bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective antagonism of capsaicin by capsazepine: evidence for a spinal receptor site in capsaicin‐induced antinociception
Authors: Dickenson A.H. , Dray A.
Published: July 2012

Capsazepine has recently been described as a competitive capsaicin antagonist. We have used this compound to test the hypotheses that the in vitro and in vivo effects of capsaicin are due to interactions with a specific receptor.

In an in vitro preparation of the neonatal rat spinal cord with functionally connected tail, the activation of nociceptive afferent fibres by the application of capsaicin, bradykinin or noxious heat (48°C) to the tail could be measured by recording a depolarizing response from a spinal ventral root. Application of capsaicin or substance P to the spinal cord also evoked a depolarizing response which was recorded in a ventral root.

When capsazepine (50 nm‐20 μm) was administered to the tail or spinal cord it did not evoke any measurable response. However on the tail, capsazepine reversibly antagonized (IC50 = 254 ± 28 nm) the responses to capsaicin but not to heat or bradykinin administered to the same site. Similarly capsazepine administration to the spinal cord antagonized the responses evoked by capsaicin (IC50 = 230 ± 20 nm) applied to the cord but not responses evoked by substance P on the cord or by noxious heat and capsaicin on the tail.

In halothane anaesthetized rats, C‐fibre responses evoked by transcutaneous electrical stimulation of the receptive field were recorded from single wide dynamic range neurones located in the spinal dorsal horn. C‐fibre evoked discharges were consistently reduced by the systemic administration of capsaicin (20 μmol kg−1, s.c.) and this action of capsaicin was antagonized by capsazepine (100 μmol kg−1) administered by the same route. In addition the systemic effect of capsaicin was antagonized by a spinal intrathecal administration of capsazepine (5–50 nmol).

Intradermal injections of capsaicin, localized to the peripheral receptive field, usually one toe of the ipsilateral hind‐paw, produced a transient increase in C‐fibre‐evoked activity followed by a prolonged period of localized insensitivity to transcutaneous C‐fibre stimulation. These effects of capsaicin were significantly reduced by the concommitant administration of capsazepine to the same site.

These data demonstrate that capsazepine is a selective antagonist of capsaicin on nociceptive neurones in vitro and in vivo and suggest that the effects of capsaicin were mediated by activation of a specific receptor. Since the antinociceptive effect produced by systemically administered capsaicin was antagonised by spinal intrathecal capsazepine this further supports the hypothesis that capsaicin exerts its antinociceptive effect by acting on specific receptors localized to sensory nerve fibres in the spinal cord.;
Enteral absorption of octreotide
Authors: Flicker G. , Drewe J. , Vonderscher J. , Kissel T. , Beglinger C.
Published: July 2012

The somatostatin octapeptide‐analogue, octreotide, is absorbed as intact peptide from the gastrointestinal (GI) tract.

In situ absorption experiments in rats confirmed our recent intubation studies in human volunteers demonstrating that the peptide has preferential absorption sites in the small intestine. Absorption of octreotide was higher in the jejunum than in the duodenum or the ileum.

Experiments with bile‐duct cannulated rats demonstrated that the absorption of octreotide decreased in the presence of bile, reflecting a negative influence of biliary components on the absorption of the peptide.

Uptake experiments using rat jejunal brush border membranes were performed to analyse the absorption mechanisms. The transport of octreotide into jejunal brush border membranes was significantly higher than the uptake into membrane vesicles isolated from rat ileum. When initial uptake (0–15 s) rates into the membrane vesicles were calculated as a function of the peptide concentration, a saturable component could be observed, indicative of transport mechanisms different from simple diffusion.
ATP overflow from the mouse isolated vas deferens
Authors: Drake M. Eliane , Petersen S.A.
Published: July 2012

A modified form of the highly specific luciferin‐luciferase assay was used to measure the overflow of adenosine 5′‐triphosphate (ATP) from the field‐stimulated mouse vas deferens in vitro. Precise timing of the stimulation, superfusate collection and assay minimized ATP degradation before assay, offering the opportunity for quantitative studies.

Stimulation with between 50 and 200 supramaximal pulses at 5 Hz increased ATP overflow by between 7 and 28 times over basal.

ATP overflow increased steadily with increasing numbers of stimuli from 50 to 200 pulses. Increasing the frequency of stimulation initially had no effect, but above 10 Hz there was increased overflow, suggesting an interaction between facilitated release and the rate of degradation by extracellular ATPases.

Contractions of the vas induced by exogenous (−)‐phenylephrine produced only a small increase in ATP overflow, suggesting that the stimulation induced increase in ATP overflow is mostly pre‐junctional in origin, though tetrodotoxin eliminated stimulation‐induced overflow only in a proportion of preparations.
Effectiveness of GABAB antagonists in inhibiting baclofen‐induced reductions in cytosolic free Ca concentration in isolated melanotrophs of rat
Authors: Shibuya I. , Kongsamut S. , Douglas W.W.
Published: July 2012

The purpose of the present experiments was to assess the activities of GABAB receptor antagonists in mammalian isolated melanotrophs.

Cytosolic free Ca concentration ([Ca2+]i) in rat melanotrophs in primary culture was monitored with the fluorescent probe, fura‐2.

(−)‐Baclofen lowered [Ca2+]i in a concentration‐dependent manner with an EC50 of 0.96 μm. The reduction in [Ca2+]i produced by (−)‐baclofen at a maximally effective concentration (100 μm) was similar to that produced by the classic transmitter inhibitory to melanotroph secretion, dopamine, at a corresponding concentration (100 nm), or by perifusion with a nominally Ca‐free solution.

The GABAB receptor antagonists, 3‐aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348), 2‐hydroxy saclofen, phaclofen and 4‐amino‐3‐(5‐methoxybenzo[b]furan‐2‐yl) butanoic acid (9H), had inhibitory effects on the reduction in [Ca2+]i produced by (−)‐baclofen (3 μm). Of the antagonists tested, CGP 35348 was the most potent with an IC50 of 60 μm, compared to 120 to 400 μm for the others. CGP 35348 acted competitively.

