bluuebunny/crossref_metadata_2025 huggingface dataset:
Virus‐induced airway hyperresponsiveness in the guinea‐pig: possible involvement of histamine and inflammatory cells
Authors: Folkerts Gert , De Clerck Fred , Reijnart Ivonne , Span Paul , Nijkamp Frans P.
Published: July 2012

Guinea‐pig tracheal contractions by histamine and by the cholinoceptor agonist, arecoline, are significantly enhanced (30% and 20%, respectively), 96 h after intra‐tracheal inoculation with Parainfluenza‐3 (PI‐3) virus.

The airway hyperresponsiveness in animals inoculated with virus coincides with a significant increase in the number of broncho‐alveolar cells (82%), and in the albumin concentration (121%) in lung lavage fluid, relative to values obtained in guinea‐pigs challenged with control solution.

The chemiluminescence production by isolated broncho‐alveolar cells, obtained from virus‐infected guinea‐pigs 96 h after inoculation stimulated with PI‐3 virus in vitro, is significantly reduced by 42% relative to broncho‐alveolar cells obtained from animals inoculated with control solution. This diminution was not specific for stimulation by PI‐3 virus since the chemiluminescence production was also significantly reduced by 30% in response to zymosan.

Pretreatment of the guinea‐pigs with the anti‐allergic drugs, oxatomide (2.5 mg kg−1) or nedocromil (2.5 mg kg−1), or the specific H1‐histamine receptor antagonist, levocabastine (0.25 mg kg−1), administered intra‐peritoneally twice a day for five successive days, inhibits the virus‐induced airway hyperresponsiveness, suppresses the influx of broncho‐alveolar cells and increase in albumin content, and corrects the reduced chemiluminescence production by broncho‐alveolar cells in response to zymosan.

In contrast, the cyclo‐oxygenase inhibitor, suprofen (5.0 mg kg−1), the 5‐HT2 receptor antagonist, ketanserin (0.63 mg kg−1), or the Ca2+ overload blocker, flunarizine (2.5 mg kg−1) do not modify the above mentioned processes.

The platelet‐activating factor receptor antagonist, WEB 2170 (10 mg kg−1), reduces virus‐induced airway hyperresponsiveness and influx of broncho‐alveolar cells into the lungs but does not attenuate the increase of albumin in the bronchial lavage fluid.

Guinea‐pigs nebulized with histamine, twice a day (30 min) during 4 successive days, do not demonstrate an increased airway responsiveness, but instead show tachyphylaxis in response to histamine in vitro. In addition, no influx of inflammatory cells is found in these animals.

These results suggest that histamine does not directly increase the responsiveness of the guinea‐pig trachea; however, histamine may be involved in a cascade of events leading to airway hyperresponsiveness after a viral infection, a process that could be related to an influx and/or an activation of broncho‐alveolar cells after PI‐3 virus stimulation.
Porcine ventricular endocardial cells in culture express the inducible form of nitric oxide synthase
Authors: Smith Jerry A. , Radomski Marek W. , Schulz Richard , Moncada Salvador , Lewis Malcolm J.
Published: July 2012

We have investigated whether porcine endocardial cells in culture express the inducible, Ca2+‐independent form of nitric oxide (NO) synthase.

NO synthase activity in cytosolic extracts of endocardial cells was measured by estimation of the rate of formation of l‐[14C]‐citrulline from l‐[14C]‐arginine.

Treatment of the cells in culture with lipopolysaccharide or cytokines induced a Ca2+‐independent NO synthase activity in the cell cytosol. The combination of tumour necrosis factor (TNFα, 10 ng ml−1) and interleukin‐1β (IL‐1β, 10 ng ml−1) induced the greatest enzyme activity.

The increased Ca2+‐independent NO synthase activity following exposure to cytokines was parallelled by an increase in guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) levels in the endocardial cell cytosol.

Simultaneous addition of dexamethasone (0.01–1 μm) or cycloheximide (0.03–3 μm) inhibited in a concentration‐dependent manner TNFα‐ and IL‐1β‐induced expression of Ca2+‐independent NO synthase activity. Neither dexamethasone (1 μm) nor cycloheximide (3 μm) had any effect on the activity of the constitutive NO synthase.

The possible pathophysiological consequences of endocardial expression of the inducible NO synthase are discussed.
Nitric oxide synthase in pig lower urinary tract: immunohistochemistry, NADPH diaphorase histochemistry and functional effects
Authors: Persson Katarina , Alm Per , Johansson Kjell , Larsson Bengt , Andersson Karl‐Erik
Published: July 2012

The distribution and colocalization of nitric oxide synthase (NOS)‐like immunoreactivity and NADPH diaphorase activity in the pig lower urinary tract were investigated by immunohistochemical and histochemical staining techniques. Functional in vitro studies were performed to correlate the presence of NOS‐immunoreactivity/NADPH diaphorase staining with smooth muscle responses involving the l‐arginine/nitric oxide (NO) pathway.

NOS‐immunoreactivity and NADPH diaphorase activity were expressed in nerve trunks and fine nerve fibres in and/or around muscular bundles in the detrusor, trigone and urethra. Thin nerve fibres that dispersed within the muscle bundles were mainly found in the urethral/trigonal area, whereas such fibres were less common in the detrusor.

Almost all neuronal structures that were NOS‐immunolabelled were also stained for NADPH diaphorase. In contrast, the urothelium, which was intensively stained by the NADPH diaphorase technique, remained unstained by immunohistochemistry.

Electrical field stimulation of pig isolated trigonal and urethral preparations induced relaxations, which were inhibited by tetrodotoxin (1 μm) and NG‐nitro‐l‐arginine (l‐NOARG, 10 μm).

l‐Arginine (1 mm), but not d‐arginine, inhibited (25–30%) electrically evoked detrusor contractions. This inhibition was reversed by l‐NOARG (0.1 mm). l‐Arginine did not inhibit detrusor contractions in the presence of scopolamine (1 μm) and had no direct smooth muscle effects per se.

