bluuebunny/crossref_metadata_2025_split huggingface dataset:
Deoxyribose analogues of N6‐cyclopentyladenosine (CPA): partial agonists at the adenosine A1 receptor in vivo
Authors: Mathôt R.A.A. , Van der Wenden E.M. , Soudijn W. , IJzerman A.P. , Danhof M. 
Published: July 2012

The purpose of the present study was to quantify the cardiovascular effects of the 2′‐, 3′‐ and 5′‐deoxyribose analogues of the selective adenosine A1 receptor agonist, N6‐cyclopentyladenosine (CPA) in vivo. The blood concentration‐effect relationships of the compounds were assessed in individual rats and correlated to their receptor binding characteristics.
The pharmacokinetics and pharmacodynamics of the compounds were determined after a single intravenous infusion of 0.80 mg kg−1 (2.5 μmol kg−1) of 5′dCPA, 1.2 mg kg−1 (3.8 μmol kg−1) of 3′dCPA or 20 mg kg−1 (63 μmol kg−1) of 2′ dCPA. The heart rate (HR) and mean arterial blood pressure (MAP) were monitored continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentration.
The relationship between blood concentrations and the reductions in both heart rate and blood pressure were described according to the sigmoidal Emax model. For the bradycardiac effect, the potencies based on free drug concentrations (EC50, u) of 5′dCPA, 3′dCPA and 2′dCPA in blood were 5.9 ± 1.7, 18 ± 4 and 260 ± 70 ng ml−1 (19 ± 6, 56 ± 11 and 830 ± 210 nM), respectively, and correlated well with the adenosine A1 receptor affinity in vitro. The Emax value of 2′dCPA was significantly less than those of the other compounds, suggesting that this compound may be regarded as a partial agonist when compared to the other analogues. The rank order of the maximal reduction in heart rate of the compounds corresponded well with the order of the GTP‐shifts, as determined in vitro.
It is concluded that deoxyribose derivatives of CPA may be partial agonists for the adenosine A1 receptor and may serve as tools for further investigation of adenosine receptor partial agonism in vivo.
Mucosa‐dependent muscarinic liberation of prostaglandins from rat isolated trachea
Authors: Brunn Gemot , Wessler Ignaz , Racke Kurt 
Published: July 2012

The present study examined whether cholinoceptor stimulation modulates the release of arachidonic acid‐derived mediators from rat isolated tracheae.
Tracheae were preincubated with [3H]‐arachidonic acid and the outflow of 3H‐compounds was determined. Acetylcholine and the muscarinic agonist, carbachol but not nicotine, increased the rate of tritium outflow maximally by about 30%. The M3 receptor‐preferring antagonist p‐fluoro‐hexahydrosiladiphenidol was more effective than pirenzepine and methoctramine in antagonizing the effect of acetylcholine.
High performance liquid chromatography analysis (methanol gradient) of the released 3H‐compounds showed that one peak, co‐eluting with [14C]‐prostaglandin E2 ([14C]‐PGE2) and [3H]‐PGD2, was enhanced almost 10 fold following muscarinic receptor activation, whereas the outflow of [3H]‐arachidonic acid remained unaffected.
Using an acetonitril gradient separation it was shown that [3H]‐PGE2, [3H]‐PGD2 and [3H]‐PGF2α are released spontaneously, but [3H]‐PGE2 represented the major fraction of 3H‐prostaglandins. Acetylcholine enhanced the release of all three 3H‐prostaglandins, but the effect on PGE2 was most pronounced and most consistent.
After removal of the mucosa the muscarinic effect of acetylcholine on total tritium and on that of the 3H‐prostaglandins ([3H]‐PGE2/PGD2 peak) was abolished.
Acetylcholine also enhanced the outflow of radioimmunologically determined PGE2 in a mucosa‐dependent manner.
After inhibition of cyclo‐oxygenase by 3 μM indomethacin, the outflow of 3H‐prostaglandins was reduced to almost undetectable levels and acetylcholine evoked a marked release of [3H]‐arachidonic acid. The phospholipase A2 inhibitor, quinacrine (up to 100 μm) also blocked the effect of acetylcholine on the outflow of 3H‐prostaglandins, but this was not followed by a compensatory increase in the outflow of [3H]‐arachidonic acid.
In conclusion, activation of muscarinic receptors which have characteristics of the M3 subtype can evoke release of prostaglandins from the airway mucosa.
Characterization of the pharmacology and regional distribution of (S)‐[3H]‐5‐fluorowillardiine binding in rat brain
Authors: Hawkins L.M. , Beaver K.M. , Jane D.E. , Taylor P.M. , Sunter D.C. , Roberts P.J. 
Published: July 2012

