bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural features important for the biological activity of the potassium channel blocking dendrotoxins
Authors: Hollecker Michelle , Marshall David L. , Harvey Alan L.
Published: July 2012

Dendrotoxins from mamba snake venoms are small proteins that block neuronal K+ channels. In order to investigate structural features associated with their biological activity, partially folded versions of dendrotoxins I and K from black mamba (Dendroaspis polylepis) were prepared by selectively reducing one or more of their three S‐S bonds.

The modified toxins were tested for ability to compete with 125I‐labelled native toxin I to high affinity binding sites on rat brain synaptosomal membranes and for the ability to increase acetylcholine release in a neuromuscular preparation.

Binding affinity increased progressively as the toxins folded to the native conformation and the most biologically active of the modified species were those in which only the disulphide bond between residues 14 and 38 was not formed. These intermediates had native‐like conformations as determined by circular dichroism but still had about 5–10 times lower affinity than native toxins.

Addition of negatively charged groups to block the free sulphydryls at positions 14 and 38 caused a further, marked loss of activity.

The results are consistent with the existence of two important regions in the dendrotoxin molecules. The region containing two of the disulphide bonds (around Cys5‐Cys55 and Cys30‐Cys51) and much of the secondary structure is essential for the binding affinity of the toxins, while the region around Cys14 and Cys38, equivalent to part of the antiprotease site of the homologous protease inhibitor from bovine pancreas (BPTI), plays an important role in the potency of dendrotoxins.
Muscarinic effect of atrial natriuretic peptide on rabbit airways
Authors: Robichaud A. , Saunier C. , Michoud M.C. , du Souich P.
Published: July 2012

The aim of the present work was to investigate under which circumstances atrial natriuretic peptide (ANP) modulates airway resistance.

Of the six groups of rabbits (n = 5) studied, three received an infusion of ANP (80 ng min−1 kg−1 i.v.) for a period of 100 min, while the other three were infused with the vehicle. Before receiving the infusion of ANP or the vehicle, the animals were pretreated with atropine (0.5 mg kg−1 i.v.), propranolol (2 mg kg−1 i.v.) or not pretreated. After 75 min of infusion of ANP, bronchoconstriction was induced by inhalation of histamine. Respiratory resistance (Rrs) was measured before and 3, 5, 10, 15 and 20 min post‐histamine challenge.

Following 75 min of ANP infusion, plasma ANP concentration increased from 153 ± 52 (mean ± s.e.mean) to 1441 ± 203 pg ml−1 (P < 0.05) without affecting baseline Rrs. Control Rrs values (12.5–20.4 cmH2O l−1 s) were significantly increased following the inhalation of histamine (P < 0.001). By themselves, atropine, propranolol or ANP did not modify the histamine‐induced increase in Rrs. However, when the animals were pretreated with atropine, ANP infusion significantly reduced the increase in Rrs induced by histamine (30 ± 2 vs 51 ± 6 cmH2O l−1 s; P < 0.05).

These data suggest that ANP has an indirect modulating effect on the airway smooth muscle and will decrease Rrs when muscarinic receptors are blocked.
Presence of NK3‐sensitive neurones in different proportions in the medial habenula of guinea‐pig, rat and gerbil
Authors: Boden P. , Woodruff G.N.
Published: July 2012

Electrophysiological recordings were made from neurones of the medial habenula (Mhb) in brain slices obtained from guinea‐pig, rat and gerbil brain. The selective NK3 agonist, senktide, was used to determine the relative number of NK3‐sensitive‐neurones in the Mhb of each species. The proportion of neurones responding to NK1 (Sar9Met(O2)11SP) and NK2 (αAla8NKA(4–10) agonists was also assessed. All (65/65) of the guinea‐pig Mhb neurones tested were excited by the NK3 agonist, but NK1 and NK2 agonists were without effect. NK3 responses in the guinea‐pig were not altered by the presence of a selective NK1 antagonist, CP‐99,994. NK1, NK2 and NK3 agonists were without effect on Mhb neurones from gerbil brain slices. In agreement with findings from previous studies, a population of rat Mhb neurones responded to NK1 or NK3 agonists alone or were excited by both. These data show that there is a difference in both the number of NK‐sensitive neurones and the type of NK response found in the medial habenula of the three species. The high sensitivity to an NK3 agonist, combined with the apparent lack of NK1 and NK2 responses in the guinea‐pig Mhb makes this preparation ideal for studies of central NK3‐mediated events.
Effects of antidepressants on the inward current mediated by 5‐HT3 receptors in rat nodose ganglion neurones
Authors: Fan Ping
Published: July 2012

Effects of three different categories of antidepressants, imipramine (tricyclic), fluoxetine (selective 5‐hydroxytryptamine (5‐HT) uptake inhibitor), phenelzine and iproniazid (monoamine oxidase (MAO) inhibitor) on the inward current mediated by 5‐HT3 receptors were investigated in rat nodose ganglion neurones. The whole‐cell patch‐clamp technique was used for recording the 5‐HT current.

All the antidepressants tested inhibited the peak 5‐HT current. The inhibition gradually reached a steady level and the recovery was incomplete when antidepressants were removed. IC50 values for imipramine, fluoxetine and phenelzine were 0.54 μm, 1.3 μm and 4.2 μm respectively. The correspondent Hill coefficients were 0.9, 0.87 and 0.92.

The antidepressants examined increased the rate of 5‐HT current desensitization. IC50 values for imipramine, fluoxetine and phenelzine on the decrease in desensitization time constant were 0.11 μm, 0.18 μm and 2.4 μm respectively. The correspondent Hill coefficients were 0.9, 1.14 and 1.06.

Intracellular applications of the protein kinase inhibitor, H‐7 (100 μm), GDP‐β‐S (2 mm) and the calcium chelator BAPTA (20 mm) did not affect the 5‐HT current and the actions of antidepressants on 5‐HT current.

These results suggest that the 5‐HT3 receptor is an acting site for the therapeutic use of antidepressants. The present observation is also helpful in explaining the analgesic effect of antidepressants seen in pain clinics.
Typical and atypical NK1 tachykinin receptor characteristics in the rabbit isolated iris sphincter
Authors: Hall Judith M. , Mitchell Debra , Morton Ian K.M.
Published: July 2012

A contraction of the rabbit isolated iris sphincter smooth muscle follows activation of either tachykinin NK1 or NK3 receptors. We have here characterized the pharmacological activity profiles of various tachykinin receptor agonists considered to have NK1‐receptor‐preferring activity in this preparation.

