bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of monovalent cations on the binding of a charged and an uncharged (‘carbo’‐)muscarinic antagonist to muscarinic receptors
Authors: Hou X. , Wehrle J. , Menge W. , Ciccarelli E. , Wess J. , Mutschler E. , Lambrecht G. , Timmerman H. , Waelbroeck M.
Published: July 2012

The effect of the buffer concentration on binding of [3H]‐N‐methylscopolamine to muscarinic receptors M2 was tested in rat heart. Tracer binding was of low affinity in a 20 mM imidazole buffer (pKD 8.3), inhibited by an increase from 10 to 100 mM of the sodium phosphate buffer concentration (pKD 9.92 to 9.22), slightly inhibited by an increase of the Tris/HCl buffer concentration from 20 to 100 mM (pKD 9.70 to 9.47) and unaffected by an increase of the histidine/HCl buffer concentration from 20 to 100 mM (pKD 9.90 to 9.82). We chose the last buffer to analyse the effect of ions on antagonists binding to cardiac M2 receptors and to transiently expressed wild‐type and (Y533→F) mutant m3 muscarinic receptors in COS‐7 cells.

Equilibrium [3H]‐N‐methylscopolamine binding to cardiac M2 receptors was inhibited, apparently competitively, by monovalent salts (LiCl≥NaCl≥KCl). In contrast, binding of the uncharged 3,3‐dimethylbutan‐l‐ol ester of diphenylglycolic acid (BS‐6181) was facilitated by addition of monovalent salts (LiCl≥NaCl≥KCl) to the binding buffer. This cation binding pattern is consistent with interaction with a large, negative field strength binding site, such as, for instance, a carboxylic acid.

In the presence of 100 mM NaCl, [3H]‐N‐methylscopolamine had a similar affinity for the wild‐type m3 receptor (pKD 9.85) and for a (Y533→F) mutant m3 receptor (pKD 9.68). However, in the absence of added salts, the tracer had a significantly lower affinity for the mutated (pKD 10.19) as compared to the wild‐type (pKD 10.70) m3 receptor. BS‐6181 had a significantly lower affinity for the (Y533→F) mutant m3 muscarinic receptor, as compared to the wild‐type m3 receptor, both in the absence (pKD 6.19‐6.72) in the presence (pKD 6.48‐7.40) of 100 mM NaCl. The effects of NaCl on binding of the uncharged ester and of [3H]‐N‐methylscopolamine to the m3 receptor were decreased by the mutation.

Taken together, these results support the hypothesis that monovalent cations from the buffer may interact with the cation binding site of the receptors (an aspartate residue in the third transmembrane helix of muscarinic receptors). Buffer cations may inhibit competitively the binding of (charged) muscarinic ligands having a tertiary amine or ammonium group, while facilitating the receptor recognition by uncharged, isosteric ‘carbo‐analogues’. Mutation of the (Y533→F) of the m3 receptor decreased the affinity of the receptor for positive charges, including the sodium ion.
KW‐4679‐induced inhibition of tachykininergic contraction in the guinea‐pig bronchi by prejunctional inhibition of peripheral sensory nerves
Authors: Ikemura Toshihide , Okarmura Kyoko , Sasaki Yasuo , Ishi Hidee , Ohmori Kenji
Published: July 2012

Sensory mechanisms play an important role in the vagal regulation of tracheobronchial smooth muscle tone. We examined the effect of KW‐4679, an anti‐allergic drug, on guinea‐pig tachykinin‐mediated contractile responses induced by electrical field stimulation (EFS) in guinea‐pig bronchial muscles.

EFS (8 Hz, 0.5 ms, 15 V, for 15 s) evoked biphasic contractile responses in the guinea‐pig isolated main bronchus in the presence of 5 μm indomethacin. The contractions consisted of a fast phase of an atropine‐sensitive transient contraction and a slow phase of a sustained contraction which was inhibited by a combination of the tachykinin NK1 receptor antagonist, (±)‐CP‐96,345 (1 μm) and the NK2 receptor antagonist, SR 48969 (0.1 μm).

KW‐4679 preferentially inhibited the slow phase in a concentration‐dependent manner by 43.2±7.7% at 10 μm, whereas the drug had no effect on the fast phase at concentrations up to 10 μm. KW‐4679, at a concentration of 100 μm, inhibited not only the slow phase by 49.2±11.4%, but also the fast phase by 36.8±8.4%.

KW‐4679 (10 μm and 100 μm) did not affect the substance P‐induced or neurokinin A‐induced contraction. Against the acetylcholine‐induced contractile responses, 100 μm KW‐4679 had a marked effect producing a 10.2 fold shift to the right in the curve.

The inhibitory effect of KW‐4679 (10 μm) on the slow phase contraction was not influenced by treatment with naloxone (100 nM), propranolol (1 μm), thioperamide (1 μm) saclofen (50 μm), yohimbine (1 μm), methiothepin (1 μm) or methysergide (1 μm).

The inhibitory effect of KW‐4679 (10 μm) on the slow phase contraction was not influenced by treatment with intermediate or large conductance Ca2+‐activated K+ channel blockers (charybdotoxin (10 nM) or iberiotoxin (10 nM)), but suppressed by treatment with small conductance Ca2+‐activated K+ channel blockers, apamin (500 nM) or scyllatoxin (300 nM). Apamin or scyllatoxin per se did not influence the slow phase contractions.

The results suggest that KW‐4679 preferentially inhibits the release of tachykinins from the bronchial sensory nerves through activation of small conductance Ca2+‐activated K+ channels.
Second messenger cascade specificity and pharmacological selectivity of the human P2Y1‐purinoceptor
Authors: Schachter Joel B. , Li Qing , Boyer José L. , Nicholas Robert A. , Harden T. Kendall
Published: July 2012

. The coding sequence of the P2Y1‐purinoceptor was cloned from a human genomic library.

