=19) which gradually declined. The EC50 for ACh was 0.13 μm. This first [Ca2+]i peak was followed, when the ACh concentration increased, in approximately 5060% of the cells, by successive peaks of decreased amplitude ([Ca2+]i oscillations) superimposed on the plateau phase. Whereas the percentage of cells exhibiting [Ca2+]i oscillations remained consistent, the frequency of these oscillations increased to up to 10 min−1 with an ACh concentration of 100 μm.

Removal of extracellular calcium (in the presence of EGTA, 0.4 mm) or addition of the voltage‐dependent Ca2+‐channel blocker verapamil (10 μm) did not alter the first [Ca2+]i peak, the plateau or the oscillations induced by ACh or carbachol. In contrast, the specific inhibitor of the sarcoplasmic Ca2+‐ATPase, thapsigargin (1 μm), completely abolished the [Ca2+]i response. Thapsigargin (1 μm) also blocked the caffeine (5 mm)‐induced transient rise in [Ca2+]i.

Atropine (a non‐selective muscarinic cholinoceptor antagonist) and 4‐diphenyl acetoxy N‐methyl piperidine (4‐DAMP, a selective M3 antagonist) inhibited the [Ca2+]i response to muscarinic cholinoceptor activation with an IC50 of 13 and 20 nm, respectively. Pirenzepine (a selective M1 antagonist) also totally inhibited the [Ca2+]i response to ACh but with a higher IC50 of 2 μm. Methoctramine (a selective M2 antagonist) up to a concentration of 10 μm caused only a 40% inhibition. The effect of muscarinic antagonists on cumulative concentration‐response curves (CCRC) for carbachol was assessed at the following concentrations: atropine and 4‐DAMP at 3, 10 and 30 nm; pirenzepine 0.3, 1 and 3 μm, and methoctramine at 1, 3 and 10 μm. For these concentrations, all of the antagonists produced a rightward shift of the CCRC for carbachol and pA2 values were 9.2, 8.8, 6.7 and 6.3, respectively.

In conclusion, the present study indicates that muscarinic stimulation of rat isolated tracheal smooth muscle cells induces [Ca2+]i oscillations. The occurrence of these oscillations depends on the graded amplitude of the first [Ca2+]i rise and their frequency may play a role in the amplitude of the mechanical activity in response to muscarinic cholinoceptor activation. Both the [Ca2+]i and the contractile responses are primarily dependent on activation of the M3 receptor subtype.

British Journal of Pharmacology (1997) 120, 1294–1301; doi:10.1038/sj.bjp.0701061
Investigation of role for oxidant stress in vascular tolerance development to glyceryl trinitrate in vitro
Authors: Laight David W. , Carrier Martin J. , Änggård Erik E.
Published: August 2006

The role of reactive oxygen species (ROS) during the development of vascular cellular tolerance to glyceryl trinitrate (GTN), was studied in the rat isolated aorta.

Nitrate tolerance induced by a 30 min incubation with GTN (30 or 100 μm) in vitro, was not affected by pretreatment with the intracellular superoxide anion scavenger, tiron (10 mm), or the intracellular scavenger of peroxynitrite anion and hydroxyl radical, dimethylsulphoxide (DMSO, 0.2% v v−1). In contrast, pretreatment with the intracellular sulphydryl donor, N‐acetyl‐l‐cysteine (NAC, 1 mm), significantly attenuated GTN‐induced tolerance.

Pretreatment with a putative inhibitor of oxidant stress‐mediated, transcription factor NF‐κ B activation, pyrrolidine dithiocarbamate (PDTC, 50 μm), an inhibitor of gene activation by NF‐κB, dexamethasone (1 μm) or an inhibitor of protein synthesis, cycloheximide (10 μm), failed to affect tolerance development to GTN.

Pretreatment with DMSO (0.2% v v−1) or PDTC (50 μm) depressed non‐tolerant vasorelaxation to GTN (1 nm1 μm) per se.

Tiron (10 mm) abolished the reduction of ferricytochrome c by a superoxide anion generating system, assessed photometrically in vitro. In contrast, DMSO (0.2% v v−1), NAC (1 mm) and PDTC (50 μm) were without effect.

Our data suggests that neither oxidant stress nor nuclear activation, is important in the development of cellular tolerance to GTN in rat isolated aortic smooth muscle.

British Journal of Pharmacology (1997) 120, 1477–1482; doi:10.1038/sj.bjp.0701078
Synergistic interaction between endothelium‐derived NO and prostacyclin in pulmonary artery: potential role for K+ATP channels
Authors: Gambone Linda M , Murray Paul A , Flavahan Nicholas A
Published: August 2006

The aim of the present study was to assess interactions between nitric oxide (NO) and prostacyclin (PGI2) during endothelium‐dependent relaxations evoked by bradykinin, calcium ionophore (A23187) and acetylcholine in canine isolated pulmonary artery.

Relaxations to low concentrations of bradykinin and A23187 were abolished by combined inhibition of NO‐synthase (by NΩ‐nitro‐L‐arginine methyl ester L‐NAME, 30 μM) and cyclo‐oxygenase (indomethacin, 10 μM), suggesting mediation by NO and PGI2. The individual contributions of NO and PGI2 to the dilator responses were quantified by use of areas above the separate indomethacin‐insensitive and L‐NAME‐insensitive components of the concentration‐effect curves, respectively. Individually, NO and PGI2 accounted for only 53±5% and 16±9% of total bradykinin‐induced relaxation, and 46±10% and 20±9% of total A23187‐induced relaxation, suggesting that NO and PGI2 acted synergistically to cause endothelium‐dependent relaxation.

Relaxation to low concentrations of acetylcholine was abolished by L‐NAME but not affected by indomethacin, suggesting the response was mediated solely by NO with no interaction from PGI2.

