bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for functional dissociation of dependence and tolerance in guinea‐pig isolated ileal segments following 20 hour exposure to morphine in vitro
Authors: David C. , Davis N. , Mason R. , Wilson V.G.
Published: July 2012

In the present study we have examined the relationship between tolerance and dependence in isolated ileal segments from the guinea‐pig under three different conditions: fresh preparations not previously exposed to morphine (fresh/morphine naive); preparations stored overnight at 4°C in modified Krebs‐Henseleit saline (overnight‐stored/morphine‐naive); preparations stored overnight at 4°C in Krebs‐Henseleit saline containing 10 μm morphine and extensively washed with modified Krebs‐Henseleit saline to remove residual morphine (overnight‐stored/morphine‐exposed).

Morphine produced a concentration‐dependent inhibition of the response of ileal segment to 0.1 Hz, 1 ms and 10 V transmural field stimulation in fresh/morphine‐naive, overnight‐stored/morphine‐naive and overnight‐stored/morphine‐exposed preparations. The maximum effect observed was similar in all three preparations—approximately 80% inhibition. Although, morphine was significantly more potent in the fresh/morphine‐naive preparations (pD2 6.72 ± 0.05, n = 8) than either the overnight‐stored/morphine‐naive (pD2 6.42 ± 0.11, n = 8) or the overnight‐stored/morphine‐exposed (pD2 6.44 ± 0.14, n = 8), there was no significant difference between the overnight models in their sensitivity to morphine. The latter observation indicates that overnight exposure to ileal segments to 10 μm morphine at 4°C failed to induce tolerance to morphine.

The μ opiate receptor antagonist, naloxone (10 μm), produced contractions in both fresh/morphine‐naive and overnight‐stored/morphine‐naive ileal segments following acute exposure to 10 μm morphine. Naloxone (10 μm) also produced contractions in 2/9 fresh/morphine‐naive, 1/9 overnight‐stored/morphine‐naive and 7/9 overnight‐stored/morphine‐exposed preparations in the absence of morphine. The greater incidence of naloxone‐induced contractions in overnight‐stored/morphine‐exposed preparations, suggests that dependence in this model is the product of adaptive changes that outlive the presence of morphine.

The selective α2‐adrenoceptor agonists, clonidine (0.3 μm) and 5‐bromo‐6‐[2‐imidazolin‐2‐ylamino]‐quinoxaline bitartrate (UK‐14304, 1 μm), inhibited naloxone‐induced contractions in overnight‐stored/morphine‐exposed preparations of ileal segments (n = 4 preparations for each agonist), suggesting that the response is due to transmitter release from the myenteric plexus.

The findings in the present study indicate that tolerance and dependence to morphine in ileal segments of the guinea‐pig can be functionally dissociated by overnight exposure to morphine at 4°C. The development of tolerance to morphine, unlike dependence, appears to be a temperature‐dependent process. This also raises the possibility that naloxone possesses intrinsic negative agonism at morphine‐sensitive receptors, which is manifested as a functional response only after adaptive changes in the myenteric plexus following exposure to morphine.
Ionotropic glutamate receptor types leading to adenosine‐mediated inhibition of electrically evoked [3H]‐noradrenaline release in rabbit brain cortex slices
Authors: von Kügelgen Ivar , Späth Leni , Starke Klaus
Published: July 2012

Glutamate inhibits the electrically evoked release of noradrenaline in rabbit brain cortex slices; the inhibition is mediated by adenyl compounds, presumably adenosine. The aim of the present study was to identify the receptors involved in this indirect inhibitory effect of glutamate. Slices of the occipitoparietal cortex were preincubated with [3H]‐noradrenaline and then superfused and stimulated by trains of 6 pulses, 100 Hz.

The ionotropic glutamate receptor agonists α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA; 10–100 μm), kainate (10–100 μm) and N‐methyl‐d‐aspartate (NMDA; 30–300 μm) but not the metabotropic glutamate receptor agonist, 1‐amino‐1,3‐cyclopentanedicarboxylate (ACPD; 10–100 μm) reduced the electrically evoked overflow of tritium.

The effects of AMPA, kainate and NMDA were attenuated or abolished by the adenosine A1‐receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) as well as by adenosine deaminase but not by the α2‐adrenoceptor antagonist yohimbine, the γ‐aminobutyric acid (GABA) receptor antagonists, bicuculline and 2‐hydroxysaclofen and the NO synthase inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME).

The NMDA receptor antagonist, 2‐amino‐5‐phosphonopentanoate (AP5) blocked the inhibitory effect of NMDA but not that of AMPA and kainate. The non‐NMDA‐receptor antagonist, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) blocked the effect of AMPA but not of kainate and NMDA.

In addition to decreasing the electrically evoked overflow of tritium, AMPA, kainate and NMDA but not ACPD caused a steep but transient rise of basal tritium efflux. This immediate releasing effect was not significantly changed by DPCPX, adenosine deaminase, yohimbine, bicuculline, 2‐hydroxy‐saclofen and l‐NAME (except that l‐NAME enhanced the effect of kainate). AP5 and CNQX antagonized the immediate releasing effects in the same way that they antagonized the inhibition by AMPA, kainate and NMDA of the electrically evoked overflow of tritium.

It is concluded that AMPA, kainate and NMDA, like glutamate, reduce the electrically evoked release of noradrenaline by releasing adenosine or an adenine nucleotide which is then degraded to adenosine. Activation of each of the three ionotropic glutamate receptors, AMPA, kainate and NMDA receptors, but not activation of metabotropic glutamate receptors can initiate this indirect inhibitory effect on the release of noradrenaline (as well as the known noradrenaline releasing effect).
The effect of defibrotide on thromboembolism in the pulmonary vasculature of mice and rabbits and in the cerebral vasculature of rabbits
Authors: Paul W. , Gresele P. , Momi S. , Bianchi G. , Page C.P.
Published: July 2012

Administration of bovine thrombin (100 u kg−1) into the carotid artery of rabbits induces a sustained accumulation of 111Indium‐labelled platelets within the cranial vasculature over the subsequent 3 h.