. The open reading frame encodes a protein of 373 amino acids that is 83% identical to the previously cloned chick and turkey P2Y1‐purinoceptor and is ≥95% homologous to the recently cloned rat, mouse, and bovine P2Y1‐purinoceptors.

. The human P2Y1‐purinoceptor was stably expressed in 1321N1 human astrocytoma cells using a retroviral vector. Although the P2Y1‐purinoceptor agonist, 2MeSATP, had no effect on inositol phosphate accumulation in 1321N1 cells infected with the control virus, this agonist markedly stimulated inositol phosphate accumulation in cells infected with the P2Y1‐purinoceptor virus. No effect of 2MeSATP on cyclic AMP accumulation was observed in P2Y1‐receptor‐expressing 1321N1 cells.

. The pharmacological selectivity of 18 purinoceptor agonists was established for the expressed human P2Y1‐purinoceptor. 2MeSATP was more potent than ATP but less potent than 2MeSADP. ADP also was more potent than ATP. A similar maximal effect was observed with most agonists tested. However, α,β‐MeATP had no effect and 3′‐NH2‐3′‐deoxyATP and A2P4 were partial agonists. The order of potency of agonists for activation of the turkey P2Y1‐purinoceptor, also stably expressed in 1321N1 cells, was identical to that observed for the human P2Y1‐purinoceptor.

. C6 glioma cells express a P2Y‐purinoceptor that inhibits adenylyl cyclase but does not activate phospholipase C. Expression of the human P2Y1‐purinoceptor in C6 cells conferred 2MeSATP‐stimulated inositol lipid hydrolysis to these cells. The phospholipase C‐activating human P2Y1‐purinoceptor could be delineated from the endogenous P2Y‐purinoceptor of C6 glioma cells by use of the P2‐purinoceptor antagonist, PPADS, which blocks the P2Y1‐purinoceptor but does not block the endogenous P2Y‐ purinoceptor of C6 cells. P2‐purinoceptor agonists also exhibited differential selectivities for activation of these two P2Y‐purinoceptors.
Antagonism of striatal muscarinic receptors inhibiting dopamine D1 receptor‐stimulated adenylyl cyclase activity by cholinoceptor antagonists used to treat Parkinson's disease
Authors: Olianas Maria C. , Onali Pierluigi
Published: July 2012

A number of cholinoceptor antagonists used in the treatment of Parkinson's disease were examined for their ability to antagonize either the muscarinic receptor‐mediated inhibition of dopamine D1 receptor‐stimulated adenylyl cyclase or the muscarinic receptor‐mediated stimulation of [3H]‐inositol phosphates ([3H]‐IPs) formation in rat striatal membranes. The drugs were found to block the receptors inhibiting adenylyl cyclase activation with high affinity and more potently than those stimulating [3H]‐IPs formation. Moreover, their rank order of potencies for the former effect showed good correlation with their clinical efficacies. These data suggest that the blockade of the muscarinic receptor‐mediated inhibition of striatal dopamine D1 receptor activation may be one of the mechanisms by which cholinoceptor blocking drugs exert their antiparkinsonian effect.
Dual regulation of cerebrovascular tone by UTP: P2U receptor‐mediated contraction and endothelium‐dependent relaxation
Authors: Miyagi Yasushi , Kobayashi Sei , Nishimura Junji , Fukui Masashi , Kanaide Hideo
Published: July 2012

The mechanisms of vascular tone regulation by extracellular uridine 5′‐triphosphate (UTP) were investigated in bovine middle cerebral arterial strips. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored by use of front‐surface fluorometry of fura‐2.

In the arterial strips without endothelium, UTP (0.1 μm‐1 mM) induced contraction in a concentration‐dependent manner. However, when the endothelium was kept intact, cumulative application of UTP (0.1–100 μm) (and only at 1 mM) induced a modest phasic contraction in arterial strips. This endothelium‐dependent reduction of the UTP‐induced contraction was abolished by 100 μm Nω‐nitro‐L‐arginine (L‐NOARG) but not by 10 μm indomethacin. In the presence of intact endothelium, UTP (30 μm) induced a transient relaxation of the strips precontracted with 30 nM U‐46619 (a stable analogue of thromboxane A2), which was completely inhibited by pretreatment with L‐NOARG but not with indomethacin.

In the endothelium‐denuded strips, the contractile response to UTP was abolished by desensitization to either ATPγS or ATP (P2U receptor agonists), but not by desensitization to α, β‐methylene‐ATP (P2X receptor agonist) or to 2‐methylthio‐ATP (P2Y receptor agonist). Desensitization to UTP abolished the contractile response to ATP.

In the endothelium‐denuded artery, a single dose application of UTP induced an initial transient, and subsequently lower but sustained increase in [Ca2+]i and force. In the absence of extracellular Ca2+, UTP induced only the initial transient increases in [Ca2+]i and force, while the sustained increases in [Ca2+]i and force were abolished. UTP (1 mM) had no effect on the basic [Ca2+]i‐ force relationship obtained on cumulative application of extracellular Ca2+ at steady state of 118 mM K+‐depolarization‐induced contraction.

We conclude that in the presence of an intact endothelium, UTP‐induced relaxation of pre‐constricted middle cerebral artery is mainly mediated indirectly, by the production of an endothelium‐derived relaxing factor, but at high doses of UTP, vascular smooth muscle contraction is mediated directly via activation of P2U purinoceptor and [Ca2+]i elevation without Ca2+‐sensitization of the contractile apparatus. UTP may thus exert a dual regulatory effect upon cerebrovascular tone, but in cases where the endothelium is impaired, it may also act as a significant vasoconstrictor.
The involvement of the release of nitric oxide in the pharmacological activity of the new furoxan derivative CHF 2363
Authors: Civelli Maurizio , Giossi Massimo , Caruso Paola , Razzetti Roberta , Bergamaschi Marco , Bongrani Stefano , Gasco Alberto
Published: July 2012

The mechanism of action and the pharmacological effects of the new furoxan derivative, CHF 2363 (4‐ethoxy‐3‐phenylsulphonylfuroxan), were investigated.

