bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological actions of the selective and non‐selective β‐adrenoceptor antagonists celiprolol, bisoprolol and propranolol on human bronchi
Authors: Hauck R.W. , Schulz Ch. , Emslander H.P. , Böhm M.
Published: July 2012

The pharmacological actions of the β‐adrenoceptor antagonists, celiprolol, bisoprolol and propranolol were investigated in human lung tissue by radioligand binding experiments as well as in human isolated bronchi by functional experiments in organ baths.

Data from lung tissue were compared to those obtained from myocardial membranes.

Lung tissue was obtained from 10 patients having undergone lung resection for bronchial carcinoma and myocardial tissue from a patient who had received a heart transplantation.

In radioligand binding experiments, celiprolol exhibited a high affinity binding to β1,‐adrenoceptors in heart and a low affinity binding to β2‐adrenoceptors in lung tissue. The selectivity obtained for the β1‐adrenoceptor was calculated to a factor of eleven.

Compared to bisoprolol and propranolol, celiprolol elicited the lowest affinity for the β‐adrenoceptor, as judged from the K1‐values.

In the absence and presence of the guanine nucleotide Gpp(NH)p celiprolol did not affect receptor binding.

In functional experiments on intact bronchi, celiprolol, bisoprolol and propranolol failed to produce relaxation (± forskolin) or a significant difference in efficacy in antagonizing the relaxant effects of isoprenaline. However, a rank order of potencies was revealed (propranolol: bisoprolol.celiprolol = 46: 12:1).

Plasma concentrations for celiprolol and bisoprolol usually achieved in vivo were below the IC50 value obtained in vitro. In contrast, for propranolol, plasma concentrations were nearly identical with the IC50 value.

It is concluded that celiprolol is a selective β1‐adrenoceptor antagonist on human heart and has no agonistic properties on intact human bronchi. Compounds such as celiprolol and bisoprolol may in comparison to propranolol, possess reasonable therapeutic advantages in the treatment of patients with obstructive lung disease due to their low affinity for β2‐adrenoceptors.
The haemodynamic actions of ZENECA ZD7288, a novel sino‐atrial node function modulator, in the exercising beagle: a comparison with zatebradine and propranolol
Authors: Rouse W. , Stafford P.J. , Johnson I.R.
Published: July 2012

ZENECA ZD7288 (4‐(N‐ethyl‐N‐phenylamino)‐1,2‐dimethyl‐6‐(methylamino) pyrimidinium chloride, formerly ICID7288) is a novel sino‐atrial node function modulator which selectively slows heart rate.

The haemodynamic effects of ZD7288 (0.1, 0.3 and 1.0 mg kg−1, i.v.) have been evaluated and compared with those of placebo (physiological saline), zatebradine (ULFS 49, 0.1, 0.3 and 1.0 mg kg−1, i.v.) and propanolol (0.03, 0.1, and 0.3 mg kg−1, i.v.) in beagles chronically instrumented for measurement of heart rate, aortic pressure, aortic flow and dPLV/dtmax. The dogs were trained to run at 6.5 k h−1 on a level treadmill for 5 min at half hourly intervals over a period of 4 h. Drugs were dosed cumulatively after the second, fourth and sixth exercise periods.

Control experiments demonstrated a degree of accommodation to repeated exercise over a period of 4 h. Resting heart rate decreased by 21 beats min−1, but heart rate response to exercise was maintained, whereas dPLV/dtmax at rest remained steady while the response to exercise decreased significantly (by 25% after 2 h, P < 0.05).

ZD7288 and zatebradine both decreased heart rate during exercise in a dose‐dependent manner, whilst heart rate at rest did not differ from resting heart rates in saline dosed control animals. In contrast, heart rate at rest and during exercise were lowered equally by the lowest doses of propranolol (approximately by 30 beats min−1), and additional doses caused only minor additional decreases. The exercise‐induced tachycardia was maintained within 12% of pre‐dose levels, presumably by withdrawal of vagal tone.

Cardiac inotropism, as indicated by dPLV/dtmax, was not affected by ZD7288 or zatebradine at rest, although the inotropic response to exercise decreased in proportion to the decreases in exercise‐induced tachycardia. Propranolol caused a marked dose‐dependent decrease in the exercise‐induced inotropic response (by 85% at 0.3 mg kg−1).

Whilst the sino‐atrial node modulators increased stroke volume at rest, and augmented increases in response to exercise, propranolol did not affect resting stroke volume and decreased the responses to exercise.

Cardiac output at rest and cardiac output increases during exercise were well maintained in the presence of ZD7288 and zatebradine in contrast to propranolol which induced a significant depression of cardiac output, both at rest and during exercise. Propranolol also caused significant systemic vasoconstriction.

In conclusion, ZD7288 has haemodynamic actions comparable to those of zatebradine despite their chemical dissimilarity. ZD7288 may be of benefit in the treatment of ischaemic heart disease by reducing heart rate without impairing cardiac function.
Contractile effects of uridine 5′‐triphosphate in the rat duodenum
Authors: Johnson C. R. , Hourani S.M.O.
Published: July 2012

Previous studies have shown that the rat duodenum relaxes to adenosine and adenosine 5′‐triphosphate (ATP) via Pi and P2Y purinoceptors respectively, but in preliminary studies uridine 5′‐triphosphate (UTP) was found to contract this tissue. The non‐selective P2 antagonist suramin and a number of nucleotides were therefore used to investigate this response further.

ATP, UTP, adenosine 5′‐diphosphate (ADP), adenosine 5′‐0‐(3‐thiotriphosphate) (ATP‐γ‐S), guanosine 5′‐triphosphate (GTP) and uridine 5′‐diphosphate (UDP) each relaxed the duodenum, with an agonist potency order of ATP = ADP > ATP‐γ‐S > > GTP > > UTP = UDP, consistent with the presence of a P2Y purinoceptor mediating relaxation.

ATP‐γ‐S, UTP and UDP each contracted the duodenum with an agonist potency order of ATP‐γ‐S > UTP > UDP, although maximal responses to these agonists were not obtained at a concentration of 267 μm (ATP‐γ‐S) and 300 μm (UTP and UDP). No contractions were observed with any of the other agonists at concentrations up to 300 μm.

