bluuebunny/crossref_metadata_2025 huggingface dataset:
The influence of the trigeminal ganglion on carotid blood flow in anaesthetized guinea‐pigs
Authors: Beattie D.T. , Connor H.E.
Published: July 2012

The influence of the trigeminal ganglion on the carotid circulation has been investigated by measuring electrical stimulation‐induced alterations in carotid arterial blood flow and resistance in anaesthetized guinea‐pigs. The effects of several receptor antagonists were assessed to determine which neurotransmitters are involved in regulating carotid blood flow.

Arterial blood pressure and carotid vascular resistance were reduced by electrical stimulation (0.5 mA, 1 ms, 5 Hz, 60 s) of the trigeminal ganglion ipsilateral to the carotid artery from which flow was measured. No consistent effect of electrical stimulation on carotid blood flow was observed. However, when guinea‐pigs were pretreated with guanethidine (30 mg kg−1, s.c, 24 h prior to experiments), stimulation produced little change in blood pressure, while carotid blood flow was increased and vascular resistance decreased, consistent with vasodilatation in the cranial circulation. Stimulation of the trigeminal ganglion contralateral to the carotid artery from which blood flow was measured, had little effect on either carotid blood flow or vascular resistance.

In animals pretreated with guanethidine, intravenous administration of the vasoactive intestinal polypeptide (VIP) receptor antagonist, [p‐Cl‐d‐Phe6,Leu17]‐VIP (50 μg kg−1) significantly attenuated the increase in carotid blood flow and decrease in carotid vascular resistance evoked by trigeminal ganglion stimulation. Responses evoked by trigeminal ganglion stimulation were, however, unaffected by intravenous injection of the tachykinin NK1 receptor antagonists, GR82334 (0.3 mg kg−1) and CP‐99,994 (0.4 mg kg−1), calcitonin gene‐related peptide (CGRP) receptor antagonist, CGRP8–37 (0.9 mg kg−1) and the ganglion blocking agent, hexamethonium (10 mg kg−1).

It is concluded that in the guanethidine‐pretreated guinea‐pig, electrical stimulation of the trigeminal ganglion increases carotid blood flow and produces an accompanying decrease in carotid vascular resistance, consistent with the dilatation of carotid blood vessels. The transmitter mediating this effect is most likely to be VIP.
Comparative effects of anti‐platelet agents as adjuncts to tissue plasminogen activator in a dog model of occlusive coronary thrombosis
Authors: McAuliffe S.J.G. , Moors J.A. , Jones H.B.
Published: July 2012

This study compares a cyclo‐oxygenase inhibitor (aspirin), a 5‐HT2 antagonist (ZM170809) and a combined thromboxane synthase inhibitor/receptor antagonist (ZD1542) as adjuncts to tissue plasminogen activator (rt‐PA).

Application of an anodal current (332 ± 4.1 μA) to the stenosed left circumflex coronary artery of 20 anaesthetized dogs produced a stable platelet‐rich occlusive thrombus.

After initial i.v. administration of recombinant human tissue type plasminogen activator (rt‐PA, 3 mg bolus + 2 mg kg−1 h−1 for 30 min) thrombolysis occurred in 15 out of 20 dogs. All 15 dogs reoccluded.

The second i.v. administration of rt‐PA in the presence of either aspirin, ZM170809, ZD1542 or saline resulted in thrombolysis in all 20 dogs.

Both the combined thromboxane synthase inhibitor/receptor antagonist (ZD1542) and 5‐HT2 antagonist (ZM170809) significantly (P < 0.05) reduced the time taken to lyse the thrombus compared with the saline group. The times were 14.4 ± 2.7 min, 18.0 ± 3.9 min and 36.8 ± 6.2 min for ZD1542, ZM170809 and saline respectively.

Aspirin did not offer any additional benefit to using rt‐PA alone. The times to thrombolysis were 36.8 ± 8.4 min for aspirin and 36.8 ± 6.2 min for the saline group.

The number of dogs in which the circumflex coronary artery reoccluded within 60 min of terminating the second infusion of rt‐PA were five for saline, four for aspirin, two for ZD1542 and two for ZM170809.

These results indicate that both ZD1542 and ZM170809 are more effective adjuncts than aspirin in thombolysis and may provide an improvement in current clinical practice.
Further characterization of 5‐hydroxytryptamine receptors (putative 5‐HT2B) in rat stomach fundus longitudinal muscle
Authors: Baxter Gordon S. , Murphy Olive E. , Blackburn Thomas P.
Published: July 2012

The present study was undertaken to isolate and characterize pharmacologically homogeneous populations of 5‐hydroxytryptamine (5‐HT) receptors from a possible mixed receptor population mediating contraction of the longitudinal muscle of rat stomach fundus. Our aim was to extend the pharmacological characterization of the 5‐HT2B receptor which is reported to be expressed in this preparation.

To minimize spontaneous activity and any influence of circular muscle on the contractile response, narrow (1–1.5 × 20 mm) segments of mucosa‐denuded longitudinal muscle were used. Under these conditions, blockade of monoamine oxidase with pargyline (100 μm for 15 min) caused a leftward displacement of concentration‐effect curves for both 5‐methoxytryptamine (5‐MeO‐T) and tryptamine. Neither pargyline nor a number of uptake inhibitors affected responses to 5‐HT.

In pargyline pretreated preparations, the order of potency of a number of tryptamine analogues was as follows: 5‐MeO‐T≥α‐Me‐5‐HT≥ 5‐HT> 5‐carboxamidotryptamine (5‐CT)> tryptamine>2‐Me‐5‐HT. In addition several ligands known to act as agonists at either 5‐HT2A or 5‐HT2C receptors including 1‐m‐chlorophenylpiperazine (m‐CPP), Ru 24969, MK 212 and SCH 23390 were also agonists in rat fundus whilst sumatriptan, renzapride and 8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT) were very weak or inactive. With the exception of 2‐Me‐5‐HT and m‐CPP, most agonists produced monophasic concentration‐effect curves consistent with an interaction at a single site. High concentrations of 2‐Me‐5‐HT evoked relaxations which were blocked by phentolamine (1 μm) suggesting an interaction with α‐adrenoceptors. m‐CPP often evoked biphasic concentration‐effect curves with a second contractile phase which was insensitive to yohimbine at concentrations higher than required for antagonism of responses to 5‐HT.