Two groups of NK1‐receptor‐preferring agonists could be distinguished in terms of a common pharmacological profile. The first group (Group 1) included [Glp6,l‐Pro9]‐SP(6–11) (septide), [Glp6]‐SP (6–11), substance P methyl ester, δ‐aminovaleryl‐[l‐Pro9, N‐MeLeu10]‐SP(7–11) (GR73632), and [Apa9–10]‐SP. The second group (Group 2) included [Pro9]‐SP, substance P, physalaemin and ranamargarin.

Under control conditions, the responses to Group 1 agonists were relatively fast in offset (time for reversal of maximal responses, 11.2–18.2 min), and were antagonized by NK1‐receptor‐selective antagonists (range of pKB estimates vs various agonists; GR82334, 7.1–8.2; (±)‐CP‐96,345, 8.9–9.5; RP67580, 7.0–7.4). Following incubation of the tissue with phenoxybenzamine (20 μm, 10 min), the affinity of GR82334, tested against the Group 1 agonists, substance P methyl ester and septide, was not significantly different (P < 0.05; n = 7–18) to that determined in untreated tissues (substance P methyl ester pKB 7.5 ± 0.1 and 7.2 ± 0.2, respectively; septide 7.7 ± 0.2 and 7.9 ± 0.2, respectively). Further, response offset times (5.0–8.5 min) were little reduced as compared to those observed in untreated tissues.

Under control conditions, the response to Group 2 agonists was markedly slow in offset (times for reversal of maximal responses, 51.4–70.4 min), and was not attenuated significantly by the NK1‐receptor‐selective antagonists GR82334 (1 μm), (±)‐CP‐96,345 (0.1 μm) or RP67580 (1 μm). In contrast, after phenoxybenzamine pretreatment, responses to Group 2 agonists reversed rapidly (times for reversal of maximal responses, 13.1–24.2 min), and were now antagonized by GR82334 (pKB estimates, 6.4–7.1).

The responses to the NK3‐receptor‐selective agonist Succ‐[Asp6,Me‐Phe8]‐SP(6–11) (senktide) were relatively fast in offset (time for reversal of maximal response was 18.6 ± 1.7 min) and were not inhibited by GR82334 (10 μm; n = 5). The contractile response resulting from co‐application of the Group 1 agonist, septide together with senktide, did not exhibit prolonged response offset kinetics.

Assuming simple competition at equilibrium, these data from the rabbit iris smooth muscle could be explained either by interaction of the various ligands with two separately‐existing NK1 receptor‐subtypes or ‐isoforms; or alternatively by a preferential interaction of the two agonist groups with different binding domains on a common NK1 receptor.
Reduction by Hoe 140, the B2 kinin receptor antagonist, of antigen‐induced nasal blockage
Authors: Austin C.E. , Foreman J.C. , Scadding G.K.
Published: July 2012

In subjects with allergic rhinitis to house‐dust mite (HDM), antigen challenge produced a significant increase in nasal blockage but had no effect on nasal vascular permeability. The B2 kinin receptor antagonist, [d‐Arg0, Hyp3, Thi5,d‐Tic7, Oic8]‐bradykinin (Hoe 140), 200 μg administered by intranasal aerosol 2 min prior to challenge with HDM, 500 u significantly reduced nasal blockage induced by the antigen challenge. The data are compatible with a role for B2 kinin receptors in the nasal response to challenge with antigen which is responsible for nasal blockage.
Augmentation by intracellular ATP of the delayed rectifier current independently of the glibenclamide‐sensitive K‐current in rabbit arterial myocytes
Authors: Evans A.M. , Clapp L.H. , Gurney A.M.
Published: July 2012

Elevation of intracellular ATP levels by flash photolysis of caged ATP augmented the delayed rectifier K‐current (IKDR) in rabbit pulmonary artery myocytes. The percentage augmentation was unaffected when IKDR was inactivated by 50% (holding potential − 40 mV), although the magnitude of the ATP‐induced current was substantially reduced. Inactivation of 90% IKDR (holding potential − 20 mV) virtually abolished the ATP‐dependent augmentation. We conclude that modulation of IKDR by ATP does not require conversion of the glibenclamide‐sensitive K‐current (IKDR(ATP).
Relaxation of guinea‐pig trachea by cyclic AMP phosphodiesterase inhibitors and their enhancement by sodium nitroprusside
Authors: Turner Nicholas C. , Lamb Janine , Worby Angela , Murray Kenneth J.
Published: July 2012

The effects of agents that elevate either cyclic AMP (the phosphodiesterase (PDE) III inhibitor siguazodan, salbutamol) or cyclic GMP (sodium nitroprusside (SNP)) on the relaxant activity of the PDE IV inhibitor, rolipram, were investigated in carbachol (0.1 μm) precontracted guinea‐pig tracheal sheets.

Rolipram, siguazodan and SNP caused concentration‐related reductions in tone of tissues precontracted with 0.1 μm carbachol (EC50 values 12.5; 2.73 and 0.35 μm respectively). Whilst the concentration‐response relationship for the PDE III inhibitor, siguazodan, was monophasic that of the PDE IV inhibitor, rolipram, was biphasic.

The relaxant activity of rolipram was markedly enhanced in the presence of 10 μm siguazodan (EC50 < 0.01 μm), 0.1 μm salbutamol (EC50 0.03 μm) and 0.3 μm SNP (EC50 0.03 μm). In contrast, the relaxant activity of siguazodan was unaffected by SNP and only modestly enhanced by rolipram (10 μm) and salbutamol (0.1 μm).

The relaxant activity of SNP was enhanced by the PDE V inhibitor SK&F 96231 (30 μm: EC50 0.06 μm) and rolipram (30 μm, EC50 0.08 μm) but was unaffected by 30 μm siguazodan.

