bluuebunny/crossref_metadata_2025 huggingface dataset:
The pharmacological evaluation of LY170680, a novel leukotriene D4 and E4 antagonist in the guinea‐pig
Authors: Boot J.R. , Bond A. , Gooderham R. , O'Brien A. , Parsons M. , Thomas K.H.
Published: July 2012

This paper describes the evaluation of LY170680 (5‐<3‐[2(R) (carboxyethylthio)‐1(S)‐hydroxypentadeca 3(E)5(Z)‐dienyl]phenyl > 1H tetrazole, as an antagonist of the cysteinyl leukotrienes C4, D4, E4, in a variety of in vitro and in vivo models.

In vitro, LY170680 was a potent, selective, and competitive antagonist of LTD4, and LTE4. It produced a concentration‐dependent rightward displacement of the concentration‐response curves elicited by either LTD4 or LTE4 on both the guinea‐pig ileal and tracheal preparation. The pA2 values for LY170680 were estimated to be 8.1 ± 0.2 (n = 8) and 8.1 ± 0.1 (n = 6) on trachea, and 8.7 ± 0.1 (n = 12) and 9.0 ± 0.3 (n = 6) on ileum for both LTD4 and LTE4 respectively. The slopes of the Schild plots in these studies were all close to unity.

LY170680 was shown to be a modest antagonist of the LTC4‐induced responses on guinea‐pig ileum (pA2 7.0 ± 0.2 n = 5), but had no discernable effects against contractions induced by histamine, prostaglandin E2 (PGE2), PGF2α or acetylcholine. The compound also reduced the LTC4‐induced responses on trachea, but in a non competitive manner.

Intravenous LY170680 reduced in a dose‐dependent manner (ED50 3.8 mg kg−1, 60 min pretreatment) the fall in compliance in the anaesthetized guinea‐pig, and the rise in total pulmonary resistance (TPR) (ED50 2.0 mg kg−1, 30min pretreatment) in the artificially ventilated guinea‐pig, produced by intraveous LTD4.

LY170680 (5 mg kg−1, i.v.) was also effective in reducing the increase in TPR to intravenous antigen in sensitized animals pretreated with mepyramine, indomethacin and propranolol.

The compound was only moderately effective (ED50 40 mg kg−1 p.o.) in preventing the rise in TPR induced by intravenous LTD4, when given orally.

Inhaled LY170680 was particularly effective in preventing the increase in TPR produced by an exposure to aerosolised LTD4. An estimated 1–2 μg of LY170680 delivered to the airways by nebuliser 1 hour beforehand, produced a 6 fold lateral displacement to the right of the dose‐response curve to LTD4.

Studies in conscious animals indicated that inhaled LY170680 produced a dose‐dependent reduction in the increased volume of gas, trapped in the lung following exposure to aerosolised LTD4. Duration studies also indicated that an estimated inhaled dose of 10 μg LY170680 significantly reduced the LTD4‐induced increase in trapped lung gas volume for at least 4 h. Inhaled LY170680 also reduced LTC4‐induced increase in gas trapping in a manner similar to LTD4. However, histamine‐induced gas trapping was unaffected.
Vasodilator responses to dopamine in rat perfused mesentery are age‐dependent
Authors: Wanstall Janet C. , O'Donnell Stella R.
Published: July 2012

Dose‐dependent vasodilator responses to dopamine, isoprenaline, noradrenaline, 3‐isobutyl‐1‐methylxanthine (IBMX) and sodium nitroprusside were obtained in isolated perfused mesentery preparations, taken from reserpine‐treated rats of different ages. The preparations were pretreated with phenoxybenzamine (1 μm) and perfused with physiological salt solution containing cocaine (10 μm), additional KCl (20 mM) and vasopressin (0.1 μm).

Vasodilator responses to dopamine were abolished by the dopamine1 (DA1)‐selective antagonist SCH 23390 (10 nM) and those to isoprenaline by propranolol (1 μm), but the vasodilator responses to noradrenaline were abolished only when SCH 23390 and propranolol were used together. This indicated that dopamine was acting via DA1‐receptors, isoprenaline via β‐adrenoceptors and that noradrenaline could act via DA1‐receptors and β‐adrenoceptors in this preparation.

Responses to all the vasodilator drugs decreased in magnitude between the ages of 1 and 2 months. Responses to dopamine declined further in 4 month‐old rats and were negligible at 6 or 22–24 months of age. Responses to isoprenaline were well maintained up to 6 months of age, but were negligible at 22–24 months.

It is concluded that, in the rat mesenteric vasculature, there is a non‐specific decline in responses to vasodilator drugs during development (1 to 2 months). Subsequently there is a specific decline in DA1‐receptor‐mediated and β‐adrenoceptor‐mediated responses; the former are lost at an earlier age than the latter. This different time course suggests that age influences receptor numbers, or their coupling to adenylate cyclase, rather than a post‐receptor event in the adenylate cyclase/cyclic AMP pathway.
Functional and binding studies with muscarinic M2‐subtype selective antagonists
Authors: Lazareno S. , Roberts F.F.
Published: July 2012

The potency of a series of selective muscarinic antagonists has been measured on two functional isolated tissue preparations (rat ileum and atria) and these compared with their potency on a range of binding preparations in order to determine whether the subtypes of M2 receptor measured functionally are the same as those measured in binding studies.

On the functional preparations pirenzepine, hexahydrosiladiphenidol (HSD) and 4‐diphenylacetoxy‐N‐methylpiperidine (4‐DAMP) were more potent on the ileum than on the atrium (3 fold, 29 fold and 5 fold respectively), whereas himbacine, AF‐DX 116 and methoctramine showed the opposite selectivity (5 fold, 3 fold and 56 fold respectively). Atropine had a similar potency on the ileum and atrium.

[3H]‐N‐methyl scopolamine was used to study M2 binding sites on membranes from rat heart and rat submandibular gland. Each preparation appeared to contain a homogeneous binding site population. The potencies of the five M2 selective antagonists (and pirenzepine) in binding studies to heart membranes were very similar to those observed in functional studies of rat atria (correlation coefficient = 0.98). Similarly the binding to submandibular gland membranes was very similar to that observed in functional studies on rat ileum (correlation coefficient = 0.97).