Pre‐incubation of CHF 2363 with human platelet‐rich plasma produced a concentration‐dependent inhibition of the platelet aggregation induced by collagen, adenosine diphosphate (ADP) and platelet activating factor (PAF). The test compound was about 5 times more potent than sodium nitroprusside. 3‐Isobutyl‐1‐methyl‐xanthine (IBMX) potentiated the antiaggregating effect of CHF 2363.

CHF 2363 was a potent inhibitor of rubbed endothelium rabbit aortic ring contraction induced by noradrenaline. Comparison of IC50 values showed that CHF 2363 was as potent as glyceryl trinitrate (GTN).

Increasing concentrations of CHF 2363 elevated platelet guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) levels. Adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) levels were unaffected.

Oxyhaemoglobin reduced all the pharmacological actions of the test compound. Moreover, CHF 2363 concentration‐dependently released nitric oxide (NO) in platelet‐rich plasma. The NO release was correlated to its ability to increase platelet cyclic GMP levels.

After exposure of rat aortic strips to supramaximal concentrations of GTN (550 μm), the vasorelaxant activity of CHF 2363 did not change, although that of GTN decreased about 55 fold.

It has been concluded that the new furoxan derivative CHF 2363 exerts a potent antiaggregating and vasorelaxant activity via NO release and increase of cyclic GMP levels. No in vitro cross tolerance between GTN and CHF 2363 was observed.
Dependence on temperature of the effect of dinitrophenol on the release of transmitter quanta at neuromuscular junctions in the mouse diaphragm
Authors: Nishimura Masakazu , Taquahashi Yuhji , Fujita Keiko , Satoh Eiki , Shimizu Yoshio
Published: July 2012

The frequencies (F, s−1) of miniature endplate potentials and the quantal content (m) of endplate potentials were measured intracellularly and simultaneously at mouse diaphragm endplates in a bathing solution that contained 0.6 mM Ca2+ ions and 5 mM Mg2+ ions.

Twin pulses at 4 ms intervals gave the quantal contents of the first (m1) and second (m2) responses. The ratio of m2/m1 was taken as an indicator of the temporal facilitation of the release of transmitter.

Dinitrophenol (DNP, 10 μm) increased the values of F and m at 36°C. This effect did not depend on extracellular Ca2+ ions.

The potentiating effect of DNP disappeared at 24°C but the value of m2/m1 remained constant.

These results suggest that the effect of DNP is modifiable by temperature which can affect systems that control the intracellular metabolism of Ca2+ ions.
Effect of niflumic acid on noradrenaline‐induced contractions of the rat aorta
Authors: Criddle D.N. , de Moura R. Soares , Greenwood I.A. , Large W.A.
Published: July 2012

The effects of niflumic acid, an inhibitor of calcium‐activated chloride channels, were compared with the actions of the calcium channel antagonist nifedipine on noradrenaline‐evoked contractions in isolated preparations of the rat aorta.

The cumulative concentration‐effect curve to noradrenaline (NA) was depressed by both nifedipine and niflumic acid in a reversible and concentration‐dependent manner. The degree of inhibition of the maximal contractile response to NA (1 μm) produced by 10 μm niflumic acid (38%) was similar to the effect of 1 μm nifedipine (39%).

Contractions to brief applications (30 s) of 1 μm NA were inhibited by 55% and 62% respectively by 10 μm niflumic acid and 1 μm nifedipine.

In the presence of 0.1 μm nifedipine, niflumic acid (10 μm) produced no further inhibition of the NA‐evoked contractions. Thus, the actions of niflumic acid and nifedipine were not additive.

In Ca‐free conditions the transient contraction induced by 1 μm NA was not inhibited by niflumic acid (10 μm) and therefore this agent does not reduce the amount of calcium released from the intracellular store or reduce the sensitivity of the contractile apparatus to calcium.

Niflumic acid 10 μm did not inhibit the contractions produced by KCl (up to 120 mM) which were totally blocked by nifedipine. Contractions induced by 25 mM KCl were completely inhibited by 1 μm levcromakalim but were unaffected by niflumic acid.

It was concluded that niflumic acid produces selective inhibition of a component of NA‐evoked contraction which is probably mediated by voltage‐gated calcium channels. These data are consistent with a model in which NA stimulates a calcium‐activated chloride conductance which leads to the opening of voltage‐gated calcium channels to produce contraction.
Bradykinin and changes in microvascular permeability in the hamster cheek pouch: role of nitric oxide
Authors: Félétou M. , Bonnardel E. , Canet E.
Published: July 2012

The objective of this study in the hamster cheek pouch was to investigate the role of nitric oxide in bradykinin‐induced microvascular leakage. The cheek pouch microcirculatory bed of the anaesthetized hamster was directly observed under microscope and vascular leakage was evidenced by dextran‐fluorescein isothiocyanate (FITC‐dextran) extravasation.

Bradykinin superfusion (but not [des‐Arg9]‐bradykinin up to 3 × 10−6 m) induced an increase in microvascular permeability (log EC50: −6.5 ± 0.4) which was exclusively located on the post‐capillary venule. Plasma extravasation was blocked by intravenous pretreatment with Hoe 140, a bradykinin B2 receptor antagonist (estimated log ID50: −9.5 ± 0.2).

The effects of bradykinin (3 × 10−7 m) superfusion were partially but significantly inhibited by indomethacin (10−5 m, P < 0.05) and abolished by pretreatment with L‐nitro‐arginine (L‐NOARG; 10−5 m).

Acetylcholine (10−6 m, which releases endothelial nitric oxide (NO)) and sodium nitroprusside (10−6 m, a nitrovasodilator) superfusion did not induce any changes in permeability, per se. Cromakalim (10−5 m, a potassium channel opener) superfusion induced a moderate but significant plasma extravasation.