At concentrations up to 10 μm, neither siguazodan nor rolipram elevated tracheal cyclic AMP levels. However, the combination of 10 μm rolipram and siguazodan caused a two fold increase in the cyclic AMP content (from 2.19 to 4.36 pmol cyclic AMP mg−1protein). SNP (0.1–10 μm) failed to produce a significant increase in tracheal cyclic AMP levels. At 0.1 μm the effect of SNP on tracheal cyclic AMP levels was significantly (P < 0.05) increased in the presence of rolipram but not siguadozan.

The results indicate that the relaxant effects of rolipram are markedly enhanced by agents that inhibit PDE III activity or elevate cyclic GMP. They support the hypothesis that SNP potentiates the effects of rolipram via the inhibitory action of cyclic GMP on hydrolysis of cyclic AMP by PDE III. The findings also suggest that whilst PDE III may be more significant in regulating basal smooth muscle tone in the absence of any exogenous stimulus to cyclic AMP accumulation, PDE IV activity may be more tightly coupled to the pool of adenylyl cyclase stimulated by β2‐adrenoceptor agonists.
Cyclic GMP‐independent relaxation and hyperpolarization with acetylcholine in guinea‐pig coronary artery
Authors: Eckman D.M. , Weinert J.S. , Buxton I.L.O. , Keef K.D.
Published: July 2012

The effects of acetylcholine (ACh) on membrane potential, relaxation and cyclic GMP levels were compared to the NO donor l‐nitrosocysteine (Cys‐NO) in segments of guinea‐pig coronary artery.

ACh and Cys‐NO produced concentration‐dependent relaxations of muscles contracted with the H1 receptor agonist, 2‐(2‐aminoethyl)pyridine (AEP, 0.35 mm). The relaxation to ACh was unchanged in the presence of NG‐monomethyl‐l‐arginine (l‐NMMA; 350 μm) or indomethacin (3 μm).

Oxyhaemoglobin (HbO; 20 μm) alone or in combination with l‐NMMA increased the EC50 for ACh‐induced relaxation whereas relaxation with Cys‐NO was almost completely abolished with HbO.

Scorpion venom (SV; 8.7 μg ml−1) increased the EC50 for relaxation with ACh but not Cys‐NO. Combined l‐NMMA, HbO and SV produced nearly complete abolition of ACh‐induced relaxations.

Basal cyclic GMP levels (i.e., 20 pmol mg−1protein) were significantly increased following addition of either ACh (190 pmol mg−1protein) or Cys‐NO (240 pmol mg−1protein). l‐NMMA significantly reduced the rise of cyclic GMP with ACh but not Cys‐NO. In contrast, SV did not significantly reduce the rise in cyclic GMP produced with ACh. In the combined presence of l‐NMMA and HbO neither ACh nor Cys‐NO produced a significant increase in cyclic GMP levels.

ACh gave rise to significantly greater membrane hyperpolarization than Cys‐NO both in the presence and absence of AEP. Combined l‐NMMA and HbO did not reduce the amplitude of hyperpolarization with ACh.

These data indicate that some but not all of the actions of ACh in the coronary artery can be mimicked by the NO donor, Cys‐NO, suggesting that ACh releases NO as well as a second hyperpolarizing factor (i.e., EDHF). Release of NO results in a large increase in tissue cyclic GMP levels and minimal change in membrane potential whereas release of EDHF results in a large membrane hyperpolarization which is independent of changes in tissue cyclic GMP levels. Both of these pathways appear to contribute to relaxation throughout the entire ACh concentration‐relaxation relationship.
Depression of high‐threshold calcium currents by activation of human D2 (short) dopamine receptors expressed in differentiated NG108‐15 cells
Authors: Seabrook G.R. , McAllister G. , Knowles M.R. , Myers J. , Sinclair H. , Patel S. , Freedman S.B. , Kemp J.A.
Published: July 2012

This study examined the regulation of calcium currents in differentiated NG108‐15 cells that had been stably transfected with cDNA encoding the short isoform of the human D2 dopamine receptor. Whole cell calcium currents were recorded by nystatin‐perforated patch clamp recording.

Transient low‐threshold calcium currents elicited by depolarizations from − 100 mV to − 20 mV were reversibly depressed by NiCl2 (84 ± 8% at 30 μm; n = 3) and by ω‐agatoxin IVA (15 ± 5%; 100 nm, n = 7). These currents were unaffected by hD2 receptor activation.

High‐threshold calcium currents elicited by depolarizations from − 80 mV to 0 mV were partly blocked by ω‐conotoxin GVIA (67 ± 6% at 100 nm, n = 4) and by the subsequent addition of the dihydropyridine, nisoldipine (94 ± 3% at 1 μm). Consistent with the presence of at least two distinct types of high‐threshold calcium channels, nisoldipine alone (38 ± 15% at 1 μm, n = 6) did not preclude the inhibition caused by ω‐conotoxin GVIA (69 ± 13% at 100 nm, n = 4). The residual current was completely blocked by 100 μm CdCl2 (98.8 ± 0.4%, n = 7).

In hD2‐transfected cells, but not untransfected cells, high‐threshold currents were depressed by quinpirole (30 ± 4% at 100 nm; n = 15) with a pEC50 of 8.61 ± 0.22 (n = 5), as well as by (−)‐noradrenaline (28 ± 5% at 1 μm, n = 9). Responses to both agonists were selectively antagonized by S‐(−)sulpiride (100 nm) but not by the α‐adrenoceptor antagonist, phentolamine (10 μm). The depression caused by (−)‐noradrenaline was positively correlated with that of quinpirole for each cell (r2= 0.91, slope = 0.99).

hD2‐receptor‐mediated inhibition of high‐threshold calcium currents was abolished by pretreatment of cells with ω‐conotoxin GVIA (100 nm; n = 4). However, a component of the high‐threshold current was reversibly depressed by ω‐conotoxin GVIA (67% to 45% depression after 10 min wash). This current was also depressed by hD2 receptor activation (59 ± 9% depression in 100 nm quinpirole, n = 3), and was completely blocked by nisoldipine (95 ± 2% at 1 μm).

