bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro modulation of the eosinophil‐dependent enhancement of the permeability of the bronchial mucosa
Authors: Herbert Carolyn A. , Edwards David , Boot John R. , Robinson Clive
Published: July 2012

Basolateral to apical albumin flux has been measured in sheets of bovine bronchial and tracheal mucosa mounted in vitro.

Addition of guinea‐pig peritoneal eosinophils or neutrophils to the basolateral side of such tissues had no significant influence on the transmucosal flux of albumin in either the bronchial or tracheal mucosa.

Stimulation of eosinophils or neutrophils by the calcium ionophore A23187, or by their presentation to an opsonized airways mucosa, resulted in a significant increase in the transbronchial flux of albumin. This effect was seen after only 60 min incubation of the leucocytes with the bronchial mucosa, and was no greater when the contact time was extended to 180 min. Incubation of bronchial mucosal tissues with 1 mg ml−1polyarginine for 3 h produced a significant increase in albumin flux, but was ineffective at 0.5 mg ml−1.

In contrast to the bronchial mucosa, the tracheal mucosa appeared resistant to the effects of stimulated eosinophils and neutrophils.

The lipoxygenase inhibitor AA‐861 failed to influence the ability of eosinophils to augment the transmembrane flux of albumin. However, insertion of a Millipore filter mask between the eosinophils and the bronchial mucosa significantly inhibited the eosinophil‐dependent enhancement of mucosal permeability.

The broad spectrum antiproteinase α2‐macroglobulin achieved almost total ablation of the action of stimulated eosinophils in the bronchial mucosa. These results suggest that proteinases may make a significant contribution to the genesis of epithelial injury, whereas leukotrienes do not.
The sources of calcium for carbachol‐induced contraction in the circular smooth muscle of guinea‐pig stomach
Authors: Parekh Anant B. , Brading Alison F.
Published: July 2012

The action of carbachol on the mechanical activity of circular muscle from guinea‐pig upper stomach was studied. High concentrations of carbachol (e.g. 10−4m) produced a rapid phasic contraction followed by a smaller, sustained tonic contraction. Low concentrations (e.g. 10−7m) caused a contraction which did not generally show marked distinction between phasic and tonic components.

The response to 10−7m carbachol was very sensitive to 10−5m nifedipine as was the phasic response to 10−4m carbachol. The tonic contraction to the latter, however, was only slightly reduced by nifedipine.

The carbachol‐induced contractions remaining in the presence of nifedipine were dose‐related and very dependent on the presence of external calcium.

Carbachol, 10−7m, did not produce a contraction after 4 min exposure to calcium‐free solution whereas 10−4m carbachol did and this was phasic in nature but much reduced relative to the control in normal Ca.

A phasic followed by a small tonic contraction to 10−4m carbachol was seen superimposed on the K contracture in tissues depolarized with 100 mm K, whereas only a small tonic response occurred for 10−7m carbachol.

In the absence of a functional carbachol‐sensitive intracellular store, 10−4m carbachol was unable to trigger a contraction in calcium‐free solution. However, when calcium was simultaneously readmitted with carbachol after exposure to calcium‐free solution, a contraction occurred.

Carbachol, 10−7m, did not significantly increase inositol polyphosphate levels, whereas 10−4m carbachol did. The increase with 10−4m occurred rapidly peaking within 2 min and was undetectable after 5 min, in the absence of lithium.

It is concluded that low concentrations of muscarinic agonist trigger a contraction predominantly through a nifedipine‐sensitive route whereas higher concentrations further utilize intracellular calcium release and a receptor‐operated extracellular calcium‐dependent pathway. The former is probably associated with the phasic component and the latter with the tonic one.
Opioid activity of dermenkephalin analogues in the guinea‐pig myenteric plexus and the hamster vas deferens
Authors: Sagan Sandrine , Corbett A.D. , Amiche M. , Delfour A. , Nicolas P. , Kosterlitz H.W.
Published: July 2012

To elucidate the structural features required for selective and potent action of dermenkephalin at the δ‐opioid receptor, a series of analogues of dermenkephalin and dermorphin were tested for their effectiveness in depressing electrically‐evoked contractions of the vas deferens of the hamster (δ‐opioid receptors) and the guinea‐pig myenteric plexus‐longitudinal muscle preparation (μ‐ and κ‐opioid receptors).

Dermenkephalin was more selective and more potent at δ‐receptors than the δ‐ligand [d‐Pen2, d‐Pen5]‐enkephalin. The responses to dermenkephalin in the hamster vas deferens were increased by addition of peptidase inhibitors; the maximum effect was obtained with 3 μm thiorphan.

[l‐Met2]‐dermenkephalin had 0.2% and [l‐Ala2]‐dermorphin 0.01% of the agonist activity of the corresponding endogenous peptides which have d‐amino acids in position 2. The pharmacological activity of these analogues was unaffected by inhibition of peptidases. This emphasizes the role that the d‐configuration plays in determining the bioactive folding of these highly active peptides.

Dermenkephalin‐(1–6)‐NH2 was more potent at δ‐receptors than at μ‐receptors whereas, dermenkephalin‐(1–4)‐NH2 is a selective μ‐agonist, having no activity at δ‐receptors.

Substitution of the C‐terminal tripeptide of dermorphin with the C‐terminal tripeptide of dermenkephalin abolished the μ‐receptor preference of dermorphin. The resulting hybrid peptide, Tyr‐d‐Ala‐Phe‐Gly‐Leu‐Met‐Asp‐NH2 was as potent as dermenkephalin at δ‐receptors. A shift towards a preference for δ‐receptors was obtained when the C‐terminal tetrapeptide of dermorphin was replaced by the C‐terminal tetrapeptide of dermenkephalin.

