bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of cyclic AMP phosphodiesterases 3, 4 and 7 in human CD4+ and CD8+ T‐lymphocytes: role in regulating proliferation and the biosynthesis of interleukin‐2
Authors: Giembycz Mark A. , Corrigan Christopher J. , Seybold Joachim , Newton Robert , Barnes Peter J.
Published: July 2012

The cyclic AMP phosphodiesterases (PDE) expressed by CD4+ and CD8+ T‐lymphocytes purified from the peripheral blood of normal adult subjects were identified and characterized, and their role in modulating proliferation and the biosynthesis of interleukin (IL)‐2 and interferon (IFN)‐γ evaluated.

In lysates prepared from both subsets, SK&F 95654 (PDE3 inhibitor) and rolipram (PDE4 inhibitor) suppressed cyclic AMP hydrolysis indicating the presence of PDE3 and PDE4 isoenzymes in these cells. Differential centrifugation and subsequent inhibitor and kinetic studies revealed that the particulate fraction contained, predominantly, a PDE3 isoenzyme. In contrast, the soluble fraction contained a PDE4 (∼65% of total activity) and, in addition, a novel enzyme that had the kinetic characteristics of the recently identified PDE7.

Reverse transcription‐polymerase chain reaction (RT‐PCR) studies with primer pairs designed to recognise unique sequences in the human PDE4 and PDE7 genes amplified cDNA fragments that corresponded to the predicted sizes of HSPDE4A, HSPDE4B, HSPDE54D and HSPDE7. No message was detected for HSPDE4C after 35 cycles of amplification.

Functionally, rolipram inhibited phytohaemagglutinin‐ (PHA) and anti‐CD3‐induced proliferation of CD4+ and CD8+ T‐lymphocytes, and the elaboration of IL‐2, which was associated with a three to four fold increase in cyclic AMP mass. In all experiments, however, rolipram was approximately 60 fold more potent at suppressing IL‐2 synthesis than at inhibiting mitogenesis. In contrast, SK&F 95654 failed to suppress proliferation and cytokine generation, and did not elevate the cyclic AMP content in T‐cells. Although inactive alone, SK&F 95654 potentiated the ability of rolipram to suppress PHA‐ and anti‐CD3‐induced T‐cell proliferation, and PHA‐induced IL‐2 release.

When a combination of phorbol myristate acetate (PMA) and ionomycin were used as a co‐mitogen, rolipram did not affect proliferation but, paradoxically, suppressed IL‐2 release indicating that cyclic AMP can inhibit mitogenesis by acting at, or proximal to, the level of inositol phospholipid hydrolysis.

Collectively, these data suggest that PDE3 and PDE4 isoenzymes regulate the cyclic AMP content, IL‐2 biosynthesis and proliferation in human CD4+ and CD8+ T‐lymphocytes. However, the ability of rolipram to suppress markedly mitogen‐induced IL‐2 generation without affecting T‐cell proliferation suggests that growth and division of T‐lymphocytes may be governed by mediators in addition to IL‐2. Finally, T‐cells have the potential to express PDE7, although elucidating the functional role of this enzyme must await the development of selective inhibitors.
Neural 5‐HT4 receptors in the human isolated detrusor muscle: effects of indole, benzimidazolone and substituted benzamide agonists and antagonists
Authors: Candura S.M. , Messori Eliana , Franceschetti G.P. , D'Agostino TG. , Vicini D. , Tagliani Monica , Tonini M.
Published: July 2012

In strips of human isolated detrusor muscle, the 5‐hydroxytryptamine (5‐HT) receptor (5‐HT4) that mediates facilitation of neuromuscular cholinergic transmission was further characterized by using 5‐HT and a series of ligands known for their 5‐HT4 agonist (5‐methoxytryptamine: 5‐MeOT, cisapride, (R,S)‐zacopride, BIMU 8) or antagonist (RS 23597, GR 125487, DAU 6285) properties.

In the presence of methysergide (1 μm) and ondansetron (3 μm) to isolate pharmacologically the 5‐HT4 receptors, 5‐HT (0.3 nM‐1 μm), 5‐MeOT (10 nM −30 μm), BIMU 8 (10 nM −3 μm), cisapride (0.1–10 μm) and (R,S)‐zacopride (0.1–30 μm) potentiated cholinergic contractions to electrical field stimulation in a concentration‐dependent manner. RS 23597 (10 nM‐10 μm), a competitive 5‐HT4 receptor antagonist in other systems, also showed agonist properties. The following rank order of potency as an agonist was obtained: 5‐HT (pEC50 = 8.0) > RS 23597 (7.0) = BIMU 8 (6.9) ≥ cisapride (6.6) > 5‐MeOT (6.0) ≥ (R,S)‐zacopride (5.7). Relative to 5‐HT (intrinsic activity = 1), 5‐MeOT acted as a full agonist (1.03), while BIMU 8 (0.76), (R,S)‐zacopride (0.61), RS 23597 (0.60) and cisapride (0.41) behaved as partial agonists.

The potentiation by 5‐HT was competitively antagonized by the selective 5‐HT4 receptor antagonist GR 125487 (0.3‐3 nM) with a pA2 estimate of 9.75 (Schild slope of 1.09), and by DAU 6285 (1 μm; pKB = 6.45). Additionally, GR 125487 (3 nM) antagonized the responses to 5‐MeOT (pKB = 9.72) and reversed the potentiation induced by RS 23597. As an antagonist, RS 23597 (10, 30 and 100 nM) inhibited the response to 5‐HT. In addition, 30 and 100 nM RS 23597 reduced the 5‐HT response maximum by 30 and 50%, respectively. The pKB value calculated at 10 nM was 8.0.