Indomethacin (25 μm) did not inhibit the contractions induced by UTP, indicating that they were not mediated via production of prostaglandins.

Suramin (100 μm and 1 μm) inhibited relaxations induced by ATP, shifting the concentration‐response curve to the right, with the maximal response to ATP being decreased by the higher concentration of suramin (1 μm). Suramin (1 μm) inhibited relaxations induced by ATP‐γ‐S, shifting the concentration‐response curve to the right, and completely abolished contractions induced by ATP‐γ‐S. In contrast, suramin (100μm and 1mM) had no effect on contractions induced by UTP. Contractions induced by UTP were, however, less sustained in the presence of suramin, which also affected the basal tone of some tissues when precontracted with carbachol (0.1 μm). In the presence of suramin (1 μm), no contractions to ATP were observed.

These results confirm that in the rat duodenum there is a P2Y purinoceptor that mediates relaxation in response to a number of purine nucleotides, and at which the pyrimidine nucleotides UTP and UDP are almost inactive. There are also receptors at which UTP and ATP‐γ‐S act to cause contraction. Suramin discriminates between the contractile effects of these two agonists, which may indicate the presence of a suramin‐insensitive pyrimidinoceptor as well as a suramin‐sensitive receptor for ATP‐γ‐S. An alternative explanation is that the differential effects of suramin are via its actions as an antagonist in addition to its action as an ectonucleotidase inhibitor.
In vitro studies of release of adenine nucleotides and adenosine from rat vascular endothelium in response to α1‐adrenoceptor stimulation
Authors: Shinozuka K. , Hashimoto M. , Masumura S. , Bjur R.A. , Westfall D.P. , Hattori K.
Published: July 2012

Noradrenaline‐induced release of endogenous adenine nucleotides (ATP, ADP, AMP) and adenosine from both rat caudal artery and thoracic aorta was characterized, using high‐performance liquid chromatography with fluorescence detection.

Noradrenaline, in a concentration‐dependent manner, increased the overflow of ATP and its metabolites from the caudal artery. The noradrenaline‐induced release of adenine nucleotides and adenosine from the caudal artery was abolished by bunazosin, an α‐adrenoceptor antagonist, but not by idazoxan, an α2‐adrenoceptor antagonist. Clonidine, α 2‐adrenoceptor agonist, contracted caudal artery smooth muscle but did not induce release of adenine nucleotides or adenosine.

Noradrenaline also significantly increased the overflow of ATP and its metabolites from the thoracic aorta in the rat; however, the amount of adenine nucleotides and adenosine released from the aorta was considerably less than that released from the caudal artery.

Noradrenaline significantly increased the overflow of ATP and its metabolites from cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the aorta was also much less than that from cultured endothelial cells from the caudal artery. In cultured smooth muscle cells from the caudal artery, a significant release of ATP or its metabolites was not observed.

These results suggest that there are vascular endothelial cells that are able to release ATP by an α1‐adrenoceptor‐mediated mechanism, but that these cells are not homogeneously distributed in the vasculature.
Effect of activity at metabotropic, as well as ionotropic (NMDA), glutamate receptors on morphine dependence
Authors: Fundytus Marian E. , Coderre Terence J.
Published: July 2012

The contribution of various excitatory amino acid (EAA) receptors (NMDA, AMPA/kainate and metabotropic) in the brain to the development of morphine dependence was examined. This was performed by measuring the severity of the precipitated withdrawal syndrome following chronic subcutaneous (s.c.) morphine and intracerebroventricular (i.c.v.) EAA antagonist treatment.

Continuous subcutaneous (s.c.) treatment with morphine sulphate (36.65 μmol day−1) produced an intense and reliable naloxone‐precipitated withdrawal syndrome.

Chronic i.c.v. treatment with antagonists selective for metabotropic and NMDA receptors, but not AMPA/kainate receptors, significantly attenuated abstinence symptoms. Conversely, EAA antagonists had very little effect on non‐withdrawal behaviours.

These results suggest that, as well as changes elicited by activation of NMDA receptors, metabotropic receptors and intracellular changes in the phosphatidylinositol (PI) second‐messenger system or the cyclic adenosine 3′,5′‐monophosphate (cAMP) second messenger system, to which EAA metabotropic receptors are linked, may be involved in the development of opioid dependence with chronic morphine treatment.
Differences in sensitivity to the specific protein kinase C inhibitor Ro31‐8220 between small and large bronchioles of the rat
Authors: Chopra L.C. , Twort C.H.C. , Ward J.P.T.
Published: July 2012

The involvement of protein kinase C (PKC) in constriction of small bronchioles has never been investigated. In this study we have examined the effects of the specific PKC inhibitors Ro31‐8220 and Ro31‐7549 and the non‐specific inhibitor H7 on carbachol‐, 5‐hydroxytryptamine (5‐HT)‐ and 4β‐phorbol dibutyrate (4β‐PDBu)‐induced contractions in large and small bronchioles.

The study was performed on isolated bronchioles of the rat with internal diameters of 574 μm ± 11 (small, n= 128), and 1475 μm ± 32 (large, n = 93), using a Mulvaney‐Halpen small vessel myograph.

In these preparations 4β‐PDBu had no effect if added on its own. However, after precontracting with 30 mm K+, 0.5 μm 4β‐PDBu caused a contractile response of 110.4 ± 7.0% TK (TK = maximum response to 75 mm K+ in small and 69.3 ± 6.5% TK in large bronchioles. Ro31‐8220, Ro31‐7549 and H7 all showed concentration‐dependent inhibition of this response.

In small bronchioles 10 μm Ro31‐8220 shifted both the carbachol and 5‐HT concentration‐ response curves to the right, and reduced the maximum response. In contrast, 10 μm Ro31‐8220 had no significant effect on the EC50 to carbachol of larger bronchioles, although the maximum response was reduced, and had no significant effect on the 5‐HT concentration‐ response curve. 200 μm H7 shifted the carbachol concentration‐ response curve to the right as well as reducing the maximal response in both small and large bronchioles.