LY 53857, methiothepin, methysergide, ritanserin and ICI 170809 were potent but non‐surmountable antagonists of 5‐HT in rat fundus. In contrast, several ligands behaved as surmountable antagonists with the following order of potency: rauwolscine > yohimbine = mesulergine ≥ mianserin = SB 204070 ≥ WY 26703 ≥ SB 200646 > pirenpirone ≥ renzapride. DAU 6285, granisetron, spiperone, ketanserin, phentolamine and GR 127935 did not affect responses to 5‐HT at concentrations up to 1 μm. The agonist and concentration independent profile of antagonism supported a single site interaction for both agonists and antagonists.

We conclude that despite small differences concerning the enantiomeric selectivity and affinity of rauwolscine and yohimbine, the close pharmacological identity of 5‐HT receptors in rat stomach fundus and the recently cloned 5‐HT2B receptor is maintained. SB 200646, which demonstrates some selectivity for 5‐HT receptors in rat stomach fundus, should provide a useful ligand for confirmation of this view and allow discrimination of 5‐HT2B function both in vitro and in vivo.
Effects of phosphodiesterase isoenzyme inhibitors on cutaneous inflammation in the guinea‐pig
Authors: Teixeira M.M. , Rossi A.G. , Williams T.J. , Hellewell P.G.
Published: July 2012

Inflammation is central to the pathophysiology of asthma. The recent findings that different inflammatory cells may express different phosphodiesterase (PDE) isoenzymes have centred attention on inhibitors of these isoenzymes as new drugs for the treatment of asthma. In this study, we investigated the effect of different PDE isoenzyme inhibitors on the accumulation of 111In‐labelled eosinophils and local oedema formation at sites of allergic‐ and mediator‐induced inflammation in guinea‐pig skin.

Systemic treatment with SK&F 94120, a type III PDE inhibitor, or zaprinast, a type V PDE inhibitor, had no effect on the 111In‐eosinophil accumulation and oedema formation induced by i.d. injection of zymosan‐activated plasma (ZAP), PAF, histamine or in a passive cutaneous anaphylaxis (PCA) reaction.

Systemic treatment with rolipram, a type IV PDE inhibitor, effectively inhibited 111In‐eosinophil accumulation induced by ZAP, PAF, histamine and in a PCA reaction. However, oedema formation measured in the same sites was not affected. Systemic administration of higher doses of theophylline produced similar results. In contrast, 111In‐neutrophil accumulation induced by ZAP or in a PCA reaction was not altered by systemic treatment with rolipram.

Locally‐injected rolipram had little effect on 111In‐eosinophil accumulation and oedema formation induced by histamine, PAF and in a PCA reaction.

These data show that systemic, but not local, treatment with rolipram effectively inhibits allergic‐ and mediator‐induced 111In‐eosinophil accumulation but not oedema formation or 111In‐neutrophil accumulation. This, taken together with the potent inhibitory effects of PDE type IV inhibitors on eosinophil function in vitro, suggest that this class of drugs may be beneficial in disease states such as asthma where eosinophils are thought to play a major pathophysiological role.
Effect of potassium channel blockade and α2‐adrenoceptor activation on the release of nitric oxide from non‐adrenergic non‐cholinergic nerves
Authors: De Man Joris G. , Boeckxstaens Guy E. , Herman Arnold G. , Pelckmans Paul A.
Published: July 2012

Using a superfusion bioassay cascade, we studied the effect of K+ channel blockers and α2‐adrenoceptor agents on the release of a transferable factor, previously characterized as nitric oxide (NO) or a nitric oxide‐related substance (NO‐R), in response to non‐adrenergic non‐cholinergic (NANC) nerve stimulation in the canine ileocolonic junction (ICJ).

The non‐selective K+ channel blockers, 4‐aminopyridine (4‐AP, 50 μm) and tetraethylammonium (TEA, 1 mm) and the more selective blocker of Ca2+‐activated K+ channels, charybdotoxin (Leiurus quinquestriatus venom (LQV), 0.4 μg ml−1), significantly enhanced the release of NO‐R induced by low frequency stimulation (2–4 Hz). In the presence of 4‐AP and TEA, the release of NO‐R was nearly abolished by tetrodotoxin (2 μm), and by l‐NG‐nitroarginine (l‐NOARG, 0.1 mm). Relaxations induced by direct injection of exogenous NO (5–50 pmol) or nitroglycerin (GTN, 10–30 pmol) onto the rabbit aortic detector ring were not affected.

The α2‐adrenoceptor agonist, UK‐14,304 (0.3 μm) inhibited the release of NO‐R induced by low (2–4 Hz), but not that induced by high (16 Hz), frequency stimulation. This inhibitory effect was completely reversed by the α2‐adrenoceptor antagonist, yohimbine (0.3 μm). Neither UK‐14,304 nor yohimbine affected the relaxations induced by exogenous NO (5 pmol) or GTN (10 pmol) on the aortic detector ring.

On the other hand, in the presence of the K+ channel blockers 4‐AP (50 μm) or charybdotoxin (LQV, 0.4 μg ml−1), UK‐14,304 (0.3 μm) failed to inhibit the electrically‐induced release of NO‐R.