[3H]‐pirenzepine was used to examine the binding of these antagonists to M1 binding sites on membranes from rat cerebral cortex. The affinities of 4‐DAMP, HSD, AF‐DX116 and himbacine at M1 sites were similar to their affinities on the gland. Only pirenzepine and methoctramine had higher affinity on M1 sites than on the gland.

Himbacine had a 20 fold lower affinity at M1 binding sites than at heart sites, and it should therefore be an important tool in identifying M1 sites.

Inhibition of [3H]‐N‐methyl scopolamine binding to rat ileum and rat brainstem by M2‐selective antagonists was best described by a two‐site model. In both cases the major population of sites (70–90%) appeared to be similar to sites found on the heart (correlation coefficients = 0.95 and 0.97). The other site appeared to be similar to that on the submandibular gland (correlation coefficients = 0.96 and 1.00).

The correlations observed in these studies in which a range of selective muscarinic antagonists was used lend weight to previous studies indicating the presence of three functionally important muscarinic receptor subtypes, typified by the binding sites studied in the cerebral cortex, submandibular gland and heart.

We propose that the sub‐classification of the M2 muscarinic receptor into M2 and M3 subtypes on the basis of ligand binding studies should be extended to cover functionally‐defined receptors as well.
NK1‐receptors mediate the proliferative response of human fibroblasts to tachykinins
Authors: Ziche Marina , Morbidelli Lucia , Pacini Marco , Dolara Piero , Maggi Carlo Alberto
Published: July 2012

The effect of synthetic tachykinin selective receptor agonists was studied on the growth of cultured human skin fibroblasts (HF).

Human fibroblasts were grown in serum‐free conditions in the presence of natural tachykinins (substance P and neurokinin A) and of three synthetic agonists, [β‐Ala4, Sar9, Met(O2)11]‐SP(4–11), [β‐Ala8]‐NKA(4–10) and [MePhe7]‐NKB selective for NK1‐, NK2‐ and NK3‐receptors respectively. Cell proliferation was measured by percentage increase in cell number and by [3H]‐thymidine uptake following 48 h exposure to agents compared to baseline condition.

Neurokinin A (NKA) and substance P (SP) significantly increased cell proliferation the threshold concentrations being 10−12 and 10−11 m, respectively. Addition of thiorphan to culture conditions enhanced the effect of SP but not of NKA.

The selective NK1‐receptor agonist produced a dose‐dependent increase in cell proliferation as judged by total cell number and [3H]‐thymidine uptake. No significant effect was observed with NK2‐ and NK3‐receptor agonists.

These data indicate that the effect of SP on fibroblast proliferation is mediated by interaction with a NK1‐receptor type and local metabolism can interfere with the full expression of this effect of SP on cell proliferation.
Inhibitory effects of MK‐886 on arachidonic acid metabolism in human phagocytes
Authors: Ménard Luc , Pilote Sylvie , Naccache Paul H. , Laviolette Michel , Borgeat Pierre
Published: July 2012

We have investigated the inhibitory activity of compound MK‐886 (formerly L‐663,536), an indole derivative, on 5‐lipoxygenase product synthesis in various human phagocytes stimulated with either the ionophore A23187, in the presence and absence of exogenous arachidonic acid, or platelet‐activating factor (PAF). The lipoxygenase products were analysed by reversed‐phase h.p.l.c.

MK‐886 inhibited the formation of 5‐hydroxy‐eicosatetraenoic acid (5‐HETE), leukotriene B4 (LTB4), its ω‐oxidation products and 6‐trans‐isomers with an IC50 value of 10–14 nm in A23187‐stimulated neutrophils. In the same system, nordihydroguaiaretic acid (NDGA), AA‐861 and L‐655,240 showed IC50 values of 250–510, 110–420 nm and 1.7–3.9 μm, respectively.

MK‐886 inhibited 5‐lipoxygenase product synthesis in A23187‐stimulated blood eosinophils and monocytes, and in neutrophils primed with granulocyte‐macrophage colony‐stimulating factor and stimulated with PAF with IC50 values of 1–13 nm.

The inhibitory activity of MK‐886 was not reversed by addition of 10 μm arachidonic acid to A23187‐stimulated neutrophils.

Compound MK‐886 had no effect on 15‐lipoxygenase product synthesis in blood eosinophils and neutrophils up to a concentration of 1 μm.

At 100 nm compound MK‐886 had no significant effects on calcium ion mobilization, superoxide anion production and actin polymerization in neutrophils.

In conclusion, MK‐886 is a very potent and specific inhibitor of both LTB4 and LTC4 synthesis in various types of human phagocytes.
Electrophysiological and mechanical effects of calcitonin gene‐related peptide on guinea‐pig atria
Authors: Ohmura Tsuyoshi , Nishio Matomo , Kigoshi Shigeru , Muramatsu Ikunobu
Published: July 2012

The effects of calcitonin gene‐related peptide (CGRP) on mechanical and electrophysiological responses were studied in the guinea‐pig atrial muscle preparations and in single cells.

CGRP (>10−9m) enhanced the twitch contraction in a concentration‐dependent manner in electrically driven left atria and increased heart rate in spontaneously beating right atria. The positive inotropic and chronotropic effects of CGRP were not inhibited by propranolol but were attenuated by reduction of the calcium concentration in the bathing medium.

In single left atrial cells, CGRP slightly hyperpolarized the resting potential but did not affect the other action potential parameters significantly.

Under whole‐cell voltage‐clamp conditions, CGRP increased the calcium inward current. The peptide also increased the steady inward current elicited by hyperpolarization and the late outward current by depolarization.

