bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effects of inhibitors of nitric oxide synthase in immune complex‐induced vasculitis
Authors: Mulligan M.S. , Moncada S. , Ward P.A.
Published: July 2012

The ability of analogues of l‐arginine (N‐iminoethyl‐l‐ornithine (l‐NIO), NG‐monomethyl‐l‐arginine (l‐NMMA), NG‐nitro‐l‐arginine methyl ester (l‐NAME) and NG‐nitro‐l‐arginine (l‐NNA)) to protect against inflammatory injury induced by activated neutrophils was investigated in rats following intradermal or intrapulmonary deposition of immune complexes.

The descending order of potency for protective effects of these analogues was: l‐NIO > l‐NMMA > l‐NNA = l‐NAME. The approximate IC50 value for l‐NIO in the dermal vasculitis model was 65 μm. For all other compounds, the IC50 values were > 5 mm.

The protective effect of l‐NIO in the skin was reversed in a dose‐dependent manner by the presence of l‐arginine, but not by d‐arginine. l‐Arginine also reversed the protective effects of l‐NIO in immune complex‐induced lung injury.

The protective effects of l‐NIO were not associated with reductions in neutrophil accumulation, as measured by extraction from tissues of myeloperoxidase.

These data demonstrate that l‐NIO has the most potent protective effects against immune complex‐induced vascular injury induced by activated macrophages. Furthermore, they indicate that this injury is dependent upon the generation of nitric oxide.
Palmitoyl‐dl‐carnitine has calcium‐dependent effects on cultured neurones from rat dorsal root ganglia
Authors: Stapleton Simon R. , Currie Kevin P.M. , Scott Roderick H. , Bell B. Anthony
Published: July 2012

The effects of palmitoyl‐dl‐carnitine (0.01 to 1 mm) on whole cell voltage‐activated calcium channel currents carried by calcium or barium and Ca2+‐activated chloride currents were studied in cultured neurones from rat dorsal root ganglia.

Palmitoyl‐dl‐carnitine applied to the extracellular environment or intracellularly via the patch solution reduced Ca2+ currents activated over a wide voltage range from a holding potential of −90 mV. Inhibition of high voltage activated Ca2+ channel currents was dependent on intracellular Ca2+ buffering and was reduced by increasing the EGTA concentration from 2 to 10 mm in the patch solution. Barium currents were significantly less sensitive to palmitoyl‐dl‐carnitine than Ca2+ currents.

The amplitude of Ca2+‐activated Cl− tail currents was reduced by palmitoyl‐dl‐carnitine. However, the duration of these Cl− currents was greatly prolonged by palmitoyl‐dl‐carnitine, suggesting slower removal of free Ca2+ from the cytoplasm following Ca2+ entry through voltage‐activated channels.

Palmitoyl‐dl‐carnitine evoked Ca2+‐dependent inward currents which could be promoted by activation of the residual voltage‐activated Ca2+ currents and attenuated by intracellular application of EGTA.

We conclude that palmitoyl‐dl‐carnitine reduced the efficiency of intracellular Ca2+ handling in cultured dorsal root ganglion neurones and resulted in enhancement of Ca2+‐dependent events including inactivation of voltage‐activated Ca2+ currents. The activation of inward currents by palmitoyl‐dl‐carnitine may involve Ca2+‐induced Ca2+ release from intracellular stores, or direct interaction of palmitoyl‐dl‐carnitine with Ca2+ stores.
Inhibition of neuropeptide Y‐induced potentiation of noradrenaline‐induced vasoconstriction by PP56 (d‐myo‐inositol 1,2,6‐tris‐phosphate)
Authors: Adamsson M. , Fallgren B. , Edvinsson L.
Published: July 2012

Although neuropeptide Y (NPY) is a potent vasoconstrictor in many vascular beds, nanomolar concentrations of this peptide potentiate the noradrenaline‐induced contractions in rabbit gastroepiploic and femoral arteries, and guinea‐pig mesenteric and uterine arteries.

The potentiating effect of NPY on noradrenaline‐induced contraction was present in endothelium‐denuded femoral arteries.

The potentiating effect of NPY on noradrenaline‐induced contraction was antagonized by PP56 (d‐myo‐inositol 1,2,6‐trisphosphate) in low concentrations (down to 0.1 nm). This antagonistic effect was observed in all four types of vessels studied. Contractions induced by noradrenaline, histamine, endothelin‐1 and potassium were not altered by PP56 in concentrations upto 1 μm in femoral artery of rabbit.

We provide evidence that a non‐peptide (PP56) can selectively antagonize NPY‐induced effects in rabbit and guinea‐pig peripheral arteries without affecting the vasoconstrictor response to noradrenaline.
Mechanisms contributing to the regional haemodynamic effects of neurotensin in conscious, unrestrained Long Evans rats
Authors: Bachelard Hélène , Gardiner Sheila M. , Kemp Philip A. , Bennett Terence
Published: July 2012

The regional haemodynamic effects of i.v. bolus doses of neurotensin (10–1000 ng) were assessed in conscious, unrestrained Long Evans rats chronically instrumented with miniaturized, pulsed Doppler probes.

Neurotensin caused increases in blood pressure, together with dose‐related tachycardias and constrictions in the renal, superior mesenteric and hindquarters vascular beds. The tachycardia elicited by the 1000 ng dose of neurotensin was preceded by a transient bradycardia.

In the presence of phentolamine, the pressor effect of neurotensin (1000 ng) was converted into a hypotensive effect, accompanied by reduced tachycardic and constrictor responses in the renal, superior mesenteric and hindquarters vascular beds. The tachycardia was not preceded by a bradycardia.

In the presence of phentolamine and propranolol, the pressor and bradycardic responses to neurotensin were unaffected, whereas the tachycardia was abolished. The renal vasconstrictor effect was smaller, while the constrictions in the superior mesenteric and hindquarters vasculars beds were not different from those in untreated rats.

In rats neonatally treated with capsaicin (50 mg kg−1, s.c.), the pressor effects elicited by neurotensin (300 and 1000 ng) were reduced as were the constrictor responses in the renal (at the dose of 300 ng), superior mesenteric (at the dose of 300 ng) and hindquarters (at both doses) vascular beds. The bradycardia elicited by neurotensin (1000 ng) was absent, whereas the tachycardia was potentiated.

