bluuebunny/crossref_metadata_2025 huggingface dataset:
Significance of nitric oxide in the stimulation of intestinal fluid absorption in the rat jejunum in vivo
Authors: Schirgi‐Degen Andrea , Beubler Eckhard
Published: July 2012

The effects of inhibiting nitric oxide (NO)‐synthase on fluid transport, mucosal cyclic GMP and cyclic AMP levels and intraluminal prostaglandin E2 (PGE2)‐release were studied in a model of ligated jejunal loops of anaesthetized rats in vivo. Experiments were performed under basal conditions as well as under conditions, when net fluid secretion was induced by Escherichia coli heat stable enterotoxin a (E. coli STa) or PGE2.

Intravenous infusion of the NO‐synthase inhibitor Nω‐nitro‐l‐arginine methyl ester (l‐NAME, 0.25–50 mg kg−1, 45 min) dose‐dependently reversed net fluid absorption to net secretion, whereas infusion of d‐NAME, the inactive enantiomer of l‐NAME, in corresponding doses did not influence net fluid transport. Nω‐nitro‐l‐arginine (l‐NOARG, 25 mg kg−1), another NO‐synthase inhibitor, also elicited net secretion of fluid.

l‐NAME (25 mg kg−1)‐induced net fluid secretion was reversed to net absorption by infusion of l‐arginine (400 mg kg−1) or sodium nitroprusside (1 mg kg−1) and s.c. administration of indomethacin (10 mg kg−1). Hexamethonium (1 mg kg−1, s.c.), a ganglionic blocker and granisetron (100 μg kg−1, s.c.), a 5‐HT3‐receptor antagonist, did not influence l‐NAME‐induced net secretion.

Net fluid secretion induced by intraluminal instillation of E. coli STa (10 units ml−1) was enhanced by infusion of l‐NAME (25 mg kg−1) and was inhibited by infusion of l‐arginine (400 mg kg−1) and sodium nitroprusside (1 mg kg−1). d‐Arginine (400 mg kg−1) did not influence E. coli STa‐induced fluid secretion. Likewise, net fluid secretion induced by i.a. infusion of PGE2 (79 ng ml−1, 30 min) was enhanced by infusion of l‐NAME and was inhibited by l‐arginine and sodium nitroprusside. d‐Arginine (400 mg kg−1) did not influence PGE2‐induced fluid secretion.

PGE2 levels in intraluminal fluid were not elevated after infusion of l‐NAME (25 mg kg−1) compared to controls.

Mucosal cyclic GMP and cyclic AMP levels after l‐NAME‐treatment were not different from control values.

These results indicate that nitric oxide plays an important role in the regulation of intestinal fluid transport. The data suggest a nitric oxide‐dependent proabsorptive tone in the intestine, which possibly involves the enteric nervous system and suppression of prostaglandin formation. This proabsorptive tone also may downregulate fluid secretion induced by E. coli STa or PGE2.
Functional characterization of the adenosine receptor mediating inhibition of intestinal secretion
Authors: Hancock Debra L. , Coupar Ian M.
Published: July 2012

Previous studies have shown that the mixed A1/A2 adenosine agonist 5′‐N‐ethylcarboxamido‐adenosine (NECA) inhibits intestinal fluid secretion which is thought to contribute to its antidiarrhoeal effect in the rat. The aim of this study was to characterize the adenosine receptor mediating this antisecretory effect via functional studies using a range of selective agonists and antagonists and by applying the pharmacological criteria of relative agonist and antagonist potencies.

Adenosine agonists and antagonists were administered i.v. to anaesthetized rats. Intestinal secretion was then stimulated by i.a. infusion of vasoactive intestinal peptide (VIP, 0.8 μg min−1) and the net fluid transport across the wall of the jejunum was measured by a recirculation technique.

The rank order of agonist potency to reduce the response to VIP was: NECA > N6‐cyclopentyladenosine (CPA) > R‐N6‐(2‐phenylisopropyladenosine) (R‐PIA) > S‐PIA > chloroadenosine (2‐CADO) > 2‐phenylaminoadenosine (CV‐1808). This order best complies with the rank order of agonist potency that represents activation of the recently described A2B receptor: NECA > 2‐CADO R‐PIA = CHA > S‐PIA > = CV‐1808 > = CGS‐21680. The most potent agonists (NECA, CPA and R‐PIA) had ED50 values in the low microgram range.

The anitsecretory action of NECA (submaximal dose of 40μg kg−1) was antagonized equally (approximately 50%) by the selective adenosine antagonists 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 0.1 mg kg−1) and 8‐phenyltheophylline (8‐PT, 0.1 mg kg−1). This equipotent activity indicates the presence of an A2 and not an A1 receptor.

It is suggested that adenosine A2B receptor agonists could be evaluated for potential use as antidiarrhoeal drugs.
Pharmacology of stretch‐activated K channels in Lymnaea neurones
Authors: Small Daniel L. , Morris Catherine E.
Published: July 2012

Single‐channel recording was used to describe the pharmacology of stretch‐activated K channels in Lymnaea neurones using channel blockers amiloride, tetraethylammonium (TEA), quinidine, gadolinium (Gd) and diltiazem.

Amiloride, TEA and quinidine applied to the outside face of the membrane all produced a fast flickery block of stretch‐activated K channels. All of these agents were without effect when applied at the inside face at concentrations as high as 10, 200 and 10 mm respectively. Neither Gd nor diltiazem had any effect on stretch‐activated K channels extracellularly (100 μm).

Amiloride, TEA and quinidine block were voltage‐independent with IC50 values at positive (and negative membrane potentials of 2.3 (and 2.0) mm, 48 (and 54) mm and 0.8 (and 0.7) mm respectively. Woodhull plots for TEA and quinidine block confirmed the voltage independence of stretch‐activated K channel block by these agents.

Hill plots of the amilorde, TEA and quinidine block yield Hill coefficients at positive (and negative) membrane potentials of 1.7 (and 1.5), 1.4 (and 1.2) and 1.5 (and 1.6 mm) respectively.