These results suggest that CGRP may produce the positive inotropic and presumably chronotropic effects by increasing calcium inward current. CGRP also increases the potassium permeability. Such effects on ionic currents may not produce any apparent change in the action potential conformation, due to their opposite directional actions and relatively weak potencies.
Reactivity and sensitivity of mesenteric vascular beds and aortic rings of spontaneously hypertensive rats to endothelin: effects of calcium entry blockers
Authors: Criscione L. , Nellis P. , Riniker B. , Thomann H. , Burdet R.
Published: July 2012

The vasoconstrictor effects of endothelin‐1 were studied in perfused mesenteric vascular beds (MVB) and aortic rings of 14–16 week‐old spontaneously hypertensive rats (SHR) and age‐matched Wistar Kyoto rats (WKY).

Reactivity to endothelin‐1 was increased in MVBs of SHR, as indicated by the maximum perfusion pressure obtained (264 ± 8 and 141 ± 9 mmHg respectively) (P < 0.001), whereas sensitivity was not significantly different between the two strains (EC50 171 ± 21 and 102 ± 19, respectively).

In aortic rings, in constrast, reactivity to endothelin‐1 was reduced in SHR as compared to WKY, whereas sensitivity was similar (EC50 0.78 ± 0.08 and 0.87 ± 0.09 nm).

As with endothelin‐1, reactivity to noradrenaline and potassium chloride was increased in MVBs, but not in aortic rings of SHR. Endothelin‐1 was 30 times more potent than noradrenaline in MVBs of SHR, and 15 times more potent than noradrenaline in aortic rings.

In both strains, nifedipine and nitrendipine almost completely blocked potassium‐induced contractions in MVB and aortic rings, respectively, whereas contractions induced by endothelin‐1 or noradrenaline were only partially inhibited.

It is concluded that calcium influx via the voltage‐operated calcium channel is only partially responsible for the vasoconstrictor action of endothelin‐1 in MVBs and aortic rings of SHR and WKY rats. The increased reactivity of the MVB of SHR to endothelin‐1 at this stage of the hypertensive process is most likely to be the result of a change in vascular structure rather than due to a primary hypertensive mechanism.
Inositol phospholipid hydrolysis in human brain; adenosine inhibition of the response to histamine
Authors: Kendall D.A. , Firth J.L.
Published: July 2012

Inositol phospholipid hydrolysis was examined in human cerebral cortex slices by a [3H]‐inositol prelabelling assay.

Enhancement of [3H]‐inositol phosphates accumulation was observed in the presence of carbachol, noradrenaline, histamine, 5‐hydroxytryptamine (5‐HT) and depolarizing concentrations of KCl.

Despite having no effect alone, adenosine (and its analogue 2‐chloroadenosine) selectively inhibited the direct response to histamine.

The inhibition due to adenosine was antagonized by theophylline, but not by 8‐cyclopropyltheophylline.
Actions of second messengers synthesized by various spasmogenic agents and their relation to mechanical responses in dog tracheal smooth muscle
Authors: Katsuyama Hideaki , Suzuki Satoshi , Nishiye Eiichiro
Published: July 2012

The effects of the spasmogenic agents, carbachol (CCh), histamine, 5‐hydroxytryptamine (5‐HT) and 9,11‐epithio‐11,12‐methano‐thromboxane A2 (STA2) were investigated on smooth muscle tissues of the dog trachea.

CCh (10 μm) produced a larger contraction than high K (128 mm), 10 μm histamine, 5‐HT or STA2. Histamine and 5‐HT produced the same amplitude of contraction as each other. In Ca‐free solution containing 0.2 mm EGTA, only a phasic contraction was evoked by the above agents (except for K which induced no contraction at all).

In skinned muscle tissues, the maximum amplitude of contraction that could be induced by Ca (10 μm) was slightly larger than the maximum CCh‐induced contraction (also at 10 μm) evoked in intact muscle tissues. Caffeine and inositol 1,4,5‐trisphosphate(IP3) both produced contraction.

CCh, histamine and 5‐HT (10 μm) produced a sustained contraction for over 30 min and also increased phosphorylation of the 20 kD protein of myosin light chain (MLC20) for over 30 min with no attenuation. Greater concentrations of the above agents caused more phosphorylation of MLC20.

CCh (above 1 nm), histamine (above 10 nm) and 5‐HT (above 100 nm) increased the amount of IP3, in a concentration‐dependent manner. Synthesis of IP3 induced by the above agents reached its peak value within 30 s and lasted for about 3 min. The potencies for the synthesis of IP3 were in the following order: CCh > histamine > 5‐HT > STA2.

Isoprenaline (10 μm) markedly enhanced but CCh (10 μm) slightly reduced the amount of cyclic AMP. 5‐HT (10 μm) and STA2 (10 μm) reduced, but histamine (10 μm) and CCh (10 μm) increased the amount of cyclic GMP.

Using fura 2, cytosolic Ca was measured by monitoring the ratio of the fluorescent signal excited at 340 and 380 nm wavelengths in the presence of extracellular Ca. CCh (10 μm) increased the Ca transient from 182 nm to 1.42 μm. When the CCh‐induced peak Ca transient (10 μm) was normalised, 10 μm histamine, 5‐HT and STA2 showed smaller values such as 0.49, 0.53 and 0.04 times the control, respectively, and these values corresponded well with the amplitudes of contraction evoked by each of the stimulants.

The results can be summarized as follows: stronger spasmogenic responses occur on application of CCh than on application of 5‐HT or histamine, and STA2 may have a minor role as a spasmogenic agent. The maximum amplitudes (peaks) of contraction evoked by the above spasmogenic agents are closely related to the maximum increase in cytosolic Ca, but sustained contraction and increased phosphorylation of myosin cannot be explained by the increased amount of Ca. In the case of 5‐HT and histamine, synthesized cyclic nucleotides may interact with the action of IP3 for the regulation of contraction in a positive or negative manner, respectively.
P2‐, but not P1‐purinoceptors mediate formation of 1,4,5‐inositol trisphosphate and its metabolites via a pertussis toxin‐insensitive pathway in the rat renal cortex
Authors: Nanoff Christian , Freissmuth Michael , Tuisl Elisabeth , Schütz Wolfgang
Published: July 2012

The adenosine receptor (P1‐purinoceptor) agonists N6‐cyclopentyladenosine and N‐5′‐ethyl‐carboxamidoadenosine at concentrations up to 10 μmol l−1 affected neither basal, nor noradrenaline‐ and angiotensin II‐stimulated formation of inositol‐1‐phosphate, inositol‐1,4‐bisphosphate, and inositol‐1,4,5‐trisphosphate in slices of rat renal cortex.