Glibenclamide (1 μM), an inhibitor of ATP‐sensitive potassium (K+ATP) channels, inhibited responses to bradykinin or A23187 but did not affect relaxations evoked by acetylcholine. Glibenclamide did not affect endothelium‐independent relaxations to PGI2 or the NO‐donor, 3‐morpholinosydnonimine (SIN‐1).

With bradykinin, glibenclamide attenuated total relaxation by 49±8%, but did not alter the individual NO and PGI2‐mediated components of the response. Glibenclamide abolished the synergistic interaction between endothelium‐derived NO and PGI2.

At high concentrations, bradykinin, A23187 or acetylcholine caused endothelium‐dependent relaxation that was insensitive to L‐NAME + indomethacin. With bradykinin or A23187, this component of relaxation was inhibited by glibenclamide, whereas with acetylcholine, glibenclamide had no effect.

The synergistic interaction between endothelium‐derived NO and PGI2 in canine pulmonary artery is mediated by activation of K+ATP channels, presumably by an endothelium‐derived hyperpolarizing factor (EDHF). The pattern of endothelial dilator mediators and the presence of this synergistic interaction is dependent on the nature of the endothelial stimulus.

British Journal of Pharmacology (1997) 121, 271–279; doi:10.1038/sj.bjp.0701082
Reduced responsiveness of rat mesenteric resistance artery smooth muscle to phenylephrine and calcium following myocardial infarction
Authors: Stassen Frank R. M. , Willemsen Maurice J. J. M. F. , Janssen Ger M. J. , Fazzi Gregorio E. , Schiffers Paul M. H. , Smits Jos F. M. , De Mey Jo G. R.
Published: August 2006

We evaluated responses of peripheral resistance arterial smooth muscle to α1‐adrenoceptor stimulation in a rat model of heart failure in relation to neurohumoral changes, wall structure, receptor density and cellular calcium handling.

Plasma samples and third order mesenteric artery side‐branches were obtained from Wistar rats after induction of left ventricular infarction (MI) or sham surgery. Vessels were denuded of endothelium, sympathectomized, depleted of neuropeptides, and mounted in a myograph for recording of isometric force development in response to calcium, agonist and high potassium. Also, the morphology of these preparations was determined. Separate vessel segments were used in radioligand binding assays with [3H]‐prazosin.

At 1 week after MI, circulating plasma levels of adrenaline, angiotensin II, atrial natriuretic factor (ANF) and vasopressin were significantly elevated. At 5 weeks only a significant elevation of ANF persisted.

At 5 weeks after MI, the structure of the vessels and responsiveness to high potassium or Bay K 8466 (10−6 mol l−1) were not modified. Yet, at this stage, sensitivity to phenylephrine was increased (pD2: 6.24±0.04 vs 5.98±0.04 for controls) while maximal contractile responses to phenylephrine in the presence of 2.5 mmol l−1 calcium (2.26±0.28 vs 3.53±0.34 N m−1) and the sensitivity to calcium in the presence of phenylephrine (pD2: 2.81±0.22 vs 3.74±0.16) were reduced. Responses to the agonist in calcium‐free solution and the calcium sensitivity in the presence of 125 mmol l−1 potassium or of phorbol myristate acetate (PMA, 10−6 mol l−1) were not altered.

At 5 weeks after MI, the density of prazosin binding sites was not reduced (4.04±1.40 vs 2.29±0.21 fmol μg−1 DNA in controls).

In conclusion, myocardial infarction leads in the rat to a reduction of contractile responses of mesenteric resistance arterial smooth muscle to α1‐adrenoceptor stimulation. This seems to involve impaired agonist‐stimulated calcium influx.

British Journal of Pharmacology (1997) 120, 1505–1512; doi:10.1038/sj.bjp.0701089
Effects of hypomagnesia on histamine H1 receptor‐mediated facilitation of NMDA responses
Authors: Payne Geoffrey W , Neuman Richard S
Published: August 2006

The ability of histamine to facilitate the N‐methyl‐D‐aspartate (NMDA) induced depolarization of cortical projection neurones was examined by use of grease‐gap recording.

Histamine (1 to 15 μM) reversibly facilitated the NMDA‐induced depolarization yielding a bell‐shaped concentration‐response relationship. The peak enhancement was 167% above the control at 10 μM histamine. Desensitization was present in 4 out of 5 slices on second exposure 40 min following the first exposure.

Histamine did not alter the depolarization induced by 10 μM kainate.

The histamine‐induced facilitation persisted in the presence of tetrodotoxin, but was reduced in a concentration‐dependent manner by diphenhydramine (IC50=7.6 nM). Cyproheptadine (10 nM) also reduced the facilitation, whereas ranitidine (200 nM) and thioperamide (10 nM) were ineffective in this regard.

Histamine (10 μM) facilitated the NMDA (25 μM)‐induced depolarization in nominally Mg2+‐free medium. The magnitude of the facilitation was smaller than that observed in Mg2+‐containing medium (17% above the control) and desensitization was not observed. This facilitation was not reduced by cyproheptadine (10 nM) or diphenhydramine (1 μM).

We conclude that histamine facilitates the NMDA depolarization at cortical neurones via two distinct mechanisms. One mechanism involves activation of the histamine H1 receptor and is sensitive to Mg2+. The second mechanism is independent of histamine cell surface receptor activation and may reflect a direct action of histamine at the NMDA receptor.

British Journal of Pharmacology (1997) 121, 199–204; doi:10.1038/sj.bjp.0701123
Comparison of the contractile effects and binding kinetics of endothelin‐1 and sarafotoxin S6b in rat isolated renal artery
Authors: Devadason Peter S S , Henry Peter J
Published: August 2006

To date, only two mammalian endothelin (ET) receptors, termed ETA and ETB, have been cloned, sequenced and characterized. However, several functional studies of isolated blood vessels suggest that ET‐1‐induced contractions may be mediated by multiple ETA receptors. In this study, the ETA receptors in renal arteries isolated from Wistar rats were characterized by isometric tension recording and radioligand binding techniques.