The results indicate that in conscious, intact rats neurotensin appears to exert cardiovascular influences through activation of sympathoadrenal mechanisms and also through non‐adrenergic effects on the heart, renal, superior mesenteric and hindquarters vascular beds. The latter effects appear to involve capsaicin‐sensitive nerves.
Mechanisms of coronary vasodilatation produced by ATP in guinea‐pig isolated perfused heart
Authors: Brown Ina P. , Thompson Carl I. , Belloni Francis L.
Published: July 2012

Isolated hearts of guinea‐pigs were perfused in vitro with a physiological salt solution via a retrograde aortic cannulation (Langendorff preparation) at constant perfusion pressure. Bolus intra‐arterial injections of various vasodilator drugs were made and the coronary flow responses were measured with an electromagnetic flow probe placed in the arterial inflow circuit. Inhibitory drugs were infused intra‐arterially.

Nitro‐l‐arginine (NLA; 500 μm), an NO synthesis inhibitor, decreased coronary baseline flow by 16 ± 0.8%, converted acetylcholine‐induced coronary vasodilatation to vasoconstriction and had no effect on coronary flow responses to adenosine or papaverine. Sodium nitroprusside‐induced responses were enhanced during NLA infusion by 46 ± 11%.

Adenosine 5′‐triphosphate (ATP) increased coronary flow but coronary flow responses to ATP were not altered by infusion of NLA.

ATP‐induced coronary dilatation was not significantly attenuated by infusion of the adenosine receptor antagonist XAC, (xanthine amine congener; 2 μm), whereas XAC decreased coronary flow responses to adenosine by 75% ± 5%.

ATP‐induced coronary flow responses were reduced by only 31 ± 4% during indomethacin infusion (2.8 μm) whereas indomethacin completely eliminated the initial vasoconstriction phase and greatly attenuated the peak flow and duration of the later vasodilatation phase seen in response to arachidonic acid (0.75 nmol). Indomethacin had no effect on vasodilatations produced by adenosine or prostaglandin I2.

These results indicate that ATP‐induced coronary dilatation in the isolated, perfused heart of the guinea‐pig is not dependent upon NO production or upon degradation of ATP to adenosine. The coronary dilator action of ATP may be partially dependent (∼30%) upon the production of vasodilator prostaglandins.
In vitro characterization of prostanoid FP‐, DP‐, IP‐ and TP‐receptors on the non‐pregnant human myometrium
Authors: Senior J. , Sangha R. , Baxter G.S. , Marshall K. , Clayton J.K.
Published: July 2012

Prostaglandin F (PGF), PGD, PGI and thromboxane A2 (TXA2) receptors have been pharmacologically characterized on the non‐pregnant human myometrium in vitro in accordance with the receptor classification proposed by Coleman et al. (1984). The tools for the classification include both natural prostanoids, synthetic, selective analogues and antagonists where available.

The potent excitatory actions of the natural FP‐receptor prostanoid, PGF2α, and the synthetic analogue, fluprostenol, indicate the presence of FP‐receptors mediating contraction on the human myometrium.

PGD2 produced a biphasic response consisting of excitation followed by relaxation of spontaneous activity of the myometrium. The selective DP‐receptor agonists, BW245C, produced purely inhibitory responses illustrating the presence of inhibitory DP‐receptors in this tissue. The inhibitory responses of both PGD2 and BW245C were antagonized by the competitive DP‐receptor antagonist, BWA 868C, providing conclusive evidence for the existence of DP‐receptors.

PGI2 produced a biphasic response similar to PGD2. Iloprost, the EP1/IP‐receptor agonist also produced a biphasic response, whilst the IP‐receptor selective agonist, cicaprost, caused inhibition only, suggesting that inhibitory IP‐receptors exist in the non‐pregnant human myometrium.

The TXA2‐mimetic, U46619, produced marked stimulation of the non‐pregnant human myometrium and was approximately equipotent to PGF2α and fluprostenol in this effect. The actions of U46619 were competitively antagonized by the TP‐receptor antagonist GR32191 showing that excitatory TP‐receptors exist in this tissue.

6 All prostanoids tested, both natural and synthetic, had activity on the non‐pregnant human myometrium in vitro, supporting the existence of a heterogeneous population of prostanoid receptors in this tissue. If the results from the present study are combined with those previously reported for EP‐receptor agonists (Senior et al., 1991), it may be concluded that excitation may occur through FP‐, TP‐, EP3‐ and few EP1‐receptors, whereas inhibition may occur through DP‐, IP‐ and EP2‐receptors.
Evidence that 5‐HT2receptor activation decreases noradrenaline release in rat hippocampusin vivo
Authors: Done C.J.G. , Sharp T.
Published: July 2012

Recent electrophysiological studies have shown that 5‐HT2/5‐HT1Creceptor agonists inhibit the electrical activity of noradrenergic neurones in the rat locus coeruleus. Here we examine the effect of various agonists and antagonists of 5‐HT2/5‐HT1Creceptors on noradrenaline release in hippocampus of anaesthetized rats using microdialysis.

Subcutaneous administration of the 5‐HT2/5‐HT1Creceptor agonist, 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI: 0.2 and 0.5 mg kg−1), caused a marked decrease (50% of pre‐drug levels 60 min after injection) of noradrenaline in hippocampal dialysates which was long‐lasting (> 120 min). Noradrenaline output also decreased in response to administration of the structural analogue of DOI, 1‐(2,5‐dimethoxy‐4‐bromophenyl)‐2‐aminopropane (DOB: 1 mg kg−1, s.c.).

The effect of DOI on noradrenaline output was prevented by pretreatment with the 5‐HT2/5‐HT1Creceptor antagonist, ritanserin (0.4 mg kg−1, s.c.). Spiperone (0.2 and 1 mg kg−1, s.c.), a 5‐HT2/dopamine D2receptor antagonist which has low affinity for 5‐HT1Creceptors, also antagonized the effect of DOI (0.5 mg kg−1, s.c.). Sulpiride (50 mg kg−1, s.c.), a dopamine D2receptor antagonist did not alter the response to DOI (0.5 mg kg−1, s.c.).