This study examined the binding of the new radioligand (S)‐[3H]‐5‐fluorowillardiine to rat brain synaptic membranes. Specific binding represented greater than 80% of the total binding and was increased by 10% in the presence of 100 mM potassium thiocyanate (KSCN).
In the absence of KSCN, (S)‐[3H]‐5‐fluorowillardiine identified two binding sites with KD1 = 22.5nM, Bmax1= 1.4 pmol mg‐1 protein and KD2=1.5 μM, Bmax2= 10.8 pmol mg−1 protein. In the presence of 100 mM KSCN the affinities of both the binding sites were increased, yielding values of KD1 = 6.9nM and KD2 = 0.4 μm KSCN was without effect on the Bmax values.
(S)‐[3H]‐5‐fluorowillardiine binding was displaced by non‐NMDA receptor ligands with the rank order of potency: 2,3‐dihydroxy‐6‐nitro‐7‐sulphamoyl‐benzo(F)quinoxaline (NBQX) > domoate > (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) ≅ L‐glutamate > 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) > kainate >> (R)‐5‐fluorowillardiine. In contrast, both N‐methyl‐D‐aspartate (NMDA) and the metabotropic glutamate receptor agonist, (lS,3R)‐l‐aminocyclopentane‐l,3‐dicarboxylic acid (ACPD) were inactive.
By use of quantitative autoradiography the regional distribution of (S)‐[3H]‐5‐fluorowillardiine binding in rat brain was assessed. The highest levels of binding were in the dentate gyrus and the CA1 region of the hippocampus. Lower levels of binding were detected in the cerebral cortex, olfactory system, lateral septum, caudate putamen and nucleus accumbens.
We conclude that the pharmacological profile and regional distribution of (S)‐[3H]‐5‐fluorowillardiine binding is consistent with its specific interaction with AMPA receptors. The advantages of high percentage specific binding and recognition of the two binding sites, in the absence of KSCN, suggest that (S)‐[3H]‐5‐fluorowillardiine may be the ligand of choice for the future study of AMPA receptors.
Mechanism of endothelium‐dependent relaxation induced by substance P in the coronary artery of the pig
Authors: Kuroiwa Mari , Aoki Hiroki , Kobayashi Sei , Nishimura Junji , Kanaide Hideo 
Published: July 2012