ET‐1, sarafotoxin S6b (StxS6b) and ET‐3 produced concentration‐dependent contraction with similar response maxima in endothelium‐denuded arteries, whereas the ETB receptor‐selective agonist StxS6c was inactive. ET‐1 and StxS6b were equipotent and 30 times more potent than ET‐3. This agonist profile, together with the findings that the ETA receptor‐selective antagonists, BQ‐123 and FR‐139317 caused concentration‐dependent, rightward shifts of the concentration‐effect curves to each agonist indicated that ET‐1‐induced contractions in rat renal artery were mediated via ETA receptors.

BQ‐123 and FR‐139317 were both significantly more potent inhibitors of contractions induced by StxS6b or ET‐3 than of responses to ET‐1, raising the possibility that a component of ET‐1‐induced contraction was mediated through atypical, BQ‐123 (or FR‐139317)‐insensitive ETA receptors. However, in competition binding studies, specific [125I]‐ET‐1 and [125I]‐StxS6b binding to rat renal artery sections was completely abolished by BQ‐123 in a manner consistent with an action at a single site. Thus, competition binding studies did not provide any supportive evidence of the existence of a BQ‐123‐insensitive ETA receptor.

Additional studies revealed marked differences in the kinetics of [125I]‐ET‐1 and [125I]‐StxS6b binding. Following a 3 h period of association of [125I]‐ET‐1 with its receptors, no significant dissociation of receptor‐bound [125I]‐ET‐1 was observed during a 4 h washout period. In stark contrast, dissociation studies revealed that specific [125I]‐StxS6b binding to ETA receptors was reversible (t0.5diss, 100 min). A series of association binding studies were also consistent with the specific binding of [125I]‐ET‐1 and [125I]‐StxS6b being irreversible and reversible processes, respectively.

Thus, differences in BQ‐123 potency against ET‐1 and StxS6b‐induced contractions in rat renal arteries might be due to differences in the kinetics of agonist binding, rather than due to the existence of atypical ETA receptors.

British Journal of Pharmacology (1997) 121, 253–263; doi:10.1038/sj.bjp.0701126
Actions of the anaesthetic Saffan on rat sympathetic preganglionic neurones in vitro
Authors: Nolan M F , Gibson I C , Logan S D
Published: August 2006

Whole‐cell patch‐clamp recordings were used to investigate the effects of the anaesthetic Saffan on the electrophysiological properties of sympathetic preganglionic neurones (SPNs) in rat spinal cord slices.

Saffan (1–54 μM) abolished or reduced the frequency of spontaneous action potential firing and abolished spontaneous, sub‐threshold membrane potential oscillations. Saffan caused dose‐dependent decreases in input resistance and depending upon the initial resting membrane potential, either a depolarization, a hyperpolarization or no change in membrane potential.

Responses to Saffan were blocked by the GABAA receptor antagonists bicuculline (5–20 μM) and picrotoxin (20 μM), but not by the glycine receptor antagonist strychnine (20 μM) indicating that they were mediated by GABAA receptors.

Changes in the properties of SPN action potentials were also observed. In the presence of Saffan the amplitude and duration of the action potential after‐hyperpolarization were reduced and larger depolarizations were required in order to evoke trains of action potentials.

To examine the effects of Saffan on electrotonic coupling between SPNs, experiments were performed with the Na+ channel blocker QX‐314 in the intracellular solution and antidromic oscillations were evoked by ventral root stimulation. Saffan failed to abolish antidromic oscillations, but reduced their amplitude and duration. This indicates that the abolition of spontaneous membrane potential oscillations was not a direct effect on the coupling between SPNs, but was a result of the abolition of spontaneous activity by Saffan.

The responses to Saffan occurred within the plasma concentration range of Saffan during anaesthesia, suggesting that the electrophysiological properties of SPNs may be altered during anaesthesia with Saffan. This would be expected to lead to changes in sympathetic tone and in the integration of sympathetic output.

British Journal of Pharmacology (1997) 121, 324–330; doi:10.1038/sj.bjp.0701127
Comparative direct electrophysiological effects of propofol on the conduction system and ionic channels of rabbit hearts
Authors: Wu Mei‐Hwan , Su Ming‐Jai , Siu‐Mun Sun Selma
Published: August 2006

Propofol, an intravenous anaesthetic agent, can affect cardiac conduction but the ionic mechanisms have not been well defined.

This study measured the direct effects of propofol on the cardiac conduction system by using intracardiac recording/stimulation in Langendorff‐perfused rabbit hearts. The underlying ionic mechanism was elucidated by using the whole‐cell voltage clamp on rabbit isolated atrial and ventricular myocytes.

Propofol prolonged significantly the AV conduction (AH) interval at a clinically relevant concentration (3 μM). This AH interval prolongation was dose‐dependent (3 to 100 μM). At higher concentrations, the AV nodal Wenckebach cycle length and its refractory period were also prolonged (10 to 100 μM). In addition, the conduction through the His‐Purkinje system (HV interval) and the atrial tissue (SA interval), as well as the spontaneous cycle length, were lengthened dose‐dependently (30 to 100 μM).

In isolated ventricular myocytes, Na current was decreased dose‐dependently by propofol. In part this was due to a negative‐shift of the steady‐state voltage‐dependent inactivation and a slowed rate of recovery from inactivation. TheINasuppression by propofol was frequency‐dependent. Propofol also blocked theICa. The ED50for peak current inhibition was 6.9±0.9 (n=6) and 8.3±1.5 μM(n=7) forINaandICa, respectively.

The transient outward potassium current (Ito) of atrial myocytes was suppressed with an ED50of 5.7±0.8 μM(n=11), which was only partly caused by a left‐shift of the steady‐state inactivation. The inward rectifier K current (IK1) of the ventricular cells was reduced somewhat by propofol.