Intracarotid (i.c.) administration of defibrotide (64 mg kg−1 bolus plus 64 mg kg−1 h−1 for 1 h) prior to i.c. thrombin (100 u kg−1) significantly reduces the ability of thrombin to induce cranial thromboembolism in rabbits.

Intravenous (i.v.) administration of thrombin (20 u kg−1) in rabbits induces a reversible accumulation of radiolabelled platelets into the thoracic circulation which is significantly reduced by i.v. administration of defibrotide (64 mg kg−1 bolus plus 64 mg kg−1 h−1 for 1 h) prior to i.v. thrombin. In contrast, platelet accumulation in response to adenosine diphosphate (ADP; 20 μg kg−1, i.v.) or platelet activating factor (PAF; 50 ng kg−1, i.v.) is not significantly affected by this treatment.

Intravenous administration of the nitric oxide (NO)‐synthase inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME; 10 mg kg−1) potentiates platelet accumulation induced by low dose thrombin (10 u kg−1, i.v.) within the pulmonary vasculature of rabbits. The potentiated response is significantly abrogated following pretreatment with defibrotide (64 mg kg−1 bolus plus 64 mg kg−1 h−1 for 1 h, i.v.).

Intravenous injection of human thrombin (1250 u kg−1) to mice induces death within the majority of animals which is significantly reduced by pretreatment with defibrotide (150–175 mg kg−1, i.v.). In contrast, death induced by i.v. collagen (1.25 mg kg−1) plus adrenaline (75 μg kg−1) is not significantly affected by defibrotide pretreatment.

The inhibitory effect of defibrotide in mice is abolished following concomitant treatment with the inhibitor of fribrinolysis, tranexamic acid (100 mg kg−1, i.v.), but is unaffected following treatment with the cyclo‐oxygenase inhibitor, aspirin (300 mg kg−1, i.p.).

The protective effect of defibrotide against thrombin‐induced thromboembolism in the mouse is potentiated by recombinant tissue‐plasminogen activator (rt‐PA; 1 mg kg−1, i.v.) or unfractionated heparin (10 u kg−1, i.v.) administration.

The results suggest that defibrotide may possess antithrombotic activity on thrombin‐induced thromboembolism which, at least in the mouse, may be partially mediated via induction of the fibrinolytic pathway.
Toxic inhibition of smooth muscle contractility by plant‐derived sesquiterpenes caused by their chemically reactive α‐methylenebutyrolactone functions
Authors: Hay Alastair J.B. , Hamburger Matthias , Hostettmann K. , Hoult J.R.S.
Published: July 2012

Previous studies have shown that extracts of feverfew (Tanacetum parthenium) and parthenolide, a sesquiterpene α‐methylenebutyrolactone obtained from it, inhibit smooth muscle contractility in a time‐dependent, non‐specific and irreversible manner.

The hypothesis that this toxic effect is due specifically to the presence in the sesquiterpene lactone of the potentially reactive α‐methylene function was tested on rabbit isolated aortic ring preparations. This was done (a) by comparing the effects of two plant‐derived sesquiterpene lactones purified from yellow star thistle (Centaurea solstitialis): cynaropicrin (an α‐methylenebutyrolactone) and solstitialin 13‐acetate (lacking the α‐methylene function), and (b) by chemically inactivating the α‐methylene functions in cynaropicrin and parthenolide by reaction with cysteine.

The results show that the characteristic smooth muscle inhibitory profile is demonstrated by the two α‐methylenebutyrolactones (parthenolide and cynaropicrin), but not by the compound lacking this functional group (solstitialin 13‐acetate), or by those previously active compounds in which it has been inactivated with cysteine.

Thus the α‐methylene function is critical for this aspect of the toxic pharmacological profile of the sesquiterpene butyrolactones, which are natural products widely distributed in the Compositae family of flowering plants.
Comparison of two new inhibitors of catechol O‐methylation on striatal dopamine metabolism: a microdialysis study in rats
Authors: Törnwall M. , Kaakkola S. , Tuomainen P. , Kask A. , Männistö P.T.
Published: July 2012

Effects of two new inhibitors of catechol O‐methylation (CGP 28014 and entacapone; 30 mg kg−1, i.p.) were compared by means of brain microdialysis in rats treated with l‐3,4‐dihydroxyphenylalanine (l‐dopa)/carbidopa (50/50 mg kg−1, i.p., respectively) or saline.

In saline‐treated rats, CGP 28014 maximally (max) increased striatal dopamine and 3,4‐dihydroxyphenylacetic acid (DOPAC) effluxes by 41% and 49%, respectively, whereas homovanillic acid (HVA) levels were decreased by 71%.

In the presence of l‐dopa/carbidopa, a peripherally active inhibitor of catechol O‐methyltransferase (COMT) entacapone had a short‐lasting increasing effect on l‐dopa efflux. Compared to the effects of l‐dopa/carbidopa alone 3‐O‐methyldopa (3‐OMD) levels were effectively reduced (max 79%) by entacapone, but not by CGP 28014.

Entacapone, in contrast to CGP 28014, increased striatal dopamine efflux (max 492% of that after l‐dopa/carbidopa alone). Also DOPAC levels were increased by entacapone (255% at 180 min), but not significantly by CGP 28014 (159% at 180 min).

Both compounds initially decreased HVA efflux. The effect of CGP 18014 was longer‐lasting. By the end of the measurement, entacapone even increased HVA levels (max 259%).

Our results demonstrate that entacapone is a peripheral COMT inhibitor and support the view that CGP 18014 is mainly a centrally acting inhibitor of O‐methylation.
Regulation of aromatic l‐amino acid decarboxylase in rat striatal synaptosomes: effects of dopamine receptor agonists and antagonists
Authors: Zhu Meng‐Yang , Juorio Augusto V. , Paterson I. Alick , Boulton Alan A.
Published: July 2012

In this study we investigated the effects of dopamine receptor agonists and antagonists on rat striatal synaptosomal aromatic l‐amino acid decarboxylase (AADC) activity.