Using front‐surface fluorometry of fura‐2‐loaded porcine coronary arterial strips with the endothelium intact, we investigated the mechanisms of vasorelaxation induced by substance P (SP). Fura‐2 fluorescence signals which indicated the cytosolic Ca2+‐concentration ([Ca2+]i), were observed to arise exclusively from the smooth muscle cells in these strips.
During the contractions induced by U46619 (100 nM), a thromboxane A2 analogue, an SP‐induced endothelium‐dependent, biphasic vasorelaxation was observed, which consisted of an initial rapid relaxation phase followed by a sustained phase, with a transient decrease in [Ca2+]i. Pretreatment with indomethacin (Ind) had no effect on the SP‐induced relaxation; however, pretreatment with NG‐nitro‐L‐arginine (l‐NOARG) partially, but significantly inhibited the decrease in both the [Ca2+]i and tension induced by SP; in addition, the sustained phase of SP‐induced relaxation was almost completely abolished. Thus, part of the relaxation was considered to be mediated by L‐NOARG‐sensitive relaxing factor (endothelium‐derived relaxing factor: EDRF).
During the 40 mM K+‐depolarization‐induced contraction which may eliminate the effects of endothelium‐derived hyperpolarizing factor (EDHF), the vasorelaxation induced by SP was completely inhibited by L‐NOARG.
During the vasorelaxation induced by SP, the [Ca2+]itension relationships shifted to the right of the contractions induced by either U46619 or high K+‐depolarization.
Using front‐surface fluorometry of fura‐2 loaded porcine aortic valvular strips, we examined the effects of SP on [Ca2+]i in endothelial cells in situ. SP induced a rapid increase in [Ca2+]i of endothelial cells in situ followed by a small sustained phase in normal PSS (5.9 mM K+). The increase in extracellular K+ had no apparent effect on the SP‐induced [Ca2+]i elevation of endothelial cells.
We thus conclude that: (1) SP‐induced vasorelaxation is mediated by an L‐NOARG‐sensitive factor (EDRF) and an L‐NOARG‐resistant factor; and (2) the first, rapid, phase of the relaxation is mediated by both factors while the sustained phase seems to be mediated mainly by EDRF. The underlying mechanisms of L‐NOARG‐resistant relaxation have yet to be elucidated, but EDHF appears to be a potentially contributing factor.
Determination of KA values by controlled receptor expression in Xenopus oocytes
Authors: Murakoshi Hideyuki , Nunoki Kazuo , Ishii Kuniaki , Taira Norio 
Published: July 2012

In the present study we estimated the KA value of endothelin‐1 (ET‐1) for ETA‐receptors by a new method in which the level of expression of ETA‐receptors in Xenopus oocytes was altered in a controlled way.
Kvl.2 (a delayed rectifier type K channel) cRNA at the fixed concentration of 0.2 μg μl−1 was mixed with ETA‐receptor cRNA at various concentration ratios (10−3‐3). Oocytes were examined 2–4 days after the injection of the cRNA mixtures.
In these oocytes, ET‐1 suppressed the amplitude of Kvl.2 current in a dose‐dependent manner in the range of 0.1–100 nM; the maximum inhibition produced by ET‐1 was larger and the EC50 value for the inhibition by ET‐1 was smaller as the mixture ratio was increased. Double‐reciprocal plots of equiactive concentrations of ET‐1 in 1/1‐ and 1/30‐injected oocytes yielded a KA for ET‐1 of 7.4 nM. The number of ETA‐receptors in 1/30‐injected oocytes was 13% of that in 1/1‐injected oocytes, whereas the inhibition of the current in 1/30‐injected oocytes was about 60% of that in 1/1‐injected oocytes. This suggests the presence of spare receptors of ETA in the latter.
A saturation binding experiment estaimated a KD value of 0.1 nM for ET‐1 at ETA‐receptors and the number of ETA‐receptors in 1/30‐injected oocytes was 23% of that in 1/1‐injected ones. This value was not significantly different from that estimated by the above new method. However, there was a discrepancy between KA and 1KD, which could be due to factors unique to the expression system employed in the present study.
Mastoparan‐induced phosphatidylcholine hydrolysis by phospholipase D activation in human astrocytoma cells
Authors: Mizuno Kouichi , Nakahata Norimichi , Ohizumi Yasushi 
Published: July 2012