In summary, propofol can cause direct dromotropic and chronotropic effects on the cardiac conduction system, especially the atrioventricular node. These changes can be attributed, at least in part, to its direct dose‐dependent suppression of the cardiacICa,INaandIto. Special concerns in the use of propofol anaesthesia for cardiac patients and the therapeutic antiarrythmic potential of propofol‐like compounds are addressed.

British Journal of Pharmacology(1997)121, 617–624; doi:10.1038/sj.bjp.0701155
Differential activation of the epithelial and smooth muscle NK1 receptors by synthetic tachykinin agonists in guinea‐pig trachea
Authors: Figini Michela , Emanueli Costanza , Bertrand Claude , Sicuteri Riccardo , Regoli Domenico , Geppetti Pierangelo
Published: August 2006

The presence of tachykinin NK1 receptors have been shown in the epithelium and smooth muscle of guinea‐pig airways. Previous data showed that substance P (SP), and the NK1 receptor agonist, [Sar9, Met (O2)11]‐SP, relax guinea‐pig tracheal tube preparations by stimulation of epithelial NK1 receptors and via nitric oxide (NO) release. However, the selective tachykinin NK1 receptor agonist, septide, was unable to produce this effect. The aim of the present study was to investigate the ability of a series of SP analogues to stimulate NK1 receptors of guinea‐pig airway epithelium.

Isometric tension was recorded in isolated tracheal tube preparations in which compounds were administered intraluminally in the presence of phosphoramidon, indomethacin (both 1 μM) and the tachykinin NK2 receptor antagonist, SR 48,968 ((S)‐N‐methyl N‐(4‐acetyl‐amino‐4‐phenylpiperidino)‐2‐(3,4‐dichlorophenyl)butyl)benzamide) (0.1 μM). Cumulative concentration‐response curves were obtained in preparations under resting tone or in preparations precontracted with acetylcholine (ACh, 10 μM).

Contractile responses to low concentrations (0.1–10 nM) of substance P (SP) and the selective agonist of NK1 receptors, [Pro9]‐SP, in non precontracted tracheae were higher in preparations pretreated with the NO‐synthase inhibitor, NG‐monomethyl L‐arginine (L‐NMMA, 100 μM) than in preparations pretreated with its inactive enantiomer D‐NMMA (100 μM). Tracheal tube preparations precontracted with ACh and pretreated with D‐NMMA were relaxed by low concentrations of SP and [Pro9]‐SP (0.1–10 nM). In contrast, after pretreatment with L‐NMMA, SP and [Pro9]‐SP contracted tracheae at all the concentrations tested.

Concentration‐response curves to the NK1 receptor agonists, SP methyl ester, [Apa9–10]‐SP and [pGlu6] SP (6–11) obtained in non‐precontracted tracheae were similar in the presence of either D‐NMMA or L‐NMMA. SP methyl ester, [Apa9–10]‐SP and [pGlu6] SP (6–11) did not produce any relaxation, but instead, cause contractions in tracheal tube preparations precontracted with ACh and pretreated with D‐NMMA. Concentration‐response curves produced by all these agonists were similar in preparations precontracted with ACh and pretreated with L‐NMMA or D‐NMMA.

In guinea‐pig tracheal tube preparations two groups of NK1 receptor agonists can be distinguished: one group, including [Pro9]‐SP, stimulator epithelial NK1 receptors, the other group, including SP methyl ester, [Apa9–10]‐SP and [pGlu6] SP (6–11), does not. One possible explanation for these findings and for the existence of compounds with a peculiar ‘septide‐like’ pharmacological profile in the guinea‐pig trachea could be the recently proposed phenomenon referred to as ‘agonist‐directed receptor trafficking’.

British Journal of Pharmacology (1997) 121, 773–781; doi:10.1038/sj.bjp.0701188
Involvement of nitric oxide synthesis in hepatic perturbations induced in rats by a necrogenic dose of thioacetamide
Authors: Díez‐Fernández Carmen , Sanz Nuria , Boscá Lisardo , Hortelano Sonsoles , Cascales María
Published: August 2006

The biological actions of nitric oxide (NO), a highly diffusible and short‐lived radical, range from signal transduction to cytotoxicity. The present study investigated whether NO is released in the course of liver necrosis and regeneration induced by a single necrogenic dose of thioacetamide (6.6 mmol kg−1 body wt) to rats. Samples of liver were obtained at 0, 3, 12, 24, 48, 72 and 96 h after thioacetamide administration.

Inducible nitric oxide synthase (iNOS) activity was determined in purified liver homogenates and a sharp 6 fold increase (P<0.001) in iNOS activity was recorded at 48 h of intoxication, followed by a slight but progressive increase at 72 and 96 h. Changes in the expression of iNOS, as detected by its mRNA levels, were parallel to the NOS enzyme activity. Hepatocyte NO synthesis showed a progressive increase at 24, 48 and 72 h, to 8 (P<0.001), 13 (P<0.001) and 13 (P<0.001) times the initial values, respectively.

In isolated Kupffer cells, where initial NO release was ten fold higher than in hepatocytes, a progressive increase was detected from 48 h which reached two fold of initial at 72 h of intoxication (192%, P<0.001). Hepatic cyclic GMP concentration did not change significantly. However, mitochondrial aconitase activity decreased markedly at 12 and 24 h of intoxication showing a sharp increase towards normal values at 48 h which was maintained at 72 and 96 h.

As protein kinase C (PKC) is one of the likely candidates to mediate iNOS expression, translocation (activation) of PKC was assayed in hepatocytes, and a significant two fold increase (P<0.001) between 48 and 96 h after thioacetamide intoxication was observed. When peritoneal macrophages from control rats were incubated with serum from thioacetamide‐treated rats, a sharp increase in NO release was detected with serum obtained at 48 h, reaching at 96 h a value four fold (P<0.001) that of the control.

