bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y‐ and P2U‐purinoceptor‐stimulated prostacyclin release
Authors: Patel Viral , Brown Colin , Boarder Michael R. 
Published: July 2012

. Enhanced synthesis of prostacyclin (PGI2) and inositol polyphosphates in bovine aortic endothelial cells in response to ATP and ADP is mediated by co‐existing P2Y‐ and P2U‐purinoceptors. Here we examine the regulation of these responses by isoforms of protein kinase C (PKC).
. Immunoblots with antisera specific for 8 different PKC isoforms revealed the presence of α, ∍ and ζ, while no immunoreactivity was found for β, γ, δ, η and θ isoforms. PKC‐α was largely cytosolic in unstimulated cells and almost all translocated to the membrane (Triton X‐100 soluble) after a 1 min treatment with the PKC activating phorbol myristate acetate (PMA); PKC‐∍ was always in a Triton X‐100 insoluble membrane fraction, while PKC‐ζ was found in both soluble and membrane bound (Triton X‐100 soluble) forms in the unstimulated cells and was unaffected by PM A.
. Treatment with PM A for 6 h led to a 90% downregulation of PKC‐α, while the immunoreactivity to the e and ζ isoforms remained largely unchanged.
. After either 10 min or 6 h exposure to PM A the PGI2 response to activation of both receptors was enhanced, while the inositol 1,4,5‐trisphosphate response to P2Y‐purinoceptor activation was substantially attenuated and the P2U‐purinoceptor response was unchanged. Thus the PGI2 response to PM A under conditions when 90% of the PKC‐α was lost resembles that seen on acute stimulation of PKC by PMA, and the PGI2 response does not correlate with the phospholipase C response.
. Inhibition of PKC with the isoform non‐selective inhibitors, Ro 31–8220 and Go 6850 abolished the PGI2 response to both P2U‐ and P2Y‐purinoceptor stimulation. However, Go 6976, which preferentially inhibits Ca2+ sensitive isoforms (such as PKC‐α) and not Ca2+ insensitive isoforms (such as PKC‐∍), had no effect on the PGI2 response.
. The results show that there is a requirement for PKC in the stimulation of PGI2 production by endothelial P2Y‐ and P2U‐purinoceptors. Both downregulation and inhibition studies show that PKC‐α is not responsible for the regulation of the response to P2‐purinergic stimulation, and imply that the response is mediated by PKC‐∍ (PKC‐ζ is unresponsive to PMA), or an as yet uncharacterized PKC isoform.
Effects of phosphorothioated neuropeptide Y Y1‐receptor antisense oligodeoxynucleotide in conscious rats and in human vessels
Authors: Sun Xiang Ying , Zhao Xiao He , Erlinge David , Edvinsson Lars , Fallgren Bo , Wahlestedt Claes , Hedner Thomas 
Published: July 2012

. Metabolically stabilized (phosphorothioate) human and rat NPY Yx receptor oligodeoxynucleotides (ODNs) complimentary to the rat or human Y1 mRNA were synthesized; [sense (rY1‐SODN, 5′‐AATTCAACTCTGTTCTCC‐3′), antisense (hY1‐ASODN, 5′‐CCTGGGAAAATAATGTTG‐3′ and rY1‐ASODN, 5′‐GGAGAACAGAGTTGAATT‐3′) and mismatches (hY1‐MMODN, 5′‐CCTGAGATAA‐TAAGGTTG‐3′ and rY1‐MM 5′‐GTAGATCAGAGATGAAGT‐3′)] and used to modulate cardiovascular function in vitro in human vessels as well as in vivo in the rat.
. The objectives of the experiments were to assess the influence of the NPY Y1 receptor on vasomotor function human resistance arteries in vitro and to investigate the contribution of the NPY receptor system to cardiovascular haemodynamics in vivo.
. Human subcutaneous resistance arteries removed from patients who underwent surgery for non‐vascular diseases were incubated in vitro with the stabilized phosphorothioated hY1‐receptor ASODN or MMODN (10−7 to 10−5 m).
. In human resistance vessels preincubated with hY1‐AS (10−7 to 10−5 m), the contractile response to NPY was significantly reduced in a dose‐dependent fashion. No effects were observed in the hY1‐MMODN‐incubated vessels at lower concentrations (10−7 m to 10−6 m).
. The haemodynamic effects of the phosphorothioated rY1‐ASODN, SODN or MMODN were investigated in conscious rats during 48 h of continuous infusions. The continuous infusion with the rY1‐ASODN did not change MAP while the rY1‐SODN unexpectedly induced an early (10–20 h) increase in ambulatory MAP and the rY1‐MMODN a late (24–44 h) increase.
. Contractile responses to NPY (2, 4, 8, 16 and 32 μg kg−1) were significantly reduced in the rats treated with long‐term infusion of rY1‐ASODN (2.1 mg kg−1 h−1, i.v. infusion for 48 h) compared with animals treated with rY1‐SODN and MMODN, as well as animals treated with saline and glucose. Notably, the group infused with the rY1‐SODN showed an exaggerated response to tested doses of NPY.
. We conclude that the incubation of human subcutaneous arteries with a metabolically stabilized 18 base pair hY1‐ASODN and long‐term infusion with a corresponding rY1‐ASODN attenuate NPY‐induced vasoconstriction.
Species specificity in the blood cholesterol‐lowering effect of YM‐16638
Authors: Goto Shoichiro , Shimokawa Teruhiko , Ugawa Toru , Hisamichi Nami , Masuyama Youichi , Iizumi Yuichi , Sato Noboru , Takenaka Toichi , Kodama Tatsuhiki 
Published: July 2012