The results show that 10−5–10−7 m cis‐flupenthixol increased the striatal synaptosomal AADC activity (by 25% to 57%) in a time‐dependent manner. SCH 23390 and remoxipride alone had little or no effect on striatal synaptosomal AADC activity, but in combination they increased AADC activity by 20%, suggesting that the increases in striatal synaptosomal AADC activity occurred only after blockade of both dopamine D1 and D2 receptors.

Treatment with (+)‐amphetamine and (±)‐2‐(N‐phenylethyl‐N‐propyl)amino‐5‐hydroxytetralin hydrochloride ((±)‐PPHT) produced a reduction of striatal synaptosomal AADC activity in a concentration‐ and time‐dependent manner. SKF 38393 and (−)‐quinpirole, however, exhibited no effect on striatal synaptosomal AADC activity, suggesting that only the mixed dopamine receptor agonists can reduce the AADC activity. Incubation with apomorphine at a concentration of 10−4 m inhibited the AADC activity by 74% and this inhibition cannot be antagonized by SCH 23390, remoxipride or cis‐flupenthixol, suggesting that apomorphine‐induced inhibition of striatal synaptosomal AADC activity was not mediated by dopamine receptors.

cis‐Flupenthixol can reverse the reduction of AADC activity induced by (+)‐amphetamine and (±)‐PPHT. The inhibition of AADC activity elicited by (±)‐PPHT also can be reversed by SCH 23390 and remoxipride.

The inhibition of striatal synaptosomal AADC activity induced by (±)‐PPHT is calcium‐dependent and protein kinase C may play a role in the regulation of striatal AADC activity.

These studies show that striatal synaptosomal AADC activity is regulated by dopamine receptors and indicate that in vitro dopamine D1 and D2 receptors have a synergistic effect in this regulation.
Regulation of baseline vascular resistance in the canine diaphragm by nitric oxide
Authors: Ward Michael E. , Hussain Sabah N.A.
Published: July 2012

The role played by nitric oxide (NO) in the regulation of blood flow to the canine isolated hemidiaphragm was evaluated by determining (a) the effects of the l‐arginine analogues NG‐nitro‐l‐arginine methyl ester (l‐NAME), NG‐nitro‐l‐arginine (l‐NOARG), and argininosuccinic acid (ArgSA) on baseline vascular resistance and of the latter two agents on endothelium‐dependent (acetylcholine, ACh) and endothelium independent (sodium nitroprusside, SNP) vasodilatation; (b) the effects of l‐ and d‐arginine on baseline vascular resistance; and (c) the effects of l‐glutamine, an inhibitor of intracellular recycling of l‐citrulline to l‐arginine, on baseline resistance and on the response to ACh and SNP.

l‐NAME, l‐NOARG and ArgSA (6 × 10−4 m final concentration) increased baseline diaphragmatic vascular resistance to a similar extent (28.6 ± 4.2%, 26.7 ± 4.3% and 32.8 ± 4.6% respectively). l‐NOARG and ArgSA reversed the vasodilator effect of ACh but not of SNP.

l‐ and d‐arginine had no effect on vascular resistance.

l‐Glutamine (10−3 m) increased baseline vascular resistance by 10 ± 1.9% (P < 0.05) but did not alter responses to either ACh or SNP.

Basal NO release plays a role in the regulation of baseline diaphragmatic vascular resistance. l‐Arginine analogues tested potently and specifically inhibited this process. Moreover, extracellular l‐arginine appears to have no effect on baseline diaphragmatic vascular resistance.
Differential effects of Na+,K+‐ATPase inhibition by ouabain on acid secretory responses to histamine and bethanechol in the mouse isolated stomach
Authors: Horie Syunji , Yano Shingo , Watanabe Kazuo
Published: July 2012

The effect of Na+,K+‐ATPase inhibition by ouabain on gastric acid secretion was studied in the mouse isolated whole stomach preparation.

Ouabain caused a transient enhancement of histamine‐induced gastric acid secretion followed by an inhibitory phase. On the other hand, ouabain caused a rapid reduction of bethanechol‐stimulated acid secretion without an enhancement phase.

In dibutyryl cyclic AMP‐induced acid secretion, ouabain led to a transient increase in acid secretion followed by a fall, as was seen with the histamine stimulation. Ouabain caused a rapid reduction of A23187‐induced acid secretion.

Ouabain by itself increased basal acid secretion, and thereafter slowly suppressed the acid secretion.

Atropine inhibited both the ouabain‐induced enhancement of the stimulated gastric acid secretion and the ouabain‐induced stimulation of basal acid secretion.

The present study showed that Na+,K+‐ATPase inhibition by ouabain caused a phasic enhancement of the stimulated gastric acid secretion through release of endogenous acetylcholine when the secretagogues act via an intracellular cyclic AMP pathway. It also inhibited the stimulated acid secretion irrespective of secretagogues, probably through its inhibitory effect on Na+,K+‐ATPase in the gastric parietal cell.
Presence of P2‐purinoceptors in the rat pineal gland
Authors: Ferreira Zulma S. , Cipolla‐Neto J. , Markus Regina P.
Published: July 2012

The effects of noradrenaline, ATP, adenylyl‐imidodiphosphate (AMP‐PNP), adenosine, α,β‐methylene‐ATP and the P2‐purinoceptor antagonist, suramin on N′‐acetyl‐5‐hydroxytryptamine production were studied in cultured denervated rat pineal glands.

Noradrenaline (3 nm – 1 μm) increased N′‐acetyl‐5‐hydroxytryptamine production as measured both in the gland and the culture medium.

In noradrenaline (10 nm)‐stimulated pineal glands, ATP (0.03 nm – 1 mm) or AMP‐PNP (0.1 μm – 1 mm) increased N′‐acetyl‐5‐hydroxytryptamine production in a concentration‐dependent manner.