Both the non‐selective 5‐HT receptor agonist, quipazine (1 mg kg−1, s.c.), and the 5‐HT‐releasing agent,p‐chloroamphetamine (2 mg kg−1, s.c.), decreased noradrenaline release in hippocampus and these effects were antagonized by pretreatment with ritanserin (0.4 mg kg−1, s.c.).

Our data suggest thatin vivo, noradrenaline release in hippocampus is inhibited by 5‐HT2receptor activation. This effect is probably associated with a decrease in noradrenergic neuronal activity.
Adenosine A1‐receptor stimulated increases in intracellular calcium in the smooth muscle cell line, DDT1MF‐2
Authors: Dickenson John M. , Hill Stephen J.
Published: July 2012

The effect of a range of adenosine receptor agonists on intracellular free calcium concentration ([Ca2+]i) has been studied in the hamster vas deferens smooth muscle cell line DDT1MF‐2.

Adenosine receptor agonists elicited a rapid and maintained increase in [Ca2+]i in fura‐2 loaded DDT1MF‐2 cells. The initial rise could be maintained in the absence of extracellular calcium, whereas the maintained or plateau phase was dependent upon the presence of extracellular calcium and appeared to be associated with calcium influx. The rank order of agonist potencies was N6‐cyclopentyladenosine > 5′‐N‐ethylcarboxamidoadenosine > 2‐chloroadenosine > adenosine.

The response to 2‐chloroadenosine was antagonized by the antagonists 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, KD 0.14 nm) and 8‐phenyltheophylline (KD 112 nm).

Pretreatment with the 5‐lipoxygenase inhibitor AA861 (20 μm) produced only a small (14 ± 2%) inhibition of the [Ca2+]i response elicted by N6‐cyclopentyladenosine (300 nm), in nominally Ca2+‐free buffer containing 0.1 mm EGTA. The cyclo‐oxygenase inhibitor, indomethacin (2 μm) was without effect.

The Ca2+‐influx associated with the plateau phase required the continued presence of agonist on the receptor. The antagonist DPCPX (100 nm) attenuated the rise in [Ca2+]i observed when extracellular Ca2+ was re‐applied after the cells had been stimulated with N6‐cyclopentyladenosine (CPA;300 nm) in experiments initiated in nominally Ca2+‐free buffer.

Pretreatment with pertussis toxin (200 ng ml−1 for 4 h) inhibited the CPA (100 nm) stimulated intracellular Ca2+ release and Ca2+ influx but was without effect on the response to histamine (100 μm).

These data suggest that adenosine A1‐receptor activation in DDT1MF‐2 cells stimulates release of Ca2+ from intracellular stores and influx of extracellular Ca2+ through Ca2+ entry pathways in the plasma membrane which required the continued presence of agonist on the receptor.
Selective inhibition of agonist‐induced but not shear stress‐dependent release of endothelial autacoids by thapsigargin
Authors: Macarthur Heather , Hecker Markus , Busse Rudi , Vane John R.
Published: July 2012

The effects of the Ca2+‐ATPase inhibitor, thapsigargin, on the shear stress‐dependent and on the agonist‐stimulated release of endothelium‐derived relaxing factor, i.e. nitric oxide (NO), and prostacyclin (PGI2) were studied in bovine and human cultured endothelial cells as well as in endothelium‐intact arterial segments of the rabbit.

Preincubation with thapsigargin (1 μm for 10 min) had no effect on the shear stress‐dependent release of NO from bovine aortic endothelial cells grown on beads, but abolished the release of NO induced by ADP, bradykinin, ionomycin or poly‐l‐lysine. Similarly, thapsigargin completely abrogated the agonist‐stimulated PGI2 release from these cells, but had no effect on the shear stress‐dependent release of PGI2.

The acetylcholine‐induced release of NO from the luminally perfused thoracic aorta and femoral artery of the rabbit was suppressed by pretreatment with thapsigargin (1 μm). In contrast, thapsigargin did not affect the shear stress‐dependent release of NO from the femoral artery.

Administration of thapsigargin to these vascular preparations or to cultured endothelial cells alone produced a substantial release of both NO and PGI2. This release declined towards previous values after washout of thapsigargin.

In human and bovine cultured endothelial cells, thapsigargin (1–1000 nm) caused a dose‐dependent sustained rise in [Ca2+]i, an effect that was abolished in the absence of extracellular Ca2+. Stimulation of these cells with bradykinin, histamine, ADP or ionomycin after previous exposure to thapsigargin (30–1000 nm) no longer caused an increase in [Ca2+]i.

These findings demonstrate that by emptying intracellular Ca2+ stores, thapsigargin selectively blocks the agonist‐stimulated release of both NO and PGI2, suggesting a principal difference in the control by [Ca2+]i of the release of these endothelial autacoids caused by shear stress or receptor‐dependent and independent agonists.
α2‐Autoreceptor subclassification in rat isolated kidney by use of short trains of electrical stimulation
Authors: Bohmann C. , Schollmeyer P. , Rump L.C.
Published: July 2012

Rat kidneys were perfused with Krebs‐Henseleit solution and incubated with [3H]‐noradrenaline. The renal nerves were electrically stimulated at either 1 Hz for 30 s or 100 Hz for 0.06 s. The stimulation induced (S‐I) outflow of radioactivity was taken as an index of endogenous noradrenaline release.

At a frequency of 1 Hz for 30 s the α‐adrenoceptor antagonists BRL 44408 (0.01, 0.1 μm) and imiloxan (0.1, 1.0 μm) enhanced S‐I outflow of radioactivity. However, at a frequency of 100 Hz for 0.06 s the α‐adrenoceptor antagonists, idazoxan (0.1, 1.0 μm), imiloxan (0.1, 1.0 μm), BRL 44408 (0.1, 1.0 μm), BRL 41992 (0.1, 1.0 μm) and prazosin (0.01 μm) failed to enhance S‐I outflow of radioactivity.