. The compound YM‐16638, [[5‐[[3‐(4‐acetyl‐3‐hydroxy‐2‐propylphenoxy)propyl]thio]‐1,3,4‐thiadiazol‐ 2‐yl]thio] acetic acid was developed in a series of in vitro and in vivo studies as a leukotriene D4 receptor antagonist.
. In a clinical trial as a leukotriene antagonist drug, this compound was found to have a potent serum cholesterol lowering effect in normolipidaemic healthy male volunteers.
. In the present study, we investigated the serum cholesterol lower effect of this compound in various species of experimental animals.
. Administration of YM‐16638 did not cause a significant decrease in serum total cholesterol (TC) in mice (up to 200 mg kg−1, body weight per day for 28 days), rats (200 mg kg−1 for 15 days) or rabbits (90 mg kg−1 for 18 days). In hamsters, administration of YM‐16638 orally or by peritoneal injection at 50 mg kg−1 or more daily for 7 days caused a significant decrease in serum TC and the rate of body weight gain. In monkeys, serum TC did not change in YM‐16638‐administered squirrel monkeys (50 mg kg−1 daily for 3 weeks), but a significant decrease in serum TC was observed in cynomolgus monkeys (33% decrease at 30 mg kg−1 for 4 weeks) and rhesus monkeys (27% decrease at 30 mg kg−1 for 3 weeks) without any serious decrease in body weight. These results were consistent with those in a phase I study in human subjects. In contrast, serum alanine aminotransferase (ALT) level decreased in all animals after YM‐16638 treatment.
. From these results, we conclude that YM‐16638 has a potent hypocholesterolaemic effect, but that this effect if species‐specific and is only recognized clearly in human subjects and old‐world monkeys.
Effect of 1‐aminocyclopropanecarboxylic acid on N‐methyl‐d‐aspartate‐stimulated [3H]‐noradrenaline release in rat hippocampal synaptosomes
Authors: Clos M.V. , Sanz A. Garcia , Trullas R. , Badia A. 
Published: July 2012

The effect of 1‐aminocyclopropanecarboxylic acid (ACPC), a partial agonist at the glycine site of the N‐methyl‐d‐aspartate (NMDA) receptor complex that exhibits neuroprotective, anxiolytic and antidepressant‐like actions, was investigated in a functional assay for presynaptic NMDA receptors.
NMDA (100 μm) produced a 36% increase of tritium efflux above basal efflux in rat hippocampal synaptosomes preincubated with [3H]‐noradrenaline ([3H]‐NA), reflecting a release of tritiated noradrenaline. This effect was prevented by 10 μm 7‐chlorokynurenic acid, an antagonist of the glycine site of the NMDA receptor.
Glycine enhanced the effect of NMDA with Emax and EC50 values of 84 ± 11% and 1.82 ± 0.04 μm, respectively. ACPC potentiated the effect of NMDA on tritium overflow with a lower EC50 (43 ± 6 nM) and a lower maximal effect (Emax = 40 ± 9%) than glycine. Furthermore, ACPC (0.1 μm) shifted the EC50 of glycine from 1.82 μm to ≥ 3 mM.
These results show that ACPC can reduce the potentiation by glycine of NMDA‐evoked [3H]‐NA release and hence, may act as an antagonist at the glycine site of presynaptic hippocampal NMDA receptors when the concentration of glycine is high.
Characterization of nitric oxide synthase activity in sheep urinary tract: functional implications
Authors: García‐Pascual A. , Costa G. , Labadia A. , Persson K. , Triguero D. 
Published: July 2012