α,β‐Methylene‐ATP at the concentration of 0.1 mm, but not 3 μm, attenuated the enhancement by ATP (0.1 mm) of noradrenaline (10 nm)‐induced N′‐acetyl‐5‐hydroxytryptamine production.

Suramin (0.1 mm) blocked the potentiating effect of ATP (0.1 mm), but not the potentiating effect of adenosine (0.1 mm) in glands incubated with noradrenaline (10 nm).

These findings suggest that the rat pineal gland possesses P2‐purinoceptors which when stimulated potentiate the effect of noradrenaline but do not, by themselves, induce an increase in N′‐acetyl‐5‐hydroxytryptamine production.
Drug modulation of antigen‐induced paw oedema in guinea‐pigs: effects of lipopolysaccharide, tumour necrosis factor and leucocyte depletion
Authors: da Motta Joelson I. Braga , Cunha Fernando Q. , Vargaftig Bernardo Boris , Ferreira Sergio H.
Published: July 2012

In guinea‐pigs previously sensitized with ovalbumin, the intra‐plantar administration of the antigen induced dose‐dependent and sustained oedema. An intense infiltrate of neutrophils and eosinophils was observed at the peak of the oedema (4 h).

Oedema induced by ovalbumin at the doses of 50 or 200 μg/paw was not inhibited by antihistamines (meclizine and cetirizine), a PAF antagonist (BN 50730), a cyclo‐oxygenase inhibitor (indomethacin), a lipoxygenase inhibitor (MK‐886), a dual type lipo‐ and cyclo‐oxygenase inhibitor (NDGA), a bradykinin antagonist (Hoe 140) or the combination of cetirizine, MK‐886, indomethacin and BN 50730. These drugs did inhibit paw oedema induced by their specific agonists or by carrageenin. These results suggest that histamine, PAF, prostaglandins, leukotrienes or bradykinin are not important in the development of immune paw oedema in guinea‐pigs.

Dexamethasone (10 mg kg−1) inhibited oedema induced by ovalbumin (50 or 200 μg/paw, P < 0.05). This effect apparently does not result from inhibition of arachidonate metabolism, since indomethacin, MK‐886 and NDGA were without effect.

Oedema induced by ovalbumin (50 or 200 μg/paw) was also inhibited by azelastine. This effect was not due to the anti‐histaminic property of azelastine since two other potent‐antihistamines, meclizine and cetirizine, were ineffective.

Intravenous injection of lipopolysaccharide (LPS) dose‐dependently inhibited the oedema induced by ovalbumin (200 μg/paw). This effect could not be attributed to hypotension or leucopenia since the maximal dose applied (81 μg kg−1) did not induce significant changes in the blood pressure or in the white blood cell levels of the animals. It is suggested that the effect of LPS is mediated by the endogenous release of cytokines, including tumour necrosis factor (TNFα). Murine TNFα dose‐dependently (9–81 μg kg−1) inhibited the paw oedema induced by ovalbumin.

The anti‐oedematogenic effects of LPS and/or TNFα are possibly associated with their capacity to inhibit leucocyte emigration. Accordingly, guinea‐pigs rendered leucopenic with vinblastine exhibited less intense oedema after ovalbumin. Vinblastine did not affect oedema induced by PAF or bradykinin, indicating that vascular responsiveness was not involved.
Subtypes of purinoceptors in rat and dog urinary bladder smooth muscles
Authors: Suzuki Hideka , Kokubun Shinichiro
Published: July 2012

Both adenosine and adenosine 5′‐triphosphate (ATP) (10 μm and 100 μm) relaxed 10 μm acetylcholine (ACh)‐induced contraction of rat bladder strips, which was completely antagonized by 100 μm 8‐(p‐sulphophenyl) theophylline. In dog bladder neither adenosine nor ATP inhibited ACh‐induced contraction.

P2x‐purinoceptor agonists contracted both rat and dog bladder strips with the potency order of α,β‐MeATP > ATP > ADP.

α,β‐MeADP (100 μm) induced a contraction of the rat bladder strip even after desensitization of P2x‐purinoceptors but failed to contract the dog bladder strip.

2‐MeSATP (1 μm to 300 μm) concentration‐dependently induced contraction of rat bladder strips; this contraction was significantly inhibited after desensitization of P2x‐purinoceptors. Cibacron blue 3GA (100 μm) antagonized the drug at concentrations lower than 30 μm, whereas it augmented the response to the drug at concentrations above 30 μm.

ADPβS (1 μm to 1 mm) concentration‐dependently induced contraction of rat bladder strips after desensitization of P2x‐purinoceptors; a contraction which was significantly antagonized by cibacron blue 3GA (100 μm).

It is concluded that three subtypes of purinoceptors, P1 (mediating relaxation), and P2x and another type of P2 (mediating contraction), exist in rat urinary bladder smooth muscle, whereas a single subtype of the receptor, P2x‐purinoceptor (mediating contraction) occurs in dog urinary bladder smooth muscle.
Cardiovascular and behavioural effects of centrally administered neuropeptide K in the rat: receptor characterization
Authors: Prat Alexandre , Picard Pierre , Couture Réjean
Published: July 2012

The cardiovascular and behavioural responses to intracerebroventricularly (i.c.v.) administered neuropeptide K (NPK) were studied in conscious rats. The central effects of NPK were characterized by pretreatment (i.c.v.) with selective antagonists for the NK1 ((±)‐CP 96345 and RP 67580), NK2 (SR 48968) and NK3 (R 487) receptors.

NPK (10–65 pmol) induced tachycardia and dose‐dependent increases of mean arterial blood pressure. The cardiovascular responses reached a maximum within 3 min post‐injection and lasted for more than 1 h. Concurrently, NPK produced dose‐dependent increases of face washing, head scratching, grooming, walking and wet dog shakes.

A desensitization of most of the behavioural responses (except head scratching) but not of the cardiovascular response was shown when two consecutive injections of 25 pmol NPK were given 24 h apart.