In contrast, adenine nucleotides (P2‐purinoceptor agonists) markedly stimulated inositol phosphate formation. The observed rank order of potency adenosine‐5′‐O‐(2‐thiodiphosphate) (EC50 39 μmol l−1) > adenosine‐5′‐O‐(3‐thio triphosphate) (587) ≥ 5′‐adenylylimidodiphosphate (App(NH)p, 899) > adenylyl‐(β,γ‐methylene)‐diphosphonate (4,181) was consistent with the interaction of the compounds with the P2y‐subtype of P2‐purinoceptors. AMP and the ADP analogue (α,β‐methylene)‐adenosine‐5′‐diphosphate were ineffective. ATP and ADP (≤ 10 mmol l−1) did not produce a consistent increase, owing to their hydrolytic degradation in the incubation medium.

Whereas the inositol phosphate response to App(NH)p was linear only up to 5 min incubation, the time‐dependent stimulation of noradrenaline declined at a slower rate. Following pre‐exposure of the renal cortical slices to App(NH)p, renewed addition of App(NH)p caused no further enhancement in the accumulation of inositol phosphates, whilst noradrenaline was still capable of eliciting a response. This suggests that the apparent loss of responsiveness to App(NH)p is not due to substrate depletion or enzymatic inactivation, but most likely attributable to homologous desensitization of the purinoceptor.

Pretreatment of the animals with pertussis toxin caused a substantial reduction of functional Gi‐protein, as indicated by the lack of [32P]‐NAD incorporation in a membrane preparation of the renal cortex. Nevertheless, the increase in inositol phosphate formation induced by noradrenaline, angiotensin II, and App(NH)p was not significantly impaired.

We conclude that P2y‐purinoceptors are present in the renal cortex; these receptors stimulate formation of inositol phosphates via a pertussis toxin‐insensitive pathway and undergo homologous desensitization. On the other hand, our results suggest that renal A1‐adenosine receptors do not use stimulation of phosphoinositide breakdown as a transmembrane signalling system.
Preferential biliary elimination of FPL 63547, a novel inhibitor of angiotensin‐converting enzyme, in the rat
Authors: Carr R.D. , Cooper A.E. , Hutchinsno R. , Mann J. , O'Connor S.E. , Robinson D.H. , Wells E.
Published: July 2012

The route of elimination of FPL 63547, a novel inhibitor of angiotensin‐converting enzyme (ACE), has been investigated in the anaesthetized rat. Comparisons have been made with other ACE inhibitors.

Bile and urine samples were collected over a 5 hour period following a single i.v. dose of ACE inhibitor (2 μmol kg−1). Samples were bioassayed for ACE inhibitory activity using affinity‐purified rabbit lung ACE and the amounts of the active form of inhibitor present in each sample were calculated by comparison with a standard curve.

FPL 63547 was rapidly and extensively excreted as the diacid in the bile but appeared in the urine in negligible amounts. The bile:urine ratio was 21.4:1 indicating a marked preference for the biliary route. A similar elimination profile was observed when the compound was dosed in its active form (FPL 63547 diacid), 87.9% of which was found in the bile over the 5 h collection period, with a bile: urine ratio of 14.6:1.

The marked preference of FPL 63547 for biliary elimination was not shared by the other ACE inhibitors tested in this study. Lisinopril demonstrated the opposite pattern, being excreted almost exclusively by the kidney (bile:urine ratio 0.06:1). Enalapril was eliminated in approximately equal amounts in bile and urine (ratio 0.7:1) while spirapril diacid showed a slight preference for the bile (ratio 2.6:1).

The physical chemical properties of FPL 63547 diacid may be responsible for its unusual preference for biliary elimination. In particular, the amphipathic character and strong acid functionality of the compound are thought to favour transport into the bile.

Elimination by the biliary route will be preferred in patients whose renal function is impaired as a result of disease or age. In such patients the elimination of renally‐excreted ACE inhibitors is known to be compromised, resulting in compound accumulation and the need for closer monitoring. Therefore, the elimination profile of FPL 63547, if confirmed in man, may prove to be clinically advantageous.
Cardiovascular responses to verapamil and nifedipine in hypoventilated and hyperventilated rats
Authors: Achike Francis I. , Dai Soter
Published: July 2012

The influence of hypoventilation or hyperventilation on blood pressure and pulse rate responses to verapamil and nifedipine was studied in chloralose‐anaesthetized rats.

Artificial ventilation with room air at a fixed volume of 10 ml kg−1 successfully induced combinations of hypoxaemia, hypercarbia and acidosis at a ventilator rate of 37 strokes min−1 and of hyperoxaemia, hypocarbia and alkalosis at 160 strokes min−1.

Hypoventilation caused significant decreases in both the blood pressure and pulse rate, whereas hyperventilation produced significant increases in these parameters.

In the controls, intravenous injections of graded doses of either verapamil or nifedipine caused dose‐dependent decreases in mean blood pressure. The effects on pulse rate were not marked.

The hypotensive effects of verapamil were significantly more intense in hyperventilated rats, whereas those of nifedipine were significantly less pronounced in hypoventilated animals. The hypoventilated rats exhibited a significant dose‐dependent decrease in pulse rate in response to verapamil administration.

It is concluded that cardiovascular responses to verapamil, nifedipine and probably other calcium antagonists are altered in the presence of blood gas abnormalities.
Role of β‐adrenoceptor‐adenylate cyclase system in the developmental decrease in sensitivity to isoprenaline in foetal and neonatal rat heart
Authors: Tanaka Hikaru , Shigenobu Koki
Published: July 2012

The inotropic and chronotropic sensitivity to noradrenaline and isoprenaline (Iso) of foetal and neonatal rat heart decreases as the heart becomes sympathetically innervated. In the present study, we have examined adenylate cyclase (AC) activation and β‐adrenoceptor binding to determine whether a developmental decrease in sensitivity was demonstrable in the β‐receptor‐AC system of atrial and ventricular membranes from the 15 day foetus and 1 day and 7 day neonates.