Substitution of Asp by Asn in position 7 of dermenkephalin caused an increase in μ‐receptor potency and a decrease in δ‐receptor potency, resulting in a 20 fold decrease in μ‐receptor selectivity. Dermenkephalin‐(1–6)‐NH2 and [Asn7]‐dermenkephalin have almost identical δ‐receptor agonist potencies and ratios of IC50 in the myenteric plexus to IC50 in the hamster vas deferens.

The results obtained emphasise the importance of a negative charge at the C‐terminus of dermenkephalin for selectivity at the δ‐opioid receptor. Furthermore, the hydrophobic residues Leu5and Met6may be critical in ensuring tight binding to the receptor which results in high agonist potency.
The long‐term effects of the rodenticide, brodifacoum, on blood coagulation and vitamin K metabolism in rats
Authors: Mosterd J.J. , Thijssen H.H.W.
Published: July 2012

The long‐term (30 days) effects of a single dose of brodifacoum (0.2 mg kg−1, orally) on blood clotting activity and on liver parameters of the vitamin K cycle were investigated in rats. Maximal effect on blood clotting activity was seen on day one. On day seven blood clotting activity had returned to normal.

Liver microsomal vitamin KO reductase activity was maximally suppressed (10% of control activity) on day one, steadily recovered to about 40% on day 15 to remain at that level. The same time course was seen for the number of microsomal warfarin binding sites.

The persistent inhibition of the vitamin K cycle was also verified in vivo; following vitamin K administration (10 mg kg−1, i.v.) on day 30, the brodifacoum‐treated rats accumulated vitamin KO in the liver.

Although clotting factor synthesis was normal, brodifacoum‐treated rats were highly sensitive to warfarin.

Brodifacoum rapidly accumulated in the liver until the saturation of the microsomal binding site. Brodifacoum binding to the target prevented its elimination from the liver; liver content on day 30 was not different from day 7.

The results show (1) an over capacity for the hepatocellular vitamin K cycle, (2) a dissociation of the vitamin K epoxidation and the vitamin K‐dependent carboxylation, (3) the ‘superwarfarin’ rodenticides to be extremely persistent due to their binding to the target.
MK801 attenuates behavioural adaptation to chronic nicotine administration in rats
Authors: Shoaib Mohammed , Stolerman Ian P.
Published: July 2012

The effect of administering MK801 (0.3 mg kg−1) during chronic nicotine (0.4 mg kg−1) treatment was studied on locomotor activity in rats. Sensitization of the locomotor activity response to nicotine was seen with chronic nicotine treatment. This sensitization was attenuated in rats that had received MK801 30 min before the daily nicotine injections during chronic treatment. These results suggest that the NMDA receptor may be involved in the adaptive processes seen with chronic nicotine administration.
Effects of BRL 38227 on potassium currents in smooth muscle cells isolated from rabbit portal vein and human mesenteric artery
Authors: Russell Scott N. , Smirnov Sergey V. , Aaronson Philip I.
Published: July 2012

Single smooth muscle cells were isolated from the rabbit portal vein and the human mesenteric artery and whole cell currents recorded at room temperature from either cell type by the whole cell voltage clamp technique.

In the rabbit portal vein cells addition of 10 μm BRL 38227 induced a quasi‐instantaneous, voltage‐insensitive and time‐independent current which had a reversal potential of −75 mV under experimental conditions where the calculated EK was −83 mV.

Cells were held at 0 mV and BRL 38227 was added cumulatively to construct a dose‐response relationship. BRL 38227 (0.03–10 μm) caused a dose‐dependent outward shift in the holding current with an EC50 of 1.3 μm.

BRL 38227 (10 μm) had no effect on the delayed rectifier K+ current measured in the presence of 5 mm tetraethylammonium and no effect on the Ca2+‐activated K+ current measured in the presence of 5 mm 4‐aminopyridine. Similarly BRL 38227 had no effect on the Ca2+ current.

The BRL 38227‐induced current was blocked by glibenclamide (10 μm) and phentolamine (100 μm), specific blockers of the ATP‐sensitive K+ current in single cells.

In human isolated mesenteric artery cells, BRL 38227 (10 μm) induced a glibenclamide‐sensitive current similar to, but smaller than, that observed in the rabbit portal vein.

We conclude that in these cells, BRL 38227 activates a potassium conductance which has the electrophysiological and pharmacological characteristics of ATP‐sensitive K+ channels.
Impaired endothelium‐dependent relaxation of dog coronary arteries after myocardial ischaemia and reperfusion: prevention by amlodipine, propranolol and allopurinol
Authors: Sobey Christopher G. , Dalipram Romina A. , Dusting Gregory J. , Woodman Owen L.
Published: July 2012

Anaesthetized, open‐chest dogs were subjected to 60 min of left circumflex coronary artery occlusion followed by 90 min of reperfusion. Endothelium‐dependent and ‐independent relaxant responses of the isolated coronary arterial rings were then investigated.

The endothelium‐dependent, acetylcholine‐induced relaxation of ischaemic/reperfused arterial rings was significantly attenuated in comparison to control rings (1.9 fold rightward shift, ischaemic/reperfused maximum relaxation = 57 ± 13% of control maximum relaxation; P < 0.05). In contrast, glyceryl trinitrate produced similar relaxant responses in control and ischaemic rings.

Pretreatment of dogs with either amlodipine (3 μg kg−1 min−1, i.v.) or propranolol (1 mg kg−1, i.v.) completely prevented the postischaemic impairment of endothelium‐dependent relaxant responses (100 ± 3% and 90 ± 5% of control maximum relaxation, respectively).

Allopurinol pretreatment (25 mg kg−1, p.o. 24 h previously, plus 50 mg kg−1 i.v. 5 min before arterial occlusion) partially protected against endothelial dysfunction by preventing the ischaemia‐induced rightward shift of the acetylcholine relaxation curve and increasing the maximum relaxation response (83 ± 7% of control rings).