Acetylcholine (1 nm − 10 μm) caused concentration‐dependent relaxations of noradrenaline‐induced contractions in pig vesical arteries. Removal of the endothelium practically abolished the acetylcholine‐induced relaxation. Pretreatment with l‐NOARG (0.1 mm and 0.3 mm) caused a rightward shift of the concentration‐response curves to acetylcholine, but the maximal relaxation obtained was significantly reduced (to 65 ± 12%; n = 6; P < 0.05) only at 0.3 mml‐NOARG.

In vessel segments contracted with K+ (60 mm), acetylcholine induced concentration‐dependent relaxations. When the vessels were incubated with 0.3 mml‐NOARG and then contracted with K+ (60 mm) all relaxant responses to acetylcholine were abolished.

The presence of NO synthesizing enzyme in nerve fibres and the pharmacological evidence for NO‐mediated relaxation of the trigone and urethra suggest that NO or a NO‐related substance may have a role in inhibitory neurotransmission in these regions. In the detrusor, the presence of NO‐synthesizing enzyme in nerves can be demonstrated, but its functional importance is unclear. NO, as well as other endothelium‐derived factors seem to be involved in the endothelium‐dependent acetylcholine‐induced relaxation of pig vesical arteries.
Differential Class III and glibenclamide effects on action potential duration in guinea‐pig papillary muscle during normoxia and hypoxia/ischaemia
Authors: MacKenzie I. , Saville V.L. , Waterfall J.F.
Published: July 2012

Microelectrode recording techniques were used to study the effects of several potassium channel blockers which are considered to be Class III antiarrhythmic compounds. The effects of (+)‐sotalol, UK‐66,914, UK‐68,798 and E‐4031 on action potential duration (APD) were determined in guinea‐pig isolated papillary muscles. The compounds were evaluated under normoxic or hypoxic/ischaemic conditions at 36.5°C and compared to glibenclamide, which is considered to be a blocker of ATP‐dependent potassium channels. Prolongation of action potential duration at 90% repolarization (APD90) was taken as an indirect measure of potassium channel blockade.

Under normoxic conditions, the Class III compounds prolonged APD in a concentration‐dependent manner. According to EC15 values, the order of potency of the Class III compounds was found to be UK‐68,798 > E‐4031 > UK‐66,914 > (+)‐sotalol. Glibenclamide did not significantly prolong APD90 under normoxic conditions.

Perfusion with an experimental hypoxic or ischaemic bathing solution produced qualitatively similar effects on action potentials. Over a period of 20–25 min in either of the experimental solutions, there was a small decrease in action potential amplitude (APA) and a prominent shortening of APD. The ischaemic solution also depolarized the resting membrane potential by about 15 mV.

(+)‐Sotalol and UK‐66,914 did not reverse the shortening of APD induced by perfusion with hypoxic Krebs solution. High concentrations of glibenclamide (10 μm) and UK‐68,798 (30 and 60 μm) partially reversed the hypoxia‐shortened APD. Glibenclamide was more potent and exhibited a greater time‐dependent action than UK‐68,798.

During experimental ischaemia, the Class III compound E‐4031 (10 μm, n = 7) produced small, but significant, increases in the APD90 (11 ± 3 ms after 20 min) which were not clearly time‐dependent (14 ± 4 ms after 30 min). UK‐68,798 (10 μm) also produced a small, but insignificant, increase in APD90 (12 ± 6 ms at 20 min, n = 4). Higher concentrations of UK‐68,798 (30 and 60 μm, n = 4) did not produce a consistently significant increase in APD90 during ischaemia: significance was only attained after 20 min in the presence of 60 μm UK‐68,798 (24 ± 12 ms). However, in marked contrast to the effects of the Class III compounds, glibenclamide (10 μm) produced large time‐dependent increases in ischaemic APD90 (34 ± 11 ms at 7 min, n = 9) which were significant 15 min or more after drug addition (52 ± 12 ms at 20 min, n = 7; 74 ± 5 ms at 30 min, n = 6).

The present microelectrode data suggest that blockers of ATP‐dependent potassium channels, such as glibenclamide, might prove to be more effective than Class III compounds against ischaemia‐induced shortening of cardiac action potentials.
Reduction in the number of thromboxane receptors on human platelets after exposure to GR32191
Authors: Armstrong Roma A. , Humphrey P.P.A. , Lumley P.
Published: July 2012

Exposure of human resuspended platelets in vitro for 30 min to the potent thromboxane A2 (TP)‐receptor blocking drug GR32191, followed by its removal by dilution‐dissociation, reduced the degree of subsequent binding to 2 nm [3H]‐GR32191 by almost 50%. Exposure for longer periods (60 min) led to a further reduction. However, no change in the Kd of the radioligand was observed.

This effect of GR32191 could not be explained by persistent binding of drug to platelets since a dilution‐dissociation stage, designed to remove all drug, was included prior to measurement of binding.

Using an alternative TP‐receptor radioligand, [3H]‐SQ29,548, to monitor receptor number, a reduction in Bmax was observed after GR32191 pre‐treatment; the Kd value of the radioligand remained unchanged.

The effect was not a common property of TP‐receptor blocking drugs since pre‐exposure of platelets in vitro for 30 min to BM13.177 or SQ29,548 did not produce a fall in subsequent Bmax to [3H]‐SQ29,548.

While the mechanism behind this apparent down‐regulation of platelet TP‐receptor is unknown, it may explain the long duration of action of GR32191 upon platelets in man which persists in the absence of detectable drug in the plasma.
The effect of nitric oxide on the efficacy of synaptic transmission through the chick ciliary ganglion
Authors: Scott T.R.D. , Bennett M.R.
Published: July 2012

The effect of nitric oxide on the efficacy of synaptic transmission in the chick ciliary ganglion of post‐hatched birds has been determined by use of the size of the postganglionic compound action potential resulting from chemical transmission through the ganglion as a measure of synaptic efficacy.

Sodium nitroprusside (100 μm) increased the synaptic efficacy by an average 26%. This is likely to be due to its ability to release nitric oxide, as potassium ferricyanide (100 μm) did not cause a potentiation. Sodium azide (100 μm), shown in sympathetic ganglia to stimulate production of cyclic GMP, did not modulate synaptic efficacy significantly.