Large bronchioles exhibited a greater rate of decay of carbachol‐induced contraction than did small bronchioles. Pretreatment with Ro31‐8220 accelerated the rate of decay.

Pretreatment with IOIIM Ro31‐8220 caused a small reduction in the response to 75 mm K+ in both small and large bronchioles (small: to 87.8 ± 3.0% TK; large: to 94.1 ± 0.8% TK). H7 at 200 μm caused a much larger reduction in both preparations (small: to 75.1 ± 3.0% TK); large: to 82.7 ± 0.6% TK).

Small bronchioles were more sensitive than larger bronchioles to agonists and phorbol ester. The protein kinase inhibitor Ro31‐8220 could reduce agonist‐induced constriction in small and large bronchioles, as well as reducing or abolishing phorbol ester‐induced contractions. Small bronchioles were more sensitive than large bronchioles to Ro31–8220. These results suggest that there is a significant PKC involvement in constriction of bronchioles to carbachol and 5‐HT, and that the proportion of the contractile response that can be attributed to PKC is greater in smaller than larger bronchioles.
Calcitonin gene‐related peptide receptors in human gastrointestinal epithelia
Authors: Cox H.M. , Tough I.R.
Published: July 2012

The secretory responses to calcitonin gene‐related peptide (CGRP) receptor agonists have been characterized in two human adenocarcinoma cell lines, namely HCA‐7 and Colony‐29 (Col‐29) epithelia. These cells form polarized epithelial layers when grown on permeable supports and allow changes in electrogenic ion transport in response to agonists to be monitored continuously.

α‐CGRP (rat and human sequences), rat β‐CGRP and human [Tyr0]CGRP applied to the basolateral surface were found to be full agonists, causing prolonged increases in short‐circuit current. Concentration‐response curves exhibited EC50 values of 0.6‐1.5 nm in HCA‐7 cells. The same agonists were less effective in Col‐29 epithelia, the EC50 values ranging from 1 to 10 nm in these cells. [Cys(ACM)2,7]CGRP was effective in both cell lines and was more potent in HCA‐7 cells.

CGRP receptors were preferentially located on the basolateral surface in both cell types. Addition of rα‐CGRP to the apical domain produced significantly smaller secretory responses (8.1% in HCA‐7 and 29.2% in Col‐29) compared with those produced following basolateral application (100%).

In both cell lines rα‐CGRP‐elevated short‐circuit current was inhibited by the loop diuretic piretanide (200μm) and by somatostatin (100 nm). Pretreating epithelia with the cyclo‐oxygenase inhibitor, piroxicam (5μm) had no significant effect upon CGRP responses in either cell line.

Rat α‐CGRP (0.2 nm) responses in HCA‐7 epithelia were inhibited by the C‐terminal fragment CGRP(8–37) (1 μm). Pretreatment of Col‐29 cells with CGRP(8–37) did not, however, alter the size or profile of responses to rα‐CGRP (1 nm).

We conclude that high‐affinity CGRP receptors exist on the basolateral surface of both cell lines, however they differ in their sensitivity to CGRP(8–37) and agonist orders of potency. Thus different CGRP receptor subtypes appear to predominate in these two epithelial cell types.
Block of Endothelin‐1‐induced release of thromboxane A2 from the guinea pig lung and nitric oxide from the rabbit kidney by a selective ETB receptor antagonist, BQ‐788
Authors: D'Orléans‐Juste Pedro , Claing Audrey , Télémaque Sabine , Maurice Marie‐Claude , Yano Mitsuo , Gratton Jean‐Philippe
Published: July 2012

The present study characterizes the receptors responsible for endothelin‐1‐induced release of thromboxane A2 from the guinea pig lung and of endothelium‐derived nitric oxide from the rabbit perfused kidney, by the use of the selective ETA receptor antagonist, BQ‐123, and a novel selective ETB receptor antagonist, BQ‐788.

In the guinea pig perfused lung, endothelin‐1 (ET‐1) (5 nm) induced a marked increase of thromboxane A2 which was reduced by 17 ± 5.0, 70 ± 1.0 and 93 ± 1.2% by BQ‐788 infused at concentrations of 1, 5 and 10 nm respectively. In contrast, BQ‐123 (0.1 and 1.0 μm) had little or no effect on the ET‐1‐induced release of thromboxane A2.

In the same perfused model, the selective ETB agonist, IRL 1620 (50 nm), stimulated the release of thromboxane A2, but not prostacyclin. The eicosanoid‐releasing properties of IRL 1620 were abolished by BQ‐788 at 10 nm, yet were unaffected by BQ‐123 (1 μm).

In the rabbit perfused kidney, BQ‐788 (10 nm) potentiated the increase of perfusion pressure induced by endothelin‐1 (1,5 and 10 nm) by approximately 90%, but not that induced by angiotensin II (1 μm). Furthermore, the selective ETB receptor antagonist did not reduce the release of prostacyclin triggered by either peptide.

In another series of experiments, pretreatment of the perfused kidney with a nitric oxide synthase inhibitor, L‐NAME (100 μm), potentiated the pressor responses to both endothelin‐1 and angiotensin II. Under L‐NAME treatment, BQ‐788 did not further potentiate the pressor response to endothelin‐1.

Our results illustrate the predominant role of ETB receptor activation in the release of thromboxane A2 and nitric oxide triggered by endothelin‐1 in the guinea pig perfused lung and rabbit kidney respectively.
Voltage‐ and time‐dependent inhibitory effects on rat aortic and porcine coronary artery contraction induced by propafenone and quinidine
Authors: Pérez‐Vizcaíno Trancisco , del Pozo Buensuceso Fernández , Zaragozá Francisco , Tamargo Juan
Published: July 2012

Class I antiarrhythmic drugs (e.g. Na+ channel blockers) such as propafenone and quinidine also inhibit voltage‐gated Ca2+ and K+ channels. In the present paper the voltage‐ and time‐dependent inhibitory effects of propafenone and quinidine were studied on depolarization‐induced vascular contractions and 45Ca2+ uptake in isolated endothelium denuded rat aorta and pig left descending coronary artery.