While the maximum activation of AC by Iso increased with age, the sensitivity expressed in terms of pD2 values decreased from the 15th foetal day to the first day after birth in the atria, and from the first day to the 7th day after birth in the ventricle.

In contrast, activation of AC by forskolin was almost identical at all ages both in atria and ventricle.

The maximum equilibrium binding of [3H]‐dihydroalprenolol decreased with age, the dissociation constant being about the same at all ages in both the atria and ventricle.

In conclusion, we have demonstrated a developmental decrease in the sensitivity of AC to Iso in myocardial membrane fractions consistent with the developmental decrease in chronotropic and inotropic sensitivity to β‐adrenoceptor agonists. Although a reduction in β‐adrenoceptor number partly accounts for the decrease in sensitivity, some other factors such as decreased coupling to AC may largely be responsible.
Effects of indomethacin on the regional haemodynamic responses to low doses of endothelins and sarafotoxin
Authors: Gardiner Sheila M. , Compton Alix M. , Bennett Terence
Published: July 2012

Regional haemodynamic responses to i.v. bolus injections of low doses (4 pmol and 40 pmol) of endothelin‐1, −2, −3 and sarafotoxin‐S6b were assessed in conscious, Long Evans rats in the absence and presence of indomethacin.

Both doses of endothelin‐3 and sarafotoxin‐S6b caused early renal vasodilatations that were not affected by indomethacin. Endothelin‐1 caused an initial renal vasodilatation only in the presence of indomethacin, indicating that this peptide produced concurrent release of cyclo‐oxygenase products that caused renal vasoconstriction. Neither dose of endothelin‐2 produced an increase in renal conductance.

The 4 pmol dose of all four peptides caused mesenteric vasoconstrictions only. With the 40 pmol dose of the peptides, none caused early mesenteric vasoconstriction except in the presence of indomethacin. Thus, in this vascular bed the primary vasoconstrictor effects of the peptides (seen with the 4 pmol dose) were offset, following the 40 pmol dose, by release of vasodilator cyclo‐oxygenase products. Indomethacin alone caused significant vasoconstriction only in the mesenteric vascular bed, indicating that in this region of the circulation, vasodilator prostanoids might be involved also in the tonic control of vascular conductance.

All four peptides at both doses caused early hindquarters vasodilatation. However, only the initial hypotensive and hindquarters vasodilator effects of the 40 pmol dose of sarafotoxin‐S6b were attenuated by indomethacin. Under these conditions the hindquarters vasodilator effects of sarafotoxin‐S6b were similar to those of the other peptides, indicating that the more marked effects of sarafotoxin‐S6b in the absence of indomethacin were contributed to by vasodilator cyclo‐oxygenase products in the hindquarters.
Influence of plasma protein content and platelet number on the potency of PAF and its antagonist RP 59227 in rabbit platelet preparations
Authors: Floch Anne , Cavero Icilio
Published: July 2012

The potency of platelet activating factor (PAF) as a pro‐aggregatory agent and of RP 59227, a selective antagonist of PAF‐induced platelet aggregation, was determined in several types of rabbit platelet preparations.

PAF produced concentration‐dependent responses irrespective of whether the suspension medium for the platelets (200,000 per μl) was undiluted plasma (PRP), saline‐diluted plasma (dil. PRP) or a salt solution (WP: washed platelets). The potency of PAF, expressed as pD2, was 3 fold higher in WP than PRP or diluted PRP (dil. PRP) for which the ratio (v/v) of total plasma to saline was 1:1.5. In PRP and WP preparations, an increase in the number of platelets in the reaction medium from 200,000 to 600,000 enhanced the potency of PAF slightly (2.3 fold). Furthermore, PAF was 3 times more potent in WP than PRP when studied in preparations containing either 200,000, 400,000 or 600,000 platelets per μl.

RP 59227, like the reference compounds WEB 2086 and CV‐6209, behaved as a competitive antagonist against PAF‐evoked platelet aggregation in PRP, WP and dil. PRP (200,000 platelets per μl). Their potency was slightly greater (1.6 to 2.6 fold more) in dil. PRP than in WP, but in PRP it was 3.5 to 4.3 times lower than in WP. RP 59227 was 2.3 and 5.0 times less potent when the platelet number of the PRP suspension was increased from 200,000 to 400,000 and 600,000 per μl, respectively, whereas the potency of RP 59227 in WP was not modified by these changes in platelet number. Furthermore, CV‐6209, RP 59227 and WEB 2086 were found to be 2.8 to 3.3 times more potent when studied after 10 rather than 1 min incubation time.

In conclusion, methodological variables such as platelet number, plasma protein content and incubation time can modify the potency not only of PAF, as an aggregatory agent, but also of its antagonists. Thus, only platelet aggregation studies carried out by using well‐defined experimental conditions afford an appropriate investigational approach to establish a potency rank order for PAF‐receptor antagonists. Furthermore, dil. PRP which can be easily and rapidly prepared appears to be as appropriate as WP to determine pA2 values for PAF antagonists.
Investigation of the mechanism(s) of 8‐OH‐DPAT‐mediated inhibition of plasma insulin in spontaneously hypertensive rats
Authors: Bouhelal Rochdi , Loubatières‐Mariani Marie‐Madelaine , Mir Anis K.
Published: July 2012

Effects of the prototype selective 5‐HT1A receptor agonist, 8‐hydroxy‐2‐(di‐n‐dipropylamino)tetralin (8‐OH‐DPAT), were studied on the glycaemia and insulinaemia in conscious spontaneously hypertensive (SH) rats concurrently with blood pressure (BP) and heart rate (HR); underlying mechanism(s) were investigated in anaesthetized and pithed SH rats and in the perfused rat pancreas.

Intravenous (i.v.) injections of 8‐OH‐DPAT (150 μg kg−1, i.v.) into fasted conscious but not anaesthetized SH rats increased glycaemia; glucose‐stimulated (i.v. glucose tolerance test) plasma insulin levels were significantly inhibited in both cases without significant changes in glucose tolerance. Metabolic changes were associated with prominent decreases in BP and HR.