CGP 35348 alone had no effect on basal [Ca2+]i, or on the changes in [Ca2+]i produced by dopamine (10 nm) or the specific GABAA receptor agonist, muscimol (10 μm).

The evidence indicates that of the antagonists tested, CGP 35348 offers the greatest promise for pharmacological analysis of the functional significance of the GABAB receptors in melanotrophs.
Cardiovascular actions of a new selective postjunctional α.‐adrenoceptor antagonist, SK&F 104856, in normotensive and hypertensive dogs
Authors: Hieble J. Paul , Nichols Andrew J. , Fredrickson Todd A. , DePalma P. Dennis , Ruffolo Robert R. , Brooks David P.
Published: July 2012

SK&F 104856 (2‐vinyl‐7‐chloro‐3,4,5,6‐tetrahydro‐4‐methylthieno[4,3,2ef][3]benzazepine) is a novel postjunctional α.1‐ and α.2‐adrenoceptor antagonist.

SK&F 104856 as well as prazosin and SK&F 86466 reduced blood pressure in the anaesthetized normotensive dog.

SK&F 86466 and rauwolscine but not SK&F 104856 or prazosin, produced a marked increase in myocardial contractility which corresponds with their ability to block prejunctional α.2‐adrenoceptors.

Intravenous or oral administration of SK&F 104856 resulted in dose‐dependent antihypertensive responses in 1‐kidney, 1‐clip (1‐K, 1‐C) Goldblatt hypertensive dogs with baseline blood pressure of approximately 140 mmHg. At 0.1 and 1 mg kg−1, i.v., mean arterial blood pressure fell by 11 ± 5 and 23 ± 5 mmHg, respectively. At 3 and 10 mg kg−1, p.o., blood pressure fell by 9 ± 3 and 22 ± 5 mmHg, respectively. At 10 mg kg−1, p.o., the antihypertensive effect of SK&F 104856 was still evident at 4 h.

The data indicate that SK&F 104856 shows selectivity in vivo for postjunctional versus prejunctional α.‐adrenoceptors and is a potent and long‐acting antihypertensive agent in 1‐K, 1‐C Goldblatt hypertensive dogs.
Pre‐clinical pharmacology of ICI D2138, a potent orally‐active non‐redox inhibitor of 5‐lipoxygenase
Authors: McMillan R.M. , Spruce K.E. , Crawley G.C. , Walker E.R.H. , Foster S.J.
Published: July 2012

This paper describes the pre‐clinical pharmacology of ICI D2138, a potent orally‐active non‐redox inhibitor of 5‐lipoxygenase which is undergoing clinical evaluation.

ICI D2138 potently inhibited leukotriene synthesis in murine peritoneal macrophages (IC50 = 3 nm) and human blood (IC50 = 20 nm). In human and dog blood, ICI D2138 did not inhibit thromboxane B2 synthesis at a concentration of 500 μm, thus the selectivity ratio (cyclo‐oxygenase: 5‐lipoxygenase) was greater than 20,000. In contrast, zileuton (a 5‐lipoxygenase inhibitor also undergoing clinical evaluation) exhibited a selectivity ratio of 15–100.

ICI D2138 potently and dose‐dependently inhibited ex vivo leukotriene B4 (LTB4) synthesis by rat blood with ED50 values of 0.9, 4.0 and 80.0 mg kg−1 p.o. at 3, 10 and 20 h respectively after dosing. Similar activity was observed for inhibition of LTB4 production in a zymosan‐inflamed rat air pouch model. Zileuton produced ED50 values of 5 and 20 mg kg−1 at 3 and 10 h respectively.

Oral administration of 1, 3 or 10 mg kg−1 ICI D2138 to dogs produced maximal inhibition of ex vivo LTB4 synthesis by blood for 5, 9 and 31 h respectively. A dose of 5 mg kg−1 p.o. of zileuton caused maximal inhibition of LTB4 for 24 h.

Oral administration of 10 mg kg−1 ICI D2138 caused total inhibition of LTB4 production in zymosan‐inflamed rabbit knee joint.

Topical administration of ICI D2138 to rabbit skin caused a dose‐related inhibition of arachidonic acid‐induced plasma extravasation with an ID30 of 1.08 nmol per site. Zileuton was approximately 40 times less potent.

Oral anti‐inflammatory activity was assessed in an arachidonic acid‐induced mouse ear oedema model in animals treated with indomethacin to block pro‐inflammatory prostanoids. ICI D2138, given orally, caused dose‐dependent inhibition of oedema with an approximate ID50 of 1.8 mg kg−1. Zileuton was approximately 10 times less potent.

ICI D2138 caused a dose‐dependent inhibition of antigen‐induced broncho‐constriction in guinea‐pigs with an approximate ID50 of 0.1 mg kg−1, i.v. Zileuton was approximately 10 times less potent.

In view of the pharmacological profile described here, ICI D2138 has the potential to provide improved clinical efficacy compared to existing lipoxygenase inhibitors such as zileuton.
Effect of a 5‐lipoxygenase inhibitor and leukotriene antagonist (PF 5901) on PAF‐induced airway responses in neonatally immunized rabbits
Authors: Herd Caroline M. , Donigi‐Gale Donna , Shoupe T. Scott , Page Clive P.
Published: July 2012

Aerosol administration of platelet activating factor (PAF) (80 μg ml−1 for 60 min) to neonatally immunized rabbits caused bronchoconstriction which was far in excess of that produced by a comparable aerosol of bovine serum albumin (BSA), the carrier molecule for PAF. Bronchoconstriction of a similar magnitude was elicited by PAF in immunized, sham‐immunized and normal rabbits.

Aerosol administration of PAF to immunized rabbits induced enhanced airway responsiveness to inhaled histamine in all animals tested, 24 h and 72 h after exposure. In not all cases had airways responsiveness returned to basal levels at 1 week following PAF challenge. In contrast, following exposure of immunized rabbits to BSA, no significant changes in airway responsiveness to histamine were evident at any of the measured time points.