8‐Br‐cyclic‐GMP (100 μm) increased synaptic efficacy by an average 61%. The addition of 8‐Br‐cyclic‐AMP (100 μm) had less effect, increasing transmission by on average 46%.

The nitric oxide synthase blocker, NG‐nitro‐l‐arginine methyl ester (l‐NAME, 100 μm) was added prior to the tetanic stimulation of the preganglionic nerves at 30 Hz for 20 s, a procedure known to produce both post‐tetanic potentiation and long‐term potentiation of synaptic transmission through the ganglion. l‐NAME reduced the long‐term potentiation by an average of 47% but did not significantly change the post‐tetanic potentiation.

Following the brief application of 8‐Br‐cyclic AMP, 8‐Br‐cyclic GMP and sodium nitroprusside there was an enhancement of the efficacy of synaptic transmission that persisted after the withdrawal of the drugs. The maximum increase in synaptic efficacy following the brief addition of 8‐Br‐cyclic GMP was 116%, sodium nitroprusside was 110% and 8‐Br‐cyclic AMP was 126%.

These results suggest that nitric oxide modulates synaptic transmission through the ganglion by acting on an endogenous guanylate cyclase that produces cyclic GMP.
Pharmacological characterization of the dopamine receptor coupled to cyclic AMP formation expressed by rat mesenteric artery vascular smooth muscle cells in culture
Authors: Hall Alistair S. , Bryson Susan E. , Vaughan Peter F.T. , Ball Stephen G. , Balmforth Anthony J.
Published: July 2012

Mesenteric artery vascular smooth muscle cells derived from male Wistar rats and grown in culture were prelabelled with [3H]‐adenine and exposed to a range of dopamine receptor agonists and antagonists. Resultant [3H]‐cyclic AMP formation was determined and concentration‐effect curves constructed, in the presence of propranolol (10−6 m) and the phosphodiesterase inhibitor IBMX (5× 10−4 m).

Ka apparent values for D1/DA1 dopamine receptor agonists SKF 38393, fenoldopam, 6,7‐ADTN, and dopamine were 0.06, 0.59, 4.06 and 5.77 × 10−6 m respectively. Although fenoldopam and SKF 38393 were more potent than dopamine, they were partial agonists with efficacies, relative to dopamine of approximately 48% and 24% respectively. 6,7‐ADTN, in contrast, behaved as a full agonist.

Dopamine‐stimulated cyclic AMP formation was inhibited in a concentration‐dependent manner by the D1/DA1 dopamine receptor selective antagonists, SCH 23390 and cis‐flupenthixol (Ki values 0.53 and 36.1 × 10−1 m respectively). In contrast, the D2/DA2 dopamine receptor selective antagonists, domperidone and (−)‐sulpiride, were less potent (Ki values 2.06 and 5.82 × 10−6 m respectively). Furthermore, the stereoisomers of SCH 23390 and cis‐flupenthixol, SCH 23388 and trans‐flupenthixol, were at least two orders of magnitude less potent (Ki values 0.14 and 13.2 × 10−6 m respectively) indicating the stereoselective nature of this receptor.

Our results indicate that rat mesenteric artery vascular smooth muscle cells in culture express a dopamine receptor coupled to cyclic AMP formation, which has the pharmacological profile, characteristic of the D1 dopamine receptor subfamily.
Identification of both NK1 and NK2 receptors in guinea‐pig airways
Authors: McKee Katherine T. , Millar Lorna , Rodger Ian W. , Metters Kathleen M.
Published: July 2012

NK1 and NK2 receptors have been characterized in guinea‐pig lung membrane preparations by use of [125I‐Tyr8]‐substance P and [125I]‐neurokinin A binding assays in conjunction with tachykinin‐receptor selective agonists ([Sar9Met(O2)11]substance P for NK1 and [βAla8]neurokinin A (4–10) for NK2) and antagonists (CP‐99,994 for NK1 and SR48968 for NK2).

The presence of high affinity, G‐protein‐coupled NK1 receptors in guinea‐pig lung parenchymal membranes has been confirmed. The rank order of affinity for competing tachykinins was as predicted for an NK1 receptor: substance P = [Sar9Met(O2)11]substance P > substance P‐methyl ester = physalaemin > neurokinin A = neurokinin B >> [βAla8]neurokinin A (4–10). The novel NK1 antagonist CP‐99,994 has a Ki of 0.4 nm at this NK1 site.

In order to characterize [125I]‐neurokinin A binding to guinea‐pig lung, the number of [125I]‐neurokinin A specific binding sites was increased 3–4 fold by purification of the parenchymal membranes over discontinuous sucrose gradients. The rank order of affinity determined for NK1‐ and NK2‐receptor agonists and antagonists in competition for these sites showed that the majority (80%) of [125I]‐neurokinin A specific binding was also to the NK1 receptor.

Under conditions where the guinea‐pig lung parenchymal NK1 receptor was fully occupied by a saturating concentration of either [Sar9Met(O2)11]substance P (1 μm) or CP‐99,994 (2.7 μm), residual [125I]‐neurokinin A specific binding was inhibited in a concentration‐dependent manner by both [βAla8]neurokinin A and SR48968. This result shows that the NK2 receptor is also present in these preparations.

Similar studies using guinea‐pig tracheal membranes demonstrated that [125I]‐neurokinin A specific binding was composed of a NK1‐receptor component (60%), inhibited by both [Sar9Met(O2)11]substance P and CP‐99,994, and a significant NK2‐receptor component, inhibited by both [βAla8]neurokinin A and SR48968.

In summary, these data demonstrate that guinea‐pig lung parenchyma and guinea‐pig trachea express both NK1 and NK2 receptors.
Enhancement of D2 receptor agonist‐induced inhibition by D1 receptor agonist in the ventral tegmental area
Authors: Momiyama Toshihiko , Sasa Masashi , Takaori Shuji
Published: July 2012

A microiontophoretic study was performed on chloral hydrate‐anaesthetized rats to examine the role of D1 receptors in the ventral tegmental area (VTA) neurones, which are inhibited by autoreceptor and D2 receptor agonists.