No inhibitory effect of 8‐OH‐DPAT, 150 μg kg−1 i.v., on glucose‐stimulated plasma insulin was observed in pithed SH rats; in contrast, clonidine (8 μg kg−1 i.v.), produced marked inhibition of insulin levels in association with glucose intolerance. Neither compound decreased BP; rather, pronounced vasopressor effects were observed.

In the isolated perfused pancreas of the rat, 8‐OH‐DPAT, at 10−8 and 10−7m, concentrations known to activate 5‐HT1A receptors in vitro, failed to modify glucose‐stimulated insulin release. Inhibition (39 ± 7%) was seen only at a high concentration of 10−6 m.

The present data suggest that like the cardiovascular effects of 8‐OH‐DPAT, the inhibition of glucose‐stimulated insulin release is mediated via the central nervous system. However, it is suggested that different mechanisms are involved in the cardiovascular actions and metabolic effects of 8‐OH‐DPAT in the SH rat; the latter are likely to reflect a consequence of activation of the hypothalamic‐adrenal axis.
Inhibitory actions of prostaglandin E1 on non‐adrenergic non‐cholinergic contraction in guinea‐pig bronchi
Authors: Aikawa Takashi , Sekizawa Kiyohisa , Itabashi Shigeru , Sasaki Hidetada , Takishima Tamotsu
Published: July 2012

We have investigated the effect of prostaglandin E1 (PGE1) on non‐adrenergic, non‐cholinergic (NANC) contraction in guinea‐pig bronchial strips. PGE1 (10 nM to 10 μm) did not alter baseline tension but reduced NANC contractions induced by electrical field stimulation (EFS) in a concentration‐dependent fashion (‐log EC50 was 6.60 ± 0.10M and maximum inhibition was 88.7 ± 2.9%). PGE1 (>0.3 μm) also reduced the contraction induced by substance P (1 μm). Removal of epithelium did not alter the effects of PGE1 on NANC contraction. These results suggest that PGE1 exerts both pre‐ and post‐junctional inhibitory actions on NANC contraction.
Anaesthetic suppression of transmitter actions in neocortex
Authors: Puil E. , El‐Beheiry H.
Published: July 2012

The effects of general anaesthetics were investigated on neuronal sensitivities to transmitter substances, which were determined by iontophoretic applications of acetylcholine, glutamate, N‐methyl‐d‐aspartate (NMDA) and γ‐aminobutyrate (GABA) during intracellular recording in in vitro slice preparations of neocortex (guinea‐pig).

In most of the 65 neurones studied, perfusion of isoflurane (0.5–2.5 minimum alveolar concentration (MAC)) or Althesin (25–200 μm) and, in some cases, halothane (0.5–2 MAC), markedly reduced the depolarizing responses and associated membrane conductance changes evoked by dendritic applications of acetylcholine, glutamate, NMDA and GABA.

The order of depression was acetylcholine > glutamate or NMDA ≫ GABA. This selectivity could also be assessed from the EC50 for the isoflurane‐induced depression of the just‐maximal responses to acetylcholine, which was 0.9 MAC compared with an EC50 =1.9 MAC for the suppression of glutamate responses. The selectivity was less pronounced in the case of the actions of Althesin, where the EC50s were 75 μm for the depression of acetylcholine responses and 90 μm for the depression of glutamate responses.

The hyperpolarizing responses observed when GABA was applied near the perikaryon in 7 neurones, were slightly reduced (∼ 15%) in 4, and unchanged in 3 neurones during anaesthetic application.

The pronounced depression of the responsiveness to the putative arousal transmitters and an observed blockade of acetylcholine‐induced potentiation of glutamate actions suggest that anaesthetics produce unconsciousness, at least in part, by interfering with subsynaptic mechanisms of neocortical activation.
Inhibition of endothelial‐bound angiotensin converting enzyme, in vivo
Authors: Catravas John D. , Ryan James W. , Chung Alfred Y.K. , Quinn Nancy E. , Anthony Betty L.
Published: July 2012

We determined apparent Ki constants of two inhibitors, captopril and CL242,817, for pulmonary endothelial‐bound angiotensin converting enzyme (ACE) in anaesthetized rabbits. [3H]‐benzoyl‐Phe‐Ala‐Pro was used as the substrate. The apparent kinetic parameters Km and Amax (product of Vmax and microvascular plasma volume) were measured, as was the ratio (Amax/Km) (measured under first order reaction conditions) before and 30s after the i.v. administration of captopril 10 nmol kg−1 or CL242,817, 35 nmol kg−1.

Under mixed order reaction conditions, ([S] ≥ Km), apparent Km values increased from 12.2 ± 1.9 μm to 32.9 ± 3.3 μm (P < 0.05) in the captopril‐treated rabbits and from 9.3 ± 2.3 μm to 45.8 ± 9.8 μm (P < 0.05) in the CL242,817‐treated rabbits, indicative of competitive inhibition. However, apparent Amax values decreased from 10.3 ± 2.1 to 4.5 ± 0.8 μmol min−1 (P < 0.05) and 8.9 ± 1.7 to 4.8 ± 0.5 μmol min−1 (P < 0.05), respectively.

Under first order reaction conditions ([S] ≪ Km), the Amax/Km ratio decreased from 763 ± 100 to 125 ± 38 ml min−1 (P < 0.05) and 1009 ± 149 to 126 ± 44 ml min−1 (P < 0.05) in the captopril‐ and CL242,817‐treated groups respectively.

When the single pass transpulmonary binding of 80 pmol [3H]‐RAC‐X‐65 (an ACE inhibitor) was measured in additional rabbits, a significant (P < 0.05) decrease in RAC‐X‐65 binding was observed 30 s after captopril (80% decrease) or CL242,817 (85% decrease), a result expected for a loss of catalytically active enzyme mass due to tightly bound captopril or CL242,817.