Thus, the rat isolated kidney stimulated at 100 Hz for 0.06 s, avoids autoinhibition by endogenous noradrenaline and α‐adrenoceptor antagonist affinities (pKB) at the prejunctional α‐autoreceptor were estimated without disturbance by the endogenous activator.

The α2‐adrenoceptor agonist, clonidine, inhibited the S‐I outflow of radioactivity with a maximum of 90% and an EC50 of 7.2 nm.

All α‐adrenoceptor antagonists used caused parallel shifts of the concentration‐response curve for clonidine to the right. The rank order of potencies was: rauwolscine (α2A/B) > idazoxan (α2A/B) > phentolamine (α2A/B) > WB 4101 (α2A) > BRL 44408 (α2A) > BRL 41992 (α2B) > prazosin (α2B) = imiloxan (α2B).

These data, when compared with binding, molecular and functional data of various other tissues and cell lines, indicate that prejunctional α2‐autoreceptors in rat kidney do not belong to the α2B‐ or α2C‐subtype. The α2‐autoreceptor of rat kidney seems to be of the α2A‐subtype. However, α2‐autoreceptor affinities of the present study correlate also well with binding affinities of the recently described α2D‐ligand binding site in bovine pineal gland.
Ion channel blockade by oximes and recovery of diaphragm muscle from soman poisoning in vitro
Authors: Tattersall John E. H.
Published: July 2012

The actions of oximes and related compounds on the nicotinic acetylcholine receptor ion channel at the adult mouse muscle endplate were investigated by use of single‐channel recording techniques. The aim of the study was to determine whether the channel‐blocking properties of the compounds could contribute to their therapeutic effectiveness against soman poisoning in vitro.

Therapeutic effectiveness was assessed in guinea‐pig phrenic nerve‐hemidiaphragm preparations by measuring the degree of recovery of neuromuscular function produced by the compounds following poisoning by soman. A number of the compounds, including some which lacked the oxime group, produced a significant recovery of neuromuscular function which was unrelated to acetylcholinesterase (AChE) reactivation; this was reversed by washing off the compound, and was therefore attributed to a direct pharmacological action on the muscle.

Single channel recordings showed that some of the compounds blocked open nicotinic receptor ion channels in preparations of mouse muscle fibres. The compounds which showed the greatest direct pharmacological actions in diaphragms produce a very fast, flickering blockade of the channels. Several quantitative measures of channel‐blocking activity correlated very well with the direct pharmacological action. Furthermore, for two compounds studied in greater detail, the direct action and channel‐blocking showed similar concentration‐response relationships.

The results of this study indicate that the direct pharmacological action of oximes and their analogues against neuromuscular blockade by soman in vitro is due to their channel‐blocking activity. The direct action does not correlate well with protection against soman poisoning in vivo, however, which suggests that additional non‐reactivating properties of these compounds, at sites other than the neuromuscular junction, may also be important for their therapeutic effectiveness.
Enhancement of airway reactivity to histamine by isoprenaline and related β‐adrenoceptor agonists in the guinea‐pig
Authors: Galland Barbara C. , Blackman J.G.
Published: July 2012

The effect of isoprenaline, adrenaline and salbutamol on airway reactivity to histamine was observed in anaesthetized, ventilated guinea‐pigs. Airway reactivity was determined before and 20 min and 90 min after a 30‐min i.v. infusion of each agonist by constructing cumulative dose‐response curves from breath‐by‐breath measurements of the effect of different rates of i.v. infusion of histamine on lung resistance (RL) and dynamic compliance (Cdyn).

(±)‐Isoprenaline infused i.v. for 30 min at a rate of 0.4 μmol h−1kg−1caused bronchodilatation and a fall in blood pressure. Recovery to starting values of RLand Cdynoccurred within 20 min of stopping the infusion.

Reactivity to histamine was greatly enhanced when measured 20 min and 90 min after stopping the infusion of (±)‐isoprenaline. This was not an effect of the prior infusion of histamine or of the dissolving solution.

Infusion of (−)‐isoprenaline for 30 min at a rate of 0.2 μmol h−1kg−1also enhanced reactivity to histamine. However, enhancement of reactivity to histamine was not demonstrable after infusion of (+)‐isoprenaline at equal or higher dose rates.

Infusions of bronchodilator concentrations of adrenaline and salbutamol also enhanced airway reactivity to histamine, but the bronchodilator effect of salbutamol lasted longer than that of isoprenaline or adrenaline and the development of hyperreactivity was delayed.

After acute bilateral vagotomy, infusion of (±)‐isoprenaline enhanced airway reactivity but only at the highest dose of histamine.

(±)‐Isoprenaline did not enhance contractile responses to histamine in isolated preparations of first branch bronchi.

We conclude that the bronchodilator effect of activating β‐adrenoceptors in the airways of guinea‐pigs is followed by a more persistent state of hyperreactivity to histamine.
Evidence for a nucleotide receptor on adrenal medullary endothelial cells linked to phospholipase C and phospholipase D
Authors: Purkiss John R. , Wilkinson Graeme F. , Boarder Michael R.
Published: July 2012

We have investigated whether the ‘atypical’ P2‐purinoceptor previously described on adrenal microvasculature endothelial cells is a nucleotide receptor (responds to pyrimidines and purines) and is linked to phospholipase D as well as phospholipase C.

Cultured bovine adrenal medullary endothelial (BAME) cells responded to the pyrimidine UTP, as well as the purines. The total [3H]‐inositol phosphate responses were with a rank order of UTP > ATP‐ = adenosine 5′‐O‐(3‐thio‐triphosphate) (ATPγS) >> 2MeSATP. The selective P2x agonist β, γ‐methylene ATP was inactive.

Construction of dose‐response curves to ATP, ATPγS and UTP in the presence and absence of additional agonists showed that responses to ATPγS and UTP were not additive, nor were those to UTP and ATP. This suggests that purines and pyrimidines acted via a common nucleotide receptor.

32P‐labelled BAME cells, in the presence of butanol, produced [32P]‐phosphatidylbutanol (PBut) when stimulated with ATPγS or the protein kinase C activator, tetradecanoyl phorbol acetate (TPA).

Cells labelled with [3H]‐palmitate and stimulated in the presence of butanol generated [3H]‐PBut with the same order of agonist potencies seen for inositol phosphate responses.