To define further the role of nitric oxide (NO) in urinary tract function, we have measured the presence of nitric oxide synthase (NOS) activity, and its relationship with functional NO‐mediated responses to electrical field stimulation (EFS) in the urethra, the detrusor and the ureter from sheep. NOS activity was assayed by the conversion of L‐[14C]‐arginine to L‐[14C]‐citrulline. Endogenous production of citrulline was confirmed by thin layer chromatography.
NOS enzymatic activity was detected in the cytosolic fraction from tissue homogenates with the following regional distribution (pmol citrulline mg−1 protein min−1): urethra (33 ± 3.3), detrusor (13.1 ± 1.1) and ureter (1.5 ± 0.2). No activity was detected in the particulate fraction of any region.
NOS activity was dependent on Ca2+‐calmodulin and required exogenously added NADPH and tetrahydrobyopterin (BH4) for maximal activity. Exclusion of calmodulin from the incubation mixture did not modify NOS activity, but it was significantly reduced in the presence of the calmodulin antagonist, calmidazolium, suggesting the presence of enough endogenous calmodulin to sustain the observed NOS activity.
NOS activity was inhibited to a greater extent by NG‐nitro‐l‐arginine (l‐NOARG) and its methyl ester (l‐NAME) than by NG‐monomethyl‐l‐arginine (l‐NMMA), while 7‐nitroindazole (7‐NI) was a weak inhibitor and L‐cannavine had no effect.
Citrulline formation could be inhibited by superoxide dismutase in an oxyhaemoglobin‐sensitive manner, suggesting feedback inhibition of NOS by NO.
EFS induced prominent NO‐mediated relaxations in the urethra while minor or no responses were observed in the detrusor and the ureter, respectively. Urethral relaxations to EFS were inhibited by NOS inhibitors with the rank order of potency: L‐NOARG = L‐NAME > 7‐NI > L‐NMMA.
In conclusion, we have demonstrated the presence of NO‐synthesizing enzymatic activity in the sheep urinary tract which shows similar characteristics to the constitutive NOS isoform found in brain. We suggest that the enzymatic activity measured in the urethral muscle layer may account for the NO‐mediated urethral relaxation during micturition whereas regulation of detrusor and ureteral motor function by NOS containing nerves is less likely.
Antiproliferative and c‐myc mRNA suppressive effects of tranilast on newborn human vascular smooth muscle cells in culture
Authors: Miyazawa Keiji , Hamano Shuichiro , Ujiie Arao 
Published: July 2012

Newborn human vascular smooth muscle cells (VSMCs) proliferated faster and were more sensitive to platelet‐derived growth factor‐BB (PDGF‐BB) than those from adults. In this study, we investigated mechanism of the inhibitory effect of tranilast on PDGF‐BB‐induced proliferation of VSMCs from newborns.
Tranilast (30–300 μm) concentration‐dependently inhibited the VSMC proliferation in randomly growing cultures stimulated with PDGF‐BB.
Tranilast (30–300 μm) concentration‐dependently inhibited the [3H]‐thymidine incorporation into DNA in VSMCs that had been synchronized by 48 h serum depletion and then stimulated by addition of PDGF‐BB. However, tranilast had little influence on unscheduled DNA synthesis in quiescent cells or on RNA and protein synthesis, unlike aphidicolin, actimomycin D, and cycloheximide.
In synchronized VSMC cultures, tranilast still inhibited the PDGF‐BB‐induced DNA synthesis even when added 18 h after stimulation of the quiescent cells. The mode of the antiproliferative action of tranilast was different from that of NiCl2, genistein, or staurosporin. In addition, flow cytometry of synchronized VSMCs treated with tranilast revealed a blockade of PDGF‐inducible cell‐cycle progression at the G1/S checkpoint.
Northern blotting showed that tranilast (30–300 μm) concentration‐dependently suppressed constitutive c‐myc mRNA expression even when added 18 h after PDGF‐BB‐stimulation of quiescent VSMCs. Tranilast still had an inhibitory effect on the induction of c‐myc mRNA when de novo protein synthesis was inhibited by cycloheximide and did not shorten the degradation of c‐myc mRNA at the post‐transcriptional level, demonstrating that tranilast directly inhibited c‐myc mRNA expression at the transcriptional level.
These results suggest that the inhibitory effect of tranilast on PDGF‐BB‐induced proliferation is due to S‐phase blockade and may be, at least in part, involved in the direct suppression of c‐myc gene expression. Tranilast did not cause cell toxicity and may therefore hold promising potential for the prevention of vascular proliferative diseases.
Reversal by naloxone of the spinal antinociceptive actions of a systemically‐administered NSAID
Authors: Herrero Juan F. , Headley P. Max 
Published: July 2012