A significant increase in the total number of inflammatory cells recovered in bronchoalveolar lavage (BAL) fluid was determined 24 h and 72 h following PAF exposure in immunized rabbits. This was associated with a significant increase in the number of neutrophils and eosinophils. Similar changes were observed following exposure of PAF to normal and sham‐immunized rabbits. No change in the total number of inflammatory cells was obtained in BAL after BSA challenge to immunized rabbits; however, neutrophil numbers were significantly increased.

PF 5901, a specific inhibitor of the 5‐lipoxygenase pathway of arachidonic acid metabolism and a leukotriene D4 antagonist, at a dose of 10 mg (direct intratracheal administration) significantly inhibited the airway resistance (RL) component of the bronchoconstriction induced by PAF in neonatally‐immunized rabbits. Doses of 10 mg, 3 mg and 1 mg PF 5901 (direct intratracheal administration) were sufficient to inhibit significantly the PAF‐induced increase in airways responsiveness to inhaled histamine in immunized rabbits. PF 5901 however, failed to alter the pulmonary cell infiltration induced by PAF, as assessed by BAL.

We suggest from the results of the present study that PAF induces consistent and long‐lasting increases in airways responsiveness to histamine in immunized rabbits, which is mediated, at least in part, by products of the 5‐lipoxygenase metabolic pathway. Furthermore, the inability of PF5901 to inhibit the influx of inflammatory cells into the airway lumen following PAF challenge may suggest that bronchial hyperresponsiveness and cellular infiltration are not strictly associated events.
The role of nitric oxide in endothelial cell damage and its inhibition by glucocorticoids
Authors: Palmer R.M.J. , Bridge L. , Foxwell N.A. , Moncada S.
Published: July 2012

Incubation of vascular endothelial cells with S.typhosa endotoxin and interferon‐γ caused a time‐ and concentration‐dependent reduction in the viability of the cells. This cytotoxic effect was inhibited in a concentration‐dependent manner by NG‐monomethyl‐l‐arginine, an inhibitor of nitric oxide (NO) synthesis, and by the glucocorticoids dexamethasone and hydrocortisone, two inhibitors of the induction of NO synthase. These findings indicate that in these cells the cytotoxic effect of endotoxin is mediated by the NO synthesized by an inducible NO synthase. This induction of NO synthase in vascular endothelial cells may represent a mechanism of local endothelial damage during endotoxin shock and other immunologically based conditions.
Alteration of the L‐type calcium current in guinea‐pig single ventricular myocytes by heptaminol hydrochloride
Authors: Peineau N. , Mongo K.G. , Le Guennec J.‐Y. , Garnier D. , Argibay J.A.
Published: July 2012

The effects of heptaminol on calcium current amplitude and characteristics were studied in single ventricular myocytes of guinea‐pig by use of the whole cell configuration of the patch clamp technique.

A concentration‐dependent decrease in ICa amplitude was observed. At heptaminol concentration as low as 10−6 m, this effect was observed in only two cells (n = 6). At 10−5 m the reduction of ICa was of 30 ± 15% (n = 11).

The current recovery from inactivation at — 40 mV holding potential (HP) seemed less sensitive to perfusion with heptaminol (> 10−6 m). However, at — 80 mV HP the overshoot of the recovery curve was decreased by heptaminol.

Both at — 40 mV and — 80 mV HP, heptaminol (10−5 m) significantly increased the steady state inactivation of ICa.

As previously proposed by others to explain the effects of membrane active substances, the effects of heptaminol may result from alterations in cell membrane properties and possibly from an increase in intracellular free calcium ion concentration.
Phosphodiesterase inhibition in ventricular cardiomyocytes from guinea‐pig hearts
Authors: Bethke Thomas , Meyer Wilfried , Schmitz Wilhelm , Scholz Hasso , Stein Birgitt , Thomas Klaus , Wenzlaff Holger
Published: July 2012

The present study compared the cyclic nucleotide phosphodiesterase (PDE) activities in cardiomyocytes and ventricular cardiac tissue from guinea‐pigs. The aim of the study was to determine whether PDE activities in ventricular tissue accurately reflect the isoenzymes present in cardiomyocytes.

In homogenates of cardiomyocytes and multicellular ventricular tissue, four distinct soluble PDE activities could be separated by DEAE‐sepharose chromatography.

In multicellular cardiac tissue as well as in cardiomyocyte preparations, adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) PDE isoenzymes I‐IV were comparable in terms of substrate affinities, and inhibition or stimulation by guanosine 3′:5′‐cyclic monophosphate (cyclic GMP). However, in cardiomyocytes the Vmax values of PDE I‐IV were lower by a factor of about 2 to 7 and the basal activities were lower by a factor of about 3 to 5 as compared to multicellular cardiac tissue.

To investigate whether the PDE I‐IV activities were similarly inhibited by PDE inhibitors in both preparations, we studied the effects of 3‐isobutyl‐1‐methylxanthine (IBMX), UD‐CG 212 Cl (2‐(4‐hydroxy‐phenyl)‐5‐(5‐methyl‐3‐oxo‐4,5‐dihydro‐2H‐6‐pyridazinyl)benzimidazole HCl) and rolipram. UD‐CG 212 Cl was a selective PDE III inhibitor in cardiomyocytes (IC50 0.3 μmol l−1) and in ventricular tissue (IC50 value 0.1 μmol l−1). Rolipram selectively inhibited PDE IV in cardiomyocytes (IC50 1.4 μmol ml−1) and in ventricular tissue (IC50 1.1 μmol l−1) whereas IBMX was a nonselective PDE inhibitor in both preparations.