Quinidine and propafenone (10−7m‐5 × 10−5m) produced a concentration‐dependent relaxation of the contractions induced by 80 mm KCl. Propafenone was significantly more potent (P < 0.05) than quinidine in both rat aorta and pig coronary arteries but both drugs were more potent (P < 0.05) in relaxing rat aorta than pig coronary arteries. In rat aortic rings, the relaxant effects of propafenone were unaffected by pretreatment with the Na+ channel blocker, tetrodotoxin.

The degree of inhibition produced after prolonged exposure (40 min) to propafenone and quinidine differed as the time of depolarization with 80 mm KCl was increased. Quinidine (3 × 10−6 m, 10−5 m and 3 × 10−5 m) not only produced an inhibition at the very early stage of contraction, but also a time‐dependent inhibition was observed. In contrast, propafenone (10−6 m, 3 × 10−6m and 10−5 m) produced a more marked concentration‐dependent early block but only a mild time‐dependent inhibittion.

The voltage‐dependence of propafenone‐ and quinidine‐induced inhibition, was studied in rat aorta and coronary arteries which had been incubated in 5 or 40 mm KCl Ca2+‐free solution and then contracted by changing the bath solution to 100 mm KCl and 2 mm CaCl2 solution. The inhibitor effects of quinidine were significantly enhanced (P < 0.05) when the preparations were preincubated in 40 mm KCl (depolarizing) solution. In contrast, the effects of propafenone were quite similar in 5 or in 40 mm KCl solution.

Quinidine, 10−5 m, produced a greater inhibition (P < 0.05) of 100 mm KCl‐stimulated 45Ca2+ uptake in aortic rings preincubated in depolarizing as compared to normal solution. In contrast, the inhibition produced by 3 × 10−6 m propafenone was similar in aortic rings incubated in 5 or 40 mm KCl solution.

It is concluded that both quinidine and propafenone inhibited vascular smooth muscle contraction which could be attributed to reduced Ca2+ entry. The voltage‐ and time‐dependent inhibitory effects of quinidine may reflect an increased binding of the drug to Ca2+ channels at depolarized potentials.
The effects of β‐adrenoceptor stimulation on adhesion of human natural killer cells to cultured endothelium
Authors: Benschop Robert J. , Nijkamp Frans P. , Ballieux Rudy E. , Heijnen Cobi J.
Published: July 2012

The circulation of natural killer (NK) cells in vivo is influenced by physical exercise, mental stress, and infusion of β‐adrenoceptor agonists. We have previously presented in vitro data, showing that β2‐adrenoceptor agonists induce detachment of NK cells from endothelial cells (EC), supporting the hypothesis that NK cells can be recruited from the marginating pool in blood vessels.

Because NK cells as well as EC express β2‐adrenoceptors, the present study was conducted to investigate whether stimulation of the β‐adrenoceptors on NK cells, EC or both cell types is required to induce detachment from EC.

Cells were pretreated (15min) with a selective β2‐adrenoceptor antagonist, GR81706, at various concentrations. The duration of β‐adrenoceptor blockade was tested by determining the adenosine 3′,5′‐cyclic monophosphate (cyclic AMP) production induced by terbutaline (a β2‐adrenoceptor specific agonist). This receptor‐mediated response was effectively inhibited for at least 4 h, whereas the cyclic AMP production in response to forskolin (a direct activator of adenylate‐cyclase) was not affected.

Functional adhesion assays were then performed to determine the role of β‐adrenoceptors on the different cell types involved (NK and EC) in catecholamine‐induced detachment. Peripheral blood mononuclear cells were allowed to adhere for 1 h to monolayers of unstimulated EC in the presence or absence of cyclic AMP inducing agents, and the percentage of NK cells in the adhering lymphocyte fraction was determined by flow cytometry.

Both adrenaline (10−5 m) and forskolin (10−5 m) caused detachment of NK cells from EC. After blockade of the β2‐adrenoceptors on NK cells by pretreatment with GR81706 (10−6 m), the effect of adrenaline on NK cells adhesion was pretented; after blockade of the β2‐adrenoceptors on EC, NK cell adhesion was still significantly reduced by adrenaline. In all cases, forskolin caused detachment of NK cells.

To establish further that stimulation of β‐adrenoceptors on NK cells is sufficient to cause detachment, we showed that adrenaline also reduced adhesion of NK cells to monolayers of Chinese hamster ovary cells, which do not express β‐adrenoceptors.

Together, these results show that stimulation of β2‐adrenoceptors on NK cells negatively influences their capacity to adhere to EC, and that β2‐adrenoceptors on EC play a negligible role in this phenomenon.
The interaction of α‐human atrial natriuretic peptide (ANP) with salbutamol, sodium nitroprusside and isosorbide dinitrate in human bronchial smooth muscle
Authors: Nally Jane E. , Clayton Robert A. , Thomson Neil C. , McGrath John C.
Published: July 2012

Contractions in human bronchial rings evoked by methacholine (10−6 m) were reversed by single contractions of α‐human atrial natriuretic peptide (10−6 m), salbutamol (10−6 m), sodium nitroprusside (10−6 m) or isosorbide dinitrate (4.2 × 10−5 m) and the extent of the relaxations compared. The activity of combinations of ANP with salbutamol, sodium nitroprusside and isosorbide dinitrate were compared with those for each agonist alone.

ANP and salbutamol were equipotent in reversing methacholine‐evoked contraction and, in combination these agonists evoked an additive response. ANP and sodium nitroprusside also evoked similar degrees of relaxation and were additive, as were ANP and isosorbide dinitrate; however, with isosorbide dinitrate a higher concentration was required to evoke the same degree of relaxation as ANP, sodium nitroprusside or salbutamol.

Cumulative concentration‐response curves to methacholine (10−9‐3 × 10−4 m) were examined in the presence and absence of the above bronchodilator substances, alone and in combination allowing their abilities to protect against contraction to be compared. ANP (10−6 m) and salbutamol (10−6 m) each attenuated subsequent contractions evoked by methacholine, an ability not shared with sodium nitroprusside (10−6 m) or isosorbide dinitrate (4.2 × 10−5 m). Indeed at lower concentrations of methacholine (< 3 × 10−7 m), sodium nitroprusside evoked a paradoxical enhancement of methacholine‐evoked contractions.