These data demonstrate that activation of hD2(short) dopamine receptors can regulate both ω‐conotoxin GVIA, and dihydropyridine‐sensitive high‐threshold calcium currents in neuroblastoma cells. Morever, the ability of human D2 dopamine receptors to regulate more than one type of calcium current supports the notion that these receptors have a diverse functional role in the central nervous system.
Effects of ethanol and other aliphatic alcohols on NO‐mediated relaxations in rat anococcygeus muscles and gastric fundus strips
Authors: Rand M.J. , Li C.G.
Published: July 2012

In anococcygeus muscles, ethanol (20–500 mm) slightly increased the tone and inhibited relaxations elicited by nitrergic nerve stimulation (0.5–5 Hz) in a concentration‐dependent manner.

Other aliphatic alcohols decreased the tone but had inhibitory effects similar to ethanol on stimulation‐induced relaxations, the EC50 (mm) values being: methanol 280, ethanol 80, propan‐1‐ol 20, propan‐2‐ol 55, propan 1,2‐diol 135, butan‐1‐ol 120, butan‐2‐ol 15 and pentan‐1‐ol 3.

Relaxations induced by sodium nitroprusside (SNP, 10 nm) were inhibited by ethanol (20–500 mm) in a concentration‐dependent manner and by propan‐2‐ol (100 mm). Relaxations induced by NO (1 μm) were inhibited by high concentrations of ethanol (200–300 mm) and by propan‐2‐ol (100 mm).

In gastric fundus strips, ethanol (60–200 mm) did not affect the resting tone but inhibited NO‐mediated relaxations elicited by low frequency (1 Hz) field stimulation and reduced the initial relaxation by high frequency field stimulation (10 Hz) and by SNP (50 nm). The relaxant action of isoprenaline (10 nm) was not reduced although it was slightly slower in onset. Other aliphatic alcohols tested decreased the tone and inhibited relaxations elicited by field stimulation.

Acetaldehyde (1–10 mm) inhibited relaxations elicited by field stimulation and SNP in both the rat anococcygeus muscles and gastric fundus strips. The tone of gastric fundus strips was decreased by acetaldehyde but it was transiently increased in anococcygeus muscles.

The dehydrogenase inhibitors, 4‐methylpyrazole (100 μm) and disulfiram (100 μm) did not significantly affect the inhibition by ethanol of nitrergic nerve stimulation‐induced relaxations in anococcygeus muscles, which suggests that ethanol acts directly.

The inhibition of NO‐mediated relaxations by alcohols is attributed to sequestration of NO to form nitroso‐alcohols.
Effect of PAF on rat lung vascular permeability: role of platelets and polymorphonuclear leucocytes
Authors: Sirois Martin G. , de Lima Wothan Tavares , Fernandes Artur José de Brum , Johnson Richard J. , Plante Gérard E. , Sirois Pierre
Published: July 2012

The objectives of the present experiments were to assess the contribution of polymorphonuclear leucocytes (PMNLs), platelets and their products such as thromboxane A2 (TxA2), histamine and 5‐hydroxytryptamine to platelet activating factor (PAF)‐mediated protein extravasation in rat lungs.

Intravenous injection of PAF (1.0 and 5.0 μg kg−1) increased dose‐dependently (up to 7.5 fold) the vascular permeability of the trachea, upper and lower bronchi to Evans blue dye (EB), a marker of albumin extravasation. The permeability of the pulmonary parenchyma was not affected significantly by PAF.

Thrombocytopenia induced by administration of the IgG fraction of goat anti‐rat platelet serum (APS; 15 mg 100 g−1, i.p., 16–18 h) reduced by 55, 58 and 40% the effects of the lower dose of PAF (1.0 μg kg−1) and by 31, 23 and 15% the effects of the higher dose of PAF (5.0 μg kg−1) on the permeability of the trachea, upper and lower bronchi respectively to albumin.

PMNL depletion induced by administration of rabbit anti‐rat polymorphonuclear serum (ANS; 2 mg kg−1, i.v., 24 h) did not reduce significantly the effects of the lower dose of PAF (1.0 μg kg−1) on the airways, however the effects of the higher dose of PAF (5.0 μg kg−1) on the permeability of the trachea, upper and lower bronchi to albumin were reduced by 43, 25 and 23% respectively.

The injection of both the anti‐platelet and the anti‐PMNL sera reduced by 61, 62 and 96% the effects of the lower dose of PAF (1.0 μg kg−1) and by 44, 39 and 47% the effects of the higher dose of PAF (5.0 μg kg−1) on the permeability of the trachea, upper and lower bronchi respectively.

The combined injection of the TxA2‐mimetic (U‐44069; 5.0 μg kg−1) and PAF (1.0 and 5.0 μg kg−1) in thrombocytopenic rats overcame the vascular permeability decrease induced by APS treatment.

Pretreatment of the animals with a combination of antagonists to histamine (mepyramine; 3.0 mg kg−1) and 5‐hydroxytryptamine (methysergide; 2.5 mg kg−1) did not cause a significant inhibition of the effect of PAF (1.0 and 5.0 μg kg−1) on EB extravasation in the airways.

These data show that the effect of intravenous PAF on rat vascular permeability is partly modulated by polymorphonuclear leucocyte and platelet activation. Our results suggest that following its release, TxA2 could increase postcapillary hydrostatic pressure by inducing a venoconstriction and potentiate the extravasation elicited by PAF. These results do not suggest a major role for histamine and/or 5‐hydroxytryptamine on PAF‐induced albumin extravasation.
Receptor for catecholamines responding to catechol which potentiates voltage‐dependent calcium current in single cells from guinea‐pig taenia caeci
Authors: Muraki Katsuhiko , Bolton Thomas B. , Imaizumi Y. , Watanabe M.
Published: July 2012

Single isolated cells were obtained from the taenia of the guinea‐pig's caecum by enzymic digestion and held under voltage clamp. The effects of various catecholamines, sympathomimetics and related compounds were tested for their ability to potentiate the voltage‐dependent calcium current (ICa) evoked in these cells by a depolarizing step.

ICa was potentiated by up to 60% by isoprenaline, adrenaline, and noradrenaline which were equipotent. The EC50 for isoprenaline was about 40 nm.

The racemic mixtures of the optical isomers of isoprenaline, adrenaline, and noradrenaline, and (+)‐isoprenaline, were equipotent with the (–)‐isomers of these drugs. Dopamine, l‐dopa, and catechol were equipotent with these catecholamines.