From these results, we conclude that the electrically‐induced release of NO‐R from NANC nerves of the canine ICJ is enhanced by K+ channel blockers but inhibited by α2‐adrenoceptor activation. In addition, these results suggest that the prejunctional modulation of NO‐R release by α2‐adrenoceptors may involve neuronal K+ channels.
Dexamethasone‐induced translocation of lipocortin (annexin) 1 to the cell membrane of U‐937 cells
Authors: Solito Egle , Nuti Sandra , Parente Luca
Published: July 2012

Lipocortin (annexin) 1 is a putative mediator of the inflammatory effects of glucocorticoids. By flow cytometric analysis (FACS) we have studied the effect of dexamethasone on the cellular localization of lipocortin 1. U‐937 cells were incubated with or without 10 nm phorbol 12‐myristate 13‐acetate (PMA) to induce cell differentiation. Then 1 μm dexamethasone was added and incubation carried out for increasing times (1–24 h). Dexamethasone caused a time‐dependent biphasic translocation of lipocortin 1 from the intracellular compartment to the cell membrane with maximal membrane expression at 4 and 24 h. In differentiated U‐937 cells the steroid‐induced membrane accumulation of lipocortin 1 was significantly higher than that of undifferentiated cells. The accumulation of the protein in the cell membrane may precede its release which is stimulated by dexamethasone in differentiated U‐937 cells. Since extracellular lipocortin 1 has anti‐inflammatory properties the modulation of the translocation/secretion process of the protein by glucocorticoids may be part of their mechanism of action.
Interdependence of contractile responses of rat small mesenteric arteries on nitric oxide and cyclo‐oxygenase and lipoxygenase products of arachidonic acid
Authors: Wu X.C. , Johns E. , Michael J. , Richards N.T.
Published: July 2012

We have examined the effects of nitric oxide inhibition, indomethacin and the dual lipoxygenase/cyclo‐oxygenase inhibitor, 3‐amino‐1‐[m‐(trifluoromethyl)‐phenyl]‐2‐pyrazoline (BW755C), on the responses of small mesenteric arteries of Wistar rats, with and without endothelium, to noradrenaline, potassium chloride, endothelin‐1, acetylcholine and sodium nitroprusside.

Noradrenaline, potassium chloride and endothelin‐1 caused concentration‐dependent contraction of small mesenteric arteries. Indomethacin (14 μm) attenuated the contractile response to both noradrenaline and potassium chloride. The inhibitory action of indomethacin persisted in vessels treated with CHAPS.

Acetylcholine produced concentration‐dependent relaxation in these vessels. Indomethacin (14 μm) had no significant effect on the acetylcholine concentration‐response relationship.

NG‐nitro‐l‐arginine methyl ester (l‐NAME, 100 μm) potentiated the contractile response to both noradrenaline and potassium chloride and inhibited acetylcholine‐induced relaxation. Indomethacin attenuated the effects of l‐NAME.

BW755C inhibited the contractile response to noradrenaline and potassium chloride but not to endothelin‐1. The inhibitory effects of BW755C persisted in the presence of indomethacin and in vessels treated with CHAPS.

BW755C enhanced endothelium‐dependent relaxation, as assessed by the response to acetylcholine. In the presence of indomethacin, BW755C produced a shift to the right of the concentration‐response curve to acetylcholine.

Inhibition of nitric oxide synthase with l‐NAME, reversed the inhibitory effect of BW755C on noradrenaline‐ and potassium‐induced contraction. l‐NAME and BW755C in combination resulted in a shift to the right of the concentration‐response curve to acetylcholine.

Sodium nitroprusside produced concentration‐dependent relaxation of the vessels. Endothelium removal reduced the maximum relaxation to nitroprusside. BW755C did not alter the response to sodium nitroprusside in vessels with or without endothelium.

These data support the existence of two vasoconstrictor products of arachidonic acid released during contraction of small mesenteric arteries with noradrenaline and potassium chloride: a cyclo‐oxygenase product and a lipoxygenase product both of which appear to be largely endothelium‐independent.
Molecular cloning and expression of human EP3 receptors: evidence of three variants with differing carboxyl termini
Authors: Regan J.W. , Bailey T.J. , Donello J.E. , Pierce K.L. , Pepperl D.J. , Zhang D. , Kedzie K.M. , Fairbairn C.E. , Bogardus A.M. , Woodward D.F. , Gil D.W.
Published: July 2012

The polymerase chain reaction (PCR) was used in combination with plaque hybridization analysis to clone four variants of the EP3 prostaglandin receptor from a human small intestine cDNA library.

Three of these variants, i.e. the EP3A, EP3E and EP3D, share the same primary amino acid sequence except for their carboxyl termini, which diverge from one another at the same point, approximately 10 amino acids away from the end of the seventh membrane spanning domain of the receptor. The fourth variant (EP3A1) has a nucleotide coding sequence identical to EP3A but has a completely different 3′;untranslated sequence.

The carboxyl termini of the three isoforms differ most obviously in length with the EP3A being the longest (41 amino acids) and the EP3E being the shortest (16 amino acids). They also differ in content with the EP3A containing 9 serine and threonines in its carboxyl terminus and the EP3E none.

Transient expression in eukaryotic cells showed that the human EP3 receptor variants had similar but not identical radioligand binding properties and differed in their functional coupling to second messenger pathways. Up to 3 pmol mg−1 protein of [3H]‐prostaglandin E2 binding could be obtained with more than 95% specific binding. Using a reporter gene assay, as a measure of intracellular cyclic AMP levels, the EP3A coupled more efficiently to the inhibition of adenylyl cyclase than did the EP3E.

PCR was used to confirm the presence of mRNAs encoding the four human EP3 receptor variants in tissues of the human small intestine, heart and pancreas. These findings indicate that the EP3 receptor variants identified here are likely to be expressed in tissues. The differences in the carboxyl termini at the protein level, and in the 3′ untranslated regions at the mRNA level, could be profound in terms of the regulation and functional coupling of these receptor isoforms.
The effect of the ETA receptor antagonist, FR 139317, on [125I]‐ET‐1 binding to the atherosclerotic human coronary artery
Authors: Dashwood Michael R. , Allen Sean P. , Luu Thin N. , Muddle John R.
Published: July 2012

The distribution of [125I]‐endothelin (ET‐1) binding sites on atherosclerotic human epicardial coronary arteries has been studied by in vitro receptor autoradiography.

[125I]‐ET‐1 binding was to the tunica media and regions of neovascularization.

Competition studies were carried out in the presence of ET‐1 and the ETA receptor antagonist, FR 139317. The IC50 values for ET‐1 at the tunica media and regions of neovascularization were similar (mean ± s.e.mean of n = 4 patients, 2.5 ± 0.9 nm and 2.9 ± 0.9 nm, respectively) whereas IC50 values for FR 139317 at regions of neovascularization (607 ± 34 nm) were significantly higher than those of the tunica media (12.6 ± 2.4 nm) (P < 0.0001).