It is concluded that the PDE isoenzymes I‐IV from multicellular ventricular tissue can be used as a representative system for investigating PDE inhibiting properties of PDE inhibitors in the myocardium since comparable PDE isoenzymes I‐IV exist in guinea‐pig ventricular cardiomyocytes and multicellular ventricular tissue.
Protein kinase C inhibitors enhance endothelin‐1 and attenuate vasopressin and angiotensin II evoked [Ca2+]i elevation in the rat cardiomyocyte
Authors: Xu Yanjun , Sandirasegarane L. , Gopalakrishnan Venkat
Published: July 2012

Primary cultures of neonatal rat cardiomyocytes were pretreated for 16 h with either nonselective (staurosporine, 100 nm) or selective (NPC15437, 20 μm) protein kinase C (PKC) inhibitors. These inhibitors did not affect the basal cytosolic free calcium, [Ca2+]i, level (106 ± 12 nm) as determined by fura‐2 fluorescence methodology. Both agents significantly enhanced the maximal [Ca2+]i responses to endothelin‐1 (ET‐1) and attenuated the peak [Ca2+]i responses to arginine vasopressin and angiotensin II. They did not alter the EC50 values of any of these agonists. Since depletion of [Ca2+]o led to only partial attenuation of the enhanced response to ET‐1 in the treatment groups, it is likely that PKC inhibition results in an exaggerated intracellular mobilization of Ca2+ to ET‐1. It is concluded that PKC modulates agonist(s)‐evoked intracellular Ca2+ mobilization and that the nature of regulation is governed by the agonist.
Chloride anion concentration as a determinant of renal vascular responsiveness to vasoconstrictor agents
Authors: Quilley Caroline P. , Lin Yu‐Shi R. , McGiff John C.
Published: July 2012

The role of chloride concentration in modulating vasoconstrictor responses of the rat isolated kidney, perfused with Krebs‐Henseleit solution, to angiotensin II (AII), arginine vasopressin (AVP) and phenylephrine (PE) was investigated.

Reduction of perfusate chloride from a high (117 mm) to low (87 mm) concentration, by substitution of sodium chloride with a mixture of sodium salts of propionate, acetate and methanesulphonate, reduced responsiveness to all three vasoconstrictors, the change for AII being most pronounced.

For AII, reduced vasoactivity with low chloride was evident both in terms of the threshold dose and on the linear part of the dose‐response curve but not for the maximum response. This attenuating effect of low chloride on the vasoconstrictor response to AII was reversed when perfusion with high chloride was reinstituted. Continuous perfusion with high chloride progressively increased the vasoconstrictor effect of low doses of AII for successive dose‐response curves.

In addition to reducing responses on the linear part of the dose‐response curve for both AVP and PE, low chloride also reduced the maximum vasoconstrictor response to PE, whereas the threshold dose for the two agonists was unchanged. In contrast to the enhanced pressor response to AII, during continuous perfusion with high chloride, tachyphylaxis occurred with AVP and PE.

The ability of chloride to modify renal responsiveness to vasoconstrictor agents may contribute to the increase in renal vascular resistance and decrease in glomerular filtration rate (GFR) which occurs during infusion of hyperchloremic solutions into the renal artery and explain the need for chloride as the anion accompanying sodium in salt‐sensitive hypertensive models.
Ionic effects on amiloride block of the mechanosensitive channel in Xenopus oocytes
Authors: Lane John W. , McBride Don W. , Hamill Owen P.
Published: July 2012

Patch clamp techniques were used to measure the ionic dependence of amiloride block of single mechanosensitive (MS) channels in frog (Xenopus laevis) oocytes.

The primary aim was to determine whether the difference in potency of amiloride block of MS channels in frog oocytes (IC50 = 0.5 mm) and chick auditory hair cells (IC50 = 50 μm) was due to the different ionic recording solutions.

Amiloride block of the oocyte MS channel does not vary significantly with complete substitution of external Na+ (i.e. 100 mm) with K+ in Ca2+‐free recording solution (in both Na+ and K+ the IC50 = 0.5 mm).

A physiological concentration (1.8 mm) of external Ca2+ blocks the oocyte MS channel and reduces the potency of amiloride block (IC50 = 1.1 mm) without altering the voltage‐dependence or the Hill coefficient (n = 1.8) of amiloride block. The reduction in potency can be explained by surface charge screening by Ca2+ which reduces the effective amiloride surface concentration.

The present results indicate that factors other than ionic recording conditions must underlie the difference in potency of amiloride block of MS channels in oocytes and auditory hair cells.
A comparison of the actions of agonists and antagonists at non‐NMDA receptors of C fibres and motoneurones of the immature rat spinal cord in vitro
Authors: Pook P. , Brugger F. , Hawkins N.S. , Clark K.C. , Watkins J.C. , Evans R.H.
Published: July 2012

The shift in d.c. potential in dorsal roots (EC50 8.0 μm ± 0.9 s.e.mean, n = 5) or depression of the C elevation of the compound action potential (EC50 3.0 μm ± 0.3, n = 7) have been used to measure the depolarizing action of kainate on dorsal root C fibres of immature (3 to 5 day old) rats. Depolarization of motoneurones was measured from the shift in d.c. potential in ventral roots.

6‐Cyano‐7‐nitroquinoxaline,2–3,dione (CNQX) (pA2 5.78 ± 0.06, n = 8) and 6‐nitro‐7‐suplhamobenzo(f)quinoxaline‐2,3‐dione (NBQX) (pA2 5.75 ± 0.04, n = 7) had similar potencies as antagonists of kainate at dorsal root fibres. The potency of NBQX as a kainate antagonist was similar also at motoneurones (pA2 5.72 ± 0.07, n = 3). At motoneurones, NBQX was less potent as an antagonist of domoate (pA2 5.29 ± 0.05) and more potent as an antagonist of S‐α‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) (pA2 6.80 ± 0.09) than as an antagonist of kainate.

Application of l‐glutamate, quisqualate and RS‐AMPA to dorsal roots produced only short lasting depolarizations but kainate concentration‐effect plots were shifted to the right in the presence of these three agonists (pA2 5.08 ± 0.08, (n = 3), 5.59 ± 0.04, (n = 4) and 4.46 ± 0.04 (n = 4) respectively). Slopes of dose‐ratio against concentration were significantly less than one for the latter antagonism.

The amplitude of depolarizations induced by l‐glutamate, AMPA and quisqualate were increased up to ten fold and those induced by kainate up to two fold following treatment of dorsal roots with concanavalin A. The duration of the responses was increased also by the latter treatment. Folowing 85 s applications of glutamate, quisqualate, AMPA and kainate the mean respective times (s ± s.e.mean (n)) taken for responses to decay to half the peak amplitude were increased from 63 ± 7 (10), 86 ± 17 (4), 95 ± 19 (4) and 135 ± 3 (12) to 202 ± 49 (10), 147 ± 7 (4), 160 ± 13 (6) and 163 ± 10 (10). Under similar conditions the mean decay time of γ‐aminobutyric acid‐induced responses was 145 ± 7 (10). This was not significantly altered by concanavalin A treatment.