In combination, ANP and salbutamol attenuated contractions evoked by methacholine to a significantly greater degree than that seen with either agonist alone, whilst a combination of ANP and sodium nitroprusside evoked no greater effect than that seen with ANP alone. By contrast, isosorbide dinitrate and ANP together evoked a greater inhibition than ANP alone.

These results suggest that a combination of agents such as ANP and salbutamol evokes a greater effect than either alone, both in reversing and protecting against methacholine‐evoked contractions. Such combinations may be of benefit in the treatment of patients, allowing lower doses of drug to be used. Combinations of ANP and isosorbide dinitrate may likewise be of interest; however, the mechanism underlying the enhancement of ANP responses by isosorbide dinitrate requires further study.
Modulation of dopamine and noradrenaline release and of intracellular Ca2+ concentration by presynaptic glutamate receptors in hippocampus
Authors: Malva João O. , Carvalho Arsélio P. , Carvalho Caetana M.
Published: July 2012

We studied the release of [3H]‐dopamine and [3H]‐noradrenaline (NA) from hippocampal synap‐tosomes induced by glutamate receptors and the associated Ca2+ influx through Ca2+ channels. The release of tritiated neurotransmitters was studied by use of superfusion system and the intracellular free Ca2+ concentration ([Ca2+]i) was determined by a fluorimetric assay with Indo‐1 as a probe for Ca2+.

Presynaptic glutamate receptor activation induced Ca2+‐dependent release of [3H]‐dopamine and [3H]‐NA from rat hippocampal synaptosomes. Thus, L‐glutamate induced the release of both neurotransmitters in a dose‐dependent manner (EC50 = 5.62 μm), and the effect of 100 μm L‐glutamate was inhibited by 83.8% in the presence of 10 μm 6‐cyano‐7‐nitroquinoxaline‐2,3‐dioxine (CNQX), but was not affected by 1 μm (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]‐cyclohepten‐5,10‐imine (MK‐801).

Other glutamate receptor agonists also stimulated the Ca2+‐dependent release of [3H]‐dopamine and [3H]‐NA as follows: N‐methyl‐D‐aspartate (NMDA), at 200 μm, released 3.65 ± 0.23% of the total 3H catecholamines, and this effect was inhibited by 81.2% in the presence of 1 μm MK‐801; quisqualate (50 μm), S‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolopropionic acid (AMPA) (100 μm) or kainate (100 μm) released 1.57 ± 0.26%, 1.93 ± 0.17% and 2.09 ± 0.22%, of the total 3H catecholamines, respectively.

The ionotropic glutamate receptor agonist, AMPA, induced an increase in the [Ca2+]i which was inhibited by 58.6% in the presence of 10 μm CNQX. In contrast, the increase in [Ca2+]i due to stimulation by glutamate was not sensitive to CNQX or MK–801.

Nitrendipine, at 1 μm, did not inhibit the neurotransmitter release induced by AMPA, but, both 0.5 μm ω‐conotoxin GVIA (μ‐CgTx) and 100 nm μ‐Aga IVA reduced catecholamine release to 49.03 ± 3.79% and 46.06 ± 10.51% of the control, respectively. In the presence of both toxins the release was reduced to 12.58 ± 4.64% of the control.

The results indicate that activation of presynaptic glutamate receptors of the NMDA and non‐NMDA type induces the release of [3H]‐dopamine and [3H]‐NA from rat hippocampal synaptosomes and that the release induced by AMPA involves the activation of N‐ and P‐type Ca2+ channels which allow the influx of Ca2+ that triggers the 3H catecholamines release.
Stimulation by extracellular ATP and UTP of the mitogen‐activated protein kinase cascade and proliferation of rat renal mesangial cells
Authors: Huwiler Andrea , Pfeilschifter Josef
Published: July 2012

Extracellular ATP and UTP have been reported to activate a nucleotide receptor that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases C and D, respectively. Here we report that ATP and UTP potently stimulate mesangial cell proliferation.

Both nucleotides stimulate phosphorylation and activation of mitogen‐activated protein kinase and a biphasic phosphorylation of the up‐stream mitogen‐activated protein kinase kinase.

When added at 100 μm, ATPγS, UTP and ATP were the most potent activators of mitogen‐activated protein kinase. βγ‐imido‐ATP was somewhat less active and ADP and 2‐methylthio‐ATP caused a weak induction of enzyme activity. Activation of mitogen‐activated protein kinase by both ATP and UTP is dose‐dependently attenuated by the P2‐receptor antagonist, suramin.

The protein kinase C activator 12‐0‐tetradecanoylphorbol 13‐acetate, but not the biologically inactive 4α‐phorbol 12,13‐didecanoate, increased mitogen‐activated protein kinase activity in mesangial cells, suggesting that protein kinase C may mediate nucleotide‐induced stimulation of mitogen‐activated protein kinase.

Down‐regulation of protein kinase C ‐α and‐θ isoenzymes by 4 h or 8 h treatment with phorbol ester partially inhibited ATP‐ and UTP‐triggered mitogen‐activated protein kinase activation. Moreover, a 24 h treatment of mesangial cells with phorbol ester, a regimen that also causes depletion of protein kinase C‐∍ did not further reduce the level of mitogen‐activated protein kinase stimulation.

The specific protein kinase C inhibitor, CGP 41251, which displayed a selectivity for the Ca2+‐dependent isoenzymes, as compared to the Ca2+‐independent isoenzymes did not inhibit nucleotide‐stimulated mitogen‐activated protein kinase phosphorylation, thus implicating the involvement of a Ca2+‐independent protein kinase C isoform.

In summary, these results suggest that ATP and UTP trigger the activation of the mitogen‐activated protein kinase signalling cascade in mesangial cells and this may be responsible for the potent mitogenic activity of both nucleotides.
GABA and glutamate release affected by GABAB receptor antagonists with similar potency: no evidence for pharmacologically different presynaptic receptors
Authors: Waldmeier Peter C. , Wicki Peter , Feldtrauer Jean‐Jacques , Mickel Stuart J. , Bittiger Helmut , Baumann Peter A.
Published: July 2012

The effects of a series of nine GABAB receptor antagonists of widely varying potencies on electrically stimulated release from cortical slices of [3H]‐GABA in the absence or presence of 10 μm of the GABABagonist, (−)−baclofen and of endogenous glutamate in the presence of (−)−baclofen were compared.