These results indicate that ETA receptors are present on the tunica media of the diseased human coronary artery whereas a different ET receptor subtype exists at regions of neovascularization.
Increase of extracellular brain calcium involved in interleukin‐1β‐induced pyresis in the rabbit: antagonism by dexamethasone
Authors: Palmi M. , Frosini M. , Becherucci C. , Sgaragli G.P. , Parente L.
Published: July 2012

This study investigates the role of extracellular brain calcium in the hyperthermia induced by interleukin‐1β (IL‐1β).

Intracerebroventricular (i.c.v.) injection of IL‐1β (12.5 ng kg−1) in rabbits caused a prompt and sustained rise in cerebrospinal fluid (CSF) Ca2+ concentration ([Ca2+]) followed by enhanced prostaglandin E2 (PGE2) release and hyperthermia.

A linear and significant correlation was observed between the increase in [Ca2+] induced by IL‐1β and the rise in body temperature.

Ventriculo‐cisternal perfusion with artificial CSF containing the calcium chelator EGTA (1.3 mm) blocked the IL‐1‐induced PGE2 release and countered the febrile response.

I.c.v. administration of dexamethasone (Dex) (2.4 and 24 μg kg−1) 100 min prior to IL‐1β, dose‐dependently antagonized the cytokine‐induced Ca2+ increase, the PGE2 release and the febrile response.

These results suggest that changes in extracellular brain calcium are involved in the regulation of body temperature. In this light, the antipyretic action of Dex may be related to its effect on Ca2+ uptake.
Triflocin, a novel inhibitor for the Na‐HCO3 symport in the proximal tubule
Authors: Belachgar Fouzia , Hulin Philippe , Anagnostopoulos Takis , Planelles Gabrielle
Published: July 2012

Triflocin, applied at millimolar concentration hyperpolarizes the basolateral membrane of Necturus proximal convoluted tubular cells, in vivo.

Barium, 2.5 × 10−3 m, ouabain, 10−3 m, or amiloride 10−4 m, fail to prevent this hyperpolarization.

Triflocin has no effect on the intracellular chloride activity.

In physiological acid base conditions, Triflocin increases intracellular pH.

Upon an acute isohydric hypercapnia, Triflocin depolarizes the basolateral membrane potential.

It is concluded that, Triflocin inhibits the basolateral electrogenic Na‐(HCO3)n > 1 cotransport in proximal tubules.
Vasoconstrictor responses after neo‐intima formation and endothelial removal in the rabbit carotid artery
Authors: De Meyer Guido R.Y. , Bult Hidde , Üstünes Levent , Kockx Mark , Jordaens François H. , Zonnekeyn Ludo L. , Herman Arnold G.
Published: July 2012

The present study examined the responses of the rabbit carotid artery to five vasoconstrictors after neo‐intima formation induced by perivascular collar treatment and evaluated the role of constitutive and inducible nitric oxide (NO) synthase and endothelial cells (ECs).

Ring segments of the rabbit carotid artery were mounted in organ chambers for isometric tension recording. Neo‐intima‐bearing vessels developed less force (Emax) in response to KCl, the thromboxanemimetic U‐46619 and 5‐hydroxytryptamine (5‐HT), but not to angiotensin I and II.

The collar‐treatment increased the sensitivity to 5‐HT, and decreased the sensitivity to angiotensin II. The sensitivity to U‐46619 and angiotensin I remained unchanged.

Mechanical removal of ECs and inhibition of NO biosynthesis by NG‐monomethyl‐l‐arginine (l‐NMMA) and NG‐nitro‐l‐arginine (l‐NOARG) increased the sensitivity to 5‐HT in sham and collar‐treated segments to the same extent. The effects of collar‐treatment and endothelial removal or treatment with inhibitors of NO biosynthesis were additive. Inhibition of NO biosynthesis failed to augment sensitivity to 5‐HT after endothelial denudation. l‐NOARG increased the force development to KCl in sham and collar‐treated segments to the same extent. However, l‐NMMA and l‐NOARG failed to augment the contractile responses of neo‐intima‐bearing vessels to 5‐HT and KCl after endothelial removal.

The responses to angiotensin I were not altered, either by the neo‐intima or by endothelial removal. In arteries with a neo‐intima the sensitivity to angiotensin II was decreased. Removal of the endothelium or incubation with l‐NOARG counteracted this rightward shift and increased Emax.

Our results demonstrate that contractions to 5‐HT, angiotensin II and KCl are modulated by NO in both sham and neo‐intima‐bearing vessels. Inhibition of NO biosynthesis and collar treatment resulted in additive effects on the EC50 values, suggesting that the 5‐HT and angiotensin (AT) receptors on the smooth muscle cells are also modified by the formation of a neo‐intima. Furthermore, the reduced contractile responses of segments with a neo‐intima are not due to NO formed by an inducible NO synthase in those vessels.
Rapid desensitization of adrenaline‐ and neuropeptide Y‐stimulated Ca2+ mobilization in HEL‐cells
Authors: Michel Martin C.
Published: July 2012

Desensitization of Gs‐coupled receptors, the β2‐adrenoceptor for example, involves rapid and slower components but little is known regarding the existence of rapid desensitization of Gi‐coupled receptors and its possible mechanisms. In HEL‐cells stimulation of α2A‐adrenoceptors by adrenaline or Y1‐like neuropeptide Y receptors by neuropeptide Y, transiently mobilizes Ca2+ from intracellular stores via a Gi‐protein. We have used this model to study the existence and possible mechanisms of rapid desensitization of a Gi‐mediated cellular response.

Following stimulation by adrenaline or neuropeptide Y Ca2+ levels returned towards baseline a few minutes after agonist addition and were refractory to a second agonist exposure demonstrating rapid desensitization. Cross‐desensitization experiments with neuropeptide Y, adrenaline and moxonidine demonstrated the presence of homologous (both receptors) and heterologous desensitization (neuropeptide Y receptors only), and that the α2A‐adrenoceptor desensitization was not specific for phenylethylamine (adrenaline) or imidazoline agonists (moxonidine).