Ethanol (3%) had no apparent effect on stretch‐activated K channel kinetics or conductance yet reduced the efficacy of quinidine block.

The above pharmacological fingerprint of the stretch‐activated K channel is discussed with reference to other K‐selective and stretch‐activated channels.
Mechanisms of pulmonary vasoconstriction and bronchoconstriction produced by PAF in the guinea‐pig: role of platelets and cyclo‐oxygenase metabolites
Authors: Argiolas Livio , Fabi Fulvia , del Basso Paola
Published: July 2012

The mechanisms of action of platelet activating factor (PAF) in the bronchial and cardiovascular systems have not yet been fully elucidated. In order to characterize better and to ascertain whether the effects of PAF in both these systems may be ascribed to the same mechanisms, we examined the actions of PAF in the heart‐lung preparation of guinea‐pig (HLP). The role of platelets and of cyclo‐oxygenase metabolites was investigated.

In HLPs perfused with autologous blood, bolus injections of PAF (4–32 ng) produced major effects at the pulmonary vascular and bronchial levels. Both dose‐related pulmonary vascular hypertension and bronchoconstriction produced by PAF were diminished to the same extent (46% and, respectively, 47%) when HLPs were perfused with a medium consisting of homologous red blood cells suspended in physiological solution containing 3.5% dextran (RBC). This suggests that the effects of PAF partially depend on the presence of formed elements.

When indomethacin (30 μm) was added to the perfusing blood, the dose‐response curve for the pulmonary hypertensive responses produced by PAF was strongly reduced (90%) in comparison to control preparations, whereas the bronchoconstrictor effects of PAF were only partially diminished (23%). These data constitute direct evidence that products of the cyclo‐oxygenase pathway exert a major role in the vascular, rather than in the bronchial actions of PAF.

In HLPs perfused with RBC containing indomethacin (30 μm), the pulmonary vascular hypertensive responses produced by PAF were almost completely abolished, thus indicating that cyclo‐oxygenase products from tissues are involved in these effects. Conversely, PAF administration continued to cause dose‐related bronchoconstrictor responses that were reduced only partially in comparison with HLPs perfused with RBC in the absence of the cyclo‐oxygenase inhibitor. This implies that PAF also has direct action on the bronchoconstriction evoked.

At the cardiac level, administration of PAF in HLPs perfused with blood caused a dose‐related increase in right atrial pressure accompanied by a decrease in left atrial pressure and cardiac output, which were completely suppressed or attenuated by the absence of formed elements and the addition of indomethacin. This suggests that the progressive heart impairment is secondary to the severe pulmonary hypertension induced by PAF.

The results of this study performed in the heart‐lung preparation of the guinea‐pig, which made it possible to simultaneously record cardiovascular and bronchial parameters, indicate that various components are involved in the responses produced by PAF. It is suggested that different mechanisms depending on the relative contribution of these components may account for the PAF‐induced effects at the pulmonary vascular and airway levels.
The effects of calyculin A upon calcium‐, guanine nucleotides‐ and phorbol 12‐myristate 13‐acetate‐stimulated ACTH secretion from AtT‐20 cells
Authors: McFerran B.W. , Guild S.B.
Published: July 2012

The mouse AtT‐20/D16‐16 anterior pituitary tumour cell line was used as a model system for the study of protein phosphatase involvement in the late stages of the secretory pathway for adrenocorticotrophin (ACTH) secretion. The effects of the type 1 and 2 phosphatase inhibitor calyculin A upon calcium‐, guanine nucleotide‐ and phorbol 12‐myristate 13‐acetate (PMA)‐stimulated ACTH secretion from electrically‐permeabilized AtT‐20 cells were studied.

Calyculin A (1 nM‐1 μm) inhibited both calcium (10 jam)‐ and guanosine 5′‐0‐(3‐thiotriphosphate) (GTP‐γ‐S) (100 μm)‐evoked ACTH secretion from permeabilized cells in a concentration‐dependent manner. These effects were maximal with 100 nM calyculin A.

ACTH secretion was stimulated from electrically‐permeabilized cells when the cytosolic free calcium ion concentration, controlled by calcium‐EGTA buffers, was raised over the concentration range of 100 nM to 10 μm. This calcium‐stimulated ACTH secretion was inhibited by co‐incubation with calyculin A (100 nM).

GTP‐γ‐S (10nM‐100μm) stimulated ACTH secretion from permeabilized cells at concentrations greater than 1 μm GTP‐γ‐S. Co‐incubation with calyculin A (100 nM) inhibited this stimulation of ACTH secretion observed at these concentrations of GTP‐γ‐S.

PMA (100 nM) significantly stimulated ACTH secretion from permeabilized cells in the absence of either calcium and guanine nucleotides and this action was enhanced by calyculin A (100 nM). Furthermore, an inhibition of GTP‐γ‐S (100 μm)‐stimulated ACTH secretion observed in the presence of calyculin A (100 nM) was not observed in the presence of PMA (100 nM).

The results of the present study indicate that dephosphorylation by phosphatases plays an important role in stimulus‐secretion coupling in AtT‐20 cells and is involved in mediating the effects of GE upon the secretory apparatus in these cells. Furthermore, the point of regulation of the secretory response by PKC which underlies the ability of PKC to amplify the calcium/GE system may lie distal to both GE and these phosphatases.
Heparin‐insensitive calcium release from intracellular stores triggered by the recombinant human parathyroid hormone receptor
Authors: Seuwen Klaus , Boddeke Hendrikus G.W.M.
Published: July 2012

In the present study we have characterized the parathyroid hormone (PTH)‐induced calcium signalling in 293 cells stably transfected with the human PTH receptor cDNA. In these cells, human PTH‐l(l‐38) strongly stimulates adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) formation (EC50 = 0.39 nM) but fails to activate phosphoinositide (PI) turnover. The latter pathway is strongly activated, however, by carbachol (CCh) acting through endogenous M3‐muscarinic receptors.