Thus, in the human isolated detrusor muscle, the 5‐HT4 receptors mediating facilitation of cholinergic neuromuscular transmission are activated by indoleamines (5‐HT, 5‐MeOT), substituted benzamide (cisapride, (R,S)‐zacopride), benzoate (RS 23597) and benzimidazolone (BIMU 8) derivatives. The activities (in terms of both potency and efficacy) of most agonists, as well as the affinity estimates of the antagonists GR 125487 and DAU 6285, are comparable to those found in other peripheral tissues. Exceptions are RS 23597, which acted either as a partial agonist or as an antagonist of the response to 5‐HT, and 5‐MeOT that showed an unusually low potency. The latter findings may be ascribed to differences in the efficiency of receptor coupling mechanisms and/or in the molecular structure (i.e. splice variants) of the 5‐HT4 receptor.
Enhancement by calcitonin gene‐related peptide of non‐contractile Ca2+‐induced nicotinic receptor desensitization at the mouse neuromuscular junction
Authors: Dezaki Katsuya , Kimura Ikuko , Tsuneki Hiroshi , Kimura Masayasu
Published: July 2012

Nicotinic acetylcholine receptor (AChR)‐operated non‐contractile Ca2+ mobilization (unaccompanied by muscle contraction) depressed contractile Ca2+ mobilization (accompanied by muscle contraction) in mouse diaphragm muscles. In the process of nicotinic AChR desensitization, the enhancing role of calcitonin gene‐related peptide (CGRP) on the non‐contractile Ca2+‐induced depression of contractile Ca2+ mobilization was investigated by measurement of Ca2+‐aequorin luminescence in the presence of neostigmine (0.1 μm).

When the phrenic nerve was stimulated with paired pulses at intervals of 150, 300, 600, 1000 and 2000 ms, contractile Ca2+ transients were elicited during the generation of non‐contractile Ca2+ mobilization. The amplitude of the contractile Ca2+ transients elicited by the second pulse (S2) was depressed at the shorter pulse intervals, but not at the longer pulse intervals.

The extent of depression of S2 was enhanced when the duration of non‐contractile Ca2+ mobilization was prolonged by CGRP (10 nM). However, CGRP failed to enhance the depression of S2 when non‐contractile Ca2+ mobilization was not observed at the low external Ca2+ concentration (1.3 mM).

The enhancing effect by CGRP on the depression of S2 was counteracted by staurosporine (3 nM), a protein kinase‐C inhibitor, despite prolongation of the duration of non‐contractile Ca2+ mobilization.

When H‐89 (1 μm), a protein kinase‐A inhibitor, completely blocked non‐contractile Ca2+ mobilization, the depression of S2 was diminished. The prolongation of the duration of non‐contractile Ca2+ mobilization by AA373 (300 μm), a protein kinase‐A activator, enhanced the depression of S2. The enhancing effect was observed neither with CGRP nor with AA373, in the presence of H‐89 (0.1 μm).

These findings suggest that the CGRP mobilizes non‐contractile Ca2+ through activation of protein kinase‐A, which in turn may activate protein kinase‐C, then enhance the desensitization of postsynaptic nicotinic AChRs at the neuromuscular junction.
Development of cholinergic and inhibitory non‐adrenergic non‐cholinergic responses in the rat gastric fundus
Authors: Smits Geert J.M. , Lefebvre Romain A.
Published: July 2012

Cholinergic contractions and inhibitory non‐adrenergic non‐cholinergic (NANC) relaxations were studied in longitudinal muscle strips of the gastric fundus of 2, 4 and 8 week old rats.

Contractions induced by electrical stimulation of the cholinergic neurones and by administration of acetylcholine decreased during development. The potentiating effect of physostigmine was similar in the 3 age groups.

Short train stimulation in NANC conditions induced fast relaxations, which were more pronounced in 4 and 8 week than in 2 week old rats. These relaxations were almost completely inhibited by NG‐nitro‐L‐arginine methyl ester (L‐NAME) in the 3 age groups. The nitric oxide‐induced relaxations did not change during development.

Sustained electrical stimulation in NANC conditions induced an initial relaxation, which was almost totally blocked by L‐NAME, followed by an almost complete recovery of tone at lower frequencies of stimulation. At higher frequencies of stimulation, the recovery of tone was incomplete or absent. This sustained relaxation was only partially reduced by L‐NAME and almost abolished by L‐NAME plus α‐chymotrypsin. The initial relaxations increased during development, while the sustained relaxations remained similar during this period. Vasoactive intestinal polypeptide‐induced relaxations were also similar in the 3 age groups.

These results show that the sensitivity of the gastric fundus to acetylcholine decreases from 2 weeks to 8 weeks postnatally, while the importance of the nitrergic innervation increases during this period.
Inhibition of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on peripheral sympathetic nerves
Authors: Ishac Edward J.N. , Jiang Liu , Lake Kristy D. , Varga Karoly , Abood Mary E. , Kunos George
Published: July 2012

Activation of CB1 receptors by plant cannabinoids or the endogenous ligand, anandamide, causes hypotension via a sympathoinhibitory action in anaesthetized rats. In mouse isolated vas deferens, activation of CB1 receptors inhibits the electrically evoked twitch response. To determine if these effects are related to presynaptic inhibition of noradrenaline (NA) release, we examined the effects of Δ9‐tetrahydrocannabinol (‡9‐THC), anandamide and the CB1 antagonist, SR141716A, on exocytotic NA release in rat isolated atria and vasa deferentia.

In isolated atria and vasa deferentia preloaded with [3H]‐NA, electrical field stimulation caused [3H]‐NA release, which was abolished by tetrodotoxin 0.5 μm and concentration‐dependently inhibited by ‡9‐THC or anandamide, 0.3–10 μm. The inhibitory effect of ‡9‐THC and anandamide was competitively antagonized by SR141716A, 1–10 μm.

Tyramine, 1 μm, also induced [3H]‐NA release, which was unaffected by tetrodotoxin, ‡9‐THC or anandamide in either atria or vasa deferentia.