Inhibition by microiontophoretic application of quinpirole (a D2 agonist) of antidromic spikes elicited by stimulation of the nucleus accumbens in dopaminergic neurones of the VTA, was significantly enhanced by simultaneous application of SKF 38393 (D1 agonist), although SKF 38393 alone had little effect on the neurones.

In addition, quinpirole‐induced inhibition was antagonized by iontophoretic application of domperidone (D2 antagonist), but was not affected by SCH 23390 (D1 antagonist).

Furthermore, SKF 38393‐induced enhancement of inhibition by quinpirole was antagonized by simultaneous application of SCH 23390.

These results suggest that activation of D1 receptors located on the VTA dopaminergic neurones or on non‐dopaminergic nerve terminals is not essential for inducing inhibition of the dopaminergic neurones, but enhances D2 receptor‐mediated inhibition directly or indirectly via inhibitory neurones.
Differential inhibition by NG‐monomethyl‐l‐arginine of vasodilator effects of acetylcholine and methacholine in human forearm vasculature
Authors: Chowienczyk P.J. , Cockcroft J.R. , Ritter J.M.
Published: July 2012

We compared the effects of NG‐monomethyl‐l‐arginine (l‐NMMA), an NO synthase inhibitor, on vasodilatation produced by acetylcholine and methacholine in human forearm vasculature.

Acetylcholine (83 nmol min−1) infused into the brachial artery of 8 healthy volunteers caused a submaximal increase in forearm blood flow, measured by venous occlusion plethysmography, from 3.3 ± 0.5 (mean ± s.e.mean) to 13.3 ± 1.7 ml min−1 100 ml−1.

Co‐infusion of l‐NMMA (4 μmol min−1) with acetylcholine (83 nmol min−1) over 6 min resulted in a 58% ± 12% fall in the response to acetylcholine whereas during co‐infusion of saline over the same time period in the same subjects (n = 8) on a different day the response to acetylcholine fell by only 9% ± 17% (P < 0.01).

Methacholine (1.5 and 15 nmol min−1) increased forearm blood flow from 2.5 ± 0.4 to 5.9 ± 0.9 and from 3.2 ± 0.4 to 17.0 ± 1.9 ml min−1 100 ml−1 respectively.

Co‐infusion of l‐NMMA (4 μmol min−1) had no significant effect on the response to methacholine (1.5 or 15 nmol min−1) when compared with saline control (n = 8). Co‐infusion of a higher dose of l‐NMMA (8 μmol min−1) with methacholine (1.5 nmol min−1) did not significantly inhibit the vasodilator response (n = 7).

These results suggest that, in human forearm vasculature, methacholine acts predominantly through mechanisms other than the l‐arginine/nitric oxide pathway.
Similar effects of ethanol and flumazenil on acquisition of a shuttle‐box avoidance response during withdrawal from chronic ethanol treatment
Authors: Criswell Hugh E. , Breese George R.
Published: July 2012

Acquisition of a two‐way shuttle‐box avoidance response is facilitated by ethanol. This facilitated acquisition of an avoidance response to ethanol was attenuated during withdrawal from chronic‐ethanol diet intake (i.e. tolerance developed by ethanol). The deficit in the avoidance task after chronic ethanol treatment could be overcome by increasing the dose of ethanol.

Flumazenil, a benzodiazepine antagonist, also facilitated acquisition of the avoidance response in control rats. This response to flumazenil was significantly reduced during withdrawal from chronic‐ethanol treatment. This reduced avoidance responding during withdrawal also could be overcome by increasing the dose of flumazenil.

The benzodiazepine‐inverse agonist, RO 15–4513, produced a deficit in avoidance responding that was antagonized by both ethanol and flumazenil in a dose‐related manner.

To determine whether flumazenil has the properties of a benzodiazepine agonist, it was established that, unlike the benzodiazepine chlordiazepoxide, flumazenil did not enhance the ethanol‐induced deficit in the aerial righting reflex. Additionally, flumazenil blocked the action of chlordiazepoxide in this procedure, consistent with the benzodiazepine antagonist action of flumazenil.

Data collected are consistent with the hypothesis that an endogenous substance with the properties of a benzodiazepine‐inverse agonist antagonizes the anticonflict actions of acutely administered ethanol during withdrawal from chronic‐ethanol exposure.
Use of the endothelin antagonists BQ‐123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF
Authors: Warner Timothy D. , Allcock Graham H. , Corder Roger , Vane John R.
Published: July 2012

We have compared the receptors mediating the contractions of rings of rat thoracic aorta or rabbit pulmonary artery and rat stomach strips in response to the endothelin/sarafotoxin (ET/SX) family of peptides and to those mediating endothelium‐dependent vasodilatations within the isolated perfused mesentery of the rat. To discriminate ETA receptors from ETB receptors we have used the criteria that ET‐1 is more active than SX6c on ETA receptors, and that the ET/SX peptides are equiactive on ETB receptors. We have also assessed the effects of the ETA receptor‐selective antagonist BQ‐123, and the non‐selective ET receptor antagonist PD 142893 on the responses of each preparation to the ET/SX peptides.

ET‐1‐induced constrictions of the rat thoracic aorta (EC50 3 × 10−10m), a prototypic ETA receptor‐mediated response, or isolated perfused mesentery of the rat were antagonized by BQ‐123 (10−5 m) or PD 142893 (10−5 m). SX6c did not constrict either the rat isolated perfused mesentery or the rat thoracic aorta. Thus, ETA receptors mediate these constrictions.

ET‐1 and SX6c were approximately equipotent in constricting rabbit pulmonary artery rings (EC50s 3–6 × 10−10 m). Neither BQ‐123 (10−5 m) nor PD 142893 antagonized the contractions induced by ET‐1. These effects suggest mediation by ETB receptors but PD 142893 (10−5 m) did give a 3 fold antagonism of constrictions induced by SX6c.