Despite the lack of detectable inositol phosphate (IP) formation, hPTH‐(l‐38) elicited calcium transients (EC50 =11.2 nM) which were comparable to the signals evoked by CCh. These signals are independent of cyclic AMP generation as cyclic AMP elevating agents did not mimic or modify the PTH response.

The PTH‐stimulated calcium signal still occurred in calcium‐free medium but was absent in cells pretreated with thapsigargin, an inhibitor of the calcium pump of the endoplasmic reticulum (ER). hPTH‐(l‐38) did not accelerate Mn2+‐influx through the plasma membrane. These data indicate that PTH releases calcium from intracellular stores.

Using heparin, an inhibitor of the IP3‐activated calcium release channel of the ER, we tested whether the formation of a low amount of IP3, escaping detection by our biochemical assay, might be the origin of the PTH‐induced calcium response. However, intracellular infusion of heparin through patch pipettes in voltage clamp experiments failed to block hPTH‐(l‐38)‐induced calcium signals, whereas it abolished the CCh response.

The PTH response, like the CCh response, was insensitive to micromolar concentrations of ryanodine and ruthenium red, eliminating the possibility that hPTH‐(l‐38) stimulates calcium‐induced calcium release through ryanodine receptors.

We conclude that the recombinant human PTH receptor stimulates calcium release from intracellular stores through a novel pathway not involving IP3‐ or ryanodine receptors.
Protein kinase A and nicotinic activation of bovine adrenal tyrosine hydroxylase
Authors: Marley Philip D. , Thomson Kerrie A. , Bralow Roger A.
Published: July 2012

Stimulation of nicotinic cholinoceptors on bovine chromaffin cells increases phosphorylation of three serine residues in tyrosine hydroxylase (TOH) and activates TOH. One of the serines is a target for protein kinase A phosphorylation, and phosphorylation of this serine is adequate alone to cause TOH activation. The role of protein kinase A in nicotinic activation of TOH was therefore investigated.

TOH activity was studied in situ in intact, cultured, bovine adrenal chromaffin cells, by measuring 14C02 evolved following the hydroxylation and rapid decarboxylation of [14C]‐tyrosine offered to the cells.

Nicotine (5 μm), forskolin (1 μm) and 8‐bromo‐cyclic AMP (8‐Br‐cyclic AMP, 1 mM) each increased TOH activity by up to 200% over 10 min. The effect of nicotine was completely abolished by removal of extracellular Ca2+.

TOH activation by all three drugs was blocked by H89 (3–20 4mUm), which inhibits protein kinase A by competing for the ATP binding site on the kinase. Adenosine 3′:5′‐cyclic monophosphorothioate Rp‐diastereomer (Rp‐cAMPS) (1 mM), an inhibitor of protein kinase A that competes with cyclic AMP for the regulatory subunit of the kinase, abolished the activation of TOH by nicotine, and reduced that by forskolin and 8‐Br‐cyclic AMP. Both H89 and Rp‐cAMPS inhibited basal TOH activity by 50–80%.

A structural analogue of H89, H85 (3–20 μm), which lacks activity as a protein kinase A inhibitor, did not inhibit either the activation of TOH by nicotine (5 μm) or basal TOH activity. Neither sodium nitroprusside (0.3–10 μm) nor 8‐Br‐cyclic GMP (1 mM) increased TOH activity.

In digitonin‐permeabilized chromaffin cells, forskolin (3 μm), cyclic AMP (10 μm) and Ca2+ (approx. 2 μm free Ca2+) each increased TOH activity. The response to all three drugs was blocked by H89 (10 μm), which also reduced basal TOH activity in the permeabilized cells.

Maximal activation of TOH by forskolin was achieved with 10 μm forskolin. This concentration was less than the EC50 for forskolin‐induced cyclic AMP accumulation in these cells. The activations of TOH by forskolin (10 μm) and nicotine (5 μm) were additive.

The results indicate that both basal TOH activity and nicotinic activation of TOH in bovine chromaffin cells require protein kinase A activity. However, it is unlikely that nicotinic activation of TOH is directly mediated by an activation of protein kinase A in response to elevated cyclic AMP levels. It is possible that protein kinase A plays a permissive role in allowing nicotinic cholinoceptors to activate TOH by another signalling pathway.
Contribution of P2‐purinoceptors to neurogenic contraction of rat urinary bladder smooth muscle
Authors: Hashimoto Minori , Kokubun Shinichiro
Published: July 2012

The contribution of P2‐purinoceptors to neurogenic contraction was investigated in rat urinary bladder smooth muscle by measurement of isotonic tension.

Contraction of rat urinary bladder smooth muscle induced by electrical stimulation was decreased to 84.19±3.90% of the control (n= 16) in the presence of atropine (1 μm), which was further decreased to 38.80 ±2.75% of the control (n = 49) in the presence of both atropine and 10 μm α,β‐methylene adenosine 5′‐triphosphate (α,β‐Me ATP).

The contractile response induced by electrical stimulation in the presence of atropine and α,β‐Me ATP was decreased to 27.81 ±4.07% (n = 23) and 26.63 ±5.01% (n = 15) of the control, by the addition of 100 μm cibacron blue 3GA and 100 μm suramin, respectively. The application of 100 μm adenosine 5′‐0–2‐thiodiphosphate (ADPβS) in the presence of atropine and α,β‐Me ATP decreased the contractile response induced by electrical stimulations to 17.15 ±3.71% (n=15) of the control.

Pretreatment of muscle strips with 100 μm ADPβS significantly reduced the response to either 200 μm α,β‐methylene adenosine 5′‐diphosphate or 200 μm ADPβS.

Uridine 5′‐triphosphate (100 μm to 1 mM) concentration‐dependently contracted muscle strips, and this contraction was significantly antagonized by desensitization of P2‐receptors with α,β‐Me ATP (10 μm), and completely antagonized by pretreatment of muscle strips with both α,β‐Me ATP and ADPβS (100 μm).

Di(adenosine‐5′) tetraphosphate (30 and 100 μm) contracted muscle strips, whereas it failed to contract after desensitization of P2‐receptors.