The concentrations of the compounds half maximally increasing [3H]‐GABA release (EC50's) at a stimulation frequency of 2 Hz correlated well with the IC50 values obtained from the inhibition of the binding of the agonist, [3H]‐CGP 27492, to GABAB receptors in rat brain membranes (rank order of potency: CGP 56999 A ≥ CGP 55845 A > CGP 52432 ≥ CGP 56433 A > CGP 57034 A > CGP 57070 A ≥ CGP 57976 > CGP 51176 > CGP 35348).

Likewise, the concentrations causing half‐maximal increases of [3H]‐GABA in the absence or presence of (−)−baclofen, and of endogenous glutamate in the presence of (−)−baclofen, correlated well with each other. Reports in the literature suggesting the CGP 35348 exhibits a 70 fold preference for inhibition of (−)−baclofen's effects on glutamate over [3H]‐GABA release, and that CGP 52432 shows a 100 fold preference in the opposite sense, could not be confirmed in our model.

Therefore, our results suggest that, if there are pharmacological differences between GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings in the rat cortex, they are not revealed by this series of compounds of widely different potencies.

In particular, our results with CGP 35348 and CGP 52432 do not support the hypothesis that GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings represent subtypes with different pharmacology.
Display of the characteristics of endothelium‐derived hyperpolarizing factor by a cytochrome P450‐derived arachidonic acid metabolite in the coronary microcirculation
Authors: Bauersachs Johann , Hecker Markus , Busse Rudi
Published: July 2012

In addition to nitric oxide (NO) and prostacyclin (PGI2) an endothelium‐derived factor, which hyperpolarizes vascular smooth muscle cells via activation of K+ channels, contributes to the dilator effect of bradykinin in different vascular beds. Since this so‐called endothelium‐derived hyperpolarizing factor (EDHF) also seems to play an important role in the coronary circulation, we investigated its nature and mechanism of action in the rat isolated perfused heart (Langendorff preparation).

Bolus injections of bradykinin (1, 10, and 100 pmol) elicited a transient dose‐dependent dilator response (e.g., 12 ± 2% decrease in coronary perfusion pressure (CPP) at 10 pmol bradykinin, n = 41). Administration of the cyclo‐oxygenase inhibitor, diclofenac (1 μm), augmented the bradykinin‐induced dilation approximately twofold (n = 9 P < 0.01). Combined treatment with the NO synthase inhibitor, NG‐nitro‐L‐arginine (30 μm) and diclofenac (1 μm) significantly reduced the duration, but increased the amplitude of the dilator response to bradykinin (27 ± 2% decrease in CPP, n = 24, P < 0.01).

The abolition of this NG‐nitro‐L‐arginine/diclofenac‐insensitive dilator response to bradykinin by tetrabutylammonium (0.3 μm), an inhibitor of Ca2+‐dependent K+ channels (4 ± 1% decrease in CPP, n = 6, P < 0.01), supports the view that the dilator compound released in the coronary microcirculation is EDHF.

This EDHF‐type dilation was reversibly inhibited by the phospholipase A2 inhibitor, quinacrine (3 μm, 9 ± 3% decrease in CPP, n = 6, P < 0.01) and by the cytochrome P450 inhibitor SKF525a (3 μm, 6 ± 1% decrease in CPP, n = 6, P < 0.01).

Tetrabutylammonium, quinacrine or SKF 525a did not affect the endothelium‐independent dilator response to sodium nitroprusside (1 nmol), indicating that these compounds did not affect smooth muscle relaxation in a non‐specific manner.

These findings suggest that in the coronary microcirculation bradykinin stimulates the release of a cytochrome P450‐derived arachidonic acid metabolite, which exhibits the characteristic features of EDHF.
Cyclic GMP‐mediated inhibition of L‐type Ca2+ channel activity by human natriuretic peptide in rabbit heart cells
Authors: Tohse Noritsugu , Nakaya Haruaki , Kanno Youji Takeda & Morio
Published: July 2012

Effects of atrial natriuretic peptide (ANP) on the L‐type Ca2+ channels were examined in rabbit isolated ventricular cells by use of whole‐cell and cell‐attached configurations of the patch clamp methods. ANP produced a concentration‐dependent decrease (10–100 nm) in amplitude of a basal Ca2+ channel current.

The inactive ANP (methionine‐oxidized ANP, 30 nm) failed to decrease the current.

8‐Bromo‐cyclic GMP (300 μm), a potent activator of cyclic GMP‐dependent protein kinase (PKG), produced the same effects on the basal Ca2+ channel current as those produced by ANP. The cyclic GMP‐induced inhibition of the Ca2+ channel current was still evoked in the presence of 1‐isobutyl‐3‐methyl‐xanthine, an inhibitor of phosphodiesterase. ANP failed to produce inhibition of the Ca2+ channel current in the presence of 8‐bromo‐cyclic GMP.

In the single channel recording, ANP and 8‐bromo‐cyclic GMP also inhibited the activities of the L‐type Ca2+ channels. Both agents decreased the open probability (NP0) without affecting the unit amplitude.

The present results suggest that ANP inhibits the cardiac L‐type Ca2+ channel activity through the intracellular production of cyclic GMP and then activation of PKG.
Endothelin ETA and ETB mRNA and receptors expressed by smooth muscle in the human vasculature: majority of the ETA sub‐type
Authors: Davenport Anthony P. , O'Reilly Gillian , Kuc Rhoda E.
Published: July 2012

1 We measured the ratio of ETA and ETB sub‐types in the media (containing mainly smooth muscle) of human cardiac arteries (aorta, pulmonary and coronary), internal mammary arteries and saphenous veins.