The effects of bradykinin, blocked by L‐NOARG pretreatment, were restored by the co‐perfusion of either sodium nitroprusside or cromakalim. Conversely vasoconstriction, produced by a stable analogue of thromboxane A2 (U46619, 3 × 10−7 m), inhibited the increase in permeability produced by bradykinin.

The measurement of arteriolar diameter showed that bradykinin induced a vasodilatation which was blocked by L‐NOARG. L‐NOARG in itself was a powerful vasoconstrictor. Sodium nitroprusside and cromakalim, in the presence of L‐NOARG, were able to restore the inhibited vasodilator response to bradykinin.

These results suggest: (1) bradykinin‐induced microvascular leakage is mediated by bradykinin B2 receptor activation; (2) the increase in permeability is due to two different independent phenomena, i.e. post‐capillary venular endothelial gap formation and arteriolar vasodilatation which increases the post‐capillary venular transmural pressure; (3) NO is only involved in the arteriolar dilatation component of the bradykinin‐induced increase in microvascular permeability.
α1a‐Adrenoceptor polymorphism: pharmacological characterization and association with benign prostatic hypertrophy
Authors: Shibata Katsushi , Hirasawa Akira , Moriyama Nobuo , Kawabe Kazuki , Ogawa Satoshi , Tsujimoto Gozoh
Published: July 2012

Two restriction fragment length polymorphisms of the human α1a‐adrenoceptor gene digested with PstI restriction enzyme exist; the nucleotide change causes the substitution of C residue for T at nucleotide 1441, thereby Arg492 to Cys492 transition, which might confer an additional putative palmitoylation site in the carboxy‐terminal segment of the α1a‐adrenoceptor. In the present study, we compared their pharmacological properties and examined whether this α1a‐adrenoceptor polymorphism is associated with benign prostatic hypertrophy (BPH).

The frequency of α1a‐adrenoceptor polymorphism was not differently distributed between patients with benign prostatic hypertrophy (BPH) and normal subjects in Japan; thus, the relative frequencies of the C and T alleles were 0.90: 0.10 in normal male subjects (n = 45) and 0.87: 0.13 in BPH patients (n = 222), respectively. However, the frequency distribution of this polymorphism was significantly different between the Japanese and U.S. populations; thus, C and T alleles were 0.34 and 0.66 in U.S. populations.

Utilizing Chinese hamster ovary (CHO) cells stably expressing the two polymorphic α1a‐adrenoceptors (Arg492 and Cys492), we compared their binding affinity and signal transduction. Radioligand binding studies with 2‐[β‐(4‐hydroxy‐3[125I]‐iodophenyl) ethylamino‐methyl]tetralone ([125I]‐HEAT) showed no marked difference in the antagonist or agonist binding affinities between the two receptors. Also, both receptors were found to be coupled to the calcium signaling, and the concentration‐cytosolic Ca2+ concentrations ([Ca2+]i response relationships for noradrenaline were similar for the two polymorphic receptors. Furthermore, the receptor‐mediated [Ca2+]i response was markedly desensitized after a 2 h exposure of phenylephrine (10 μm), and the extent of the desensitization was not significantly different between the two receptors.

In summary, the results showed that the two α1a‐adrenoceptors generated by genetic polymorphism have similar pharmacological characteristics, and the receptor‐mediated [Ca2+]i response can be desensitized in a similar manner. The study did not provide any evidence to support the hypothesis that α1a‐adrenoceptor gene polymorphism is associated with BPH.
P2‐purinoceptor‐mediated formation of inositol phosphates and intracellular Ca2+ transients in human coronary artery smooth muscle cells
Authors: Strøbæk Dorte , Olesen Søren‐Peter , Christophersen Palle , Dissing Steen
Published: July 2012

. The effects of extracellular adenosine 5′‐triphosphate (ATP) on smooth muscles are mediated by a variety of purinoceptors. In this study we addressed the identity of the purinoceptors on smooth muscle cells (SMC) cultured from human large coronary arteries. Purinoceptor‐mediated increases in [Ca2+]i were measured in single fura‐2 loaded cells by applying a digital imaging technique, and the formation of inositol phosphate compounds was quantified after separation on an anion exchange column.

. Stimulation of the human coronary artery SMC (HCASMC) with extracellular ATP at concentrations of 0.1–100 μ induced a transient increase in [Ca2+]i from a resting level of 49 ± 21 nM to a maximum of 436 ± 19 nM. The effect was dose‐dependent with an EC50 value for ATP of 2.2 μm.

. The rise in [Ca2+]i was independent of the presence of external Ca2+, but was abolished after depletion of intracellular stores by incubation with 100 nM thapsigargin.

. [Ca2+]i was measured upon stimulation of the cells with 0.1–100 μm of the more specific P2‐purinoceptor agonists α,β‐methyleneadenosine 5′‐triphosphate (α,β‐MeATP), 2‐methylthioadenosine 5′‐triphosphate (2MeSATP) and uridine 5′‐triphosphate (UTP). α,β‐MeATP was without effect, whereas 2MeSATP and UTP induced release of Ca2+ from internal stores with 2MeSATP being the most potent agonist (EC50 = 0.17 μ), and UTP having a potency similar to ATP. The P1 purinoceptor agonist adenosine (100 μm) did not induce any changes in [Ca2+]i.

. Stimulation with a submaximal concentration of UTP (10 μm) abolished a subsequent ATP‐induced increase in [Ca2+]i, whereas an increase was induced by ATP after stimulation with 10 μm 2MeSATP.

. The phospholipase C (PLC) inhibitor U73122 (5 μm) abolished the purinoceptor‐activated rise in [Ca2+]i, whereas pretreatment with the Gi protein inhibitor pertussis toxin (PTX, 500 ng ml−1) was without effect on ATP‐evoked [Ca2+]i increases.

. Receptor activation with UTP and ATP resulted in formation of inositol phosphates with peak levels of inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) observed 5–20 s after stimulation.