It is suggested that about 20% of the neurogenic contraction of rat urinary bladder smooth muscle is mediated via ADPβS‐sensitive purinoceptors.
Contribution of P1 (A2b subtype) and P2‐purinoceptors to the control of vascular tone in the rat isolated mesenteric arterial bed
Authors: Rubino Annalisa , Ralevic Vera , Burnstock Geoffrey
Published: July 2012

The direct vascular effects of adenosine and ATP were compared in the isolated and perfused mesenteric arterial bed of the rat. The actions of analogues of adenosine and ATP were also examined.

In preparations at basal tone, adenosine lacked vasoconstrictor actions, while ATP elicited dose‐dependent vasoconstrictor responses. When the tone of preparations was raised by adding methoxamine to the perfusate, adenosine and its stable analogue, 2‐chloroadenosine (2‐CADO) elicited dose‐dependent vasodilatation. The A2 adenosine receptor agonist, 5′‐N‐ethylcarboxamidoadenosine (NECA) was active at lower doses than adenosine, while the A2a‐selective agonist, CGS 21680 and the selective A1 agonist, N6‐cyclopentyladenosine (CPA) failed to induce vasodilatation. ATP and its analogue, 2‐methylthio ATP, elicited dose‐dependent vasodilatation at doses 400 fold lower than adenosine.

Vasodilator responses to adenosine and 2‐CADO were sensitive to antagonism by 1 μm 8‐sulphophenyltheophylline (8‐SPT) and were unaffected by inhibition of nitric oxide synthase by Nω‐nitro‐L‐arginine methyl ester (L‐NAME). In contrast, vasodilator responses to ATP were not sensitive to antagonism by 8‐SPT and were almost abolished by L‐NAME treatment.

These results indicate that in the rat mesenteric arterial bed, while both adenosine and ATP participate in the purinergic control of vascular tone, adenosine appears to be a weaker vasodilator than ATP and lacks vasoconstrictor action. A2b adenosine receptors account for the adenosine‐induced vasodilatation which is independent of the production of nitric oxide.
Actions of endothelins and sarafotoxin 6c in the rat isolated perfused lung
Authors: Lal H. , Woodward B. , Williams K.I.
Published: July 2012

Endothelin (ET) receptors within the vasculature and airways were studied in a rat perfused lung model in which pulmonary perfusion pressure (PPP), pulmonary inflation pressure (PIP) and lung weight were continuously monitored.

The vascular potencies of ETs (ET‐l>ET‐2>ET‐3) suggest an action via ETA receptors. This was confirmed by use of the antagonist, BQ123 (2 μm). The vasoconstrictor effects of sarafotoxin 6c (SX6C) also indicated the presence of ETB receptors.

Lung weight increases induced by ETs appeared to be a consequence of their vasoconstrictor potencies. The mixed ET receptor antagonist, bosentan (5 μm), markedly attenuated the responses of ET‐1 and SX6C on PPP and lung weight, further implicating activation of both ETA and ETB receptors in these responses.

Endothelin‐1 (ET‐1) induced an accumulation of albumin‐bound Evans blue dye in orthogradely perfused lungs. Retrograde perfusion attenuated the extravasation and increase in lung weight due to ET‐1 but significantly augmented those induced by SX6C.

The bronchoconstrictor actions of ETs (ET‐l = ET‐2 = ET‐3) and SX6C suggest this is an ETB‐mediated response. However SX6C was more potent than ETs and the dose‐response curve was significantly steeper and achieved a higher maximum.

Indomethacin did not affect the vascular or bronchial responses to ET‐1 or SX6C.

These findings indicate that rat pulmonary vasculature contains both ETA and ETB receptors. Retrograde perfusion suggests that ETB receptors are located arterially whereas ETA receptors are predominantly venous in distribution. Differences in the bronchoconstrictor potency of SX6C (compared to ETs) and the antagonism by bosentan may indicate ETB receptor heterogeneity in the airways.
Functional characterization of the adenosine receptor mediating inhibition of peristalsis in the rat jejunum
Authors: Hancock Debra L. , Coupar Ian M.
Published: July 2012

The non‐selective adenosine agonist, 5′‐N‐ethylcarboxamidoadenosine (NECA), is a potent inhibitor of morphine withdrawal diarrhoea in rats. More recently we found that NECA exerts its antidiarrhoeal effect by inhibiting secretion in both the jejunum and ileum and also by inhibiting peristalsis in the ileum. The specific aim of this study was to characterize the receptor in the rat jejunum mediating inhibition of peristalsis via functional studies using a range of metabolically stable adenosine analogues based on the pharmacological criteria of relative agonist and antagonist potencies.

Peristalsis in the rat isolated jejunum was achieved by raising the pressure to between 7–11 cmH2O for 3 min followed by a 3 min rest period (pressure at zero). The mean rate of peristalsis during inflation was 7.3 ± 0.1 peristaltic waves per 3 min and this rate remained consistent for up to 30 min, in 5 separate tissues. The inhibitory effects of the adenosine analogues were quantified by expressing their effects as a % reduction in the mean number of peristaltic contractions derived from the control tissues.

The rank order of agonist potency to reduce the rate of peristalsis was: N6‐cyclopentyladenosine (CPA) > NECA > R(−)−N6‐(2‐phenylisopropyl)adenosine (R‐PIA) > chloroadenosine (2‐CADO) > S‐PIA > 2‐phenylaminoadenosine (CV‐1808). This order complies well with the rank order of agonist potency that represents the activation of the A1 receptor subtype (CPA > R‐PIA = CHA = > NECA > 2‐CADO > S‐PIA > CV‐1808).

The selective A1 adenosine antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX) and the nonselective adenosine antagonist 8‐phenyltheophylline (8‐PT) at their respective concentrations of 10 nM and 2 μm caused parallel rightward shifts in the concentration‐response curve to the non‐selective Al/A2 agonist NECA. DPCPX was significantly more potent at inhibiting NECA than 8‐PT as revealed by their apparent pA2 values; DPCPX (9.5) and 8‐PT (7.26). The high affinity of DPCPX relative to that of 8‐PT suggests the presence of an A1 and not an A2B receptor. In addition, the high affinity of DPCPX (pA2:9.37) against the selective A2 agonist CPA, further confirms the presence of the A1 receptor subtype.