Removal or substitution of one or more of the hydroxy groups of the catechol moiety, as in phenylephrine, salbutamol, procaterol, methoxamine, terbutaline, BRL 37344, ICI 215001 or tyramine substantially reduced efficacy and/or potency.

The adrenoceptor blockers propranolol, phentolamine, dihydroergotamine, atenolol, CGP 20712A and ICI 118551, or the dopamine receptor blockers, haloperidol or flupenthixol, did not block the potentiating action of catechol or the catecholamines.

The receptor activated by catecholamines to increase ICa we suggest should be called a C‐receptor in view of its sensitivity to catechol. It may arise by enzymic modification of a conventional adrenoceptor but its transduction also involves a novel mechanism which might indicate that it is present in the muscle cells before enzyme treatment.
Characterization of 5‐hydroxytryptamine receptors mediating mucosal secretion in guinea‐pig ileum
Authors: Johnson P.J. , Bornstein J.C. , Furness J.B. , Woollard D.J. , Orrman‐Rossiter S.L.
Published: July 2012

The receptor subtypes through which 5‐hydroxytryptamine (5‐HT) increases electrolyte secretion across the mucosa of guinea‐pig ileum were studied.

Flat sheep preparations of guinea‐pig mucosa plus submucosa were placed in Ussing chambers and the short circuit current (ISC), an index of net electrogenic electrolyte transport across the mucosa, was measured under voltage clamp conditions.

Low concentrations of 5‐HT (10–300 nm) evoked monophasic increases in ISC which were significantly reduced by hyoscine (100 nm), tetrodotoxin (TTX, 300 nm) and the 5‐HT2 receptor antagonist, ketanserin (3–300 nm).

Higher concentrations of 5‐HT (1–10 μm) produced biphasic responses which were reduced by hyoscine (100 nm), TTX (300 nm), ketanserin (3–300 nm) and also by the 5‐HT3 receptor antagonists, granisetron (1 μm) and ICS 205–930 (100 nm).

2‐Methyl‐5‐HT (1–100 μm) and α‐methyl‐5‐HT (30 nm‐30 μm), agonists at 5‐HT3 and 5‐HT2 receptors respectively, also evoked ISC increases. These responses were reduced by hyoscine (100 nm) and abolished by TTX (300 nm) and the respective receptor antagonists, granisetron (1 μm) and ketanserin (30 nm).

The 5‐HT4 receptor antagonist, SDZ 205–557 (300 nm) had no effect on the response to 5‐HT.

The TTX‐resistant response to 5‐HT was not affected by 5‐HT23 or 4 receptor antagonists.

These results indicate that 5‐HT mediates secretion partly by an action on 5‐HT3 receptors located on cholinergic and noncholinergic secretomotor neurones, partly by an action on higher affinity ‘5‐HT2‐like’ receptors predominantly on noncholinergic neurones, and partly by a direct action on the epithelium.
Interactions between indomethacin, noradrenaline and vasodilators in the fetal rabbit ductus arteriosus
Authors: Smith Gordon C.S. , McGrath John C.
Published: July 2012

Interactions between indomethacin, noradrenaline and vasodilators were studied in rings of ductus arteriosus isolated from fetal rabbits. The effect of incubation with prostaglandin E2 (PGE2) on the noradrenaline concentration‐contraction response curve was studied in the presence and absence of indomethacin. Also, the ductus was pre‐contracted with 10 μm noradrenaline and concentration‐relaxation response curves (CRRC) to PGE2, cicaprost, cromakalim and forskolin were obtained in the presence and absence of indomethacin.

In the absence of indomethacin, PGE2 (from 1 nm to 100 nm) decreased the pEC50 to noradrenaline to a maximum of 0.4 to 0.5 log units (i.e. an approximately three fold increase in EC50 [m]). In the presence of 1 μm indomethacin, PGE2 (0.1 nm to 100 nm) decreased the pEC50 to noradrenaline by 2.39 log units (i.e. a 245 fold increase in EC50). By comparing the control pEC50 to noradrenaline with the relationship between the pEC50 to noradrenaline and [PGE2] in 1 μm indomethacin, the effect of endogenous PGE2 synthesized in the vessel wall was estimated as being equivalent to a bath concentration of approximately 1 nm exogenous PGE2.

When the vessel was pre‐contracted with 10 μm noradrenaline, indomethacin had no effect on the CRRC to PGE2 but did alter the CRRC to other vasodilators. The sensitivity of the vessel to cicaprost, cromakalim and forskolin was decreased in 1 μm indomethacin compared with control. Forskolin caused complete relaxation in the presence and absence of indomethacin. Indomethacin decreased the maximum response to cromakalim but increased the maximum response to cicaprost. PGE2, 0.3 nm, partially reversed the effect of indomethacin on the sensitivity of the vessel to forskolin.

We conclude that under varying experimental conditions, indomethacin increased the sensitivity of the ductus to the effects of PGE2 but decreased its sensitivity to other vasodilators. Both effects can be explained by elimination of endogenous PGE2. However, indomethacin increased the maximum response to cicaprost, which cannot be explained by elimination of endogenous PGE2 but may be due to elimination of endogenous prostacyclin.
Coupling of a transfected human brain A1 adenosine receptor in CHO‐K1 cells to calcium mobilisation via a pertussis toxin‐sensitive mechanism
Authors: Iredale Philip A. , Alexander Stephen P.H. , Hill Stephen J.
Published: July 2012

The presence of A1 adenosine receptors in CHO‐K1 cells transfected with the human brain A1 sequence was confirmed by ligand binding studies using 8‐cyclopentyl‐[3H] 1,3‐dipropylxanthine ([3H]‐DPCPX).

Alterations in intracellular calcium ([Ca2+]i) were measured with the calcium‐sensitive dye, fura‐2.

N6‐cyclopentyladenosine (CPA), the selective A1 agonist, and 5′‐N‐ethylcarboxaminoadenosine (NECA), a relatively non‐selective adenosine receptor agonist, elicited rapid, biphasic increases in [Ca2+]i which involved both mobilisation from intracellular stores and calcium entry.

The calcium response to CPA was significantly inhibited by the selective A1 antagonist DPCPX. The non‐selective adenosine receptor, xanthine amino congener (XAC), was less potent.