Both the cardiovascular and behavioural responses (except the head scratching) to 25 pmol NPK were blocked by pre‐administration (i.c.v.) of 6.5 nmol (±)‐CP 96345 or RP 67580 given 5 min earlier. No inhibition of NPK responses was observed when 6.5 nmol SR 48968 or R 487 were used in a similar study. Additionally, NPK effects were significantly reduced 24 h after the prior injection of (±)‐CP 96345 but not of RP 67580.

These results support the involvement of NK1 receptors in the cardiovascular and behavioural effects of i.c.v. NPK. Thus, this peptide may play a putative role in central cardiovascular regulation as it is the most potent endogenous tachykinin described centrally, to date.
Differential effects of lumenal l‐arginine and NG‐nitro l‐arginine on blood flow and water fluxes in rat ileum
Authors: Mailman David
Published: July 2012

The role of endogenous mucosal nitric oxide (NO) in the local regulation of H2O absorption and blood flow in rat ileum was studied by perfusing l‐arginine (l‐Arg) (0.1–1.0 mm) and NG‐nitro l‐arginine (l‐NOARG) (0.01–1.0 mm) through the lumen. d‐Arginine (d‐Arg) or l‐Arg (1 mm), combined with l‐NOARG, were used to determine if any of the measured intestinal effects of l‐NOARG were exerted through NO formation.

Net and unidirectional H2O fluxes and effective mucosal blood flow were measured using 3H2O and [14C]‐inulin in the perfusate. Mucosal NO formation was measured as the appearance of lumenal NO2−.

l‐NOARG, beginning at a concentration of 0.1 mm, decreased net H2O absorption, but had only minor effects on unidirectional H2O fluxes or on blood flow. l‐NOARG increased blood pressure, beginning at a concentration of 0.5 mm.

l‐Arg had no significant effects on net H2O absorption or blood pressure, and only minor effects on unidirectional H2O fluxes and blood flow.

NO appearance in the lumen was marginally decreased by 1.0 mm l‐NOARG, but not increased by l‐Arg.

Mucosal blood flow resistance paralleled systemic blood pressure suggesting that vascular effects on the mucosa were exerted only after l‐NOARG had reached the general circulation.

Lumenal l‐Arg reversed the effects of lumenal l‐NOARG on net H2O absorption and blood pressure, but d‐Arg did not.

It was concluded that there is tonic NO production by the rat intestinal mucosa that promotes H2O absorption, but does not affect blood flow resistance. Mucosal NO production was not related to the observed effects on mucosal function.
Evidence from desensitization studies for distinct receptors for ATP and UTP on the rat superior cervical ganglion
Authors: Connolly Gerald P.
Published: July 2012

Prolonged contact of the rat superior cervical ganglia (SCG) with the purine α,β‐methylene‐ATP (α,β‐Me‐ATP) selectively depressed responses to ATP and α,β‐Me‐ATP but not responses to uridine 5′‐triphosphate (UTP), potassium or adenosine. Prolonged contact with the pyrimidine UTP selectively depressed responses to UTP but not responses evoked by α,β‐Me‐ATP, potassium or adenosine. These results are consistent with the presence of P2‐purinoceptors and pyrimidinoceptors on the rat SCG and the hypothesis that pyrimidinoceptors exist within the nervous system.
Lithium‐induced decrease in spontaneous Ca2+ oscillations in single GH3 rat pituitary cells
Authors: Varney Mark A. , Galione Antony , Watson Steve P.
Published: July 2012

Measurement of [Ca2+]i in single rat pituitary GH3 cells by dynamic single cell imaging techniques demonstrated that under basal conditions there is a large variation in the temporal pattern of [Ca2+]i signalling between individual cells ranging from high frequency asynchronous oscillations to quiescence.

We have reported previously that treatment of GH3 cells with 1 mm Li+ (a concentration used therapeutically in the treatment of manic depression) for 7 days reduces basal and thyrotrophin‐releasing hormone (TRH)‐stimulated levels of mass inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3]. In the present study, we show that this is associated with a reduction in the number of cells exhibiting basal Ca2+ oscillations over a sampling period of 60 s, whereas the maximum amplitude of oscillations is unaffected.

The pattern of [Ca2+]i responses to the agonist TRH varied considerably between individual cells, making quantitation of the responses difficult; however, data obtained from measurements made on a population of cells showed that increases in peak [Ca2+]i induced by high concentrations of TRH were reduced in cells treated with 1 mm Li+ for 7 days relative to control cells.

The sensitivity of the phosphoinositide pathway to [Ca2+]i was investigated by loading GH3 cells with BAPTA/AM at a concentration sufficient to lower ‘basal’ [Ca2+]i in a population of cells and to inhibit agonist‐stimulated increases in [Ca2+]i. Under these conditions, basal and TRH‐stimulated mass Ins(1,4,5)P3 levels were unaffected.

These results demonstrate that a 7‐day Li+ treatment leads to an alteration in Ca2+ signalling, in particular by reducing the number of cells exhibiting high frequency Ca2+ oscillations under basal conditions. The significance of these results to the clinical effectiveness of Li+ in the treatment of manic depression is discussed.
Noradrenergic‐nitrergic interactions in the rat anococcygeus muscle: evidence for postjunctional modulation by nitric oxide
Authors: Kasakov Lübomir , Belai Abebech , Vlaskovska Mila , Burnstock Geoffrey
Published: July 2012

The distribution of NADPH‐diaphorase positive and catecholamine‐containing nerve structures, and functional noradrenergic‐nitrergic interactions, were studied in the rat anococcygeus muscle.

The morphological findings demonstrated NADPH‐diaphorase positive neurones mostly as aggregates in intramural ganglia, nerve tracts and few single nerve fibres forming plexus‐like structures.

The nitric oxide synthase inhibitor NG‐nitro‐l‐arginine (l‐NOARG) inhibited concentration‐dependently the nitrergic relaxation, an effect reversed by l‐arginine. The drug had dual effects on noradrenergic contractile responses: at lower concentrations (0.1–10 μm) it decreased the amplitude of contractions and this was not affected by l‐arginine; higher concentrations (50–500 μm) potentiated the contractions, an effect that was prevented by l‐arginine.