In this study we found that the A1 adenosine receptor is involved in regulating in vitro peristalsis which is different from the adenosine receptor regulating inhibition of secretion (A2B) in the same region of intestine of the same species. We propose that A2B adenosine agonists could be of clinical value in the management of diarrhoea that is due to microbiological organisms where antimotility effects are not desired.
Mechanisms of angiotensin II chronotropic effect in anaesthetized dogs
Authors: Lambert C.
Published: July 2012

The chronotropic effect of angiotensin II (5 μg in 1 ml of Tyrode solution), injected directly into the sinus node artery of 24 anaesthestized and vagotomized dogs pretreated with a β‐adrenoceptor antagonist, was evaluated before and after the administration of: (a) an angiotensin II AT1 receptor antagonist (losartan, 50 μg kg−1 min−1 infused i.v. for 120 min), (b) an α‐adrenoceptor antagonist (prazosin, 1 mg kg−1 i.v. bolus injected), (c) a Ca2+ channel blocker (nifedipine 50, 100 and 200 μg kg−1 i.v. bolus injected) and (d) a protein kinase inhibitor (staurosporine, 800 nM infused via the sinus node artery at 0.6 ml min−1 for 15 min).

Losartan and staurosporine by themselves had no effect on basal systemic arterial pressure and heart rate, whereas prazosin and nifedipine caused significant diminutions of both parameters.

Angiotensin II induced significant increases in heart rate, the mean augmentations being 29 ±2 beats min−1. Losartan, nifedipine and staurosporine significantly decreased the chronotropic effect of angiotensin II, the mean respective diminutions being 65 ±8, 40 ±9 and 64 ±10%, whereas prazosin had no effect.

This work has demonstrated that angiotensin II exerts in vivo a significant positive chronotropic effect that is mediated via AT1 receptors located in the region of the sinoatrial node. This effect is independent of the adrenergic system. It is decreased by the inhibition of the production of protein kinases, most probably of protein kinase C, and by the blockade of the voltage‐sensitive L‐type Ca2+ channels. Other studies are obviously needed to ascertain the role of angiotensin II in the control of heart rate and/or the genesis of arrhythmias.
BaCl2‐ and 4‐aminopyridine‐evoked phasic contractions in the rat vas deferens
Authors: Huang Yu
Published: July 2012

The actions of BaCl2 and 4‐aminopyridine, blockers of K+ channels, on the mechanical activity of the epididymal half of the rat vas deferens were investigated.

Both BaCl2 and 4‐aminopyridine dose‐dependently evoked phasic contractions. High extracellular potassium (35–40 mM) caused a tonic contraction but abolished the BaCl2‐ and 4‐aminopyridine‐induced phasic activity and reduced the BaCl2‐induced sustained component of contraction, but increased the 4‐aminopyridine‐induced tonic contraction.

Omission of calcium from the extracellular medium totally abolished the 4‐aminopyridine‐induced response but only reduced the mean amplitude of phasic contractions induced by BaCl.

Procaine (10 mM), an inhibitor of internal calcium release, completely abolished the phasic activity and reduced the sustained contraction induced by BaCl2. The remaining tone was abolished by nifedipine (1 μm).

Tetraethylammonium (1 mM) suppressed the amplitude of the BaCl2‐induced phasic contractions, and induced a biphasic increase in tonic tension.

The BaCl2‐induced responses were resistant to prazosin (1 μm), yohimbine (3 μm), propranolol (3 μm) or atropine (3 μm); in contrast, the 4‐aminopyridine‐induced activity was effectively inhibited by prazosin (Éμm) attenuated by yohimbine (Éμm) and atropine (Éμm) but not by propranolol (3μm). The 4‐aminopyridine‐induced response was abolished by pretreatment of the vas deferens with 6‐hydroxydopamine (0.5 mM).

The results indicate that the BaCl2‐evoked activity in the vas deferens was mainly due to blockade of Ba2+‐sensitive K+ channels on the smooth muscle plasma membrane. Subsequent calcium entry through the depolarized plasma membrane was needed to trigger generation of phasic contractions. 4‐Aminopyridine‐induced action, however, was largely mediated by neurotransmitters released from the depolarized nerve terminals as a result of blockade of K+ channels.
Nitroglycerin‐induced direct protection of the ischaemic myocardium in isolated working hearts of rats with vascular tolerance to nitroglycerin
Authors: Ferdinandy P. , Szilvassy Z. , Csont T. , Nagy E. , Dux L. , Csonka C. , Koltai M.
Published: July 2012

We investigated whether nitroglycerin (NTG) was able to produce an anti‐ischaemic effect in isolated working hearts of rats with vascular tolerance to NTG. Hearts isolated from tolerant and non‐tolerant rats were subjected to 10 min coronary occlusion in the presence of 10−7 m NTG and/or its solvent. NTG alleviated ischaemia‐induced deterioration of cardiac function and decreased lactate dehydrogenase release whilst having no effect on coronary flow nor the area of the ischaemic zone both in hearts isolated from NTG‐tolerant and non‐tolerant rats. The magnitude of the effect was similar in the two groups. These results suggest that the anti‐ischaemic effect of NTG involves direct myocardial mechanisms independent of its vascular action and that vascular tolerance to NTG does not affect this direct protective action.
Potent block of potassium currents in rat isolated sympathetic neurones by the uncharged form of amitriptyline and related tricyclic compounds
Authors: Wooltorton Julian R.A. , Mathie Alistair
Published: July 2012

The block of K+currents by amitriptyline and the related tricyclic compounds cyproheptadine and dizocilpine was studied in dissociated rat sympathetic neurones by whole‐cell voltage‐clamp recording.