The calcium response to CPA was completely prevented by pretreatment of the cells with pertussis toxin implicating the involvement of Gi in the receptor‐mediated response.

In summary, we present evidence for the coupling of transfected human brain A1 adenosine receptors in CHO‐K1 cells to mobilisation of [Ca2+]i via a pertussis toxin‐sensitive G protein.
Evidence for a cooperation between adenosine A2 receptors and β1‐adrenoceptors on cardiac automaticity in the isolated right ventricle of the rat
Authors: Hernandez J. , Figueira M.A. , Ribeiro J.A. , Pinto F.
Published: July 2012

The effects of the adenosine receptor agonists, 5′‐N‐ethyl‐carboxamidoadenosine (NECA) and 2‐[4‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS‐21680) on ventricular automaticity induced by a local injury in the isolated right ventricle of the rat were studied.

In concentrations ranging from 0.1 to 100 nm, NECA significantly increased ventricular automaticity. This effect was more reproducible when the adenosine receptor antagonist 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX) was present at 5 nm, a concentration that blocks A1 adenosine receptors.

The excitatory effect of NECA was not observed when DPCPX was present at a concentration of 10 μm, which antagonizes both A1 and A2 adenosine receptors, as well as when rats were reserpinized.

In reserpinized rats, NECA increased ventricular automaticity in the presence of isoprenaline and the β2‐adrenoceptor antagonist, ICI‐118,551, but not in the presence of the βradrenoceptor antagonists, bisoprolol or atenolol.

The A2a‐selective adenosine receptor agonist, CGS‐21680 (0.1 nm‐10 μm) was devoid of excitatory effect on ventricular automaticity. Binding studies of this compound to the rat ventricular membranes revealed that in the preparation there was no specific binding.

These results suggest that the excitatory effect of NECA on ectopic ventricular automaticity is dependent on endogenous catecholamines and is mediated by an A2 adenosine receptor which is in some way ‘linked’ to the β1‐adrenoceptor. These A2 receptors do not appear to be of the A2a adenosine receptor subtype.
Mediation by nitric oxide formation in the preoptic area of endotoxin and tumour necrosis factor‐induced inhibition of water intake in the rat
Authors: Calapai G. , Mazzaglia G. , Cilia M. , Zingarelli B. , Squadrito F. , Caputi A.P.
Published: July 2012

Drinking was induced in rats by 24 h of water deprivation. Water intake (ml) was evaluated for a 1 h period.

NG‐nitro‐l‐arginine methyl ester (l‐NAME, 5–10 μg, i.c.v., 50–100 ng into the preoptic area (POA)), an inhibitor of nitric oxide (NO) synthase, and methylene blue (50–100 ng into POA), an inhibitor of guanylate cyclase activation, antagonized the inhibition of drinking induced by E. coli endotoxin (LPS, 640 μg kg−1, i.v.) and tumour necrosis factor (TNFα, 40 ng, i.c.v.) in 24 h water‐deprived rats.

l‐Arginine (25, 50 and 100 ng), the precursor aminoacid of NO, but not the stereoisomer d‐arginine (100 ng), inhibited drinking induced by water deprivation when injected into the POA 30 min before water presentation (74.4% of inhibition with the highest dose). A dose of 12.5 ng l‐arginine into the POA did not exhibit antidipsogenic effects.

TNFα (20 and 40 ng, i.c.v.; 1.25, 2.5 and 5 ng into the POA) showed a dose‐dependent and powerful inhibition of drinking behaviour in water‐deprived rats (70.4% and 80.8%, i.c.v. and into POA, with the highest doses, respectively). A dose of 10 ng of TNFα given i.c.v. had no effect on the intake of water.

LPS and TNFα, given at doses (160 μg kg−1, i.v. and 10 ng, i.c.v., respectively) that did not influence drinking in water‐deprived rats, exhibited a strong antidipsogenic effect in water‐deprived rats treated with a dose of l‐arginine (12.5 ng, into the POA) which did not modify drinking by itself. (LPS + l‐arginine: 53.6% of inhibition; TNFα + l‐arginine: 52.0% of inhibition).

These results suggest that NO into the POA: (1) acts as an inhibitory mechanism on thirst and (2) plays a role in the antidipsogenic effect of LPS and TNFα.
On the nature of the 5‐HT receptor subtype inhibiting acetylcholine release in the guinea‐pig ileum
Authors: Ramírez M.J. , Del Río J. , Cenarruzabeitia E. , Lasheras B.
Published: July 2012

The nature of the 5‐hydroxytryptamine (5‐HT) receptor subtype controlling acetylcholine release and contraction induced by stimulation of the neurokinin NK3 receptor has been studied in the longitudinal muscle‐myenteric plexus preparation from guinea‐pig ileum.

In preparations preloaded with [3H]‐choline, the selective NK3 agonist, senktide, produced a concentration‐dependent increase in tritium overflow, an index of [3H]‐acetylcholine release. Low concentrations of neurokinin B, also markedly increased tritium efflux.

The senktide‐induced acetylcholine release was markedly increased by the same concentration of methysergide and mesulergine. The 5‐HT2A/2C agonist DOI (1 μm) inhibited the tritium overflow while 8‐OH‐DPAT, sumatriptan and ketanserin (1 μm each) were without effect on the senktide‐induced tritium efflux.

The contractile response to senktide in the guinea‐pig ileum was attenuated by atropine, 0.1 μm. Methysergide, a 5‐HT1/2 receptor antagonist, and mesulergine, a 5‐HT2A/2B/2C receptor antagonist, (1 μm each), enhanced the contractile effect of the NK3 receptor agonist.

It is concluded that the acetylcholine release induced by a NK3 receptor agonist is inhibited by stimulation of a 5‐HT receptor, possibly of the 5‐HT2C or 5‐HT2B subtype.
Pharmacological characterization of rabbit corpus cavernosum relaxation mediated by the tissue kallikrein‐kinin system
Authors: Lopes‐Martins Rodrigo A.B. , Antunes Edson , Oliva Maria Luiza V. , Sampaio Cláudio A.M. , Burton James , de Nucci Gilberto
Published: July 2012

The roles of the tissue kallikrein‐kinin system and nitric oxide (NO) release in Phoneutria nigriventer venom‐induced relaxations of rabbit corpus cavernosum (RbCC) smooth muscle have been investigated by use of a bioassay cascade.