The protein kinase C inhibitor, Ro 31–8220, abolished TPA and agonist stimulation of [3H]‐PBut production.

These observations, and our related studies on bovine aortic endothelial cells, provide the first demonstration of a phospholipase C linked nucleotide receptor on vascular endothelial cells. It is concluded that BAME cells express a nucleotide receptor linked to phospholipase C and phospholipase D, but that activation of phospholipase D is probably down‐stream of phospholipase C.
Ap4A and ADP‐β‐S binding to P2 purinoceptors present on rat brain synaptic terminals
Authors: Pintor Jesús , Díaz‐Rey M. Angel , Miras‐Portugal M. Teresa
Published: July 2012

Diadenosine tetraphosphate (Ap4A) a dinucleotide stored and released from rat brain synaptic terminals presents two types of affinity binding sites in synaptosomes. When [3H]‐Ap4A was used for binding studies a Kd> value of 0.10 ± 0.014 nm and a Bmax value of 16.6 ± 1.2 fmol mg−1 protein were obtained for the high affinity binding site from the Scatchard analysis. The second binding site, obtained by displacement studies, showed a Ki value of 0.57 ± 0.09 μm.

Displacement of [3H]‐Ap4A by non‐labelled Ap4A and P2‐purinoceptor ligands showed a displacement order of Ap4A > adenosine 5′‐O‐(2‐thiodiphosphate) (ADP‐β‐S) > 5′‐adenylyl‐imidodiphosphate (AMP‐PNP) > α,β‐methylene adenosine 5′‐triphosphate (α,β‐MeATP) in both sites revealed by the Ki values of 0.017 nm, 0.030 nm, 0.058 nm and 0.147 nm respectively for the high affinity binding site and values of 0.57 μm, 0.87 μm, 2.20 μm and 4.28 μm respectively for the second binding site.

Studies of the P2‐purinoceptors present in synaptosomes were also performed with [35S]‐ADP‐β‐S. This radioligand showed two binding sites the first with Kd and Bmax values of 0.11 ± 0.022 nm and 3.9 ± 2.1 fmol mg−1 of protein respectively for the high affinity binding site obtained from the Scatchard plot. The second binding site showed a Ki of 0.018 ± 0.0035 μm obtained from displacement curves.

Competition studies with diadenosine polyphosphates of [35S]‐ADP‐β‐S binding showed a displacement order of Ap4A > Ap5A > Ap6A in the high affinity binding site and Ki values of 0.023 nm, 0.081 nm and 5.72 nm respectively. The second binding site potency order was Ap5A > Ap4A > Ap6A, with Ki values of 0.28 μm, 0.53 μm and 5.32 μm respectively.

Displacement studies of [35S]‐ADP‐β‐S with P2‐purinoceptor agonists showed the following potency pattern: ADP‐β‐S > AMP‐PNP > α,β‐MeATP with Ki values of 0.021 nm, 0.029 nm 0.215 nm respectively in the high affinity binding site. 2‐Methylthio‐adenosine 5′‐triphosphate (2MeSATP) was unable to displace [35S]‐ADP‐β‐S in this binding site. The second binding site showed a profile of ADP‐β‐S > α,β‐MeATP> AMP‐PNP > 2MeSATP and Ki values of 0.018 μm, 0.212 μm, 0.481 μm and 18.04 μm respectively.

These studies suggest the presence of a new P2‐purinoceptor in rat brain synaptosomes with high affinity for diadenosine polyphosphates which we tentatively designate as P2d.
Inhibition by recombinant SLPI and half‐SLPI (Asn55‐Ala107) of elastase and cathepsin G activities: consequence for neutrophil‐platelet cooperation
Authors: Renesto Patricia , Balloy Viviane , Kamimura Takashi , Masuda Ken‐ichi , Imaizumi Atsushi , Chignard Michel
Published: July 2012

The capacity of recombinant human secretory leukocyte proteinase inhibitor (SLPI) to inhibit human leukocyte elastase (HLE) and cathespin G (Cat G) was investigated and compared with a recombinant truncated form (carboxyl‐terminal domain, Asn55‐Ala107) called 1/2 SLPI.

Both compounds were efficient when tested against enzymatic activities of purified HLE and Cat G indicating that the HLE‐ and Cat G‐inhibitory sites were preserved in the truncated form. SLPI and 1/2 SLPI also affected platelet activation induced by 0.2 μm Cat G (IC50 = 112 ± 13 nm for SLPI and 280 ± 12 nm for 1/2 SLPI).

The effects of SLPI and 1/2 SLPI were then tested against polymorphonuclear neutrophil (PMN)‐mediated platelet activation, a cell‐to‐cell interaction mediated by HLE and Cat G released from PMN. In this experimental system, addition of SLPI or 1/2 SLPI before N‐formyl‐Met‐Leu‐Phe (fMLP) led to the inhibition of the resulting platelet activation. As was the case for Cat G enzymatic activity and Cat G‐induced platelet activation, SLPI was more efficient than 1/2 SLPI (IC50 = 676 ± 69 nm vs 1121 ± 150 nm).

The ratio of the IC50 against PMN‐mediated platelet activation compared to purified Cat G‐mediated platelet activation was 6.03 for SLPI and 4.32 for 1/2 SLPI. This difference may be due to the smaller size of the truncated form which could allow this molecule to diffuse more easily between PMN and platelets.

In conclusion, 1/2 SLPI could be a promising candidate in the treatment of pathological states linked to inflammation in which participation of HLE and Cat G has been evoked.
Inhibition of spinal opioid antinociception by intrathecal β‐endorphin1–27 in the rat
Authors: Hong M. , Sutak M. , Jhamandas K.
Published: July 2012

The effects of intrathecal (i.t.) administration of β‐endorphin and two shorter fragments, human and ovine β‐endorphin1–27, were examined for antinociceptive activity in the tail‐flick and paw‐pressure tests in the rat. Additionally, the ability of ovine β‐endorphin1–27 to influence the action of i.t. β‐endorphin, morphine and [d‐Pen2‐d‐Pen5]enkephalin (DPDPE) was also examined in these tests.