Cyproheptadine (30 μm) inhibited the delayed‐rectifier current (Kv) by 92% and the transient current (KA) by 43%. For inhibition of KV, cyproheptadine had a KDof 2.2 μm. Dizocilpine (30 μm) inhibited KVby 26% and KAby 22%. The stereoisomers of dizocilpine were equally potent at blocking KVand KA.

Amitriptyline, a weak base, was significantly more effective in blocking KVat pH 9.4 (KD=0.46 μm) where the ratio of charged to uncharged drug was 50:50 compared with pH 7.4 (KD=11.9 μm) where the ratio was 99:1.

N‐methylamitriptyline (10 μm), the permanently charged analogue of amitriptyline, inhibited Kvby only 2% whereas in the same cells amitriptyline (10 μm) inhibited Kvby 36%.

Neither amitriptyline nor N‐methylamitriptyline had a detectable effect on Kvwhen added to the intracellular solution.

It is concluded that the uncharged form of amitriptyline is approximately one hundred times more potent in blocking Kvthan the charged form. However, this does not seem to be due to uncharged amitriptyline having better access to an intracellular binding site.
Sumatriptan‐induced saphenous venoconstriction in the anaesthetized dog through 5‐HT1‐like receptor activation
Authors: la Rochelle C. Drieu , O'Connor S.E.
Published: July 2012

The role of vasoconstrictor 5‐HT1‐like receptors in the control of vascular reactivity in vivo has been relatively little studied, particularly with regards to venous function. Using an anaesthetized dog model, we have investigated the haemodynamic profile of the selective 5‐HT, ‐like agonist, sumatriptan, focussing on the reactivity of the saphenous venous bed. The key feature of our experimental model was the implantation of ultrasonic crystals on the adventitial surface of the lateral saphenous vein to provide direct and continuous measurement of drug‐induced changes in vein diameter. Saphenous vein pressure was measured simultaneously via a proximal branch.

Sumatriptan 1–30 μg kg−1, i.v., produced pronounced dose‐related reductions in saphenous vein diameter which reached ⋍40% at the highest dose tested. Sumatriptan also produced modest increases in mean blood pressure, total peripheral resistance and left ventricular end diastolic pressure but had little or no effect on cardiac output, heart rate, cardiac contractility or saphenous venous pressure. Sumatriptan‐induced reductions in saphenous vein diameter were strongly antagonized by the 5‐HT1‐receptor antagonist, methiothepin (0.3 mg kg−1, i.v.) but were unaffected by the 5‐HT2 antagonist, ketanserin (0.3 mg kg−1, i.v.).

Hence, 5‐HT1‐like receptor stimulation in vivo can result in a powerful local venoconstrictor effect.
Haemodynamic effects of losartan and the endothelin antagonist, SB 209670, in conscious, transgenic ((mRen‐2)27), hypertensive rats
Authors: Gardiner S.M. , March J.E. , Kemp P.A. , Mullins J.J. , Bennett T.
Published: July 2012

Hypertensive transgenic (TGR(mRen‐2)27) (abbreviated to TG) rats (n = 6) and their normotensive Sprague‐Dawley (SD) control strain (n = 7) were chronically instrumented for the measurement of cardiac haemodynamics. The hypertension in TG rats (mean blood pressure 181±9 mmHg) was entirely attributable to a reduction in total peripheral conductance (TG rats=169 ± 7, SD rats=292 ± 15 μl min−1 mmHg−1 100g−1) since cardiac index was not different in the two strains (TG rats = 30.5±1.2, SD rats = 29.5 ± 1.6 ml min−1 100g−1).

In other animals instrumented for the assessment of regional haemodynamics, the extent of peripheral vasoconstriction was similar in renal, mesenteric and hindquarters vascular beds in the TG rats (reduction in vascular conductance relative to SD rats = 42%, 46% and 49%, respectively).

During an 8 h observation period with saline infusion, or following injection of losartan (10 mg kg−') in SD rats there was no hypotension or regional vasodilatation. With infusion of the endothelin antagonist, SB 209670 (10 μg kg−1 min−), there was a slight hypotension, but no significant vasodilatation; co‐administration of losartan and SB 209670 caused a similar profile of effect, although the hypotension was increased 4 With the same experimental protocol in TG rats, losartan caused a biphasic, progressive fall in mean arterial blood pressure accompanied by renal, mesenteric and hindquarters vasodilatation. Although the response to SB 209670 was not biphasic, its hypotensive and vasodilator effects were not different from those of losartan after 8 h. In the combined presence of losartan and SB 209670, mean arterial blood pressure (116 ± 5 mmHg) was significantly lower than with SB 209670 (132±4mmHg) or losartan (136 ± 6 mmHg) alone, and renal, mesenteric and hindquarters vascular conductances (61 + 3, 90+14 and 52+4 [kHz mmHg−1]103, respectively) were higher than the corresponding values following either SB 209670 (49±4, 52±4 and 34±3 [kHz mmHg−1]103, respectively) or losartan (43±5, 59±3 and 35±4 [kHz mmHg−1]103, respectively) alone. These results indicate the maintenance of hypertension in TG rats is dependent upon renal, mesenteric and hindquarters vasoconstriction, mediated by angiotensin II (AII) and endothelin (ET). Since we found that plasma ET‐1 levels in TG rats (12.06±2.87 pmol 1−1) were lower than in SD rats (21.53±3.94 pmol 1−1), then it is possible that locally‐generated, rather than circulating ET‐1 contributes to the widespread vasoconstriction in TG rats.
Stimulation by endothelin‐1 of mitogen‐activated protein kinases and DNA synthesis in bovine tracheal smooth muscle cells
Authors: Malarkey Kevin , Chilvers Edwin R. , Lawson Mark F. , Plevin Robin
Published: July 2012

In cultures of bovine tracheal smooth muscle cells, platelet‐derived growth factor‐BB (PDGF), bradykinin (BK) and endothelin‐1 (ET‐1) stimulated the tyrosine phosphorylation and activation of both pp42 and pp44 kDa forms of mitogen‐activated protein (MAP) kinase.

Both ET‐1 and PDGF stimulated a sustained activation of MAP kinase whilst the response to BK was transient.