These results suggest that iNOS activity and NO release play a role in the pathophysiological mechanisms that trigger post‐necrotic hepatocellular regeneration following thioacetamide administration.

British Journal of Pharmacology (1997) 121, 820–826; doi:10.1038/sj.bjp.0701191
(+)‐WAY 100135, a partial agonist, at native and recombinant 5‐HT1B/1D receptors
Authors: Davidson Colin , Ho Michael , Price Gary W. , Jones Brian J. , Stamford Jonathan A.
Published: August 2006

We have studied the effects of the purportedly selective 5‐HT1A receptor antagonist (+)‐WAY100135 on electrically stimulated 5‐hydroxytryptamine (5‐HT) efflux in the ventrolateral geniculate nucleus (vLGN), and its affinity at human 5‐HT1B and 5‐HT1D receptors stably expressed in Chinese hamster ovary (CHO) cells.

On short ‘pseudo single pulse’ stimulations (20 pulses at 100 Hz, 190 ms train duration), (+)‐WAY100135 (1.0 μM) decreased 5‐HT efflux in the vLGN to 68±8% of pre‐drug values (P<0.01). This decrease could be blocked by the 5‐HT1D/1B receptor antagonist GR 127935 (50 nM). Conversely, when long stimulations (20 pulses at 20 Hz, 950 ms train) were used, (+)‐WAY100135 had no effect on 5‐HT efflux (84±8% of pre‐drug values) although both methiothepin (200nM) and GR 127935 (50 nM) caused significant increases (to 175±18 and 130±10% of pre‐drug values, respectively).

Paroxetine (100 nM), the selective 5‐HT reuptake inhibitor, increased stimulated 5‐HT efflux and re‐uptake half‐life (to 145±18% and 649±121%, respectively) on pseudo single pulse stimulations. When (+)‐WAY 100135 was added in combination with the uptake blocker, the effect of paroxetine on stimulated 5‐HT efflux was potentiated to 282±48% (P<0.01) without further effect on the 5‐HT re‐uptake half‐life.

The affinity and intrinsic activity of (+)‐WAY 100135 were determined at recombinant human 5‐HT1B and 5‐HT1D receptors expressed in CHO cells, by use of radioligand binding and [35S]‐GTPγS binding. (+)‐WAY 100135 was a partial agonist at human 5‐HT1B and 5‐HT1D receptors with moderately high affinity for 5‐HT1D receptors (pEC50=7.61).

In conclusion, (+)‐WAY 100135 was found to be not a selective 5‐HT1A autoreceptor antagonist but may act as a partial agonist at the 5‐HT1B/1D receptor, displaying agonist or antagonist properties depending on the stimulation protocol used and the resultant 5‐HT ‘tone’ at the receptor.

British Journal of Pharmacology (1997) 121, 737–742; doi:10.1038/sj.bjp.0701197
The mechanisms of the relaxation induced by vasoactive intestinal peptide in the porcine coronary artery
Authors: Kawasaki Junya , Kobayashi Sei , Miyagi Yasushi , Nishimura Junji , Fujishima Masatoshi , Kanaide Hideo
Published: August 2006

This study was designed to investigate the mechanism of the relaxation induced by vasoactive intestinal peptide (VIP) in medial strips of the porcine coronary artery, by determining the effect on the cytosolic Ca2+ concentration ([Ca2+]i), the [Ca2+]i‐force relation and the involvement of G‐protein.

Front‐surface fluorometry of fura‐2 revealed that U46619, a thromboxane A2 analogue, and the high K+‐depolarization induced increases in both the [Ca2+]i and force of the medial strips. At a steady state of contraction, the extent of an increase in [Ca2+]i induced by 100 nM U46619 was similar to that induced by 30 mM K+‐depolarization. VIP concentration‐dependently (1 nM–1 μM) induced transient decreases in both the [Ca2+]i and force of the medial strips precontracted with 100 nM U46619. The decreases in the [Ca2+]i and force induced by VIP during the contraction with U46619 were much greater than those with 30 mM K+‐depolarization.

The VIP‐induced decreases in the [Ca2+]i and force were attenuated by K+ channel blockers such as tetrabutylammonium (TBA: non‐selective K+ channel blocker), charybdotoxin (large conductance Ca2+‐activated K+ channel blocker), and 4‐aminopyridine (4‐AP: voltage‐dependent K+ channel blocker). However, neither glibenclamide (ATP‐sensitive K+ channel blocker) nor apamin (small conductance Ca2+‐activated K+ channel blocker) had any significant inhibitory effect.

In the 30 mM K+‐depolarized strips, pretreatment with thapsigargin, a specific Ca2+‐ATPase inhibitor of the Ca2+ store sites, completely abolished the VIP‐induced decrease in [Ca2+]i, but partially attenuated the VIP‐induced decrease in force.

VIP shifted the [Ca2+]i‐force relation of the U46619‐induced contractions to the right in a concentration‐dependent manner. In the α‐toxin‐permeabilized strips, VIP decreased the force development at a constant [Ca2+]i level (pCa=6.5) in a GTP‐dependent manner, which was antagonized by guanosine‐5′‐O‐(β‐thiodiphosphate) (GDPβS).

We thus conclude that VIP relaxes the coronary artery via three mechanisms: (1) a decrease in [Ca2+]i by inhibiting the Ca2+ influx presumably through the membrane hyperpolarization mediated by the activation of the large conductance Ca2+‐activated (charybdotoxin‐sensitive) K+ channels and voltage‐dependent (4‐AP‐sensitive) K+ channels; (2) a decrease in [Ca2+]i by sequestrating cytosolic Ca2+ into thapsigargin‐sensitive Ca2+ store sites; and (3) a decrease in the Ca2+‐sensitivity of the contractile apparatus through the activation of G‐protein.
Inhibition by nociceptin of neurogenic inflammation and the release of SP and CGRP from sensory nerve terminals
Authors: Helyes Zsuzsanna , Németh József , Pintér Erika , Szolcsányi János
Published: August 2006

Pretreatment with the novel neuropeptide nociceptin (20 μg kg−1, i.p.) caused an inhibition of plasma extravasation evoked by antidromic stimulation of the saphenous nerve or by topical application of 1% mustard oil on the skin of the acutely denervated hindleg of the rat. In contrast, it did not affect non‐neurogenic inflammation evoked by s.c. injection of bradykinin after chronic denervation. Release of substance P (SP) and calcitonin gene‐related peptide (CGRP) from rat isolated tracheae in response to electrical field stimulation was diminished by nociceptin (100 nM). It is concluded that nociceptin inhibits the release of sensory neuropeptides from terminals of nociceptive neurones.