After i.t. injection, β‐endorphin produced potent dose‐related antinociception in the tail‐flick and paw‐pressure tests. Shorter endorphins produced much weaker effects. The order of antinociceptive efficacy was β‐endorphin > human β‐endorphin1_27 > ovine β‐endorphin1–27.

Administration of ovine β‐endorphin1–27 (0.72, 1.44 nmol, i.t.) significantly attenuated the antinociceptive effect of β‐endorphin (2.88 nmol, i.t.) in the tail‐flick and paw‐pressure tests.

Both i.t. morphine and DPDPE produced dose‐related antinociception in the tail‐flick and paw‐pressure tests. The potency of DPDPE was lower than that of morphine in both tests; however, the effect of DPDPE was weaker in the paw‐pressure test.

Administration of ovine β‐endorphin1–27 (1.44 nmol, i.t.) significantly attenuated the antinociceptive effect of morphine (14.9 nmol, i.t.) in both tests and the effect of DPDPE (38.7 nmol) in the tail‐flick test.

The results show that β‐endorphin1–27 acts as an opioid antagonist at the spinal level in the rat. Its ability to inhibit the action of morphine and DPDPE suggests that it may attenuate β‐endorphin action by an interaction with μ‐ and/or δ‐opioid receptors.
Pharmacological characterization of the contractile responses to angiotensin analogues in guinea‐pig isolated longitudinal muscle of small intestine
Authors: Hawcock A.B. , Barnes J.C.
Published: July 2012

The contractile responses to angiotensin II, angiotensin III and two synthetic analogues, [Lys2] angiotensin II and [Sar1]angiotensin II, in the guinea‐pig isolated longitudinal muscle preparation of small intestine have been characterized in vitro.

Tachyphylaxis to the angiotensin analogues was reduced by use of a Krebs‐Henseleit solution containing a raised (sub‐contractile) concentration of potassium (11.2 mm). Under these conditions, reproducible cumulative concentration‐response curves to all agonists were established. The pD2 estimates for angiotensin II, [Lys2]angiotensin II, angiotensin III and [Sar1]angiotensin II were 9.15 ± 0.14, 7.42 ± 0.06, 7.69 ± 0.18 and 9.50 ± 0.15 respectively and the maximum responses achieved were not significantly different.

The contractile responses to angiotensin II, angiotensin III and [Sar1]angiotensin II were reduced by greater than 80% by tetrodotoxin (TTX; 0.1 μm). However, the responses to [Lys2]angiotensin II were reduced by only 63 ± 5%. Atropine (0.1 μm) also reduced the responses to angiotensin II, angiotensin III and [Lys2]angiotensin II, although its effect was less than that produced by TTX. Furthermore, while responses to these agonists were not significantly modified by the NK1 receptor antagonist (±)‐CP‐96,345 (30 nm) alone, the combined pre‐incubation with both atropine and (±)‐CP‐96,345 reduced maximum agonist responses to a level not significantly different from those produced by TTX.

Indirect and direct contractile responses to angiotensin II and [Lys2]angiotensin II (in the presence of TTX) respectively were characterized by use of the selective AT1 receptor antagonist, losartan and the AT2 receptor antagonist, PD123177. Losartan produced parallel rightward displacement of the concentration‐response curve to angiotensin II and [Lys2]angiotensin II, with an estimated pKB of 8.56 (8.42–8.68) and 9.18 (8.63–9.50) respectively. The AT2 receptor antagonist, PD123177 (3 μm) failed to modify the contractile responses to either angiotensin II or [Lys2]angiotensin II.

We conclude that two populations of angiotensin II receptors exist in the guinea‐pig longitudinal muscle of small intestine, one located neuronally mediating the release of both acetylcholine and substance P and the other located on the smooth muscle mediating direct contractile responses. The neuronal component provides the major contribution to the agonist responses. Both receptor populations are of the AT1 receptor subtype.
The glycine/NMDA receptor antagonist, R‐(+)‐HA‐966, blocks activation of the mesolimbic dopaminergic system induced by phencyclidine and dizocilpine (MK‐801) in rodents
Authors: Bristow L.J. , Hutson P.H. , Thorn L. , Tricklebank M.D.
Published: July 2012

The effects of the glycine/N‐methyl‐d‐aspartate (NMDA) receptor antagonist, R‐(+)‐HA‐966 on the neurochemical and behavioural responses to phencyclidine (PCP) and dizocilpine (MK‐801) have been determined in rodents.

In rats, pretreatment with PCP (5 and 10 mg kg−1) or MK‐801 (0.25 and 0.5 mg kg−1) dose‐dependently stimulated dopamine turnover in nucleus accumbens, amygdala and medial prefrontal cortex, but had no effect in striatum. In contrast, pretreatment with (+)‐HA‐966 (10 and 30 mg kg−1) did not affect dopamine turnover in any brain region investigated.

Pretreatment with (+)‐HA‐966 (10 and 30 mg kg−1) significantly antagonized the stimulation of dopamine turnover induced by both PCP (10 mg kg−1) and MK‐801 (0.5 mg kg−1) in rat nucleus accumbens, amygdala and medial prefrontal cortex.

Intracerebral dialysis studies in conscious rats demonstrated that systemic injection of PCP (10 mg kg−1) markedly stimulated dopamine release from the nucleus accumbens, an effect that was abolished by pretreatment with (+)‐HA‐966 (30 mg kg−1).

Pretreatment with PCP (3–30 mg kg−1) or MK‐801 (0.1–1.6 mg kg−1) significantly increased locomotor activity in mice. In contrast, subcutaneous injection of (+)‐HA‐966 (10–100 mg kg−1) failed to stimulate activity.

Pretreatment with (+)‐HA‐966 (10 and 30 mg kg−1) dose‐dependently antagonized both PCP (10 mg kg−1) and MK‐801 (0.4 mg kg−1) induced hyperactivity in mice.

Blockade of PCP‐induced hyperactivity by (+)‐HA‐966 is unlikely to be explained by the induction or potentiation of sedation/ataxia since PCP‐induced rotarod deficits were not significantly different in mice pretreated with (+)‐HA‐966 (30 mg kg−1) or saline.