. These findings show, that cultured HCASMC express G protein‐coupled purinoceptors, which upon stimulation activate PLC to induce enhanced Ins(1,4,5)P3 production causing release of Ca2+ from internal stores. Since a release of Ca2+ was induced by 2MeSATP as well as by UTP, the data indicate that P2Y‐ as well as P2U‐purinoceptors are expressed by the HCASMC.
Influence of dopamine as noradrenaline precursor on the secretory function of the bovine corpus luteum in vitro
Authors: Kotwica J. , Skarzynski D. , Bogacki M. , Miszkiel G.
Published: July 2012

. Dopamine is assumed to affect the ovary function after its conversion into noradrenaline (NA).

. To study this bovine luteal slices from 11–14 days of the oestrous cycle were preincubated for 24 h to recover β‐receptors and next they were incubated for 1, 2 or 4 h with (a) different doses of dopamine; (b) dopamine together with a β‐antagonist (propranolol) or with a dopamine receptor blocker (droperidol); (c) dopamine with a dopamine‐β‐hydroxylase inhibitor.

. Dopamine stimulated the luteal content of oxytocin (OT) and progesterone. This effect was inhibited by propanolol but not by droperidol.

. Dopamine added to the medium was followed by an increase of noradrenaline there. This rise was dose and time‐dependent.

. The dopamine‐β‐hydroxylase inhibitor, inhibited the stimulating effect of dopamine on luteal progesterone and OT content.

. Bovine corpus luteum can synthesize de novo NA from dopamine as a precursor.
Carbon monoxide‐induced relaxation of the ductus arteriosus in the lamb: evidence against the prime role of guanylyl cyclase
Authors: Coceani F. , Kelsey L. , Seidlitz E.
Published: July 2012

. We have previously found that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450‐based mono‐oxygenase reaction which sustains contractile tone. Our proposal, however, has been questioned on the evidence of findings in other blood vessels implicating the guanylyl cyclase‐based relaxing mechanism as the target for CO. To investigate this issue further, we have carried out experiments in the isolated ductus from near‐term foetal lambs and have examined the effect of CO concomitantly on muscle tone and cyclic GMP content, both in the absence and presence of guanylyl cyclase inhibitors, or during exposure to monochromatic light at 450 nm.

. CO (65 μm) reversed completely, or nearly completely, the tone developed by the vessel in the presence of oxygen (30%) and indomethacin (2.8 μm). Cyclic GMP content tended to increase with the relaxation, but the change did not reach significance. Sodium nitroprusside (SNP), a NO donor, mimicked CO in relaxing the ductus. Contrary to CO, however, SNP caused a marked accumulation of cyclic GMP with levels being positively correlated with the relaxation.

. Methylene blue (10 μm) reduced marginally the CO relaxation, whilst LY‐83583 (10 μm) had an obvious, albeit variable, inhibitory effect. Basal cyclic GMP content was lower in tissues treated with either compound and rose upon exposure to CO. However, the levels attained were still within the range of values for tissues prior to any treatment. Furthermore, the elevation in cyclic GMP was not related to the magnitude of the CO relaxation.

. Illumination of the ductus with monochromatic light at 450 nm reversed the CO relaxation and any concomitant increase in cyclic GMP. In the absence of CO, light by itself had no effect.

. Ductal preparations with only muscle behaved as the intact preparations in reacting to CO, both in the absence and presence of guanylyl cyclase inhibitors, or during illumination.

. We conclude that the primary action of CO in the ductus arteriosus is not exerted on the guanylyl cyclase heme and that cyclic GMP may only have an accessory role in the relaxation to this agent. This finding reasserts the importance of a cytochrom P450‐based mono‐oxygenase reaction for generation of tone and as a target for CO in the ductus.
Modulation of the hepatic α1‐adrenoceptor responsiveness by colchicine: dissociation of free cytosolic Ca2+‐dependent and independent responses
Authors: Butta Nora , Martin‐Requero Angeles , Urcelay Elena , Parrilla Roberto , Ayuso Matilde S.
Published: July 2012

. The cytoskeletal depolymerizing agent, colchicine, prevents the hepatic α1‐adrenoceptor‐mediated stimulation of respiration, H+ and Ca2+ release to the effluent perfusate, intracellular alkalosis, and glycogenolysis. Unlike the other parameters, colchicine does not perturb the α1‐agonist‐induced stimulation of gluconeogenesis or phosphorylase ‘a’ activation, and enhances the increase in portal pressure response. The lack of effect of colchicine on the hepatic α2‐adrenoceptor‐mediated effects indicates that its actions are α1‐specific.

. Colchicine enhances the acute α1‐adrenoceptor‐mediated intracellular Ca2+ mobilization and prevents the activation of protein kinase C. This differential effect on the two branches of the α1‐adrenoceptor signalling pathway is a distinctive feature of the colchicine action.

. The lack of effect of colchicine in altering the α1‐adrenoceptor ligand binding affinity suggests that it might interact with some receptor‐coupled regulatory element(s).

. The acuteness of the colchicine effect and the ability of its isomer β‐lumicolchicine to prevent all the α1‐adrenoceptor‐mediated responses but the increase in vascular resistance, indicate that its action cannot be merely ascribed to its effects in depolymerizing tubulin.

. Colchicine perturbs the hepatic responses to vasoactive peptides. It enhances the vasopressin‐induced rise of cytosolic free Ca2+ in isolated hepatocytes and prevents the sustained decrease of Ca2+ in the effluent perfusate. It also inhibits the stimulation of glycogenolysis, without altering the stimulation of gluconeogenesis.

. It is concluded that there are at least two major α1‐adrenoceptor signalling pathways. One is colchicine‐sensitive, independent of variations in free cytosolic Ca2+, and protein kinase C‐independent; the other one is colchicine‐insensitive, dependent on variations in free cytosolic Ca2+, and protein kinase C‐independent.
Differential presynaptic modulation of noradrenaline release in human atrial tissue in normoxia and anoxia
Authors: Münch Götz , Kurz Thomas , Urlbauer Thomas , Seyfarth Melchior , Richardt Gert
Published: July 2012

. Presynaptic modulation of noradrenaline release in human atrial tissue specimens was investigated under normoxic and anoxic conditions.