British Journal of Pharmacology (1997) 121, 613–615; doi:10.1038/sj.bjp.0701209
Nitric oxide‐dependent and ‐independent vascular hyporeactivity in mesenteric arteries of portal hypertensive rats
Authors: Heinemann Akos , Wachter Christof H , Holzer Peter , Fickert Peter , Stauber Rudolf E
Published: August 2006

Increased production of nitric oxide (NO) has been suggested to underlie both the vascular hyporeactivity to vasoconstrictors and the splanchnic vasodilatation seen in portal hypertension. This study assessed the role of NO in the vasoconstrictor hyporeactivity of portal vein‐ligated (PVL) rats in isolated and in situ perfused mesenteric arterial beds.

Isolated perfused mesenteric arteries of PVL rats were significantly less reactive to noradrenaline (NA), methoxamine (METH), arginine vasopressin (AVP) and endothelin‐1 (ET‐1) than those from sham‐operated (Sham) rats.

Blockade of NO synthesis with NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM) in isolated perfused mesenteric arteries from PVL rats restored the reactivity to bolus injections of AVP and ET‐1, but had little effect on the hyporeactivity to NA or METH. Cyclo‐oxygenase inhibition with indomethacin (5 μM) likewise did not restore reactivity to METH of isolated perfused mesenteric arteries of PVL rats.

The hyporeactivity to METH seen in isolated perfused mesenteric arteries from PVL rats was reduced by low concentrations of AVP (20 nM) or ET‐1 (1 nM) which per se caused only a slight increase in perfusion pressure. When L‐NAME (100 μM) was combined with AVP (20 nM) or ET‐1 (1 nM), respectively, reactivity to METH of isolated perfused mesenteric arteries of PVL rats was restored to the level seen in Sham rats. These effects of AVP and ET‐1 were not mimicked by precontracting the vessels with 5‐hydroxytryptamine (5 μM).

The differential effects of L‐NAME and AVP on the hyporesponsiveness to methoxamine and AVP were corroborated by experiments performed with the in situ perfused mesenteric vascular bed preparation.

These data indicate that both NO‐dependent and NO‐independent mechanisms are involved in the vasoconstrictor hyporesponsiveness of mesenteric arteries from portal hypertensive rats. The hyporeactivity to AVP and ET‐1 is mediated by NO whereas the reduced responsiveness to adrenoceptor agonists appears to be predominantly NO‐independent. AVP and ET‐1, in addition, seem to inhibit the NO‐independent mechanism of vascular hyporeactivity, since the hyporesponsiveness to METH was reduced in the presence of AVP or ET‐1 and abolished by the combination of these peptides with L‐NAME.
Induction of bradykinin B1 receptors in rat colonic epithelium
Authors: Teather Sarah , Cuthbert Alan W
Published: August 2006

Des‐Arg9 bradykinin (DAB), a classical B1‐kinin receptor agonist was without effect when applied to the basolateral surface of rat isolated colon epithelium. Three hours after tissues were isolated DAB caused, after a delay of up to 2 min, a maintained increase of short circuit current (SCC).

The SCC increase in colonic epithelia, mounted in vitro for three hours, caused by DAB was due to electrogenic chloride secretion as the current increase was reversed by frusemide and did not occur in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. The EC50 for DAB was approximately 50 nM.

An inhibitor of transcription (actinomycin D) and of translation (cycloheximide) prevented the appearance of DAB sensitivity without affecting the responses to another secretagogue (forskolin).

The classical B1‐kinin receptor antagonist, Leu8‐des‐Arg9 bradykinin, was shown to be an agonist in rat colon epithelium. Other B1‐kinin receptor antagonists (des‐Arg10‐Hoe 140 and R‐715) inhibited the responses to DAB in ‘aged’ colonic epithelia, and the inhibition was easily surmounted by increasing the concentration of DAB.

Response to DAB did not appear to involve to any significant extent, the formation of prostaglandins, leukotrienes, histamine or nitric oxide. Furthermore, no neuronal involvement was apparent in the stripped colonic preparations. The responses to DAB were not significantly different in epithelia taken from different parts of the distal colon.

The differences between the responses of the colonic epithelium to B1‐ and B2‐kinin receptor agonists are discussed.
Involvement of calyculin A inhibitable protein phosphatases in the cyclic AMP signal transduction pathway of mouse corticotroph tumour (AtT20) cells
Authors: Antaraki A , Ang K L , Antoni F A
Published: August 2006

The role of non‐calcineurin protein phosphatases in the cyclic AMP signal transduction pathway was examined in mouse pituitary corticotroph tumour (AtT20) cells.

Blockers of protein phosphatases, calyculin A and okadaic acid, were applied in AtT20 cells depleted of rapidly mobilizable pools of intracellular calcium and activated by various cyclic AMP generating agonists. Inhibitors of cyclic nucleotide phosphodiesterases were present throughout. The accumulation of cyclic AMP was monitored by radioimmunoassay, phosphodiesterase activity in cell homogenates was measured by radiometric assay.

Neither calyculin A nor okadaic acid altered basal cyclic AMP levels but cyclic AMP formation induced by 41 amino acid residue corticotrophin releasing‐factor (CRF) was strongly inhibited (up to 80%). 1‐Norokadaone was inactive. Similar data were also obtained when isoprenaline or pituitary adenylate cyclase activating peptide1–38 were used as agonists.