The protein kinase C activator, phorbol ester, rapidly desensitized the hormonal Ca2+ responses and inhibitors of protein kinase C enhanced the hormonal responses inconsistently. The tyrosine kinase inhibitor, herbimycin, enhanced Ca2+ mobilization by adrenaline and neuropeptide Y, whereas the protein phosphatase inhibitor, okdadaic acid, did not affect Ca2+ mobilization or its desensitization.

In the absence of extracellular Ca2+ the endoplasmic reticulum Ca2+‐ATPase inhibitor, thapsigargin, reduced hormone‐stimulated Ca2+ elevations, demonstrating that mobilization occurs from a thapsigargin‐sensitive pool in the endoplasmic reticulum. The inositol phosphate‐independent Ca2+ release modulator, ryanodine, significantly enhanced adrenaline‐ and neuropeptide Y‐stimulated Ca2+ elevations. Blockade of the endoplasmic reticulum Ca2+‐ATPase by thapsigargin in the presence of extracellular Ca2+ enhanced hormone‐stimulated Ca2+ increases, demonstrating the importance of this enzyme for the termination of the Ca2+ signal.

It is concluded that adrenaline and neuropeptide Y‐stimulated Ca2+ mobilization in HEL‐cells occurs from a thapsigargin‐ and ryanodine‐sensitive store in the endoplasmic reticulum and desensitizes rapidly; this appears to involve multiple mechanisms including protein kinases, possibly acting on receptors, and Ca2+ release and sequestration mechanisms.
Nitrovasodilator‐induced relaxation and tolerance development in porcine vena cordis magna: dependence on intact endothelium
Authors: Kojda Georg , Beck Jan Klaus , Meyer Wilfried , Noack Eike
Published: July 2012

Isolated segments of porcine vena cordis magna exhibited a reproducible contractile activity upon application of prostaglandin F2α (PGF2α) or KCl, that was independent of the presence of intact endothelium. Substance P (3 nm) elicited strictly endothelium‐dependent relaxations amounting to 46.1 ± 1.4% (n = 206) of contractions induced by 10 μm PGF2α.

S‐nitroso‐N‐acetyl‐d,l‐penicillamine (SNAP), a compound that spontaneously liberates nitric oxide, concentration‐dependently relaxed PGF2α‐precontracted (50 μm) venous segments. Tolerance induction (incubation with 100 μm SNAP for 30 min) within the same segments resulted in a 3 fold attenuation of this effect, which was not further reduced after additional preincubation with glyceryl trinitrate (GTN). Removal of endothelium or the presence of Nω‐nitro‐l‐arginine methylester (l‐NAME) significantly improved the potency of SNAP before and after tolerance induction.

Concentration‐dependent relaxations induced by GTN in non‐tolerant veins were similar in the presence and absence of endothelium but much more reduced in tolerant endothelium‐denuded (75 fold) compared to intact (20 fold) segments. In contrast, the presence of l‐NAME significantly improved GTN‐activity solely in non‐tolerant veins, which, therefore, also resulted in a more pronounced attenuation of activity due to tolerance induction (100 fold). Preincubation of intact veins with SNAP also reduced GTN‐activity but to a lesser extent (10 fold).

The more delayed but much longer, and compared to GTN somewhat weaker, acting new nitrovasodilator N‐(3‐nitrato‐pivaloyl)‐1‐cysteineethylester (SPM 3672) was more potent in denuded than intact non‐tolerant venous segments. Induction of tolerance by GTN resulted in a 2 fold‐attenuation of potency. This effect was increased to 15 fold in denuded veins but solely due to enhanced potency of SPM 3672 caused by removal of endothelium.

These data demonstrate that intact endothelium of porcine vena cordis magna attenuates the relaxant potency of nitrovasodilators but also probably participates in vascular bioactivation of GTN. We suggest that the reduced potency of nitrovasodilators is due to endogenous production of nitric oxide, which may affect the soluble guanylate cyclase/cyclic GMP‐system or inhibit nitrate bioactivation pathways.
The effect of leukocyte depletion on pulmonary M2 muscarinic receptor function in parainfluenza virus‐infected guinea‐pigs
Authors: Fryer Allison D. , Yarkony Kathryn A. , Jacoby David B.
Published: July 2012

Parainfluenza infections of the airways cause dysfunction of inhibitory M2 muscarinic receptors on the pulmonary parasympathetic nerves. To distinguish the direct effects of virus from the effects of virus‐induced airway inflammation on M2 muscarinic receptor function, guinea‐pigs were depleted of leukocytes by pretreating with cyclophosphamide (30 mg kg−1, i.p. daily for 7 days) after which they were infected with parainfluenza virus type 1 (Sendai virus).

Guinea‐pigs were anaesthetized, tracheotomized, and ventilated. The vagus nerves were isolated and cut, and the distal ends were electrically stimulated causing bronchoconstriction. In control animals, pilocarpine (1–100 μg kg−1, i.v.) inhibited and gallamine (0.1–10 mg kg−1, i.v.) potentiated vagally‐induced bronchoconstriction by stimulating or blocking M2 muscarinic receptors on the vagus. These effects of pilocarpine and gallamine were almost completely lost in virus‐infected animals, demonstrating loss of M2 receptor function.

Cyclophosphamide depleted peripheral blood leukocytes and inhibited the virus‐induced influx of inflammatory cells into the lung. Depletion of leukocytes protected M2 receptor function from viral infection in some, but not all, guinea‐pigs tested.

Among infected animals that had been depleted of leukocytes, the viral content (expressed as the log of the number of tissue culture infectious doses per g lung tissue) of those that retained normal M2 receptor function was 4.29 ± 0.05 (mean ± s.e.mean), while the viral content of those that lost M2 receptor function despite leukocyte depletion was 5.45 ± 0.20 (P = 0.011). Thus the viral content of the lungs in which M2 receptor function was lost was 16 times greater than that of the lungs in which M2 receptor function was preserved. Viral content correlated with the inhibition of vagally‐mediated bronchoconstriction after the maximum dose of pilocarpine (100 μg kg−1; r2 = 0.81, P = 0.0004).

In antigen‐challenged animals, inhibitory M2 muscarinic receptor function is restored when positively charged inflammatory cell proteins are bound and neutralized by heparin. However, heparin (2000 u kg−1, i.v.) did not reverse virus‐induced loss of M2 muscarinic receptor function, even in those guinea‐pigs with a lower viral titer.