The effect of mastoparan on phosphatidylcholine hydrolysis was examined in 1321N1 human astrocytoma cells. Mastoparan (3–30 μm) caused an accumulation of diacylglycerol (DG) and phosphatidic acid (PA) accompanied by choline release in a concentration‐ and time‐dependent manner.
In the presence of 2% n‐butanol, mastoparan (3–100 μM) induced phosphatidylbutanol (PBut) accumulation in a concentration‐ and time‐dependent manner, suggesting that mastoparan activates phospholipase D (PLD). Propranolol (30–300 μM), a phosphatidate phosphohydrolase inhibitor, inhibited DG accumulation induced by mastoparan, supporting this idea.
Depletion of extracellular free calcium ion did not alter the effect of mastoparan on PLD activity.
A protein kinase C (PKC) inhibitor, calphostin C (1 μm), did not inhibit mastoparan‐induced PLD activation but the ability of mastoparan to stimulate phospholipase D activity was decreased in the PKC down regulated cells.
PLD activity stimulated by mastoparan was not prevented by pretreatment of the cells with pertussis toxin (PT) or C3 ADP‐ribosyltransferase. Furthermore, guanine nucleotides did not affect PLD activity stimulation by mastoparan in membrane preparations.
Mastoparan stimulated PLD in several cell lines such as RBL‐2H3, RBL‐1, HL‐60, P388, endothelial cells, as well as 1321N1 human astrocytoma cells.
These results suggest that mastoparan induces phosphatidylcholine (PC) hydrolysis by activation of PLD, not by activation of phosphatidylcholine‐specific phospholipase C (PC‐PLC); mastoparan‐induced PLD activation is not mediated by G proteins.
Pharmacological characterization of behavioural responses to SK&F 83959 in relation to ‘D1‐like’ dopamine receptors not linked to adenylyl cyclase
Authors: Deveney Aaron M. , Waddington John L. 
Published: July 2012

Behavioural responses to the new benzazepine derivative, SK&F 83959, a compound that both fails to stimulate adenylyl cyclase and inhibits the stimulation of adenylyl cyclase induced by dopamine, were characterized in detail.
In rat striatal membrane preparations, radioligand binding studies with [3H]‐SCH 23390 and [3H]‐spiperone indicated SK&F 83959 had a high affinity and > 250 fold selectivity for D1 over D2 receptors.
Using a rapid time‐sampling behavioural check list technique, SK&F 83959 (0.01‐1.25 mg kg−1) induced grooming in the manner of all known D1 receptor agonists, together with some vacuous chewing, which declined at higher doses with the emergence of directed chewing and rearing as an adjunct to prominent sniffing; no stereotyped behaviour was evident.
Grooming to SK&F 83959 (0.05 mg kg−1) was blocked by the selective D1 receptor antagonists, SCH 23390 (0.01‐1.0 mg kg−1) and BW 737C (0.04–5.0 mg kg−1) and was attenuated by the selective D2 receptor antagonist, YM 09151‐2 (0.005‐0.5 mg kg−1); vacuous chewing to SK&F 83959 was not influenced by either SCH 23390 or BW 737C and was enhanced by YM 09151‐2.
The paradoxical induction of typical D1 receptor agonist‐induced grooming by SK&F 83959, an agent satisfying criteria for a D1 receptor antagonist as classically defined, together with its blockade by typical D1 antagonists, strongly suggests mediation via a ‘D1‐like’ site that appears to respond similarly to agents independent of whether they exert agonist or antagonist actions at the classical adenylyl cyclase‐coupled D1 receptor. This direct functional evidence for a ‘D1‐like’ site that is not linked to adenylyl cyclase readily complements neurochemical data suggesting the existence of a cyclase‐independent ‘D1‐like’ receptor that may be coupled to phosphoinositide hydrolysis.
Relationship between agonist binding, phosphorylation and immunoprecipitation of the m3‐muscarinic receptor, and second messenger responses
Authors: Tobin A.B. , Willars G.B. , Burford N.T. , Nahorski S.R. 
Published: July 2012

Phosphoinositidase C‐linked m3‐muscarinic receptors expressed in Chinese hamster ovary cells (CHO‐m3 cells) are phosphorylated on serine following agonist stimulation
m3‐Muscarinic receptor phosphorylation is concentration‐dependent requiring a carbachol concentration of 13.2 μm for half maximal stimulation
The phosphorylation concentration‐response curve lies to the left of the curve for carbachol binding to muscarinic receptors (KD=100μm) in membranes from CHO‐m3 cells. In contrast, receptor phosphorylation closely correlates with receptor‐mediated phosphoinositidase C activation (EC50 for inositol 1,4,5 trisphosphate accumulation during the peak and plateau phases were 7.14 μm and 5.92 μm respectively) but not with rapid agonist‐mediated calcium elevation (EC50 = 0.32 μm) measured in fura‐2‐AM loaded cells
These data suggest a dissociation of receptor phosphorylation from agonist occupation. Such an apparent ‘receptor reserve’ for m3‐muscarinic receptor phosphorylation may be indicative of a mechanism that is dependent on a small amplification of the receptor signal, though probably dissociated from the calcium signal.
[3H]‐RS‐45041‐190: a selective high‐affinity radioligand for I2 imidazoline receptors
Authors: MacKinnon Alison C. , Redfern William S. , Brown Christine M. 
Published: July 2012