Pertussis toxin did not modify the inhibition of CRF‐induced cyclic AMP production by calyculin A.

Pretreatment with calyculin A completely prevented the stimulation of cyclic AMP formation by cholera toxin even in the presence of 0.5 mM isobutylmethylxanthine (IBMX) and 0.1 mM rolipram. Cholera toxin mediated ADP‐ribosylation of the 45K and 52K molecular weight Gsα isoforms in membranes from calyculin A‐pretreated cells was enhanced to 150–200% when compared with controls.

Cholera toxin‐induced cyclic AMP was reduced by calyculin A within 10 min when calyculin A was applied after a 90 min pretreatment with cholera toxin. Under these conditions the effect of calyculin A could be blocked by the combination of 0.5 mM IBMX and 0.1 mM rolipram, but not by 0.5 mM IBMX alone.

Phosphodiesterase activity in AtT20 cell homogenates showed a significant, 2.7 fold increase after treatment with calyculin A. In control cells phosphodiesterase activity was blocked by 80% in the presence of IBMX (0.5 mM), or IBMX plus rolipram (0.1 mM). In calyculin A‐treated cells phosphodiesterase activity was also strongly inhibited by IBMX, but because of the stimulating effect of calyculin A, the activity remaining was still 55% of that found in control homogenates. This activity was reduced to 5% of control by using IBMX and rolipram in combination. Assay of phosphodiesterase in Ca2+ free conditions showed that calyculin A markedly increases the activity of rolipram sensitive (type 4) phosphodiesterase.

Taken together, blockers of protein phosphatases (PPases) impaired signal transduction through Gs‐mediated pathways and activated cyclic AMP degrading phosphodiesterase(s), indicating that PPases 1 and/or 2A are essential for agonist‐mediated regulation of cyclic AMP levels in AtT20 cells, and are thus important in maintaining the secretory phenotype of the cells.
NMDA receptor‐mediated pilocarpine‐induced seizures: characterization in freely moving rats by microdialysis
Authors: Smolders Ilse , Khan Ghous M , Manil Jacqueline , Ebinger Guy , Michotte Yvette
Published: August 2006

Pilocarpine administration has been used as an animal model for temporal lobe epilepsy since it produces several morphological and synaptic features in common with human complex partial seizures. Little is known about changes in extracellular neurotransmitter concentrations during the seizures provoked by pilocarpine, a non‐selective muscarinic agonist.

Focally evoked pilocarpine‐induced seizures in freely moving rats were provoked by intrahippocampal pilocarpine (10mm for 40min at a flow rate of 2μl min−1) administration via a microdialysis probe. Concomitant changes in extracellular hippocampal glutamate, γ‐aminobutyric acid (GABA) and dopamine levels were monitored and simultaneous electrocorticography was performed. The animal model was characterized by intrahippocampal perfusion with the muscarinic receptor antagonist atropine (20mm), the sodium channel blocker tetrodotoxin (1μm) and the N‐methyl‐d‐aspartate (NMDA) receptor antagonist MK‐801 (dizocilpine maleate, 100μm). The effectiveness of locally (600μm) or systemically (10mg kg−1 day−1) applied lamotrigine against the pilocarpine‐induced convulsions was evaluated.

Pilocarpine initially decreased extracellular hippocampal glutamate and GABA levels. During the subsequent pilocarpine‐induced limbic convulsions extracellular glutamate, GABA and dopamine concentrations in hippocampus were significantly increased. Atropine blocked all changes in extracellular transmitter levels during and after co‐administration of pilocarpine. All pilocarpine‐induced increases were completely prevented by simultaneous tetrodotoxin perfusion. Intrahippocampal administration of MK‐801 and lamotrigine resulted in an elevation of hippocampal dopamine levels and protected the rats from the pilocarpine‐induced seizures. Pilocarpine‐induced convulsions developed in the rats which received lamotrigine perorally.

Pilocarpine‐induced seizures are initiated via muscarinic receptors and further mediated via NMDA receptors. Sustained increases in extracellular glutamate levels after pilocarpine perfusion are related to the limbic seizures. These are arguments in favour of earlier described NMDA receptor‐mediated excitotoxicity. Hippocampal dopamine release may be functionally important in epileptogenesis and may participate in the anticonvulsant effects of MK‐801 and lamotrigine. The pilocarpine‐stimulated hippocampal GABA, glutamate and dopamine levels reflect neuronal vesicular release.
Differential effects of the neuropeptide galanin on striatal acetylcholine release in anaesthetized and awake rats
Authors: Antoniou K , Kehr J , Snitt K , Ögren S O
Published: August 2006

In the present study the mechanisms were examined by which the neuropeptide galanin modulates the extracellular concentrations of striatal acetylcholine (ACh) in enflurane anaesthetized and in freely moving male rats by use of in vivo microdialysis and high performance liquid chromatography.

The perfusion of galanin through the microdialysis probe (0.3nmolμl−1, flow rate: 2μlmin−1) caused a statistically significant increase in the basal striatal ACh levels in anaesthetized but a decrease in awake animals. No significant effect was revealed after a low dose (0.1nmolμl−1, flow rate: 2μlmin−1) of galanin perfusion. Both the stimulating and inhibitory effects of galanin on basal ACh release were reversible.

The muscarinic antagonist scopolamine (0.1mgkg−1, subcutaneously (s.c.)) caused a significant increase in ACh release in both anaesthetized and awake animals.

The combination of galanin plus scopolamine attenuated the stimulant effect on ACh release caused by scopolamine alone in awake animals.

The putative galanin receptor antagonist M35 at 0.3nmolμl−1 but not at 0.1nmolμl−1 caused a significant reduction (20%) in ACh release, supporting the view that M35 at higher concentrations behaves as a partial agonist at the galanin receptor. When M35 (0.1nmolμl−1) was co‐infused with galanin (0.3nmolμl−1) the galanin‐evoked decrease in ACh release was completely blocked.