Because leukocyte depletion protected M2 muscarinic receptor function only in animals with mild viral infections, it appears that viruses have both an indirect, leukocyte‐dependent effect on M2 receptors and, in animals with more severe infections, a leukocyte‐independent effect on M2 receptors. The failure of heparin to restore M2 receptor function demonstrates that the leukocyte‐dependent loss of M2 receptor function is not mediated by positively charged inflammatory cell proteins.
Metabolic disposition of leukotriene B4 (LTB4) and oxidation‐resistant analogues of LTB4 in conscious rabbits
Authors: Marleau Sylvie , Dallaire Nancy , Poubelle Patrice E. , Borgeat Pierre
Published: July 2012

The kinetics of leukotriene B4 (LTB4), after single i.v. injections of doses of 0.1 to 1 μg kg−1, were investigated in conscious rabbits and compared with those of the Ω‐ and β‐oxidation resistant bioactive analogues, 20, 20, 20‐trifluoro‐LTB4 (20‐F3‐LTB4) and 3‐thio‐LTB4, respectively.

Immunoreactive LTB4 (IR‐LTB4) elimination was first‐order, as shown by a constant systemic clearance (ClLTB4) and a proportional increase in the area under the curve (AUC) of the plasma concentration versus time curve over the dose‐range studied. Our results showed a good correlation between observed steady‐state plasma concentrations (Css) of IR‐LTB4 after continuous infusion of LTB4 and those predicted by using the mean estimated ClLTB4 of 93 ± 4 ml min−1 kg−1, further confirming the linearity of IR‐LTB4 elimination.

The half‐life (t½) or IR‐LTB4 increased from 0.47 ± 0.02 to 0.63 ± 0.04 min as a consequence of a change in the apparent volume of distribution (Vd) from 72 ± 5 to 109 ± 13 ml kg−1, for the 0.1 and 1 μg kg−1 doses injected, respectively.

Single i.v. injections of [3H]‐LTB4 (4.7 ng kg−1) were administered, and the decay of plasma [3H]‐LTB4 following h.p.l.c. purification was used to estimate the kinetic parameters. The kinetic parameters of [3H]‐LTB4 were characterized by a mean systemic clearance (Cl) of 96 ± 11 ml min−1 kg−1, a t½ of 0.53 ± 0.03 min, and an apparent Vd of 85 ± 9 ml kg−1, similar to the parameters obtained after LTB4 boluses.

The disposition of LTB4 analogues, whether resistant to β‐ or to β‐oxidation in vitro, did not differ significantly from the disposition of the LTB4 molecule. The half‐lives of 20‐F3‐LTB4 and 3‐thio‐LTB4 in the circulation were 0.52 ± 0.07 min and 0.70 ± 0.11 min, respectively.

In summary, our results showed that LTB4, as well as Ω‐oxidation‐ and β‐oxidation‐resistant analogues were cleared very rapidly from the rabbit circulation and indicate that in situ, metabolism in blood is not a rate‐limiting factor for the elimination of LTB4.
Effects of the new A2 adenosine receptor antagonist 8FB‐PTP, an 8 substituted pyrazolo‐triazolo‐pyrimidine, on in vitro functional models
Authors: Dionisotti Silvio , Conti Annamaria , Sandoli Daniele , Zocchi Cristina , Gatta Franco , Ongini Ennio
Published: July 2012

We have characterized the in vitro pharmacological profile of putative A2 adenosine antagonists, two non‐xanthine compounds, 5‐amino‐8‐(4‐fluorobenzyl)‐2‐(2‐furyl)‐pyrazolo [4,3‐e]‐1,2,4‐triazolo[1,5‐c] pyrimidine (8FB‐PTP) and 5‐amino‐9‐chloro‐2‐(2‐furyl 1,2,4‐triazolo [1,5‐c] quinazoline (CGS 15943), and the xanthine derivative (E)7‐methyl‐8‐(3,4‐dimethoxystyryl)‐1,3‐dipropyl‐xanthine (KF 17837).

In binding studies on bovine brain, 8FB‐PTP was the most potent (Ki = 0.074 nm) and selective (28 fold) drug on A2 receptors, whereas CGS 15943 and KF 17837 exhibited affinity in the low and high nanomolar range, respectively, and showed little selectivity.

In functional studies, 8FB‐PTP antagonized 5′‐N‐ethyl‐carboxamidoadenosine (NECA)‐induced vasorelaxation of bovine coronary artery (pA2 = 7.98) and NECA‐induced inhibition of rabbit platelet aggregation (pA2 = 8.20). CGS 15943 snowed weak activity in the platelet aggregation model (pA2 = 7.43) and failed to antagonize NECA‐induced vasodilatation. KF 17837 was ineffective in both models up to micromolar concentrations.

Antagonism of A1‐mediated responses was tested versus 2‐chloro‐N6‐cyclopentyladenosine (CCPA) in rat atria. 8FB‐PTP and CGS 15943 also antagonized competitively the negative chronotropic response induced by CCPA. Conversely, KF 17837 was unable to reverse A1‐mediated responses.

8FB‐PTP is a potent and competitive antagonist of responses mediated by A2 adenosine receptors. The data provided a basis to reduce, by further chemical modifications, the affinity at A1 receptor and therefore enhance A2 receptor selectivity.
Importance of the intestine as a site of metformin‐stimulated glucose utilization
Authors: Bailey C.J. , Mynett K.J. , Page T.
Published: July 2012

The intestine has been implicated as a site of increased glucose utilization by the antihyperglycaemic drug, metformin. This study makes a quantitative assessment of this effect.

Glucose utilization by the intestine and hind limb region was determined by arterial‐venous glucose difference adjusted for blood flow rate in fasted rats receiving a hyperglycaemic hyperinsulinaemic infusion.

Intrajejunal administration of metformin, 250 mg kg−1, increased glucose disposal during the infusion procedure, associated with increased glucose utilization in the intestine by 69% and in the hind limb region by 40%.