RS‐45041‐190 (4‐chloro‐2‐(imidazolin‐2‐yl)isoindoline) is an I2 imidazoline receptor ligand with the highest affinity and selectivity so far described; [3H]‐RS‐45041‐190 has a tritium atom attached to the 7‐position on the isoindoline ring
[3H]‐RS‐45041‐190 binding to rat kidney membranes was saturable (Bmax = 223.1 ± 18.4 fmol mg−1 protein) and of high affinity (Kd = 2.71 ±0.59 nM). Kinetic studies revealed that the binding was rapid and reversible, with [3H]‐RS‐45041‐190 interacting with two sites or two affinity states
Competition studies showed that 60–70% of [3H]‐RS‐45041‐190 binding (1 nM) was specifically to imidazoline binding sites of the I2 subtype, characterized by high affinity for idazoxan (pIC50 7.85 ±0.03) and cirazoline (pIC50 8.16 ±0.05). The remaining 30–40% was displaced specifically by the monoamine oxidase A inhibitors, clorgyline and pargyline
α1 and α2‐adrenoceptor, Ii imidazoline, histamine, 5‐hydroxytryptamine or dopamine receptor ligands had low affinity suggesting that [3H]‐RS‐45041‐190 did not label receptors of these classes
In autoradiography studies, [3H]‐RS‐45041‐190 labelled discrete regions of rat brain corresponding to the distribution of I2 subtypes, notably the subfornical organ, arcuate nucleus, interpeduncular nucleus, medial habenular nucleus and lateral mammillary nucleus, and additional sites in the locus coeruleus, dorsal raphe and dorsomedial hypothalamic nucleus
[3H]‐RS‐45041‐190 therefore labels I2 receptors with high affinity, and an additional site which has high affinity for some monoamine oxidase inhibitors.
Evidence that activation of 5‐HT2 receptors in the forebrain of anaesthetized cats causes sympathoexcitation
Authors: Anderson Ian K. , Martin Graeme R. , Ramage Andrew G. 
Published: July 2012