CB1 receptor mRNA is present in the superior cervical ganglion, as well as in whole brain, cerebellum, hypothalamus, spleen, and vas deferens and absent in medulla oblongata and atria, as demonstrated by reverse transcription‐polymerase chain reaction. There was no evidence of the presence of CB1A receptor mRNA in ganglia, brain, or cerebellum. These results suggest that activation of presynaptic CB1 receptors located on peripheral sympathetic nerve terminals mediate sympathoinhibitory effects in vitro and in vivo.
Pharmacological modulation of immunoreactive imidazoline receptor proteins in rat brain: relationship with non‐adrenoceptor [3H]‐idazoxan binding sites
Authors: Escribá Pablo V. , Alemany Regina , Sastre Magdalena , Olmos Gabriel , Ozaita Andrés , García‐Sevilla Jesús A.
Published: July 2012

The densities of various imidazoline receptor proteins (with apparent molecular masses of ≅29/30 −45‐ and 66‐kDa) were quantitated by immunoblotting in the rat cerebral cortex after various drug treatments. The modulation of these imidazoline receptor proteins was then compared with the changes in the density of non‐adrenoceptor [3H]‐idazoxan binding sites (I2‐sites) induced by the same drug treatments.

Chronic treatment (7 days) with the I2‐selective imidazol(in)e drugs idazoxan (10 mg kg−1), cirazoline (1 mg kg−1) and LSL 60101 (10 mg kg−1) differentially increased the immunoreactivity of imidazoline receptor proteins. The levels of the 29/30‐kDa protein were increased by idazoxan and LSL 60101 (23%), the levels of the 45‐kDa protein only by cirazoline (44%) and those of the 66‐kDa protein only by idazoxan (50%). These drug treatments also increased the density of I2‐sites (32–42%).

Chronic treatment (7 days) with efaroxan (10 mg kg−1), RX821002 (10 mg kg−1) and yohimbine (10 mg kg−1), which possess very low affinity for I2‐imidazoline receptors, did not alter either the immunoreactivity of imidazoline receptor proteins or the density of I2‐sites.

Chronic treatment (7 days) with the monoamine oxidase (MAO) inhibitors clorgyline (10 mg kg−1) and phenelzine (10 mg kg−1) decreased the immunoreactivity of the 29/30‐kDa (17–24%), 45‐kDa (19%) and 66‐kDa (23–31%) imidazoline receptor proteins. The alkylating agent N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (1.6 mg kg−1, 6 h) also decreased the levels of the three imidazoline receptor proteins (20–47%). These drug treatments consistently decreased the density of I2‐sites (31–57%).

Significant correlations were found when the mean percentage changes in immunoreactivity of imidazoline receptor proteins were related to the mean percentage changes in the density of I2‐sites after the various drug treatments (r = 0.92 for the 29/30‐kDa protein, r = 0.69 for the 45‐kDa protein and r = 0.75 for the 66‐kDa protein).

In the rat cerebral cortex the I2‐imidazoline receptor labelled by [3H]‐idazoxan is heterogeneous in nature and the related imidazoline receptor proteins (29/30‐, 45‐ and 66‐kDa) detected by immunoblotting contribute differentially to the modulation of I2‐sites after drug treatment.
Evidence for the presence of cannabinoid CB1 receptors in mouse urinary bladder
Authors: Pertwee Roger G. , Fernando Susanthi R.
Published: July 2012

CP 55,244, (−)−11‐hydroxy‐dimethylheptyl‐Δ8‐tetrahydrocannabinol, WIN 55,212‐2, Δ9‐tetrahydro‐cannabinol, nabilone and anandamide each inhibited electrically‐evoked contractions of the mouse isolated urinary bladder in a concentration‐related manner, their EC50 values being respectively 15.9, 18.27, 27.23, 1327.6, 1341.5 and 4950.3 nM. (+)‐11‐hydroxy‐dimethylheptyl‐Δ8‐tetrahydrocannabinol was inactive at the highest concentration used (10 μm).

SR141716A (31.62 or 100 nM) produced parallel rightward shifts in the log concentration‐response curves of CP 55,244, (−)−11‐hydroxy‐dimethylheptyl‐Δ8‐tetrahydrocannabinol, WIN 55,212‐2, Δ9‐tetrahydrocannabinol and anandamide for inhibition of electrically‐evoked bladder contractions. The effect of the antagonist on the log concentration‐response curve of CP 55,244 was shown to depend on the concentration of SR141716A used (31.62 to 1000 nM).

The amplitudes of contractions evoked by acetylcholine or β, γ‐methylene‐L‐ATP were not decreased by 316.2 nM CP 55,244 or 3162 nM Δ9‐tetrahydrocannabinol. Electrically‐evoked contractions were almost completely abolished by 200 nM tetrodotoxin.

The above results support the hypothesis that mouse urinary bladder contains prejunctional CB1 cannabinoid receptors which can mediate inhibition of electrically‐evoked contractions, probably by reducing contractile transmitter release.

AM 630 which behaves as a cannabinoid receptor antagonist in the mouse isolated vas deferens did not antagonize the ability of CP 55,244 or Δ9‐tetrahydrocannabinol to inhibit electrically‐evoked contractions of the mouse bladder.

SR141716A produced small but significant increases in the amplitude of electrically‐evoked contractions of the bladder suggesting that this tissue may release an endogenous cannabinoid receptor agonist or that some cannabinoid receptors in this tissue are precoupled to the effector system.
Synergistic inhibition of thrombin‐induced platelet aggregation by the novel nitric oxide‐donor GEA 3175 and adenosine
Authors: Grenegård M. , Gustafsson M.C. , Andersson R.G.G. , Bengtsson T.
Published: July 2012

The influence of the novel nitric oxide‐donor GEA 3175 on thrombin‐ and ionomycin‐stimulated human platelets was investigated. The effect of GEA 3175 was compared with that of adenosine, an activator of platelet adenylyl cyclase.

GEA 3175 inhibited thrombin‐induced secretion of ATP but did not affect aggregation; similar results were obtained with adenosine.

Thrombin‐stimulated rises in the cytosolic free Ca2+ concentration, [Ca2+]i, were dose‐dependently inhibited by GEA 3175 and adenosine. GEA 3175 and adenosine maximally reduced the initial rise in [Ca2+]i by 41% and 35%, respectively.

Simultaneous exposure to GEA 3175 and adenosine nearly abolished both the functional responses (i.e. aggregation and degranulation) and the rises in [Ca2+]i in thrombin‐stimulated platelets.