The electron acceptor, nitro blue tetrazolium (NBT) produced a rapid inhibition of the noradrenergic contractile responses (EC50 0.178 ± 0.041 μm). The drug decreased the tone of the preparations. However, it potentiated concentration‐dependently the nitrergic relaxations.

NBT (1 μm) had no significant effect on the relaxations induced by exogenously applied nitric oxide (NO)‐donor sodium nitroprusside (SNP, 0.01–50 μm). However, the effect of NBT (0.1–10 μm) on the electrically induced relaxation was significantly decreased by l‐NOARG (10 and 50 μm). The inhibition was of a non‐competitive type.

Neither l‐NOARG (100 μm) nor NBT (1 μm) had any effect on the spontaneous or electrically‐induced release of 3H‐radioactivity from the tissues preincubated in [3H]‐noradrenaline.

It is concluded that l‐arginine‐NO pathway can modulate noradrenergic transmission in the rat anococcygeus muscle at postjunctional, but not prejunctional site(s).
Mechanisms of sympathetic enhancement and inhibition of parasympathetically induced salivary secretion in anaesthetized dogs
Authors: Lung Mary A.
Published: July 2012

The effects of superimposed and continuous sympathetic nerve stimulation on submandibular parasympathetic salivation were investigated in anaesthetized dogs.

Superimposed sympathetic nerve stimulation (1–2 min) initially enhanced and later inhibited salivary secretion induced by parasympathetic nerve stimulation (2–8 Hz) in glands with uncontrolled blood supply or constant‐flow vascular perfusion. Propranolol (0.05 mg kg−1, i.a.) did not affect the diphasic sympathetic action whereas phentolamine (0.1 mg kg−1, i.a.) abolished it. Prazosin (0.025 mg kg−1, i.a.) greatly lessened the initial enhancement while yohimbine (0.025 mg kg−1, i.a.) alleviated the late inhibition.

Salivary secretion, induced by parasympathetic nerve stimulation (4 Hz) or acetylcholine infusion (10 μg kg−1 min−1, i.a.), was abolished by atropine (0.05 mg kg−1, i.a.), increased by phenylephrine infusion (0.25 μg kg−1 min−1, i.a.) and depressed by clonidine infusion (0.75 μg kg−1 min−1, i.a.). Hexamethionium (12.5 mg kg−1, i.a.) abolished the nerve‐induced secretion but had no effect on the acetylcholine‐induced secretion.

Continuous background sympathetic nerve stimulation decreased parasympathetic nerve‐induced salivary secretion in glands with uncontrolled blood supply or constant‐flow vascular perfusion.

These results show that parasympathetic salivation can be modified by the sympathetic system at the postsynaptic level;
AT1 receptor characteristics of angiotensin analogue binding in human synovium
Authors: Walsh David A. , Suzuki Takahiro , Knock Gregory A. , Blake David R. , Polak Julia M. , Wharton John
Published: July 2012

Angiotensin II (AII) reduces blood flow, modulates vascular remodelling and is a growth factor. Human inflammatory arthritides are characterized by synovial hypoperfusion, hypoxia and proliferation. We aimed to localize and characterize receptors for AII in human synovium.

We used quantitative in vitro receptor autoradiography with [125I]‐(Sar1, Ile8)AII and [125I]‐AII on human synovium from patients with chondromalacia patellae, osteoarthritis and rheumatoid arthritis.

[125I]‐(Sar1, Ile8)AII and [125I]‐AII bound to similar sites on synovial blood vessels, lining cells and stroma. Binding to microvessels (< 100 μm diameter) was more dense than to arteriolar media, and vascular binding was more dense than that to lining cells and stroma.

Microvessels and arterioles which displayed angiotensin converting enzyme‐like immunoreactivity also displayed specific binding of [125I]‐(Sar1, Ile8)AII.

Specific binding of [125I]‐(Sar1, Ile8)AII to each structure was completely inhibited by 10 μm dithiothreitol and was inhibited by unlabelled ligands with the rank order of potency (Sar1, Ile8)AII > AII > losartan = SKF108566 > PD123319 indicating an AT1 subclass of angiotensin receptor.

GTPγS (1 μm) abolished specific binding of [125I]‐AII and abolished the high affinity component of the binding inhibition curve for AII against [125I]‐(Sar1, Ile8)AII, indicating G protein coupling.

The distribution of [125I]‐(Sar1, Ile8)AII binding sites was similar in all disease groups and no significant differences in binding densities, affinities or specificities were observed between disease groups.

Locally generated AII may act on synovial AT1 receptors to modulate synovial perfusion and growth. Specific AT1 receptor antagonists should help elucidate the role of angiotensins in human arthritis.
Requirement for endothelium‐derived nitric oxide in vasodilatation produced by stimulation of cholinergic nerves in rat hindquarters
Authors: Loke K.E. , Sobey C.G. , Dusting G.J. , Woodman O.L.
Published: July 2012

We aimed to determine whether nitric oxide (NO) and/or the endothelium is involved in cholinergic neurogenic vasodilatation in the rat isolated hindquarters.

The abdominal aorta was cannulated for perfusion of the rat hindquarters with Krebs bicarbonate solution containing phenylephrine, to induce basal constrictor tone. In the presence of noradrenergic neurone blockade with guanethidine (200 mg kg−1, i.p.) electrical stimulation of peri‐aortic nerves induced frequency‐dependent decreases in hindquarters perfusion pressure, indicating vasodilatation. Both the endothelium‐dependent vasodilator, acetylcholine (ACh) and the endothelium‐independent vasodilator, sodium nitroprusside (SNP) induced dose‐dependent decreases in perfusion pressure. In each experiment, responses to either nerve stimulation, ACh or SNP were recorded before and after treatment with saline vehicle, atropine (1 μm), NG‐nitro‐l‐arginine (l‐NOARG, 100 μm), l‐arginine (1 mm), l‐arginine plus l‐NOARG, or 3–3 cholamidopropyl dimethylammonio 1‐propanesulphonate (CHAPS, 30 mg). Hindquarters dilatation after each treatment was expressed as a percentage of the control response.