. Noradrenaline release was induced by electrical stimulation and release during experimental intervention (S2) was compared with release during a preceding control stimulation (S1). The results were expressed as the geometric means and 95% confidence intervals of the S2/S1 ratio.

. The α2‐adrenoceptor agonist, UK 14304 (0.1 μmol 1−1) significantly inhibited noradrenaline release, resulting in a S2/S1 ratio of 0.49 (0.40‐0.59), and the α2‐adrenoceptor antagonist, yohimbine (1 μmol 1−1) increased noradrenaline release (S2/S1 1.83 [1.43‐2.35]) during normoxia. Both compounds were ineffective during anoxia.

. Adenosine (30 μmol 1−1) inhibited noradrenaline release with a S2/S1 ratio of 0.54 (0.42‐0.66). The adenosine antagonist, 8‐phenyltheophylline, alone had no effect during normoxia. During anoxia, neither adenosine nor 8‐phenyltheophylline altered noradrenaline release.

. The β2‐adrenoceptor agonist, terbutaline (1 μmol 1−1) increased (1.53 [1.14‐2.01]) and the β‐adrenoceptor antagonist, pindolol (1 μmol 1−1) suppressed noradrenaline release (0.62 [0.49‐0.79]) under normoxic conditions. During anoxia, pindolol significantly inhibited noradrenaline release with a S2/S1 ratio of 0.66 (0.51‐0.85), whereas terbutaline did not influence noradrenaline release.

. Angiotensin II (0.1 μmol 1−1) enhanced noradrenaline release resulting in a S2/S1 ratio of 1.44 (1.34‐1.54), while the angiotensin II antagonist, losartan (1 μmol 1−1) had no effect on noradrenaline release during normoxia. Conversely, angiotensin II did not increase noradrenaline release and losartan significantly inhibited noradrenaline release to a S2/S1 ratio of 0.60 (0.46‐0.77) during anoxia.

. In conclusion, human cardiac tissue possesses presynaptic inhibitory α2‐adrenoceptors and adenosine receptors, as well as facilitatory β2‐adrenoceptors and angiotensin II receptors regulating noradrenaline release under normoxic conditions. During anoxia the responses to α2‐adrenoceptors and adenosine receptor stimulation are lost, whereas facilitatory responses to β2‐adrenoceptors and angiotensin II receptor stimulation are maintained and these receptors appear to be maximally stimulated. This differential presynaptic modulation in anoxia may contribute to enhanced sympathetic activity in ischaemia.
Differences in hypolipidaemic effects of two statins on Hep G2 cells or human hepatocytes in primary culture
Authors: Clerc Thierry , Sbarra Véronique , Domingo Nicole , Rault Jean Philippe , Diaconescu Nicolas , Moutardier Vincent , Hasselot Nicole , Lafont Huguette , Jadot Guy , Laruelle Claude , Chanussot Françoise
Published: July 2012

. The objective of this study was to compare in cultured human hepatocytes or Hep G2 cells, changes in the fate of unesterified low density lipoprotein (LDL)‐cholesterol induced by crilvastatin, a new cholesterol lowering drug and a reference statin, simvastatin.

. The experiments were carried out for 20 h, each well contained 4.2 × 105/cm2 Hep G2 cells or 0.5 × 105/cm2 human hepatocytes, 130 μm ursodeoxycholate, 0.68 μCi or 1.59 μCi unesterified human [14C]‐LDL‐cholesterol, crilvastatin or simvastatin at 0 or 50 μm (both cell types) or 300 μm (Hep‐G2 cells). Incubation with the two drugs resulted in increased amounts of unesterified [14C]‐LDL‐cholesterol taken by the two cell types, compared to control.

. Crilvastatin 50 μm led to significantly higher quantities of [14C]‐glyco‐ and [14C]‐tauro‐conjugated bile salts, compared to simvastatin. Statins reduced the apo B100 level secreted by the two cell types (simvastatin) or human hepatocytes (crilvastatin). Crilvastatin enhanced both the level of apo A1 secreted by the Hep G2 cells and the level of APF, a high density lipoprotein (HDL) and biliary apoprotein.

. Crilvastatin not only acts by stimulating LDL‐cholesterol uptake by hepatocytes, but also by enhancing the catabolism of LDL‐cholesterol in bile salts and probably by stimulating HDL and/or bile component secretion. Such a mechanism was not previously described for HMG CoA reductase inhibitors. Our results on APF show that this apoprotein could be considered also as an indicator of changes in bile and/or HDL compartments.

. The human hepatocyte model appeared to be a suitable and relevant model in the pharmacological***metabolic experiments carried out in this study. It led to more consistent data than those obtained with Hep G2 cells.
The role of TNF‐α in fever: opposing actions of human and murine TNF‐α and interactions with IL‐β in the rat
Authors: Stefferl Andreas , Hopkins Stephen J. , Rothwell Nancy J. , Luheshi Giamal N.
Published: July 2012

The role of tumour necrosis factor‐α (TNF‐α) in fever is controversial. Some studies have indicated that TNF‐α acts as a cryogen to inhibit fever, while others suggest that TNF‐α is an endogenous pyrogen which mediates fever. The majority of studies in experimental animals supporting a cryogenic action have been conducted using human (h)TNF‐α, which has been shown to bind only to one (p55) of the two TNF‐α receptors in rodents.

The aim of the present investigation was to study the role of TNF‐α in fever by comparing effects of hTNF‐α, which binds only to the p55 receptor, with those of murine (m)TNF‐α, which binds to both p55 and p75 TNF‐α receptors, and to investigate the relationship between TNF‐α and interleukin‐1 (IL‐1), an important endogenous pyrogen.