Application to dorsal roots of l‐aspartate at concentrations up to 5 mm (with or without concanavalin A treatment), the selective metabotropic agonist 1S,3R‐trans‐1‐aminocyclopentane‐1,3‐dicarboxylate (1 mm,) and d‐serine (20 μm) in the presence or absence of N‐methyl‐d‐aspartate (NMDA, 500 μm) neither depolarized the preparations nor shifted the kainate concentration‐effect plot.

It is concluded that primary afferent C fibres possess only one type of non‐NMDA receptor which is activated strongly by domoate or kainate but only weakly by AMPA. This receptor is readily desensitized by glutamate, quisqualate or AMPA and it is less readily desensitized by kainate.
Renal selective N‐acetyl‐l‐γ‐glutamyl prodrugs: studies on the selectivity of some model prodrugs
Authors: Drieman J.C. , Thijssen H.H.W. , Struyker‐Boudier H.A.J.
Published: July 2012

In this study, a number of structurally different N‐acetyl‐l‐γ‐glutamyl prodrugs were investigated with respect to selective uptake by the kidney in male Wistar rats.

All prodrugs were tested in vitro in rat kidney slices and kidney homogenate to study their uptake and conversion. It was found that the prodrugs of para‐nitroaniline (agPNA), aminophenyl acetic acid (agAFA), sulphamethoxazole (agSM), sulphadimethoxine (agSDM), propranolol (agPP) and metoprolol (agMP) were accumulated by a probenecid‐sensitive carrier. The prodrug of 4′‐aminoantipyrine (agAAP) was not accumulated by a probenecid‐ or buthionine sulphoximine‐sensitive carrier. Unlike all other prodrugs, agAAP and agMP were not, or only a very limited extent converted to the parent compound in vitro.

agPNA, agAFA and agPP were also investigated in vivo. The tissue distribution of the prodrugs and the parent drugs was established, as was their urinary excretion and pharmacokinetic behaviour. agPNA and agAFA showed selective uptake by the kidney, in contrast to agPP which accumulated in the liver. The distribution of the parent compounds following prodrug administration was as follows: agPNA was found in kidney and plasma: agAFA in kidney only; agPP in liver only.

The factors which determine the selectivity of N‐acetyl‐l‐γ‐glutamyl prodrugs are discussed. The main factors are: the transport into the kidney, the conversion rate, the residence time of the prodrug in the kidney and the presence or absence of competition for uptake and conversation by other tissues, e.g. the liver. It is concluded that this prodrug approach offers the possibility of delivering drugs selectively to the kidney, but also that it is not universally applicable.
The effect of cyclic AMP and cyclic GMP phosphodiesterase inhibitors on the superoxide burst of guinea‐pig peritoneal macrophages
Authors: Turner Nicholas C. , Wood Lorna J. , Burns Fiona M. , Gueremy Thomas , Souness John E.
Published: July 2012

The cyclic nucleotide phosphodiesterase (PDE) activity of guinea‐pig peritoneal macrophages was partially characterized and the effects of selective and non‐selective inhibitors of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP PDE) and guanosine 3′:5′‐cyclic monophosphate (cyclic GMP PDE) phosphodiesterases on superoxide generation were investigated using peritoneal macrophages from horse‐serum pretreated guinea‐pigs.

The non‐selective PDE inhibitor, 3‐isobutyl‐1‐methylxanthine (IBMX) and the PDE I/V selective inhibitor, zaprinast, inhibited spontaneous superoxide generation with IC50s of 30.7 ± 11.3 μm and 145 ± 17 μm respectively (n = 6 and 5). The concentration‐response curves for the PDE IV selective inhibitors rolipram and Ro20–1724 were biphasic; mean maximum inhibitions were 56.9 ± 5.9% and 66.8 ± 10.5% respectively at 300 μm, but in 2 out of 6 (rolipram) and 2 out of 5 (Ro20–1724) experiments inhibition was < 50%. The PDE III inhibitor SK&F 94120 was without effect. Spontaneous superoxide generation was reduced 57 ± 10% by 1 μm prostaglandin E2 (PGE2) and 62.6 ± 3.76% by 1 μm salbutamol.

The increase in superoxide generation elicited by FMLP (10−9−10−5 m) was unaffected by any of the PDE inhibitors studied. Inhibition of FMLP‐stimulated superoxide generation by PGE2 was enhanced in the presence of 10 μm IBMX.

Macrophages were found to contain a predominantly membrane bound cyclic AMP PDE (90% of total activity) which was unaffected by cyclic GMP or calcium/calmodulin. The cyclic AMP PDE activity in the cytosolic fraction was enhanced in the presence of calcium/calmodulin. Selective inhibitors of PDE IV inhibited the particulate cyclic AMP PDE activity (IC50s rolipram 1.5 ± 0.3 μm, Ro 20–1724 4.1 ± 0.6 μm) as did the non‐selective inhibitor IBMX (IC50 22 ± 8 μm). The macrophage particulate PDE activity was resistant to inhibition by the PDE III inhibitor SK&F 94836 and the PDE I/V inhibitor, zaprinast. The cytosolic calcium/calmodulin stimulated cyclic AMP hydrolytic activity was inhibited by zaprinast (IC50 ‐ calcium/calmodulin 123 ± 39 μm; + calcium/calmodulin IC50 17.7 ± 6.3 μm).

The results indicate that guinea‐pig peritoneal macrophages contain a type IV cyclic AMP PDE which is predominantly membrane associated and a predominantly cytosolic calcium/calmodulin stimulated cyclic AMP PDE. Functional studies suggest that both of these PDE activities contribute to cyclic AMP hydrolysis and regulation of superoxide generation in these cells. Inhibition of spontaneous superoxide generation, but not that stimulated by FMLP, suggests that the activity of PDE inhibitors is subject to functional antagonism but that this can be overcome by enhancing cyclic AMP formation.
Effects of the immunosuppressants cyclosporin A and FK 506 on exocytosis in the rat exocrine pancreas in vitro
Authors: Waschulewski Ingo H. , Hall Diane V. , Kern Horst F. , Edwardson J. Michael
Published: July 2012

We have examined the effects of the immunosuppressive drugs cyclosporin A (CsA) and FK 506 on exocytosis in two in vitro preparations of the exocrine pancreas—lobules and dispersed acini.