Aggregation and increases in [Ca2+]i triggered in platelets by the Ca2+‐ionophore ionomycin were only marginally affected by a combination of GEA 3175 and adenosine.

GEA 3175 potently increased the guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) content in platelets but did not affect adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) levels. Adenosine did not increase either the cyclic AMP or the cyclic GMP levels in platelets. However, adenosine and GEA 3175 combined significantly elevated the platelet cyclic AMP content.

The results show that simultaneous exposure to GEA 3175 and adenosine promotes potent anti‐aggregatory properties in platelets in vitro. The findings suggest that blockage of the cytosolic Ca2+‐signal, which is probably mediated by an amplified cyclic nucleotide response, is an important event during the synergistic inhibition of thrombin‐induced aggregation.
Role of sarcoplasmic reticulum in inhibitory junction potentials and hyperpolarizations by nitric oxide donors in opossum oesophagus
Authors: Cayabyab Francisco S. , Daniel Edwin E.
Published: July 2012

Previous patch clamp studies of oesophageal circular muscle cells showed that nitric oxide (NO) modulated the opening of Ca2+‐activated K+ channels involved in mediating the inhibitory junction potentials (i.j.ps). This study clarified the role of Ca2+ release from the superficial sarcoplasmic reticulum (SR) in the mechanism of i.j.ps or hyperpolarizing responses to NO‐releasing compounds. Electrical and mechanical activities were simultaneously recorded by intracellular microelectrode or double sucrose gap techniques.

The NO‐donors, sydnonimine (SIN‐1) and sodium nitroprusside, each at 500 μm, hyperpolarized oesophageal circular muscle cells by 15–20 mV, like i.j.ps.

The selective inhibitors of SR Ca2+‐ATPase (cyclopiazonic acid 10–30 μm and thapsigargin 5 μm) and the SR Ca2+ release channel activator (ryanodine 30 μm) caused depolarization and spontaneous contractions which were diminished after prolonged (>30 min) incubation with these agents in Ca2+‐containing medium. Moreover, these agents inhibited both the i.j.p. and NO‐donor hyperpolarizations, suggesting that a functional SR Ca2+ uptake is necessary for the response to endogenous or exogenous NO.

These results, along with our previous findings of the dependence of i.j.ps and NO‐donor hyperpolarizations on K+ channel activation and cyclic GMP elevation, support the hypothesis that subplasmalemmal Cai2+ elevation, via vectorial Ca2+ release from superficial SR toward the plasmalemma, may be an important mechanism by which NO, from NO‐liberating compounds or released from inhibitory neurones induces relaxation and i.j.ps in opossum oesophagus.
The effect of insulin treatment and of islet transplantation on the resistance artery function in the STZ‐induced diabetic rat
Authors: Heygate Katherine M. , Davies Joanne , Holmes Michael , James Roger F.L. , Thurston Herbert
Published: July 2012

This study was designed to investigate the influence of insulin treatment and islet transplantation on the smooth muscle contractility and endothelium‐dependent and independent relaxation of resistance arteries in the chemically induced streptozotocin (STZ) diabetic rat after 6–8 weeks, and 12–14 weeks of diabetes, compared to non‐diabetic age‐matched controls.

The morphology, and contractile responses to high potassium physiological salt solution (KPSS), KPSS containing 10−5 m noradrenaline (NAK), and concentration‐response curves to noradrenaline (NA) of mesenteric resistance arteries were recorded, along with the endothelium‐dependent relaxation responses to acetylcholine (ACh) and bradykinin (BK), and endothelium‐independent relaxation to sodium nitroprusside (SNP). Concentration‐response curves were then repeated in the presence of a nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine (L‐NOARG).

Insulin‐treated diabetic rats in the 12 week study demonstrated enhanced vascular contractility to KPSS, NAK and NA, compared to age‐matched non‐diabetic controls.

Incubation with L‐NOARG resulted in both a significant increase in maximum contractile response, and sensitivity (pD2) to NA in the untreated diabetic group (6 weeks). A significant shift in sensitivity was also seen in the insulin‐treated diabetic group. In the 12 week study, incubation with L‐NOARG resulted in an increased maximum contractile response and sensitivity to NA in the insulin‐treated diabetics. An increase in sensitivity was also observed in the untreated diabetic group.

Endothelium‐dependent relaxation to ACh was significantly augmented in the untreated diabetics (6‐weeks), compared to the control group. In the 12‐week study, relaxation to both ACh and BK was not significantly different in any of the experimental groups when compared to the sham‐operated non‐diabetic controls.

Incubation with L‐NOARG resulted in a significant attenuation of the maximum relaxation response to ACh and BK in all of the experimental groups, in the 6‐ and the 12‐week study.

There was no significant difference in the maximum relaxation response or sensitivity to sodium nitroprusside between the diabetic groups and their age‐matched controls in either the 6‐week or the 12‐week study.

The results of this study suggest an enhanced release of nitric oxide in the early stages of diabetes, which is more evident in the untreated diabetic rats than the insulin treated, and appears to normalize as the duration of diabetes progresses. This study also shows that the alteration in vascular reactivity of the resistance arteries can be restored to within normal limits by the transplantation of islets of Langerhans, and that islet transplantation is an effective strategy in the correction of the metabolic abnormalities associated with insulin‐dependent diabetes.
Effects of purinoceptor agonists on cytosolic Ca2+ concentration in swine tracheal smooth muscle cells in culture
Authors: Sawai Hiroko , Wang Ruixia , Yamashita Toshikazu , Kokubun Shinichiro
Published: July 2012

The effects of various purinoceptor agonists on intracellular Ca2+ concentration ([Ca2+]i) in swine tracheal smooth muscle cells in primary culture were examined to investigate the subtype of purinoceptors in these cells.

ATP (1 μm to 1 mM) concentration‐dependently increased [Ca2+]i which was measured by monitoring the fluorescence signal of fura2.