Injection of hTNF‐α (0.3–10 μg kg−1, i.p.) had no effect on core temperature in conscious rats (measured by remote radiotelemetry), whereas mTNF‐α (3 μg kg−1) induced fever which was maximal 1 h after the injection (38.2 ± 0.2°C compared to 37.3 ± 0.1°C in controls). Intracerebroventricular (i.c.v.) administration of either form of TNF‐α elicited dose‐dependent fever at doses higher than 0.12 μg kg−1.

Peripheral injection of hIL‐1β (1 μg kg−1) resulted in fever (38.3 ± 0.2°C compared to 37.2 ± 0.1°C in controls at 2 h), which was significantly attenuated (P < 0.01) by co‐administration of a sub‐pyrogenic dose of hTNF‐α (1 μg kg−1), but was unaffected by co‐administration of mTNF‐α (0.1 or 0.3 μg kg−1, i.p.). In contrast, intracerebroventricular (i.c.v.) co‐administration of a sub‐pyrogenic dose (0.12 μg kg−1) of hTNF‐α did not attenuate fever induced by intraperitoneal (i.p.) injection of IL‐1β, and sub‐pyrogenic dose (0.12 μg kg−1, i.c.v.) of mTNF‐α significantly prolonged the febrile response to IL‐1β. Pretreatment of animals with anti‐TNF‐α antiserum (i.c.v.) did not affect the febrile response to systemic IL‐1β.

Animals injected i.p. with a pyrogenic dose of mTNF‐α developed fever (38.2 ± 0.2°C compared to 37.3 ± 0.1°C in controls 2 h after the injection) that was completely abolished by peripheral administration of IL‐***1ra (2 mg kg−1, P < 0.001), while i.c.v. administration of IL‐***1ra (400 μg/rat) did not affect mTNF‐α‐induced fever.

These data indicate that endogenous TNF‐α is probably a pyrogen and that previous results suggesting cryogenic actions of TNF‐α resulted from the use of a heterologous protein in the rat. The markedly contrasting effects of mTNF‐α and hTNF‐α could result from different interactions with the two TNF‐α receptor subtypes. The data also suggest that fever induced by exogenous TNF‐α is mediated via release of IL‐1β in peripheral tissues, but not in the brain.
Investigation of the contributions of nitric oxide and prostaglandins to the actions of endothelins and sarafotoxin 6c in rat isolated perfused lungs
Authors: Lal H. , Woodward B. , Williams K.I.
Published: July 2012

The aims of the study were to assess the contribution of prostaglandins and nitric oxide (NO) to the effects of endothelin (ETs) and sarafotoxin 6c (SX6c) in perfused rat lungs. This was carried out by using indomethacin, a cyclo‐oxygenase inhibitor and NG‐nitro‐L‐arginine (L‐NOARG), a NO synthase inhibitor. Responses were studied under basal perfusion conditions and in other experiments after the elevation of vascular tone with the thromboxane‐mimetic, U46619. The sub‐types of ET receptors involved were characterized by use of ET receptor antagonists and cross‐tachyphylaxis.

Pulmonary perfusion pressure (PPP), lung weight and pulmonary inflation pressure (PIP), were continuously recorded. Although L‐NOARG (100 μm) did not alter basal parameters it markedly augmented the vasoconstriction and lung weight increases induced by ET‐1 (50–400 pmol) or SX6C (25–200 pmol) while vasoconstrictor responses to phenylephrine were not affected by L‐NOARG.

L‐NOARG markedly potentiated the bronchoconstriction induced by ET‐1 or SX6C whereas it had no effect on responses to carbachol.

When vascular tone was elevated, low doses (1.25–40 pmol) of ET‐1, ET‐3 and SX6C produced falls in PPP. The vasodilator potencies were SX6C > ET‐1 = ET‐3. The ETA receptor antagonist, BQ123, did not affect these depressor responses, whereas the mixed ETA/ETB antagonist, bosentan, blocked them.

Indomethacin (10 μm) partially inhibited vasodilator response to ET‐1, whereas it had no effect on SX6C‐induced vasodilatation.

L‐NOARG plus indomethacin completely blocked ET‐1 induced vasodilatation, whereas responses to SX6C were blocked by L‐NOARG alone.

Repeated injections of submaximal doses of ET‐1 or SX6C caused tachyphylaxis to vasodilator responses. Subsequent injections of SX6C or ET‐1 did not elicit depressor responses showing cross tachyphylaxis had occurred.

These findings indicate that under basal conditions the pulmonary vasoconstrictor, lung weight and bronchoconstrictor responses to ET‐1 and SX6C are attenuated by evoked release of nitric oxide (NO). When vascular tone was elevated, lower doses of ETs and SX6C produced vasodilatation. These vasodilator responses are indirect, those to SX6C being mediated via NO production, whereas those to ET‐1 involve both NO and prostanoid(s). Tachyphylaxis and ET antagonist experiments indicate that the same receptor subtype is involved in mediating the vasodilatation and that this is of the ETB type located on the endothelium. However the post‐receptor vasodilator events triggered by ET‐1 or SX6C appear to be different.
Agmatine, an endogenous modulator of noradrenergic neurotransmission in the rat tail artery
Authors: González C. , Regunathan S. , Reis D.J. , Estrada C.
Published: July 2012

We investigated the vascular effects of agmatine (decarboxylated arginine), an endogenous ligand for α2‐adrenoceptors and non‐adrenoceptor imidazoline (I‐) receptors, present in endothelium, smooth muscle and plasma, using the rat tail artery as a model.

While by itself agmatine (10 nM‐1 mM) was without effect on isolated arterial rings, at the highest concentration used (1 mM) it slightly increased EC50 values for contractions elicited respectively by the α1‐ and α2‐adrenoceptor agonists methoxamine and clonidine.

Agmatine (0.03‐1 mM) produced a concentration‐dependent transient inhibition of the contractions induced by transmural nerve stimulation (TNS; 200 mA, 0.2 ms, 1 Hz, 10 s). This effect was abolished by the α2‐adrenoceptor antagonists, rawolscine and idazoxan.