Metformin, 250 mg kg−1, increased glucose disappearance during an intravenous glucose tolerance test. This was accompanied by increased uptake of tritiated 2‐deoxy‐d‐glucose into the intestinal mucosa to a greater extent than into skeletal muscles (per unit wet weight of tissue).

The results demonstrate that the intestinal mucosa is a quantitatively important site of increased glucose utilization during the blood glucose‐lowering effect of metformin.
Nitric oxide‐mediated inhibitory response of rat proximal colon: independence from changes in membrane potential
Authors: Suthamnatpong Naowarat , Hosokawa Muneaki , Takeuchi Tadayoshi , Hata Fumiaki , Takewaki Tadashi
Published: July 2012

We studied the relation of nitric oxide‐mediated relaxation of smooth muscle to changes in membrane potential of cells in the proximal colon of rats.

The resting membrane potential and electrical field stimulation (EFS)‐induced junction potentials were recorded from the circular and longitudinal muscle cells.

Localized distension with a small balloon caused relaxation of the circular muscle on the anal side of the distended region (descending relaxation). Relaxation of the longitudinal muscle was also induced by EFS.

Inhibitory junction potentials (i.j.ps) were recorded from all circular muscle cells tested, but rarely from the longitudinal muscle cells.

The i.j.ps were recorded only in the presence of atropine but relaxations of both muscles were induced even in the absence of atropine.

Apamin (100 nm) completely abolished the i.j.ps recorded in both circular and longitudinal muscle cells, but had no significant effect on the relaxations of either.

In contrast to apamin, NG nitro‐l‐arginine (10 μm) inhibited the relaxations of both muscles, but did not affect the i.j.ps.

Exogenously added nitric oxide (0.1–10 μm) induced relaxations of both muscles concentration‐dependently, but did not affect the membrane potentials at these concentrations.

These data strongly suggest that nitric oxide‐mediated relaxation of rat proximal colon is not associated with the i.j.ps of the cell membrane.
Evidence for a differential location of vasoconstrictor endothelin receptors in the vasculature
Authors: Moreland Suzanne , McMullen Diane , Abboa‐Offei Benoni , Seymour Andrea
Published: July 2012

There are at least two subtypes of vascular endothelin (ET) receptors. Stimulation of the ETA receptors on vascular smooth muscle cells leads to vasoconstriction, whereas activation of the ETB receptors on endothelial cells elicits vasodilatation. Several reports in the literature have suggested the presence of a vasoconstrictor non‐ETA receptor on vascular smooth muscle which has pharmacological similarities to the ETB receptor. The present study was undertaken to determine the location of this ETB‐like receptor within the vascular system.

Fourteen vascular smooth muscle preparations from six species were used to determine the effect of the ETA receptor antagonist, BQ‐123, on concentration‐response curves elicited by ET‐1 and the ability of the ETB receptor agonist, sarafotoxin S6c, to cause contraction. The vessels fell into two categories. One group was sensitive to BQ‐123 and insensitive to sarafotoxin S6c and, thus, probably contained ETA receptors. The other group, with vasoconstrictor ETB‐like receptors, was insensitive to BQ‐123 and sensitive to sarafotoxin S6c.

Vessels from cynomolgus monkeys, when studied in vitro, appeared to contain primarily ETA receptors, although the potency of BQ‐123 was quite variable, suggesting the possibility of ETA receptor subtypes. In contrast, both ET‐1 and sarafotoxin S6c, given as intravenous injections in conscious monkeys, produced transient, equipotent, and dose‐related increases in blood pressure. The highest dose of sarafotoxin S6c (1 nmol kg−1, i.v.) also caused a marked secondary depressor response (‐80± 6 mmHg) that lasted approximately 10 min. The pressor responses suggest that the vasoconstrictor ETB‐like receptors are present in cynomolgus monkeys.

The data suggest the presence of two distinct vasoconstrictor ET receptor subtypes on smooth muscle cells. The ETA receptors are primarily located on the high pressure side of the circulation. The vasoconstrictor ETB‐like receptors appear to be concentrated on the low pressure side.
Adenosine‐sensitive α1‐adrenoceptor effects on reperfused ischaemic hearts: comparison with phorbol ester
Authors: Khandoudi Nassirah , Moffat Margaret P. , Karmazyn Morris
Published: July 2012

We have examined the effects of the α1‐adrenoceptor agonists, phenylephrine or methoxamine, on contractility in rat and rabbit isolated hearts as well as their effects on postischaemic ventricular recovery. We compared these effects to those of 12‐phorbol 13‐myristate acetate (PMA), a direct activator of protein kinase C (PKC).

The positive inotropic effect of α1‐receptor agonists was significantly attenuated in the presence of the Na/H exchange inhibitor, methylisobutyl amiloride (MIA, 1 μm), whereas the positive inotropic effect of PMA was unaffected.

Reperfusion of rat hearts subjected to either 30 or 60 min of zero‐flow ischaemia, resulted in recovery of contractility to 91 ± 2% and 57 ± 7% of the preischaemic values, respectively which was unaffected by phenylephrine. In contrast, PMA at a concentration (10 pM) devoid of direct depressant effects, significantly decreased recovery following 60 min of ischaemia to 31 ± 4% of pre‐ischaemic value (P < 0.05 from control); an effect which was completely prevented by the PKC inhibitor, bisindolyl‐maleimide. A similar inhibitory effect of PMA and lack of effect of phenylephrine were seen in reperfused rabbit hearts.

As α1‐receptor activation has been shown previously to stimulate cardiac adenosine production, we assessed whether blockade of adenosine A1 receptors with the specific antagonist, 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX, 0.5 μm) would unmask the actions of phenylephrine in hearts subjected to 30 min ischaemia and reperfusion. In the presence of DPCPX, phenylephrine reduced recovery to 44 ± 9% compared to 82 ± 10% recovery in the absence of phenylephrine (P < 0.05). Identical results were observed in rabbit hearts treated with DPCPX in which recovery was reduced from 57.1 ± 11.2% to 17.8 ± 6.8% by phenylephrine (P < 0.05). Another A1 receptor antagonist, (±)‐N6‐endonorbornan‐2‐yl‐9‐methyladenine (N‐0861, 0.5 μm) produced virtually identical results to those observed with DPCPX.