SX6c was more potent than ET‐1 in contracting the rat stomach strip (threshold concentrations 10−10 and 3 × 10−10 m). Contractions to ET‐1 or SX6c were unaffected by BQ‐123 (10−5 m), again indicative of ETB receptor‐mediated events. PD 142893 (10−5 m) was ineffective against ET‐1 but produced a 3 fold antagonism of SX6c.

In the rat isolated perfused mesentery ET‐1 or SX6c (0.3–300 pmol) were equipotent in producing dose‐related vasodilatations that were unaffected by BQ‐123 (10−6 m), indicative of an ETB receptor‐mediated response. In contrast to the other ETB‐mediated responses, PD 142893 (10−6 m) strongly antagonized these vasodilatations.

Thus, ETA receptors mediate constrictions of the rat thoracic aorta and rat isolated perfused mesentery whereas ETB receptors mediate constrictions of the rabbit pulmonary artery and rat stomach strip and endothelium‐dependent dilatations within the mesentery. However, within the group of ETB receptor‐mediated responses, endothelium‐dependent vasodilatations are sensitive to PD 142893, whereas contractions of the isolated smooth muscle preparations are not. Thus, the receptor present on the endothelium responsible for the release of nitric oxide in response to the ET/SX peptides is most probably different from that present on smooth muscle that mediates BQ‐123‐insensitive contractions.
Characterization of endothelin receptors mediating the effects of the endothelin/sarafotoxin peptides on autonomic neurotransmission in the rat vas deferens and guinea‐pig ileum
Authors: Warner Timothy D. , Allcock Graham H. , Mickley Emma J. , Vane John R.
Published: July 2012

To characterize the receptors mediating the effects of the endothelin/sarafotoxin family of peptides on the responses to electrical stimulation of the rat vas deferens (RVD) and guinea‐pig ileum (GPI) we have used endothelin‐1 (ET‐1), ET‐3, sarafotoxin 6b (SX6b) and SX6c as agonists and the endothelin‐receptor antagonists BQ‐123 (ETA receptor selective) and PD 142893 (non‐selective).

In the RVD, ET‐1 and SX6b increased the twitches induced by field stimulation starting at a threshold concentration of 10−10 m while the threshold concentration for ET‐3 was 3 × 10−9 m. SX6c (up to 3 × 10−8 m) did not potentiate the twitches. SX6b produced significantly (P < 0.05) greater potentiations than ET‐1 at concentrations of 3 × 10−9 m and higher, and 10−7 m ET‐3 also produced a significantly greater effect than ET‐1 at the same concentration. Thus, at threshold the rank order of peptides was ET‐1 = SX6b > ET‐3 >> SX6c, and at concentrations of 3 × 10−8 m and higher, SX6b > ET‐3 > ET‐1 >>> SX6c.

In the presence of BQ‐123 or PD 142893 (10−5 m) the threshold concentrations for ET‐1 to augment the twitches were increased 30 fold. In the same conditions neither SX6b nor ET‐3 potentiated the responses. The relative activities of the endothelin/sarafotoxin peptides and the effectiveness of the endothelin receptor antagonists are consistent with postjunctional ETA receptors mediating these effects.

In the transmurally stimulated GPI the endothelin/sarafotoxin peptides produced two effects; an increase in the basal tension of the tissues and an inhibition of the twitch responses. To increase the basal tension the peptides had the order of potency ET‐1 > SX6b >> ET‐3 = SX6c. These direct effects of ET‐1 or SX6b were strongly antagonized (100 fold) by either BQ‐123 (10−5 m) or PD 142893 (10−5 m). Thus, ETA receptors mediate contractions of the GPI induced by these peptides.

The endothelin/sarafotoxin peptides were approximately equipotent at depressing twitches of the GPI in response to transmural stimulation (EC50S, 4 × 10−11 to 1.5 × 10−10 m). The depressions induced by ET‐1 were unaffected by either BQ‐123 (10−5 m) or PD 142893 (10−5 m). BQ‐123 produced a small (three fold) antagonism of the inhibitory effects of ET‐3 or SX6c. These results indicate that a receptor of the ETB type mediates the inhibitory effects of the endothelin/sarafotoxin peptides on neurotransmission in the GPI.

Thus, both ETA receptors and ETB receptors mediate the effects of the endothelin/sarafotoxin peptides on neurotransmission.
Role of oxygen radicals and arachidonic acid metabolites in the reverse passive Arthus reaction and carrageenin paw oedema in the rat
Authors: Boughton‐Smith N.K. , Deakin A.M. , Follenfant R.L. , Whittle B.J.R. , Garland L.G.
Published: July 2012

The role of arachidonic acid metabolites and oxygen radicals in carrageenin‐induced rat paw oedema and dermal reverse passive Arthus reaction (RPA) have been investigated.

Indomethacin (10 mg kg−1, p.o.) inhibited carrageenin paw oedema when administered 30 min before, but not 2 h after carrageenin. BWB70C (10 mg kg−1, p.o.), a selective inhibitor of 5‐lipoxygenase, had no effect whether administered before or after carrageenin. Administration of both indomethacin and BWB70C had no greater anti‐inflammatory effect than indomethacin alone.

BW755C (20 mg kg−1, p.o.), which inhibits the cyclo‐oxygenase and lipoxygenase pathways of arachidonic acid metabolism, or superoxide dismutase‐polyethylene glycol conjugate (SOD‐PEG, 3000 u, i.v.) inhibited carrageenin paw oedema whether administered either 30 min before, or 2 h after carrageenin.

Pretreatment with dexamethasone (0.1 mg kg−1) or colchicine (2 mg kg−1), likewise suppressed carrageenin paw oedema.

BW755C (25–100 mg kg−1, p.o.) dose‐dependently reduced plasma leakage in the RPA, whereas indomethacin (5 mg kg−1, p.o.) or BWB70C either alone or in combination, did not.

SOD‐PEG (300–3000 u, i.v.) dose‐dependently inhibited plasma leakage in the RPA. In addition, the iron chelator and peroxyl radical scavenger, desferrioxamine (200 mg kg−1, s.c.) also inhibited plasma leakage.