These results confirm that endothelium‐dependent coronary vascular relaxation is impaired by ischaemia and reperfusion, and that the ischaemia‐induced impairment is reduced by pretreatment with amlodipine, propranolol or allopurinol.
Effect of sulphur containing amino acids on [3H]‐acetylcholine release from amacrine cells of the rabbit retina
Authors: Neal Michael J. , Cunningham Jo R.
Published: July 2012

The effects of the sulphur containing amino acids, homocysteic acid, homocysteine sulphinic acid, cysteic acid and cysteine sulphinic acid on the release of [3H]‐acetylcholine ([3H]‐ACh) from the cholinergic amacrine cells of the rabbit retina were examined.

All the compounds stimulated the spontaneous resting release and abolished the light‐evoked release of [3H]‐ACh. Except for homocysteine sulphinic acid these actions occurred at concentrations that did not affect the erg b‐wave amplitude, indicating a site of action at the inner retina.

N‐methyl‐d‐aspartate (in Mg2+‐containing medium) clearly blocked the effects of homocysteic acid and homocysteine sulphinic acid on the resting release of [3H]‐ACh but had no effect on the actions of cysteic acid and cysteine sulphinic acid.

Since N‐methyl‐d‐aspartate is an antagonist of the light‐evoked endogenous bipolar cell transmitter released onto cholinergic cells, these results are consistent with the suggestion that homocysteic acid or homocysteine sulphinic acid may be a transmitter released from this subpopulation of bipolar cells.

The present experiments indicate the existence of excitatory amino acids that have closer pharmacological properties to a bipolar cell transmitter than glutamate but it remains to be seen whether homocysteic acid or homocysteine sulphinic acid occur in these particular bipolar cells.
Studies on the mechanism of 5‐HT1 receptor‐induced smooth muscle contraction in dog saphenous vein
Authors: Sumner Michael J. , Feniuk Wasyl , McCormick Julie D. , Humphrey Patrick P. A.
Published: July 2012

We have investigated the mechanism of smooth muscle contraction evoked by activation of 5‐HT1‐like receptors in dog isolated saphenous vein.

In the presence of the 5‐HT2 receptor antagonist, ritanserin (0.1 μm), concentration‐effect curves (10 nm‐300 μm) for 5‐hydroxytryptamine (5‐HT)‐induced smooth muscle contraction were biphasic. This could be attributed to a direct action on 5‐HT1‐like receptors at low concentrations of 5‐HT (10 nm‐10 μm) and an indirect (through the release of noradrenaline from sympathetic neurones) activation of postjunctional α‐adrenoceptors at higher 5‐HT concentrations. In contrast, concentration‐effect curves (100 nm–100 μm) for sumatriptan‐induced contractions were not biphasic, and were due solely to activation of 5‐HT1‐like receptors.

Smooth muscle contractions evoked either by low concentrations of 5‐HT or by sumatriptan were abolished by removal of extracellular calcium and were markedly inhibited, but not abolished, by the calcium channel blocker, verapamil (1–30 μm). In contrast, contractions evoked by high concentrations of 5‐HT were markedly less sensitive to removal of extracellular calcium or to verapamil.

5‐HT and sumatriptan also inhibited (to a maximum of about 50%) prostaglandin E2 (PGE2,5 μm)‐stimulated adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) formation. This effect was mimicked by the α2‐adrenoceptor agonist, azepexole (B‐HT933) but not by the α1‐adrenoceptor agonist, methoxamine.

In contrast to mediation of smooth muscle contraction, the 5‐HT1‐like receptor‐mediated inhibition of PGE2‐stimulated cyclic AMP formation evoked by 5‐HT or sumatriptan was not attenuated by removal of extracellular calcium or by verapamil (1 μm).

A directly‐acting inhibitor of adenylyl cyclase, 2′,3′‐dideoxyadenosine (1 mm) inhibited PGE2‐stimulated cyclic AMP formation but did not produce smooth muscle contraction.

These results suggest that contractile responses of dog isolated saphenous vein arising through activation of 5‐HT1‐like receptors are associated with both an influx of extracellular calcium ions (to a large extent via voltage‐dependent channels) and an inhibition of adenylyl cyclase. However, although these two responses are coupled to the same receptor, they appear to be independent.
The effect of lipopolysaccharide, interleukin‐1 and tumour necrosis factor on the hepatic accumulation of 5‐hydroxytryptamine and platelets in the mouse
Authors: Endo Yasuo , Nakamura Masanori
Published: July 2012

Injection of lipopolysaccharide (LPS; 0.5–500 μg kg−1) into mice induced a dose‐dependent, slowly developing increase in hepatic content of 5‐hydroxytryptamine (5‐HT). This sustained increase could not be attributed to an LPS‐induced alteration of the pharmacokinetic handling of 5‐HT by stimulation of its uptake or inhibition of its degradation.

Regional differences were apparent in the tissue content of histamine and 5‐HT between mast cell‐deficient (W/Wv) and normal (+/ +) mice. LPS administration (0.5 mg kg−1) gave comparable increases in the hepatic level of 5‐HT in mast cell‐deficient and normal mice.

Reserpine pretreatment (1 mg kg−1) selectively reduced 5‐HT levels in the blood, spleen, liver, brain and lung of normal mice. Prior treatment with this agent also abolished the LPS (0.5 mg kg−1)‐induced hepatic accumulation of 5‐HT.

Accumulation of 5‐HT in the liver by LPS (0.1 mg kg−1) was temporally associated with both a fall in the levels of circulating platelets, and a reduction in the concentration of 5‐HT in the blood. The LPS dose‐dependent (0.5–500 μg kg−1) increase in hepatic 5‐HT content was associated with a similar dose‐dependent reduction in the circulating levels of 5‐HT.