Following treatment with saline, responses to nerve stimulation and ACh were 99 ± 9% and 107 ± 10% of control, respectively demonstrating the reproducibility of these responses. Nerve stimulation‐induced dilatation was abolished by atropine (0 ± 0% of control, P < 0.05) or reduced to 14 ± 10% of control by NO synthase inhibition with l‐NOARG (P < 0.05). Dilator responses to ACh were also abolished by atropine (0 ± 0% of control, P < 0.05) or inhibited by l‐NOARG (59 ± 10% of control, P < 0.05), indicating that the neurogenic dilatation is cholinergic and is mediated by NO. The administration of the NO precursor, l‐arginine, prevented the inhibitory effect of l‐NOARG on dilator responses to nerve stimulation and ACh (l‐arginine plus l‐NOARG: 89 ± 13% and 122 ± 24% of control, respectively). In addition CHAPS, which removes endothelial cells, inhibited responses to both nerve stimulation (0 ± 0% of control, P < 0.05) and ACh (33 ± 8% of control, P<0.05). In contrast, no treatment significantly reduced the vasodilator responses to SNP.

These observations suggest that cholinergic neurogenic vasodilatation in the rat isolated hindquarters requires the synthesis and release of NO from the endothelium.
Effects of glibenclamide on systemic and splanchnic haemodynamics in conscious rats
Authors: Moreau Richard , Komeichi Hirokazu , Kirstetter Philippe , Yang Song , Aupetit‐Faisant Brigitte , Cailmail Stéphane , Lebrec Didier
Published: July 2012

The effects of the sulphonylurea, glibenclamide (20 mg kg−1, i.v.), at a dose that blocks vascular potassium channels, on systemic and splanchnic haemodynamics (radioactive microspheres) were studied in conscious rats.

Glibenclamide significantly decreased cardiac index and hepatic artery blood flow while it significantly increased vascular resistance in systemic, portal and hepatic arterial territories.

In rats with suppressed cardiovascular reflexes, glibenclamide induced vasoconstriction in systemic, portal and hepatic arterial territories.

Intracerebroventricular administration of glibenclamide did not alter systemic or regional vascular tone.

Glibenclamide blunted the vasodilator effect of the potassium channel opener, diazoxide but not that of the L‐type calcium channel blocker, nicardipine.

Another sulphonylurea, glipizide (20 mg kg−1, i.v.), induced significant systemic and splanchnic vasoconstriction.

Thus, the glibenclamide‐induced blockade of vascular potassium channels caused a vasoconstriction in the systemic and splanchnic vascular beds. In these territories, therefore, the opening of glibenclamide‐sensitive potassium channels might be responsible for a basal vasodilator tone.
Blockade by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonate (DIDS) of P2X‐purinoceptors in rat vas deferens
Authors: Bültmann Ralph , Starke Klaus
Published: July 2012

The possibility of an antagonist effect of 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonate (DIDS) at P2X‐purinoceptors was studied in rat vas deferens.

DIDS reduced contractions elicited by α,β‐methylene ATP 3 μm, IC50 1.6 μm, but did not change contractions elicited by K+ 35 mm. DIDS 3.2 μm slightly shifted the concentration‐response curve of α,β‐methylene ATP to the right and reduced the maximum. DIDS 10 μm markedly decreased and DIDS 32 μm abolished contractions over the entire range of the α,β‐methylene ATP concentration‐response curve. DIDS 32 μm also abolished contractions elicited by ATP but did not change contractions elicited by noradrenaline. The antagonist effect of DIDS was only slowly reversible.

The presence of either suramin 320 μm or α,β‐methylene ATP 10 μm during the exposure to DIDS protected the tissue from the long‐lasting blocking effect of DIDS.

4,4′‐Diisothiocyanatodihydrostilbene‐2,2′‐disulphonate (H2DIDS) was equipotent with DIDS whereas several analogues in which one or both of the isothiocyanate residues were replaced were less effective or without effect against α,β‐methylene ATP.

DIDS attenuated the purinergic component of neurogenic contractions elicited by electrical field stimulation, IC50 3.9 μm, but did not change the adrenergic component.

It is concluded that DIDS causes a selective, long‐lasting, non‐equilibrium blockade of P2X‐purinoceptors in rat vas deferens. Due to this effect it also selectively blocks the purinergic component of neurogenic contractions.
Effects of GABA and various allosteric ligands on TBPS binding to cloned rat GABAA receptor subtypes
Authors: Im Wha Bin , Pregenzer Jeffrey F. , Thomsen Darrel R.
Published: July 2012

[35S]t‐butylbicyclophosphorothionate (TBPS) is a high affinity ligand for the picrotoxin site of GABAA receptors. Here we examined TBPS binding to the cloned receptors made of α1, α3 or α6 in combination with β2 or β2 and γ2 subunits, in the presence of GABA and several allosteric ligands (diazepam, methyl 6,7‐dimethoxy‐4‐methyl‐β‐carboline‐3‐carboxylate (DMCM), 3α,21‐dihydroxy‐5α‐pregnan‐20‐one (5α‐THDOC), pentobarbitone and Zn). The cloned receptors were transiently expressed in SF‐9 insect cells by infecting with recombinant baculoviruses.

In αβ subtypes, GABA at nanomolar concentrations enhanced TBPS binding but inhibited binding at micromolar concentrations. Half maximal GABA concentrations for enhancement or inhibition of TBPS binding were correlated with high and low affinity GABA binding sites, respectively, in individual subtypes. The maximal enhancement of binding also varied according to the α isoform (α3β2>>α1β2>α6β2). In αβγ subtypes, TBPS binding was unaffected by GABA at nanomolar concentrations, but was inhibited by GABA at micromolar concentrations. Addition of γ2 thus appeared to abolish conformational coupling between high affinity GABA sites and TBPS sites, and also altered low affinity GABA sites; in particular, the half maximal GABA concentration for inhibition of TBPS binding changed from > 100 (α6β2) to 1 μm (α6β2γ2).