Pretreatment with dexamethasone (0.1 mg kg−1) or colchicine (1 mg kg−1) reduced the plasma leakage in RPA, whereas MK‐886 (10 mg kg−1) had no effect.

These results indicate an important role for oxygen radicals but not arachidonic acid metabolites in the maintenance of carrageenin paw oedema and the plasma leakage in RPA. Furthermore, the results suggest that the anti‐inflammatory actions of BW755C can be dissociated from its effects on arachidonic acid metabolism and are attributed to its anti‐oxidant activity.
Electrophysiological actions of alfentanil: intracellular studies in the rat locus coeruleus neurones
Authors: Chiu T.H. , Yeh M.H. , Tsai S.K. , Mok M.S.
Published: July 2012

The electrophysiological effects of alfentanil on 156 neurones of the rat locus coeruleus were investigated by use of intracellular recordings from the in vitro brain slice preparation.

Bath application of alfentanil (5–100 nm) reversibly decreased the firing rate of all neurones tested in a dose‐dependent manner, with an IC50 4.1 nm.

Based on inhibition of the spontaneous firing rate, alfentanil was 22 times more potent than morphine.

At 100 nm, alfentanil produced a complete inhibition of firing of all neurones tested (n = 62); the inhibition was accompanied by a membrane hyperpolarization 17.0 ± 0.8 mV (range 6.1–30.3 mV, n = 62) and a reduction in input resistance 26.4 ± 1.7% (range 6.5−53%, n = 51).

The effects of alfentanil were antagonized by naloxone, with a dissociation equilibrium constant of 2.7 ± 0.4 nm (n = 6).

The reversal potential for the alfentanil‐induced hyperpolarization was −; 110 ± 2 mV (n = 9), which is approximately the potassium equilibrium potential.

The alfentanil‐induced hyperpolarization was blocked by caesium chloride and barium chloride.

These results indicate that alfentanil binds to μ‐opioid receptors on the cell membrane of neurones of the locus coeruleus. This leads to opening of the inward‐going rectification potassium channels, resulting in the observed hyperpolarization of the membrane.
Expression of inducible nitric oxide synthase by neurones following exposure to endotoxin and cytokine
Authors: Minc‐Golomb Dahlia , Tsarfaty Ilan , Schwartz Joan P.
Published: July 2012

In the CNS, nitric oxide (NO) has been implicated as both a mediator of neurotoxicity and a neuromodulator. The inducible NO synthase (iNOS), thought to mediate toxic effects of NO, has been attributed to glial cells in the CNS. We now report that cerebellar granule cell neurones can be stimulated by lipopolysaccharide and interferon‐γ to express iNOS in vitro, as demonstrated by reverse transcription‐polymerase chain reaction and fluorescent in situ hybridisation. The expression of both constitutive NO synthase (cNOS) and iNOS by neurones suggests that NO has diverse functions in the brain, and supports the possibility that iNOS plays a role in neuronal damage and inflammation following activation of brain microglia and production of cytokines.
A pharmacological analysis of receptors mediating the excitatory response to 5‐hydroxytryptamine in the guinea‐pig isolated trachea
Authors: Lucchelli Adele , Santagostino‐Barbone Maria Grazia , Barbieri Annalisa , Tonini Marcello
Published: July 2012

Experiments were carried out to characterize the receptors mediating the indirect excitatory response to 5‐hydroxytryptamine (5‐HT) in the guinea‐pig isolated trachea.

5‐HT caused concentration‐dependent contractions of tracheal strips, and the resulting concentration‐response curve was biphasic in nature. The first phase was obtained with agonist concentrations in the range of 0.01–3 nm and achieved a maximum which was 30% of the total 5‐HT response, while the second phase was in the range 10 nm–1 μm.

Atropine (0.1 μm) and tetrodotoxin (TTX: 0.3 μm) significantly reduced both phases of the 5‐HT curve. Morphine (10 μm), which can act to inhibit neuronal acetylcholine release, abolished the first phase and reduced the second phase. This suggests that the first phase is mainly neurogenic (cholinergic) in nature, while the second phase is in part neurogenic and in part due to direct activation of the effector cells.

The 5‐HT2A receptor antagonist, ketanserin (0.01, 0.1 μm) markedly depressed the first phase and shifted the second phase to the right in a parallel manner, with some depression of the 5‐HT response maximum. The less selective (5‐HT1/5‐HT2A) antagonist, methiothepin (0.1 μm) mimicked the action of ketanserin, albeit with less potency. Concomitant administration of ketanserin and methiothepin (each at 0.1 μm) produced an antagonism similar to that caused by ketanserin (0.1 μm) alone.

The 5‐HT3 receptor antagonists, ondansetron (0.1 μm) and granisetron (0.01 μm) slightly but significantly inhibited the first phase of the 5‐HT curve without altering the second phase. SDZ 205,557 (0.3 μm), a 5‐HT4 receptor antagonist, was ineffective.

Our results suggest that neural 5‐HT2A and, to a lesser extent, 5‐HT3 receptor subtypes mediate the first phase of the 5‐HT curve in the guinea‐pig trachea. The second phase is mediated by 5‐HT2A receptors, which are probably located at both the neural and muscular level. No evidence for the participation of 5‐HT1 receptors in the 5‐HT response has been obtained.
Peripheral and central adrenoceptor modulation of the behavioural effects of clozapine in the paw test
Authors: Prinssen Eric P.M. , Ellenbroek Bart A. , Cools Alexander R.
Published: July 2012

In rats, the atypical neuroleptic, clozapine, has been found to increase the hindlimb retraction time but not the forelimb retraction time, in the paw test. These parameters have predictive validity for the antipsychotic efficacy and extrapyramidal side‐effects of drugs, respectively. The present study analysed to what extent drugs acting on adrenoceptors affect the behavioural effect of clozapine in the paw test.

The α1‐adrenoceptor agonist, ST 587 but not the peripherally working α1‐agonist, methoxamine, decreased the effect of clozapine on the hindlimb retraction time. The α1‐antagonist phenoxybenzamine increased this effect of clozapine, and blocked the effect of ST 587 on clozapine at low doses. Only the combination of phenoxybenzamine with clozapine produced an increase in forelimb retraction time.