These results indicate that, in vivo, both captopril and CL242,817 are competitive, tight binding inhibitors of lung ACE. Furthermore, they suggest means for evaluating the interaction of other potential ACE inhibitors with the pulmonary endothelial membrane‐bound enzyme, in vivo, possibly in phase I clinical trials.
Studies of receptor‐mediated inhibition of 45Ca accumulation into synaptosomes
Authors: Xiang Jian‐Zhong , Brammer Michael J. , Campbell Iain C.
Published: July 2012

The effects of α2‐adrenoceptor and κ‐opiate receptor activation on 45Ca accumulation into rat cortical synaptosomes were examined.

Clonidine (1μm) and U50488H (1μm) significantly reduced 45Ca accumulation under both resting (5 mM K+) and depolarizing (15–30 mM K+) conditions.

The inhibitory effects of the agonists on 45Ca accumulation into synaptosomes were enhanced in the presence of the Na+‐Ca2+ exchange inhibitor sodium orthovanadate (vanadate, 2mm), and were not present in mitochondrial preparations.

When the agonists were used together, their inhibitory effects were not additive but were, in fact, attenuated.

In the presence of the α2‐adrenoceptor antagonist idazoxan (1 μm), the inhibitory effect of U50488H on 45Ca accumulation was enhanced. A similar increase in the inhibitory effectiveness of clonidine was observed in the presence of naloxone (20 μm).

When synaptosomes were pretreated with 3‐isobutyl‐1‐methylxanthine (IBMX, 0.5 mm), dibutyrylcyclic AMP (db‐cyclic AMP, 10 μm) or 8‐bromo‐cyclic AMP (8Br‐cyclic AMP, 10μm), the inhibitory effects of clonidine and U50488H were abolished, suggesting that a decrease in cyclic AMP production is part of the receptor‐effector coupling mechanism of both receptor systems.

The phorbol ester, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA, 0.05 μm) increased 45Ca accumulation but did not alter the inhibitory effects of clonidine or U50488H, thus showing that the effects of the agonists are not mediated by protein kinase C.

We conclude that a2‐adrenoceptor and κ‐opiate receptor activation dramatically reduce 45Ca influx through Ca2+ channels (e.g., by 50%), that there is a functional antagonism between the two receptor systems and that in both cases, the receptor effector mechanism involves a decrease in cyclic AMP production.
l‐Arginine and arginine analogues: effects on isolated blood vessels and cultured endothelial cells
Authors: Schmidt Harald H.H.W. , Baeblich Sören E. , Zernikow Bettina C. , Klein Marcus M. , Böhme Eycke
Published: July 2012

The present study examined effects of arginine (Arg) and various Arg analogues on the vascular tone of rabbit and rat aortic rings, the release of nitrite from cultured bovine aortic endothelial cells and the metabolism of l‐Arg in bovine and porcine endothelial cell homogenates. The respective d‐enantiomers or N‐α‐benzoyl‐l‐arginine ethyl ester did not substitute for l‐Arg.

In bovine aortic endothelial cells, the release of nitrite was only observed in the presence of l‐Arg or l‐Arg methyl ester in the cell culture medium.

In dialyzed homogenates of porcine and bovine aortic endothelial cells, l‐Arg was metabolized independently of NADPH and Ca2+ to yield l‐ornithine (l‐Orn) and l‐citrulline (l‐Cit). No concomitant nitrite formation was detected.

Pretreatment of rabbit and rat aortic rings with l‐canavanine (l‐Can) or NG‐monomethyl‐l‐Arg (l‐NMMA) inhibited ATP‐ and acetylcholine‐induced relaxations (endothelium‐dependent) but not glyceryltrinitrate‐induced relaxations (endothelium‐independent).

In rabbit aortic rings, Arg and monomeric Arg analogues induced endothelium‐independent relaxations. l‐Arg methyl ester induced an endothelium‐independent contraction, and l‐NMMA induced a relaxation in the absence of endothelium and a contraction in the presence of endothelium. Polymeric basic amino acids such as poly l‐Arg induced endothelium‐dependent relaxations (inhibited by l‐Can), a subsequent refractoriness to endothelium‐dependent vasodilatators (not prevented by l‐Can) and endothelial cell death.

We suggest that extracellular l‐Arg is essential for the formation of endothelium‐derived nitrogen oxides (EDNO). However, Arg and Arg analogues do not exert endothelium‐dependent relaxation. l‐Can and l‐NMMA inhibit endothelium‐dependent relaxation, consistent with an inhibition of EDNO formation from l‐Arg, but also exert endothelium‐independent effects on vascular tone.
Cytoplasmic calcium and the relaxation of canine coronary arterial smooth muscle produced by cromakalim, pinacidil and nicorandil
Authors: Yanagisawa Teruyuki , Teshigawara Tadashi , Taira Norio
Published: July 2012

In order to investigate the vasodilator mechanisms of the K+ channel openers, cromakalim, pinacidil and nicorandil, we measured changes in cytoplasmic Ca2+ concentration ([Ca2+]i) simultaneously with force by a microfluorimetric method using fura‐2, a calcium indicator, in canine coronary arterial smooth muscle cells.

The three K+ channel openers all produced a concentration‐dependent reduction of [Ca2+]j in 5 and 30mM KCl physiological salt solution (PSS) but failed to affect [Ca2+]i in 45 and 90 mM KC1‐PSS.

Cromakalim only partly inhibited (‐45%) the 30 mM KCl‐induced contractures, whereas pinacidil and nicorandil nearly abolished contractions produced by 45 mM, 90 mM and 30 mM KC1‐PSS.

Tetrabutylammonium (TBA), a nonselective K+ channel blocker, or glibenclamide, a supposed adenosine 5′‐triphosphate (ATP)‐sensitive K+ channel blocker, abolished the reduction of [Ca2+]i caused by the three K+ channel openers and the relaxant effect of cromakalim, whereas they only slightly attenuated the relaxant effects of pinacidil and nicorandil.

The increase in [Ca2+]i produced by 45 or 90 mM KCl‐PSS in the presence of pinacidil or nicorandil was abolished by 10−5m verapamil, indicating that the increase in [Ca2+]i was caused by the influx of extracellular Ca2+ and that pinacidil and nicorandil did not affect the voltage‐dependent Ca2+ channel directly.