Phoneutria nigriventer venom (10–30 μg), porcine pancreatic kallikrein (100 mu), rabbit urinary kallikrein (10 mu), bradykinin (BK, 0.3–3 nmol), acetylcholine (ACh, 0.3–30 nmol) and glyceryl trinitrate (GTN, 0.5–10 nmol) caused relaxations of the RbCC strips. Captopril (1 μm) substantially potentiated Phoneutria nigriventer venom‐ and BK‐induced RbCC relaxations without affecting those elicited by GTN.

The bradykinin B2 receptor antagonist, Hoe 140 (d‐Arg‐[Hyp3, Thi5,d‐Tic7, Oic8]‐BK, 50 nm), aprotinin (10 μg ml−1) and the tissue kallikrein inhibitor, Pro‐Phe‐Aph‐Ser‐Val‐Gln‐NH2 (KIZD‐06, 1.3 μm) significantly inhibited Phoneutria nigriventer venom‐induced RbCC relaxations, without affecting those provoked by GTN and ACh. The B1 receptor antagonist, [Leu9]des Arg10BK (0.5 μm) and soybean trypsin inhibitor (SBTI, 10 μg ml−1) had no effect on Phoneutria nigriventer venom‐induced RbCC relaxations.

The relaxations induced by Phoneutria nigriventer venom, porcine pancreas kallikrein, BK and ACh were significantly inhibited by Nω‐nitro‐l‐arginine methyl ester (l‐NAME, 10 μm) but not by d‐NAME (10 μm). l‐NAME did not affect GTN‐induced relaxations. l‐Arginine (300 μm), but not d‐arginine (300 μm), significantly reversed the inhibitory effect of l‐NAME.

Our results indicate that Phoneutria nigriventer venom activates the tissue kallikrein‐kininogen‐kinin system in RbCC strips leading to NO release and suggest a functional role for this system in penile erection.
Generation by the phosphoramidon‐sensitive peptidases, endopeptidase‐24.11 and thermolysin, of endothelin‐1 and C‐terminal fragment from big endothelin‐1
Authors: Murphy Leonard J. , Corder Roger , Mallet Anthony I. , Turner Anthony J.
Published: July 2012

Phosphoramidon, a potent inhibitor of endopeptidase‐24.11 (E‐24.11) and thermolysin, has been shown to reduce the hypertensive effect of exogenous big endothelin‐1 (big ET‐1) in rats. To examine whether E‐24.11 or thermolysin convert big ET‐1 to endothelin‐1 (ET‐1) and C‐terminal fragment (CTF), the effects on porcine and human big ET‐1 of each of the purified enzymes were compared in vitro.

For E‐24.11, the relative rates of hydrolysis were ET‐1 > CTF >> big ET‐1. The relative half‐lives for hydrolysis of 3 nmol of each peptide by 200 ng enzyme were: big ET‐1 > 24 h; ET‐1, 37 min; CTF, 57 min. For comparison, the half‐life for hydrolysis of substance P under similar conditions was 2.1 min.

For thermolysin the relative rates of hydrolysis were found to be big ET‐1 > CTF > ET‐1. The relative half‐lives for hydrolysis of 3 nmol peptide by 50 ng enzyme were: big ET‐1, 25 min; ET‐1, 56 min; CTF, 47 min.

Because the low rate of conversion of big ET‐1 to ET‐1 by E‐24.11 did not yield sufficient ET‐1 for h.p.l.c. quantification a RIA specific for ET‐1 (16–21) was used to study further the hydrolysis of big ET‐1 by E‐24.11. Incubation of big ET‐1 (0.2–2 nmol) with E‐24.11 (4–400 ng) generated ET‐1 levels of between 1.7 and 33 pmol measured by RIA. Incubation of big ET‐1 (2 nmol) with E‐24.11 (40 ng) for 8 h showed that steady state levels of ET‐1 were achieved after 4 h indicating that the rate of ET‐1 degradation was then equal to the formation of new ET‐1. Characterization of the immunoreactivity by h.p.l.c. and RIA confirmed that authentic ET‐1 had been produced, but the yield was insufficient for verification by mass spectrometry.

Both ET‐1‐like and CTF‐like peaks were detected at 214 nm when the products of big ET‐1 hydrolysis by thermolysin were resolved by h.p.l.c. RIA and mass spectrometry confirmed the production of ET‐1 with amounts in the range 120–160 pmol.

The hydrolysis profile of ET‐1 by E‐24.11 and thermolysin shows that both enzymes have some common cleavage sites consistent with their similar specificities hydrolysing on the amino side of a hydrophobic residue.

Thermolysin, for which 3D structural information is available, may represent a better model for endothelin converting enzyme (ECE) action than E‐24.11 and could be useful for the design of ECE inhibitors. Since E‐24.11 can both synthesize and hydrolyse ET‐1, the presence of E‐24.11 in membrane fractions or in partially purified ECE preparations may produce misleading estimates of ECE activity.
Mutations in transmembrane segment VIJ of the AT1 receptor differentiate between closely related insurmountable and competitive angiotensin antagonists
Authors: Schambye Hans T. , v. Wijk Bjørn , Hjorth Siv A. , Wienen Wolfgang , Entzeroth Michael , Bergsma Derk J. , Schwartz Thue W.
Published: July 2012

Chimeric constructs between the human and the Xenopus laevis AT1 receptor have demonstrated, that the binding of non‐peptide angiotensin antagonists is dependent on non‐conserved residues located deep in transmembrane segment VII of the AT1 receptor. Here we have studied four pairs of closely related antagonists each consisting of a competitive and an insurmountable compound differentiated by one out of three different types of minor chemical modifications. None of the antagonists bound to the Xenopus receptor and the binding of all of the compounds to the human receptor was severely impaired by the introduction of non‐conserved residues from transmembrane segment VII of the Xenopus receptor. In all four pairs of antagonists the competitive compound was affected more by these substitutions than the corresponding insurmountable one (209 vs. 22, 281 vs. 29, 290 vs. 29 and 992 vs. 325‐fold increase in K i values). A similar pattern was observed in response to substitution of a single non‐conserved residue in transmembrane segment VII, Asn295 to Ser. These results indicate that a common molecular mechanism distinguishes the interaction of insurmountable and competitive antagonists with the AT1 receptor.
Local cardiac effects of substance P: roles of acetylcholine and noradrenaline
Authors: Chiao Hsi , Caldwell Robert W.
Published: July 2012

The local cardiac actions of substance P were examined in isolated perfused hearts and atria of the guinea‐pig.