The α1‐adrenoceptor agonist, clonidine, decreased the effect of clozapine on the hindlimb retraction time. This effect was neither antagonized by the α1‐antagonist rauwolscine nor by the α1‐antagonist phenoxybenzamine. Rauwolscine or the peripherally working α2‐antagonist L‐659,066 did not influence the effect of clozapine on the hindlimb retraction time. The forelimb retraction time was not affected by any of the drug combinations.

In contrast to the β2‐adrenoceptor agonist, clenbuterol, which was ineffective, the peripherally acting β‐agonist, (−)‐isoprenaline, increased the effects of clozapine on the hindlimb retraction time. The β‐antagonist, (−)‐propranolol as well as the peripherally acting β‐antagonist, nadolol decreased this effect of clozapine. Low doses of the peripherally acting β1‐antagonist, atenolol, as well as low doses of the β2‐antagonist, ICI‐118,551, decreased the effect of clozapine. A low dose of nadolol blocked the effect of (−)‐isoprenaline on clozapine. Only the combination of clenbuterol with clozapine produced an increase in forelimb retraction time.

It is concluded that blockade of central α1‐adrenoceptors plays an important role in the effect of clozapine on the hindlimb retraction time. Furthermore, the effect of clozapine on the hindlimb retraction time is strongly modulated by peripheral β1 and/or β2‐adrenoceptors. Given the predictive validity of the paw test, the presented data suggest that the α1‐adrenoceptor antagonist properties of clozapine are important for its therapeutic effects, but not for its lack of extrapyramidal side‐effects.
Actions of two new antagonists showing selectivity for different sub‐types of metabotropic glutamate receptor in the neonatal rat spinal cord
Authors: Jane D.E. , Jones P.L.St.J. , Pook P.C‐K. , Tse H‐W. , Watkins J.C.
Published: July 2012

The presynaptic depressant action of l‐2‐amino‐4‐phosphonobutyrate (l‐AP4) on the monosynaptic excitation of neonatal rat motoneurones has been differentiated from the similar effects produced by (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylate ((1S,3R)‐ACPD), (1S,3S)‐ACPD and (2S,3S,4S)‐α‐(carboxycyclopropyl)glycine (l‐CCG‐I), and from the postsynaptic motoneuronal depolarization produced by (1S,3R)‐ACPD, by the actions of two new antagonists, α‐methyl‐l‐AP4 (MAP4) and α‐methyl‐l‐CCG‐I (MCCG). Such selectivity was not seen with a previously reported antagonist, (+)‐α‐methyl‐4‐carboxyphenylglycine (MCPG).

MAP4 selectively and competitively antagonized the depression of monosynaptic excitation produced by l‐AP4 (KD 22 μm). At ten fold higher concentrations, MAP4 also antagonized synaptic depression produced by l‐CCG‐I but in an apparently non‐competitive manner. MAP4 was virtually without effect on depression produced by (1S,3R)‐ or (1S,3S)‐ACPD.

MCCG differentially antagonized the presynaptic depression produced by the range of agonists used. This antagonist had minimal effect on l‐AP4‐induced depression. The antagonism of the synaptic depression effected by (1S,3S)‐ACPD and l‐CCG‐I was apparently competitive in each case but of varying effectiveness, with apparent KD values for the interaction between MCCG and the receptors activated by the two depressants calculated as 103 and 259 μm, respectively. MCCG also antagonized the presynaptic depression produced by (1S,3R)‐ACPD.

Neither MAP4 nor MCCG (200–500 μm) significantly affected motoneuronal depolarizations produced by (1S,3R)‐ACPD. At the same concentrations the two antagonists produced only very weak and variable effects (slight antagonism or potentiation) on depolarizations produced by (S)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) and N‐methyl‐d‐aspartate (NMDA).

It is concluded that MAP4 is a potent and selective antagonist for those excitatory amino acid (EAA) receptors on neonatal rat primary afferent terminals that are preferentially activated by l‐AP4, and that MCCG is a relatively selective antagonist for different presynaptic EAA receptors that are preferentially activated by (1S,3S)‐ACPD and (perhaps less selectively) by l‐CCG‐I. These receptors probably comprise two sub‐types of metabotropic glutamate receptors negatively linked to adenylyl cyclase activity.
Contribution of both nitric oxide and a change in membrane potential to acetylcholine‐induced relaxation in the rat small mesenteric artery
Authors: Waldron Gareth J. , Garland Christopher J.
Published: July 2012

Acetylcholine stimulated repolarization and relaxation in isolated segments of rat small mesenteric artery (D100 = 325 ± 9 μm) in which the smooth muscle cells were depolarized and contracted by submaximal concentrations of noradrenaline (0.75–2.5 μm). There was no significant difference either in the time taken to initiate relaxation or hyperpolarization, or for these parameters to reach maximum in response to acetylcholine.

The nitric oxide synthase inhibitor, NG‐nitro l‐arginine methyl ester (l‐NAME, 100 μm) reduced the pD2 for acetylcholine‐induced relaxation from 7.5 to 7 and depressed the maximum relaxation from 89% to 68% in tissues stimulated with noradrenaline. The pD2 for smooth muscle repolarization in these experiments was also reduced (7.4 to 6.6) but the maximum change in membrane potential in response to acetylcholine was unaltered. The increase in potential now clearly preceded relaxation by 3.7 s (to initiation) and 4.7 s (to maximum).

In the presence of noradrenaline and a raised potassium concentration (25 mm), the repolarization to acetylcholine was markedly attenuated. Simultaneous tension measurements also revealed a marked reduction in the maximal relaxation to acetylcholine, but the pD2 was unchanged at 7.4.

The residual relaxation recorded in the absence of marked repolarization (in the presence of noradrenaline and 25 mm potassium) was abolished by the addition of 100 μm l‐NAME.

Nitric oxide gas in solution (0.2–2.2 μm; NOg) relaxed artery segments precontracted with noradrenaline. The magnitude of relaxation to NOg was not altered in the presence of noradrenaline and 25 mm potassium.