The [Ca2+]i‐force relationship in the presence of cromakalim was not distinguishable from that of control.

The [Ca2+]i‐force curve was shifted to the right by pinacidil and nicorandil.

These results show that cromakalim is a more specific K+ channel opener than pinacidil and nicorandil, and that vasodilatation produced by cromakalim in this study is predominantly a result of a reduction of [Ca2+]i due to the closure of voltage‐dependent Ca2+ channels by hyperpolarization. In contrast, additional mechanisms are involved in the vasodilator actions of pinacidil and nicorandil. One of these is related to a reduction in the sensitivity of contractile proteins to Ca2+. The latter mechanism of nicorandil is akin to that of nitroglycerin. K+ channels opened by these K+ channel openers may be ATP‐sensitive ones which are blocked by glibenclamide.
Inhibitory role of endothelium‐derived relaxing factor in rat and human pulmonary arteries
Authors: Crawley D.E. , Liu S.F. , Evans T.W. , Barnes P.J.
Published: July 2012

The inhibitory role of endothelium‐derived relaxing factor was studied in both rat and human pulmonary arteries in vitro by inhibiting its synthesis with the L‐arginine analogue NG‐monomethyl‐l‐arginine (l‐NMMA).

In rat pulmonary arteries, l‐NMMA pretreatment (10–300 μm) dose‐dependently inhibited acetylcholine‐induced relaxation (which is endothelium‐dependent). NG‐monomethyl‐d‐arginine (d‐NMMA, 100 μm) was without effect, l‐Arginine, but not d‐arginine, dose‐dependently reversed this inhibition. l‐NMMA had no effect on relaxation induced by sodium nitroprusside.

In human small pulmonary arteries l‐NMMA (100 μm) pretreatment similarly inhibited the acetylcholine‐induced relaxation but had no effect on the sodium nitroprusside‐induced relaxation.

In both rat and human pulmonary arteries, l‐NMMA, but not d‐NMMA, always caused contraction of preconstricted tissues whereas it had no effect on baseline tone. In the rat this contraction was completely prevented by prior treatment with l‐arginine.

l‐NMMA (100 μm) pretreatment mimicked the effect of endothelium removal on phenylephrine‐induced vasoconstriction, both resulting in an increase in tension development at each concentration of phenylephrine. This enhancement was greatest at low concentrations of phenylephrine but was still present even at the highest concentrations. Pretreatment with l‐NMMA (100 μm) also significantly increased the responses to single doses of phenylephrine.

These results suggest that endothelium‐derived relaxing factor from endothelial cells both mediates the relaxation response to acetylcholine and also acts as a physiological brake against vasoconstriction in pulmonary vessels.
Reduction of NMDA receptors with dithiothreitol increases [3H]‐MK‐801 binding and NMDA‐induced Ca2+ fluxes
Authors: Reynolds Ian J. , Rush Elizabeth A. , Aizenman Elias
Published: July 2012

We have investigated the modulation of N‐methyl‐d‐aspartate (NMDA) receptor activation by the sulphydryl redox reagents dithiothreitol (DTT) and 5,5‐dithio‐bis‐2‐nitrobenzoic acid (DTNB).

Increases in [3H]‐MK‐801 binding produced by glutamate, glycine and spermidine were enhanced by DTT (2 mm) and diminished by DTNB (0.5 mm).

The inhibition of [3H]‐MK‐801 binding by CGS 19755 and 7‐chlorokynurenate was not altered by 2 mM DTT. However, the potency of the competitive polyamine antagonist, arcaine, was decreased by DTT.

NMDA‐induced Ca2+ fluxes into primary cultures of rat forebrain neurones were enhanced by DTT in a DTNB‐reversible fashion. In addition to augmenting the magnitude of NMDA‐induced increase in intracellular free Ca2+, 10mM DTT also prolonged the duration of the Ca2+ signal. However, DTT had no effect on the increase in Ca2+ produced by depolarizing neurones with 50 mM KCl.

These studies show that the reduction of disulphide bonds on the NMDA receptor complex by DTT increases activation. The precise site of these groups remains unclear but they are unlikely to form an integral part of the glutamate, glycine or polyamine binding domains. The enhancement of the activation of the NMDA receptor by DTT is associated with increased Ca2+ fluxes. The possible pathophysiological consequences of receptor reduction are discussed.
Carbamylcholine‐ and 5‐hydroxytryptamine‐induced contraction in rat isolated airways: inhibition by calcitonin gene‐related peptide
Authors: Cadieux Alain , Lanoue Chantal , Sirois Pierre , Barabe Jean
Published: July 2012

The effects of rat and human α‐calcitonin gene‐related peptide (α‐CGRP) were investigated in isolated smooth muscle preparations obtained from three levels of the rat respiratory tract.

Neither peptide (10−10–10−6m) had any effect on resting tension or on carbamylcholine (10−6m)‐induced tone of trachea or main bronchus. In contrast, CGRP sometimes reduced spontaneous or carbamylcholine‐induced tone of lung parenchymal strips.

CGRP produced a significant rightward shift of the log concentration‐response curves to carbamylcholine and 5‐hydroxytryptamine (5‐HT) in the main bronchus. A rightward shift was also seen in trachea and parenchymal strips but this did not achieve the level of significance. The maximal response to 5‐HT was reduced in the main bronchus and lung parenchyma whereas the maximal contraction to carbamylcholine was decreased in parenchymal strip only.

In all three airway preparations, CGRP caused concentration‐dependent inhibition of responses elicited by challenges with 10−7m carbamylcholine or 5 × 10−7m 5‐HT. The inhibitory effect of the peptide was inversely related to the size of the airways: the smaller the calibre, the greater the inhibition.

The inhibitory action of CGRP was not modified by pretreatment with tetrodotoxin (10−6m), propranolol (10−6m) or indomethacin (10−6m).

The results strongly suggest that (a) CGRP has a nonspecific inhibitory action on airway smooth muscle cells, (b) CGRP may act as a potent inhibitor of responses elicited by bronchoconstrictor substances and (c) its inhibitory activity may be most powerfully expressed in peripheral regions of the respiratory tract.