Interleukin‐1, α and β (10 μg kg−1) and tumour necrosis factor α (TNFα) (1 mg kg−1) significantly enhanced the accumulation of 5‐HT within the liver. Administration of TNFα (10 μg kg−1) potentiated the increase in hepatic 5‐HT content seen with IL‐1β (10 μg kg−1).

Electron microscopy revealed numerous platelets in the sinusoidal and perisinusoidal Disse spaces within the liver, in animals pretreated with LPS (0.1 mg kg−1). The platelets retained their intact structure and showed no evidence of degranulation.

These data suggest that the LPS and cytokine‐induced mobilization of 5‐HT in the liver is associated with the hepatic translocation of platelets. This migration appears to be independent of platelet aggregation.
The influence of atropine and atenolol on the cardiac haemodynamic effects of NG‐nitro‐l‐arginine methyl ester in conscious, Long Evans rats
Authors: Widdop R.E. , Gardiner S.M. , Kemp P.A. , Bennett T.
Published: July 2012

In the present study, the extent to which baroreflexes contribute to the cardiac effects of NG‐nitro‐l‐arginine methyl ester (l‐NAME) was assessed in conscious, Long Evans rats chronically instrumented with thoracic electromagnetic flow probes for the measurement of cardiac haemodynamics.

l‐NAME (10 mg kg−1, i.v.) was administered in the absence (n = 6) and in the presence (n = 7) of atropine (1 mg kg−1) and atenolol (1 mg kg−1).

l‐NAME caused a marked increase in mean arterial pressure and marked reductions in total peripheral conductance, cardiac output, heart rate, stroke volume, peak thoracic flow and the maximum rate of rise of aortic flow.

Administration of atropine, after the maximal bradycardic effect of l‐NAME was established, restored the heart rate to resting levels. Concurrently, there was a reduction in stroke volume, such that cardiac output, although transiently elevated, did not show a sustained increase. No other variables were significantly affected by atropine. Additional administration of atenolol had no effect other than to cause a slight bradycardia, such that in the presence of atropine and atenolol, heart rate was not different from that in animals receiving atropine and atenolol before l‐NAME.

In the presence of atropine and atenolol, l‐NAME had similar pressor, vasoconstrictor and cardiac haemodynamic effects to those in untreated animals, although the bradycardia was significantly attenuated. However, there was still a significant reduction in heart rate following l‐NAME in the presence of atropine and atenolol.

These results indicate that the major component of the bradycardia following l‐NAME is indirect and mediated through an increase in vagal efferent activity. However, the substantial reduction in cardiac function caused by l‐NAME is not dependent on the autonomic control of the heart but rather, may depend on the increase in afterload and/or a direct effect of l‐NAME on the heart and/or its vasculature.
α4‐β2 and other nicotinic acetylcholine receptor subtypes as targets of psychoactive and addictive drugs
Authors: Connolly John , Boulter Jim , Heinemann Stephen F.
Published: July 2012

Xenopusoocytes were injected with various muscle and neuronal nicotinic acetylcholine receptor (ACh receptor, cholinoceptor) subunit RNA combinations and their pharmacological properties studied using two‐electrode voltage clamp. The functional expression of one of these combinations, rat α4–2β2, has not been previously described. The α4–2 mRNA is a splicing variant transcribed from the α4 gene. In the experiments reported here, the α4–2β2 subtype was functionally indistinguishable from the α4–1β2 subtype.

For each subtype, the relative potency of nicotine compared with acetylcholine was obtained by estimating the relative concentration of nicotine which would elicit the same current response as 0.1 μmACh. The ratios of these concentrations (nicotine: ACh) for the mouse muscle ACh receptor‐(α1β1γδ) was 96.1:1. In contrast, the ratios for the rat neuronal subtypes were: α2β2, 1.01:1; α3β2, 2.01:1; α4β2, 0.76:1 and α4–2β2, 0.76:1. The much greater relative nicotine sensitivity of the neuronal subtypes as compared with muscle receptors illustrates their potential to mediate the psychoactive and addictive effects of nicotine. However, it does not appear that the differences in relative nicotinic sensitivity among the neuronal receptors themselves can be used as a simple discriminative tool in neuronal tissue.

The slopes of the log dose‐log response curves at low ACh concentrations were all greater than 1 but less than 2, suggesting that at least two agonist binding sites mediate the functional response of each hetero‐oligomer.

The response of all the neuronal subtypes to ACh could be inhibited by the psychoactive drugs mecamylamine, amitriptyline, phencyclidine, trifluoperazine and promethazine. With the exception of the very potent antagonist, mecamylamine, the degree of block of the peak current to ACh produced by 10 μmconcentrations of these drugs was remarkably similar (around 50%).

The degree of inhibition produced when the antipsychotic drug, trifluoperazine, was co‐applied with ACh increased as the duration of application increased. Such an effect was not observed with promethazine, a related phenothiazine derivative which does not have antipsychotic actions.
8‐Hydroxy‐2‐(di‐n‐propylamino)tetralin impairs spatial learning in a water maze: role of postsynaptic 5‐HT1A receptors
Authors: Carli M. , Samanin R.
Published: July 2012

The effect of 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), a 5‐HT1A receptor agonist, on place navigation was studied by use of two spatial tasks in a water maze.

In the first experiment, rats treated subcutaneously with 100 and 300 (but not 30) μg kg−1 8‐OH‐DPAT were impaired in their ability to locate a hidden platform. The probe test confirmed the impairment of spatial navigation but the effect (time spent in the training quadrant) was quantitatively different, depending on whether 8‐OH‐DPAT was administered only before each training session, only before the probe test or in both conditions.

In the second experiment, rats received 150 μg 5,7‐dihydroxytryptamine (5,7‐DHT) intracerebroventricularly to destroy 5‐hydroxytryptamine (5‐HT)‐containing neurones and 24 days later were examined for choice accuracy in a two‐platform spatial discrimination task.