α, β‐Me ATP at concentrations higher than 10 μm increased [Ca2+]i in the presence of extracellular Ca2+. Responses to the drug were 12 ± 5 and 61 ± 4% of responses to ATP (100 μm) at 100 μm and 1 mM, respectively (n = 7). The response to 100 μm ATP was inhibited by 62% in the presence of 1 mM α, β‐Me ATP (n = 8), though the drug at concentrations lower than that did not affect the response to ATP.

ATP increased [Ca2+]i in the absence of extracellular Ca2+. The response to ATP in this condition was 40% of that in the presence of extracellular Ca2+ (n = 8).

Neither cibacron blue 3GA (10 μm) (n = 8) nor suramin (10 and 100 μm) (n = 10) affected the response to ATP (1 μm to 100 μm).

The rank order of potency in the absence of extracellular Ca2+ was UTP> ATP> adenosine 5′‐o‐(3‐thiotriphosphate)>> ADP = α, β‐methylene adenosine 5′‐triphosphate > 2‐(methylthio)‐adenosine 5′‐(tetrahydrogen triphosphate).

UTP (1 μm to 100 μm) concentration‐dependently increased inositol 1,4,5‐triphosphate (IP3) production.

These results suggest that the increase in [Ca2+]i induced by purinoceptor agonists is mediated mainly via a nucleotide receptor in swine tracheal smooth muscle cells in primary culture.
Effects of cantharidin on force of contraction and phosphatase activity in nonfailing and failing human hearts
Authors: Linck Bettina , Boknik Peter , Knapp Jörg , Müller Frank U. , Neumann Joachim , Schmitz Wilhelm , Vahlensieck Ute
Published: July 2012

The effect of the phosphatase inhibitor, cantharidin (3–300 μm) on force of contraction was studied in isolated electrically driven right ventricular trabeculae carneae from human myocardium.

The positive inotropic effect of cantharidin started at a concentration of 100 μm with a positive inotropic effect to 199% and to 276% of the predrug value in nonfailing and failing human hearts, respectively.

Under basal conditions the contraction time parameters were prolonged in human heart failure vs. nonfailing preparations. However, the positive inotropic effect of cantharidin did not affect contraction time parameters. Thus, time to peak tension, time of relaxation and total contraction time were not shortened by cantharidin in nonfailing and failing preparations.

The phosphatase activity was unchanged in preparations from failing hearts compared to nonfailing hearts.

Cantharidin inhibited phosphatase activity in a concentration‐dependent manner. The IC50 value of cantharidin was about 3 μm in both nonfailing and failing human myocardium.

The positive inotropic effect of cantharidin was similar in nonfailing and failing human hearts, accompanied by a similar inhibitory effect of cantharidin on the phosphatase activity. The positive inotropic effect of cantharidin in failing hearts was as strong as the effect of isoprenaline in nonfailing hearts.

It is concluded that the treatment with a phosphatase inhibitor may offer a new positive inotropic modality for the treatment of human heart failure.
Effects of memantine on recombinant rat NMDA receptors expressed in HEK 293 cells
Authors: Bresink Iris , Benke Timothy A. , Collett Valerie J. , Seal Andy J. , Parsons Chris G. , Henley Jeremy M. , Collingridge Graham L.
Published: July 2012

The actions of the uncompetitive N‐methyl‐d‐aspartate (NMDA) receptor antagonists, memantine (1‐amino‐3,5‐dimethyladamantane) and (+)‐MK‐801 ((+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate, dizocilpine), on recombinant NMDA receptors has been studied by use of the whole‐cell patch clamp technique.

Human embryonic kidney (HEK) 293 cells were transiently transfected with different NMDA receptor subunit combinations (NR1a/NR2A, NR1a/NR2B and NR1a/NR2D). A mutant form of the green fluorescent protein (GFP) was cotransfected with the NMDA receptor subunits to enable the visualization of transfected cells.

Memantine (0.3–30 μm) blocked L‐glutamate (100 μm)‐mediated currents in a concentration‐dependent manner in NR1a/NR2A, NR1a/NR2B and NR1a/NR2D transfected cells with IC50 values (at −70 mV) of 0.93±0.15 μm, 0.82±0.12 μm and 0.47±0.06 μm (mean±s.e. mean), respectively.

The memantine‐induced block was strongly voltage‐dependent. Alteration of the holding potential from −70 mV to +60 mV resulted in an e‐fold increase in the IC50 values per 30–33 mV change in membrane potential, for all 3 subunit combinations investigated.

The kinetics of the actions of memantine (30 μm) were investigated for the NR1a/2A combination, in 6 cells (13–15 determinations). At −70 mV, the block and recovery from block were both best described by two exponentials with time‐constants of 201±23 ms (81±2%) and 3.9±0.6 s and 597±94 ms (18±1%) and 18.6±2.4 s, respectively. The predominant effect of depolarization was to increase the weight of the faster recovery time‐constant. Kinetic analysis suggests that these results are consistent with previously proposed Markov models.

(+)‐MK‐801 was studied briefly for comparative purposes. (+)‐MK‐801 (200 nM) preferentially blocked NMDA receptor currents (at −70 mV) in NR1a/NR2A and NR1a/NR2B (82±10% and 93±2% depressions) compared to NR1a/NR2D (38±7%) transfected cells. (+)‐MK‐801 appeared to be less voltage‐dependent than memantine on all three receptor combinations.

In conclusion, memantine was a voltage‐dependent antagonist of recombinant rat NMDA receptors expressed in HEK 293 cells but showed little selectivity between the subunits investigated. Its actions on these recombinant receptor combinations are similar to its actions on native NMDA receptors.
Neuropeptide Y Y2 receptor‐mediated attenuation of neurogenic plasma extravasation acting through pertussis toxin‐sensitive mechanisms
Authors: Yu Xian‐Jie , Moskowitz Michael A.
Published: July 2012

The effects of neuropeptide Y (NPY) receptor agonists (administered intravenously) were examined on plasma protein ([125I]‐bovine serum albumin) leakage within dura mater evoked by unilateral trigeminal ganglion stimulation (0.6 mA, 5 ms, 5 Hz, 5 min), capsaicin (1 μmol kg−1, i.v.) or substance P (1 nmol kg−1, i.v.) in anaesthetized Sprague‐Dawley rats.