The aim of the present experiments was to determine whether the effects of lateral ventricular application of 5‐HT on cardiovascular and respiratory variables in anaesthetized cats are mediated by forebrain 5‐HT2 receptors. This was carried out by determining whether the effects of 5‐HT are blocked by the 5‐HT2 antagonist, cinanserin and if they are mimicked by the selective 5‐HT2 agonist, 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI)
Cats were anaesthetized with a mixture of oc‐chloralose and pentobarbitone sodium, neuromuscularly blocked and artificially ventilated. The following cardiovascular and respiratory variables were recorded: renal, splanchnic and cardiac sympathetic nerve activities, phrenic nerve activity, heart rate, arterial blood pressure, femoral arterial conductance and tracheal pressure. All drugs were administered via the lateral ventricle and the action of these agonists was restricted to forebrain sites by a cannula placed in the Aqueduct of Sylvius
Cumulative doses of 5‐HT (10–160 nmol kg−1) and DOI (80–320 nmol kg−1) injected into the lateral ventricle caused significant increases in blood pressure, heart rate, cardiac and splanchnic sympathetic nerve activity and a decrease in femoral arterial conductance. DOI and 5‐HT caused a greater increase in cardiac compared with splanchnic nerve activity and failed to change renal nerve activity
‐HT but not DOI significantly increased the magnitude and the number of phrenic bursts as well as significantly increasing tracheal pressure. The effects of 5‐HT also differed from DOI in that 5‐HT evoked maximal pressor and near maximal sympathoexcitatory effects after the first dose, whereas the pressor and sympathoexcitatory effects of DOI were graded over the complete dose‐range
The 5‐HT2 antagonist, cinanserin (265 nmol kg−1, i.c.v.) caused significant falls in blood pressure, heart rate and cardiac nerve activity and an increase in femoral arterial conductance. Splanchnic and renal sympathetic nerve activity, phrenic nerve activity and tracheal pressure were unaffected by cinanserin. After pretreatment with cinanserin all cardiovascular and respiratory effects of 5‐HT were significantly attenuated
It is concluded that in the cat, as DOI and 5‐HT have similar effects on the cardiovascular variables recorded and as the effects of 5‐HT are blocked by cinanserin, 5‐HT can act on 5‐HT2 receptors located in the forebrain to cause differential sympathoexcitation and a rise in arterial blood pressure. Further, the sympathoexcitatory effects mediated by 5‐HT2 receptors located in the forebrain differ from those located in the hindbrain in that they mediate increases in cardiac nerve activity and heart rate and also have no effect on renal nerve activity.
Activation of phospholipase C in SH‐SY5Y neuroblastoma cells by potassium‐induced calcium entry
Authors: Smart D. , Wandless A. , Lambert D.G. 
Published: July 2012

We used SH‐SY5Y human neuroblastoma cells to investigate whether depolarization with high K+ could stimulate inositol (1,4,5)trisphosphate (Ins(1,4,5)P3) formation and, if so, the mechanism involved
Ins(1,4,5)P3 was measured by a specific radioreceptor mass assay, whilst [Ca2+]i was measured fluorimetrically with the Ca2+ indicator dye, Fura‐2
Depolarization with K+ caused a time‐ and dose‐dependent increase in [Ca2+]j (peak at 27 s, EC50 of 50.0±9.0 mM) and Ins(1,4,5)P3 formation (peak at 30 s, EC50 of 47.4±1.1 mM)
Both the K+‐induced Ins(1,4,5)P3 formation and increase in [Ca2+]i were inhibited dose‐dependently by the L‐type voltage‐sensitive Ca2+ channel closer(R +)‐BayK8644, with IC50 values of 53.4 nM and 87.9 nM respectively.
These data show a close temporal and dose‐response relationship between Ca2+ entry via L‐type voltage‐sensitive Ca2+ channels and Ins(1,4,5)P3 formation following depolarization with K+, indicating that Ca2+ influx can activate phospholipase C in SH‐SY5Y cells
Modulation of calcium currents by G‐proteins and adenosine receptors in myenteric neurones cultured from adult guinea‐pig small intestine
Authors: Baidan Lessia V. , Zholos Alexander V. , Wood Jackie D. 
Published: July 2012

Whole‐cell patch clamp methods were used to analyse voltage‐dependent calcium currents in cultured myenteric neurones enzymatically isolated from adult guinea‐pig small intestine
Activation of G‐proteins by intracellular administration of GTP‐γ‐S (100–200 μm in pipette) decreased the amplitude of high voltage activated Ca2+ current (ICa) by more than 50%. Residual ICa was activated more slowly and was non‐inactivating during 500 ms test pulses when GTP‐γ‐S was included in the pipette solution
Inclusion of 500 μm GDP‐β‐S in the patch pipettes increased the amplitude of ICa by over 30% without altering the voltage‐dependency
Extracellular application of 2‐chloroadenosine suppressed ICa dose‐dependently by reducing both transient and sustained components of the current
Pretreatment of the neurones with cholera toxin or forskolin did not alter the actions of GTP‐γ‐S or GDP‐β‐S or 2‐chloroadenosine
The results suggest that high threshold calcium channels in myenteric neurones are influenced by G‐proteins and that the inhibitory action of 2‐chloroadenosine on ICa involves G‐protein coupling of the adenosine receptors to the Ca2+ channel.