In lobules taken from starved rats and stimulated with the secretagogue caerulein, both CsA and FK 506, given shortly before stimulation, caused a dose‐dependent inhibition of amylase secretion. In lobules from rats that had been pretreated in vivo with the protease inhibitor FOY‐305 to stimulate secretion maximally, both CsA and FK 506 inhibited secretion of newly synthesized proteins, whereas only FK 506 inhibited caerulein‐stimulated amylase release.

These different effects of the immunosuppressants on amylase release were reflected in their effects on degranulation, as revealed by electron microscopy. Control acinar cells in lobules from FOY‐305‐treated rats were almost completely degranulated, whereas treatment with FK 506, but not CsA, caused the accumulation of zymogen granules close to the apical plasma membrane.

In dispersed acini, stimulated with the cholinomimetic secretagogue bethanechol, both CsA and FK 506 reduced the secretory response, to about 45% of control; IC50 values were 50 nm and 3 nm, respectively. A similar partial inhibition of exocytosis was seen in acini permeabilized with the bacterial toxin streptolysin O and stimulated with 10 μm Ca2+.

These results demonstrate that the immunosuppressants cause an inhibition of exocytosis in the exocrine pancreas that is both rapid in onset and potent. The loss of the inhibitory effect of CsA on amylase release in lobules taken from FOY‐305‐treated rats may reveal a change in the characteristics of exocytosis as a consequence of the high level of stimulation, and also indicates that CsA and FK 506 have subtly different effects on secretion. We suggest that these drugs might be useful tools in the dissection of the molecular mechanisms of exocytosis.
α2‐Adrenoceptor antagonist potencies of two hydroxylated metabolites of yohimbine
Authors: Berlan Michel , Verge Roger Le , Galitzky Jean , Corre Pascal Le
Published: July 2012

The α2‐adrenoceptor antagonist capacities of two hydroxylated metabolites of yohimbine in man (10‐OH‐yohimbine and 11‐OH‐yohimbine) were investigated on the α2‐adrenoceptors of human platelets and adipocytes and compared to those of yohimbine.

Yohimbine and 11‐OH‐yohimbine exhibited similar α2‐adrenoceptor affinity in biological studies i.e. inhibition of adrenaline‐induced platelet aggregation and inhibition of UK14304‐induced antilipolysis in adipocytes.

Yohimbine and the two metabolites displaced [3H]‐RX 821002 binding with equivalent affinities in platelet and adipocyte membranes with the following order of potency: yohimbine > 11‐OH‐yohimbine > 10‐OH‐yohimbine. However, when binding studies were carried out in binding buffer supplemented with 5% albumin, the apparent affinity of yohimbine was reduced about 10 fold and was similar to that of 11‐OH‐yohimbine.

Yohimbine and its metabolites were bound to different extents to plasma proteins, the bound fraction being 82%, 43% and 32% respectively for yohimbine, 11‐OH‐yohimbine and 10‐OH‐yohimbine.

These results show that the main hydroxylated metabolite of yohimbine in man (11‐OH‐yohimbine) possesses α2‐adrenoceptor antagonist properties. The discrepancies found in binding studies (i.e. 10 fold lower affinity of 11‐OH‐yohimbine than yohimbine for α2‐adrenoceptors but similar capacities in blocking biological α2‐adrenoceptor effects in cells) are attributable to the higher degree of binding of yohimbine to plasma protein.
Effect of dexamethasone on neutrophil accumulation and oedema formation in rabbit skin: an investigation of site of action
Authors: Yarwood Helen , Nourshargh Sussan , Brain Susan , Williams Timothy J.
Published: July 2012

The anti‐inflammatory actions of dexamethasone on vascular and leukocyte responses in rabbit skin were investigated.

Neutrophil accumulation and oedema formation were simultaneously measured as the local accumulation of i.v. administered 111In‐labelled neutrophils and 125I‐labelled albumin. Systemically administered dexamethasone (3 mg kg−1) inhibited neutrophil accumulation induced by i.d. zymosan activated plasma (ZAP), N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) and leukotriene B4 (LTB4) when co‐injected with prostaglandin E2 (PGE2). Dexamethasone also inhibited oedema formation elicited by these stimuli and the responses induced by i.d. platelet activating factor (PAF) + PGE2 and bradykinin (BK) + PGE2.

Intradermal dexamethasone (2 × 10−10 mol per site) but not indomethacin (10−8 mol per site) inhibited oedema formation induced by i.d. ZAP + PGE2 and BK + PGE2. This inhibitory effect of dexamethasone was significant only with pretreatment periods of 4 h, shorter pretreatment periods resulting in greatly reduced effects. Intradermal dexamethasone had no effect on neutrophil accumulation induced by ZAP + PGE2.

Intradermal dexamethasone (2 × 10−10 mol per site) had no effect on increase in blood flow induced by PGE2 as measured by 133Xenon clearance.

The accumulation of neutrophils isolated from donor rabbits pretreated with i.v. saline or dexamethasone (3 mg kg−1) was investigated in untreated recipient rabbits. The accumulation of neutrophils, induced by ZAP + PGE2, FMLP + PGE2 and LTB4 + PGE2, from dexamethasone‐pretreated donors was significantly smaller than the accumulation of neutrophils from saline‐pretreated donors.

The results of this study suggest that dexamethasone can have a direct effect on vascular endothelial cells resulting in an inhibition of oedema formation.