Activation of MAP kinase occurred in a concentration‐dependent manner (EC50 values: ET‐1, 2.3±1.3 nM; BK, 8.7±4.1 nM, PDGF, 9.7±3.2 ng ml−1).

Pretreatment with the protein kinase C (PKC) inhibitor Ro‐318220, significantly reduced ET‐1 activation of MAP kinase at 2 and 5 min but enhanced MAP kinase activation at 60 min.

Following chronic phorbol ester pretreatment, BK‐stimulated activation of MAP kinase was abolished whilst the responses to PDGF and ET‐1 were only partly reduced (80 and 45% inhibition respectively).

Pretreatment with pertussis toxin reduced ET‐1 stimulated activation of MAP kinase particularly at later times (60 min), but left the responses to both PDGF and BK unaffected.

ET‐1 also stimulated a 3 fold increase in [3H]‐thymidine incorporation which was abolished by pertussis toxin pretreatment. In contrast, PDGF stimulated a 131 fold increase in [3H]‐thymidine incorporation which was not affected by pertussis toxin.

These results suggest that a pertussis toxin‐sensitive activation of MAP kinase may play an important role in ET‐1‐stimulated DNA synthesis but that activation of MAP kinase alone is not sufficient to induce the magnitude of DNA synthesis observed in response to PDGF.
The effect of a synthetic 7‐thiaprostaglandin E1 derivative, TEI‐6122, on monocyte chemoattractant protein‐1 induced chemotaxis in THP‐1 cells
Authors: Tanaka Hiroko , Minoshima Toru , Endo Noriaki
Published: July 2012

The ability of various prostaglandins (PGs) to inhibit monocyte chemotaxis induced by monocyte chemoattractant protein‐1 (MCP‐1) was investigated with a human monocytic leukaemia cell line, THP‐1. Moreover, to investigate the mechanism of the inhibitory action of PGs the involvement of either intracellular adenosine 3″:5″‐cyclic monosphosphate (cyclic AMP) accumulation or intracellular Ca2+ mobilization was studied.

TEI‐6122, a synthetic 7‐thia‐PGE, derivative, inhibited chemotaxis of THP‐1 cells induced by MCP‐1 with an IC50 of 1.5 pM. Its inhibitory activity was 1000 fold more than that of PGE1 and PGE2 (IC50=2.8 nM and 0.9 nM, respectively), which were more potent than other PGs such as PGA1, PGA2, PGF2α and PGI2 (IC50≥1 μm).

With respect to the effect on intracellular cyclic AMP accumulation in THP‐1 cells, TEI‐6122 was as potent as PGEI and PGE2, which were approximately 100 to 1000 fold more potent than the other PGs such as PGA1, PGA2 and PGI2. The minimum concentration of TEI‐6122 required to increase intracellular cyclic AMP accumulation in THP‐1 cells was 1 nM.

TEI‐6122 and PGE1 (4 μm) transiently increased intracellular calcium levels in THP‐1 cells. When added prior to MCP‐1, both PGs partially suppressed the increased in Ca2+ caused by this cytokine. There were no significant differences between the activity of TEI‐6122 and PGEI in either respect.

It is concluded that TEI‐6122, a synthetic 7‐thia‐PGE, derivative is a much more potent inhibitor of MCP‐1‐induced THP‐1 cell chemotaxis than PGEI and PGE2 which are the best inhibitors among the natural PGs tested, while neither intracellular cyclic AMP accumulation nor effects on Ca2+ mobilization account for the extremely potent inhibitory activity of TEI‐6122. Thus, either a novel PGE2 receptor (EP receptor) or a novel intracellular signal transduction system may be involved in the extremely potent chemotaxis inhibitory activity of TEI‐6122.
Loss of NADPH‐diaphorase containing neurones after reversible focal ischaemia in rats delayed by L‐NAME
Authors: Margaill Isabelle , Allix Monique , Charriaut‐Marlangue Christiane , Boulu Roger G. , Plotkine Michel
Published: July 2012

In the present study, NADPH‐diaphorase histochemistry was used to assess the temporal evolution of the number of nitric oxide (NO)‐synthase containing neurones after reversible focal cerebral ischaemia in rats. The number of NADPH‐diaphorase containing neurones was reduced by 50% and 90% respectively 6 and 24 h after ischaemia. L‐NAME, a NO‐synthase inhibitor, prevented the loss of NADPH‐diaphorase containing neurones observed 6 h after ischaemia but not 24 h after ischaemia, suggesting that in the early phase, nitric oxide is involved in this phenomenon.
2‐Naphthalenesulphanyl‐L‐aspartyl‐2‐(phenethyl) amide (2‐NAP) and food intake in rats: evidence that endogenous peripheral CCK does not play a major role as a satiety factor
Authors: Ebenezer I.S. , Baldwin B.A.
Published: July 2012

The demonstration that systemic administration of the CCKA receptor antagonist, devazepide, increases food intake in rats has provided the strongest support for the hypothesis that endogenous peripherally released cholecystokinin (CCK) acts as a satiety factor. However, interpretation of these results has been confounded by the fact that devazepide can enter the brain from the systemic circulation and may increase food intake by a central action. The present study was therefore undertaken to confirm the hypothesis that endogenous peripheral CCK is a satiety factor by investigating the effects of a novel CCKA receptor antagonist, 2‐NAP, which is unlikely to cross the blood brain barrier, on food intake in rats.

2‐NAP (1–16 mg kg−1, i.p.) had no significant effects on the intake of a test meal in rats.

Pretreatment of rats with 2‐NAP (2 mg kg−1, s.c.) abolished the inhibitory effects of exogenous peripheral CCK (5 μg kg−1, i.p.) on food intake.

In agreement with previous results, devazepide (50–200 μg kg−1, i.p.) significantly increased the intake of a test meal in rats.