These data provide additional evidence that acetylcholine‐evoked endothelium‐dependent increases in membrane potential provide a major mechanism for smooth muscle relaxation in the mesenteric artery. They also show that voltage‐dependent and independent (initiated by NO) mechanisms can both contribute to relaxation, and suggest that NO may modulate the increase in membrane potential or the release of a hyperpolarizing factor.
Effects of cyclic GMP and analogues on neurogenic transmission in the rat tail artery
Authors: Ouedraogo Sylvin , Tschöpl Martin , Stoclet Jean‐Claude , Bucher Bernard
Published: July 2012

The effects of membrane permeable analogues of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP), and of the NO donor, 3‐morpholinosydnonimine‐N‐ethylcarbamide (SIN‐1) were investigated on [3H]‐noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries.

Two 8‐substituted analogues of cyclic GMP (8‐bromoguanosine 3′:5′‐cyclic monophosphate; 8‐bromo‐cyclic GMP and 8‐(4‐chlorophenylthio)‐guanosine 3′:5′‐cyclic monophosphate; 8‐pCPT‐cyclic GMP) concentration‐dependently enhanced stimulation‐induced [3H]‐noradrenaline release. These prejunctional effects were antagonized by the cyclic AMP‐dependent protein kinase (PKA) inhibitor N‐[2‐((3‐(4‐bromophenyl)‐2‐propenyl)‐amino)‐ethyl]‐5 isoquinolinesulphonamide dihydrochloride (H‐89; 100 nm) but not by the cyclic GMP‐dependent protein kinase (PKG) inhibitors, Rp‐8‐bromoguanosine 3′:5′‐cyclic monophosphorothioate (Rp‐8‐bromo‐cyclic GMPS; 10 μm) or Rp‐8‐(4‐chlorophenylthio)‐guanosine 3′:5′‐cyclic monophosphorothioate (Rp‐8‐pCPT‐cyclic GMPS; 10 μm).

β‐Phenyl‐1,N2‐ethenoguanosine 3′:5′‐cyclic monophosphate (PET‐cyclic GMP) had no effect on stimulation‐induced [3H]‐noradrenaline release but concentration‐dependently decreased the stimulation‐induced vasoconstriction.

The two 8‐substituted cyclic GMP derivatives, PET‐cyclic GMP and SIN‐1, both decreased stimulation‐induced vasoconstriction. In addition, SIN‐1 relaxed rat tail arteries precontracted with phenylephrine (1 μm). The SIN‐1 concentration‐relaxation curve was shifted in parallel manner to the right by Rp‐8‐bromo‐cyclic GMPS (10 μm) and Rp‐8‐pCPT‐cyclic GMPS (10 μm) with no change in the maximum effect, showing that the relaxation was mediated by a cyclic GMP/PKG‐dependent mechanism.

It is concluded that PKA activation is involved in the noradrenaline release enhancing effect of the two 8‐substituted cyclic GMP analogues, whereas a cyclic GMP/PKG‐operated pathway accounts for the inhibitory effects of the cyclic GMP and its analogues on vascular smooth muscle contraction.
Comparison between the pharmacology of dopamine receptors mediating the inhibition of cell firing in rat brain slices through the substantia nigra pars compacta and ventral tegmental area
Authors: Bowery B. , Rothwell L.A. , Seabrook G.R.
Published: July 2012

Electrophysiological recordings were made from presumed dopaminergic neurones in the substantia nigra pars compacta and ventral tegmental area of rat brain slices. The ability of selective dopamine receptor agonists to hyperpolarize neurones and inhibit cell firing, as well as the ability of dopamine receptor antagonists to block responses to quinpirole were compared.

Six dopamine receptor agonists were examined for their ability to hyperpolarize neurones within the substantia nigra pars compacta. Of these, the most potent ligand tested was naxagolide with an EC50 value of 20 nm and estimated maximum of 10 mV. The rank order of agonist potency was naxagolide> quinpirole>apomorphine>dopamine.

Quinpirole was more potent at inhibiting cell firing in the substantia nigra pars compacta (pIC50 = 7.65 ± 0.06, n = 35) than in the ventral tegmental area (pIC50 = 7.24 ± 0.06, n = 32; P < 0.01, Student's t test). 7‐Hydroxy‐N,N‐di‐n‐propyl‐2‐aminotetralin (7‐OH‐DPAT), a putative D3 selective agonist, had a comparable potency to quinpirole in both the ventral tegmental area (pIC50 = 7.39 ± 0.26, n = 4), and substantia nigra pars compacta (pIC50 = 7.71 ± 0.20; n = 4).

The inhibition of cell firing by quinpirole was antagonized by haloperidol, S(−)−sulpiride, clozapine, and ritanserin. S(−)‐sulpiride and haloperidol had the highest estimated affinities in the substantia nigra, with pA2 values of 8.97 (slope = 0.85) and 8.20 (slope = 2.09) respectively. The pA2 values for S(−)‐sulpiride and haloperidol in the ventral tegmental area were 8.07 (slope =0.87) and 8.11 (slope= 1.48) respectively. Clozapine had a lower functional affinity than S(−)‐sulpiride and haloperidol in both the substantia nigra (pA2 = 6.47, slope = 1.19) and ventral tegmental area (pA2 = 6.53, slope 0.87). Ritanserin, a 5‐HT2 receptor antagonist that also binds to D2‐like dopamine receptors, caused a slight but significant shift in the concentration‐effect curve to quinpirole with an estimated pKA of 6.97 ± 0.13 (n = 4) in the substantia nigra and pKA of 7.12 ± 0.22 (n = 4) in the ventral tegmental area.

Comparison of these data with the binding affinity for cloned dopamine receptors demonstrates that the responses to quinpirole on dopaminergic neurones in both the A9 (substantia nigra) and A10 (ventral tegmental area) brain areas are consistent with the activation of predominantly D2, and not D3 or D4 dopamine receptors. Furthermore, the similarity in functional affinity of antagonists for these receptors suggest that the mesolimbic selectivity of atypical neuroleptics, like clozapine, may be a consequence of their actions on other receptors or their effects elsewhere in the brain.