Allosteric ligands also altered TBPS binding to sensitive GABAA receptor subtypes. For instance, diazepam only in the α1β2γ2 and α3β2γ2 subtypes, and 5α‐THDOC in all the subtypes enhanced TBPS binding in the absence of GABA, and intensified the inhibitory effect of GABA. Pentobarbitone exhibited only the latter effect in all the subtypes we examined.

DMCM and Zn, inhibitors of GABA‐induced Cl currents in αβγ and αβ subtypes, respectively, produced opposite effects to agonists, decreasing TBPS binding in the absence of GABA and attenuating (or eliminating in the case of Zn) the inhibitory effect of GABA on TBPS binding.

These results show that GABA binding sites and their conformational coupling with TBPS sites are differentially affected by the α isoform (particularly α6 as compared to α1 or α3) and by quaternary interactions involving the γ2 subunit. Moreover, changes in TBPS binding by allosteric ligands include not only direct (allosteric) effects on TBPS sites but also indirect effects via GABA sites, and are consistent with their known subtype selectivity and functionality from previous studies.
Antiarrhythmic effects of BN‐063, a newly synthesized adenosine A1 agonist, on myocardial ischaemia in rats
Authors: Lee Yen‐Mei , Chern Ji‐Wang , Yen Mao‐Hsiung
Published: July 2012

It has been shown that adenosine is able to reduce the severity of arrhythmias induced by myocardial ischaemia. In isolated preparations, the antiarrhythmic effect of adenosine on ventricular myocardium is known to antagonize the catecholamine‐induced stimulation of intracellular cyclic AMP production, an effect mediated via adenosine A1 receptors.

The aim of this study was to evaluate the antiarrhythmic effect of BN‐063 (1‐cyclo‐propylisoguanosine), a newly synthesized selective adenosine A1 agonist, on ventricular arrhythmias in rats.

Arrhythmias were induced by left coronary artery ligation or by administration of isoprenaline (7 mg kg−1) subcutaneously. Pretreatment with BN‐063 (0.25, 0.5 and 1.0 mg kg−1) 10 min prior to occlusion significantly delayed the onset of ventricular arrhythmias, reduced the total number of ventricular premature contraction (VPC) and ventricular tachycardia (VT), decreased the incidence of VT and ventricular fibrillation (VF) and mortality during the first 30 min following left coronary artery ligation. In contrast, pretreatment with 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), an adenosine A1 antagonist, was arrhythmogenic during the ischaemic period. The rate‐pressure product, an index for indirect measurement of myocardial oxygen consumption, was also significantly reduced by BN‐063 during ligation time.

The incidence of VT, VF and mortality was also significantly reduced when BN‐063 was administered after left coronary artery ligation.

BN‐063 converted the VF induced by isoprenaline to normal sinus rhythm and improved the survival rate.

It is concluded that, through activation of adenosine A1 receptors, BN‐063 can suppress ventricular arrhythmias induced by myocardial ischaemia and catecholamines. The antiarrhythmic actions of BN‐063 may be mediated by reducing heart rate and antagonizing the stimulatory effects of catecholamine in myocardial ischaemia.
Novel histamine H3 receptor antagonists: affinities in an H3 receptor binding assay and potencies in two functional H3 receptor models
Authors: Schlicker E. , Kathmann M. , Reidemeister S. , Stark H. , Schunack W.
Published: July 2012

We determined the affinities of ten novel H3 receptor antagonists in an H3 receptor binding assay and their potencies in two functional H3 receptor models. The novel compounds differ from histamine in that the aminoethyl side chain is replaced by a propyl or butyl chain linked to a polar group (amide, thioamide, ester, guanidine, guanidine ester or urea) which, in turn, is connected to a hexocyclic ring or to an alicyclic ring containing an alkyl residue.

The specific binding of [3H]‐Nα‐methylhistamine to rat brain cortex membranes was monophasically displaced by each of the ten compounds at pKi values ranging from 7.56 to 8.68.

Inhibition by histamine of the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]‐noradrenaline was antagonized by the ten compounds and the concentration‐response curve was shifted to the right with apparent pA2 values ranging from 7.07 to 9.20.

The electrically induced contraction in guinea‐pig ileum strips (which was abolished by atropine) was inhibited by the H3 receptor agonists R‐(−)‐α‐methylhistamine (pEC50 7.76), Nα‐methylhistamine (7.90) and imetit (8.18). The concentration‐response curve of R‐(−)‐α‐methylhistamine was shifted to the right by thioperamide (apparent pA2 8.79) and by the ten novel compounds (range of pA2 values 6.64–8.81).

The affinities and potencies were compared by linear regression analysis. This analysis was extended to thioperamide, the standard H3 receptor antagonist, which is also capable of differentiating between H3A and H3B sites. Comparison of the apparent pA2 values in the two functional H3 receptor models yielded a regression coefficient of 0.77 (P < 0.02). When the pA2 of the drugs in the mouse brain cortex were compared to the pKi for H3 sites (ten novel compounds) and for H3A sites (thioperamide), a significant correlation (r = 0.87; P < 0.001) was obtained. There was, however, no significant correlation when the pKi of thioperamide for H3B sites was used instead (r = 0.52). In a similar manner, comparison of the pA2 in the guinea‐pig ileum with the pKi in the binding assay yielded a significant correlation (r = 0.70, P < 0.05) only when the pKi of thioperamide for H3A sites was used but not when its pKi for H3B sites was considered (r = 0.17, NS).

On the basis of these results, structure‐activity relationships for the novel H3 receptor antagonists, and the nature of the H3 receptors in the guinea‐pig ileum and mouse brain, are considered.