Possible interactions between non‐steroidal anti‐inflammatory drugs (NSAIDs) and endogenous opioids were examined in electrophysiological experiments in α‐chloralose anaesthetized spinalized rats without or with carrageenan‐induced acute inflammation of one hindpaw. Spinal reflex responses, monitored as single motor unit discharges, were elicited by noxious pinch and electrical stimuli.
The μ‐opioid agonist, fentanyl, was an effective depressant of reflexes under all conditions (ED50 6–14 μg kg−1, i.v.). In rats without peripheral inflammation the NSAID, flunixin, a niflumic acid derivative, had only a small effect that was not dose‐dependent. However, in animals with unilateral inflammation, flunixin reduced spinal reflexes evoked both by noxious pinch stimuli (that activate peripheral nociceptors; ID50 4 mg kg−1, i.v.) and by electrical stimuli (that bypass nociceptor endings; ID50 6.5–11 mg kg−1, i.v.), indicating that it has a central site of action at doses comparable to those used clinically.
The opioid antagonist, naloxone (1 mg kg−1, i.v.), reversed all actions of fentanyl. It did not reverse the small effects that flunixin had in rats without inflammation, showing that the NSAID is not a direct opioid agonist. In rats with carrageenan‐induced inflammation of the hindpaw, however, naloxone fully reversed or prevented the antinociception by flunixin, but not that by the α2‐adrenoceptor agonist, medetomidine.
We conclude that under conditions of peripheral inflammation and the resultant central changes, the NSAID, flunixin, has antinociceptive actions that are mediated by endogenous opioids acting within the spinal cord.
Tyrphostin inhibition of ATP‐stimulated DNA synthesis, cell proliferation and Fos‐protein expression in vascular smooth muscle cells
Authors: Erlinge David , Heilig Markus , Edvinsson Lars 
Published: July 2012

We and others have shown that extracellular ATP (adenosine triphosphate), released from sympathetic nerves and platelets, stimulates growth of vascular smooth muscle cells (SMC). To study the importance of tyrosine kinases for ATP‐mediated proliferation in vascular smooth muscle cells we used tyrphostins, a recently developed group of highly specific inhibitors of tyrosine kinases.
ATP induced a powerful concentration‐dependent increase in DNA synthesis measured by [3H]‐thymidine incorporation in rat aorta SMC (RASMC) and an increase in total cell number after 72 h of incubation as measured by an enzymatic cell proliferation assay. Tyrphostin 25 (10−5 m) had no effect per se on basal DNA synthesis but reduced ATP‐stimulated DNA synthesis and increase in cell number in a dose‐dependent manner. Higher concentrations of ATP could not reverse the inhibitory effect of tyrphostin 25. The potency of several (six) other tyrphostins was also examined and found to be slightly greater than tyrphostin 25 with equal efficacy.
When RASMC were incubated with 10−5 m ATP for 2 h, nearly all of the cells (87±5%) were intensely stained with an antibody to the Fos protein while in the controls only 1±2% of the cells were weakly stained. Tyrphostin 25 greatly reduced the Fos‐protein staining (14±2%).
ATP induced a concentration‐dependent increase in 45Ca2+‐influx and formation of inositol phosphates (IPtotal) in RASMC. These effects were not inhibited by tyrphostin 25.
Tyrphostin 25 did not alter ATP‐induced contraction in ring segments of rat aorta.
In conclusion, tyrphostin 25 inhibited ATP‐induced DNA synthesis, cell proliferation and Fosprotein expression, but not ATP‐induced 45Ca2+‐influx, inositolphosphate‐production or vasoconstriction. This indicates that the mitogenic effect of ATP on vascular smooth muscle cells is dependent on tyrosine kinases in contrast to the contractile effect of ATP in blood vessels.
Regulation of histamine H1 receptor coupling by dexamethasone in human cultured airway smooth muscle
Authors: Hardy Elaine , Farahani Mosavar , Hall Ian P. 
Published: July 2012