NPY (EC50: 5.6 nmol kg−1) and NPY fragment 13–36 [NPY (13–36)] (ED50: 4.3 nmol kg−1), an NPY Y2 receptor agonist, dose‐dependently attenuated [125I]‐bovine serum albumin extravasation from meningeal vessels when administered 10 min prior to electrical stimulation. [Leu31, Pro34]‐NPY, an NPY Y1 and Y3 receptor agonist, inhibited the response at a higher dose only (23 nmol kg−1) (P < 0.05).

NPY also significantly decreased plasma protein extravasation induced by capsaicin (1 μmol kg−1) but not by substance P (1 nmol kg−1).

Pertussis toxin (20 μg kg−1, administered intracisternally 48 h prior to stimulation) blocked completely the inhibitory effect of NPY and NPY (13–36) but did not inhibit extravasation alone.

We conclude that NPY inhibits neurogenically‐mediated plasma protein extravasation acting through presynaptic pertussis toxin‐sensitive NPY Y2 receptors, possibly by inhibition of neuropeptide release from perivascular trigeminovascular afferents.
Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides
Authors: Cunha Rodrigo A. , Correia‐de‐Sá Paulo , Sebastião Ana M. , Ribeiro J. Alexandre
Published: July 2012

In the present work, we investigated the action of adenosine originating from extracellular catabolism of adenine nucleotides, in two preparations where synaptic transmission is modulated by both inhibitory A1 and excitatory A2a‐adenosine receptors, the rat hippocampal Schaffer fibres/CA1 pyramid synapses and the rat innervated hemidiaphragm.

Endogenous adenosine tonically inhibited synaptic transmission, since 0.5‐2 u ml−1 of adenosine deaminase increased both the population spike amplitude (30 ± 4%) and field excitatory post‐synaptic potential (f.e.p.s.p.) slope (27 ± 4%) recorded from hippocampal slices and the evoked [3H]‐acetylcholine ([3H]‐ACh) release from the motor nerve terminals (25 ± 2%).

a, b̊‐Methylene adenosine diphosphate (AOPCP) in concentrations (100–200 μm) that almost completely inhibited the formation of adenosine from the extracellular catabolism of AMP, decreased population spike amplitude by 39 ± 5% and f.e.p.s.p. slope by 32 ± 3% in hippocampal slices and [3H]‐ACh release from motor nerve terminals by 27 ± 3%.

Addition of exogenous 5′‐nucleotidase (5 u Ml−1) prevented the inhibitory effect of AOPCP on population spike amplitude and f.e.p.s.p. slope by 43–57%, whereas the P2 antagonist, suramin (100 μm), did not modify the effect of AOPCP.

In both preparations, the effect of AOPCP resulted from prevention of adenosine formation since it was no longer evident when accumulation of extracellular adenosine was hindered by adenosine deaminase (0.5‐2 u ml−1). The inhibitory effect of AOPCP was still evident when A1 receptors were blocked by 1,3‐dipropyl‐8‐cyclopentylxanthine (2.5‐5 nM), but was abolished by the A2 antagonist, 3,7‐dimethyl‐1‐propargylxanthine (10 μm).

These results suggest that adenosine originating from catabolism of released adenine nucleotides preferentially activates excitatory A2 receptors in hippocampal CA1 pyramid synapses and in phrenic motor nerve endings.
Evidence for 5‐HT1‐like receptor‐mediated vasoconstriction in human pulmonary artery
Authors: MacLean Margaret R. , Clayton Robin A. , Templeton Alison G.B. , Morecroft Ian
Published: July 2012

The 5‐hydroxytryptamine (5‐HT) receptors mediating contraction of human isolated pulmonary artery rings were investigated. Responses to the agonists 5‐carboximidotryptamine (5‐CT, non‐selective 5‐HT1 agonist), sumatriptan (5‐HT1D‐like receptor agonist), 5‐HT and 8‐hydroxy‐2‐(di‐n‐propylamino)‐tetralin (8‐OH‐DPAT, 5‐HT1A receptor agonist) were studied. Responses to 5‐HT and sumatriptan in the presence of the antagonists, methiothepin (non‐selective 5‐HT1+2‐receptor antagonist), ketanserin (5‐HT2A receptor antagonist) and the novel antagonist, GR55562 (5‐HT1D receptor antagonist) were also studied.

All agonists contracted human pulmonary artery ring preparations in the following order of potency 5‐CT > 5‐HT = sumatriptan > 8‐OH‐DPAT. Maximum responses to 5‐HT, 5‐CT and sumatriptan were not significantly different.

Methiothepin 1 nM and 10 nM, but not 0.1 nM reduced the maximum contractile responses to 5‐HT but did not alter tissue sensitivity to 5‐HT. Methiothepin 0.1 nM, 1 nM and 10 nM had a similar effect on responses to sumatriptan.

The 5‐HT2A receptor antagonist ketanserin (10 nM, 100 nM and 1 μm) also reduced the maximum contractile response to both 5‐HT and sumatriptan without affecting tissue sensitivity to these agonists.

The novel 5‐HT1D receptor antagonist, GR55562, inhibited responses to 5‐HT and sumatriptan in a true competitive fashion.

The results suggest that the human pulmonary artery has a functional population of 5‐HT1D‐like receptors which are involved in the contractile response to 5‐HT.
Pharmacological characterization of the 5‐hydroxytryptamine receptor mediating relaxation in the rat isolated ileum
Authors: Tuladhar B.R. , Costal B. , Naylor R.J.
Published: July 2012

The aim of the present study was to investigate a 5‐HT4 receptor involvement in the mediation of a 5‐HT‐induced relaxation response in the rat isolated ileum in vitro.

Ileal segments were taken at regular intervals from the ileo‐caecal junction to duodenum. 5‐HT (1 μm) induced a relaxation or contraction response in segments taken from the terminal ileum: the relaxation decreased and finally disappeared as contractions dominated in the proximal tissues. The 5‐HT‐induced relaxations were enhanced in the terminal segments and the contractions attenuated in both terminal and proximal segments, in the presence of methysergide (1 μm) and atropine (0.1 μm).