2 In saturation experiments, [125I]‐endothelin‐1 ([125I]‐ET‐1) bound with high affinity to the media of each vessel (n = 3 individuals or homogenate preparations ± s.e.mean): coronary artery, KD = 0.14 ± 0.02 nm, Bmax = 71.0 ± 21.0 fmol mg−1 protein; pulmonary artery, KD = 0.85 ± 0.25 nm, Bmax = 15.2 ± 10.3 fmol mg−1 protein; aorta, KD = 0.51 ± 0.02 nm, Bmax = 9.4 ± 4.4 fmol mg−1 protein; internal mammary artery, KD = 0.34 ± 0.31 nm, Bmax = 2.0 ± 0.5 fmol mg−1 protein and saphenous vein, KD = 0.28 ± 0.05 nm, Bmax = 52.8 ± 1.0 fmol mg−1 protein. In each vessel, over the concentration‐range tested, Hill slopes were close to unity and a one site fit was preferred to a two site model.

3 In competition binding assays, the ETA selective ligand, BQ123 inhibited the binding of 0.1 nm [125I]‐ET‐1 to the media in a biphasic manner. In each case, a two site fit was preferred to a one or three site model: coronary artery, KDETA = 0.85 ± 0.03 nm, KDETB = 7.58 ± 2.27 μm, ratio = 89:11%; pulmonary artery, KDETA = 0.27 ± 0.05 nm, KDETB = 24.60 ± 5.34 μm, ratio = 92:8%; aorta, KDETA = 0.80 ± 0.40 nm, KDETB = 2.67 ± 2.60 μm ratio = 89:11%; saphenous vein, KDETA = 0.55 ± 0.17 nm, KDETB = 14.4 ± 0.26 μm, 85:15% (n = 3 individuals or homogenate preparations ± s.e.mean). BQ123 showed up to 18000 fold selectivity for the ETA over the ETB sub‐type. The ETA‐selective ligand, [125I]‐PD151242 labelled 85% of the receptors detected by a fixed concentration of [125I]‐ET‐1 in media of internal mammary artery, measured by quantitative autoradiography. In contrast, the density of ETB receptors detected with [125I]‐BQ3020 was 7.0 ± 1.5 amol mm−2, representing about 8% of [125I]‐ET‐1.

4 A single band corresponding to the expected position for mRNA encoding the ETA receptor (299 base pairs) was found in the media in each of the five vessels (n = 3 individuals) using reverse‐transcriptase polymerase chain reaction assays. A single band corresponding to the ETB sub‐type (428 base pairs) was also always detected.

5 35S‐labelled antisense probes to ETA and ETB hybridised to the media of epicardial coronary arteries as well as intramyocardial vessels, confirming the presence of mRNA encoding both sub‐types in the vascular smooth muscle of the vessel wall.

6 Although mRNA for both receptors was detected, competition binding using BQ123 demonstrated that the majority (at least 85%) of ET receptors present in smooth muscle are the ETA sub‐type. These results provide further support for the hypothesis that the ETA sub‐type is the receptor that must be blocked in humans to produce a beneficial vasodilatation in pathophysiological conditions where there is an increase in peptide concentration or receptor density.
Effects of suramin on contractions of the guinea‐pig vas deferens induced by analogues of adenosine 5′‐triphosphate
Authors: Bailey S.J. , Hourani S.M.O.
Published: July 2012

1 Adenosine 5′‐triphosphate (ATP) and some of its analogues contract the guinea‐pig vas deferens, acting via receptors which have been classified as P2X‐purinoceptors. We have recently shown, however, that the effects of ATP are enhanced, rather than inhibited, by the non‐selective P2 antagonist, suramin, and that this enhancement could not easily be explained in terms of inhibition by suramin of the breakdown of ATP. We therefore investigated the effects of suramin on contractions induced by ATP analogues, to define the structure‐activity relationships of the suramin‐resistant response.

2 In the absence of suramin, the order of potency for ATP analogues was adenosine 5′‐(α,β‐methylene)triphosphonate (AMPCPP) = P1,P5‐diadenosine pentaphosphate (Ap5A) = adenosine 5′‐tetraphosphate (Ap4) > adenosine 5′‐O‐(3‐thiotriphosphate) (ATPγS) = adenylyl 5′‐(β,γ‐methylene) diphosphonate (AMPPCP)>P1,P5‐diadenosine tetraphosphate (Ap4A) > adenosine 5′‐O‐(2‐thiodiphos‐phate) (ADPβS)>2‐methylthioadenosine 5′‐triphosphate (MeSATP) ≥ ATP > adenosine 5′‐diphosphate (ADP). This is generally in agreement with previously reported structure‐activity relationships in this tissue.

3 In the presence of suramin (1 mm), responses to Ap5A, Ap4A, AMPPCP, ADPβS and ADP were abolished or greatly reduced, and contractions induced by AMPCPP, Ap4 and ATPγS were inhibited. Contractions induced by MeSATP however, like those induced by ATP itself, were not reduced, but at concentrations above 100 μm were enhanced. In the presence of suramin (1 mm) the order of potency of analogues was therefore AMPCPP = Ap4 > ATP = MeSATP > ATPγS, with all other analogues tested being essentially inactive at concentrations up to 500 μm.

4 Contractile responses of the vas deferens to transmural nerve stimulation (1–50 Hz) in the presence of the α‐adrenoceptor antagonist, phentolamine (10 μm), were abolished by suramin (1 mm). This is in agreement with previous reports that suramin inhibits the excitatory junction potential, a response thought to be mediated by P2 purinoceptors. It is however hard to reconcile the evidence implicating ATP as the non‐adrenergic transmitter responsible for this response with the failure of suramin to inhibit the contractions induced by ATP itself while abolishing nerve‐mediated contractions.