The results demonstrate that (+)‐HA‐966 antagonizes both the neurochemical and behavioural effects of PCP and MK‐801, possibly through interactions at the glycine/NMDA receptor.
PAF activation of a voltage‐gated R‐type Ca2+ channel in human and canine aortic endothelial cells
Authors: Bkaily Ghassan , D'Orléans‐Juste Pedro , Naik Radha , Pérodin Jacqueline , Stankova Jana , Abdulnour Elias , Rola‐Pleszczynski Marek
Published: July 2012

By the use of fura‐2 and digital imaging techniques, [K]o depolarization or PAF (10−9 m) were shown to induce a sustained increase of [Ca]i in human or canine single aortic vascular endothelial cells (VEC) that was insensitive to nifedipine but sensitive to (−)‐PN200–110 or to lowering of [Ca]o. The PAF‐induced effect on [Ca]i was blocked by the PAF receptor antagonist, WEB2170. Our results suggest that [K]o depolarization and PAF increase [Ca]i via the activation of R‐type Ca2+ channels.
Prejunctional modulation of the nitrergic innervation of the canine ileocolonic junction via potassium channels
Authors: De Man Joris G. , Boeckxstaens Guy E. , Pelckmans Paul P. , De Winter Benedicte Y. , Herman Arnold G. , Van Maercke Yvan M.
Published: July 2012

The effects of different K+ channel blockers were studied on nitric oxide (NO)‐mediated non‐adrenergic non‐cholinergic (NANC) relaxations in the canine ileocolonic junction.

The non‐selective blockers of K+ channels, 4‐aminopyridine (4‐AP) and tetraethylammonium (TEA) and the blocker of large conductance Ca2+‐activated K+ channels, charybdotoxin, potently enhanced the NANC relaxations induced by low frequency stimulation. The blocker of small conductance Ca2+‐activated K+ channels, apamin, had no effect on electrically‐induced NANC relaxations.

NANC nerve‐mediated relaxations induced by adenosine 5′‐triphosphate (ATP), acetylcholine (ACh) and γ‐aminobutyric acid (GABA) were significantly enhanced by 4‐AP and charybdotoxin but not by apamin. TEA significantly enhanced the NANC relaxations in response to GABA and ATP while that in response to ACh was abolished.

None of the K+ channel blockers had an effect on the dose‐response curve to NO, on the noradrenaline‐induced contraction or on the relaxation to nitroglycerine (GTN).

From these results we conclude that inhibition of prejunctional K+ channels increases the nitrergic relaxations induced by electrical and chemical receptor stimulation of NANC nerves and thus suggests a regulatory role for these prejunctional K+ channels in the release of NO from NANC nerves in the canine ileocolonic junction.
Adenylate cyclase‐mediated vascular responses of rabbit aorta, mesenteric artery and skin microcirculation
Authors: Wilson A.J. , Warren J.B.
Published: July 2012

The importance of adenylate cyclase‐mediated vascular relaxation in the macro and microcirculation was assessed in rabbit aortic and coeliac artery bioassay rings in vitro and skin microvessels in vivo.

The neuropeptide pituitary adenylate cyclase‐activating polypeptide (PACAP38), the β‐agonist, isoprenaline, and the prostaglandins, PGE1 and PGE2, were compared with the activity of nitroprusside, which acts by stimulating guanylate cyclase.

In aortic tissue the relative relaxant potencies were (−log m EC50, 100% = response to nitroprusside 10−6 m): nitroprusside 7.0, PACAP38 6.8, isoprenaline 6.3; PGE1 and PGE2 were weak constrictors. In coeliac artery rings relative potencies were (−log m EC50, 100% = response to nitroprusside 10−5 m): PACAP38 6.6, PGE1 6.6, nitroprusside 6.5, PGE2 4.9, and isoprenaline 4.3.

Comparative potencies when injected into anaesthetized rabbit skin in vivo were (−log mol/site required to increase blood red cell flux by 75%): PACAP38 13.0, PGE2 10.7, isoprenaline 9.7, PGE1 9.1, nitroprusside < 7.

Nitroprusside, the most effective relaxant tested in the aorta, was 107 fold less potent than PACAP in its effect on skin blood flow. PGE1 and PGE2 were constrictors of the aorta, of intermediate effect in the coeliac artery, but potent vasodilators of the microcirculation.

In this model, the importance of adenylate cyclase‐mediated vascular relaxation increases with decreasing vessel size.
Enhancement by calcitonin gene‐related peptide of nicotinic receptor‐operated noncontractile Ca2+ mobilization at the mouse neuromuscular junction
Authors: Kimura Ikuko , Tsuneki Hiroshi , Dezaki Katsuya , Kimura Masayasu
Published: July 2012

The involvement of calcitonin gene‐related peptide (CGRP) in the mechanism of nicotinic acetylcholine receptor‐operated noncontractile Ca2+ mobilization (not accompanied by twitch tension) was investigated by measuring Ca2+‐aequorin luminescence at the neuromuscular junction of mouse diaphragm muscle treated with neostigmine.

Noncontractile Ca2+ transients were enhanced by 4‐aminopyridine (100 μm), a K+ channel blocker, and inhibited by botulinum toxin (1–100 μg, i.p.) and hexamethonium (10–100 μm), a neuronal nicotinic receptor antagonist.

Noncontractile Ca2+ transients were diminished by CGRP8–37 (10–20 μm), a CGRP antagonist. CGRP (0.3–10 nm) prolonged the duration of noncontractile Ca2+ transients. The effect of CGRP was suppressed by CGRP8_37 (0.1 μm).

Noncontractile Ca2+ transients were inhibited by H‐89 (0.1–1 μm), a protein kinase‐A inhibitor. The catalytic subunit of protein kinase‐A and AA373 (300 μm), a protein kinase‐A activator, prolonged the duration of noncontractile transients. The prolongations either by CGRP or by AA373 were not observed in the presence of H‐89 (0.1 μm).

Contractile (accompanied by twitch tension) but not noncontractile Ca2+ transients were decreased by 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA, 0.3–1 μm), a protein kinase‐C activator. Phospholipase A2 increased only contractile Ca2+ transients. Calmodulin‐related agents affected neither type of Ca2+ transients.