At 100 (but not 30) μg kg−1 8‐OH‐DPAT impaired rats' accuracy with no effect on latency and no errors of omission. In 5,7‐DHT‐treated rats, this dose had a greater effect, including errors of omission. Sham‐operated rats injected with 300 μg kg−1 8‐OH‐DPAT were markedly impaired in accuracy but they had longer latencies and made more errors than controls. All the effects were increased in 5,7‐DHT treated rats.

The results suggest that, at doses causing no apparent changes in motor behaviour or motivation, 8‐OH‐DPAT impairs spatial navigation by stimulating postsynaptic 5‐HT1A receptors in the rat brain.
Effect of K+ channel‐modulating drugs on the vasoconstrictor responses of leukotrienes C4, D4 and angiotensin II in the guinea‐pig isolated perfused heart
Authors: McLeod J.D. , Piper P.J.
Published: July 2012

The vascular actions of leukotrienes C4 (LTC4) and LTD4 in the guinea‐pig isolated perfused heart were studied in the presence of potassium (K+) channel modulatory compounds.

Cromakalim (0.35–10 μm), a K+ channel activator, inhibited the vasoconstrictor responses of LTC4 (30 pmol), LTD4 (30 pmol) and angiotensin II (AII) (100 pmol) in a concentration‐dependent manner.

Glyceryl trinitrate (10 mgl−1) and vasoactive intestinal peptide (10 nm) induced a similar vasodilator action to cromakalim in the isolated heart but had no effect on responses to LTC4 and LTD4.

The inhibitory action by cromakalim (10 μm) on the LTC4 (30 pmol) response could be reversed in the presence of an equimolar concentration of glibenclamide. However, glibenclamide (10 μm) only partially restored the LTD4 (30 pmol) actions.

Galanin (10 nm) and charybdotoxin (60 nm) had no effect on the vascular responses to LTC4 and LTD4 (30 pmol).

Inhibition by cromakalim of coronary vasospasm induced by vascular LTC4, LTD4 and AII appears to be separate from its vasodilator action and it is postulated that a cromakalim‐sensitive mechanism in the coronary vasculature is important in the vasoconstrictor responses to LTC4, LTD4 and AII.
The effect of adrenoceptor antagonists on the ileal brake mechanism in the rat
Authors: Brown Nicola J. , Rumsey R.D.E. , Bogentoft C. , Read N.W.
Published: July 2012

Studies were carried out in the rat to investigate the effect of adrenoceptor antagonists on stomach to caecum transit time under control conditions and during ileal infusion of Intralipid. Stomach to caecum transit time (SCTT) of the head of the meal was measured by use of environmental hydrogen analysis and the distribution of the meal was assessed by a scintigraphic technique.

Four adrenoceptor antagonists were used in these studies, the α1 antagonist prazosin, the α2 antagonist, idazoxan, the β1 antagonist atenolol and the β2 antagonist ICI 118551.

None of the antagonists affected SCTT of the head of the meal during ileal infusion of saline. However, the α1 and β1 antagonists significantly reversed (P < 0.05) the delay in SCTT induced by ileal infusion of Intralipid whereas the α2 antagonist, idazoxan, potentiated this delay (P < 0.05).

Study of the distribution of the radiolabelled meal showed that the Intralipid delayed SCTT by slowing both gastric emptying (P < 0.05) and small bowel transit (P < 0.05).

Prazosin delayed gastric emptying under control conditions (P < 0.001) but did not alter significantly the effect of ileal lipid on the distribution of the meal, 100 min or 200 min after gavage. Idazoxan delayed gastric emptying during ileal infusion of saline (P < 0.05) and produced a proximal shift in the geometric centre of the meal in the small intestine (P < 0.001) and despite the apparent potentiation of the ileal brake effect indicated by the hydrogen analysis, idazoxan increased the amount of radioactivity in the colon 200 min after gavage (P < 0.05).

The meal distribution was more compatible with the hydrogen analysis after administration of the β‐adrenoceptor antagonists. The reversal of the lipid‐induced delay in SCTT caused by atenolol was associated with more radioactivity in the large intestine 200 min after the gavage. ICI 118551 had no significant effects on either the distribution of the meal or the SCTT of the head of the meal.

In conclusion, the data confirm that the sympathetic nervous system normally modulates or mediates the mechanisms that influence gastrointestinal transit in the rat and suggest that these mechanisms may be involved in the ileal brake effect. Nevertheless the data also suggest that simple measurement of the transit of the head of the meal by use of environmental hydrogen analysis may sometimes give a misleading impression of the action of drugs on gastrointestinal transit of the bulk of a test meal.
Short‐term desensitization of the histamine H1 receptor in human HeLa cells: involvement of protein kinase C dependent and independent pathways
Authors: Smit M.J. , Bloemers S.M. , Leurs R. , Tertoolen L.G.J. , Bast A. , de Laat S.W. , Timmerman H.
Published: July 2012

In this study we have investigated the effects of short‐term exposure of cells to histamine on the subsequent H1 receptor responsiveness in HeLa cells, using Ca2+ fluorescence microscopy and video digital imaging.

In HeLa cells, histamine (100 μm) induces an immediate H1 receptor‐mediated biphasic elevation of the intracellular Ca2+ concentration ([Ca2+]i) (basal [Ca2+]i: 81 ± 30 nm, histamine‐induced Ca2+ response: first phase: 1135 ± 79 nm; second phase: 601 ± 52 nm, n = 11).

The histamine H1 receptors on HeLa cells are readily susceptible to desensitization since repetitive exposure of the same group of cells to histamine (100 μm) markedly affected the release and influx component of the induced Ca2+ response (second application of histamine: first phase: 590 ± 92 nm, second phase: 279 ± 47 nm; third application of histamine: first phase: 454 ± 127 nm, second phase: 240 ± 45 nm, n = 6). Video digital imaging revealed an increase in the lag time between stimulation and monitoring of the Ca2+ response and a reduced increase in [Ca2+]i after desensitization with histamine.