In the presence of rawolscine or idazoxan, agmatine produced a concentration‐dependent delayed facilitation of TNS‐induced contractions, which was prevented by cocaine.

Neither inhibitory nor potentiating actions were produced by agmatine on contractions induced by noradrenaline (NA) administration.

Agmatine did not directly affect [3H]‐NA uptake in bovine cultured chromaffin cells.

Agmatine can regulate vascular function by two opposing actions at sympathetic nerve terminals, with different latencies: a transient inhibition of NA release mediated by prejunctional α2‐adrenoceptors and a cocaine‐sensitive delayed facilitation the mechanism of which is undetermined at present.

The results reveal the existence of a novel endogenous amine modulating NA release in the perivascular sympathetic terminals.
Some evidence against the involvement of arachidonic acid in muscarinic suppression of voltage‐gated calcium channel current in guinea‐pig ileal smooth muscle cells
Authors: Unno T. , Komori S. , Ohashi H.
Published: July 2012

To see if arachidonic acid (AA) plays a role in the sustained suppression of voltage‐gated calcium channel currents produced by muscarinic receptor stimulation by carbachol (CCh), the effects of AA on membrane currents were examined in whole‐cell voltage‐clamped smooth muscle cells of the guinea‐pig ileum.

In cells bathed in Ba2+ PSS and dialysed with Cs+‐based low EGTA (0.05 mM) pipette solution, and in which Ba2+ current (IBa) flowing through voltage‐gated calcium channels was evoked repeatedly by stepping to 0 mV from the holding potential of −60 mV, AA (1–30 μm), applied extracellularly, gradually suppressed IBa in a concentration‐dependent manner. The IBa suppression was observed even with 20 mM EGTA in the pipette.

AA (3 μm) and CCh (10 μm) shifted the voltage‐dependent inactivation curve of IBa in the negative potential direction, but the effect of AA differed from that of CCh in that an accompanying appreciable decrease in the slope was observed.

The sustained suppression of IBa induced by CCh (10 μm) remained almost unaltered after pretreatment with 4‐bromophenacyl bromide (10 μm), an inhibitor of phospholipase A2, or a combination of indomethacin (10 μm), an inhibitor of the cyclo‐oxygenase pathway, and nordihydroguaiaretic acid (10 μm), an inhibitor of the lipoxygenase pathway.

In cells bathed in Ca2+ PSS and dialysed with K+‐based pCa 6.5 pipette solution, voltage‐dependent Ca2+ current (ICa) and K+ current (IK) were recorded simultaneously. AA (3 μm) suppressed IK as well as ICa, whereas CCh (10 μm)) suppressed ICa but not IK.

We conclude from these results that AA or its metabolite is unlikely to be involved in the sustained suppression of voltage‐gated calcium channel current induced by muscarinic receptor stimulation in guinea‐pig ileal smooth muscle cells.
Mediation by neurotensin‐receptors of effects of neurotensin on self‐stimulation of the medial prefrontal cortex
Authors: Fernández R. , Sabater R. , Sáez J.A. , Montes R. , Alba F. , Ferrer J.M.R.
Published: July 2012

Intracortical microinjections of neurotensin (NT) selectively decreased intracranial self‐stimulation (ICSS) of the medial prefrontal cortex in the rat.

To elucidate whether this effect is mediated by NT receptors or by the formation of NT‐dopamine complexes, we investigated the effects on ICSS of intracortical microinjections of neurotensin (1–11), an NT fragment that forms extracellular complexes with dopamine but does not bind to NT receptors.

We also studied the effects of the peripheral administration of SR 48692, a selective antagonist of NT receptors, on the inhibition of ICSS produced by the intracortical administration of NT.

Unilateral microinjections of neurotensin (1–11) at doses of 10, 20 and 40 nmol into the medial prefrontal cortex did not change the basal ICSS rate of this area.

The intraperitoneal administration of SR 48692 at doses of 0.08 and 0.16 mg kg−1 30 min before microinjection of 10 nmol of NT into the medial prefrontal cortex, antagonized the inhibition of ICSS produced by the neuropeptide.

These results demonstrate that the inhibitory effect of NT on ICSS is mediated by NT receptors.
Increased interleukin‐8 release by β‐adrenoceptor activation in human transformed bronchial epithelial cells
Authors: Lindén Anders
Published: July 2012

The effect of β‐adrenoceptor activation on release of the neutrophil chemoattractant, interleukin‐8 (IL‐8), was examined in human transformed bronchial epithelial cells (16HBE cells).

The combined β1‐ and β2‐adrenoceptor agonist, isoprenaline, time‐ (100 nM, 2–18 h) and concentration‐ (1–30 nM) dependently increased IL‐8 protein content in the cell culture supernatant as measured by an enzyme immunosorbent assay standardized for DNA by fluoro‐colorimetry.

Isoprenaline (1–100 nM, 15 min) increased cyclic AMP concentration‐dependently.

The effect of isoprenaline (100 nM) was inhibited by the β‐adrenoceptor blocker propranolol (10 μm). The maximum magnitude of IL‐8 increase caused by β‐adrenoceptor activation was 40% of that caused by the pro‐inflammatory cytokine tumor necrosis factor‐alpha (TNF‐α: 100 ng ml−1).

The selective β2‐adrenoceptor agonist salbutamol (1 μm), increased IL‐8 protein similarily to isoprenaline and the cyclic AMP analogue, dibutyryl cyclic AMP (1 mM) produced a corresponding effect.

Pretreatment with isoprenaline (100 nM) followed by TNF‐α (20 ng ml−1) increased IL‐8 additively.

In conclusion, β‐adrenoceptor stimulation increased the release of the neutrophil chemoattractant, IL‐8 in 16HBE cells, via an increase in intracellular cyclic AMP. β‐adrenoceptor stimulation adds to the IL‐8 increase caused by the pro‐inflammatory cytokine TNF‐α. If this mechanism exists in vivo, β‐adrenoceptor activation may increase neutrophil chemotaxis into the airways.