In the presence of methysergide (1 μm) and atropine (0.1 μm), a cumulative addition of 5‐HT (0.01‐1 μm) induced a concentration‐dependent relaxation in the terminal (1–20 cm from the ileo‐ceacal junction) ileal segments which at higher concentrations of 5‐HT (3–30 μm) reverted to contraction.

The rank order of potency of indole agonists in inducing a concentration‐related relaxation response in tissues of the terminal ileum (pretreated with pargyline (100 μm) and in the presence of methysergide (1 or 100 μm) and atropine (0.1 μm)) was 5‐hydroxytryptamine (6.97 ± 0.06), 5‐methoxytryptamine (6.50 ± 0.07), α‐methyl‐5‐hydroxytryptamine (5.53 ± 0.17), 5‐carboxamidotryptamine (5.51 ± 0.12) and 2‐methyl‐5‐hydroxytryptamine (< 5), the pEC50 values (mean ± s.e.mean) being shown in parentheses.

Pretreatment of tissues with pargyline (100 μm) selectively enhanced the potency of 5‐methoxytryptamine by a factor of 19 but failed to modify the potency of the other indole agonists.

The 5‐HT4 receptor antagonists, tropisetron, SDZ 205–557 and GR 113808 antagonized the relaxation response to 5‐HT (in the presence of methysergide (1 or 10 μm) and atropine (0.1 μm)) with pKB values (95% CL) of 6.09 (5.94‐6.24), 7.0 (6.9‐7.09) and 8.95 (8.81‐9.1) respectively. Apparent pKB values estimations for tropisetron (1 μm) and GR 113808 (10 nM) using the agonists 5‐methoxytryptamine and 5‐carboxamidotryptamine were 6.37 ± 0.31, 5.91 ± 0.38 and 8.83 ± 0.11, 8.82 ± 0.22 respectively.

Tropisetron (10 μm), SDZ 205–557 (3 μm) and GR 113808 (10–100 nM) caused an increase in basal tone of the rat terminal ileum when administered in the presence of methysergide and atropine.

The relaxation response to 5‐HT in the rat terminal ileum was not antagonized by ritanserin (1 μm), ondansetron (1 μm) or Nω‐nitro‐l‐arginine methyl ester (100 μm) and with only a twofold dextral shift of the concentration‐response curve by tetrodotoxin (1 μm).

It is concluded that the relaxant response to 5‐HT in the terminal region of the ileum is mediated directly at the smooth muscle; a ranked indole agonist potency and selective antagonism by 5‐HT4 receptor antagonists tropisetron, SDZ 205–557 and GR 113808 indicate a 5‐HT4 receptor involvement in the relaxation response.
The increase in human plasma immunoreactive endothelin but not big endothelin‐1 or its C‐terminal fragment induced by systemic administration of the endothelin antagonist TAK‐044
Authors: Plumpton Christopher , Ferro Charles J. , Haynes William G. , Webb David J. , Davenport Anthony P.
Published: July 2012

We examined the effects of systemic infusion, in healthy human volunteers, of the endothelin antagonist TAK‐044 on the plasma concentrations of mature endothelin, big endothelin‐1 and the C‐terminal fragment of big endothelin‐1, by selective solid‐phase extraction and specific radioimmunoassays.

Unlabelled TAK‐044 competed with specific [125I]‐endothelin‐1 binding to human left ventricle tissue in a biphasic manner giving KD values of 0.11 nM and 26.8 nM at the ETA and ETB receptor subtypes, respectively, indicating a 244 fold selectivity for the ETA receptor subtype.

A 15 min intravenous infusion of placebo or 30 mg TAK‐044 (giving a serum concentration of 2 nM, calculated to block > 95% of ETA but <5% ETB receptors) had no effect on the immunoreactive plasma concentrations of the three peptides.

At the higher dose of 750 mg TAK‐044 (giving a serum concentration of 80 nM, calculated to block > 99% of ETA and > 75% ETB receptors), the immunoreactive plasma endothelin concentrations were increased 3.3 fold over basal levels (P < 0.01). The concentrations of big endothelin‐1 or C‐terminal fragment of big endothelin‐1 were unchanged.

At both doses of TAK‐044, there were significant decreases in diastolic blood pressure, and peripheral vascular resistance, with corresponding increases in cardiac index and stroke index. There were no changes in systolic or mean arterial blood pressures or heart rate.

Since only the concentrations of the mature peptide were increased, we conclude that the most likely sources of endothelin contributing to the observed rise were displacement of receptor‐bound peptide and reduction in plasma clearance rather than peptide synthesis.
Endothelial 5‐HT receptors mediate relaxation of porcine pulmonary arteries in response to ergotamine and dihydroergotamine
Authors: Glusa E. , Roos A.
Published: July 2012

The aim of the present study was to investigate whether antimigraine ergot compounds may act at endothelial 5‐hydroxytryptamine (5‐HT) receptors which trigger the release of endothelium‐derived relaxing factor (EDRF). Changes in tone of porcine isolated pulmonary arteries were measured isometrically. The integrity of the endothelium was assessed by the bradykinin‐induced relaxation of prostaglandin F2α (PGF2α, 3 μm)‐precontracted vessels.

The ergot derivatives ergotamine, dihydroergotamine (DHE) and dihydroergocristine, as well as 5‐HT and (±)‐α‐methyl‐5‐HT, elicited a reversible endothelium‐dependent relaxation of PGF2α‐precontracted arterial ring segments. The relaxation to both ergotamine and 5‐HT was associated with an increase in cyclic GMP. After pretreatment of the vessels with NG‐nitro‐L‐arginine methyl ester (200 μm), or removal of endothelium by mechanical rubbing, the relaxant responses were abolished.

The mean pEC50 values for relaxant responses followed the order: (±)‐α‐methyl‐5‐HT (8.80) > 5‐HT (8.75) > ergotamine (8.17) > DHE (7.70) > 5‐carboxamidotryptamine (7.62) > dihydroergocristine (7.17).