Neither the release component of the ATP response (50 μm) nor the caffeine (3 mm)‐induced Ca2+ release were found to be affected by desensitization with 100 μm histamine. However, the second phase of the ATP response was significantly reduced after desensitization with histamine (control cells: 516 ± 33 nm; desensitized cells: 331 ± 96 nm, n = 4, P < 0.05).

Activation of protein kinase C (PKC) by phorbol‐12‐myristate‐13‐acetate was found to inhibit the histamine as well as ATP‐induced Ca2+ response in a dose‐dependent manner.

6 In PKC downregulated cells the second phase of the histamine‐induced Ca2+ response was significantly elevated, indicating the involvement of PKC in the negative feedback on the Ca2+ influx (control cells: second phase: 601 ± 52 nm (n = 11); PKC downregulated cells: second phase: 890 ± 90 nm, n = 10, P < 0.05).

7 Homologous desensitization of H1 receptor responsiveness was still observed in PKC downregulated cells, implying the rapid activation of a regulatory mechanism other than PKC.

8 Based on our experimental data we suggest that short‐term desensitization of the histamine H1 receptor evolves from two different processes: a selective reduction of the histamine‐induced Ca2+ release, mediated by a PKC‐independent pathway, and a non‐selective inhibition of the receptor‐mediated Ca2+ influx activated by a PKC‐dependent pathway.
Pharmacological modulation of inhaled sodium metabisulphite‐induced airway microvascular leakage and bronchoconstriction in the guinea‐pig
Authors: Sakamoto Tatsuo , Elwood Wayne , Barnes Peter J. , Chung K. Fan
Published: July 2012

We have investigated the effects of chlorpheniramine, atropine and capsaicin pretreatment on inhaled sodium metabisulphite (MBS)‐induced airway microvascular leakage and bronchoconstriction in anaesthetized guinea‐pigs in order to clarify the mechanisms involved in these responses. The effects of frusemide and nedocromil sodium were also examined.

Lung resistance (RL) was measured for 6 min after inhalation of MBS (20, 40, 80 and 200 mm; 30 breaths), followed by measurement of extravasation of Evans blue dye into airway tissues, used as an index of airway microvascular leakage. MBS caused an increase in RL and leakage of dye at all airway levels in a dose‐dependent manner.

Chlorpheniramine (10 mg kg−1, i.v.), atropine (1 mg kg−1, i.v.), their combination or inhaled nedocromil sodium (10 mg ml−1, 7 min) had no effect against the airway microvascular leakage induced by 80 mm MBS (30 breaths). Capsaicin pretreatment (50 mg kg−1, s.c.) caused a significant decrease in the leakage of dye in the main bronchi and inhaled frusemide (10 mg ml−1, 7 min) also in the main bronchi and proximal intrapulmonary airway.

Chlorpheniramine, atropine, their combination, capsaicin pretreatment and frusemide, but not nedocromil sodium, inhibited significantly the peak RL induced by 80 mm MBS (30 breaths) by approximately 50%.

We conclude that a cholinergic reflex and neuropeptides released from sensory nerve endings may participate in the mechanisms of MBS‐induced airway responses. Frusemide but not nedocromil sodium may have an inhibitory effect on these neural mechanisms. The inhibitory effect of nedocromil sodium against lower doses of MBS is not excluded.
Lack of effect of the antimigraine drugs, sumatriptan, ergotamine and dihydroergotamine on arteriovenous anastomotic shunting in the dura mater of the pig
Authors: den Boer Marinus O. , Somers Judith A.E. , Saxena Pramod R.
Published: July 2012

In anaesthetized animals, the antimigraine drugs, sumatriptan, ergotamine and dihydroergotamine, reduce carotid arteriovenous anastomotic shunting. Within the carotid vascular bed arteriovenous anastomoses are located, amongst other places in the dura mater, which is a putative site of the pain during a migraine attack.

In this investigation, we have localized and measured the arteriovenous shunting within the carotid vascular bed of the pig by using simultaneous intracarotid injections of radiolabelled microspheres of three different sizes (10, 15 and 50 μm), which provides an index of blood flow via arteriovenous anastomoses larger than approximately 14, 27 and 90 μm diameter, respectively. The effects of sumatriptan (0.3 mg kg−1), ergotamine (0.02 mg kg−1), dihydroergotamine (0.1 mg kg−1) and saline were studied by repeating the injections of 15 and 50 μm spheres after the treatments.

There was no difference in shunting or entrapment between the 10 and 15 μm microsphere, indicating the absence of arteriovenous anastomoses with a diameter between 14 and 27 μm.

Arteriovenous anastomoses with a diameter between 27 and 90 μm, as indicated by the difference in blood flow measured by 15 and 50 μm spheres, were located in the dura mater, ears, skin, fat and, to a lesser extent, in the skeletal muscles and eyes.

Sumatriptan, ergotamine and dihydroergotamine reduced the overall flow in the smaller arteriovenous anastomoses (diameter between 27 and 90 μm), and even more in larger shunts (wider than 90 μm).

Locally, blood flow in the smaller arteriovenous shunts was reduced in the skin and fat, but not in the dura mater, ears, eyes and muscles. It is not possible to determine in which tissues blood flow in the larger arteriovenous anastomoses was reduced.

Tissue blood flow measured with 15 μm microspheres remained unchanged after the three antimigraine drugs, implying a lack of effect on capillary flow.