Neutrophil accumulation can be inhibited by an effect of dexamethasone on the neutrophil itself or on the vascular endothelium. These results indicate that at least part of the inhibitory effect is on the circulating neutrophil induced by dexamethasone or a dexamethasone‐induced product.
Effects of P1060 and aprikalim on whole‐cell currents in rat portal vein; inhibition by glibenclamide and phentolamine
Authors: Ibbotson T. , Edwards G. , Noack Th. , Weston A.H.
Published: July 2012

Smooth muscle cells of the rat portal vein were dispersed by enzymatic treatment and recordings of whole‐cell currents were made by the voltage‐clamp technique. The effects of the potassium (K) channel openers, P1060 (0.3–10 μm) and aprikalim (3–30 μm) on these currents were investigated. Antagonism of these agents by glibenclamide and phentolamine was also studied.

When cells were clamped at — 10 mV, P1060 (1 μm) and aprikalim (3 μm) each induced a slowly‐developing K‐current (IKCO), the noise of which gradually increased. The rate of onset of IKCO was greater for P1060 than for aprikalim. Current‐voltage plots showed that P1060 and aprikalim each caused an approximately 25 mV negative shift of the reversal potential at zero current.

P1060 (1 μm) and aprikalim (3 μm) each inhibited the slowly activating, slowly inactivating delayed rectifier current, ITO.

Addition of MgATP (5 mm) to the recording pipette inhibited the generation of IKCO by P1060 (1 μm) and reduced the accompanying inhibition of ITO.

Stationary fluctuation analysis of the current noise associated with IKCO induced by P1060 (1 μm) or aprikalim (3 μm) at a holding potential of — 10 mV indicated that the unitary conductance of the underlying K‐channels was 10.5 pS at 0 mV under the quasi‐physiological conditions of the experiment.

In the absence of K‐channel openers, neither phentolamine (30–100 μm) nor glibenclamide (1 μm) affected the magnitude of control non‐inactivating currents. However, phentolamine (30–100 μm), but not glibenclamide (1 μm) inhibited the control delayed rectifier current, ITO.

After induction of IKCO by P1060 (1 μm) or aprikalim (3 μm), subsequent exposure to glibenclamide (1 μm) or phentolamine (30 μm) inhibited this current. After aprikalim‐induced reduction of ITO had developed, subsequent exposure to glibenclamide was able partially to reverse the inhibition of ITO whereas phentolamine was without effect. Pre‐exposure to glibenclamide (1 μm) prevented both the generation of IKCO by aprikalim (3 μm) and the inhibitory effect of this agent on ITO.

It is concluded that P1060 and aprikalim each induce the current IKCO by opening the same small conductance, ATP‐sensitive K‐channel (KATP), an effect which can be inhibited by glibenclamide or phentolamine. The opening of KATP by both P1060 and aprikalim probably involves competition between these agents and ATP for the ATP‐control site associated with the channel. Inhibition of the delayed rectifier current, ITO, by P1060 and aprikalim was glibenclamide‐sensitive and may be caused by the induction of a state of run‐down in the channel which underlies this current.
Modulation of adjuvant arthritis by endogenous nitric oxide
Authors: Ialenti Armando , Moncada Salvador , Di Rosa Massimo
Published: July 2012

The role of endogenous nitric oxide (NO) in adjuvant arthritis in Lewis rats has been studied by use of l‐arginine, the amino acid from which NO is synthesized, and NG‐nitro‐l‐arginine methyl ester (l‐NAME), an inhibitor of NO synthase. Prolonged modulation (35 days) of the l‐arginine: NO pathway in rats was achieved by dissolving test compounds in the drinking water (l‐arginine: 3, 10 and 30 mg ml−1; l‐NAME: 0.1, 1 and 10 mg ml−1).

Arthritis was exacerbated by l‐arginine and suppressed by l‐NAME in a dose‐related fashion. Combined treatment with l‐NAME (1 mg ml−1) and l‐arginine (30 mg ml−1) did not modify the arthritis.

Reduced weight gain, which is a feature of adjuvant arthritis, was modified by these compounds so that l‐arginine reduced weight gain whereas l‐NAME increased weight gain compared with that in control animals.

d‐Arginine (30 mg ml−1), NG‐nitro‐d‐arginine methyl ester (d‐NAME: 1 mg ml−1) and l‐lysine (30 mg ml−1), an amino acid not involved in the generation of NO, were without effect on either arthritis or body weight gain.

. Antigen‐stimulated proliferation of T‐lymphocytes as well as generation of nitrite (NO2−) and release of acid phosphatase from macrophages were all enhanced in l‐arginine‐treated arthritic rats and reduced in l‐NAME‐treated animals.

These results suggest that endogenous NO modulates adjuvant arthritis, possibly by interfering with the activation of T‐lymphocytes and/or macrophages.
The role of nitric oxide in cholinergic neurotransmission in rat trachea
Authors: Sekizawa Kiyohisa , Fukushima Takeyasu , Ikarashi Yasushi , Maruyama Yuji , Sasaki Hidetada
Published: July 2012

We have investigated the role of nitric oxide (NO) in cholinergic contraction in rat trachea.

Methylene blue (10 nm to 30 μm) potentiated cholinergic contraction induced by electrical field stimulation (EFS) at 5 Hz in a concentration‐dependent fashion. At a concentration of 30 μm, methylene blue decreased responses to log EFS frequency, producing 50% of maximum contraction from a control value of 0.74 ± 0.09 Hz to 0.30 ± 0.05 Hz without a significant effect on concentration‐response curves to acetylcholine (ACh).

NG‐monomethyl‐l‐arginine (l‐NMMA; 100 μm) also potentiated cholinergic contraction induced by EFS at 5 Hz (131.5 ± 4.6% of control) without having any effect against ACh (3 μm)‐induced contractions. Likewise, l‐NMMA (100 μm) significantly increased EFS (5 Hz)‐evoked release of ACh from tracheal segments into the bath solution (51.4 ± 4.0 pmol ml−1 in the presence of l‐NMMA and 35.0 ± 1.8 pmol ml−1 in the absence of l‐NMMA, respectively).

Administration of NO (present in acidified soluton of NaNO2) (1 nm to 10 μm) and sodium nitroprusside (100 nm to 10 μm) concentration‐dependently reduced EFS (5 Hz)‐induced cholinergic contractions without having a significant effect on ACh (3 μm)‐induced contractions. These results were unaffected by prior exposure of the tissues to l‐NMMA (100 μm).