These results provide the first evidence that nicotinic acetylcholine receptor‐operated noncontractile Ca2+ mobilization is promoted by nerve‐released CGRP activating protein kinase‐A, and is dependent on the accumulated amounts of acetylcholine at the neuromuscular junction where desensitization might readily develop.
Differential effects of acetylcholine, nitric oxide and levcromakalim on smooth muscle membrane potential and tone in the rabbit basilar artery
Authors: Plane Trances , Garland Christopher J.
Published: July 2012

Endothelium‐dependent hyperpolarization of smooth muscle cells in isolated, pre‐contracted segments of rabbit basilar artery in response to acetycholine (100 μm) was abolished in the presence of glibenclamide (10 μm).

Acetylcholine‐evoked relaxation was unaffected by either glibenclamide or 65 mm potassium chloride, indicating that the change in membrane potential did not form an essential component of relaxation and that high concentrations of potassium did not inhibit the release or action of endothelium‐derived relaxing factor in this vessel.

Saturated solutions of nitric oxide (NO) gas in solution (150 μm), which evoked maximal relaxation of arterial segments pre‐contracted and depolarized by noradrenaline (10–100 μm), did not alter the membrane potential of either unstimulated or depolarized smooth muscle cells.

The potassium channel opener levcromakalim, evoked concentration‐dependent relaxation and hyperpolarization in pre‐constricted smooth muscle cells. The threshold concentrations for hyperpolarization and relaxation, the EC50 values and the maximally effective concentration of levcromakalim (around 30 nm, 150 nm and 10 μm, respectively) were not significantly different, and both components of the response were inhibited by glibenclamide (10 μm), indicating a close coupling between the two responses.

In the presence of 65 mm potassium chloride, the hyperpolarization to levcromakalim was abolished, while a small relaxation (25 ± 4%) persisted, indicating an additional mechanism for relaxation to this agent.

These results show that different mechanisms underlie the relaxant action of potassium channel openers, NO and endothelium‐derived factors in cerebral arteries and provide further evidence that in the basilar artery, in contrast to some other vessels, endothelium‐dependent hyperpolarization to acetylcholine is not important for smooth muscle relaxation.
Characterization of receptors for endothelins in the perfused arterial and venous mesenteric vasculatures of the rat
Authors: D'Orléans‐Juste Pedro , Claing Audrey , Warner Timothy D. , Yano Mitsuo , Télémaque Sabine
Published: July 2012

Endothelin−1 and −3 induced marked arterial and venous constrictions in the perfused mesenteric vasculature of the rat with endothelin‐3 being at least 20 times less active than endothelin‐1, on both arterial and venous sides of the vasculature.

Two ETB selective agonists, BQ‐3020 and IRL 1620 (500 pmol), induced weak constrictions of the venous mesenteric vasculature and were inactive in the arterial side at doses up to 1000 pmol.

In mesenteric vasculatures precontracted with either methoxamine (arterial side) or the thromboxane A2‐mimetic, U46619 (venous side), acetylcholine or bradykinin produced vasodilatations of both arterial and venous vessels, whereas endothelin‐3 induced vasodilatations only on the arterial side.

A selective ETA receptor antagonist, BQ‐123, blocked, in a concentration‐dependent and reversible fashion, the vasoconstrictions induced by endothelin‐1 on both sides of the mesenteric circulation (IC50; arterial side: 0.013 μm; venous side: 0.032 μm).

In contrast, the vasodilator responses induced by endothelin‐3 on the arterial side of the precontracted mesenteric vasculature were not affected by BQ‐123.

The present study illustrates the presence of ETA receptors which are responsible for vasoconstriction by endothelins in the arterial and venous mesenteric vasculatures. Furthermore, we suggest that the vasodilatations induced by endothelin‐3 in the arterial vasculature uniquely, are ETB receptor‐mediated.
Effect of a calcitonin gene‐related peptide antagonist (CGRP8–37) on skin vasodilatation and oedema induced by stimulation of the rat saphenous nerve
Authors: Escott K. Jane , Brain Susan D.
Published: July 2012

The effect of the calcitonin gene‐related peptide antagonist (CGRP8–37, 400 nmol kg−1, i.v.) on the increased blood flow induced by calcitonin gene related peptide (CGRP), vasodilator prostaglandins, and topical capsaicin was measured with a laser Doppler blood flow meter in rat abdominal skin.

The saphenous nerve was electrically stimulated and the effect of CGRP8–37 (400 nmol kg−1, i.v.) on the increased blood flow (measured by laser Doppler flowmetry) and oedema formation (measured by the extravascular accumulation of [125I]‐albumin) was investigated in the rat hind paw.

CGRP8–37 (400 nmol kg−1, i.v.) had no effect on basal cutaneous blood flow at uninjected sites and sites injected with Tyrode buffer, but acted selectively to inhibit the increased blood flow induced by intradermal CGRP (10 pmol/site, P < 0.05), but not that induced by prostaglandin E2 (PGE2, 300 pmol/site) or carba‐prostacyclin (cPGI2, 100 pmol/site).

Capsaicin (0.1–33 mm), applied topically, acted in a dose‐related manner to increase blood flow. CGRP8–37 (400 nmol kg−1, i.v.) almost totally inhibited blood flow induced by capsaicin (10 mm; P < 0.05) but did not significantly inhibit blood flow induced by a higher dose of capsaicin (33 mm).

The increased blood flow induced by short stimulation of the saphenous nerve (10 V, 1 ms, 2 Hz for 30 s) was inhibited by 76%, 5 min after i.v. CGRP8–37 (400 nmol kg−1, i.V., P < 0.05).

A longer (5 min) electrical stimulation of the saphenous nerve caused oedema formation, in addition to increased blood flow. The oedema formation was significantly inhibited by CGRP8–37 (400 nmol kg−1, i.v., P < 0.05).

The results suggest that the potent microvascular vasodilator neuropeptide, CGRP, is responsible for the increased blood flow observed after short stimulation of the saphenous nerve and that endogenous CGRP contributes in a pro‐inflammatory manner to neurogenic oedema formation in the rat hind paw.