MIA failed to modulate the inhibition of postischaemic recovery by phenylephrine. Bisindolyl‐maleimide, on the other hand, partially prevented the effects of phenylephrine on postischaemic contractile dysfunction. The inhibitory effect of either PMA or phenylephrine on postischaemic recovery of both rat and rabbit hearts was generally dissociated from alterations in energy metabolism, although in the case of rat hearts, inhibition by phenylephrine was associated with diminished high energy phosphate content.

Our results demonstrate that both α1‐receptor activation as well as direct activation of PKC with phorbol ester can attenuate post‐ischaemic ventricular recovery. Moreover, our results strongly suggest that endogenous adenosine protects the heart against the deleterious effects of α1‐receptor activation during ischaemia and reperfusion.
Permeation properties and modulation of volume‐activated Cl−‐currents in human endothelial cells
Authors: Nilius Bernd , Sehrer Jan , Droogmans Guy
Published: July 2012

We have studied the permeation and pharmacological properties of a recently described volume‐activated, calcium‐insensitive, small‐conductance Cl−‐channel in endothelial cells from human umbilical vein.

The relative permeability for various anions was I− > Cl− ∼ Br− > F− > gluconate− (1.63 ± 0.36: 1:0.95 ± 0.16:0.46 ± 0.04:0.19 ± 0.07, n = 10).

5‐Nitro‐2‐(3‐phenylpropylamino)‐benzoic acid (NPPB) induced a fast and reversible block of the current (K1 = 29 μmol l−1).

Extracellular ATP induced a low‐affinity block of the current, that showed a small voltage‐dependence (K1 = 4.9 mmol l−1 at + 80 mV and K1 = 8.2 mmol l−1 at − 80 mV).

Extracellularly applied arachidonic acid (10 μmol l−1) irreversibly blocked the current in 5 out of 9 cells. This block seems to be non‐specific, because other ionic currents, e.g. inwardly rectifying K+ currents, were blocked as well.

Tamoxifen induced a high affinity block of the current (K1 = 2.9 μmol l−1). Block and reversal of block were however much slower than with NPPB.

Cytotoxic compounds, which are substrates of the P‐glycoprotein multidrug transporter, loaded into endothelial cells via the patch pipette, exerted only minor effects on the volume‐activated current. Vinblastine and colcemid did not affect the volume‐activated current, whereas daunomycin and vincristine induced a slow ‘run‐down’ of the current.

The similarity between permeation and pharmacological properties of volume‐activated Cl−‐currents in endothelial cells and those in many other cell types may suggest that they all belong to the same family of volume‐activated small‐conductance Cl−‐channels. Evidence that they belong to the class of P‐glycoprotein associated Cl−‐channels is however only marginal, whereas their biophysical characteristics differ significantly from those of the CIC‐2 volume‐activated Cl−‐channels.
Blockade of the inward rectifier potassium current by the Ca2+‐ATPase inhibitor 2′,5′‐di(tert‐butyl)‐1,4‐benzohydroquinone (BHQ)
Authors: Hasséssian Haroutioun , Vaca Luis , Kunze Diana L.
Published: July 2012

We have investigated the effect of 2′,5′‐di (tert‐butyl)‐1,4‐benzohydroquinone (BHQ) and thapsigargin, inhibitors of the intracellular Ca2+‐ATPase, on ionic currents in rat basophilic leukaemia (RBL‐2H3) cells under whole cell voltage clamp.

The whole cell current was inwardly rectifying and reversed at − 35 ± 6 mV (n = 16). The conductance of the inward current increased as the concentration of extracellular K+ was raised from 2.7 to 5.4, 10.8 and 21.6 mM. BaCl2 (100 μm) reduced the current to a small linear component and shifted the reversal potential to − 4 ± 3 mV (n = 6). A concentration of 50 μm BaCl2 produced 45 ± 10% (n = 4) blockade of the inward current.

BHQ and thapsigargin were examined for their effects on the inwardly rectifying current. A maximal blockade of inward current was obtained within 6 min after perfusion with 10 μm BHQ. The small current remaining after blockade with BHQ had a linear voltage‐dependence and reversed direction at − 6 ± 9 mV (n = 6). Thapsigargin (up to 3 μm) was without effect on the inward rectifier.

In contrast to the blockade of the inward rectifier produced by BaCl2 which was predominantly on the steady state current, particularly at the very hyperpolarized holding potentials (− 120 mV), blockade by BHQ was equally strong on the instantaneous as well as the steady state current.

Blockade of the inward rectifier by BHQ may cause depolarization of the cell which will affect Ca2+ influx during investigations with BHQ. Thapsigargin does not block the inward rectifier and will not inhibit Ca2+ influx in this way.
Effects of bipyridylium compounds on calcium release from triadic vesicles isolated from rabbit skeletal muscle
Authors: Kang J.J. , Hsu K.S. , Lin‐Shiau S.Y.
Published: July 2012

The effects of 1,1′‐diheptyl‐4,4′‐bipyridinium dibromide (DHBP), a viologen for electrochromic memory display agent, on calcium release and ryanodine binding were studied with triad‐rich sarcoplasmic reticulum (SR) vesicles isolated from rabbit skeletal muscle.

DHBP inhibited the calcium release induced by 2 mM caffeine and 2 μg ml−1 polylysine with an IC50 value of 5 μg ml−1 and 4 μg ml−1 respectively.

DHBP inhibited [3H]‐ryanodine binding in a dose‐dependent manner with an IC50 of 2.5 μg ml−1 and 90–100% inhibition at 20–30 μg ml−1.

Calcium uptake by SR was inhibited in the presence of caffeine and this inhibition was antagonized by concomitant addition of DHBP.

The effect of DHBP on muscle twitches was studied on the mouse diaphragm. Muscle twitches elicited by direct electrical muscle stimulation and contractions induced by either 10 mM caffeine or 1 μm ryanodine were blocked by pretreatment with DHBP.

Data from this study provided evidence that DHBP blocked the calcium release from SR by direct interaction with the calcium release channel, also known as the ryanodine receptor. A possible use of this agent as a specific inhibitor for calcium release and as a muscle relaxant was suggested.