5 In conclusion, these results confirm our previous findings of a suramin‐resistant component to the ATP‐induced contraction in the guinea‐pig vas deferens, and show that the structure‐activity relationships of this response are not identical to those of any known P2‐purinoceptor subclass. Although the inhibition by suramin of the breakdown of ATP may contribute to the suramin‐resistance of some of the ATP analogues, it does not appear to provide the full explanation.
Rapid, agonist‐induced desensitization of α2‐autoreceptors modulating transmitter release
Authors: Boehm Stefan , Huck Sigismund , Schwarz Karin , Agneter Ernst , Drobny Helmut , Singer Erast A.
Published: July 2012

1 The release of previously incorporated [3H]‐noradrenaline was investigated in cultures of dissociated chick or rat sympathetic neurones and in cerebrocortical slices from neonatal or adult rats. Noradrenaline, in the presence of 10 μmol l−1 of the uptake inhibitor, cocaine, or the selective α2‐adrenoceptor agonist, 5‐bromo‐N‐(4,5‐dihydro‐1H‐imidazol‐2‐yl)‐6‐quinoxalinamine (UK 14,304), was applied for different periods of time in order to detect a possible time‐dependence of the α2‐adrenoceptor‐mediated inhibition of electrically evoked tritium outflow.

2 In chick sympathetic neurones, stimulation‐evoked overflow was reduced to 30%, 42%, or 56% of control when noradrenaline (1 μmol l−1) was present for 2, 8, or 16 min, respectively. Likewise, UK 14,304 (1 μmol l−1) present for these periods of time reduced 3H overflow to 35%, 51%, and 53% of control, respectively. Addition of 1 nmol l−1 to 10 μmol l−1 UK 14,304 for either 2 or 16 min did not produce significantly different IC50 values, but the inhibitory effects were smaller with 16 min as compared to 2 min exposure at concentrations ≥ 10 nmol l−1.

3 In rat sympathetic neurones, noradrenaline (100 nmol l−1) reduced stimulation‐evoked overflow to 33%, 56%, or 57% of control, when present for 2, 8, or 16 min, respectively. Addition of UK 14,304 (1 μmol l−1) for these periods of time caused inhibition to 11%, 41%, and 46% of control. Applying UK 14,304 for either 2 or 16 min did not result in significantly different IC50 values, but the inhibition induced by 16 min as compared to 2 min exposure was smaller at concentrations ≥ 10 nmol l−1.

4 In cerebrocortical slices from either neonatal or adult rats, exposure to 0.1 to 1.0 μmol l−1 UK 14,304 for 16 min never caused a smaller inhibition than a corresponding 3 min exposure, although various experimental conditions were investigated.

5 The results demonstrate that α2‐adrenoceptors which regulate noradrenaline release from sympathetic neurones undergo agonist‐induced desensitization within minutes. Such rapid desensitization of α2‐autoreceptors was not detected in brain slice preparations.
A novel enhancing effect of clofilium on transient outward‐type cloned cardiac K+ channel currents
Authors: Kobayashi Tsutomu , Mikala Gabor , Yatani Atsuko
Published: July 2012

1 The antiarrythmic drug, clofilium, has been shown to block several types of K+ channel currents. To investigate the effects of clofilium on the transient outward K+ current (Ito), a cloned Ito‐type cardiac K+ channel (RHK1) was expressed in Xenopus oocytes and the drug effects were examined on whole cell currents.

2 Extracellular application of clofilium slightly inhibited the current at + 60 mV from a holding potential of −80 mV. However, it unexpectedly enhanced the current from a holding potential of −60 mV in a dose‐dependent manner (219 ± 39% of control at 100 μm).

3 This enhancement is probably due to an increase in the ratio of channels in the resting state during steady depolarization, since clofilium shifted the inactivation curve in the depolarizing direction.

4 LY97119, a tertiary ammonium analogue of clofilium, did not exhibit this enhancing effect but only inhibited the current.

5 Clofilium may be useful for the study of channel inactivation because this type of phenomenon has not been reported for any other drug.
Delayed circulatory failure due to the induction of nitric oxide synthase by lipoteichoic acid from Staphylococcus aureus in anaesthetized rats
Authors: De Kimpe Sjef J. , Hunter Melanie L. , Bryant Clare E. , Thiemermann Christoph , Vane John R.
Published: July 2012

1 This study investigates the effect of lipoteichoic acid (LTA) from the cell wall of Staphylococcus aureus, a micro‐organism without endotoxin, on haemodynamics and induction of nitric oxide synthase (iNOS) in the anaesthetized rat.

2 Intravenous injection of LTA (10 mg kg−1) resulted in a decrease in blood pressure from 123 ± 1 mmHg to 83 ± 7 mmHg after 270 min (P< 0.001) and a reduction of the pressor response to noradrenaline (1 μg kg−1) from 33 ± 1 mmHg.min to 23 ± 3 mmHg.mm after 270 min (P>0.05).

3 The delayed circulatory failure (hypotension and vascular hyporeactivity) caused by LTA was prevented by pretreatment of rats with dexamethasone (10 mg kg−1, 60 min prior to LTA) or the nitric oxide synthase inhibitor NG‐monomethyl‐l‐arginine (l‐NMMA, 10 mg kg−1h−1, i.v. infusion starting 30 min prior to LTA).

4 In contrast, treatment of rats with polymyxin B (0.05 mg kg−1), an agent which binds endotoxin (lipopolysaccharides, LPS), did not affect the delayed circulatory failure caused by LTA. Polymyxin B, however, attenuated the hypotension and vascular hyporeactivity to noradrenaline afforded by endotoxaemia (2 mg kg−1 LPS, i.v.) for 270 min.

5 The delayed circulatory failure caused by LTA was associated with a time‐dependent increase in (i) the expression of iNOS protein in the lung (Western blot analysis), and (ii) iNOS activity. This increase in iNOS protein and activity was prevented by pretreatment of LTA‐rats with dexamethasone (10 mg kg−1).

6 Intravenous injection of LTA resulted in an increase in serum tumour necrosis factor (TNF)‐α (maximum at 90 min after LTA), which was attenuated by pretreatment of rats with dexamethasone (10 mg kg−1, 60 min prior to LTA). The magnitude of the rise in TNF‐α caused by LTA was similar to the one elicited by LPS (10 mg kg−1, i.v.).

7 Thus, an enhanced formation of nitric oxide following the induction of iNOS contributes importantly to the delayed vascular failure (hypotension and vascular hyporeactivity) caused by LTA in the anaesthetized rat. We suggest that the endogenous release of TNF‐α contributes to the induction of iNOS caused by LTA in vivo.