The regulation of histamine‐induced [3H]‐inositol phosphate and intracellular calcium responses in human cultured airway smooth muscle cells was studied.
Histamine induced concentration‐dependent [3H]‐inositol phosphate formation (EC50 4 μm). This response was inhibited by a range of selective H1 receptor antagonists but not by the H2‐selective antagonist, tiotidone or the H3 receptor‐selective antagonist, thioperamide, indicating that an H1 receptor is involved in this response in human cultured airway smooth muscle cells.
Preincubation of human cultured airway smooth muscle cells with concentrations of dexamethasone > 10 nM for 22 h produced concentration‐dependent inhibition of histamine‐induced inositol phosphate formation. The maximum inhibition observed was 45% of the response in control cells. The inhibitory effect of dexamethasone was itself reversed by prior exposure to the glucocorticoid receptor antagonist, RU38486 (10 μm). Preincubation for 22 h with 1 μm dexamethasone produced inhibition of the inositol phosphate response to histamine at all concentrations of histamine inducing significant inositol phosphate formation in these cells. In contrast, the response to the G protein activator, NaF (0.1–20 mM) was unaltered by preincubation with dexamethasone.
Preincubation of human airway smooth muscle cells with 1 μm dexamethasone for time periods of < 6 h failed to inhibit histamine‐induced inositol phosphate formation in human airway smooth muscle cells.
Histamine also induced concentration‐dependent elevation of intracellular calcium levels in Fura 2‐loaded human airway smooth muscle cells. This response was inhibited by preincubation with 1 μm dexamethasone.
We conclude that signal transduction through the H1 receptor in human airway smooth muscle is subject to regulation by dexamethasone and that this may in part account for the protective effect of dexamethasone against spasmogen‐induced contractile responses in the airways.
Electrophysiological mechanisms for antiarrhythmic efficacy and positive inotropy of liriodenine, a natural aporphine alkaloid from Fissistigma glaucescens
Authors: Chang Gwo‐Jyh , Wu Mei‐Hwan , Wu Yang‐Chang , Su Ming‐Jai 
Published: July 2012

. The antiarrhythmic potential and electromechanical effects of liriodenine, an aporphine alkaloid isolated from the plant, Fissistigma glaucescens, were examined.
. In the Langendorff perfused (with constant pressure) rat heart, at a concentration of 0.3 to 3 μm, liriodenine was able to convert a polymorphic ventricular tachyrhythmia induced by the ischaemia‐reperfusion (EC50 = 0.3 μm).
. In isolated atrial and ventricular muscle, liriodenine increased the contractile force and slowed the spontaneous beating of the right atrium.
. The liriodenine‐induced positive inotropy was markedly attenuated by a transient outward K+ channel blocker, 4‐aminopyridine (4‐AP) but was not significantly affected by prazosin, propranolol, verapamil or carbachol.
. In rat isolated ventricular myocytes, liriodenine prolonged action potential duration and decreased the maximal upstroke velocity of phase 0 depolarization (V̇max) and resting membrane potential in a concentration‐dependent manner. The action potential amplitude was not significantly changed.
. Whole‐cell voltage clamp study revealed that liriodenine blocked the Na+ channel (INa) concentration‐dependently (IC50 = 0.7 μm) and caused a leftward shift of its steady‐state inactivation curve. However, its recovery rate from the inactivated state was not affected. The L‐type Ca2+ currents (ICa) were also decreased, but to a lesser degree (IC50 = 2.5 μm, maximal inhibition = 35%).
. Liriodenine inhibited the 4‐AP‐sensitive transient outward current (Ito) (IC50 = 2.8 μm) and moderately accelerated its rate of decay. The block of Ito was not associated with changes in the voltage‐dependence of the steady‐state inactivation curve or in the process of recovery from inactivation of the current. Liriodenine also reduced the amplitude of a slowly inactivating, steady‐state outward current (Iss) (IC50 = 1.9 μm). These effects were consistent with its prolonging effect on action potential duration. The inwardly rectifying background K+ current (IK***1), was also decreased but to a less degree.
. Compared to quinidine, liriodenine exerted a stronger degree of block on INa, comparable degree of block on IK1, and lesser extent of block on ICa and Ito.
. It is concluded that, through inhibition of Na+ and the Ito channel, liriodenine can suppress ventricular arrhythmias induced by myocardial ischaemia reperfusion. The positive inotropic effect can be explained by inhibition of the Ito channel and the subsequent prolongation of action potential duration. These results provide a satisfactory therapeutic potential for the treatment of cardiac arrhythmias.