In both hearts and right atria, substance P caused negative inotropic and chronotropic effects.

Atropine (10−6 m) or depletion of acetylcholine, by electrical stimulation and hemicholinium‐3 perfusion, significantly attenuated the negative inotropic and chronotropic effects of substance P. α‐ and β‐adrenoceptor blockade by nadolol and phentolamine (10−6 m each) did not prevent the negative inotropic and chronotropic effects of substance P. This indicates that cholinergic neurones, but not adrenergic neurones, partially mediate the effects of substance P.

There was no significant difference in the effects of substance P observed between groups with acetylcholine depletion and with cholinoceptor blockade. This suggests that substance P elicits its effects mainly through release of acetylcholine.

These results indicate that substance P has negative inotropic and chronotropic effects in guinea‐pig hearts and right atria mediated partly by release of acetylcholine. Substance P also appears to have direct effects on cardiac tissue.
Modulation by nitric oxide of prostaglandin biosynthesis in the rat
Authors: Sautebin Lidia , Ialenti Armando , Ianaro Angela , Di Rosa Massimo
Published: July 2012

Modulation of prostaglandin biosynthesis in vivo by either exogenous or endogenous nitric oxide (NO) has been studied in the rat using arachidonic acid (AA)‐induced paw oedema and measuring both the foot volume and the amount of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α), the stable metabolite of prostacyclin (PGI2), in the oedematous fluid recovered from inflamed paws.

Paw injections of 150 or 300 nmol of AA were virtually inactive whereas 600 nmol produced a moderate oedema which was greatly reduced by the NO synthase inhibitor l‐NG‐nitro arginine methyl ester (l‐NAME, 100 nmol/paw) and the NO scavenger haemoglobin (Hb, 30 μmol/paw), but unaffected by the inhibitor of the soluble guanylate cyclase, methylene blue (Mb, 3 μmol/paw) and L‐arginine (15 μmol/paw).

The NO‐donors (10 μmol/paw) 3‐morpholino‐sydnonimine‐hydrochloride (SIN‐1), S‐nitroso‐N‐acetyl‐d, l‐penicillamine (SNAP) and sodium nitroprusside (SNP) significantly potentiated the paw oedema induced by AA (300 nmol/paw).

SIN‐1 (2.5, 5 and 10 μmol/paw) produced a significant dose‐dependent increase of the oedema induced by AA which was correlated with increased amounts of 6‐keto‐PGF1α in the fluid recovered from inflamed paws.

Both oedema and prostaglandin biosynthesis induced by the combination AA + SIN‐1 were greatly suppressed by either Hb (30 μmol/paw) or indomethacin (3 μmol/paw or 5 mg kg−1 s.c.) but unaffected by Mb (3 μmol/paw).

In LPS‐treated rats (6 mg kg−1, i.p.) doses of AA inactive in normal animals produced a remarkable oedema which was reduced by l‐NAME or Hb, unaffected by Mb and increased by l‐arginine.

These results demonstrate that NO increases prostaglandin biosynthesis in vivo through a guanosine 3′:5′‐cyclic monophosphate (cyclic GMP)‐independent mechanism and suggest that the interaction between NO synthase and cyclo‐oxygenase (COX) pathways may represent an important mechanism for the modulation of the inflammatory response.
Effects of nitric oxide synthase inhibition combined with nitric oxide inhalation in a porcine model of endotoxin shock
Authors: Klemm Peter , Thiemermann Christoph , Winklmaier Gisela , Martorana Piero A. , Henning Rainer
Published: July 2012

The present investigation compares the effects of intravenous infusion of the NO synthase inhibitor NG‐monomethyl‐l‐arginine (l‐NMMA) with that of an inhalation with NO gas in a porcine model of endotoxin (lipopolysaccharide, LPS) shock. In addition, the effects of the combination of these two treatments were also investigated.

Male pigs were anaesthetized and instrumented for the measurement of haemodynamic parameters. Blood samples were withdrawn at different time intervals for determination of blood gases, pH, and plasma levels of nitrite/nitrate and tumour necrosis factor.

Endotoxin infusion (15 μg kg−1 h−1 for 3 h) caused a progressive fall in mean arterial blood pressure (MABP) and cardiac output (CO) and a biphasic increase in mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistence (PVR). A continuous infusion of l‐NMMA (0.1 mg kg−1 min−1) significantly attenuated the fall in MABP, but did not affect MPAP, CO and PVR. NO‐inhalation (50 p.p.m.) did not affect MABP, but significantly blunted the biphasic increase in MPAP and PVR and significantly delayed the fall in CO. The combination of l‐NMMA infusion (0.1 mg kg−1 min−1) with NO‐inhalation (50 p.p.m.) completely prevented the fall in MABP, significantly improved CO, and attenuated the biphasic increase in MPAP and PVR.

Endotoxin also caused a decline in Pao2 and a rise of Paco2. Infusion of l‐NMMA neither affected the fall in Pao2 nor the increase in Paco2. In contrast, inhalation with NO gas alone as well as the combined administration of l‐NMMA infusion and NO‐inhalation completely prevented the fall in Pao2 and significantly protected against the increase in Paco2.

Infusion of endotoxin for 180 min resulted in a mortality of 58%, which was not affected by l‐NMMA (63%). In contrast, treatment of LPS‐animals with either NO‐inhalation alone or NO‐inhalation plus l‐NMMA completely prevented mortality.

This investigation demonstrates that treatment with NO‐inhalation, in order to prevent the dramatic increase in MPAP, PVR and the alterations in peripheral blood gases combined with systemic l‐NMMA to improve systemic MABP and thus organ perfusion, may be a new therapeutic regimen in the treatment of septic shock.