The relaxant effects of both ergotamine and dihydroergotamine were resistant to block by indomethacin (3 μm), prazosin (1 μm) and ketanserin (1 μm). However, the ergotamine‐induced relaxation was highly susceptible to block by pizotifen (pA2 = 8.23), norclozapine (pA2 = 8.20), methiothepin (‐log IC50 = 7.26), rauwolscine (pA2 = 7.24) and mesulergine (pA2 = 6.64). Each antagonist inhibited the relaxant responses to (±)‐α‐methyl‐5‐HT in the same manner with similar potency as that determined against ergotamine.

Recently, mRNA transcripts for 5‐HT1Dβ and 5‐HT2B receptors have been demonstrated in porcine pulmonary arteries. The rank order of potencies of agonists and antagonists in the present study suggests that the relaxant responses to 5‐HT and ergot derivatives are mediated through activation of endothelial 5‐HT receptors which are similar to the 5‐HT2B receptor subtypes.
Role of bradykinin receptors in the renal effects of inhibition of angiotensin converting enzyme and endopeptidases 24.11 and 24.15 in conscious rabbits
Authors: Tomoda Fumihiro , Lew Rebecca A. , Smith A. Ian , Madden Anna C. , Evans Roger G.
Published: July 2012

We tested the effects on systemic haemodynamics and renal function, of inhibition of endopeptidase (EP) 24.15 (E.C. 3.4.24.15), in conscious uninephrectomized rabbits in which the activities of angiotensin converting enzyme (ACE, E.C. 3.4.15.1) and neutral endopeptidase (EP 24.11, E.C. 3.4.24.11) were already inhibited. To test the role of bradykinin B2‐receptors in mediating the effects following inhibition of these enzymes, the antagonist Hoe 140 was used.

Hoe 140 (0.1 mg kg−1, i.v.) did not affect resting mean arterial pressure or heart rate, but antagonized the depressor effect of right atrial administration of bradykinin. The dose‐response curve for bradykinin was shifted more than 1000 fold to the right for more than 4 h. Hoe 140 approximately doubled resting urine flow and increased fractional Na+ excretion from 4.2 to 6.0%; consistent with the hypothesis that it exerts a partial agonist effect on the kidney.

Combined inhibition of ACE (captopril; 0.25 mg kg−1 plus 0.2 mg kg−1h−1) and EP 24.11 (SCH 39370; 3 mg kg−1 plus 3 mg kg−1h−1) was followed by a sustained reduction in arterial pressure (−6 ± 2 mmHg) and increase in heart rate (35 ± 7 beats min−1). There was a small increase in renal blood flow (by 6.5 ± 3.2% relative to vehicle‐treatment) without a change in glomerular filtration rate, and about a 150% increase in Na+ excretion. Hoe 140 (0.1 mg kg−1, i.v.) pretreatment did not influence the renal effects of captopril and SCH 39370, although it did appear to blunt their hypotensive and tachycardic effects.

When EP 24.15 was inhibited with N‐[1‐(R,S)‐carboxy‐3‐phenylpropyl]‐Ala‐Ala‐Tyr‐p‐aminobenzoate (cFP‐AAY‐pAB; 5 mg kg−1 plus 3 mg kg−1h−1, i.v.) in rabbits pretreated with captopril and SCH 39370, no changes in systemic haemodynamics or renal function were observed.

We concluded that in conscious uninephrectomized rabbits, EP 24.15 does not play a major role in modulating renal function, at least under conditions where ACE and EP 24.11 are already inhibited. In contrast, ACE and/or EP 24.11 do modulate renal function in this model, but their influences are mediated chiefly through metabolism of peptides other than bradykinin.
Different subtypes of α1A‐adrenoceptor mediating contraction of rat epididymal vas deferens, rat hepatic portal vein and human prostate distinguished by the antagonist RS 17053
Authors: Marshall Ian , Burt Richard P. , Green G. Mark , Hussain Monira B. , Chapple Christopher R.
Published: July 2012

The α1‐adrenoceptor subtype mediating contraction of the rat hepatic portal vein to phenylephrine was characterized by use of competitive antagonists previously shown to have selectivity between the expressed α1‐subtype clones. Prazosin competitively antagonized the phenylephrine contractions with a pA2 value of 9.2, as did WB 4101 (pA2 9.4), 5‐methyl urapidil (pA2 8.6), indoramin (pA2 8.4) and BMY 7378 (pA2 6.5).

The pA2 values on the rat portal vein correlated highly with their previously published pA2 values for the α1A‐adrenoceptors mediating contraction of the rat epididymal vas deferens and human prostate and poorly with those for the α1B‐ and α1D‐adrenoceptors mediating contraction of the rat spleen and aorta, respectively. The antagonist pA2 values on the rat portal vein correlated highly with their previously published pK1 values for the expressed α1a‐clone and poorly with those for the expressed α1b‐ and α1d‐clones. Therefore the results show that contraction of the rat portal vein to phenylephrine is mediated by α1A‐adrenoceptors.

The novel α1‐adrenoceptor antagonist RS 17053 had a relatively high affinity for the α1A‐adrenoceptors mediating contraction of the rat epididymal vas deferens (pA2 9.5) compared with the α1B‐adrenoceptors in the rat spleen (pA2 7.2) or the α1D‐adrenoceptors in the rat aorta (pKB 7.1), in agreement with its selectivity for the expressed α1a‐clone. However, RS 17053 had over 100 fold lower affinity for the α1A‐adrenoceptors mediating contraction of the rat portal vein (pKB 7.1) and human prostate (pKB 7.1) compared with its affinity for the α1A‐adrenoceptors in the rat epididymal vas deferens or the expressed α1a‐clone.

The difference in affinity of RS 17053 between the rat epididymal vas deferens and rat portal vein cannot be explained by a species difference in the receptor. Therefore RS 17053 may distinguish between subtypes of the α1a‐adrenoceptor in the rat portal vein and human prostate compared with those in the rat epididymal vas deferens or the expressed α1a‐clone.