It is concluded that in the anaesthetized pigs the only evident effect of these antimigraine drugs on carotid haemodynamics is a decrease in blood flow in both smaller and larger arteriovenous anastomoses; the smaller arteriovenous anastomoses were affected in the skin and fat, but not in other tissues.
Endothelial‐dependent sexual dimorphism in vascular smooth muscle: role of Mg2+ and Na+
Authors: Zhang Aimin , Altura Bella T. , Altura Burton M.
Published: July 2012

. In isolated aortae of the male rat [Mg2+]o withdrawal and concomitant reduction in [Na+]o (to 84 mm) induced significant increases of basal tone, but, surprisingly, this did not occur in intact aortae removed from female rats. Such tension development, however, was observed in endothelium‐denuded aortic preparations from both sexes. These observed gender‐related differences were not dependent on animal strain or types of tissue preparations.

. No tension development was observed in aortae obtained from castrated males treated with oestradiol. Aortic tissues of sexually‐immature male and female rats exhibited marked tension development when exposed to 0 mm [Mg2+]o and low [Na+]o.

. Tension development in Mg2+‐free, low‐Na+ media was not tachyphylactic and completely dependent on extracellular Ca2+; addition of 1.2 mm Mg2+ to the Mg2+ and Na+‐deficient incubation media relaxed the increase in tension to a normal basal level.

. Two known endothelial‐derived relaxant factor (EDRF) inhibitors, methylene blue and haemoglobin, induced tension development in female aortae with intact endothelium exposed to Mg2+‐Na+ deficient media, while use of a specific inhibitor of EDRF‐derived nitric oxide, viz., NG‐monomethyl‐l‐arginine (l‐NMMA), resulted in potentiation of tension development in male, but not in female, aortae. This effect of l‐NMMA was antagonized by l‐arginine.

. The Ca ionophore, A23187, partially relaxed contractile responses in male aortae (with intact endothelium) which were followed by potentiated contractions. Endothelium‐dependent vasodilator responses to A23187 (10−10−10−6 m) of aortic rings from male or female rats in normal Krebs‐Ringer bicarbonate solution were not different.

. These results suggest that: (a) as in vascular smooth muscle cells, Mg2+ plays an important role in Ca2+ homeostasis in endothelial cells, probably via Na+‐Ca2+ exchange; and (b) sex steroid hormones, probably the female sex hormone, 17‐β‐oestradiol, may regulate contractile responses of intact vascular smooth muscle by modifying endothelium functions through such Mg2+‐regulated internal Na+‐dependent Ca2+ entry. These data may help to explain why female subjects, despite Mg deficiency, unlike male subjects, are protected against ischaemic heart disease and cerebrovascular disease until menopause.
M‐currents in frog sympathetic ganglion cells: manipulation of membrane phosphorylation
Authors: Chen Hsinyo , Smith Peter A.
Published: July 2012

. The inward current and the M‐current (IM) suppression produced when muscarine is applied to frog sympathetic ganglion cells was recorded by means of the whole‐cell patch‐clamp technique. The holding potential was −30 mV and [K+]o was 6 mm.

. The steady‐state IM was maintained for at least 20 min when the patch pipette contained neither adenosine 5′‐triphosphate (ATP) nor adenosine 3′:5′‐cyclic monophosphate (cyclic AMP). Inclusion of these substances or the ATP antagonst, β,γ‐methyleneadenosine 5′‐triphosphate (β,γ‐MethATP; 1 or 2 nm) (failed to alter the rate of IM ‘run down’. By contrast, inclusion of adenosine‐5′‐O‐(3‐thiotriphosphate) (ATP‐γ‐S, 1 or 2 mm) resulted in a 60% reduction of the current within 18 min.

. Despite the inability of ATP‐γ‐S to maintain steady‐state IM, it had no effect on the ability of muscarine (2–100 μm) to suppress a constant fraction of the available current. ATP‐γ‐S and β,γ‐MethATP increased the rise time and duration of the response to muscarine.

. Inclusion of a phosphatase inhibitor, diphosphoglyceric acid (DPG, 1–2.5 mm) or alkaline phosphatase (100 μg ml−1) failed to affect the amplitude of muscarinic responses.

. These results question the role of the phosphorylation and/or dephosphorylation reactions in the transduction mechanism for muscarine‐induced IM suppression but are consistent with the possibility that M‐channels are ‘directly coupled’ via G‐protein to the muscarinic receptor.
An ubiquitous modulating function of rabbit tracheal epithelium: degradation of tachykinins
Authors: Inoue Kazuaki , Sakai Yasushi , Homma Ikuo
Published: July 2012

To examine the role of epithelium in the responsiveness of tracheal smooth muscle in rabbit, we measured the contractile responses to acetylcholine (ACh), KCl, 5‐hydroxytryptamine (5‐HT), histamine, substance P (SP), neurokinin A (NKA), and electrical field stimulation (EFS) in intact and epithelium‐denuded preparations.

Removal of epithelium did not alter the contractile response to any agonist examined, except SP.

Removal of epithelium enhances the contractile response to SP. In the presence of phosphoramidon, the contractile response to SP was not significantly different in either group. The results suggest that the effect of epithelium is largely due to degradation of SP by enzymes in the epithelium.

Arachidonic acid metabolites did not seem to be related to the responses induced by contractile agonists or EFS.

In the presence of SP, the contractile responses and [3H]‐choline outflow evoked by EFS were dose‐dependently enhanced. Contractile responses to EFS and [3H]‐choline outflow evoked by EFS were enhanced by SP significantly more than by NKA. Both were abolished by atropine or tetrodotoxin.

These results suggest that rabbit tracheal epithelium may modulate SP‐induced contractions, both direct and indirect, by inactivation of SP. This phenomenon is widespread in mammals. The rabbit may be a useful model to examine airway cholinergic functions.
Cytoprotection by iloprost against paracetamol‐induced toxicity in hamster isolated hepatocytes
Authors: Nasseri‐Sina P. , Fawthrop D.J. , Wilson J. , Boobis A.R. , Davies D.S.
Published: July 2012

The ability of iloprost (ZK36374) to protect hamster isolated hepatocytes from the toxic effects of paracetamol and its reactive metabolite N‐acetyl‐p‐benzoquinoneimine (NABQI) was investigated. The cytoprotection provided by iloprost was compared with that of N‐acetyl‐l‐cysteine.