Taken together, these results indicate that galanin affects basal ACh release via stimulation of galanin receptors within the striatum. The mechanism involved is dependent on the anaesthesia procedure which may act via enhancement of γ‐aminobutyric acidA (GABAA) mediated transmission within striatal and/or output neurones. In addition, anaesthesia may also decrease the activity of glutamatergic striatal afferents. The results with M35 indicate that the role of galanin perfused in striatum is permissive in the normal rat. Furthermore, galanin is a potent inhibitory modulator of basal ACh release also in the striatum, as recently was shown in the ventral hippocampus in awake animals.
Differential antinociceptive effects of spinal opioids on foot withdrawal responses evoked by C fibre or Aδ nociceptor activation
Authors: Lu Y , Pirec V , Yeomans D C
Published: August 2006

Intrathecal application of μ, δ, and κ opioids attenuate responses on several tests of animal nociception. However, the potency of these opioids differ depending on which tests were used. One factor contributing to these discrepancies is that different types of noxious stimuli activate different sets of nociceptor types, which may be differentially sensitive to opiate inhibition. To examine this hypothesis, we used a recently developed behavioural test which allows for differential assessment of nociception evoked by the activation of myelinated (Aδ) and unmyelinated C thermonociceptors.

Administration of a κ‐selective agonist was ineffective on either type of response. δ1 drugs were slightly more potent on C fibre‐mediated responses than on Aδ‐mediated responses.

Intrathecal μ and δ2 drugs were antinociceptive on both Aδ and C nociceptor‐mediated responses. However, unlike the δ1 effects, the dose‐response curves for μ and δ2 drugs were significantly more steep for Aδ than for C fibre‐mediated responses, potentially indicating differences in the mechanisms by which the drugs act on these 2 response types.
Involvement of vasoactive intestinal polypeptide in nicotine‐induced relaxation of the rat gastric fundus
Authors: Curròi Diego , Preziosi Paolo
Published: August 2006

Nicotine‐induced relaxation and release of vasoactive intestinal polypeptide (VIP)‐ and peptide histidine isoleucine (PHI)‐like immunoreactivity (LI) were measured in longitudinal muscle strips from the rat gastric fundus.

Under non‐cholinergic conditions (0.3μm atropine), nicotine (3–300 μm) produced concentration‐dependent relaxations of the 5‐hydroxytryptamine (3μm)‐precontracted strips. Under non‐adrenergic non‐cholinergic (NANC) conditions (0.3μm atropine+1μm phentolamine+1μm nadolol), relaxations induced by sub‐maximal nicotine concentrations (10 and 30μm) were significantly smaller, while that produced by the highest concentration used (300μm) was similar to that seen under non‐cholinergic conditions.

Re‐exposure to the same nicotine concentration 1h later induced smaller relaxations, indicating desensitization. The reductions seen in the second responses were proportional to the concentration used.

Under non‐cholinergic conditions, the relaxant response to 30μm nicotine was abolished by hexamethonium (100μm) and significantly reduced by tetrodotoxin (TTX, 3μm). The TTX‐resistant component was not observed under NANC conditions.

NANC relaxation induced by 30μm nicotine was significantly reduced by a specific anti‐VIP serum (approximately 35% less than that seen with normal rabbit serum).

Nicotine (30–300μm) caused significant, concentration‐dependent increases in the outflow of VIP‐ and PHI‐LI from the strips; these effects were also diminished with re‐exposure. The increases in both types of immunoreactivity evoked by nicotine (300μm) were abolished by hexamethonium (300μm), TTX (3μm) and a calcium‐free medium.

These findings indicate that VIP and possibly PHI are involved in NANC relaxation of the rat gastric fundus induced by nicotine.
Tolerance to μ‐opioid agonists in human neuroblastoma SH‐SY5Y cells as determined by changes in guanosine‐5′‐O‐(3‐[35S]‐thio)triphosphate binding
Authors: Elliott Jackie , Guo Li , Traynor John R
Published: August 2006

The agonist action of morphine on membranes prepared from human neuroblastoma SH‐SY5Y cells was measured by an increase in the binding of the GTP analogue [35S]‐GTPγS. Morphine increased the binding of [35S]‐GTPγS to SH‐SY5Y cell membranes by 30 fmol mg−1 protein with an EC50 value of 76±10 nM.

Incubation of SH‐SY5Y cells with 10 μM morphine for 48 h caused a tolerance to morphine manifested by a 2.5 fold shift to the right in the EC50 value with a 31±6% decrease in the maximum stimulation of [35 S]‐GTPγS binding. The response caused by the partial agonist pentazocine was reduced to a greater extent.

Chronic treatment of the cells with the more efficacious μ‐ligand [D‐Ala2, MePhe4, Gly‐ol5]enkephalin (DAMGO, 10 μM) for 48 h afforded a greater effect than treatment with morphine. The maximal agonist effect of morphine was reduced to 58.9±6% of that seen in control cells while the maximal effect of DAMGO was reduced to 62.8±4%. There was a complete loss of agonist activity for pentazocine.

The development of tolerance was complete within 24 h and was blocked by naloxone and by the nonselective protein kinase inhibitor H7, but not by the putative β‐adrenoceptor kinase (β‐ARK) inhibitor suramin.

The observed tolerance effect was accompanied by a down‐regulation of μ‐opioid receptors determined by a decrease in the maximal binding capacity for the opioid antagonist [3H]‐diprenorphine of 66±4%, but with no change in binding affinity. Binding of the agonist [3H]‐DAMGO was similarly reduced.

The modulation of [35S]‐GTPγS binding in SH‐SY5Y cell membranes by opioids provides a simple method for the study of opioid tolerance at a site early in the signal transduction cascade.