Dibutyryl cyclic GMP (3 mm) also reduced cholinergic contractions induced by EFS at 5 Hz (70.1 ± 3.6% of control) without any significant effect on ACh (3 μm)‐induced contractions.

Pretreatment of tissues with capsaicin (30 μm) or α‐chymotrypsin (1 u ml−1) failed to inhibit methylene blue (30 μm)‐induced potentiation of responses to EFS at 5 Hz.

These results suggest that an endogenous NO‐like factor may mediate prejunctional inhibition of cholinergic contraction through a cyclic GMP‐dependent mechanism in rat trachea.
The effects of a converting enzyme inhibitor (Captopril) and angiotensin II on fetal renal function
Authors: Lumbers E.R. , Burrell J.H. , Menzies R.I. , Stevens A.D.
Published: July 2012

Renal function was studied in chronically catheterized fetal sheep (119–128 days gestation), before and during treatment of the ewe with the angiotensin converting enzyme (ACE) inhibitor, Captopril, which crosses the placenta and blocks the fetal renin angiotensin system.

An i.v. dose of 15 mg (about 319 μg kg−1) of Captopril to salt‐replete ewes followed by an infusion to the ewe of 6 mg h−1 (about 128 μg kg−1 h−1) caused a fall in fetal arterial pressure (P < 0.01), and a rise in fetal renal blood flow (RBF) from 67.9 ± 5.6 to 84.9 ± 8.3 ml min−1 (mean ± s.e.mean) (P < 0.05). Renal vascular resistance and glomerular filtration rate (GFR) fell (P < 0.01); fetal urine flow (P < 0.01) and sodium excretion declined (P < 0.05).

Ewes were treated for the next 2 days with 15 mg Captopril twice daily. On the 4th day, 15 mg was given to the ewe and fetal renal function studied for 2 h during the infusion of Captopril (6 mg h−1) to the ewe. Of the 9 surviving fetuses, 3 were anuric and 3 had low urine flow rates. When 6 μg kg−1 h−1 of angiotensin II was infused directly into the fetus RBF fell from 69 ± 10.1 ml min−1 to 31 ± 13.9 ml min−1, GFR rose (P < 0.05) and urine flow (P < 0.01) and sodium excretion increased in all fetuses.

It is concluded that the small fall in fetal arterial pressure partly contributed to the fall in fetal GFR but in addition, efferent arteriolar tone fell so that the filtration pressure fell further. Thus maintenance of fetal renal function depends on the integrity of the fetal renin angiotensin system. These findings explain why use of ACE inhibitors in human pregnancy is associated with neonatal anuria.
A comparison of intravenous NBQX and GYKI 53655 as AMPA antagonists in the rat spinal cord
Authors: Chizh Boris A. , Cumberbatch Michael J. , Headley P. Max
Published: July 2012

The effects of intravenous administration of two α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) antagonists were studied on responses of single neurones to iontophoretically applied excitatory amino acids. The tests were performed on spinal neurones in α‐chloralose anaesthetized, spinalized rats.

Both the quinoxaline, NBQX (2–16 mg kg−1) and the 2,3‐benzodiazepine, GYKI 53655 (2–8 mg kg−1) dose‐dependently decreased responses to AMPA.

Both compounds were short acting, with half‐recovery times of 15 min for NBQX and 7 min for GYKI 53655.

The selectivity for responses to AMPA over those to N‐methyl‐d‐aspartate (NMDA) was significantly poorer for systemic NBQX than for either systemic GYKI 53655 or iontophoretic NBQX, suggesting that systemic NBQX may be converted to a less selective metabolite.

GYKI 53655 is therefore likely to be a more valuable tool than NBQX for the study of AMPA receptor‐mediated processes in vivo.
Pharmacological characterization of the human histamine H2 receptor stably expressed in Chinese hamster ovary cells
Authors: Leurs Rob , Smit Martine J. , Menge Wiro M.B.P. , Timmerman Hendrik
Published: July 2012

The gene for the human histamine H2 receptor was stably expressed in Chinese hamster ovary (CHO) cells and characterized by [125I]‐iodoaminopotentidine binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated.

After cotransfection of CHO cells with pCMVhumH2 and pUT626, a phleomycine‐resistant clonal cell line (CHOhumH2) was isolated that expressed 565 ± 35 fmol kg−1 protein binding sites with high affinity (0.21 ± 0.02 nm) for the H2 antagonist, [125I]‐iodoaminopotentidine.

Displacement studies with a variety of H2 antagonists indicated that the encoded protein was indistinguishable from the H2 receptor identified in human brain membranes and guinea‐pig right atrium. The Ki‐values observed in the various preparations correlated very well (r2 = 0.996–0.920).

Displacement studies with histamine showed that a limited fraction (32 ± 6%) of the binding sites showed a high affinity for histamine (2 ± 1.2 μm); the shallow displacement curves were reflected by a Hill‐coefficient significantly different from unity (nH = 0.58 ± 0.09). The addition of 100 μm Gpp(NH)p resulted in a steepening of the displacement curve (nH = 0.79 ± 0.02) and a loss of high affinity sites for histamine.

Displacement studies with other agonists indicated that the recently developed specific H2 agonists, amthamine and amselamine, showed an approximately 4–5 fold higher affinity for the human H2 receptor than histamine.

Stimulation of CHOhumH2 cells with histamine resulted in a rapid rise of the intracellular cyclic AMP levels. After 10 min an approximately 10 fold increase in cyclic AMP could be measured. The EC50 value for this response was 7 ± 1 nm for histamine. This response was effectively blocked by tiotidine and cimetidine, resulting in Ki values of 8 ± 1 nm and 0.56 ± 0.24 μm respectively.

Stimulation of CHOhumH2 cells with histamine neither inhibited the A23187‐induced release of [3H]‐arachidonic acid nor changed the intracellular IP3 levels.

These results show that the cloned human gene encodes a histamine H2 receptor that is indistinguishable from the H2 receptor identified in human brain tissue. This receptor is functionally coupled to the adenylate cyclase in CHO cells, but does not influence the inositolphosphate turnover or arachidonic acid release.