The observations that 2‐NAP, which is unlikely to penetrate the blood brain barrier, had no effect on food intake, but that 2‐NAP abolished the suppressant effect of exogenous peripheral CCK, suggest that endogenously released peripheral CCK is not important as a satiety factor in rats.
Changes in benzodiazepine‐GABA receptor coupling in an accumbens‐habenula circuit after chronic diazepam treatment
Authors: Brett R.R. , Pratt J.A.
Published: July 2012

The effects of subacute and of chronic diazepam treatment upon binding to the GABAA receptor have been examined by use of receptor autoradiography for determining flunitrazepam (FNZP) binding, GABA enhancement of FNZP binding, SR 95531 2‐(3″‐carboxy‐2″, propyl)‐3‐amino‐6‐p‐methoxyphe‐nylpyridazinium bromide) binding and GABA binding in parallel sections from rat brain. Prior to the autoradiographic procedures, a behavioural assessment of the rats was made in the elevated plus‐maze test of anxiety.

Rats receiving diazepam either subacutely (3 days) or chronically (28 days) by both continuous release, from previously implanted subcutaneous silastic capsules, or by daily injection (5 mg kg−1) did not display changes in FNZP or GABA binding in any of the 47 brain structures analysed. Similarly, there were no significant effects of treatment upon mean total entries or on the open:total ratio for entries in the elevated plus‐maze.

There were reductions in the GABA enhancement of FNZP binding in the nucleus accumbens and central grey after subacute diazepam treatment. This effect persisted in the nucleus accumbens after chronic treatment. Less marked effects occurred in the lateral habenula, dorsal raphe and substantia nigra pars compacta. In the dorsal tegmental nucleus, GABA enhancement of FNZP binding was enhanced after chronic treatment and this was accompanied by reductions in SR 95531 binding. Treatment did not otherwise affect SR 95531 binding, with the exception of the dorsal raphe where binding was decreased after subacute treatment.

In general, the patterns of binding produced by the two different treatment routes were very similar. However, SR 95531 binding was lower in certain hippocampal fields in the i.p. treated animals compared to the rats implanted with silastic capsules.

It is concluded that repeated administration of diazepam evokes changes in benzodiazepine and GABA receptor coupling, and to a lesser extent changes in low affinity GABA binding, in certain interrelated brain structures of which an accumbens‐habenula circuit is a central feature. These changes occur soon after the initiation of diazepam treatment, suggesting that they are unlikely to account for tolerance to the anxiolytic effects of diazepam but may trigger and/or accompany other critical neurochemical events.
Comparison between novel steroid‐like and conventional nonsteroidal antioestrogens in inhibiting oestradiol‐ and IGF‐I‐induced proliferation of human breast cancer‐derived cells
Authors: de Cupis Alessandra , Noonan Douglas , Pirani Paolo , Ferrera Alessandra , Clerico Luana , Favoni Roberto E.
Published: July 2012

This study has two specific aims: (a) to compare the antioestrogenic activity of two steroidal analogues of 17β‐oestradiol, the 7α‐alkylamide, ICI 164, 384 and the 7α‐alkylsulphinylamide, ICI 182, 780, with that of the triphenylethylene‐derived compound 4OH‐tamoxifen on a pool of human breast cancer cell lines (HBCCL) with a range of hormonal responsiveness and acquired antioestrogen resistance and (b) to investigate the ability of such antioestrogens to modulate the potent breast carcinoma growth‐stimulatory activity of the ‘IGF‐I system’.

For the chemosensitivity investigations we used a long‐term colorimetric and the short‐term thymidine incorporation assay; we analysed IGF‐I in conditioned media by a radioimmunoassay, IGF‐I mRNA in the cells by RT‐PCR and molecular species of IGF‐I‐binding proteins, secreted in conditioned media, by Western ligand blot. IGF‐I receptors were assayed on cell monolayers by binding studies and by Scatchard analysis, we calculated KD, Bmax and sites/cell.

Our results indicate that ICI 182, 780 and ICI 164, 384 are 1.5‐5.5 fold more potent than 40H‐tamoxifen in inhibiting the basal proliferation of oestrogen‐receptor positive (ER +) breast cancer cell lines. Moreover we demonstrate the capacity of ICI 182, 780 and ICI 164, 384 to reduce, in a time‐dependent fashion, oestrogen‐ and/or IGF‐I‐stimulated growth of ER + cell lines, possibly by negatively interfering with an IGF‐I‐like material secretion and IGF‐I‐receptor number.

Our data provide the first evidence that, on ER+ human breast carcinoma cell lines, steroidal antioestrogens inhibit cell growth and modulate the IGF‐I mitogenic system. The mechanism of this latter effect has yet to be identified.
Adenosine receptor‐mediated relaxation of guinea‐pig precontracted, isolated trachea
Authors: Losinski Amanda , Alexander Stephen P.H.
Published: July 2012

We have investigated the pharmacological profile of the adenosine receptor mediating relaxation of the carbachol pre‐contracted guinea‐pig trachea.

5″‐N‐Ethylcarboxamidoadenosine (NECA) and 2‐chloroadenosine elicited concentration‐dependent relaxations with pD2 (‐log10 half‐maximal values) of 6.37±0.04 and 5.25±0.09, with maximal relaxations of 73±7 and 208±38%, respectively. In the presence of 10 μm NECA, 2‐chloroadenosine was able to relax the tissue further with a pD2 value of 4.74±0.11 and a maximal response of 252±68%.

CGS 21680, APEC and adenosine failed to elicit significant relaxations of precontracted tracheal rings at concentrations below 10 μm. At 10 μm, adenosine analogues elicited relaxations with the following order of magnitude (% relaxation): 2‐chloroadenosine (75±16%)=NECA (69±16%)>APEC (25±8%)>CGS 21680 (11±2%)>adenosine (6±4%).

NECA‐induced relaxation of precontracted trachea was antagonized by adenosine receptor antagonists with the rank order of apparent affinity (Ki, nM): PD 115, 199 (27±8) = XAC (43±11)> CP 66, 713(285±89) = DPCPX (316±114).

We conclude that the adenosine analogue‐induced relaxation of guinea‐pig tracheal rings fails to fit into the current classification of A2 adenosine receptors.