Treatment of hepatocytes with either NABQI (0.4 mm) or paracetamol (2 mm) alone resulted in a considerable loss of cell viability, as assessed by trypan blue exclusion or leakage of lactate dehydrogenase, accompanied by an increase in the percentage of viable cells that were blebbed. N‐acetyl‐l‐cysteine (1.25 mm) pretreatment diminished the loss of cell viability and the percentage of blebbed cells resulting from exposure to NABQI or paracetamol, whereas iloprost (10−16 m to 10−10 m) pretreatment reduced only the loss of cell viability, not the percentage of viable cells exhibiting blebbing. Pretreatment with N‐acetyl‐l‐cysteine significantly attenuated the depletion by paracetamol of glutathione and decreased the covalent binding of [14C]‐paracetamol to cellular proteins, whereas iloprost was without any such effects.

The effects of iloprost and N‐acetyl‐l‐cysteine were also investigated by use of a model of paracetamol toxicity in which it is possible to study the biochemical events leading to cell injury separate from the generation of toxic metabolites. Hamster hepatocytes were incubated with paracetamol (4 mm) for 90 min at 37°C during which metabolism of paracetamol occurs with minimal loss of cell viability. Following washing of cells, to remove paracetamol and its metabolites, there was a progressive loss of viability and increase in the percentage of cells exhibiting blebbing when incubated in buffer alone. Addition of either N‐acetyl‐l‐cysteine (1.25 mm) or iloprost (10−14 m to 10−8 m), following washing, significantly reduced the expected loss of cell viability. Iloprost at concentrations outside this range was without effect.

Paracetamol toxicity to isolated hepatocytes could be prevented or delayed by treatment with either N‐acetyl‐l‐cysteine or iloprost, but whereas the former prevented or even reversed plasma membrane blebbing with a resultant reduction in the percentage of viable cells that were blebbed, the prostanoid appeared only to delay the progression from plasma membrane blebbing to loss of viability. Hence, the percentage of viable cells that were ultimately blebbed following exposure to paracetamol was not significantly reduced by addition of iloprost.

Aspirin or ibuprofen exacerbated the loss of viability induced by prior incubation with paracetamol. Thus, there may be a role for endogenous prostaglandins in protecting hepatocytes from paracetamol toxicity.

Iloprost is cytoprotective without any effect upon toxin metabolism or detoxication. The mechanism of action of iloprost probably does not involve induction of prostaglandin synthesis or activation of the previously‐characterized prostacyclin receptor.
Pharmacological characterization of a receptor for calcitonin gene‐related peptide on rat, L6 myocytes
Authors: Poyner David R. , Andrew David P. , Brown Derek , Bose Christopher , Hanley Michael R.
Published: July 2012

The L6 myocyte cell line expresses high affinity receptors for calcitonin gene‐related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) accumulation.

In intact cells at 37°C, human and rat α‐ and β‐CGRP all activated adenylyl cyclase with EC50s of about 1.5 nm. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22°C in 1 mmMg2+, the above all bound to a single site with IC50s of 0.1–0.4 nm.

The fragment CGRP(8–37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculatedKdof 5 nm. TheKddetermined in membrane binding assays was lower (0.5 nm).

The N‐terminal extended human α‐CGRP analogue Tyro‐CGRP activated adenylyl cyclase and inhibited [125I]‐iodohistidyl‐CGRP binding less potently than human α‐CGRP (EC50for cyclase = 12 nm, IC50for binding = 4 nm).

The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology.
Different patterns of release of endothelium‐derived relaxing factor and prostacyclin
Authors: Mitchell Jane A. , DE Nucci Gilberto , Warner Timothy D. , Vane John R.
Published: July 2012

Release of endothelium derived relaxing factor (EDRF) and prostacyclin (PGI2) from endothelial cells (EC) cultured from bovine aortae was measured by bioassay and radioimmunoassay, respectively, during infusions (10 min) of bradykinin (BK), adenosine diphosphate (ADP), arachidonic acid (AA), alkaline buffers and the free‐bases (FB) of l‐arginine or d‐arginine. Release of EDRF from the luminally perfused rabbit aorta was also measured during infusions (10 min) of acetylcholine (ACh), substance P and ADP.

Bradykinin (10 or 30 nm) infused through the column of EC induced release of both EDRF and PGI2, neither of which was maintained for the duration of the infusion.

ADP (1.6 or 4 μm) infused through the column of EC induced release of a EDRF which was maintained for the duration of the infusion and a release of PGI2 which lasted for a much shorter period.

Arachdonic acid (30 or 90 μm) infused through the column of EC caused a sustained release of EDRF and PGI2, both of which outlasted the infusion of AA.

l‐Arginine FB, d‐arginine FB or alkaline buffer infused through the column of EC released EDRF, but only small amounts of PGI2. The release of EDRF outlasted the period of infusion and was due to an increase in the pH of the Krebs solution perfusing the EC.

Infusions of ACh (0.25–1 μm) or ADP (4–16 μm) caused a sustained release of EDRF from the luminally‐perfused rabbit aorta, whereas infusion of substance P (3.3–10 μm) caused only a transient release of EDRF.

These results show that distinct patterns of EDRF release exist to different agonists in both cultured and in situ EC, and that EDRF and PGI2 do not necessarily follow the same time course of release. Furthermore, sustained release of EDRF does not require the constant infusion of the precursor, l‐arginine, whereas sustained release of PGI2 only occurs when AA, the precursor of PGI2, is present in the extracellular medium.